Pediatric Bone, Second Edition: Biology & Diseases

PEDIATRIC BONE BIOLOGY & DISEASES SECOND EDITION Edited by

FRANCIS H. GLORIEUX Genetics Unit, Shriners Hospital, McGill University, Montreal, Quebec, Canada

JOHN M. PETTIFOR Mineral Metabolism Research Unit. Department of Pediatrics, Chris Hani Baragwanath Hospital, Soweto, Johannesburg, South Africa

HARALD JU¨PPNER Endocrine Unit, Department of Medical and Pediatrics, Massachusetts General Hospital, and Harvard Medical School, Boston, Massachusetts, USA

AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier

Academic Press is an imprint of Elsevier 32 Jamestown Road, London NW1 7BY, UK 225 Wyman Street, Waltham, MA 02451, USA 525 B Street, Suite 1800, San Diego, CA 92101-4495, USA First Edition 2003 Second Edition 2012 Copyright Ó 2012, 2003 Elsevier Inc. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: [email protected]. Alternatively, visit the Science and Technology Books website at www.elsevierdirect.com/rights for further information Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made Medicine is an ever-changing field. Standard safety precautions must be followed, but as new research and clinical experience broaden our knowledge, changes in treatment and drug therapy may become necessary or appropriate. Readers are advised to check the most current product information provided by the manufacturer of each drug to be administered to verify the recommended dose, the method and duration of administrations, and contraindications. It is the responsibility of the treating physician, relying on experience and knowledge of the patient, to determine dosages and the best treatment for each individual patient. Neither the publisher nor the authors assume any liability for any injury and/or damage to persons or property arising from this publication British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN : 978-0-12-382040-2 For information on all Academic Press publications visit our website at www.elsevierdirect.com Typeset by TNQ Books and Journals Pvt. Ltd. Printed and bound in United States of America 12 13 14 15 10 9 8 7 6 5 4 3 2 1

Preface to the Second Edition

The first edition of this book, published in 2003, was sufficiently well received that we find it timely to publish a second edition 8 years later. A great deal of new knowledge has enriched the field of pediatric bone health and diseases during this period. We have kept in this second edition the same overall organization set in the first one. There have been changes in the list of contributors with a few declining and new ones embarking with enthusiasm. The first part of the book deals with basic information on the major systems controlling the development, structure and homeostasis of bone tissue, with one chapter added on dental development and maturation. The second part describes the tools and techniques validated for the evaluation of bone development and its abnormalities. To follow suggestions received, a new chapter on radiographic imaging completes this section. The third part deals with specific disorders or groups of disorders with emphasis where appropriate on basic aberrations and their clinical expression. As was requested by several readers, we have tried to provide more information on management of the various diseases. We hope that the new edition will be well received, as pediatricians and researchers working in the field of pediatric bone diseases see the need for such a textbook. The realm of pediatric bone diseases continues to rapidly expand and we hope that the book will be useful

to those interested in it, to the scientists at the bench to understand the clinical expression of the biological pathways and functions they study in the laboratory, and to the clinicians who wish to connect their observations with complex biological mechanisms and to the underlying genetic defects as they evaluate their patients’ response to established or new therapies. We wish to express our gratitude to Mara Conner and Megan Wickline at Elsevier/Academic Press for their understanding, patience and support. We also extend our appreciation to all the contributors to this large effort. In the preface of the first edition we expressed the hope that the content of the book would give substance to the recognition that “pediatric osteology” had come of age as a medical specialty, which has frequently provide substantial new insights into biology by the fact that an increasing number of these pediatric diseases has now been defined at the molecular level and thus may have significant implications also for the understanding of adult bone diseases. Although those who have invested their professional life into it come from a spectrum of different backgrounds, they now identify themselves more clearly with this emerging field. May the book be of help in that effort, and also entice younger colleagues to join in an exciting journey.

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List of Contributors

Judith E. Adams MBBS, FRCR, FRCP, FBIR Consultant Radiologist, Manchester Royal Infirmary & Professor of Diagnostic Radiology, University of Manchester, UK

John Damilakis Associate Professor, University of Crete, Faculty of Medicine, Department of Medical Physics, Iraklion, Crete, Greece

Laura K. Bachrach MD Professor of Pediatrics, Department of Pediatrics, Stanford University School of Medicine, Palo Alto, CA, USA

Marie B. Demay MD Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

Murat Bastepe MD, PhD Assistant Professor of Medicine, Endocrine Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA

Michael J. Econs MD Professor of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA Serge Ferrari MD Division of Bone Diseases, University Hospitals and Medical Faculty of Geneva, Switzerland

Clemens Bergwitz MD Assistant Professor of Medicine, Massachusetts General Hospital, Department of Medicine, Division Endocrinology, Massachusetts General Hospital, Boston, MA, USA

Mary Fewtrell Reader in Childhood Nutrition, Honorary Consultant Paediatrician, Childhood Nutrition Research Centre, UCL Institute of Child Health, London, UK

Maria Luisa Bianchi MD Bone Metabolism Unit, Istituto Auxologico Italiano IRCCS, Milan, Italy

Thomas J. Gardella PhD Associate Professor of Medicine, Massachusetts General Hospital, Department of Medicine, Division of Endocrinology, Massachusetts General Hospital, Endocrine Unit, Boston, MA, USA

Paolo Bianco MD Professor of Pathology, Sapienza University of Rome, Director, Anatomic Pathology, Umberto I University Hospital, Rome, Italy

Francis H. Glorieux OC, MD, PhD Professor of Surgery, Pediatrics and Human Genetics, McGill University, Adjunct Professor of Pediatrics, University of Montre´al, Director of Research, Shriners Hospital for Children, Montreal, Canada

Nick Bishop MD Head, Academic Unit of Child Health, Professor of Paediatric Bone Disease, University of Sheffield, Sheffield Children’s Hospital, Sheffield, UK Lynda F. Bonewald PhD Vice Chancellor for Research Interim, Curator’s Professor, Lee M and William Lefkowitz Professor, Director, Bone Biology Research Program Director, UMKC Center of Excellence in Mineralized Tissues University of Missouri at Kansas City School of Dentistry, Department of Oral Biology, Kansas City, MO, USA

Michel Goldberg DDS Professeur e´merite, UMR-S 747INSERM Universite´ Paris Descartes Nick Harvey MD Lecturer and Honorary Consultant in Rheumatology, The MRC Lifecourse Epidemiology Unit, University of Southampton, Southampton General Hospital, Southampton, UK

Jean-Philippe Bonjour MD Division of Bone Diseases, University Hospitals and Medical Faculty of Geneva, Switzerland

Priv.Dozent Dr Wolfgang Ho¨gler Consultant Paediatric Endocrinologist, Birmingham Children’s Hospital, Honorary Senior Lecturer, University of Birmingham, UK

Noe¨l Cameron Professor of Human Biology, Centre for Global Health and Human Development, Loughborough University, Loughborough, Leicestershire, UK

Ingrid A. Holm MD, MPH Division of Genetics, Program in Genomics, and the Manton Center for Orphan Disease Research, Children’s Hospital Boston and Harvard Medical School, Boston, MA, USA

Thomas O. Carpenter MD Departments of Pediatrics (Endocrinology) and Orthopaedics and Rehabilitation, Yale University School of Medicine, New Haven, CT, USA

Katharina Ja¨hn PhD University of Missouri-Kansas City, School of Dentistry, Kansas City, MO, USA

Thierry Chevalley MD Division of Bone Diseases, University Hospitals and Medical Faculty of Geneva, Switzerland

Harald Ju¨ppner MD Professor of Pediatrics, Endocrine Unit and Pediatric Nephrology Unit, Department of Medicine and Pediatrics, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA

William G. Cole MD, PhD Chief of Pediatric Surgery, University of Alberta, Canada

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xii

LIST OF CONTRIBUTORS

Frederick S. Kaplan MD Departments of Orthopedic Surgery, Medicine, and the Center for Research in FOP and Related Disorders, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Christopher S. Kovacs MD, FRCPC, FACP Professor of Medicine (Endocrinology and Metabolism), Obstetrics & Gynecology, and BioMedical Sciences, Memorial University of Newfoundland, Attending and Consultant Specialist (Endocrinology and Metabolism), Health Sciences Centre and Eastern Health Corporation, St. John’s, Newfoundland, Canada Henry M. Kronenberg MD, PhD Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA Mary B. Leonard MD, MSCE Associate Professor of Pediatrics & Epidemiology, The Children’s Hospital of Philadelphia, Department of Pediatrics, Department of Biostatistics and Epidemiology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Fanxin Long PhD Associate Professor of Medicine, and Associate Professor of Developmental Biology, Division of Endocrinology, Metabolism and Lipid Research, Department of Medicine, Washington University in St Louis, MO, USA Christa Maes PhD Laboratory of Experimental Medicine and Endocrinology (LEGENDO), Katholieke Universiteit Leuven, Belgium

John M. Pettifor MBBCh, PhD Mineral Metabolism Research Unit, Department of Paediatrics, University of the Witwatersrand and Chris Hani Baragwanath, South Africa Anthony A. Portale MD Professor of Pediatrics, University of California San Francisco, San Francisco, CA, USA John T. Potts MD Endocrine Unit, Department of Medicine, The Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA Ann Prentice PhD Director, MRC Human Nutrition Research, Elsie Widdowson Laboratory, Fulbourn Road, Cambridge, UK Frank Rauch MD Associate Professor, Genetics Unit, Shriners Hospital for Children, Montreal, Canada Jean-Marc Retrouvey Division of Orthodontics, McGill University, Montreal, Quebec, Canada David L. Rimoin MD, PhD Medical Genetics Birth Defects Center, Cedars-Sinai Health System and Department of Pediatrics and Medicine, UCLA School of Medicine, Los Angeles, CA, USA Rene Rizzoli MD Division of Bone Diseases, University Hospitals and Medical Faculty of Geneva, Switzerland David Rowe Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT, USA Isidro B. Salusky MD Professor of Pediatrics, Division of Pediatric Nephrology, Director, Pediatric Dialysis Program, Director, General Clinical Research Center, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

Daniella Magen MD Division of Pediatric Nephrology, Meyer Children’s Hospital, Rambam Health Care Campus and Laboratory of Molecular Medicine, Rappaport Faculty of Medicine and Research Institute Technion e Israel Institute of Technology, Haifa, Israel

Ste´phane Schwartz DDS Assistant Director, Dental Clinic Montreal Children’s Hospital MUHC (McGill University Health Center) Associate Professor, McGill University

David D. Martin MD Paediatric Endocrinology and Diabetology, University Children’s Hospital, Tu¨bingen University, Tu¨bingen, Germany

Nick Shaw MD Consultant Paediatric Endocrinologist, Birmingham Children’s Hospital, Honorary Senior Lecturer, University of Birmingham, UK

Marc D. McKee PhD Professor, Faculty of Dentistry, Department of Anatomy & Cell Biology and Faculty of Medicine, McGill University, Montreal, Quebec, Canada

Eileen M. Shore PhD Departments of Orthopedic Surgery, Genetics, and the Center for Research in FOP and Related Disorders, University of Pennsylvania School of Medicine, Philadelphia, PA, USA

Walter L. Miller MD Distinguished Professor of Pediatrics, University of California San Francisco, San Francisco, CA, USA Geert Mortier MD Department of Medical Genetics, University of Antwerp, Antwerp, Belgium Zulf Mughal Consultant in Paediatric Bone Disorders & Honorary Senior Lecturer in Child Health, Royal Manchester Children’s Hospital, Central Manchester University Hospitals NHS Foundation Trust, Manchester, UK Amaka C. Offiah HEFCE Clinical Senior Lecturer, Academic Unit of Child Health, Sheffield Children’s NHS Foundation Trust, Sheffield, UK Bram Perdu MD Department of Medical University of Antwerp, Antwerp, Belgium

Genetics,

Farzana Perwad MD Assistant Professor of Pediatrics, University of California San Francisco, San Francisco, CA, USA

Rene´ St-Arnaud PhD Genetics Unit, Shriners Hospital for Children, Montre´al (Que´bec) and Departments of Surgery and Human Genetics, McGill University, Montre´al (Que´bec), Canada Andrea Superti-Furga Professor, University of Lausanne, Division of Pediatrics, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland Rajesh V. Thakker MD, ScD, FRCP, FRCPath, FMedSci Professor of Medicine, Academic Endocrine Unit, Nuffield Department of Medicine, Oxford Centre for Diabetes, Endocrinology and Metabolism (OCDEM), Churchill Hospital, Headington, Oxford, UK Sheila Unger MD, FRCP University of Lausanne, Service of Medical Genetics, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland Filip Vanhoenacker MD Department of University Hospital of Antwerp, Belgium

Radiology,

LIST OF CONTRIBUTORS

Wim Van Hul MD Department of Medical Genetics, University and University Hospital Antwerp, Belgium Katherine Wesseling Perry MD David Geffen School of Medicine at University of California, Los Angeles, CA, USA Michael P. Whyte MD Medical-Scientific Director, Center for Metabolic Bone Disease and Molecular Research, Shriners Hospital for Children, Professor of Medicine, Pediatrics, and Genetics, Washington University School of Medicine, St. Louis, MO, USA

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Shlomo Wientroub MD Department of Pediatric Orthopedic Surgery, Dana Children’s Hospital, Tel Aviv Medical School, Tel Aviv, Israel Israel Zelikovic MD Division of Pediatric Nephrology, Meyer Children’s Hospital, Rambam Health Care Campus, and Laboratory of Developmental Nephrology, Department of Physiology and Biophysics, Rappaport Faculty of Medicine and Research Institute, Technion e Israel Institute of Technology, Haifa, Israel

C H A P T E R

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Bone Cell Biology: Osteoclasts, Osteoblasts, Osteocytes K. Ja¨hn, L.F. Bonewald University of Missouri-Kansas City, School of Dentistry, 650 E 25th Street, Kansas City, MO 64108, USA

INTRODUCTION TO BONE

RANKL, ephrins, and sclerostin as described in this chapter. Moreover, the extracellular matrix (ECM) on which osteoblasts and osteoclasts attach and in which osteocytes reside, influences the function of these cells. Although previously viewed as mainly a support structure for bone cells, it is now clear that the bone ECM controls and directs bone cell function. This chapter will provide an overview of how bone cells coordinate their actions to generate, maintain and remove bone mass.

Depending upon field of study, bone is described very differently. From a material, biochemical, or physical point of view, bone is described as a composite material that is made up of an organic component consisting of collagenous and non-collagenous proteins and a mineral phase consisting of calcium and phosphate [1]. Often bone is viewed as a dead, hard material with only a structural purpose, to hold the body in place, to protect internal organs, and to serve as attachment sites for skeletal muscles to achieve movement. Yet bone is far from being either dead or static. From a biological point of view, bone is a complex living tissue in which this composite material of organic and mineral components is created and maintained by at least three major cell types, namely osteoblasts, osteocytes and osteoclasts (see schematic bone remodeling unit in Figure 1.1) [2]. In the first and second decades of human life, bone is constantly growing. Towards the end of the second and into the third decades, dynamic remodeling takes place to maintain the skeleton with the potential for increasing bone mass. After the third decade, bone mass starts its inevitable decline. Considering this simplified time course of human bone development, it is obvious that bone is dynamically controlled and remodeled. As a dynamic connective tissue, bone is constantly responding to external forces such as loading of the skeleton and to internal and external signals such as cytokines, growth factors and hormones. Several hormones have been shown to play important roles in the skeleton such as the estrogens and androgens, parathyroid hormone (PTH) and 1,25-dihydroxyvitamin vitamin D3 (1,25,D3). In addition to these external signals, bone cells are in constant communication with each other and cells of the immune and hematopoietic systems, through factors such as osteoprotogerin,

Pediatric Bone, Second Edition DOI: 10.1016/B978-0-12-382040-2.10001-2

OSTEOCLASTS The cells that resorb the mineralized ECM of bone are called osteoclasts. They arise from hematopoietic progenitors that also give rise to macrophages. The osteoclast precursor cells are recruited to the bone surface where they fuse to form multinucleated cells [3] (see schematic image in Figure 1.2). The co-stimulating molecule RANKL (RANK ligand), which is expressed by osteoblasts, stromal cells and osteocytes, is the most commonly known cellular activation pathway of osteoclast-precursors and the osteoclastogenic cascade of transcription factors [3,4]. Osteoclasts are very rare in bone, only two to three cells per mm3 bone can be found [5]. The active form of these multinucleated giant cells is present in specialized cavities on the bone surface, known as Howship’s lacunae. Osteoclasts show a high level of polarization when attached to the bone surface. The most characteristic feature in this state is the ruffled border, which consists of finger-like cytoplasmic projections [6] and is turned towards the bone surface. The osteoclast seals the cavity around its ruffled border and then secretes protons and a variety of proteolytic enzymes, i.e. collagenases, gelatinases, into the cavity, to carry out the organic breakdown of the bone ECM [7].

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Copyright Ó 2012 Elsevier Inc. All rights reserved.

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1. BONE CELL BIOLOGY: OSTEOCLASTS, OSTEOBLASTS, OSTEOCYTES

FIGURE 1.1

The bone remodeling unit. This drawing of a bone remodeling unit depicts an active osteoclast sitting in its Howship’s lacunae releasing protons and enzymes into a sealed zone to degrade and digest the mineralized bone matrix. Cuboidal-shaped osteoblasts are positioned behind the osteoclasts secreting type I collagen and other non-collagenous proteins to form the osteoid e the newly deposited, unmineralized bone matrix. Several differentiation stages of the osteoblast phenotype are illustrated. The spindle-shaped bone lining cells are covering all inactive bone surfaces, as well as the bone remodeling unit to create a micromilieu where theoretically cross-talk takes place between osteoclasts and osteoblasts. Transition stages from the osteoblast to the osteocyte are shown including the early osteoid osteocyte to the mature embedded osteocyte. The role of the osteocyte to create a communication network between practically all cells on the bone surface is demonstrated by the extensive cell processes connecting osteocytes, osteoblasts, bone lining cells and osteoclasts.

The sole function of the osteoclast is to resorb bone. The mature osteoclast is described histologically as a multinucleated, tartrate resistant, acid phosphatase positive cell. However, macrophage polykaryons can have these same characteristics; so the “gold standard” for identifying an osteoclast is the formation of resorption lacunae or “pits” on a mineralized surface (see histological image in Figure 1.2). Other characteristics of the osteoclast include the expression of calcitonin receptors, enzymes such as cathepsin K and matrix metalloprotein-9 (MMP-9) that play a role in matrix degradation, and the vacuolar proton pump for the transport of protons to the resorption lacunae. For the osteoclast to resorb, it must form a “sealing zone” around the periphery of its attached area to concentrate not only proteases but also protons into this limited area. Underneath the cell, within the ruffled border, the pH is reduced to approximately 2e3 which enhances the degradation of mineralized matrix [8]. As osteoclast precursors are derived from hematopoietic precursors, the same stem cells that become granulocytes and monocytes/macrophages, cell lines such as RAW 267.4 and MOPC-5, are available that represent osteoclast precursors for experimental studies. These

cells can form TRAP positive multinucleated cells that resorb bone and form resorption lacunae on dentin [9]. It has been well known for the last 10e15 years in the bone field that osteoclast precursors require supporting cells for osteoclast formation. The importance of factors produced by supporting cells such as macrophage colony stimulating factor (M-CSF) to induce proliferation of osteoclast precursors has been validated [10]. Critical factors and cell surface molecules involved in osteoclast formation have only recently been elucidated with the discovery of RANK ligand (RANKL) and osteoprotogerin (OPG) [11,12]. The osteoclast precursor expresses a receptor known as RANK (receptor activator of NFkB) that signals through the NF-kB pathway. The binding of the cell membrane bound ligand, RANKL, activates RANK receptor. However, the soluble factor, OPG, acting as a “decoy” receptor can bind to RANKL, preventing osteoclast formation. The expression of RANKL on the surface of supporting cells occurs when these cells are exposed to bone resorbing cytokines, hormones, and factors such as interleukins 1, 6, 11, PTH, PTH-related protein (PTHrp), oncostatin M, leukemia inhibitory factor, prostaglandin E2, or 1,25 D3 [13]. These factors upregulate RANKL to a level

PEDIATRIC BONE

THE OSTEOBLAST CELL LINEAGE

FIGURE 1.2 Osteoclast differentiation and activity. The activation of osteoclast precursors to fuse and form active osteoclasts is shown in the schematic image (upper panel). Arising from a hematopoietic precursor that is activated by M-CSF and RANKL, the pre-osteoclast is formed and fuses with others to produce multinucleated TRAP positive cells, which finally form a sealing zone to attach to the bone matrix to form the bone resorption cavity underneath a ruffled border. Active, TRAP positive stained osteoclasts (red) can be seen on the histological image taken from the distal region of a mouse femur. Methyl green was used as counterstaining to label all cell nuclei. (See color plate section.)

capable of overcoming the effects of circulating OPG, thereby resulting in osteoclast formation. Efforts to generate osteoclasts without supporting cells have only recently been accomplished in vitro by using an artificial, soluble form of RANKL [14]. A new therapeutic called Denosumab which is an anti-RANKL antibody has been approved for the treatment of osteoporosis [15,16].

THE OSTEOBLAST CELL LINEAGE Osteoblasts are known as the cells that form bone, characterized by their unique ability to secrete a type I collagen-rich ECM that eventually mineralizes.

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Osteoblasts were first named by Gegenbaur in 1864 [17], and since then their function as the major proteinexpressing cells in bone has been widely studied. All of the cells and stages of differentiation with the osteoblast lineage are often quickly summarized as “the osteoblast”. However, at least four to five major maturation stages of the osteoblast cell lineage are commonly accepted, the immature osteoblast referred to as preosteoblasts, mature osteoblasts, osteoid ostecytes, early osteocyte, and mature osteocytes as terminally differentiated osteoblasts embedded in the bone matrix [18]. Franz-Odendaal et al. in 2006 postulated a total of eight different maturation stages for the transition of osteoblasts to osteocytes based on morphological features as well as expression profiles of the cells [19]. The recent classification of osteoblast-lineage clearly distinguishes between active surface osteoblasts that secrete the ECM and several maturation stages of osteoid osteoblasts and young osteocytes to mature and deeply embedded osteocytes. Lining cells on the bone surface are thought also to be terminally differentiated osteoblasts on the bone surface, but the lineage of these cells has not been completely validated (a short summary of osteoblast maturation stages and markers can be found in Figure 1.3). Osteoblasts arise from multipotent progenitor cells of mesenchyme origin, mesenchymal stem cells. Friedenstein and co-workers were the first to characterize these clonogenic cells by their adherence to tissue culture plastic surfaces, and discovered that this isolated population is able to differentiate into a variety of mature cell types ranging from osteoblasts, to chondrocytes or adipocytes, under the appropriate conditions [20,21]. The niche for osteoprogenitors in bone is the periosteum, the endosteum, and the marrow stroma [22,23]. With bone trauma, fracture, or other conditions requiring bone repair, mesenchymal progenitor cells serve as a stem cell reservoir that are recruited to the injury site. However, unlike the hematopoietic stem cell, the identification of mesenchymal stem cells has not been as thoroughly characterized. One can identify single hematopoietic stem cells, but this has not been accomplished for mesenchymal stem cells. At present, a population of cells with a series and panel of markers has been identified with the capacity to differentiate into osteoblasts [24]. Chemotactic molecules such as osteopontin [25] and various members of the transforming growth factor-b family [26,27], which are stored in great amounts in the bone ECM, when released attract progenitor cells to the injured site or to a previously resorbed, remodeling site. These progenitors differentiate into matrix-producing osteoblasts. A “master control gene” necessary for progenitor cells to differentiate into osteoblasts is the transcription factor Runx2 (Runt-related transcription factor-2; also

PEDIATRIC BONE

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1. BONE CELL BIOLOGY: OSTEOCLASTS, OSTEOBLASTS, OSTEOCYTES

FIGURE 1.3 The osteoblast lineage. The upper panel represents the differentiation of a pre-osteoblast into an osteocyte. While the preosteoblast descended from the mesenchymal progenitor cell is mainly but not entirely expressing type I collagen, the mature osteoblast also expresses a variety of non-collagenous proteins for ECM synthesis, including bone sialoprotein and osteocalcin, a bone specific marker. During the differentiation into mature osteocytes, several proteins are upregulated such as E11/gp38, a marker for embedding and early osteocytes and sclerostin a marker for late, mature osteocytes. The histological image from the distal region of a mouse femur (stained with hematoxylin and eosin) shows an active cuboidal osteoblast on the bone surface (purple arrow), an early osteocyte (blue arrow) and a mature, stellar-shaped osteocyte deeply embedded in the bone matrix (green arrow). (See color plate section.)

named Cbfa1, Osf2, Pebp2a1, or Aml3) [28,29]. Runx2 is of crucial importance during osteoblast differentiation. This was dramatically demonstrated by deleting or “knocking-out” the gene in mice. Newborn mice possessed a cartilagenous, but non-mineralized skeleton and died upon birth due to inability to breathe. The importance of this “master control gene” was highlighted in 1997, when a total of five papers were

published in Cell describing the role of Runx2 during osteoblast differentiation and the dramatic effects of its targeted deletion in mice models leading to the complete absence of bone formation [28,30e33]. Runx2 was the first true osteoblast-specific transcription factor identified through its binding site within the promotor of the osteocalcin gene [34]. Runx2 is known to control osteoblast differentiation by subsequently activating the expression of osteoblast phenotype-specific genes such as type I collagen and osteocalcin [34,35]. While Runx2 is the earliest and most specific marker of osteogenesis identified to date [36], several homeodomain transcription factors, such as Msx2, Dlx5, Bapx1, and Hoxa-2 have been suggested to regulate Runx2 expression. A second crucial transcription factor required for osteoblast differentiation during development that acts downstream of Runx2 is osterix (Osx) [35]. Mice lacking Osx have a similar phenotype to those lacking Runx2. Nakashima et al. demonstrated that osterix-null mice do not show signs of intramembranous or endochondral bone formation with the absence of bone markers such as osteocalcin, osteopontin, or osteonectin [35]. The active, mature osteoblasts exhibit a cuboidal or polygonal cell shape. As these cells are responsible for the secretion of the bone matrix or osteoid, osteoblasts contain abundant endoplasmatic reticula and enlarged Golgi apparatus [37]. The major protein secreted by osteoblasts is type I collagen, a fibrillar extracellular matrix protein that determines the tensile strength of bone. Type I collagen is not exclusively found within bone, it is in fact one of the most abundant proteins in vertebrates and is present in skin, tendons and ligaments. Each type I collagen molecule is composed of three polypeptide chains (one a2 and two a1 chains) that are organized as a right-handed triple helix [38e40]. Osteoblasts further express a variety of so-called non-collagenous proteins to form the unmineralized matrix in the osteoid seam [41,42]. The specific function of most of these proteins is still not completely understood. Osteocalcin (OCN) is one of the most prominent non-collagenous proteins that is bone-specific in expression. This small 49 amino acid protein contains, in most species, uncommon post-transcriptional modification, gammacarboxylation, of three glutamic acids (Gla). These calcium-binding residues lead to the formation of “Gla”-helices which bind to hydroxyapatite (the inorganic component of the ECM of bone) [38]. OCN is expressed during the late stage of osteoblast differentiation in vitro [43]. Moreover, in embryonic bone, it is upregulated with the early onset of hydroxyapatite crystal formation [40]. Therefore, OCN has been suggested to be one of the main regulators of bone turnover, and mineralization [44]. Recent findings also imply a role for this calcium-binding protein in its undercarboxylated form in the hormonal regulation of glucose

PEDIATRIC BONE

THE OSTEOCYTE

and fat metabolism [45]. This has opened a whole new area of investigation and supports the novel hypothesis that bone can function as an endocrine organ. Other cells derived from the osteoblastic-lineage are bone lining cells. This origin of lining cells has been very controversial. Bone lining cells are thought to be resting osteoblasts, pre-osteoblasts or post-osteoblasts. We know that these cells cover almost all surfaces in adult bone to build a connective tissue barrier. More recently, bone lining cells were acknowledged as active cells that participate in bone resorption and formation. Moreover, these cells appear to be involved in homeostatic processes as they are in communication with the osteocyte network and are an element of the strain sensing network [37]. Bone lining cells may also be one of the major sources for active osteoblasts and could be seen as a reservoir for pre-osteoblasts if subjected to the right stimulus. This theory was proposed by Dobnig et al., where intermittent treatment of adult rats with PTH led to a reactivation of bone lining cells to bone forming osteoblasts that led to increased bone formation [46].

THE OSTEOCYTE The mature differentiated stage of the osteoblast lineage becomes apparent when osteoblasts are surrounded by the ECM of bone. Osteoblasts then undergo morphological changes; they develop long slender-like cell processes, lose many of their cytoplasmic organelles and develop into stellar-shaped cells [18]. This very distinctive osteoblast maturation stage is then called an osteocyte. Osteocytes possess a very unique location in bone, being trapped within lacunae similar to small “caves” inside the bone matrix, where these cells form a connective network by sending their dendritic processes through small “tunnels”, called canaliculi, that connect and span throughout the whole bone volume. It is unclear why, considering that osteocytes are also the most abundant cell type found in bone [47], their specific role within bone biology was overlooked for decades. This is thought to be due to the difficulty of isolating and removing mature osteocytes from the mineralized matrix. The proposed main function of osteocytes was to sense distribution and amount of mechanical strain that is applied to the bone [48,49]. A key factor that led to the discovery of this particular function of osteocytes is their location within the load-bearing ECM of bone [50]. Moreover, the importance of the integrity of the osteocytic network is highlighted in aging or microdamaged bone or with certain treatments, i.e. glucocorticoids, where the loss of viable osteocytes causes bone

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tissue to become a truly inactive, dead tissue [51,52]. The transfer of the strain information between osteocytes is realized through their long slender-like processes e the most dramatic morphological feature of osteocytes. Whereas numerous osteoblast cell lines have been generated for experimental studies, only one osteocyte cell line has been generated to date, the MLO-Y4 cell that possesses dendritic processes and early markers for osteocytes. This cell line has been and is being used extensively to elucidate osteocyte function [53e59]. Gap junctions at the end of these processes enable the osteocytes to communicate. Gap junctions are specialized cellecell contact points which are formed by members of the connexin protein family [60,61]. Six of these transmembrane proteins are joined across the extracellular gap between two adjacent cells to form a channel structure allowing the passage of small metabolites, ions and intracellular signaling molecules below a size of 1 kDa. But what causes this form of intracellular signaling due to mechanical stimulation? In theory, there are at least three different forms of mechanical strain on bone cells e hydrostatic pressure, direct cell strain and fluid flow-induced shear stress. The most commonly accepted theory is load-induced fluid flow [48,49,60]. Interstitial fluid is squeezed through the porous ECM and through the lacunarecanaliculi system in response to bone deformations by physiological loading and the shear stresses then act directly on the outer cellular structures of the osteocytes [62]. The most prominent molecules that have been involved in mechanotransduction in bone are nitric oxide [63], adenosine triphosphate (ATP) [64,65], prostaglandin (PGE2) [49,66] and calcium [67]. Osteocytes have recently come into the spotlight as they appear to be multifunctional [68]. One function is to contribute to the regulation of mineral homeostasis. It has been shown that osteocytes can regulate calcium availability by removing and replacing their mineralized matrix under normal, healthy conditions such as lactation [68,69]. In addition to the regulation of calcium, osteocytes have a major role in the regulation of phosphate homeostasis. Osteocytes regulate both biomineralization and phosphate through molecules such as phosphate regulating factor with homologies to endopeptidase on chromosome X (Phex), dentin matrix protein-1 (Dmp1), and fibroblast growth factor (FGF23), all expressed by osteocytes [70e73]. Both Dmp1 and Phex appear to downregulate the expression of FGF-23, which allows more efficient reabsorption of phosphate by the kidney thereby maintaining sufficient circulating phosphate to maintain normal bone mineral content. Dmp1 null mice have a similar phenotype to hyp mice carrying a Phex mutation, that of osteomalacia and rickets due to elevated FGF-23 levels in osteocytes

PEDIATRIC BONE

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1. BONE CELL BIOLOGY: OSTEOCLASTS, OSTEOBLASTS, OSTEOCYTES

[73,74]. While autosomal dominant hypophosphatemic rickets in patients is due to mutations in Phex [75], autosomal recessive hypophosphatemic rickets is due to mutations in Dmp1 [74] and both show elevation of FGF-23 in osteocytes. Based on these observations, we proposed that the osteocyte lacunocanalicular network can function as an endocrine system, targeting distant organs such as kidney [74]. Another function of osteocytes is to act as orchestrators of both bone formation and bone resorption. Osteocytes produce a factor, sclerostin, which inhibits osteoblastic bone formation [76,77]. This molecule is now the target of therapeutics as an antibody to sclerostin has been shown to enhance bone mass and promote bone healing [78e81]. Conversely, osteocytes can support osteoclast formation, especially in their dying or apoptotic state [82,83]. Osteocytes appear to express both RANKL and OPG [84]. What is not clear is how factors produced by osteocytes can reach the bone surface, but Dallas and co-workers have shown by dynamic imaging that osteocytes can extend their dendritic processes into marrow and vascular spaces suggesting a mean for the delivery of factors [85,86].

CONCLUSION Not only are bone cells in constant communication with each other, but the ECM can also influence the function of bone cells. Bone is a reservoir of factors ready to be released during resorption that can modify the bone coupling process or provide circulating growth factors. A number of transcription factors as described above have been identified that are specific for bone induction and development. Clearly, these growth factors and transcription factors are regulated by a number of circulating hormones such as parathyroid hormone, estrogen, and 1,25(OH)2D3. As outlined in this chapter, another layer of complexity is added due to the fact that bone structure is also regulated by mechanical load or lack of mechanical loading. Understanding the normal physiology of bone and its diseases has led to the generation of therapeutics important in the prevention and treatment of bone disease and acceleration and initiation of bone repair. Studies are also underway to identify means to treat or reverse abnormal bone development based on studies of bone cell origin, function, and their regulation.

References [1] Cowin SC. Bone Mechanics Handbook. New York: CRC Press; 2001. [2] Bilezikian JP, Lawrence GR, Rodan GA. Principles of Bone Biology. Academic Press; 2002.

[3] Takahashi N, Udagawa N, Takami M, Suda T. Cells of bone: osteoclast generation. In: Bilezikian JP, Raisz LG, Rodan GA, editors. Principles of Bone Biology. Academic Press; 2002. p. 109e26. [4] Matsuo K, Irie N. Osteoclast-osteoblast communication. Arch Biochem Biophys 2008;473:201e9. [5] Meunier PJ, Coindre JM, Edouard CM, Arlot ME. Bone histomorphometry in Paget’s disease. Quantitative and dynamic analysis of pagetic and nonpagetic bone tissue. Arthritis Rheum 1980;23:1095e103. [6] Roodman GD. Advances in bone biology: the osteoclast. Endocr Rev 1996;17:308e32. [7] Hill PA. Bone remodelling. Br J Orthod 1998;25:101e7. [8] Roodman GD. Advances in bone biology: The osteoclast. Endocr Rev 1996;17:308e32. [9] Chen W, Li YP. Generation of mouse osteoclastogenic cell lines immortalized with SV40 large T antigen. J Bone Miner Res 1998;13:1112e23. [10] Kodama H, Nose M, Niida S, Yamasaki A. Essential role of macrophage colony-stimulating factor in the osteoclast differentiation supported by stromal cells. J Exp Med 1991;173:1291e4. [11] Yoshizawa T, Handa Y, Uematsu Y, et al. Mice lacking the vitamin D receptor exhibit impaired bone formation, uterine hypoplasia and growth retardation after weaning. Nat Genet 1997;16:391e6. [12] Simonet WS, Lacey DL, Dunstan CR, et al. Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 1997;89:309e19. [13] Aubin JE, Bonnelye E. Osteoprotegerin and its ligand: A new paradigm for regulation of osteoclastogenesis and bone resorption. Medscape Womens Health 2000;5:5. [14] Quinn JM, Elliott J, Gillespie MT, Martin TJ. A combination of osteoclast differentiation factor and macrophage-colony stimulating factor is sufficient for both human and mouse osteoclast formation in vitro. Endocrinology 1998;139:4424e7. [15] Bekker PJHD, Rasmussen AS, Murphy R, et al. A single-dose placebo-controlled study of AMG 162, a fully human monoclonal antibody to RANKL, in postmenopausal women. J Bone Miner Res 2004;19:1059e66. [16] Rizzoli RYU, Kirkpatrick P. Denosumab. Nat Rev Drug Discov 2010;9:591e2. ¨ ber die Bildung des Knochengewebes. Natur[17] Gegenbaur C. U wissenschaften 1864. [18] Aubin JE, Liu F. The osteoblast lineage. In: Bilezikian JP, Raisz LG, Rodan GA, editors. Principles in Bone Biology. Academic Press; 1996. p. 51e67. [19] Franz-Odendaal TA, Hall BK, Witten PE. Buried alive: how osteoblasts become osteocytes. Dev Dyn 2006;235:176e90. [20] Friedenstein AY, Lalykina KS. Lymphoid cell populations are competent systems for induced osteogenesis. Calcif Tiss Res 1970;(Suppl):105e6. [21] Friedenstein AJ, Chailakhyan RK, Gerasimov UV. Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers. Cell Tiss Kinet 1987;20:263e72. [22] Burger EH, Boonekamp PM, Nijweide PJ. Osteoblast and osteoclast precursors in primary cultures of calvarial bone cells. Anat Rec 1986;214:32e40. [23] Aubin JE, Triffitt JT. Mesenchymal stem cells and osteoblast differentiation. In: Bilezikian JP, Raisz LG, Rodan GA, editors. Principles of Bone Biology. Academic Press; 2002. p. 59e82. [24] Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006;8:315e7.

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REFERENCES

[25] Forquer Raheja L, Geneto DC, Yellowley CE. Hypoxic osteocytes recruit human MSCs through an OPN/CD44-mediated pathway. Biochem Biophys Res Commun 2008;366:393e403. [26] Postlethwaite AE, Seyer JM. Identification of a chemotactic epitope in human transforming growth factor-beta 1 spanning amino acid residues 368-374. J Cell Physiol 1995;164:587e92. [27] Cho TJ, Gerstenfeld LC, Einhorn TA. Differential temporal expression of members of the transforming growth factor beta superfamily during murine fracture healing. J Bone Miner Res 2002;17:513e20. [28] Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsenty G. Osf2/ Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 1997;89:747e54. [29] Schinke T, Karsenty G. Transcriptional control of osteoblast differentiation and function. In: Bilezikian JP, Raisz LG, Rodan GA, editors. Principles of Bone Biology. Academic Press; 2002. p. 83e91. [30] Komori T, Yagi H, Nomura S, et al. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 1997;89:755e64. [31] Mundlos S, Otto F, Mundlos C, et al. Mutations involving the transcription factor CBFA1 cause cleidocranial dysplasia. Cell 1997;89:773e9. [32] Otto F, Thornell AP, Crompton T, et al. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 1997;89:765e71. [33] Rodan GA, Harada S. The missing bone. Cell 1997;89:677e80. [34] Ducy P. Cbfa1: a molecular switch in osteoblast biology. Dev Dyn 2000;219:461e71. [35] Nakashima K, Zhou X, Kunkel G, et al. The novel zinc fingercontaining transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 2002;108:17e29. [36] Karsenty G. How many factors are required to remodel bone? Nat Med 2000;6:970e1. [37] Majeska RJ. Cell biology of bone. In: Cowin SC, editor. Bone Mechanics Handbook. CRC Press; 2001. p. 1e24. [38] Calvo MS, Eyre DR, Gundberg CM. Molecular basis and clinical application of biological markers of bone turnover. Endocr Rev 1996;17:333e68. [39] Prockop DJ. Mutations that alter the primary structure of type I collagen. The perils of a system for generating large structures by the principle of nucleated growth. J Biol Chem 1990;265:15349e52. [40] Rossert J, de Crombrugghe B. Type I collagen. In: Bilezikian JP, Raisz LG, Rodan GA, editors. Principles in Bone Biology. Academic Press; 2002. p. 189e210. [41] Hauschka PV, Lian JB, Cole DE, Gundberg CM. Osteocalcin and matrix Gla protein: vitamin K-dependent proteins in bone. Physiol Rev 1989;69:990e1047. [42] Oldberg A, Franzen A, Heinegard D. Cloning and sequence analysis of rat bone sialoprotein (osteopontin) cDNA reveals an Arg-Gly-Asp cell-binding sequence. Proc Natl Acad Sci USA 1986;83:8819e23. [43] Lian JB, Stein GS. Concepts of osteoblast growth and differentiation: basis for modulation of bone cell development and tissue formation. Crit Rev Oral Biol Med 1992;3:269e305. [44] Ivaska KK, Hentunen TA, Vaaraniemi J, Ylipahkala H, Pettersson K, Vaananen HK. Release of intact and fragmented osteocalcin molecules from bone matrix during bone resorption in vitro. J Biol Chem 2004;279:18361e9. [45] Ferron M, Hinoi E, Karsenty G, Ducy P. Osteocalcin differentially regulates beta cell and adipocyte gene expression and affects the development of metabolic diseases in wild-type mice. Proc Natl Acad Sci USA 2008;105:5266e70.

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[46] Dobnig H, Turner RT. Evidence that intermittent treatment with parathyroid hormone increases bone formation in adult rats by activation of bone lining cells. Endocrinology 1995;136:3632e8. [47] Parfitt AM. The cellular basis of bone turnover and bone loss: a rebuttal of the osteocytic resorption-bone flow theory. Clin Orthop Relat Res 1977;127:236e47. [48] Knothe Tate ML. Whither flows the fluid in bone? An osteocyte’s perspective. J Biomech 2003;36:1409e24. [49] Klein-Nulend J, van der PA, Semeins CM, et al. Sensitivity of osteocytes to biomechanical stress in vitro. FASEB J 1995;9:441e5. [50] Lanyon LE. Osteocytes, strain detection, bone modeling and remodeling. Calcif Tissue Int 1993;53(Suppl.1):S102e6. [51] Dunstan CR, Somers NM, Evans RA. Osteocyte death and hip fracture. Calcif Tissue Int 1993;53(Suppl.1):S113e6. [52] Frost HM. In vivo osteocyte death. J Bone Joint Surg Am 1960;42-A:138e43. [53] Kato Y, Windle JJ, Koop BA, Mundy GR, Bonewald LF. Establishment of an osteocyte-like cell line, MLO-Y4. J Bone Miner Res 1997;12:2014e23. [54] Bonewald L. Establishment and characterization of an osteocyte-like cell line, MLO-Y4. J Bone Miner Metab 1999;17:61e5. [55] Cheng BZ, Luo J, Sprague E, Bonewald LF, Jiang JX. Expression of functional gap junctions and regulation by fluid flow in osteocyte-like MLO-Y4 cells. J Bone Miner Res 2001;16:249e59. [56] Plotkin LI, Bellido T. Glucocorticoids induce osteocyte apoptosis by blocking focal adhesion kinase-mediated survival. Evidence for inside-out signaling leading to anoikis. J Biol Chem 2007;282:24120e30. [57] Kogianni GMV, Noble BS. Apoptotic bodies convey activity capable of initiating osteoclastogenesis and localized bone destruction. J Bone Miner Res 2008;23:915e27. [58] Bakker ADSV, Krishnan R, Bacabac RG, et al. Tumor necrosis factor alpha and interleukin-1beta modulate calcium and nitric oxide signaling in mechanically stimulated osteocytes. Arthritis Rheum 2009;60:3336e45. [59] Guo DKA, Guthrie J, Veno PA, Harris SE, Bonewald LF. Identification of osteocyte-selective proteins. Proteomics 2010;10:3688e98. [60] Cherian PP, Cheng B, Gu S, Sprague E, Bonewald LF, Jiang JX. Effects of mechanical strain on the function of Gap junctions in osteocytes are mediated through the prostaglandin EP2 receptor. J Biol Chem 2003;278:43146e56. [61] Jiang JX, Siller-Jackson AJ, Burra S. Roles of gap junctions and hemichannels in bone cell functions and in signal transmission of mechanical stress. Front Biosci 2007;12:1450e62. [62] Weinbaum S, Cowin SC, Zeng Y. A model for the excitation of osteocytes by mechanical loading-induced bone fluid shear stresses. J Biomech 1994;27:339e60. [63] Vatsa A, Mizuno D, Smit TH, Schmidt CF, MacKintosh FC, Klein-Nulend J. Bioimaging of intracellular NO production in single bone cells after mechanical stimulation. J Bone Miner Res 2006;21:1722e8. [64] Jørgensen NRGS, Civitelli R, Steinberg TH. ATP- and gap junction-dependent intercellular calcium signaling in osteoblastic cells. J Cell Biol 1997;139:497e506. [65] Genetos DCKC, Zhang Y, Yellowley CE, Donahue HJ. Oscillating fluid flow activation of gap junction hemichannels induces ATP release from MLO-Y4 osteocytes. J Cell Physiol 2007;212:207e14. [66] Robinson JA, Chatterjee-Kishore M, Yaworsky PJ, et al. Wnt/ beta-catenin signaling is a normal physiological response to mechanical loading in bone. J Biol Chem 2006;281:31720e8.

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[67] Hung CT, Pollack SR, Reilly TM, Brighton CT. Real-time calcium response of cultured bone cells to fluid flow. Clin Orthop Relat Res 1995:256e69. [68] Bonewald LF. The amazing osteocyte. J Bone Miner Res 2011;26:229e38. [69] Qing H, Bonewald LF. Osteocyte remodeling of the perilacunar and pericanalicular matrix. Int J Oral Sci 2009;1:59e65. [70] Bonewald LF. Osteocytes as dynamic, multifunctional cells. Ann NY Acad Sci 2007;1116:281e90. [71] Thompson DL, Sabbagh Y, Tenenhouse HS, et al. Ontogeny of Phex/PHEX protein expression in mouse embryo and subcellular localization in osteoblasts. J Bone Miner Res 2002;17: 311e20. [72] Nampei A, Hashimoto J, Hayashida K, et al. Matrix extracellular phosphoglycoprotein (MEPE) is highly expressed in osteocytes in human bone. J Bone Miner Metab 2004;22: 176e84. [73] Liu S, Zhou J, Tang W, Jiang X, Rowe DW, Quarles LD. Pathogenic role of Fgf23 in Hyp mice. Am J Physiol 2006;291:E38e49. [74] Feng JQ, Ward LM, Liu S, et al. Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism. Nat Genet 2006;38:1310e5. [75] Autosomal dominant hypophosphataemic rickets is associated with mutations in FGF23. Nat Genet 2000;26:345e8. [76] Winkler DGSM, Geoghegan JC, Yu C, et al. Osteocyte control of bone formation via sclerostin, a novel BMP antagonist. EMBO J 2003;22:6267e76. [77] Beighton P. Sclerosteosis. J Med Genet 1988;25:200e3. [78] Li X OM, Warmington KS, Morony S, et al. Sclerostin antibody treatment increases bone formation, bone mass, and bone strength in a rat model of postmenopausal osteoporosis. J Bone Miner Res 2009;24:578e88.

[79] Li X WK, Niu QT, Asuncion FJ, et al. Inhibition of sclerostin by monoclonal antibody increases bone formation, bone mass and bone strength in aged male rats. J Bone Miner Res 2010 Jul [Epub ahead of print]. [80] Tian XJW, Li X, Paszty C, Ke HZ. Sclerostin antibody increases bone mass by stimulating bone formation and inhibiting bone resorption in a hindlimb-immobilization rat model. Bone 2010 Sep; [Epub ahead of print]. [81] Agholme FLX, Isaksson H, Ke HZ, Aspenberg P. Sclerostin antibody treatment enhances metaphyseal bone healing in rats. J Bone Miner Res 2010;25:2412e8. [82] Noble BS, Peet N, Stevens HY, et al. Mechanical loading: biphasic osteocyte survival and targeting of osteoclasts for bone destruction in rat cortical bone. Am J Physiol Cell Physiol 2003;284; C934eC43. [83] Tatsumi SIK, Amizuka N, Li M, et al. Targeted ablation of osteocytes induces osteoporosis with defective mechanotransduction. Cell Metab 2007;5:464e75. [84] Kramer IHC, Keller H, Pegurri M, et al. Osteocyte Wnt/betacatenin signaling is required for normal bone homeostasis. Mol Cell Biol 2010;30:3071e85. [85] Kamel SAVP, Jiang J, Bonewald LF, Dallas SL. Comprehensive imaging of osteocytes and their dendrites in situ using widefield fluorescence, confocal microscopy and time lapse imaging. 31st Annual Meeting of the American Society for Bone and Mineral Research (abstract) 2009 Sep. [86] Veno PALY, Kamel SA, Feng JQ, Bonewald LF, Dallas SL. A membrane-targeted GFP selectively expressed in osteocytes reveals cell and membrane dynamics in living osteocytes. 32nd Annual Meeting of the American Society for Bone and Mineral Research (abstract) 2010 October 16.

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Bone Matrix and Mineralization Marc D. McKee 1, William G. Cole 2 1

Faculty of Dentistry, and Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada 2 Divisions of Orthopaedics and Pediatric Surgery, The Stollery Children’s Hospital, and University of Alberta, Edmonton, Alberta, Canada

STRUCTURAL HIERARCHY IN BONE

intimate intercalation of nanocrystals of mineral with the organic scaffolding extracellular matrix [1]. In fact, it is precisely this nanoscale deposition of trillions of inorganic, tiny plate-like crystallites within a softer organic matrix that provides for the inherent flexibility of bones that allows them to be deformed during mechanical challenge, but then return to their original organization characteristic of any given skeletal element. Beyond the macroscale, bone tissue is largely organized as a mosaic of different connected geometric subunits. These are generally referred to as cylindrical osteons (Haversian systems), half-cylindrical hemiosteons, or generally as irregularly disposed intervening bone tissue having a variety of forms related to the forces placed upon it. These subunits are constructed together either loosely as cancellous (spongy) trabecular bone, or densely as compact, generally cortical bone. Collectively, these osseous subunits are “welded” together at their adhesive interfaces by so-called cement lines (really cement planes) [4]. The organic and inorganic components of cement lines likely ensure molecular bonding across this adhesive interface, safeguarding tissue cohesion during bone remodeling as defective, fatigued (microfractured) bone is resorbed, and new bone is deposited to fill the resorption site leaving an intervening cement line at this temporospatially distinct boundary [5]. The cell teams responsible for these alternating cycles of bone resorption followed by formation are called bone multicellular/metabolic units (BMUs) and consist of osteoblasts, osteocytes and osteoclasts that leave behind nascent bone packets bonded to the residual bone [6]. Collectively, this cellular activity e together with the abundant mineralized matrix e creates the necessary physiologic and functional properties of bone, and allow for its carefully orchestrated removal and replacement otherwise know as bone turnover. Figure 2.1 presents a montage of light and transmission

Remarkable in its structural complexity and function, bone tissue serves multiple purposes depending on its anatomic location and on its surrounding and attached tissues. Indeed, hierarchical organization of bone extending from the macroscale to the nanoscale provides for a variety of functions and biomechanical properties that result entirely from its basic structural elements and how they are assembled and interact with one another [1]. Bone tissue is a hard, tough and durable material, able to withstand substantial loads and impacts, and able to respond dynamically e through the actions of cells e to a variety of mechanical challenges. In doing so, the skeleton ultimately repairs and replaces itself throughout the lifespan. Further to this, bone tissue also serves as an important vital ion reservoir, harboring abundant quantities of key mineral ions such as calcium and phosphate which can be mobilized upon demand and released for systemic use by cells throughout the body, and for something no less important than providing nutrient mineral ions to breastfeeding infants during lactation [2]. Bone’s hardness and toughness come from the fact that it is a composite material, consisting primarily of a protein-rich, interconnected organic matrix network whose macromolecular assemblies are interwoven in a way that provides a scaffold for mineral deposition [1]. Mineralization of this extracellular matrix by calcium and phosphate complexation to form crystalline salts that harden bones (and teeth) is not haphazard, but in fact occurs in a way that is almost unimaginably controlled at the molecular level e indeed, like most cellular processes. The inorganic phase (mostly crystalline, as a substituted form of hydroxyapatite [3]) that forms in the skeleton provides exactly the required hardness, while at the same time ensuring requisite toughness by

Pediatric Bone, Second Edition DOI: 10.1016/B978-0-12-382040-2.10002-4

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Copyright Ó 2012 Elsevier Inc. All rights reserved.

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2. BONE MATRIX AND MINERALIZATION

(A)

(C)

(B)

(E)

(D)

FIGURE 2.1 Light (A) and transmission electron (BeE) micrographs showing osteoblast-lineage bone cells (osteoblasts, Ob; osteocytes, Oc), unmineralized bone extracellular matrix (Osteoid), and the mineralized matrix (MM) of bone. (A) Cuboidal osteoblasts align at forming bone surfaces, with some becoming incorporated into the bone as osteocytes. (B, C) Osteoblasts secrete a collagen fibril-rich layer of extracellular matrix known as the osteoid which in its deeper regions show patches of mineralized matrix. Mineralization becomes more confluent at the mineralization front where it permeates throughout the fibrillar and interfibrillar matrix compartments. (D, E) Some osteoblasts become trapped within the extracellular matrix eventually to become embedded within mineralized bone as osteocytes. Osteocyte cell processes extend into the mineralized bone matrix within small channels termed canaliculi (arrows).

electron micrographs illustrating the cells, matrix and mineral of bone, and their relationships at the ultrastructural level. The subsequent sections of this chapter provide details on the composition of the extracellular matrix of bone, with a section at the end providing a discussion of matrix mineralization, and how proteins in the matrix are thought to regulate this process.

ORGANIC MATRIX OF BONE This section focuses on the organic matrix which constitutes approximately 20e30% by weight of bone [7]. The organic matrix contains approximately 90% collagen (by weight) and 10% non-collagenous proteins, proteoglycans, and lipids; although on a molar basis,

the latter category roughly equals collagen. Plasma proteins are also present in the organic matrix. Many plasma proteins permeate bone deriving from its rich vasculature, but have little or no affinity for bone, whereas others such as a2-HS glycoprotein (fetuin A) have a higher affinity for the matrix and/or mineral of bone [7]. A description of the primary structure, synthesis, and assembly of the major macromolecules of bone follows. Major bone collagens are described first, followed by the small, leucine-rich, interstitial proteoglycans (SLRPs), and then by non-collagenous proteins of bone. Some macromolecules are not discussed, including the microfibrillar proteins, such as type VI collagen and the fibrillins, as well as the lipoproteins. Details concerning the latter macromolecules as well as other macromolecules of the extracellular matrix and further details about the

PEDIATRIC BONE

ORGANIC MATRIX OF BONE

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 e the most abundant protein of bone e is also present in periosteum, perichondrium, ligaments, tendons, annulus fibrosus, menisci, dermis, sclera, dentin, fascia, and the adventitial layers of viscera [8,9]. 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 [10]. 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 a1(I) chains and one a2(I) chain [10]. A small number of molecules contain three a1(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 a1(I) chains. Homozygous loss in the Mov 13 mouse, in which the a1(I) gene is inactivated by insertional mutagenesis, results in a prenatal lethal phenotype because of the lack of type I collagen in the tissues [11]. Heterozygous loss of one a1(I) allele, either in the Mov 13 mouse or in humans, yields the osteogenesis imperfecta type IA phenotype [12]. The a1(I) and a2(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 [13,14]. 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 contains 52 exons [15,16]. The differences of 18 kb for COL1A1 and 38 kb for COL1A2 are attributable to differences in the sizes of their introns. For the pre-pro-a2(I) chain, the signal peptide is encoded by part of exon 1; the N-propeptide is encoded by part of exon 1, exons 2e5, 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 7e48, and part of the junctional exon 49; the Ctelopeptide is encoded by part of exon 49; the C-propeptide is encoded by part of exon 49 and exons 50e52 [10,15]. The same arrangement exists for the pre-proa1(I) chain, except that the exon numbering is reduced by one beyond exons 33 and 34, which are fused in COL1A1 [10]. The exons encoding the main triple helix of the two chains have similar sizes [14]. Each exon encodes complete GlyeXeY triplets, where X and Y are often proline so that they commence with a codon for Gly

11

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 of introns. The 45- and 99-bp exons result from recombinations between two 54-bp exons [17]. The pre-pro-a1(I) chain contains 1464 amino acid residues [10,15]. It contains a signal peptide of 22 residues, an N-propeptide of 139 residues, an N-non-helical telopeptide of 17 residues, a main triple helix of 1014 residues, a C-non-helical telopeptide of 26 residues, and a C-propeptide of 246 residues. The mature tissue form of the a1(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 Pro161eGln162 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 Gly953e Ile954 bond. Proline residue 1164 may be 3-hydroxylated. There are two potential RGD (Arg-Gly-Asp) cell attachment sites at residues 745e747 and 1093e1095. Procollagen C-proteinase cleaves at Ala1218eAsp1219. The globular C-propeptide contains four cysteine residues at positions 1259, 1265, 1282, and 1291 that are involved in interchain disulfide bonding [18]. It also contains cysteine residues that are involved in intrachain disulfide bonding between residues 1299 and 1462 and between residues 1370 and 1415. There is also a putative Asn1365 site for attachment of an N-linked oligosaccharide. The pre-pro-a2(I) chain is shorter than the prepro-a1(I) chain, although the main triple-helical domains are the same size [16,19]. The pre-pro-a2(I) chain has a signal peptide of 22 amino acid residues, an N-propeptide of 57 residues, an N-non-helical telopeptide of 11 residues, a main helical domain of 1014 residues, a C-terminal non-helical telopeptide of 15 residues, and a globular C-propeptide of 247 residues. The very short globular domain of the N-propeptide contains only two 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 Asn79eGln80 bond. Lysine residues 177 and 1023 are potential sites for hydroxylation and glycosylation. There are potential cell attachment sites at RGD sequences at positions 777e779, 822e824, and 1005e1007. The procollagen C-proteinase cleavage site is at the Ala1119eAsp1120 bond. Three sites for interchain disulfide bonds are at residues 1163, 1186, and 1195.

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2. BONE MATRIX AND MINERALIZATION

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 Asn1267. The structures of the C-propeptide of human type III procollagen and the C-telopeptides of type I collagen have been described [20,21]. The structures of the Cpropeptides of types I and III procollagen are likely homologous. In contrast to the elongated structure of the C-terminal triple-helical region and the Ctelopeptide, the C-propeptide has a low-resolution structure composed of three large lobes and one small lobe [21]. This structure is readily interpretable in terms of the subunit composition and known positions of inter- and intrachain disulfide bonds. Among the eight cysteines found in the C-propeptide domains of type III procollagen, cysteines 1e4 are involved in interchain disulfide bonding, whereas cysteines 5e8 form intrachain disulfide bonds [22]. 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 chainechain interactions [21]. The C-telopeptide of type I collagen had a hairpin conformation, with the C terminus folded back onto the triple helix [20]. After being transcribed, the pre-mRNA for the prepro-a1(I) and pre-pro-a2(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 [23]. Formation of the triple-helical heterotrimeric type I procollagen molecule requires the synthesis of the constituent pro-a1(I) and pro-a2(I) chains from their distinct mRNAs. Transfer to the Golgi apparatus and movement into the secretory pathway require completion of both post-translational modifications and triple helix formation. The [pro-a1(I)]2[proa2(I)] heterotrimer is the major product, whereas stable [pro-a1(I)]3 homotrimer is formed in only small amounts. A [pro-a1(I)][pro-a2(I)]2 heterotrimer or a [pro-a2(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-a1(I) and pro-a2(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 [24e26]. However, the ratios of the pro-a1(I):pro-a2(I) mRNAs vary widely, even though the resultant heterotrimers maintain the protein chain ratio of 2:1 [27]. Consequently, chain selection probably plays an important role in molecular assembly [28]. In support of the latter proposal, distinct chain recognition sequences of 15 amino acid residues have been identified within the Cpropeptides of the type I procollagen chains [29]. The interactions between the C-propeptides that lead to registration of nascent pro-a1(I) chains occur while the chains are still associated with polysomes [30e32]. 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 mRNA translocons is a critical factor in molecular assembly [23]. Elongation pauses are also a prominent feature of synthesis of pro-a1(I) and proa2(I) chains [33]. There also appears to be an interaction between the translation complexes for the synthesis of the two chain types [28]. These interactions may serve two purposes: to bring the translation complexes to the same regions of the endoplasmic reticular membrane to ensure the co-localization of the nascent chains for interaction and to regulate or coordinate the rates of synthesis of the two pro-a chains. In many systems producing type I collagen, the pro-a1(I):pro-a2(I) ratio is greater than 2:1 [34]. In such situations, it is likely that all of the pro-a2(I) mRNA is engaged in heterotrimer synthesis, whereas the excess pro-a1(I) chains may either produce [pro-a1(I)]3 trimer or be degraded [35]. 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 co-translational events. Approximately 100 proline residues in the Y position of GlyeXeY repeats undergo 4-hydroxylation by prolyl 4-hydroxylase. A single proline residue in the X position undergoes 3-hydroxylation by prolyl 3hydroxylase. 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 co-factors need to be present, such as ferrous ions, molecular oxygen, a-ketoglutarate, and ascorbate [36]. Prolyly 4-hydroxylase, also called procollagen proline 2 oxoglutarate 4-dioxygenase, is a tetramer consisting of two a and two b subunits with a molecular weight of 240 kDa. The b subunit is also known as protein

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ORGANIC MATRIX OF BONE

disulfide isomerase. The gene for the b subunit, P4HB, is located on chromosome 17q25 [37]. The gene is expressed ubiquitously. There are two a subunits whose genes are named P4HA1 and P4HA2 [38]. The P4HA1 gene is located on chromosome 10q21.3-23.1 [39], and the P4HA2 gene is located on chromosome 5q31 [38]. The mRNAs encoded by P4HA1 and P4HA2 are expressed ubiquitously, although the ratios of the mRNAs vary between tissues. There are two a (I) subunit mRNA isoforms that result from mutually exclusive alternative splicing of exons 9 or 10 of P4HA1 [40]. Tetramers containing either two a (I)/two b subunits or two a1(II)/two b subunits have similar enzyme activities [38]. Co-expression of recombinant P4HA1, P4HA2, and P4HB in insect cells did not show any tetramers that contained both P4HA1 and P4HA2 protein chains [41]. Proline 986 of the triple helical region of pro a1(I) chain is the single proline residue that undergoes 3hydroxylation. Cartilage-associated protein (CRTAP), encoded by CRTAP, forms a complex with prolyl-3hydroxylase-1 (P3H1), encoded by LEPRE1 and the prolyl cis-trans isomerase cyclophilin B (CyPB), encoded by PPIB that is required for type I collagen 3-prolyl hydroxylation [42]. There appear to be at least four lysyl hydroxylases. The enzyme is also called procollagen-lysine 2 oxoglutarate 5-dioxygenease. Lysyl hydroxylase 1, an 85-kDa membrane-bound homodimeric protein, is localized to the cisternae of the rough endoplasmic reticulum [43]. It hydroxylates specific lysine residues in XeLyseGly sequences. Its gene, PLOD1, is located on chromosome 1p36.3-p36.2 [43]. 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 [44]. It also forms homodimers. It is highly expressed in non-skeletal tissues as well as in bone [45]. Lysyl hydroxylase 2 modifies lysyl residues in the telopeptides of type I collagen involved in intermolecular cross-link formation. Lysyl hydroxylase 3 is encoded by PLOD3, which is located on chromosome 7q36 [46,47]. It is also expressed mostly in non-skeletal tissues. There is also a putative telopeptide lysyl hydroxylase that is specific for bone. The gene called either BRKS or TLH1 was predicted from linkage studies of patients with Bruck syndrome type I to be located on chromosome 17p12 [48]. Recently, FKBP10, which encodes FKBP65, which has prolyl cis-trans isomerase activity, was identified as a possible cause of some cases of Bruck syndrome type I e the gene is located on chromosome 17q21.2 [49]. 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

13

state. Increased levels of glycosylation tend to reduce the size of the collagen fibrils presumably caused by interference with packing of the molecules. A mannose-rich oligosaccharide may also attach to an asparagine residue in the C-propeptide. 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 [50,51]. Release of prolyl 4-hydroxylase, including its b-subunit protein disulfide isomerase, from the triple-helical domain coincides with the assembly of thermally stable triple-helical 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 associate independently 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 [50]. It is also able to catalyze disulfide bond formation within and between the C-propeptides. HSP47, also called collagen-binding protein 2, is a heat shock protein that resides in the endoplasmic reticulum [52]. 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. HSP47 may form a chaperone complex with FK506binding protein 10 (FKBP10) that interacts with procollagen [49]. The importance of HSP47 in normal type I collagen biosynthesis is highlighted by the phenotype of mice lacking the heat shock protein [52]. 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 triple-helical structure without the assistance of HSP47 and that HSP47 was essential for normal development. HSP47 is encoded by SERPINH1 which is located on chromosome 11q13.5 [53]. 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 b-subunit of prolyl 4-hydroxylase. This process

PEDIATRIC BONE

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2. BONE MATRIX AND MINERALIZATION

is critical for the correct alignment of the main triplehelical 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-a chains and after the formation of the interchain disulfide bonds within the C-propeptide [54,55]. It is likely that formation of the triple helix is a posttranslational event because the production of triplehelical molecules requires approximately 8 or 9 minutes after completion of the synthesis of the pro-a 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 [56]. However, a minimum of two hydroxyproline-containing GlyeXeY triplets at the C-terminal end of the triple helix are required for nucleation to occur [56]. Direct nuclear magnetic resonance measurements of chick calvarial collagen showed that approximately 16% of the XePro and 8% of XeHyp bonds were cis in the unfolded collagen [57]. Many studies have shown that the rate-limiting step in the zipper-like propagation of the helix is the process of cis-trans isomerization [54,55,57,58]. Peptidyl prolyl cis-trans isomerase catalyzes the cis-trans isomerization of XePro peptide bonds in collagen [59]. FKBP65, encoded by FKBP10, also has prolyl cis-trans isomerase activity [60]. Protein disulfide isomerase is also present in the rough endoplasmic reticulum. However, protein disulfide isomerase does not appear to act as a cisetrans isomerase [59]. Full hydroxylation of proline residues in the Y position of GlyeXeY triplets also enhances the rate of propagation of the triple helix from the site of nucleation to the N terminus [61]. Biophysical studies using model collagen peptides have also shown that the folding of GlyeXeY peptides is best described as an all-or-none, third-order reaction [62e64]. 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 N- and C-propeptides are rapidly cleaved. The N-propeptide is specifically cleaved by procollagen 1 N-endoproteinase. The enzyme is encoded by the ADAMTS2 gene, which is an abbreviation for a disintegrin-like and metalloproteinase with thrombospondin type I motif [65]. The enzyme exists in a long and a short form as a result of alternative splicing [66]. The C-propeptide is cleaved by procollagen C-endoproteinase, which is the same as bone morphogenetic protein-1 (BMP-1) [67]. The gene is located on chromosome 8q21 [68]. 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 [69]. The long form appears to be an inactive proenzyme that can be activated by removal of the pro-domain. There are four mammalian BMP-1/TLD-like proteases [70]. One of them, tolloid-like-1 (TLL1), is also an astracin-like metalloproteinase. Its gene, TLL1, is located on chromosome 4q32-q33 [70]. 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 Cproteinase enzyme [71]. Its gene, PCOLCE, is located on chromosome 7q21.3-q22 approximately 6 Mb from the COL1A2 gene that encodes pro-a2(I) chains of type I procollagen [72]. A second procollagen C-endopeptidase enhancer has been isolated. Its gene, PCOLCE2, is located on chromosome 3q21-q24 [73]. A number of functions have been proposed for the released a1(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 [74]. However, there is little evidence to support these proposals. For example, deletion of exon 2 of COL1A1 in mice, which deleted the 65-amino acid cysteine-rich globular domain of the N-propeptide of pro-a1(I) chains, did not produce any demonstrable anomalies in type I collagen biosynthesis, collagen cross-linking, or collagen fibrillogenesis [74]. 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 [75,76]. 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 300 nm and a diameter of approximately 1.4 nm. The overall helical symmetry is left-handed, 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 10 nm [77,78]. 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 (eGlyeXeYe)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 eXe and eYe positions protrude from the chain, and this arrangement allows a variety of amino acid residues to be accommodated in the molecule. The high imino acid content, particularly the high 4-hydroxyproline

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ORGANIC MATRIX OF BONE

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 one-quarter of their length. The periodicity of the cross-striated fibril is a result of each monomer having five highly charged regions at approximately 67-nm intervals. The repeat period e called a D period e 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 non-integral 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 protein-lysine 6-oxidase, is encoded by LOX, which is located on chromosome 5q23.3-q31.2 [79]. Alternative splicing produces three mRNA isoforms [79]. There are also four lysyl oxidase-like loci. LOXL1 is located on chromosome 15q22, LOXL2 on chromosome 8p21-p21.2, LOXL3 on chromosome 2p13.3, and LOXL4 on chromosome 10q24 [80,81]. 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 3-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

15

a hydroxylysine residue to form a reducible cross-link that can undergo an Amadori rearrangement to form hydroxylysino-5-oxo-norleucine. The hydroxylysinederived 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 [82]. It includes hydroxylysylpyridinoline, derived from three hydroxylysine residues, and lysylpyridinoline, 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. Insights into the importance of posttranslational modifications and preferred intermolecular binding partners for telopeptide and helical cross-linking domains in regulating cross-link type and placement are being obtained using ion-trap mass spectrometry and peptide-specific antibodies [83]. 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 Nand C-proteases [84,85]. 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 [76,85]. 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 [85]. Newly formed fibrils with characteristic D periodicity have been isolated from various embyronic tissues [86]. These fibrils also frequently show a single polarity reversal [87]. Fibrils may increase in size by the fusion of small (1e10 mm) segments, and the lateral association of long or short fibrils may lead to thicker fibrils [86,88]. The growth of type I collagen fibrils appears to be partly regulated by other collagen molecules that are included within the heterotypic fibers [89]. For example, other collagens in the heterotypic type I collagen fibrils of dermis include types III, V, XII, and possibly XIV collagen [90]. In bone, the other collagens are type V and type V/XI hybrid molecules [91]. 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 [90]. 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 [92]. The molecular packing of collagen fibrils has been determined mainly in tissues such as tendon. X-ray diffraction studies indicate the presence of three-

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16

2. BONE MATRIX AND MINERALIZATION

dimensional crystallinity admixed with liquid-like lateral order [93,94]. 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 (z4 nm) orientated radially with respect to the fibril center. A feature of the model is that molecules are tilted obliquely in a plane oriented 30
to the fibril surface [94], 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 [93]. An alternative molecular packing model is the fivestranded Smith microfibril [94]. 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 [93]. 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 gapeoverlap junctions. Bone and other connective tissues have distinctive collagen fiber sizes and distinctive suprafibrillar architectures, as seen by polarized light microscopy [95]. In woven bone, the collagen fibrils are generally 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 [96,97]. The type V collagen molecules exist as heterotrimers a1(V)2a2(V) a1(V) a2(V) a3(V) and as homotrimers a1(V)3 [98,99]. An apparently distinct a4(V) chain is synthesized by Schwann cells [100]. Type V collagen chains also form heterotypic molecules with type XI collagen chains. For example, the highly homologous a1(V) and a1(XI) chains may yield an a1(V) a1(XI) a2(V) trimer in bone and cartilage [91]. The following description of type V collagen is limited to the a1(V) and a2(V) chains because the a3(V) and a4(V) genes are not expressed in bone, although the a3(V) gene is expressed in ligament attachments to bone [101].

The gene for the a1(V) chain, COL5A1, is located on chromosome 9q34.2-q34.3 [102]. The gene has 66 exons, more than the number of exons in type I and II collagens. Exon 1 encodes the signal peptide of 36 amino acid residues and one base of the N-propeptide. Exons 2e14 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-a1(V) N-propeptide is similar in size and domain structure to the N-propeptides of the proa1(XI) and pro-a2(XI) chains [103e105]. The Npropeptides 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 type IeIII collagens. The globular domains of the pro-a1(V), pro-a1(XI), and pro-a2(XI) chains are bisected by a cluster of two cysteines into a basic Nterminal subdomain and a C-terminal subdomain rich in acidic residues and tyrosines [103e106]. The latter region contains 27 tyrosine residues and 73% of the total number of tyrosine residues in the pre-pro-a1(V) chain. Approximately 40% of the N-propeptide tyrosine residues are sulfated [107]. The N-terminal subdomain is analogous to the thrombospondin 1 motif and is identical to the proline- and arginine-rich peptide (PARP) motif [108]. The derived three-dimensional structure of PARP suggests a conserved nine b-stranded structure [109,110]. The PARP motif is also present in the N-propeptides of the pro-a1(XI) and pro-a2(XI) chains but not in the pro-a2(V) chains. The globular domain of the N-propeptide of the pro-a1(V) chain is followed by an interrupted triple-helical domain of 25 GlyeXeY repeats and then another short non-collagenous region prior to the main triple-helical domain. Pro-a1(V), proa1(XI), and pro-a2(XI) sequences share similarities in all subdomains of their N-propeptides, with the exception of the acidic globular variable region [104]. There is no evidence of alternative splicing in the Npropeptide of pro-a1(V) chains, although it occurs in the pro-a1(XI) and pro-a2(XI) N-propeptides [103]. There are two hypothetical N-proteinase cleavage sites (Ala-Gln) at positions 541e542 and 546e547. The main triple-helical domain of a1(V) chains is similar to those of type IeIII collagens in that each non-junctional exon begins with a complete codon for glycine and ends with a complete codon for a residue in the Y position of GlyeXeY triplets [102,111]. The codons are commonly 45 or 54 bps. The main triplehelical domain is encoded by exons 14e62 [102]. 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

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cross-linkages, are preserved. The helical domain lacks the Gly-Ile/Leu mammalian cleavage site at position 775e776 of type IeIII collagens [112]. The absence of this sequence explains the lack of cleavage of the a1(V) chains by mammalian collagenase. Carboxyl terminal of the main triple helix is the C-telopeptide of 33 amino acid residues and the Cpropeptide of 233 residues. They are encoded by exons 62e66. The putative C-proteinase cleavage site is located at position 1605e1606 (AlaeAsp). The C-propeptide has a high homology with the same region of pro-a1(XI) chains. Seven cysteine residues and their vicinities are conserved. The pro-a chains that can form homotrimers or both homo- and heterotrimers of pro-a chains have eight cysteine residues, but the pro-a chains that form heterotrimers only have seven cysteine residues [17]. The C-propeptide of pro-a1(V) chains has eight cysteine residues, whereas the C-propeptide of the pro-a1(XI) chains has seven cysteine residues [113]. The pro-a1(V) chain contains numerous sites for potential attachment of N-linked oligosaccharides. It has a heparin binding site at residues 897e929 [114]. Heparin binding sites are also present in a1(XI) and a2(XI) chains but not a2(V) chains [115]. Binding of extracellular chondroitin sulfate E to type V collagen may facilitate cell binding and matrix assembly [116]. There are RGD cell attachment sites at positions 645e647 and 663e665. The gene for the a2(V) chain, COL5A2, resides on chromosome 2q14-q32 [117,118]. 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. COL5A2 encodes a pre-pro-a2(V) chain of 1496 amino acid residues [119,120], and it includes a signal peptide of 26 residues. The pro-a2(V) N-propeptide is encoded by four complete exons and partially by the junctional exon. The N-propeptide of 167 residues includes an aminoterminal 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 Npropeptide of pro-a1(I) chains but dissimilar to the equivalent subdomain of the pro-a1(V) chain [111,121]. The N-propeptide also includes an interrupted helical subdomain of 78 residues and a non-helical subdomain of seven residues. The N-proteinase cleavage site is at the AsneGln bond at position 193e194 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 Cpropeptide of 246 residues. The C-proteinase cleavage

17

site is located at the GlyeAsp bond at position 1250e1251. There are seven RGD sequences that are potential cell binding sites. There are several sites for the attachment of N-linked oligosaccharides. Also, there are three cysteine residues, at positions 1293, 1299, and 1325, for intermolecular cross-linking and sites for intrachain disulfide cross-links at positions 1333e1494 and 1402e1447. 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 Npropeptides. 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 [109,122,123]. Rotary shadowing confirmed the retention of the N-propeptides on some of the type V collagen molecules extracted from tissues [124]. Nonetheless, within pro-a1(V)2pro-a2(V) heterotrimers, some proa1(V) N-propeptides and pro-a2(V) C-propeptides are enzymatically removed by bone morphogenetic protein-1-like enzymes. Pro-a1(V) C-propeptides are processed by furin-like proprotein convertases in vivo [125]. When type V collagen epitopes are unmasked in tissues, this collagen is found to co-localize with type I collagen [126]. The exact spatial relationship between these collagens in the type I collagen fibrils is unclear. However, co-polymeric assembly is likely because cross-linkages between types I and V collagens have been isolated from bone [127]. It is also likely that type V collagen regulates the formation of the type I collagen fibrils [128]. Type V collagen molecules are capable of forming homotypic fibrils in vitro with or without an apparent 67-nm cross-striation pattern [129]. Increasing the quantity of type V collagen relative to type I collagen decreased the final fibril diameter [130]. 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 [89,130]. Confirmation of the importance of the amino-terminal extension of the a2(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 a2(V) chains. The mice showed abnormal type I collagen fibrillogenesis in the dermis [131]. Although type V collagen, with the exception of its N-propeptides, is buried within the type I collagen fibrils, its main triple-helical domain is known to bind to thrombospondin, heparin, heparan sulfate, decorin, and biglycan [114,132e135]. Type V collagen contains seven RGD sequences on the a2(V) and two on the a1(V) chains that may enable type V collagen to attach to various cell types. These interactions may involve the a1b1 and a2b1 integrins [136].

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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 [137]. Many of the family members bind to various collagens and to growth factors such as TGF-b1 [138]. 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 [139,140]. 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 [141,142]. Other members of the latter family include the proline- and arginine-rich and leucine-rich repeat protein (PRELP) and osteomodulin. The class III SLRP expressed in cartilage is epiphycan, also called PG-Lb [143]. Chondroadherin represents its own subfamily, with a different gene organization and a different amino acid composition [144]. The leucine-rich repeating extracellular glycoproteins/proteoglycans have core proteins of 32e42 kDa [145]. 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 four Cys residues, which form intrachain disulfide bonds. 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. The different leucine-rich repeat proteoglycans and glycoproteins have similar C-terminal domains, which contain approximately 50 amino acid residues. This domain contains two 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 60e80% 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 nine to 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, phenylalanine, tyrosine, or methionine. The asparagine

residue at position 9 may be replaced by cysteine or threonine. 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. 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 a-helices and b-sheets stabilized by interchain hydrogen bonds [146]. Results of structural studies of decorin and biglycan are in accordance with the latter x-ray crystallographic findings [147]. Decorin, fibromodulin, and lumican bind to fibrillar collagens in vitro, leading to delayed fibril formation and the formation of thinner fibrils [148]. These changes are likely attributable 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 [85]. 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 [149]. Decorin and fibromodulin bind to distinct and apparently separate sites in the gap region of the D period of the collagen fibril in vivo [150,151]. Decorin has been shown to bind to a small region of the C-terminus of the triple-helical domain of a1(I) chains, close to one of the intermolecular crosslinking sites [152]. This region corresponds to the c1 band of the collagen fibril D period. Proteoglycan core proteins of decorin, biglycan, and fibromodulin, prepared as fusion proteins, each bound TGF-b1 [153]. 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-b1 binding. Affinity measurements suggest a two-site binding model with KD values ranging from 1 to 20 nM for the high-affinity binding site and from 50 to 200 nM for the low-affinity binding site. Stoichiometry indicated that the highaffinity binding site was present in one of 10 proteoglycan core molecules and that each molecule contained a low-affinity binding site. Tissue-derived biglycan and decorin were less effective competitors for TGF-b binding than fibromodulin or the non-glycosylated 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 biglycan, suggesting that the glycosaminoglycan chains may hinder the interaction of the core proteins with TGF-b. The fusion proteins competed for the binding of radiolabeled TGF-b to Mv1Lu cells and

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endothelial cells. Affinity labeling showed that the binding of TGF-b to betaglycan and the type-I receptors in Mv1Lu cells and to endoglin in endothelial cells was reduced, but the binding to the type II receptors was unaffected. Latent recombinant TGF-b1 precursor bound slightly to fibromodulin and not at all to decorin and biglycan. These findings indicate that the three decorin-type proteoglycans each bind TGF-b isoforms and that slight differences exist in their binding properties. They, and possibly other members of this family of proteoglycans, may regulate TGF-b activities by sequestering TGF-b in the extracellular matrix. Representative members of the subfamilies of small, leucine-rich interstitial proteoglycans are described next. Decorin and Biglycan The decorin gene, DCN, and the biglycan gene, BGN, are located on chromosomes 12ql3.2 and Xq28, respectively [154]. Both genes have a similar eight exon gene structure. The decorin gene is approximately 45 kbs, whereas the biglycan gene is approximately 8 kbs. Exon 1 of decorin, which encodes the 50 untranslated region, exists as exon 1a and exon 1b, which undergo alternative splicing [140]. The decorin transcript can also undergo additional alternative splicing to yield five isoforms, AeE. Isoform A, which contains the full amino acid coding sequence, is the product of transcript variants Al from exon 1a and A2 from exon 2b. Isoform B lacks the exons 3 and 4 sequence, isoform C lacks the exons 3e5 sequence, isoform D lacks the exons 4e7 sequence, and isoform E lacks the exons 3e7 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 [140]. The biglycan mRNA sizes, also related to the use of different polyadenylation sites, are 2.1 and 2.6 kbs [139]. The BGN gene is subject to X inactivation because there is no homologous gene on the Y chromosome. However, the pseudoautosomal expression of BGN probably results from the regulation of the BGN gene by a gene or genes that escape X inactivation [155]. Preprodecorin contains 359 amino acid residues [156]. It includes a signal peptide of 16 residues, an Npropeptide of 14 residues, and a mature chain of 329 residues. Intrachain disulfide bonds are present between Cys54 and Cys67 as well as between Cys313 and Cys346. 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 weight of 39 kDa, whereas the protein extracted from tissues has a molecular weight of

19

approximately 130 kDa, of which the chondroitin sulfate chain contributes approximately 40 kDa [157]. Preprobiglycan contains 368 amino acid residues [139]. 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 316e368 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 40 kDa [158]. 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 [159]. The BMP-1-related proteases, mammalian Tolloid and mammalian Tolloidlike 1 (mTLL-1), have low levels of probiglycan-cleaving activity. Wild-type mouse embryo fibroblasts produce only fully processed biglycan, whereas the fibroblasts derived from embryos homozygous null for the Bmp1 gene, which encodes both BMP-1 and mammalian Tolloid, produce predominantly unprocessed probiglycan, and fibroblasts homozygous null for both the Bmp1 gene and the mTLL-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. 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 attributable to coarse and irregular collagen fibrils, confirming the importance of decorin in normal collagen fibrillogenesis [160]. Biglycan-deficient mice show reduced longitudinal growth and decreased bone mass, indicating an important role for biglycan in bone health [161]. Mice lacking both decorin and biglycan have more profound osteopenia compared to mice deficient in only one of these SLRPs [162]. Fibromodulin and Related Small Proteoglycans 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 [163]. The following descriptions will be restricted to fibromodulin and osteomodulin which are both present in bone.

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The fibromodulin gene, FMOD, is approximately 8.5 kbs and is located on chromosome 1q32 [164]. The gene contains three exons with conserved exon/intron junctions. Exon 1 encodes the 50 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 30 untranslated region. The fibromodulin mRNA is approximately 3 kbs. The highest expression of the gene is in hyaline cartilages, tendon, and ligament, whereas lower levels of expression occur in most other connective tissues [163]. Fibromodulin is a 59-kDa protein [163]. 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. 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 [141]. The keratan sulfate chains are attached by N-glycosidic linkages from Nacetylglucosamine to asparagine residues in the central domain of the molecule [165]. 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. Mice lacking fibromodulin showed altered tendon structure because of the high proportion of thin, irregular collagen fibrils [145]. These relatively mild changes confirmed the important role of fibromodulin in regulating collagen fibrillogenesis in vivo. The gene for osteomodulin (also known as osteoadherin), OMD, is located on chromosome 9q22.31. 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 [166,167]. The entire translated primary sequence corresponds to a 49,116-Da protein with a calculated 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 Nlinked glycosylation, some of which are substituted with keratan sulfate chains. Osteomodulin shows 42% sequence identity to bovine keratocan and 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 mRNA for osteomodulin to be expressed in bone only. Osteomodulin mRNA 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 avb3 [167].

Thrombospondins The five members of the thrombospondin family are designated thrombospondin-1 to thrombospondin-5 [168]. 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 matricellular functions. The thrombospondin-1 gene, THBS1, is located on chromosome 15ql5 [169]. 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 [170]. The full-length protein contains 1172 amino acids, including a signal peptide of 18 amino acid residues. The thrombospondin-3 gene, THBS3, on chromosome 1q21 encodes a protein monomer of 956 amino acids, including a signal peptide of 36 amino acid residues [171]. The thrombospondin-4 gene, THBS4, on chromosome 5ql3 encodes a full-length protein monomer of 961 amino acid residues, including a signal peptide of 21 residues [172]. Finally, the thrombospondin-5 gene, COMP, located on chromosome 19pl3.1 encodes a fulllength monomer of 757 amino acids, including a signal peptide of 20 amino acid residues [173]. Each thrombospondin is expressed in multiple tissues, particularly during fetal life. Similarly, most tissues express multiple members of the thrombospondin family [174]. Consequently, most of the thrombospondins are expressed in bone and cartilage during embryogenesis [175]. COMP is the most abundant form in postnatal growth plates [176]. Thrombospondin-2 and COMP are also present in postnatal bone and bone marrow stromal cells [176]. The thrombospondins are divided into subfamily A and subfamily B [174]. 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-3e5 are in subfamily B. They have unique N-terminal regions and lack the procollagen homology domain and type 1 repeats, but they contain

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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 [177]. The conformations of the stalk regions and the termini are determined, at least in part, by the calcium ion concentration [178]. 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 [179]. The procollagen homology region of thrombospondin-1 also folds into a stable, compact, and disulfide-bonded monomer. Isolated type 3 repeats of COMP produce a 14.2-nm rod-like structure [180]. 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 [180]. Thrombospondin-1 and thrombospondin-2 can form homotrimers and heterotrimers [181]. Thrombospondin trimers and pentamers are stabilized by the formation of interchain disulfide bonds between their corresponding oligomerization domains. Pentamerization of COMP involves the formation of interchain disulfide bonds between cysteine residues 68 and 71 [182]. Intrachain disulfide bonds are also present in the type 3 repeats. There are also specific sites for the addition of N- and O-linked sugars and for the C-mannosylation of tryptophan [183]. 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 that number of calcium ions [184]. 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 [183]. 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 [185]. The small latent TGF-b1 complex with the latencyassociated peptide binds to the WSIIWSPW motif in the second type 1 repeat of thrombospondin-1 [186]. An intermolecular activation effect of the KRFK motif

21

in the first type 1 repeat of thrombospondin-1 releases mature, active TGF-b1. Thrombospondin-2, which lacks the KRFK motif, can bind the latent TGF-b1 complex but cannot activate it [186]. Thrombospondin-4 and COMP bind to type I and II collagens [187]. COMP also binds type IX collagen and MMP-19 and -20, whereas thrombospondin-4 also binds to laminin, fibronectin, and matrilin-2 [187]. Cell surface interactions with thrombospondin-1 have been studied in detail [174]. The amino-terminal domain, type 1 repeats, type 3 repeats, and the Cterminal 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 [188]. The C-terminal domains of thrombospondin-4 and COMP bind types I, II, and IX collagens in a zinc iondependent manner [187,189]. The binding of the 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. Further insights into the functions of thrombospondin (TSP)-1, -2, -3 and -5 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-b1 activation in the inflamed lungs and pancreas [190]. TSP1-deficient mice have no reported appendicular skeletal defects but have craniofacial dysmorphism, that is more pronounced in compound TSP1/2-deficient mice, spinal lordosis and altered rates of wound healing and tissue remodeling in various injury models. Mice lacking thrombospondin-2 have increased thickness and density of the longbone cortices, attributed to increased bone formation [191]. The TSP2-deficient mice also show resistance to ovariectomy-induced bone loss, differential surface responsiveness to mechanical loading, increased bone formation and reduced chondrogenesis in response to fracture, and altered bone mass and geometry associated with aging [168]. The TSP2-deficient mice have fragile skin, lax tendons, abnormal fibrillar collagen organization, increased vascular density, prolonged bleeding time, and accelerated skin wound healing [191]. The changes in the skin and tendons 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 TSP2-deficient mice.

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2. BONE MATRIX AND MINERALIZATION

In the TSP3-deficient mice, skeletal development was initially normal but, by 9 weeks of age, there was accelerated ossification of the secondary ossification centers of the femoral heads [192]. The TSP3-null mice also had an increased femoral cortical bone periosteal circumference and parameters of mechanical strength. The phenotype of TSP4-deficient mice has not been reported to date. However, the C-terminal domain of TSP4 binds to collagens I, II, III and V, as well as laminin, fibronectin and matrilin-2 [174]. TSP5-deficient mice have a mild skeletal phenotype with growth plate disorganization and mild exercise-induced articular cartilage flattening [193]. These skeletal changes are worse in compound TSP1/5 and TSP3/5-deficient mice [193]. Missense mutations of THBS5, particularly those that affect calcium binding and protein folding, are associated with the pseudoachondroplasia and multiple epiphyseal dysplasia phenotypes in mice and humans [194].

Osteonectin Osteonectin, initially isolated from demineralized bone matrix, was named according to its ability to bind to calcium, hydroxyapatite, and collagen, and to nucleate hydroxyapatite deposition [195,196]. The protein has also been called secreted protein, acidic and rich in cysteine (SPARC), basement membrane 40, or “culture shock” protein [197]. Osteonectin is an antiadhesive protein because it inhibits cell spreading, induces rounding of cells, and disassembles focal adhesions [198]. Other activities of osteonectin include calcium-dependent binding to collagens and thrombospondin, binding to platelet-derived growth factor-AB and -BB, and regulation of cell proliferation and MMP expression [199]. The gene SPARC or ON, which contains 10 exons, is located on chromosome 5q31.3-q32 [200]. 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 [201]. The SPARC gene is also expressed in several postnatal non-skeletal tissues, including salivary and renal tubular epithelium [202]. Osteonectin is the most abundant non-collagenous protein in mineralized bone matrix in some species [196]. The SPARC gene encodes a full-length protein of 303 amino acids, including the signal peptide of 17 amino acid residues. The core molecular weight is 33 kDa [203]. The protein extracted from bone has an apparent molecular weight of 43 kDa attributable to post-translational modifications such as glycosylation. The mature human protein consists of 286 amino acid residues divided into three domains [204]. An aminoterminal acidic segment (residues 1e52) binds five to eight calcium ions with low affinity and mediates

interactions with hydroxyapatite. A follistatin-like domain (residues 53e137) contains five disulfides and an N-linked oligosaccharide at Asn [99]. Finally, an ahelical domain contains two EF hand, high-affinity, extracellular calcium binding sites (EC domain, residues 138e286). 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 [205]. 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 Nlinked oligosaccharide. Crystal structure analysis also has shown that the collagen-binding epitope in the helix aA is partially masked by helix aC [205]. Deletion of helix aC produced a 10-fold increase in collagen affinity, similar to that seen after proteolytic cleavage of this helix [206]. Five residues were crucial for collagen binding: R149 and N156 in helix aA and L242, M245, and E246 in a loop region connecting the two EF hands of osteonectin. These residues were spatially close and formed ˚ , which matches the diameter of a flat ring of 15 A a triple-helical collagen domain. Nearly identical binding characteristics were displayed by type I and IV collagens. The absence of Sparc in transgenic mice gives rise to aberrations in the structure and composition of the extracellular matrix that result in cataracts, severe osteopenia, and impaired wound healing [207e209]. Sparcnull mice have decreased trabecular bone volume and fail to demonstrate an increase in bone mineral density in response to a bone-anabolic parathyroid hormone treatment regimen [209,210]. By 6 months of age, the mice develop severe eye pathology [207]. Sparc-null mice have greater deposits of subcutaneous fat and larger epididymal fat pads in comparison with wildtype mice. The dermis and fat pads of the SPARCdeficient mice contain less collagen I and the fibers are of smaller diameter than in controls [211e213].

Osteocalcin Osteocalcin is a small protein that accounts for approximately 10% of the non-collagenous protein of bone [214]. It is also called bone g-carboxyglutamate or bone Gla protein. It is one of several Gla proteins found in the skeleton. Other Gla proteins, such as protein S, are made elsewhere and are deposited in the bone matrix from the circulation [215]. The Gla proteins have in common the presence of glutamic acid residues that have been g-carboxylated by a specific g-carboxylase that requires vitamin K as a co-factor [216]. These

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g-carboxyglutamate residues have a high affinity for mineral ions such as Ca2þ and for hydroxyapatite crystals. The osteocalcin gene, BGLAP, is located on chromosome 1q25-q31 [217,218]. The gene is very small (<1 kb) and contains only four exons [219]. Exons 1e3 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 [220]. Mice lacking osteocalcin develop a phenotype marked by higher bone mass and bones of improved functional quality [221]. 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 [221,222]. 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 [223]. The genes were named osteocalcin gene 1, osteocalcin gene 2, and osteocalcinrelated gene from the 50 end of the osteocalcin cluster. The osteocalcin 1 and osteocalcin 2 genes each contain four exons, whereas the osteocalcin-related gene contains, in addition to the four exons typical of osteocalcin, exon 1 that is not translated. Each of these genes can be transcribed. The osteocalcin 1 and osteocalcin 2 genes are expressed only in bone, whereas osteocalcinrelated gene is expressed in kidney but not in bone. The protein encoded by the osteocalcin-related gene is the same as nephrocalcin, a calcium-binding protein of the kidney [224]. The osteocalcin promoter has been studied in detail. Responsive elements for 1,25-dihydroxyvitamin D3, glucocorticoids, and tumor necrosis factor-a have been identified [225e227]. Other transcriptional regulatory sites such as one that binds MSX1 and MSX2, two homeodomain-containing proteins, have also been identified [228]. 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 OSE1 appears to regulate expression of the gene by immature osteoblasts. The full-length protein contains 100 amino acid residues [219]. 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 two rather than three Gla residues seen in other species [229]. The predicted structure of

23

the protein consists of two antiparallel a-helical domains connected by a b-turn [230]. A disulfide bond between cysteines 23 and 29 stabilizes the structure. The x-ray crystal structure of porcine osteocalcin at 2.0-angstrom resolution, revealed a negatively charged protein surface that coordinates five calcium ions in a spatial orientation that is complementary to calcium ions in a hydroxyapatite crystal lattice [231]. The Gla residues have affinity for free Ca2þ and Ca2þ-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 [232]. Since 2007, osteocalcin has been identified as playing an important role in a reciprocal relationship between bones and energy metabolism [233]. This relationship involves the effects of leptin on osteoblast function and bone remodeling and osteocalcin in turn influencing energy metabolism apparently through the levels of uncarboxylated and undercarboxylated osteocalcin in the circulation [233,234]. Not all of the three glutamic acid residues in osteocalcin are fully carboxylated and bind to hydroxyapatite crystals. Uncarboxylated or undercarboxylated glutamic acid residues make osteocalcin susceptible to release from osteoblasts into the circulation [234]. Leptin may regulate the levels of gcarboxylation via regulating the levels of expression of Esp by osteoblasts [235]. The Esp gene, which encodes osteotesticular protein tyrosine phosphatase (OST-PTP), may contribute to the g-carboxylation of osteocalcin.

Small, Integrin-binding Ligand N-linked Glycoproteins This class of glycoproteins, also called SIBLINGs [236], includes a family of five proteins encoded by identically orientated tandem genes clustered within a 375 kb region on chromosome 4 [237]. The genes in order are dentin sialophosphoprotein (DSPP), dentin matrix acidic phosphoprotein 1 (DMP1), bone sialoprotein (IBSP), matrix extracellular phosphoglycoprotein (MEPE), and secreted phosphoprotein 1 (SPP1, also called osteopontin, OPN). Except for an apparent pseudogene between MEPE and SPP1 in humans, there are no other significant open reading frames in this region of chromosome 4 [237]. These genes have similar structures with either six or seven exons. It is likely that they arose by early gene duplication and subsequent divergence. The three-dimensional structures of bone sialoprotein and osteopontin, as determined by NMR analysis, shows that the proteins have an extended and flexible structure in solution [237]. The latter structure would be expected to facilitate binding to, and bridging between, multiple partners; for example, RGD and non-RGD integrins, other cell surface proteins,

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matrix metalloproteinases and complement factor H [237]. The extended structure could also be important for increasing binding to different crystallographic faces of bone mineral, and to different mineral phases [238]. The following sections will describe the members of the SIBLING family with the exception of DSPP which is present primarily in dentin rather than in bone. Bone Sialoprotein Bone integrin-binding sialoprotein, usually referred to as bone sialoprotein (BSP), is the second major sialoprotein of bone [7]. It constitutes approximately 12% of the non-collagenous protein of human bone. It binds to calcium and hydroxyapatite, cells, and collagens. The gene for bone integrin-binding sialoprotein, IBSP is located on chromosome 4q21-q25 between the DMP1 and MEPE genes [237]. The gene of about 15 kb includes seven exons and the promoter contains motifs that determine developmental regulation and tissue-specific expression [239]. As with the other members of the SIBLING family, exon 1 is non-coding while exon 2 includes the signal peptide and the first two codons of the mature protein. Exons 3 and 5 contain serine phosphorylation consensus sequences while exon 4 is proline rich. Exon 6 contains a glycosaminoglycan attachment site and a specific protease cleavage site. Exon 7 contains an RGD motif. Gene expression is more limited than that for osteopontin [240]. 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 [241]. Outside of the skeleton, IBSP is expressed in tooth odontoblasts and cementoblasts, and in trophoblasts of the placenta. The IBSP mRNA 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 [242]. Bone sialoprotein also contains stretches of polyglutamic acid as opposed to polyaspartic acid as found in osteopontin. The stretches of up to 10 glutamic acid residues provide high-affinity binding to Ca2þ. This characteristic is likely important in the role of integrin-binding sialoprotein in matrix mineralization. Bone sialoprotein, purified from bovine bone, has a molecular weight of 59 kDa due to its high content of carbohydrate. Bovine bone sialoprotein contains 5.8 phosphates that are added by casein kinase II to serine residues [243]. Mass spectrometry, combined with deglycosylation procedures, showed that bone sialoprotein contains 33.8% oligosaccharides, with 12.3% being N-linked and 21.5% O-linked [244,245]. An RGD sequence is located at the C-terminus of bone sialoprotein, in contrast to the more central location in osteopontin. It enables bone sialoprotein to bind to cells via an integrin receptor of the vitronectin

type (avb3). 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 RGDdependent manner, but fragments of bone sialoprotein can bind to cells in an RGD-independent manner. BSP knockout mice have a higher bone mass than wild-type littermates, with very low bone formation activity and reduced osteoclast surfaces and numbers [246]. BSP/ preosteoclast cultures display impaired proliferation and enhanced apoptosis. The expression of osteoclast-associated genes is markedly altered in BSP/ osteoclasts, with reduced expression of cell adhesion and migration genes (aV integrin chain and OPN) and increased expression of resorptive enzymes (TRACP and cathepsin K) [247]. Despite the low basal turnover, both catabolic (ovariectomy) and anabolic (intermittent PTH) challenges increased bone formation and resorption in BSP/ mice, suggesting that compensatory pathways are operative in the skeleton of BSP-deficient mice [248]. Cortical bone repair and mineralization are also impaired by the absence of BSP [247,249]. Dentin Matrix Acidic Phosphoprotein-1 Dentin matrix acidic phosphoprotein-1 (DMP1) was first isolated by cDNA cloning using a rat odontoblast mRNA library [250]. It was initially postulated to be specific to dentin but later was detected in calvaria and long bones [251]. The gene encoding dentin matrix acidic phosphoprotein-1, DMP1, is located between DSPP and IBSP in the SIBLING gene cluster on chromosome 4q21 [237]. The gene contains six exons [252]. Exons 1e5 are similar in size to those of IBSP and SPP1. Exon 1 is non-coding, while exon 2 encodes the signal peptide and first two amino acids of the mature protein chain. Exons 3 and 5 contain serine phosphorylation sequences and exon 4 is proline rich. Exon 5 can be alternatively spliced. Exon 6, which encodes most of the protein, is equivalent to exons 6 and 7 of SPP1 and IBSP. Exon 6 of DMP1 contains a glycosaminoglycan attachment site, a specific protease cleavage site, and a more distal RGD motif. The mouse gene encodes a protein chain of 513 amino acids, including a signal peptide [253]. The amino acid sequence is highly acidic and serine rich. Phosphate analysis indicates that the NH2-terminal 37-kDa region contains 12 phosphates/mol, while the COOH-terminal 57-kDa region contains 41 [254]. The phosphates may serve as sequestering groups for recruiting calcium ions [255], or for binding to mineral. The cDNAdeduced amino acid sequence indicates that full-length rat DMP1 has three putative AsneXe-Ser/Thr N-linked glycosylation sites that are located within the 57-kDa

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proteolytic fragment, COOH-terminal to the RGD motif [250]. Full-length DMP1 is an inactive precursor which is proteolytically cleaved into two fragments. Extracts of bone and dentin contain two proteolytic fragments of DMP1 e an NH2-terminal 37-kDa fragment and a COOH-terminal 57-kDa fragment [254]. Sequence analysis revealed that DMP1 is proteolytically cleaved at four bonds: Phe173eAsp174, Ser180eAsp181, Ser217e Asp218, and Gln221eAsp222. BMP-1/Tolloid-like proteinase and the PHEX protein (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) are candidate enzymes responsible for the proteolytic processing of DMP1 [254,256]. DMP1-null mice and humans with autosomal recessive hypophosphatasia manifest rickets and osteomalacia with isolated renal phosphate wasting associated with elevated serum fibroblast growth factor 23 (FGF23) levels and normocalciuria [257,258]. Osteopontin Osteopontin, also known as secreted phosphoprotein 1, is a secreted glycoprotein with the characteristics of a matricellular protein [259]. It was named osteopontin because it was proposed to act as a “bone bridge” from bone cells to hydroxyapatite [260]. It is highly expressed by bone cells but also by many other cell types, including hypertrophic chondrocytes, odontoblasts, cementoblasts, macrophages, as well as endothelial, smooth muscle, and epithelial cells [157,261]. 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 [262]; the inhibition is dose dependent and is abolished when phosphate groups were removed from osteopontin [262e265]. The osteopontin gene, SPP1 (secreted phosphoprotein 1), maps to chromosome 4q21-q25 and is located at the end of the cluster of the five genes of the SIBLING family [266]. It contains seven exons spanning approximately 9 kbs [267,268]. Exon 1 is non-coding while exon 2 encodes the signal peptide and the first two amino acids of the mature protein. Exons 3 and 5 contain phosphorylation sequences while exon 3 also contains a glycosaminoglycan attachment site. Exons 3 and 5 can be alternatively spliced. Exon 4 is proline rich. Exon 6 contains an RGD site and a specific proteolytic site [237]. Differential RNA splicing involving 42 nucleotides of exon 5 generates two alternatively spliced products in humans [266]. Mouse Spp1 mRNA contains two translational start sites which can produce a fulllength protein of 75 kDa and a 70 kDa short form that lacks the signal peptide [269].

25

Specific sequences in the 50 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-b1, TGF-b2, retinoic acid, PTH, endothelin, proinflammatory cytokines, some BMPs, and 1,25-dihydroxyvitamin D3 [270e275]. Bone cells are a major site of synthesis and secretion of phosphorylated osteopontin [276]. In bone, osteopontin has been localized to osteoblasts, bone-lining cells, osteocytes, and cells with fibroblastic morphology associated with the periosteum [5,277,278]. In addition to being found throughout the bone matrix, osteopontin accumulates at various cellematrix interfaces (as a lamina limitans) and at matrixematrix interfaces (as a cement line) [1,4]. This protein is also synthesized by hypertrophic chondrocytes [276]. The full-length human osteopontin protein contains 300 amino acid residues, including a signal peptide of 16 amino acid residues [279]. The protein is acidic, can be highly phosphorylated [280], and is rich in sialic acid [274]. Phosphorylation of osteopontin occurs mainly on serine residues and is principally catalyzed by casein kinases [281]. Rat bone osteopontin has an average of 12 phosphoserine and one phosphothreonine residues, with considerable heterogeneity [282e284]. The variable level of phosphorylation of osteopontin observed in transformed osteogenic cells was probably attributable to modulation by 1,25-dihydroxyvitamin D3 [285]. In rat bone osteopontin, a single N-linked and 5-6 O-linked oligosaccharides have been identified, representing about 16.6% of the protein weight [282]. Three regions of the protein are highly conserved, including the N-terminal one-fourth of the chain, a segment around the RGD integrin-binding sequence, and the extreme C-terminus [279]. Four of the nine or 10 residues in the poly-Asp region (residues 70e80) are strictly conserved. In addition to the phosphate groups, this sequence may be involved in the attachment of the protein to mineral and in the regulation of growth of the bone crystals [263]. Osteopontin has an RGD cell-binding site, calciumbinding sites, and two heparin-binding domains [286]. The RGD domain (residues 128e130) interacts with cell surface integrins avb3, avb1, and avb5 to regulate cell attachment and spreading, intracellular signaling, and cell migration [287,288]. A cryptic SVVYGLR-containing domain interacts with a9b1 integrins after thrombin cleavage at residues 137 and 138 [289]. Osteopontin does not bind the standard form of CD44 (hyaluronic acid receptor) but may bind various isoforms of CD44 [290]. Osteopontin contains two conserved amino-terminal domains with heparin-binding homology that are likely to regulate its binding to the extracellular matrix.

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Osteopontin also binds directly to fibronectin, collagen, and osteocalcin [259]. Extracted osteopontin peptides from bone show that the protein exists as a heterogeneous mixture of molecules that differ in their extent of post-translational modification [283]. In particular, bone contains osteopontin molecules that differ widely in the extent of serine and threonine phosphorylation. Similar findings have been observed for osteopontin 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 [291]. 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 enzymes [292]. Transglutaminasepromoted cross-linking may account for the highermolecular-weight aggregates of osteopontin found in bone extracts [293e295]. Mice that lack osteopontin show no evidence of skeletal developmental anomalies in the neonatal period [296]. 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 4e6 months of age, the osteopontin-deficient mice had two times the trabecular bone volume and three times the number of osteoclasts than wildtype mice [297]. 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 [297].

Matrix, Extracellular Phosphoglycoprotein Matrix, extracellular phosphoglycoprotein, MEPE, was first identified using expression screening of cDNA libraries prepared from tumors removed from patients with oncogenic hypophosphatemic osteomalacia [298]. The MEPE gene is located between IBSP and SPP1 in the SIBLING domain of chromosome 4q21.1 [237]. MEPE contains five exons. Exon 1 is noncoding while exon 2 encodes the signal peptide and the first two amino acids of the main protein. Exons 3 and 4 are similar to the equivalent exons of SPP1, IBSP and DMP1. Exon 5 of SPP1, IBSP and DMP1 is absent in MEPE. Exon 5 of MEPE encodes most of the protein. A 2-kb MEPE transcipt was detected in oncogenic hypophosphatemic osteomalacia tumors [299]. The predicted MEPE protein contains 525 amino acids, including

a signal peptide, and has two N-glycosylation motifs, phosphorylation and glycosaminoglycan attachment sites, an RGD motif and a specific protease cleavage site [298]. Mouse Mepe, also called osteoblast/osteocyte factor 45 (OF45), contains three methionine translational start codons. The first of them predicts a protein containing 441 amino acids. Northern and Western blot analyses confirmed expression in mouse bone [299]. The expression of Mepe was increased during mineralization in osteoblast cultures and in osteocytes. MEPE (and other SIBLING proteins) contains a conserved amino acid sequence called the ASARM peptide (acidic, serineand aspartate-rich motif) with potent mineralization inhibition properties [300e302]. ASARM interactions with (or cleavage by) PHEX can release this mineralization inhibition [300,303]. Patients with inactivating mutations of PHEX develop osteomalacia (hypomineralization, osteoidosis) as part of X-linked hyphosphatemia in the absence of functional PHEX [304]. Mice lacking one or two alleles of Mepe had increased bone mass [299]. The Mepe-null mice were resistant to bone loss during aging. Increased bone mass appeared to be attributable to increased osteoblast numbers and activity while osteoclast number and bone resorption surface area were unchanged. These findings suggested that Mepe normally plays an inhibitory role in bone formation in mice [299].

MINERALIZATION OF BONE The mineralization of vertebrate bone is a remarkable melding of geological and biological processes. Not surprisingly, Nature makes good use of terrestrial elements for tissue hardening and combines them in such a way to form generally insoluble (at physiologic neutral pH) calciumephosphate salts whose nanocrystals solidify bone, cartilage and tooth extracellular matrices. Particularly intriguing is the biological scaling down of geological, rock-forming processes such that bone nano-“biorocks” are single crystals that attain a remarkable regularity in size, shape, orientation and number within the extracellular matrix. The location of mineral within bone extracellular matrix clearly resides both within, and between, the collagen fibrils [1,305,306]. These distinct ultrastructural compartments for the mineral phase invariably provide different spatial and chemical environments for crystal growth, affecting crystal size and orientation. Whereas a reasonable amount of published information exists on how crystal growth e once initiated e is regulated in bone, there is a relative paucity of information on the nucleation process by which clusters of mineral ions reach a critical, stable nucleus size to

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impart the crystalline, long-range order characteristic of mature bone mineral crystals. Indeed, precursor phases e such as amorphous calcium phosphate, and octacalcium phosphate e have long been proposed to exist prior to mineral maturation to form the crystalline apatitic phase, but compelling evidence for this remains scant [3]. Regardless of this mineral progression, key early facilitators of bone matrix mineralization have recently been identified, largely revolving around the handling and processing of the potent mineralization inhibitor pyrophosphate. Pyrophosphate (P2O7) is a ubiquitous metabolic byproduct of many intracellular processes found in most cells, and it also can be produced extracellularly [307]. Tissue fluid pyrophosphate found throughout the body prevents mineral deposition by binding either ionic or crystalline (in lattice) calcium such that mineralization is prevented. Tissue-non-specific alkaline phosphatase (ALPL, TNAP; the bone/kidney/liver isoform of alkaline phosphatase) [308] is an enzyme highly expressed by mineralized tissue cells, where it enzymatically degrades inhibitory pyrophosphate to promote mineralization, and where the resultant two orthophosphate ions produced by the hydrolysis might additionally promote mineralization by elevating local phosphate concentrations. Such an elegant, relatively simple way of initiating mineralization in bones and teeth (via high cellular ALPL expression to remove pyrophosphate), while preventing mineralization in soft tissues (low, or absent, ALPL expression resulting in persistent pyrophosphate), is supported by recent compelling mouse genetics studies [309]. Transgenic mice deficient in either ENPP1 (ectonucleotide pyrophosphatase phosphodiesterase 1; also called PC1, plasma cell membrane glycoprotein 1) that produces pyrophosphate directly extracellularly from nucleotides such as ATP, or ANK (ankylosis protein) which transports pyrophosphate out of the cell and into the extracellular compartment, both show decreased extracellular pyrophosphate and abundant ectopic calcification [310,311]. Overexpression of ALPL in the dermis of the skin in transgenic mice intentionally to remove pyrophosphate from a soft connective tissue results in dermal collagen mineralization [312]. Importantly, inactivating mutations in the ALPL gene e where ALPL enzymatic activity is reduced and extracellular inhibitory pyrophosphate levels are thus elevated e causes hypophosphatasia with characteristic osteomalacia (hypomineralization of bone, osteoidosis) [313]. Equally important effectors of bone mineralization are serum and tissue fluid levels of calcium and phosphate. The endocrinology underlying mineral ion homeostasis is both critical and complex, and essential for maintaining circulating and tissuespecific levels of mineral ions appropriate for extracellular matrix mineralization [314]. Probably the single

27

biggest advance in these combined fields is the realization that phosphate plays a far more critical role in physiologic and pathologic calcification than ever imagined (whereas previously the emphasis was on calcium), and that the phosphate:pyrophosphate ratio (modulated in large measure by the actions of ALPL) provides key molecular regulation maintaining the equilibrium between early promoters and inhibitors necessary for the initiation of normal bone matrix mineralization [315]. Superimposed on these determinants are the matrix vesicles released by osteoblasts (and maybe osteocytes) into the extracellular matrix [316]. Matrix vesicles contain much of the molecular machinery for initiation of mineralization as described above e within a shed, membrane-circumscribed “cytoplasmic droplet” e while additionally having a concentrated phospholipid-rich membrane component from which enzymatically released phosphate (by PHOSPHO1) [317] may likewise be a critical factor regulating the phosphate:pyrophosphate balance within and immediately surrounding matrix vesicles. Once matrix vesicle integrity is lost by rupture of the vesicles, molecular determinants within the extracellular milieu, as described above and below, take further control of the mineralization process. Beyond the initiation of mineralization in bone, crystal growth is regulated by a subcategory of the secreted, calcium-binding phosphoprotein (SCPP) family [318] called the small, integrin-binding ligand, N-linked glycoproteins (SIBLINGs) that includes OPN, MEPE, BSP, DMP1 and DSPP [236]. Details for most of these proteins have already been provided in the preceding sections, and here we focus on a broader discussion of the role bone matrix proteins in general, and their peptides, in the regulation of bone mineralization. The larger family of SCPPs are calcium-binding proteins secreted into tissue fluids where calcium is particularly abundant (e.g. milk and saliva) where they are thought to bind and transport calcium while preventing crystal formation [318]. They are also prominent proteins in skeletal and dental mineralized tissues where they regulate crystal growth e again, usually as inhibitors of mineralization [236]. The need for crystal growth inhibitors in tissues destined for mineralization may not at first glance be readily apparent, but the massive mineralization events that occur in bones and teeth e induced by the processes described above e require a significant level of SIBLING protein regulation at the organiceinorganic interface as crystals must attain tissue-specific size, shape and orientation with the extracellular matrix [1]. For bone extracellular matrix, crystal growth regulation occurs in two ultrastructurally distinct compartments e within collagen fibrils, and between collagen fibrils. While biochemically and structurally distinct,

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each becomes fully calcified such that mineralization ultimately permeates and extends throughout the entire volume of assembled matrix. Early mineralization events occur as small calcification foci in the generally unmineralized, preassembled matrix termed the osteoid, although it is not unambiguously clear where the initial mineralization occurs, be it in matrix vesicles, within collagen fibrils or between collagen fibrils. Beyond the osteoid deeper in the matrix, there is a massive propagation of crystal growth at a planar zone called the mineralization front; here, bone matrix incorporates its major hardening event forming the mineralized matrix proper of bone which, over time, undergoes a slower maturation gradually to become even more mineralized [319]. Figure 2.1 illustrates generally locations of mineralization within bone extracellular matrix, and ultrastructural matrixemineral relationships within the collagenous matrix. Closely correlated with mineralization are osteoblast and osteocyte expression of tissue-non-specific alkaline phosphatase and SIBLING proteins, all of which initiate and regulate this key extracellular event. The SIBLING proteins have common features that include their being highly negatively charged (important in calcium binding) and extensively post-translationally modified, particularly by heavy phosphorylation in bone [284] which, in turn, adds additional negative charge to the proteins. Specific peptide motifs within the SIBLING proteins have been identified as being particularly effective in inhibiting crystal growth [320e322], and enzymatic processing of parent SIBLING proteins releasing smaller, highly active inhibitory peptides is thought to be an important step in how these proteins regulate mineralization [302,303]. In turn, enzymatic degradation of the inhibitory peptides releases crystals from growth inhibition [300,321]. Such a proteolytic processing cascade involved in the direct regulation of bone mineralization and acting locally at the level of the extracellular matrix (as compared to circulating factors modulating mineral ion homeostasis systemically) is perhaps best exemplified by work done on the ASARM peptide which is highly conserved among the SIBLING proteins [303]. Accumulating evidence for the mineralization-inhibiting ASARM peptide (from MEPE and OPN) [300,302,321] shows that it is a binding partner and substrate for PHEX e an enzyme whose inactivating mutations lead to X-linked hypophosphatemia (XLH) showing hallmark osteomalacia. In the absence of PHEX, renal phosphate wasting and accumulated ASARM peptides residing in the bone matrix likely act together in preventing adequate skeletal mineralization in XLH. In summary, bone extracellular matrix mineralization occurs as the net result of both systemic circulating factors regulating mineral ion homeostasis in blood

plasma and bone tissue fluids, together with local factors acting at the organiceinorganic interface [323]. Collectively, these determinants of mineralization induce and control crystal growth, and ultimately terminate the mineralization process, in a manner appropriate for the biomechanical demands placed on the skeleton.

References [1] McKee MD, Addison WN, Kaartinen MT. Hierarchies of extracellular matrix and mineral organization in bone of the craniofacial complex and skeleton. Cells Tissues Organs 2005;181: 176e88. [2] Wysolmerski JJ. Interactions between breast, bone, and brain regulate mineral and skeletal metabolism during lactation. Ann NY Acad Sci 2010;1192:161e9. [3] Rey C, Combes C, Drouet C, Glimcher M. Bone mineral: update on chemical composition and structure. Osteoporos Int 2009;20: 1013e21. [4] McKee MD, Nanci A. Osteopontin: an interfacial extracellular matrix protein in mineralized tissues. Connect Tissue Res 1996;35:197e205. [5] McKee MD, Nanci A. Osteopontin at mineralized tissue interfaces in bone, teeth, and osseointegrated implants: ultrastructural distribution and implications for mineralized tissue formation, turnover, and repair. Microsc Res Tech 1996;33:141e64. [6] Parfitt AM. Targeted and nontargeted bone remodeling: relationship to basic multicellular unit origination and progression. Bone 2002;30:5e7. [7] Herring G, editor. The Organic Matrix of Bone. New York: Academic Press; 1972. [8] Click EM, Bornstein P. Isolation and characterization of the cyanogen bromide peptides from the alpha 1 and alpha 2 chains of human skin collagen. Biochemistry 1970;9:4699e706. [9] Fietzek PP, Furthmayr H, Kuhn K. Comparative sequence studies on alpha2-CB2 from calf, human, rabbit and pig-skin collagen. Eur J Biochem 1974;47:257e61. [10] Bernard MP, Chu ML, Myers JC, Ramirez F, Eikenberry EF, Prockop DJ. Nucleotide sequences of complementary deoxyribonucleic acids for the pro alpha 1 chain of human type I procollagen. Statistical evaluation of structures that are conserved during evolution. Biochemistry 1983;22:5213e23. [11] Hartung S, Jaenisch R, Breindl M. Retrovirus insertion inactivates mouse alpha 1(I) collagen gene by blocking initiation of transcription. Nature 1986;320:365e7. [12] Bonadio J, Jepsen KJ, Mansoura MK, Jaenisch R, Kuhn JL, Goldstein SA. A murine skeletal adaptation that significantly increases cortical bone mechanical properties. Implications for human skeletal fragility. J Clin Invest 1993;92:1697e705. [13] Retief E, Parker MI, Retief AE. Regional chromosome mapping of human collagen genes alpha 2(I) and alpha 1(I) (COLIA2 and COLIA1). Hum Genet 1985;69:304e8. [14] de Wet W, Bernard M, Benson-Chanda V, et al. Organization of the human pro-alpha 2(I) collagen gene. J Biol Chem 1987;262: 16032e6. [15] Tromp G, Kuivaniemi H, Stacey A, et al. Structure of a fulllength cDNA clone for the prepro alpha 1(I) chain of human type I procollagen. Biochem J 1988;253:919e22. [16] Kuivaniemi H, Tromp G, Chu ML, Prockop DJ. Structure of a full-length cDNA clone for the prepro alpha 2(I) chain of human type I procollagen. Comparison with the chicken gene confirms unusual patterns of gene conservation. Biochem J 1988;252:633e40.

PEDIATRIC BONE

29

REFERENCES

[17] Vuorio E, de Crombrugghe B. The family of collagen genes. Annu Rev Biochem 1990;59:837e72. [18] Makela JK, Raassina M, Virta A, Vuorio E. Human pro alpha 1(I) collagen: cDNA sequence for the C-propeptide domain. Nucleic Acids Res 1988;16:349. [19] Bernard MP, Myers JC, Chu ML, Ramirez F, Eikenberry EF, Prockop DJ. Structure of a cDNA for the pro alpha 2 chain of human type I procollagen. Comparison with chick cDNA for pro alpha 2(I) identifies structurally conserved features of the protein and the gene. Biochemistry 1983;22:1139e45. [20] Orgel JP, Wess TJ, Miller A. The in situ conformation and axial location of the intermolecular cross-linked non-helical telopeptides of type I collagen. Structure Fold Des 2000;8:137e42. [21] Bernocco S, Finet S, Ebel C, et al. Biophysical characterization of the C-propeptide trimer from human procollagen III reveals a tri-lobed structure. J Biol Chem 2001;276:48930e6. [22] Lees JF, Bulleid NJ. The role of cysteine residues in the folding and association of the COOH-terminal propeptide of types I and III procollagen. J Biol Chem 1994;269:24354e60. [23] Veis A, Leibovich SJ, Evans J, Kirk TZ. Supramolecular assemblies of mRNA direct the coordinated synthesis of type I procollagen chains. Proc Natl Acad Sci USA 1985;82:3693e7. [24] Vuust J. Procollagen biosynthesis by embryonic-chick-bone polysomes. Estimation of the relative numbers of active proalpha1 and proalpha2 messenger ribonucleic acids. Eur J Biochem 1975;60:41e50. [25] Vuust J, Piez KA. A kinetic study of collagen biosynthesis. J Biol Chem 1972;247:856e62. [26] Vuust J, Sobel ME, Martin GR. Regulation of type I collagen synthesis. Total pro alpha 1(I) and pro alpha 2(I) mRNAs are maintained in a 2:1 ratio under varying rates of collagen synthesis. Eur J Biochem 1985;151:449e53. [27] Olsen AS, Prockop DJ. Transcription of human type I collagen genes. Variation in the relative rates of transcription of the pro alpha 1 and pro alpha 2 genes. Matrix 1989;9:73e81. [28] Hu G, Tylzanowski P, Inoue H, Veis A. Relationships between translation of pro alpha1(I) and pro alpha2(I) mRNAs during synthesis of the type I procollagen heterotrimer. J Cell Biochem 1995;59:214e34. [29] Lees JF, Tasab M, Bulleid NJ. Identification of the molecular recognition sequence which determines the type-specific assembly of procollagen. Embo J 1997;16:908e16. [30] Veis A, Brownell AG. Triple-helix formation on ribosome-bound nascent chains of procollagen: deuterium-hydrogen exchange studies. Proc Natl Acad Sci USA 1977;74:902e5. [31] Brownell AG, Veis A. Intracellular location of triple helix formation of collagen. Enzyme probe studies. J Biol Chem 1976;251:7137e43. [32] Brownell AG, Veis A. The intracellular location of the glycosylation of hydroxylysine of collagen. Biochem Biophys Res Commun 1975;63:371e7. [33] Veis A, Kirk TZ. The coordinate synthesis and cotranslational assembly of type I procollagen. J Biol Chem 1989;264:3884e9. [34] Kosher RA, Kulyk WM, Gay SW. Collagen gene expression during limb cartilage differentiation. J Cell Biol 1986;102:1151e6. [35] Bienkowski RS, Baum BJ, Crystal RG. Fibroblasts degrade newly synthesised collagen within the cell before secretion. Nature 1978;276:413e6. [36] Prockop DJ, Kivirikko KI. Heritable diseases of collagen. N Engl J Med 1984;311:376e86. [37] Popescu NC, Cheng SY, Pastan I. Chromosomal localization of the gene for a human thyroid hormone-binding protein. Am J Hum Genet 1988;42:560e4. [38] Helaakoski T, Annunen P, Vuori K, MacNeil IA, Pihlajaniemi T, Kivirikko KI. Cloning, baculovirus expression, and

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54]

characterization of a second mouse prolyl 4-hydroxylase alphasubunit isoform: formation of an alpha 2 beta 2 tetramer with the protein disulfide-isomerase/beta subunit. Proc Natl Acad Sci USA 1995;92:4427e31. Pajunen L, Jones TA, Helaakoski T, et al. Assignment of the gene coding for the alpha-subunit of prolyl 4-hydroxylase to human chromosome region 10q21.3-23.1. Am J Hum Genet 1989;45: 829e34. Helaakoski T, Veijola J, Vuori K, et al. Structure and expression of the human gene for the alpha subunit of prolyl 4-hydroxylase. The two alternatively spliced types of mRNA correspond to two homologous exons the sequences of which are expressed in a variety of tissues. J Biol Chem 1994;269:27847e54. Annunen P, Helaakoski T, Myllyharju J, Veijola J, Pihlajaniemi T, Kivirikko KI. Cloning of the human prolyl 4-hydroxylase alpha subunit isoform alpha(II) and characterization of the type II enzyme tetramer. The alpha(I) and alpha(II) subunits do not form a mixed alpha(I)alpha(II)beta2 tetramer. J Biol Chem 1997;272:17342e8. Ishikawa Y, Wirz J, Vranka JA, Nagata K, Bachinger HP. Biochemical characterization of the prolyl 3-hydroxylase 1.cartilage-associated protein.cyclophilin B complex. J Biol Chem 2009;284:17641e7. Hautala T, Byers MG, Eddy RL, Shows TB, Kivirikko KI, Myllyla R. Cloning of human lysyl hydroxylase: complete cDNA-derived amino acid sequence and assignment of the gene (PLOD) to chromosome 1p36.3ep36.2. Genomics 1992;13:62e9. Szpirer C, Szpirer J, Riviere M, Vanvooren P, Valtavaara M, Myllyla R. Localization of the gene encoding a novel isoform of lysyl hydroxylase. Mamm Genome 1997;8:707e8. Valtavaara M, Papponen H, Pirttila AM, Hiltunen K, Helander H, Myllyla R. Cloning and characterization of a novel human lysyl hydroxylase isoform highly expressed in pancreas and muscle. J Biol Chem 1997;272:6831e4. Passoja K, Rautavuoma K, Ala-Kokko L, Kosonen T, Kivirikko KI. Cloning and characterization of a third human lysyl hydroxylase isoform. Proc Natl Acad Sci USA 1998;95:10482e6. Valtavaara M, Szpirer C, Szpirer J, Myllyla R. Primary structure, tissue distribution, and chromosomal localization of a novel isoform of lysyl hydroxylase (lysyl hydroxylase 3). J Biol Chem 1998;273:12881e6. Breslau-Siderius EJ, Engelbert RH, Pals G, van der Sluijs JA. Bruck syndrome: a rare combination of bone fragility and multiple congenital joint contractures. J Pediatr Orthop B 1998;7: 35e8. Kelley BP, Malfait F, Bonafe L, et al. Mutations in FKBP10 cause recessive osteogenesis imperfecta and Bruck syndrome. J Bone Miner Res 2010;26:666e72. Bottomley MJ, Batten MR, Lumb RA, Bulleid NJ. Quality control in the endoplasmic reticulum: PDI mediates the ER retention of unassembled procollagen C-propeptides. Curr Biol 2001;11: 1114e8. Wilson R, Lees JF, Bulleid NJ. Protein disulfide isomerase acts as a molecular chaperone during the assembly of procollagen. J Biol Chem 1998;273:9637e43. Nagai N, Hosokawa M, Itohara S, et al. Embryonic lethality of molecular chaperone hsp47 knockout mice is associated with defects in collagen biosynthesis. J Cell Biol 2000;150:1499e506. Ikegawa S, Sudo K, Okui K, Nakamura Y. Isolation, characterization and chromosomal assignment of human colligin-2 gene (CBP2). Cytogenet Cell Genet 1995;71:182e6. Bruckner P, Eikenberry EF. Formation of the triple helix of type I procollagen in cellulo. Temperature-dependent kinetics support a model based on cis in equilibrium trans isomerization of peptide bonds. Eur J Biochem 1984;140:391e5.

PEDIATRIC BONE

30

2. BONE MATRIX AND MINERALIZATION

[55] Bruckner P, Eikenberry EF, Prockop DJ. Formation of the triple helix of type I procollagen in cellulo. A kinetic model based on cis-trans isomerization of peptide bonds. Eur J Biochem 1981;118:607e13. [56] Bulleid NJ, Dalley JA, Lees JF. The C-propeptide domain of procollagen can be replaced with a transmembrane domain without affecting trimer formation or collagen triple helix folding during biosynthesis. Embo J 1997;16:6694e701. [57] Sarkar SK, Young PE, Sullivan CE, Torchia DA. Detection of cis and trans X-Pro peptide bonds in proteins by 13C NMR: application to collagen. Proc Natl Acad Sci USA 1984;81:4800e3. [58] Buevich AV, Dai QH, Liu X, Brodsky B, Baum J. Site-specific NMR monitoring of cis-trans isomerization in the folding of the proline-rich collagen triple helix. Biochemistry 2000;39:4299e308. [59] Lang K, Schmid FX. Protein-disulphide isomerase and prolyl isomerase act differently and independently as catalysts of protein folding. Nature 1988;331:453e5. [60] Alanay Y, Avaygan H, Camacho N, et al. Mutations in the gene encoding the RER protein FKBP65 cause autosomal-recessive osteogenesis imperfecta. Am J Hum Genet 2010;86:551e9. [61] Perret S, Merle C, Bernocco S, et al. Unhydroxylated triple helical collagen I produced in transgenic plants provides new clues on the role of hydroxyproline in collagen folding and fibril formation. J Biol Chem 2001;276:43693e8. [62] Xu Y, Bhate M, Brodsky B. Characterization of the nucleation step and folding of a collagen triple-helix peptide. Biochemistry 2002;41:8143e51. [63] Boudko S, Frank S, Kammerer RA, et al. Nucleation and propagation of the collagen triple helix in single-chain and trimerized peptides: transition from third to first order kinetics. J Mol Biol 2002;317:459e70. [64] Persikov AV, Ramshaw JA, Kirkpatrick A, Brodsky B. Peptide investigations of pairwise interactions in the collagen triplehelix. J Mol Biol 2002;316:385e94. [65] Hurskainen TL, Hirohata S, Seldin MF, Apte SS. ADAM-TS5, ADAM-TS6, and ADAM-TS7, novel members of a new family of zinc metalloproteases. General features and genomic distribution of the ADAM-TS family. J Biol Chem 1999;274:25555e63. [66] Colige A, Sieron AL, Li SW, et al. Human Ehlers-Danlos syndrome type VII C and bovine dermatosparaxis are caused by mutations in the procollagen I N-proteinase gene. Am J Hum Genet 1999;65:308e17. [67] Kessler E, Takahara K, Biniaminov L, Brusel M, Greenspan DS. Bone morphogenetic protein-1: the type I procollagen Cproteinase. Science 1996;271:360e2. [68] Yoshiura K, Tamura T, Hong HS, et al. Mapping of the bone morphogenetic protein 1 gene (BMP1) to 8p21: removal of BMP1 from candidacy for the bone disorder in Langer-Giedion syndrome. Cytogenet Cell Genet 1993;64:208e9. [69] Sieron AL, Tretiakova A, Jameson BA, et al. Structure and function of procollagen C-proteinase (mTolloid) domains determined by protease digestion, circular dichroism, binding to procollagen type I, and computer modeling. Biochemistry 2000;39:3231e9. [70] Scott IC, Clark TG, Takahara K, et al. Assignment of TLL1 and TLL2, which encode human BMP-1/Tolloid-related metalloproteases, to chromosomes 4q32e>q33 and 10q23e>q24 and assignment of murine Tll2 to chromosome 19. Cytogenet Cell Genet 1999;86:64e5. [71] Scott IC, Clark TG, Takahara K, Hoffman GG, Greenspan DS. Structural organization and expression patterns of the human and mouse genes for the type I procollagen COOH-terminal proteinase enhancer protein. Genomics 1999;55:229e34. [72] Takahara K, Osborne L, Elliott RW, Tsui LC, Scherer SW, Greenspan DS. Fine mapping of the human and mouse genes

[73]

[74]

[75]

[76]

[77] [78] [79]

[80]

[81]

[82]

[83] [84]

[85] [86]

[87]

[88]

[89]

[90]

[91]

for the type I procollagen COOH-terminal proteinase enhancer protein. Genomics 1996;31:253e6. Xu H, Acott TS, Wirtz MK. Identification and expression of a novel type I procollagen C-proteinase enhancer protein gene from the glaucoma candidate region on 3q21-q24. Genomics 2000;66:264e73. Bornstein P, Walsh V, Tullis J, Stainbrook E, Bateman JF, Hormuzdi SG. The globular domain of the proalpha 1(I) Npropeptide is not required for secretion, processing by procollagen N-proteinase, or fibrillogenesis of type I collagen in mice. J Biol Chem 2002;277:2605e13. Birk DE, Trelstad RL. Extracellular compartments in matrix morphogenesis: collagen fibril, bundle, and lamellar formation by corneal fibroblasts. J Cell Biol 1984;99:2024e33. Silver D, Miller J, Harrison R, Prockop DJ. Helical model of nucleation and propagation to account for the growth of type I collagen fibrils from symmetrical pointed tips: a special example of self-assembly of rod-like monomers. Proc Natl Acad Sci USA 1992;89:9860e4. Ramachandran G, Kartha G. Structure of collagen. Nature 1954;174:269e70. Rich A, Crick F. The structure of collagen. Nature 1955;176: 915e6. Mariani TJ, Trackman PC, Kagan HM, et al. The complete derived amino acid sequence of human lysyl oxidase and assignment of the gene to chromosome 5 (extensive sequence homology with the murine ras recision gene). Matrix 1992;12:242e8. Kenyon K, Modi WS, Contente S, Friedman RM. A novel human cDNA with a predicted protein similar to lysyl oxidase maps to chromosome 15q24-q25. J Biol Chem 1993;268:18435e7. Jourdan-Le Saux C, Le Saux O, Donlon T, Boyd CD, Csiszar K. The human lysyl oxidase-related gene (LOXL2) maps between markers D8S280 and D8S278 on chromosome 8p21.2-p21.3. Genomics 1998;51:305e7. Eyre DR, Oguchi H. The hydroxypyridinium crosslinks of skeletal collagens: their measurement, properties and a proposed pathway of formation. Biochem Biophys Res Commun 1980;92:403e10. Eyre DR, Weis MA, Wu JJ. Advances in collagen cross-link analysis. Methods 2008;45:65e74. Miyahara M, Hayashi K, Berger J, et al. Formation of collagen fibrils by enzymic cleavage of precursors of type I collagen in vitro. J Biol Chem 1984;259:9891e8. Kadler KE, Holmes DF, Trotter JA, Chapman JA. Collagen fibril formation. Biochem J 1996;316:1e11. Birk DE, Nurminskaya MV, Zycband EI. Collagen fibrillogenesis in situ: fibril segments undergo post-depositional modifications resulting in linear and lateral growth during matrix development. Dev Dyn 1995;202:229e43. Holmes DF, Lowe MP, Chapman JA. Vertebrate (chick) collagen fibrils formed in vivo can exhibit a reversal in molecular polarity. J Mol Biol 1994;235:80e3. Parkinson J, Kadler KE, Brass A. Self-assembly of rodlike particles in two dimensions: A simple model for collagen fibrillogenesis. Physical Review E Stat Phys Plasmas Fluids Relat Interdisciplinary Top 1994;50:2963e6. Birk DE. Type V collagen: heterotypic type I/V collagen interactions in the regulation of fibril assembly. Micron 2001;32: 223e37. White JF, Werkmeister JA, Darby IA, Bisucci T, Birk DE, Ramshaw JA. Collagen fibril formation in a wound healing model. J Struct Biol 2002;137:23e30. Niyibizi C, Eyre DR. Identification of the cartilage alpha 1(XI) chain in type V collagen from bovine bone. FEBS Lett 1989;242: 314e8.

PEDIATRIC BONE

REFERENCES

[92] Scott JE, Parry DA. Control of collagen fibril diameters in tissues. Int J Biol Macromol 1992;14:292e3. [93] Hulmes DJ. Building collagen molecules, fibrils, and suprafibrillar structures. J Struct Biol 2002;137:2e10. [94] Orgel JP, Miller A, Irving TC, Fischetti RF, Hammersley AP, Wess TJ. The in situ supermolecular structure of type I collagen. Structure (Camb) 2001;9:1061e9. [95] Giraud-Guille MM. Twisted liquid crystalline supramolecular arrangements in morphogenesis. Int Rev Cytol 1996;166:59e101. [96] Chung E, Rhodes K, Miller EJ. Isolation of three collagenous components of probable basement membrane origin from several tissues. Biochem Biophys Res Commun 1976;71: 1167e74. [97] Rhodes RK, Miller EJ. Physicochemical characterization and molecular organization of the collagen A and B chains. Biochemistry 1978;17:3442e8. [98] Fessler LI, Robinson WJ, Fessler JH. Biosynthesis of procollagen [(pro alpha 1 V)2 (pro alpha 2 V)] by chick tendon fibroblasts and procollagen (pro alpha 1 V)3 by hamster lung cell cultures. J Biol Chem 1981;256:9646e51. [99] Morris NP, Watt SL, Davis JM, Bachinger HP. Unfolding intermediates in the triple helix to coil transition of bovine type XI collagen and human type V collagens alpha 1(2) alpha 2 and alpha 1 alpha 2 alpha 3. J Biol Chem 1990;265:10081e7. [100] Chernousov MA, Rothblum K, Tyler WA, Stahl RC, Carey DJ. Schwann cells synthesize type V collagen that contains a novel alpha 4 chain. Molecular cloning, biochemical characterization, and high affinity heparin binding of alpha 4(V) collagen. J Biol Chem 2000;275:28208e15. [101] Imamura Y, Scott IC, Greenspan DS. The pro-alpha3(V) collagen chain. Complete primary structure, expression domains in adult and developing tissues, and comparison to the structures and expression domains of the other types V and XI procollagen chains. J Biol Chem 2000;275:8749e59. [102] Takahara K, Hoffman GG, Greenspan DS. Complete structural organization of the human alpha 1 (V) collagen gene (COL5A1): divergence from the conserved organization of other characterized fibrillar collagen genes. Genomics 1995;29:588e97. [103] Greenspan DS, Cheng W, Hoffman GG. The pro-alpha 1(V) collagen chain. Complete primary structure, distribution of expression, and comparison with the pro-alpha 1(XI) collagen chain. J Biol Chem 1991;266:24727e33. [104] Zhidkova NI, Brewton RG, Mayne R. Molecular cloning of PARP (proline/arginine-rich protein) from human cartilage and subsequent demonstration that PARP is a fragment of the NH2terminal domain of the collagen alpha 2(XI) chain. FEBS Lett 1993;326:25e8. [105] Tsumaki N, Kimura T. Differential expression of an acidic domain in the amino-terminal propeptide of mouse pro-alpha 2(XI) collagen by complex alternative splicing. J Biol Chem 1995;270:2372e8. [106] Yoshioka H, Ramirez F. Pro-alpha 1(XI) collagen. Structure of the amino-terminal propeptide and expression of the gene in tumor cell lines. J Biol Chem 1990;265:6423e6. [107] Fessler LI, Chapin S, Brosh S, Fessler JH. Intracellular transport and tyrosine sulfation of procollagens V. Eur J Biochem 1986;158:511e8. [108] Neame PJ, Young CN, Treep JT. Isolation and primary structure of PARP, a 24-kDa proline- and arginine-rich protein from bovine cartilage closely related to the NH2-terminal domain in collagen alpha 1 (XI). J Biol Chem 1990;265:20401e8. [109] Moradi-Ameli M, Rousseau JC, Kleman JP, Champliaud MF, Boutillon MM, Bernillon J, et al. Diversity in the processing events at the N-terminus of type-V collagen. Eur J Biochem 1994;221:987e95.

31

[110] Moradi-Ameli M, Deleage G, Geourjon C, van der Rest M. Common topology within a non-collagenous domain of several different collagen types. Matrix Biol 1994;14:233e9. [111] Takahara K, Sato Y, Okazawa K, et al. Complete primary structure of human collagen alpha 1 (V) chain. J Biol Chem 1991;266:13124e9. [112] Fields GB, Van Wart HE, Birkedal-Hansen H. Sequence specificity of human skin fibroblast collagenase. Evidence for the role of collagen structure in determining the collagenase cleavage site. J Biol Chem 1987;262:6221e6. [113] Bernard M, Yoshioka H, Rodriguez E, et al. Cloning and sequencing of pro-alpha 1 (XI) collagen cDNA demonstrates that type XI belongs to the fibrillar class of collagens and reveals that the expression of the gene is not restricted to cartilagenous tissue. J Biol Chem 1988;263:17159e66. [114] Yaoi Y, Hashimoto K, Koitabashi H, Takahara K, Ito M, Kato I. Primary structure of the heparin-binding site of type V collagen. Biochim Biophys Acta 1990;1035:139e45. [115] Fichard A, Kleman JP, Ruggiero F. Another look at collagen V and XI molecules. Matrix Biol 1995;14:515e31. [116] Takagaki K, Munakata H, Kakizaki I, Iwafune M, Itabashi T, Endo M. Domain structure of chondroitin sulfate E octasaccharides binding to type V collagen. J Biol Chem 2002;277: 8882e9. [117] Weil D, Bernard M, Gargano S, Ramirez F. The pro alpha 2(V) collagen gene is evolutionarily related to the major fibrillarforming collagens. Nucleic Acids Res 1987;15:181e98. [118] Emanuel BS, Cannizzaro LA, Seyer JM, Myers JC. Human alpha 1(III) and alpha 2(V) procollagen genes are located on the long arm of chromosome 2. Proc Natl Acad Sci USA 1985;82:3385e9. [119] Myers JC, Loidl HR, Seyer JM, Dion AS. Complete primary structure of the human alpha 2 type V procollagen COOHterminal propeptide. J Biol Chem 1985;260:11216e22. [120] Valkkila M, Melkoniemi M, Kvist L, Kuivaniemi H, Tromp G, Ala-Kokko L. Genomic organization of the human COL3A1 and COL5A2 genes: COL5A2 has evolved differently than the other minor fibrillar collagen genes. Matrix Biol 2001;20:357e66. [121] Woodbury D, Benson-Chanda V, Ramirez F. Amino-terminal propeptide of human pro-alpha 2(V) collagen conforms to the structural criteria of a fibrillar procollagen molecule. J Biol Chem 1989;264:2735e8. [122] Niyibizi C, Eyre DR. Structural analysis of the extension peptides on matrix forms of type V collagen in fetal calf bone and skin. Biochim Biophys Acta 1993;1203:304e9. [123] Fichard A, Tillet E, Delacoux F, Garrone R, Ruggiero F. Human recombinant alpha1(V) collagen chain. Homotrimeric assembly and subsequent processing. J Biol Chem 1997;272:30083e7. [124] Bachinger HP, Doege KJ, Petschek JP, Fessler LI, Fessler JH. Structural implications from an electronmicroscopic comparison of procollagen V with procollagen I, pC-collagen I, procollagen IV, and a Drosophila procollagen. J Biol Chem 1982;257:14590e2. [125] Unsold C, Pappano WN, Imamura Y, Steiglitz BM, Greenspan DS. Biosynthetic processing of the pro-alpha 1(V) 2pro-alpha 2(V) collagen heterotrimer by bone morphogenetic protein-1 and furin-like proprotein convertases. J Biol Chem 2002;277:5596e602. [126] Fitch JM, Gross J, Mayne R, Johnson-Wint B, Linsenmayer TF. Organization of collagen types I and V in the embryonic chicken cornea: monoclonal antibody studies. Proc Natl Acad Sci USA 1984;81:2791e5. [127] Niyibizi C, Eyre DR. Structural characteristics of cross-linking sites in type V collagen of bone. Chain specificities and heterotypic links to type I collagen. Eur J Biochem 1994;224:943e50. [128] Kypreos KE, Birk D, Trinkaus-Randall V, Hartmann DJ, Sonenshein GE. Type V collagen regulates the assembly of

PEDIATRIC BONE

32

[129]

[130]

[131]

[132]

[133]

[134]

[135] [136]

[137]

[138]

[139]

[140]

[141]

[142]

[143]

[144]

[145]

[146]

[147]

2. BONE MATRIX AND MINERALIZATION

collagen fibrils in cultures of bovine vascular smooth muscle cells. J Cell Biochem 2000;80:146e55. Adachi E, Hayashi T, Hashimoto PH. Immunoelectron microscopical evidence that type V collagen is a fibrillar collagen: importance for an aggregating capability of the preparation for reconstituting banding fibrils. Matrix 1989;9:232e7. Birk DE, Fitch JM, Babiarz JP, Doane KJ, Linsenmayer TF. Collagen fibrillogenesis in vitro: interaction of types I and V collagen regulates fibril diameter. J Cell Sci 1990;95:649e57. Andrikopoulos K, Liu X, Keene DR, Jaenisch R, Ramirez F. Targeted mutation in the col5a2 gene reveals a regulatory role for type V collagen during matrix assembly. Nat Genet 1995;9: 31e6. Mumby SM, Raugi GJ, Bornstein P. Interactions of thrombospondin with extracellular matrix proteins: selective binding to type V collagen. J Cell Biol 1984;98:646e52. Koda JE, Rapraeger A, Bernfield M. Heparan sulfate proteoglycans from mouse mammary epithelial cells. Cell surface proteoglycan as a receptor for interstitial collagens. J Biol Chem 1985;260:8157e62. Whinna HC, Choi HU, Rosenberg LC, Church FC. Interaction of heparin cofactor II with biglycan and decorin. J Biol Chem 1993;268:3920e4. Aho S, Uitto J. Two-hybrid analysis reveals multiple direct interactions for thrombospondin 1. Matrix Biol 1998;17:401e12. Ruggiero F, Champliaud MF, Garrone R, Aumailley M. Interactions between cells and collagen V molecules or single chains involve distinct mechanisms. Exp Cell Res 1994;210:215e23. Iozzo RV. The family of the small leucine-rich proteoglycans: key regulators of matrix assembly and cellular growth. Crit Rev Biochem Mol Biol 1997;32:141e74. Fleischmajer R, Fisher LW, MacDonald ED, Jacobs Jr L, Perlish JS, Termine JD. Decorin interacts with fibrillar collagen of embryonic and adult human skin. J Struct Biol 1991;106: 82e90. Fisher LW, Heegaard AM, Vetter U, et al. Human biglycan gene. Putative promoter, intron-exon junctions, and chromosomal localization. J Biol Chem 1991;266:14371e7. Danielson KG, Fazzio A, Cohen I, Cannizzaro LA, Eichstetter I, Iozzo RV. The human decorin gene: intron-exon organization, discovery of two alternatively spliced exons in the 50 untranslated region, and mapping of the gene to chromosome 12q23. Genomics 1993;15:146e60. Antonsson P, Heinegard D, Oldberg A. Structure and deduced amino acid sequence of the human fibromodulin gene. Biochim Biophys Acta 1993;1174:204e6. Grover J, Chen XN, Korenberg JR, Roughley PJ. The human lumican gene. Organization, chromosomal location, and expression in articular cartilage. J Biol Chem 1995;270:21942e9. Johnson HJ, Rosenberg L, Choi HU, Garza S, Hook M, Neame PJ. Characterization of epiphycan, a small proteoglycan with a leucine-rich repeat core protein. J Biol Chem 1997;272: 18709e17. Grover J, Chen XN, Korenberg JR, Roughley PJ. The structure and chromosome location of the human chondroadherin gene (CHAD). Genomics 1997;45:379e85. Svensson L, Aszodi A, Reinholt FP, Fassler R, Heinegard D, Oldberg A. Fibromodulin-null mice have abnormal collagen fibrils, tissue organization, and altered lumican deposition in tendon. J Biol Chem 1999;274:9636e47. Kobe B, Deisenhofer J. Crystal structure of porcine ribonuclease inhibitor, a protein with leucine-rich repeats. Nature 1993;366: 751e6. Krishnan P, Hocking AM, Scholtz JM, Pace CN, Holik KK, McQuillan DJ. Distinct secondary structures of the leucine-rich

[148]

[149]

[150]

[151]

[152] [153]

[154]

[155]

[156]

[157]

[158]

[159] [160]

[161]

[162]

[163]

[164]

[165]

repeat proteoglycans decorin and biglycan. Glycosylationdependent conformational stability. J Biol Chem 1999;274: 10945e50. Vogel KG, Trotter JA. The effect of proteoglycans on the morphology of collagen fibrils formed in vitro. Coll Relat Res 1987;7:105e14. Scott JE, Haigh M. Identification of specific binding sites for keratan sulphate proteoglycans and chondroitin-dermatan sulphate proteoglycans on collagen fibrils in cornea by the use of cupromeronic blue in ’critical-electrolyte-concentration’ techniques. Biochem J 1988;253:607e10. Pringle GA, Dodd CM. Immunoelectron microscopic localization of the core protein of decorin near the d and e bands of tendon collagen fibrils by use of monoclonal antibodies. J Histochem Cytochem 1990;38:1405e11. Hedlund H, Mengarelli-Widholm S, Heinegard D, Reinholt FP, Svensson O. Fibromodulin distribution and association with collagen. Matrix Biol 1994;14:227e32. Keene DR, San Antonio JD, Mayne R, et al. Decorin binds near the C terminus of type I collagen. J Biol Chem 2000;275:21801e4. Hildebrand A, Romaris M, Rasmussen LM, et al. Interaction of the small interstitial proteoglycans biglycan, decorin and fibromodulin with transforming growth factor beta. Biochem J 1994;302:527e34. McBride OW, Fisher LW, Young MF. Localization of PGI (biglycan, BGN) and PGII (decorin, DCN, PG-40) genes on human chromosomes Xq13-qter and 12q, respectively. Genomics 1990;6(2):219e25. Geerkens C, Vetter U, Just W, et al. The X-chromosomal human biglycan gene BGN is subject to X inactivation but is transcribed like an X-Y homologous gene. Hum Genet 1995;96:44e52. Krusius T, Ruoslahti E. Primary structure of an extracellular matrix proteoglycan core protein deduced from cloned cDNA. Proc Natl Acad Sci USA 1986;83:7683e7. Fisher LW, Hawkins GR, Tuross N, Termine JD. Purification and partial characterization of small proteoglycans I and II, bone sialoproteins I and II, and osteonectin from the mineral compartment of developing human bone. J Biol Chem 1987;262:9702e8. Roughley PJ, White RJ. Dermatan sulphate proteoglycans of human articular cartilage. The properties of dermatan sulphate proteoglycans I and II. Biochem J 1989;262:823e7. Scott IC, Imamura Y, Pappano WN, et al. Bone morphogenetic protein-1 processes probiglycan. J Biol Chem 2000;275:30504e11. Danielson KG, Baribault H, Holmes DF, Graham H, Kadler KE, Iozzo RV. Targeted disruption of decorin leads to abnormal collagen fibril morphology and skin fragility. J Cell Biol 1997;136:729e43. Xu T, Bianco P, Fisher LW, et al. Targeted disruption of the biglycan gene leads to an osteoporosis-like phenotype in mice. Nat Genet 1998;20:78e82. Corsi A, Xu T, Chen XD, et al. Phenotypic effects of biglycan deficiency are linked to collagen fibril abnormalities, are synergized by decorin deficiency, and mimic Ehlers-Danlos-like changes in bone and other connective tissues. J Bone Miner Res 2002;17:1180e9. Heinegard D, Larsson T, Sommarin Y, Franzen A, Paulsson M, Hedbom E. Two novel matrix proteins isolated from articular cartilage show wide distributions among connective tissues. J Biol Chem 1986;261:13866e72. Sztrolovics R, Chen XN, Grover J, Roughley PJ, Korenberg JR. Localization of the human fibromodulin gene (FMOD) to chromosome 1q32 and completion of the cDNA sequence. Genomics 1994;23:715e7. Antonsson P, Heinegard D, Oldberg A. Posttranslational modifications of fibromodulin. J Biol Chem 1991;266:16859e61.

PEDIATRIC BONE

REFERENCES

[166] Sommarin Y, Wendel M, Shen Z, Hellman U, Heinegard D. Osteoadherin, a cell-binding keratan sulfate proteoglycan in bone, belongs to the family of leucine-rich repeat proteins of the extracellular matrix. J Biol Chem 1998;273:16723e9. [167] Wendel M, Sommarin Y, Heinegard D. Bone matrix proteins: isolation and characterization of a novel cell-binding keratan sulfate proteoglycan (osteoadherin) from bovine bone. J Cell Biol 1998;141:839e47. [168] Hankenson KD, Sweetwyne MT, Shitaye H, Posey KL. Thrombospondins and novel TSR-containing proteins, R-spondins, regulate bone formation and remodeling. Curr Osteoporos Rep 2010;8:68e76. [169] Dixit VM, Hennessy SW, Grant GA, Rotwein P, Frazier WA. Characterization of a cDNA encoding the heparin and collagen binding domains of human thrombospondin. Proc Natl Acad Sci USA 1986;83:5449e53. [170] LaBell TL, Milewicz DJ, Disteche CM, Byers PH. Thrombospondin II: partial cDNA sequence, chromosome location, and expression of a second member of the thrombospondin gene family in humans. Genomics 1992;12:421e9. [171] Adolph KW, Long GL, Winfield S, Ginns EI, Bornstein P. Structure and organization of the human thrombospondin 3 gene (THBS3). Genomics 1995;27:329e36. [172] Newton G, Weremowicz S, Morton CC, et al. The thrombospondin-4 gene. Mamm Genome 1999;10:1010e6. [173] Newton G, Weremowicz S, Morton CC, et al. Characterization of human and mouse cartilage oligomeric matrix protein. Genomics 1994;24:435e9. [174] Adams JC. Thrombospondins: multifunctional regulators of cell interactions. Annu Rev Cell Dev Biol 2001;17:25e51. [175] Di Cesare PE, Fang C, Leslie MP, Tulli H, Perris R, Carlson CS. Expression of cartilage oligomeric matrix protein (COMP) by embryonic and adult osteoblasts. J Orthop Res 2000;18:713e20. [176] Carron JA, Bowler WB, Wagstaff SC, Gallagher JA. Expression of members of the thrombospondin family by human skeletal tissues and cultured cells. Biochem Biophys Res Commun 1999;263:389e91. [177] Morgelin M, Heinegard D, Engel J, Paulsson M. Electron microscopy of native cartilage oligomeric matrix protein purified from the Swarm rat chondrosarcoma reveals a five-armed structure. J Biol Chem 1992;267:6137e41. [178] Lawler J, Derick LH, Connolly JE, Chen JH, Chao FC. The structure of human platelet thrombospondin. J Biol Chem 1985;260:3762e72. [179] Misenheimer TM, Huwiler KG, Annis DS, Mosher DF. Physical characterization of the procollagen module of human thrombospondin 1 expressed in insect cells. J Biol Chem 2000;275: 40938e45. [180] Maddox BK, Mokashi A, Keene DR, Bachinger HP. A cartilage oligomeric matrix protein mutation associated with pseudoachondroplasia changes the structural and functional properties of the type 3 domain. J Biol Chem 2000;275:11412e7. [181] O’Rourke KM, Laherty CD, Dixit VM. Thrombospondin 1 and thrombospondin 2 are expressed as both homo- and heterotrimers. J Biol Chem 1992;267:24921e4. [182] Efimov VP, Lustig A, Engel J. The thrombospondin-like chains of cartilage oligomeric matrix protein are assembled by a fivestranded alpha-helical bundle between residues 20 and 83. FEBS Lett 1994;341:54e8. [183] Hofsteenge J, Huwiler KG, Macek B, et al. C-mannosylation and O-fucosylation of the thrombospondin type 1 module. J Biol Chem 2001;276:6485e98. [184] Misenheimer TM, Mosher DF. Calcium ion binding to thrombospondin 1. J Biol Chem 1995;270:1729e33.

33

[185] Hogg PJ. Thrombospondin 1 as an enzyme inhibitor. Thromb Haemostat 1994;72:787e92. [186] Schultz-Cherry S, Chen H, Mosher DF, et al. Regulation of transforming growth factor-beta activation by discrete sequences of thrombospondin 1. J Biol Chem 1995;270:7304e10. [187] Narouz-Ott L, Maurer P, Nitsche DP, Smyth N, Paulsson M. Thrombospondin-4 binds specifically to both collagenous and non-collagenous extracellular matrix proteins via its C-terminal domains. J Biol Chem 2000;275:37110e7. [188] DiCesare PE, Morgelin M, Mann K, Paulsson M. Cartilage oligomeric matrix protein and thrombospondin 1. Purification from articular cartilage, electron microscopic structure, and chondrocyte binding. Eur J Biochem 1994;223:927e37. [189] Rosenberg K, Olsson H, Morgelin M, Heinegard D. Cartilage oligomeric matrix protein shows high affinity zinc-dependent interaction with triple helical collagen. J Biol Chem 1998;273: 20397e403. [190] Lawler J, Sunday M, Thibert V, et al. Thrombospondin-1 is required for normal murine pulmonary homeostasis and its absence causes pneumonia. J Clin Invest 1998;101:982e92. [191] Kyriakides TR, Zhu YH, Smith LT, et al. Mice that lack thrombospondin 2 display connective tissue abnormalities that are associated with disordered collagen fibrillogenesis, an increased vascular density, and a bleeding diathesis. J Cell Biol 1998;140: 419e30. [192] Hankenson KD, Hormuzdi SG, Meganck JA, Bornstein P. Mice with a disruption of the thrombospondin 3 gene differ in geometric and biomechanical properties of bone and have accelerated development of the femoral head. Mol Cell Biol 2005;25:5599e606. [193] Posey KL, Hankenson K, Veerisetty AC, Bornstein P, Lawler J, Hecht JT. Skeletal abnormalities in mice lacking extracellular matrix proteins, thrombospondin-1, thrombospondin-3, thrombospondin-5, and type IX collagen. Am J Pathol 2008;172: 1664e74. [194] Hecht JT, Hayes E, Haynes R, Cole WG. COMP mutations, chondrocyte function and cartilage matrix. Matrix Biol 2005;23: 525e33. [195] Romberg RW, Werness PG, Lollar P, Riggs BL, Mann KG. Isolation and characterization of native adult osteonectin. J Biol Chem 1985;260:2728e36. [196] Termine JD, Kleinman HK, Whitson SW, Conn KM, McGarvey ML, Martin GR. Osteonectin, a bone-specific protein linking mineral to collagen. Cell 1981;26:99e105. [197] Sage H, Tupper J, Bramson R. Endothelial cell injury in vitro is associated with increased secretion of an Mr 43,000 glycoprotein ligand. J Cell Physiol 1986;127:373e87. [198] Workman G, Sage EH. Identification of a sequence in the matricellular protein sparc that interacts with the scavenger receptor stabilin-1. J Cell Biochem 2011;112:1003e8. [199] Clark CJ, Sage EH. A prototypic matricellular protein in the tumor microenvironmentewhere there’s SPARC, there’s fire. J Cell Biochem 2008;104:721e32. [200] Swaroop A, Hogan BL, Francke U. Molecular analysis of the cDNA for human SPARC/osteonectin/BM-40: sequence, expression, and localization of the gene to chromosome 5q31q33. Genomics 1988;2:37e47. [201] Metsaranta M, Young MF, Sandberg M, Termine J, Vuorio E. Localization of osteonectin expression in human fetal skeletal tissues by in situ hybridization. Calcif Tissue Int 1989;45:146e52. [202] Young MF, Day AA, Dominquez P, McQuillan CI, Fisher LW, Termine JD. Structure and expression of osteonectin mRNA in human tissue. Connect Tissue Res 1990;24:17e28. [203] Bolander ME, Young MF, Fisher LW, Yamada Y, Termine JD. Osteonectin cDNA sequence reveals potential binding regions

PEDIATRIC BONE

34

[204]

[205]

[206]

[207]

[208]

[209]

[210]

[211]

[212]

[213]

[214] [215]

[216] [217]

[218]

[219]

[220]

[221]

2. BONE MATRIX AND MINERALIZATION

for calcium and hydroxyapatite and shows homologies with both a basement membrane protein (SPARC) and a serine proteinase inhibitor (ovomucoid). Proc Natl Acad Sci USA 1988;85:2919e23. Hohenester E, Maurer P, Hohenadl C, Timpl R, Jansonius JN, Engel J. Structure of a novel extracellular Ca(2þ)-binding module in BM-40. Nat Struct Biol 1996;3:67e73. Sasaki T, Hohenester E, Gohring W, Timpl R. Crystal structure and mapping by site-directed mutagenesis of the collagenbinding epitope of an activated form of BM-40/SPARC/osteonectin. Embo J 1998;17:1625e34. Sasaki T, Gohring W, Mann K, et al. Limited cleavage of extracellular matrix protein BM-40 by matrix metalloproteinases increases its affinity for collagens. J Biol Chem 1997;272:9237e43. Gilmour DT, Lyon GJ, Carlton MB, et al. Mice deficient for the secreted glycoprotein SPARC/osteonectin/BM40 develop normally but show severe age-onset cataract formation and disruption of the lens. Embo J 1998;17:1860e70. Basu A, Kligman LH, Samulewicz SJ, Howe CC. Impaired wound healing in mice deficient in a matricellular protein SPARC (osteonectin, BM-40). BMC Cell Biol 2001;2:15. Boskey AL, Moore DJ, Amling M, Canalis E, Delany AM. Infrared analysis of the mineral and matrix in bones of osteonectin-null mice and their wildtype controls. J Bone Miner Res 2003;18:1005e11. Delany AM, Hankenson KD. Thrombospondin-2 and SPARC/ osteonectin are critical regulators of bone remodeling. J Cell Commun Signal 2009;3:227e38. Bradshaw AD, Graves DC, Motamed K, Sage EH. SPARC-null mice exhibit increased adiposity without significant differences in overall body weight. Proc Natl Acad Sci USA 2003;100: 6045e50. Bradshaw AD, Puolakkainen P, Dasgupta J, Davidson JM, Wight TN. Helene Sage E. SPARC-null mice display abnormalities in the dermis characterized by decreased collagen fibril diameter and reduced tensile strength. J Invest Dermatol 2003;120:949e55. Puolakkainen P, Bradshaw AD, Kyriakides TR, et al. Compromised production of extracellular matrix in mice lacking secreted protein, acidic and rich in cysteine (SPARC) leads to a reduced foreign body reaction to implanted biomaterials. Am J Pathol 2003;162:627e35. Gallop PM, Lian JB, Hauschka PV. Carboxylated calciumbinding proteins and vitamin K. N Engl J Med 1980;302:1460e6. Maillard C, Berruyer M, Serre CM, Dechavanne M, Delmas PD. Protein-S, a vitamin K-dependent protein, is a bone matrix component synthesized and secreted by osteoblasts. Endocrinology 1992;130:1599e604. Suttie JW. Vitamin K-dependent carboxylase. Annu Rev Biochem 1985;54:459e77. Puchacz E, Lian JB, Stein GS, Wozney J, Huebner K, Croce C. Chromosomal localization of the human osteocalcin gene. Endocrinology 1989;124:2648e50. Cancela L, Hsieh CL, Francke U, Price PA. Molecular structure, chromosome assignment, and promoter organization of the human matrix Gla protein gene. J Biol Chem 1990;265:15040e8. Celeste AJ, Rosen V, Buecker JL, Kriz R, Wang EA, Wozney JM. Isolation of the human gene for bone gla protein utilizing mouse and rat cDNA clones. Embo J 1986;5:1885e90. Bronckers AL, Gay S, Finkelman RD, Butler WT. Developmental appearance of Gla proteins (osteocalcin) and alkaline phosphatase in tooth germs and bones of the rat. Bone Miner 1987;2: 361e73. Ducy P, Desbois C, Boyce B, et al. Increased bone formation in osteocalcin-deficient mice. Nature 1996;382:448e52.

[222] Boskey AL, Gadaleta S, Gundberg C, Doty SB, Ducy P, Karsenty G. Fourier transform infrared microspectroscopic analysis of bones of osteocalcin-deficient mice provides insight into the function of osteocalcin. Bone 1998;23:187e96. [223] Desbois C, Hogue DA, Karsenty G. The mouse osteocalcin gene cluster contains three genes with two separate spatial and temporal patterns of expression. J Biol Chem 1994;269:1183e90. [224] Deyl Z, Vancikova C, Macek K. gamma-Carboxyglutamic acidcontaining protein of rat kidney cortex. Changes with high fat diet, and molecular parameters. Hoppe Seylers Z Physiol Chem 1980;361:1767e72. [225] Yu VC, Delsert C, Andersen B, et al. RXR beta: a coregulator that enhances binding of retinoic acid, thyroid hormone, and vitamin D receptors to their cognate response elements. Cell 1991;67:1251e66. [226] Morrison NA, Shine J, Fragonas JC, Verkest V, McMenemy ML, Eisman JA. 1,25-dihydroxyvitamin D-responsive element and glucocorticoid repression in the osteocalcin gene. Science 1989;246:1158e61. [227] Li YP, Stashenko P. Characterization of a tumor necrosis factorresponsive element which down-regulates the human osteocalcin gene. Mol Cell Biol 1993;13:3714e21. [228] Hoffmann HM, Catron KM, van Wijnen AJ, et al. Transcriptional control of the tissue-specific, developmentally regulated osteocalcin gene requires a binding motif for the Msx family of homeodomain proteins. Proc Natl Acad Sci USA 1994;91: 12887e91. [229] Poser JW, Esch FS, Ling NC, Price PA. Isolation and sequence of the vitamin K-dependent protein from human bone. Undercarboxylation of the first glutamic acid residue. J Biol Chem 1980;255:8685e91. [230] Hauschka PV, Lian JB, Cole DE, Gundberg CM. Osteocalcin and matrix Gla protein: vitamin K-dependent proteins in bone. Physiol Rev 1989;69:990e1047. [231] Hoang QQ, Sicheri F, Howard AJ, Yang DS. Bone recognition mechanism of porcine osteocalcin from crystal structure. Nature 2003;425:977e80. [232] Hauschka PV, Carr SA. Calcium-dependent alpha-helical structure in osteocalcin. Biochemistry 1982;21:2538e47. [233] Lee NK, Sowa H, Hinoi E, et al. Endocrine regulation of energy metabolism by the skeleton. Cell 2007;130:456e69. [234] Berkner KL. The vitamin K-dependent carboxylase. Annu Rev Nutr 2005;25:127e49. [235] Hinoi E, Gao N, Jung DY, et al. The sympathetic tone mediates leptin’s inhibition of insulin secretion by modulating osteocalcin bioactivity. J Cell Biol 2008;183:1235e42. [236] Fisher LW, Fedarko NS. Six genes expressed in bones and teeth encode the current members of the SIBLING family of proteins. Connect Tissue Res 2003;44:33e40. [237] Bellahcene A, Castronovo V, Ogbureke KU, Fisher LW, Fedarko NS. Small integrin-binding ligand N-linked glycoproteins (SIBLINGs): multifunctional proteins in cancer. Nat Rev Cancer 2008;8:212e26. [238] Hunter GK, O’Young J, Grohe B, Karttunen M, Goldberg HA. The flexible polyelectrolyte hypothesis of protein-biomineral interaction. Langmuir 2010;26:18639e46. [239] Kim RH, Shapiro HS, Li JJ, Wrana JL, Sodek J. Characterization of the human bone sialoprotein (BSP) gene and its promoter sequence. Matrix Biol 1994;14:31e40. [240] Heinegard D, Oldberg A. Structure and biology of cartilage and bone matrix noncollagenous macromolecules. Faseb J 1989;3: 2042e51. [241] Bianco P, Fisher LW, Young MF, Termine JD, Robey PG. Expression of bone sialoprotein (BSP) in developing human tissues. Calcif Tissue Int 1991;49:421e6.

PEDIATRIC BONE

REFERENCES

[242] Fisher LW, McBride OW, Termine JD, Young MF. Human bone sialoprotein. Deduced protein sequence and chromosomal localization. J Biol Chem 1990;265:2347e51. [243] Salih E. In vivo and in vitro phosphorylation regions of bone sialoprotein. Connect Tissue Res 2003;44:223e9. [244] Zaia J, Boynton R, Heinegard D, Barry F. Posttranslational modifications to human bone sialoprotein determined by mass spectrometry. Biochemistry 2001;40:12983e91. [245] Wuttke M, Muller S, Nitsche DP, Paulsson M, Hanisch FG, Maurer P. Structural characterization of human recombinant and bone-derived bone sialoprotein. Functional implications for cell attachment and hydroxyapatite binding. J Biol Chem 2001;276:36839e48. [246] Malaval L, Wade-Gueye NM, Boudiffa M, et al. Bone sialoprotein plays a functional role in bone formation and osteoclastogenesis. J Exp Med 2008;205:1145e53. [247] Boudiffa M, Wade-Gueye NM, Guignandon A, et al. Bone sialoprotein deficiency impairs osteoclastogenesis and mineral resorption in vitro. J Bone Miner Res 2010;25:2393e403. [248] Wade-Gueye NM, Boudiffa M, Laroche N, et al. Mice lacking bone sialoprotein (BSP) lose bone after ovariectomy and display skeletal site-specific response to intermittent PTH treatment. Endocrinology 2010;151:5103e13. [249] Malaval L, Monfoulet L, Fabre T, et al. Absence of bone sialoprotein (BSP) impairs cortical defect repair in mouse long bone. Bone 2009;45:853e61. [250] George A, Sabsay B, Simonian PA, Veis A. Characterization of a novel dentin matrix acidic phosphoprotein. Implications for induction of biomineralization. J Biol Chem 1993;268:12624e30. [251] MacDougall M, Gu TT, Luan X, Simmons D, Chen J. Identification of a novel isoform of mouse dentin matrix protein 1: spatial expression in mineralized tissues. J Bone Miner Res 1998;13:422e31. [252] Qin C, Brunn JC, Jones J, et al. A comparative study of sialic acid-rich proteins in rat bone and dentin. Eur J Oral Sci 2001;109: 133e41. [253] Hirst KL, Simmons D, Feng J, Aplin H, Dixon MJ, MacDougall M. Elucidation of the sequence and the genomic organization of the human dentin matrix acidic phosphoprotein 1 (DMP1) gene: exclusion of the locus from a causative role in the pathogenesis of dentinogenesis imperfecta type II. Genomics 1997;42:38e45. [254] Qin C, Brunn JC, Cook RG, et al. Evidence for the proteolytic processing of dentin matrix protein 1. Identification and characterization of processed fragments and cleavage sites. J Biol Chem 2003;278:34700e8. [255] Qin C, Baba O, Butler WT. Post-translational modifications of sibling proteins and their roles in osteogenesis and dentinogenesis. Crit Rev Oral Biol Med 2004;15:126e36. [256] Steiglitz BM, Ayala M, Narayanan K, George A, Greenspan DS. Bone morphogenetic protein-1/Tolloid-like proteinases process dentin matrix protein-1. J Biol Chem 2004;279:980e6. [257] Ling Y, Rios HF, Myers ER, Lu Y, Feng JQ, Boskey AL. DMP1 depletion decreases bone mineralization in vivo: an FTIR imaging analysis. J Bone Miner Res 2005;20:2169e77. [258] Feng JQ, Ward LM, Liu S, et al. Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism. Nat Genet 2006;38:1310e5. [259] Giachelli CM, Steitz S. Osteopontin: a versatile regulator of inflammation and biomineralization. Matrix Biol 2000;19: 615e22. [260] Oldberg A, Franzen A, Heinegard D. Cloning and sequence analysis of rat bone sialoprotein (osteopontin) cDNA reveals an Arg-Gly-Asp cell-binding sequence. Proc Natl Acad Sci USA 1986;83:8819e23.

35

[261] O’Brien ER, Garvin MR, Stewart DK, et al. Osteopontin is synthesized by macrophage, smooth muscle, and endothelial cells in primary and restenotic human coronary atherosclerotic plaques. Arterioscler Thromb 1994;14:1648e56. [262] Boskey AL, Maresca M, Ullrich W, Doty SB, Butler WT, Prince CW. Osteopontin-hydroxyapatite interactions in vitro: inhibition of hydroxyapatite formation and growth in a gelatingel. Bone Miner 1993;22:147e59. [263] Hunter GK, Kyle CL, Goldberg HA. Modulation of crystal formation by bone phosphoproteins: structural specificity of the osteopontin-mediated inhibition of hydroxyapatite formation. Biochem J 1994;300:723e8. [264] Addison WN, Azari F, Sorensen ES, Kaartinen MT, McKee MD. Pyrophosphate inhibits mineralization of osteoblast cultures by binding to mineral, up-regulating osteopontin, and inhibiting alkaline phosphatase activity. J Biol Chem 2007;282:15872e83. [265] Jono S, Peinado C, Giachelli CM. Phosphorylation of osteopontin is required for inhibition of vascular smooth muscle cell calcification. J Biol Chem 2000;275:20197e203. [266] Young MF, Kerr JM, Termine JD, et al. cDNA cloning, mRNA distribution and heterogeneity, chromosomal location, and RFLP analysis of human osteopontin (OPN). Genomics 1990;7: 491e502. [267] Hijiya N, Setoguchi M, Matsuura K, Higuchi Y, Akizuki S, Yamamoto S. Cloning and characterization of the human osteopontin gene and its promoter. Biochem J 1994;303:255e62. [268] Crosby AH, Lyu MS, Lin K, et al. Mapping of the human and mouse bone sialoprotein and osteopontin loci. Mamm Genome 1996;7:149e51. [269] Inoue M, Shinohara ML. Intracellular osteopontin (iOPN) and immunity. Immunol Res 2011;49:160e72. [270] Kaji H, Sugimoto T, Kanatani M, Fukase M, Kumegawa M, Chihara K. Retinoic acid induces osteoclast-like cell formation by directly acting on hemopoietic blast cells and stimulates osteopontin mRNA expression in isolated osteoclasts. Life Sci 1995;56:1903e13. [271] Kubota T, Zhang Q, Wrana JL, et al. Multiple forms of SppI (secreted phosphoprotein, osteopontin) synthesized by normal and transformed rat bone cell populations: regulation by TGFbeta. Biochem Biophys Res Commun 1989;162:1453e9. [272] Noda M, Rodan GA. Transcriptional regulation of osteopontin production in rat osteoblast-like cells by parathyroid hormone. J Cell Biol 1989;108:713e8. [273] Noda M, Rodan GA. Type beta transforming growth factor regulates expression of genes encoding bone matrix proteins. Connect Tissue Res 1989;21:71e5. [274] Prince CW, Butler WT. 1,25-Dihydroxyvitamin D3 regulates the biosynthesis of osteopontin, a bone-derived cell attachment protein, in clonal osteoblast-like osteosarcoma cells. Coll Relat Res 1987;7:305e13. [275] Shioide M, Noda M. Endothelin modulates osteopontin and osteocalcin messenger ribonucleic acid expression in rat osteoblastic osteosarcoma cells. J Cell Biochem 1993;53:176e80. [276] Mark MP, Butler WT, Prince CW, Finkelman RD, Ruch JV. Developmental expression of 44-kDa bone phosphoprotein (osteopontin) and bone gamma-carboxyglutamic acid (Gla)containing protein (osteocalcin) in calcifying tissues of rat. Differentiation 1988;37:123e36. [277] Chen J, Singh K, Mukherjee BB, Sodek J. Developmental expression of osteopontin (OPN) mRNA in rat tissues: evidence for a role for OPN in bone formation and resorption. Matrix 1993;13:113e23. [278] Chen J, Zhang Q, McCulloch CA, Sodek J. Immunohistochemical localization of bone sialoprotein in foetal porcine bone tissues: comparisons with secreted phosphoprotein 1 (SPP-1,

PEDIATRIC BONE

36

[279]

[280]

[281]

[282]

[283] [284]

[285]

[286]

[287]

[288]

[289]

[290]

[291] [292]

[293]

[294]

[295] [296]

[297]

2. BONE MATRIX AND MINERALIZATION

osteopontin) and SPARC (osteonectin). Histochem J 1991;23: 281e9. Kiefer MC, Bauer DM, Barr PJ. The cDNA and derived amino acid sequence for human osteopontin. Nucleic Acids Res 1989;17:3306. Christensen B, Nielsen MS, Haselmann KF, Petersen TE, Sorensen ES. Post-translationally modified residues of native human osteopontin are located in clusters: identification of 36 phosphorylation and five O-glycosylation sites and their biological implications. Biochem J 2005;390:285e92. Lasa M, Chang PL, Prince CW, Pinna LA. Phosphorylation of osteopontin by Golgi apparatus casein kinase. Biochem Biophys Res Commun 1997;240:602e5. Prince CW, Oosawa T, Butler WT, et al. Isolation, characterization, and biosynthesis of a phosphorylated glycoprotein from rat bone. J Biol Chem 1987;262:2900e7. Neame PJ, Butler WT. Posttranslational modification in rat bone osteopontin. Connect Tissue Res 1996;35:145e50. Keykhosravani M, Doherty-Kirby A, Zhang C, et al. Comprehensive identification of post-translational modifications of rat bone osteopontin by mass spectrometry. Biochemistry 2005;44: 6990e7003. Safran JB, Butler WT, Farach-Carson MC. Modulation of osteopontin post-translational state by 1,25-(OH)2-vitamin D3. Dependence on Ca2þ influx. J Biol Chem 1998;273:29935e41. Mazzali M, Kipari T, Ophascharoensuk V, Wesson JA, Johnson R, Hughes J. Osteopontin e a molecule for all seasons. Q J Med 2002;95:3e13. Liaw L, Lindner V, Schwartz SM, Chambers AF, Giachelli CM. Osteopontin and beta 3 integrin are coordinately expressed in regenerating endothelium in vivo and stimulate Arg-Gly-Aspdependent endothelial migration in vitro. Circ Res 1995;77: 665e72. Liaw L, Skinner MP, Raines EW, et al. The adhesive and migratory effects of osteopontin are mediated via distinct cell surface integrins. Role of alpha v beta 3 in smooth muscle cell migration to osteopontin in vitro. J Clin Invest 1995;95:713e24. Smith LL, Cheung HK, Ling LE, et al. Osteopontin N-terminal domain contains a cryptic adhesive sequence recognized by alpha9beta1 integrin. J Biol Chem 1996;271:28485e91. Weber GF, Ashkar S, Glimcher MJ, Cantor H. Receptor-ligand interaction between CD44 and osteopontin (Eta-1). Science 1996;271:509e12. Sodek J, Chen J, Nagata T, et al. Regulation of osteopontin expression in osteoblasts. Ann NY Acad Sci 1995;760:223e41. Prince CW, Dickie D, Krumdieck CL. Osteopontin, a substrate for transglutaminase and factor XIII activity. Biochem Biophys Res Commun 1991;177:1205e10. Sorensen ES, Rasmussen LK, Moller L, Jensen PH, Hojrup P, Petersen TE. Localization of transglutaminase-reactive glutamine residues in bovine osteopontin. Biochem J 1994;304:13e6. Kaartinen MT, El-Maadawy S, Rasanen NH, McKee MD. Tissue transglutaminase and its substrates in bone. J Bone Miner Res 2002;17:2161e73. Nurminskaya M, Kaartinen MT. Transglutaminases in mineralized tissues. Front Biosci 2006;11:1591e606. Rittling SR, Matsumoto HN, McKee MD, et al. Mice lacking osteopontin show normal development and bone structure but display altered osteoclast formation in vitro. J Bone Miner Res 1998;13:1101e11. Yoshitake H, Rittling SR, Denhardt DT, Noda M. Osteopontindeficient mice are resistant to ovariectomy-induced bone resorption. Proc Natl Acad Sci USA 1999;96:8156e60.

[298] Rowe PS, de Zoysa PA, Dong R, et al. MEPE, a new gene expressed in bone marrow and tumors causing osteomalacia. Genomics 2000;67:54e68. [299] Gowen LC, Petersen DN, Mansolf AL, et al. Targeted disruption of the osteoblast/osteocyte factor 45 gene (OF45) results in increased bone formation and bone mass. J Biol Chem 2003;278: 1998e2007. [300] Addison WN, Nakano Y, Loisel T, Crine P, McKee MD. MEPEASARM peptides control extracellular matrix mineralization by binding to hydroxyapatite: an inhibition regulated by PHEX cleavage of ASARM. J Bone Miner Res 2008;23:1638e49. [301] Boskey AL, Chiang P, Fermanis A, et al. MEPE’s diverse effects on mineralization. Calcif Tissue Int 2010;86:42e6. [302] Martin A, David V, Laurence JS, et al. Degradation of MEPE, DMP1, and release of SIBLING ASARM-peptides (minhibins): ASARM-peptide(s) are directly responsible for defective mineralization in HYP. Endocrinology 2008;149:1757e72. [303] David V, Martin A, Hedge AM, Drezner MK, Rowe PS. ASARM peptides: PHEX-dependent and -independent regulation of serum phosphate. Am J Physiol Renal Physiol 2011;300: F783e91. [304] Drezner MK. PHEX gene and hypophosphatemia. Kidney Int 2000;57:9e18. [305] Su X, Sun K, Cui FZ, Landis WJ. Organization of apatite crystals in human woven bone. Bone 2003;32:150e62. [306] Rubin MA, Jasiuk I, Taylor J, Rubin J, Ganey T, Apkarian RP. TEM analysis of the nanostructure of normal and osteoporotic human trabecular bone. Bone 2003;33:270e82. [307] Russell RG, Fleisch H. Pyrophosphate and diphosphonates in skeletal metabolism. Physiological, clinical and therapeutic aspects. Clin Orthop Relat Res 1975;108:241e63. [308] Millan JL. Alkaline phosphatases: structure, substrate specificity and functional relatedness to other members of a large superfamily of enzymes. Purinergic Signal 2006;2:335e41. [309] Narisawa S, Frohlander N, Millan JL. Inactivation of two mouse alkaline phosphatase genes and establishment of a model of infantile hypophosphatasia. Dev Dyn 1997;208:432e46. [310] Gurley KA, Chen H, Guenther C, et al. Mineral formation in joints caused by complete or joint-specific loss of ANK function. J Bone Miner Res 2006;21:1238e47. [311] Hessle L, Johnson KA, Anderson HC, et al. Tissue-nonspecific alkaline phosphatase and plasma cell membrane glycoprotein-1 are central antagonistic regulators of bone mineralization. Proc Natl Acad Sci USA 2002;99:9445e9. [312] Murshed M, Harmey D, Millan JL, McKee MD, Karsenty G. Unique coexpression in osteoblasts of broadly expressed genes accounts for the spatial restriction of ECM mineralization to bone. Genes Dev 2005;19:1093e104. [313] Whyte MP. Physiological role of alkaline phosphatase explored in hypophosphatasia. Ann NY Acad Sci. 2010;1192:190e200. [314] Quarles LD. Endocrine functions of bone in mineral metabolism regulation. J Clin Invest 2008;118:3820e8. [315] Murshed M, McKee MD. Molecular determinants of extracellular matrix mineralization in bone and blood vessels. Curr Opin Nephrol Hypertens 2010;19:359e65. [316] Anderson HC. Matrix vesicles and calcification. Curr Rheumatol Rep 2003;5:222e6. [317] Roberts S, Narisawa S, Harmey D, Millan JL, Farquharson C. Functional involvement of PHOSPHO1 in matrix vesicle-mediated skeletal mineralization. J Bone Miner Res 2007;22:617e27. [318] Kawasaki K, Weiss KM. Evolutionary genetics of vertebrate tissue mineralization: the origin and evolution of the secretory calcium-binding phosphoprotein family. J Exp Zool B Mol Dev Evol 2006;306:295e316.

PEDIATRIC BONE

REFERENCES

[319] Roschger P, Paschalis EP, Fratzl P, Klaushofer K. Bone mineralization density distribution in health and disease. Bone 2008;42:456e66. [320] Bresler D, Bruder J, Mohnike K, Fraser WD, Rowe PS. Serum MEPE-ASARM-peptides are elevated in X-linked rickets (HYP): implications for phosphaturia and rickets. J Endocrinol 2004;183:R1e9. [321] Addison WN, Masica DL, Gray JJ, McKee MD. Phosphorylation-dependent inhibition of mineralization by osteopontin

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ASARM peptides is regulated by PHEX cleavage. J Bone Miner Res 2010;25:695e705. [322] Pampena DA, Robertson KA, Litvinova O, Lajoie G, Goldberg HA, Hunter GK. Inhibition of hydroxyapatite formation by osteopontin phosphopeptides. Biochem J 2004;378: 1083e7. [323] Addison WN, McKee MD. ASARM mineralization hypothesis: a bridge to progress. J Bone Miner Res. 2010;25: 1191e2.

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C H A P T E R

3

Prenatal Bone Development Fanxin Long Department of Medicine, Department of Developmental Biology, Washington University School of Medicine, Washington University in St Louis, Missouri, USA

Intramembranous Ossification

Proper formation of the skeleton requires precise control of both skeletal patterning and skeletal cell differentiation. Skeletal patterning usually refers to the specification of position, number, and shape of the skeletal elements, parameters believed to be determined before the appearance of skeletal cell types. This very important aspect was dealt with extensively in the previous edition of this chapter by St.-Jacques and Helms, but will not be discussed here. Instead, this update will focus on the more recent findings, especially those from mouse genetic studies, regarding the molecules that control the formation of chondrocytes and osteoblasts. Before this discussion, however, the chapter will briefly reiterate the different modes and the time line of bone formation in the human embryo, largely based on the previous edition of the chapter by St.-Jacques and Helms.

Intramembranous ossification begins toward the end of the second month of gestation in humans. The process has been most extensively studied in the developing cranium, where it is often preceded by a cellular proliferation at specific sites in the mesenchyme, and becomes histologically evident when a cluster of pale-staining stellate cells aggregate and take on a rounded basophilic appearance [3]. The aggregated mesenchymal cells gradually differentiate into mature secretory osteoblasts that actively produce the collagen I-rich extracellular matrix that is characteristic of bone. This differentiation process occurs in a small number of sites within the territory of each intramembranous bone, and these sites are called ossification 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 [4]. The inital bone tissue (spicule) is irregularly shaped and completely surrounded by the osteoblasts that secreted it. Some of the osteoblasts soon become encased in the matrix and 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 and eventually differentiate into new osteoblasts, which continue to add bone to form a well-defined longer structure called a trabecula (beam). Multiple trabeculae soon connect and form a scaffolding characteristic of cancellous (or spongy) bone [3]. The first bone formed in the embryo is of an immature type, displaying relatively high cellularity and an almost random orientation of collagen fibers [5]. At approximately the time of birth,

MODES OF BONE FORMATION Most skeletal elements of modern vertebrates can be traced to the endoskeleton (skeleton formed inside the body) found in primitive vertebrates [1]. Vestiges of the exoskeleton (skeleton formed outside the body), 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 differently from the endoskeleton during embryogenesis. These bones, termed dermal because of their historical association with the skin, form by direct differentiation of mesenchymal cells into osteoblasts through intramembranous ossification. On the other hand, bones derived from the primitive endoskeleton develop first into cartilage templates but are subsequently replaced by bone through endochondral ossification [2].

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this woven bone is gradually replaced by the more mature lamellar bone characterized by successive layers of uniformly oriented collagen fibers.

Endochondral Ossification This process also initiates with the aggregation of undifferentiated mesenchymal cells to form condensations (Fig. 3.1). However, unlike in intramembranous ossification, cellular proliferation does not appear to play an important part in this process, as these condensations are the result of an increase in cell packing mediated by changes in the extracellular matrix and cellecell adhesion molecules [6]. By their positions, shapes, and sizes, these prechondrogenic condensations prefigure the different skeletal elements [7]. 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 chondrocytes 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 [8]. Perichondrial cells retain chondrogenic potential and probably contribute to the radial expansion of the cartilage by appositional growth. Initially, 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 specific for each element, chondrocytes in the center E10.5

E12.5

E14.5

E15.5 – 18.5

FIGURE 3.1 Endochondral bone formation. Time line is based on mouse embryonic days, and the events are depicted for the tibia. The timing of events may differ in other skeletal elements. Cartilage initially develops from mesenchymal condensation within an avascular environment. Vascularization occurs following the hypertrophy of cartilage. (Courtesy of Dr Matthew Hilton.)

undergo progressive maturation. They acquire a flattened appearance and become organized in columns along the longitudinal axis of the developing skeletal element. Columns are separated by relatively thick lateral partitions of extracellular matrix, the longitudinal septa, whereas chondrocytes within a column are separated by thin transverse septa. Further maturation of these cells leads to hypertrophy characterized by cell enlargement, cessation of proliferation, and secretion of a distinct extracellular matrix rich in type X collagen that becomes progressively calcified [9]. These changes are accompanied by vascular invasion of the hypertrophic cartilage, and by differentiation of the inner perichondrium cells into osteoblasts, which secrete a layer of primary bone to form the bone collar [8]. At this stage, the thin layer of tissue 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 [10]. This results in the death of the terminally differentiated hypertrophic chondrocytes and degradation of the uncalcified transverse cartilage septa by invading “chondroclasts”, a cell type of ill-defined origin associated with the invading capillary sprouts. The invading blood vessels are believed to carry in osteoprogenitors that eventually differentiate into osteoblasts. Remnants of the calcified longitudinal septa act as templates on which the osteoblasts secrete bone matrix and establish the primary ossification center. Osteoclasts, which are bone-resorbing cells of hematopoietic origin, also appear within the ossifcation center, contributing to the formation of the marrow cavity. The synchronous maturation and columnar organization of the chondrocytes result in a characteristic histological structure known as the embryonic growth plate in which zones of proliferation, maturation, hypertrophy, and bone formation can be identified, linearly progressing from the articular ends (epiphysis) towards the midshaft (diaphysis) of the element. Continued proliferation of the less mature chondrocytes at the epiphysis, followed by their hypertrophy and their eventual replacement by trabecular bone near the diaphysis results in a distal displacement of the growth plate and longitudinal growth of the skeletal element. In a typical long bone, this growth is accompanied by resorption of the older trabeculae by osteoclasts, thus leaving significant trabecular bone only at the region immediately adjacent to the growth plate (metaphysis). Continued deposition of cortical bone by the periosteum (subperiosteal bone) leads to radial growth. Eventually, a secondary ossification center forms at the center of the epiphysis distal to the growth plate, from which

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ossification proceeds radially in all directions. Formation of the secondary ossification center does not modify the general organization of the growth plate, which remains active until cessation of growth after puberty. In the long bones of humans, the growth plate eventually disappears with the only cartilage remaining at the articular surface. As in intramembranous ossification, the first bone tissue produced in the trabeculae and the diaphysis of the long bones is of a woven type. Remodeling of this immature bone following its resorption by osteoclasts leads progressively to replacement by mature lamellar bone.

TIMING AND SEQUENCE OF BONE FORMATION IN HUMANS All skeletal elements do not form simultaneously in the embryo. Generally, chondrification (cartilage formation) proceeds in a rostralecaudal (head-to-tail) fashion in the axial skeleton and in a proximaledistal (shoulderto-fingertip) fashion in the limb skeleton. 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 [11]. In the cephalic region, cartilage formation proceeds in a caudal-to-rostral direction. It starts at the beginning of week 7 and continues well into week 8 [12]. 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. Almost all primary ossification centers appear between weeks 7 and 12 of embryonic life in humans. In contrast, the secondary ossification centers such as those 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. Generally, the facial and calvarial centers appear before the basicranial centers, followed by the hyoid centers [13]. More than 100 centers of ossification appear during human skull formation, but extensive fusion takes place between many of them, reducing the number to 45 by the time of birth [14]. 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

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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. Ossification centers in the neural arches appear in a cephalocaudal sequence except in the atlas and axis, in which ossification is slightly delayed [13]. 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 [15]. 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 [13]. 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 [3].

MOLECULAR REGULATION OF SKELETAL DEVELOPMENT EpithelialeMesenchymal Interaction The requirement for an early tissue interaction to induce mesenchymal condensation is best demonstrated by tissue dissociationerecombination experiments with craniofacial tissues. 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 epithelialemesenchymal contact [16]. 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

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produced is independent of the source of epithelium but is dictated by the nature of the mesenchyme [16]. Finally, an epithelium such as the mandibular is incapable of inducing bone formation in a mesenchyme that is normally non-osteogenic. Such interactions have been studied in detail mostly in craniofacial structures and have been shown to be essential to induce formation of scierai ossicles and scierai cartilage, mandible and Meckel’s cartilage, maxilla, palatine bone, otic vesicle, and calvarial bones, and also clavicle and limb cartilage [17,18]. The molecular nature of the epithelial signal(s) is unclear but probably involves in many cases the bone morphogenic proteins (BMPs), which form a subfamily of secreted proteins among the TGF-b 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 to bind a type II transmembrane receptor and induce recruitment and phosphorylation of the cytoplasmic tail of one of the type I receptors. 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 [19]. 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 bone formation in subcutaneous implants [20,21]. Purified recombinant BMPs share this capacity and are able to potentiate chondrocyte and osteoblast differentiation of cells in vitro [22]. Consequently, BMPs have long been considered in vivo “bone inducers”. However, BMPs induce ectopic bone by recapitulating the events occurring during endochondral bone formation [23], and therefore may not act by inducing ossification (production of bone matrix) per se. Instead, the bone-forming ability of BMPs probably reflects their role in the induction of mesenchymal condensations [24]. Several BMPs known to have cartilage/boneinducing potential accumulate in the extracellular material (ECM), including epithelial basal lamina [25,26]. In the case of the mandible, an epithelialemesenchymal interaction involving BMP signaling has been documented. BMPs-2, -4, and -7 are found in the distal mandibular epithelium whereas the homeobox-containing transcription factor Msx1 is present in the mandibular mesenchyme. Msx1 function in the mesenchyme was demonstrated by Msx1 knockout mice, which exhibited mandibular defects [27]. Expression of Msx1 requires mandibular epithelium [28] and can be induced by ectopic BMPs [29], thus implicating these molecules

in the early epithelialemesenchymal required for mandible formation.

interaction

Condensation 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 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 [30]. Cell proliferation plays a role in condensation of the calvarial, scierai, and mandibular primordia but not in the precartilaginous condensations of the limb, where cell movements appear to be more important [7]. Regardless of the mechanism, the high cell density generated in the condensations leads to increased cellecell contact as well as the formation of gap junctions facilitating intercellular communication [31,32]. These appear to be essential for differentiation to take place, and the extent of cellular condensation correlates with the level of subsequent chondrogenesis [33]. Two cell adhesion molecules implicated in the condensation process are N-cadherin and N-CAM. Ncadherin belongs to a group of calcium-dependent transmembrane glycoproteins that mediate cellecell 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 cellecell 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 [33]. Conversely, overexpression of N-cadherin and N-CAM in micromass cultures stimulates chondrogenesis [34,35]. Cellecell adhesion in the digit condensations was also shown to depend on Epheephrins interactions. The Eph receptors belong to the family of receptor tyrosine kinases. They are characterized by a unique cysteine-rich motif in their extracellular domain, followed by two fibronectin type III motifs. They interact with a family of at least eight ephrin ligands associated

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with the cell membranes via a transmembrane domain or a GPI anchor [36]. EphrineEph signaling has been implicated in the sorting and adhesion of mesenchymal cells in both mouse and chicken [37e39]. Interestingly, in Hoxa13 mutant mice, expression of EphA7 was reduced in mesenchymal condensations correlating with altered adhesiveness, poorly resolved condensations, and defective chondrogenesis [38], whereas misexpression of Hoxd11 or Hoxa13 in the developing chick limb affected condensation size and cell adhesiveness [40,41]. Thus, Hox transcription factors may control skeletal patterning via the regulation of ephrineEph signaling, 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 cellecell interactions. As condensation begins, a transient increase in hyaluronidase activity leads to controlled hydrolysis of HA and reduced intercellular space, thus favoring cellecell interactions [42,43]. Condensation also coincides with upregulation of a number of ECM proteins, including type I collagen, fibronectin, and various proteoglycans [33]. Syndecan-3 is an integral membrane protein that is also a heparan sulfate proteoglycan that interacts with ECM components and heparin-binding growth factors [44]. It is transiently expressed at high levels during formation of the precartilage condensations and downregulated (except in the perichondrium) after chondrocyte differentiation. Antibodies against syndecan-3 impair the formation of precartilaginous condensations in micromass cultures of chick limb mesenchyme [45]. Fibronectin is a dimeric proteoglycan present in the ECM of many tissues and 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, fibronection is distributed throughout the intercellular space of the mesenchyme, but it accumulates in the condensations and reaches its maximal level of expression just prior to overt chondrogenesis [46]. Interaction between extracellular fibronectin and heparin-like molecules of the mesenchymal cell surface is crucial for the formation of precartilage condensations [47,48]. Antibodies specific to a certain isoform of fibronectin also perturbed chondrogenesis [49]. Expression of the aforementioned cellecell and cellematrix adhesion molecules is modulated by signaling factors. For instance, treatment of pluripotential cells or limb bud mesenchyme with TGF-b stimulated expression of fibronectin, N-CAM, N-cadherin, and tenascin [50,51]. Exposure of limb bud mesenchymal cells to certain BMPs also correlated with an increase in N-cadherin and N-CAM expression and

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stimulates chondrogenesis [33]. Functionally, BMP/ GDF gain-of-function experiments in chick [52,53] and mouse embryos [54,55] led to hyperplasia and, in some cases, fusion of limb cartilage elements. Conversely, broad repression of BMP signaling inhibited cartilage development [54,56]. The important role of BMP signaling in chondrogenesis has been further demonstrated by genetic experiments in the mouse. Earlier gene deletion studies of BMPs in the mouse have been complicated by either early lethality or functional redundancy among the family members. For instance, null mutations in Bmp-2 or -4, as well as in the BMP receptor type 1A gene (Bmp-r1A), lead to early embryonic lethality [57e59], whereas mice null for Bmp-3 or Bmp-3b (also known as Gdf10) display no obvious embryonic phenotype in the skeleton [60,61]. However, by using the Cre-loxP technology, simultaneous deletion of Bmp2 and Bmp4 in the prechondrogenic limb mesenchyme with Prx1-Cre led to the failure of the formation of certain chondrogenic condensations [62]. These results indicate that a threshold of Bmp signaling is necessary for chondrogenesis. The fact that all chondrogenic condensations were not equally affected by the loss of Bmp2 and Bmp4 suggests that the less affected skeletal element may require a different set of Bmp family members. As in the mouse, mutations affecting BMP signaling in humans also lead to defects in skeletal elements. For instance, mutations in GDF5 have been found to cause brachypodism (bp) in the mouse, and HuntereThompson acromesomelic dysplasia as well as Grebe syndrome in humans [63]. Moreover, mutations in the gene encoding the BMP antagonist Noggin (NOG) also cause two autosomal dominant disorders e proximal symphalangism (SYM1) and multiple synostose syndrome (SYNS1), both characterized by multiple joint fusions [64]. Although the precise function of BMP signaling during mesenchymal condensation remains to be fully elucidated, studies have suggested that BMP/GDF signaling controls recruitment of cells into the early condensations [52,55]. Consistent with this view, genetic deletion of noggin, an extracellular antagonist of BMP proteins, resulted in enlarged cartilage elements and the lack of joints in the mouse [65]. 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 [66,67]. More recently, by using an imaging system that dynamically visualizes limb mesenchymal cells undergoing successive phases of cartilage formation in vitro, Barna and Niswander found that BMP signaling was required for the “compaction” event that coalesces the small cellular aggregates into a round cluster of tightly associated cells with a distinct outer boundary,

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a prerequisite for chondrogenic differentiation [68]. Importantly, the compaction event appeared to be independent of Sox9 function, as Sox9-null cells exhibited no defect up to this point, although they subsequently became segregated from the chondrogenic mesenchymal condensations and adopted a “fibroblastoid” morphology distinct from the round morphology typical of the cells that remained within the condensation. Thus, BMP signaling appears to play a critical role in the formation of mesenchymal condensation before Sox9 becomes important. The molecular nature for this early requirement of BMP however, is not known at present. 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. It was shown that this vascular regression is essential for the condensation step and subsequent chondrogenesis in the developing limb of chick embryos, although the molecular mechanism was unclear. Interestingly, a more recent study showed that the chondrogenic primordia played a role in patterning the limb vasculature through the expression of vascular endothelial growth factor (VEGF) [69]. Thus, early chondrogenic events may trigger the vascular regression, which may in turn reinforce chondrogenesis.

Chondrocyte Differentiation The overt differentiation of condensed mesenchymal cells into chondrocytes is characterized by a shift in production of ECM and adhesion molecules. Whereas N-CAM, N-cadherin, and type I collagen are downregulated, collagen types II, IX, and XI, as well as the large proteoglycan aggrecan, are upregulated. Sox9, an HMG domain DNA-binding protein, is a key transcription factor regulating chondrocyte differentiation. Mutations in this gene cause the rare and severe dwarfism campomelic dysplasia in humans [70]. During embryogenesis, Sox9 is expressed in all prechondrogenic condensations, where it precedes the expression of the Col2a1 gene, which encodes the major cartilage matrix protein type II collagen. Because Sox9þ/ males were sterile, the function of Sox9 in chondrogenesis was initially 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 chondrocytespecific markers [71]. Interestingly, a more recent study, by applying live imaging techniques to limb mesenchymal condensations in vitro, demonstrated that the Sox9/ cells participated in the initial mesenchymal

condensations normally, but later segregated from the prechondrogenic clusters [68]. Tissue-specific knockout of Sox9 by the Cre-loxP technology confirmed that Sox9 is indispensable for chondrogenesis [72]. Sox9 likely performs multiple functions during chondrocyte development. Live imaging of mesenchymal condensation in vitro with Sox9/ cells indicated that Sox9 was required not for the initial mesenchymal condensation, but rather performed a subsequent role in maintaining the round morphology typical of the prechondrogenic cells [68]. Because Col2a1, one of the best known target genes of Sox9, was not yet activated in a majority of the prechondrogenic cells, the function of Sox9 at this stage was likely to be independent of type II collagen. Thus, in addition to activating chondrocyte-specific genes, Sox9 appears to play an earlier role that remains to be elucidated. Two other members of the Sox family, L-Sox5 and Sox6, interact with the same sequences and together with Sox9 activate transcription of the Col2a1 and aggrecan genes. L-Sox5 and Sox6 have partly redundant functions and single mutants displayed limited skeletal abnormalities, but the Sox5/Sox6 double mutants showed severe generalized chondrodysplasia characterized by poor differentiation of chondrocytes [73]. Thus, L-Sox5 and Sox6 are potent enhancers of chondrocyte differentiation. In addition to its role in mesenchymal condensation as discussed above, BMP signaling also stimulates chondrocyte differentiation after the condensation stage. BMPs transduce signals by binding to complexes of type I and II serine/threonine kinase receptors. In the most extensively studied mechanism, known as the “canonical” pathway, ligand binding induces phosphorylation of the receptors which, in turn, phosphorylates and activates receptor Smads (R-Smads) 1, 5 and 8 [74]. The activated R-Smads then complex with Smad4 to enter the nucleus, eventually regulating gene expression. Simultaneous deletion of the type I receptors Bmpr1a and Bmpr1b in the chondrogenic lineage with Col2-Cre resulted in a severe form of general chondrodysplasia where a majority of the cartilage promodia that prefigure the endochondral bones were absent [75]. Moreover, simultaneous removal of Smad1 and Smad5 in chondrogenic cells, regardless the status of Smad8, essentially abolished cartilage formation [76]. These findings therefore establish that canonical BMP signaling through R-Smads is indispensable for the formation of cartilage following the activation of the Col2a1 gene. Interestingly, deletion of Smad4, the binding partner for Smad1, 5 or 8, with a similar Col2Cre line, did not severely affect cartilage formation [77]. The discrepancy between the R-Smad- and Smad4-deficient mice raises the possibility that BMPSmad1/5/8 signaling may control cartilage development in a Smad4-independent manner. Indeed, BMPs

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were shown to induce R-Smad nuclear localization in Smad4-null colon cancer cells [78], but whether this occurs during cartilage development is currently unknown. In contrast to BMP, some members of the Wnt family inhibit chondrocyte differentiation. The Wnt genes encode a large family (19 members in mouse and humans) of cysteine-rich secreted glycoproteins sharing homology with the Drosophila signaling factor wingless. Wnt proteins control morphogenesis and tissue patterning in a wide variety of organs [79]. Depending on the cell context, Wnt ligands interact with several types of membrane receptors including those of the Frizzled (Fz) family and the low-density lipoprotein receptor related proteins Lrp5 and 6 to activate a variety of intracellular signaling cascades. Among the most extensively studied is the b-catenin-dependent, canonical pathway. In this mechanism, binding of Wnt ligands stabilizes bcatenin, which enters the nucleus to interact with the LEF/TCF family of transcription factors, resulting in transcriptional activation of downstream target genes. Other b-catenin-independent pathways include those mediated by PKC or the Rho family of small GTPases, although the latter was recently shown also to participate in b-catenin signaling [80]. Studies in chick embryonic limbs or limb bud micromass cultures showed that ectopic expression of Wnt1 or Wnt7a inhibited chondrocyte differentiation [81,82]. Consistent with the view that the antichondrogenic role of Wnt proteins is mediated through b-catenin, overexpression of a stabilized form of b-catenin in embryonic limb mesenchyme resulted in a near complete loss of all limb cartilage elements in the mouse, due to an early differentiation defect reflected by the loss of Sox9 expression in the limb mesenchyme [83]. In addition, canonical Wnt signaling also plays further inhibitory roles at subsequent stages after Col2a1 expression is activated, as conditional overexpression of the stabilized b-catenin by Col2-Cre led to severe achondrodysplasia [84]. Similar to canonical Wnt signaling, Notch signaling also suppresses chondrogenesis. Notch signaling mediates communication between neighboring cells to control cell fate decisions in all metazoans [85,86]. The mammalian genome encodes four Notch receptors (Notch1e4) and at least five ligands (Jagged1, 2 and Delta-like 1, 3, 4). In the canonical Notch pathway, binding of the ligands to the Notch receptors present on the neighboring cell surface triggers two successive intramembrane proteolytic cleavages of the receptors mediated by the g-secretase complex and resulting in the release of the Notch intracellular domain (NICD) [87e89]. Upon its release from the plasma membrane, NICD translocates to the nucleus where it interacts with a transcription factor of the CSL family (RBPJk/ CBF-1 in mammals) to activate transcription of target

45

genes [90]. Expression studies revealed that multiple ligands and receptors are expressed in the prechondrogenic mesenchyme. Abolition of Notch signaling in the embryonic limb mesenchyme by the removal of either the g-secretase activity or the transcriptional effector RBPjk resulted in an acceleration of chondrocyte differentiation [91]. Conversely, forced expression of the constitutively active NICD in the limb mesenchyme completely abolished chondrogenesis. Importantly, the antichondrogenic effect of NICD was dependent on RBPjk, as simultaneous removal of the latter fully rescued the chondrogenic defect caused by NICD overexpression [91]. In addition to the direct suppressive role in chondrogenesis, Notch signaling is also known to control axial skeletal patterning through the regulation of somitogenesis. This was evidenced by the findings that Delta-like 3 (Dll3) [92], presenilin 1 (PS1) [93,94], a catalytic subunit of the g-secretase complex, or lunatic fringe, a glycosyltransferase that modifies Notch proteins [95] exhibited defects in the axial skeleton. Consistent with the mouse studies, human mutations in Dll3 [96] were found to cause spondylocostal dysostosis. Retinoid signaling via the retinoic acid receptor a (RARa) also acts to inhibit early chondrocyte differentiation [67,97]. In these studies, transgenic mice were generated to express a constitutively active RARa 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 an RARa-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 [67]. 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 [98]. 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 [99]. It is not known whether the antagonistic interaction between Sox9 and Msx2 is direct or mediated by other factors.

Chondrocyte Proliferation and Maturation PTHrP (parathyroid hormone-related peptide) is a paracrine factor that shares homology with PTH (parathyroid hormone) in its N-terminal domain and binds

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a common receptor, the PTH/PTHrP receptor 1 (PTHR1). In developing long bones in rodents, Pthrp is expressed at highest levels by cells of the periarticular perichondrium, and at lower levels by the proliferating chondrocytes near the articular surface [100]. The Pth-r1 gene is expressed at low levels by proliferating chondrocytes and at high levels by the maturing chondrocytes adjacent to the Ihh-secreting cells, as well as osteoblastic cells in the perichondrium [101,102]. Targeting inactivation of the gene encoding either PTHrP or its receptor in the mouse resulted in neonatal lethality due to severe skeletal dysplasia in which premature maturation of chondrocytes led to short-limb dwarfism and excessive bone formation at birth [103]. Moreover, Pthr1/ chondrocytes present in the growth plate of chimeric mouse embryos developed from both Pthr1/eand wild-type ES cells underwent hypertrophy prematurely in a cell autonomous manner [104]. These studies identified a primary function for PTH signaling in the growth plate, which is to suppress the rate of chondrocyte hypertrophy. PTHrP acts by activating cAMP-dependent signaling pathways, in part by increasing PKA-dependent phosphorylation and transcriptional activity of Sox9 [105,106]. In keeping with the negative role of PTHrP in hypertrophy, misexpression of PTHrP in chondrocytes through a transgene markedly delayed chondrocyte maturation and bone formation, resulting in a completely cartilaginous endochondral skeleton at birth [107]. In humans, inactivating mutations in PTH-R1 cause Blomstrand chondrodysplasia characterized by advanced skeletal maturation with shortness of long bones and increased bone density [108e110]. Conversely, gain-of-function mutations in PTH-R1 cause Jansen metaphyseal chondrodysplasia [111,112], which was recapitulated in mice overexpressing such a mutant receptor [113]. Yet another activating mutation in PTH-R1 was identified in some enchondromatosis, a condition characterized by the presence of benign cartilage tumors adjoining the growth plate [114]. However, another research group could not confirm these findings [115], while others identified additional novel PTH-R1 mutations [116]. The expression of Pthrp in the periarticular region is strictly dependent on Indian hedgehog (Ihh), a member of the Hh family that includes additionally Sonic hedgehog (Shh) and Desert hedgehog (Dhh) in mammals. The Hedgehog (Hh) family of proteins plays fundamental roles in animal development conserved from flies to humans [117e119]. Smoothened (Smo), a seven-pass transmembrane protein is indispensable for transducing the Hh signal. Hh signaling ultimately controls the processing and subcellular localization of the Gli transcription factors (Gli1e3) that regulate expression of downstream target genes. In the developing cartilage, Ihh is primarily expressed by

prehypertrophic chondrocytes (chondrocytes immediately prior to hypertrophy) and early hypertrophic chondrocytes; Ihh signals to both immature chondrocytes and the overlying perichondrial cells [101,102]. Definitive evidence for the physiological function of Ihh signaling came from mouse genetic knockout studies. Ihh/ embryos exhibited multiple defects in the developing cartilage of the endochondral skeleton, these including a 50% reduction in chondrocyte proliferation and premature hypertrophy of chondrocytes [101, 120]. Subsequent genetic studies of Smo, revealed that whereas direct Ihh input was required for proper proliferation of chondrocytes [120,121], it did not appear to be critical for the regulation of chondrocyte hypertrophy, which instead depended primarily on PTHrP whose expression was induced by Ihh [101,120,122]. Later experiments however, did reveal that direct Ihh signaling does play a role within the proliferating population, in controlling the formation of the columnar from the round chondrocytes [123,124]. The control of PTHrP by Ihh appears to be through direct Hh signaling in the target cells, as localized removal of Smo led to a corresponding loss of PTHrP expression within the periarticular region [123]. Finally, the role of Ihh in chondrocyte proliferation and PTHrP expression is mainly mediated through the derepression of Gli3 repressor function, as simultaneous deleleton of both Gli3 and Ihh restored normal proliferation and hypertrophy [125,126]. Overall, these studies have led to a model in which Ihh and PTHrP jointly regulate chondrocyte proliferation and maturation. The insulin-like growth factors 1 and 2 (IGF-1 and IGF-2) are general growth-promoting factors that also directly regulate chondrocytes. These factors and their receptor (IGF-1R) are closely related 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-activated protein kinase or the phosphatidyl-inositol 3 kinase pathways [127]. 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). These receptors are ubiquitously expressed, indicating the wide spectrum of cell types that insulin/IGF signaling acts on. IGF-1 and -2 are both locally produced by chondrocytes, in addition to the circulating IGF-1 produced in the liver that acts as an endocrine factor. Gene inactivation studies in the mouse have confirmed the essential role of the IGF signaling pathway in overall growth of the mouse embryo development, primarily through the control of cell proliferation [128e130]. Genetic experiments with

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single and compound mutant embryos in IGF and Ihh signaling indicated that the two signaling pathways appear to stimulate chondrocyte proliferation in parallel mechanisms [131]. Fibroblast growth factor (FGF) signaling is also recognized as an essential component of the control of chondrocyte proliferation [132]. FGFs are polypeptide growth factors with diverse biological functions [133]. The human and mouse genomes contain 22 FGF and four FGFR genes. Most FGFs function by binding to and activating cell surface tyrosine kinase FGF receptors (FGFR). FGFs also bind to heparin or heparan sulfate proteoglycans that facilitate the FGF-binding and activation of FGFRs. Signaling via FGFRs is propagated through recruitment and phosphorylation of a variety of signaling proteins, that is triggered by the autophosphorylation of the activated receptors and tyrosinephosphorylation of the closely linked docking proteins [134]. As a result, multiple signaling modules including MAPK, PI3K, STAT1 and PKC are activated. Activating mutations in the transmembrane domain of the receptor FGFR-3 cause a spectrum of chondrodysplasia including hypochondroplasia, achondroplasia and thanatophoric dysplasia, depending on the degree of activation of the receptor [132,135,136]. Similarly, mouse models harboring the activating mutations in FGFR3 phenocopied many aspects of these diseases and revealed that reduced chondrocyte proliferation was the main cellular defect [137,138]. Conversely, FGFR3 deletion in the mouse led to increased proliferation and postnatal skeletal overgrowth [139,140]. Thus, FGFR-3 signaling normally suppresses chondrocyte proliferation in the growth plate. FGFR-3 signaling in chondrocytes is likely to be activated by FGF-18, as FGF-18/ embryos exhibited a similar overproliferation phenotype in chondrocytes [141,142]. The intracellular signaling may involve the signal transducer and activator of transcription (STAT) family of transcription factors [143,144]. Others have shown that FGFs activated the MAPK pathway to upregulate Sox9 expression and activity during chondrogenesis [145].

Osteoblast Differentiation Regardless of their origin from either the intramembranous or the endochondral process, osteoblast differentiation requires the same key transcription factors. One such factor is Runt domain factor 2/core binding factor a1 (Runx2/Cbfa1). Runx2 is a transcription factor isolated by virtue of its binding to cis-regulatory elements of the gene encoding osteocalcin, an osteoblast-specific protein. Runx2 is first expressed in cells of the mesenchymal condensations believed to contain common osteochondroprogenitor cells. With development, its expression progressively increases in

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preosteoblasts and then in mature osteoblasts but is downregulated in chondrocytes except for the hypertrophic chondrocytes where Runx2 is expressed. Runx2/ Cbfa1 is indispensable for osteoblast differentiation, as demonstrated by knockout studies in the mouse. The skeleton of Runx2/ mice, which die at birth, is completely devoid of osteoblasts due to a differentiation arrest [146,147]. Runx2 is also required for osteoblast function beyond differentiation [148]. However, ectopic expression of Runx2 was not sufficient to induce osteoblast differentiation in the limb mesenchyme [149], and overexpression in osteoblasts caused osteopenia due to a blockage in osteoblast maturation [150]. Consistent with the fundamental importance of Runx2 in osteoblast differentiation and function, haploinsufficiency of Runx2 in humans causes cleidocranial dysplasia (CCD), an autosomal dominant disorder characterized primarily by a delay in closure of cranial sutures, reduced or absent clavicle, and dental anomalies [151]. Osterix (Osx, Sp7) is another transcription factor essential for osteoblast differentiation. Osx was discovered as upregulated in C2C12 cells by BMP during osteoblast differentiation [152]. Genetic deletion of Osx led to a complete lack of osteoblasts in the mouse embryo. The relatively normal Runx2 expression in the Osx/ embryos indicates that Osx functions in osteoblast differentiation downstream of Runx2. Furthermore, in the long bones of these mutant embryos, some cells normally destined to become osteoblasts adopted the chondrocyte phenotype, indicating that the Runx2-positive cells are bipotential and can differentiate into chondrocytes as opposed to osteoblasts in the absence of Osx. ATF4, a member of the basic leucine zipper family of transcription factors, plays an important role at a later stage than Runx2 and Osx during osteoblast differentiation [153]. Atf4/ embryos exhibited a severe deficit in the formation of mature osteoblasts even though Runx2 and Osx were expressed. In addition to its role in directly regulating the transcription of the mature osteoblast marker osteocalcin, Atf4 also controls the osteoblast phenotype by stimulating amino acid import to ensure a high level of protein translation of type I collagen, the main constituent of the bone matrix. Besides transcription factors, a number of developmental signals have been identified to control osteoblast differentiation in the embryo [154] (Fig. 3.2). Deletion of Ihh in the mice caused profound defects not only in chondrocytes, as discussed earlier, but also in osteoblast development in the endochondral skeleton [101,120]. In particular, loss of Ihh arrested osteoblast development at a primitive stage prior to expression of the earliest known markers including Cola1(I), AP and Runx2, and before the activation of canonical Wnt signaling in the lineage [155]. Thus, Ihh functions genetically upstream of canonical Wnt signaling during osteoblast

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Fgf, Bmp Hh

MSC

Wnt, Fgf

Runx2+

Wnt, Bmp

Osx+

OB

Notch

FIGURE 3.2 Multiple developmental signals control osteoblast

differentiation. Y: stimulation; t: inhibition. MSC: mesenchymal stem cells; OB: mature osteoblasts. (Figure modified from [154].)

development. Through genetic deletion of Smo to cellautonomously remove Hh responsiveness, it was shown that direct Ihh input was required in the perichondrial osteoprogenitors for development of the osteoblast lineage [120,121]. The control of Ihh over osteoblast differentiation requires both derepression of Gli3 repressor and activation of the Gli2 activator [156,157]. The importance of Gli2 activity is consistent with the finding that Gli2-null embryos (lethal at birth) showed impaired osteoblast formation [158]. The importance of canonical Wnt signaling in osteoblast differentiation during embryogenesis was suggested by genetic deletion of b-catenin from early osteogenic progenitors in the mouse [83,155,159,160]. These studies demonstrated that b-catenin is required for the progression both from Runx2- to Osterix-positive stage [155], and from Osx-positive cells to mature osteoblasts [160]. However, b-catenin appears to be dispensable in mature osteoblasts, as its deletion by Col1-Cre or OC-Cre did not affect osteoblasts per se, but instead decreased the expression of Opg by osteoblasts, and therefore indirectly enhanced osteoclast formation [161,162]. Additionally, non-canonical Wnt signaling mechanisms have also been shown to stimulate osteoblast differentiation [163,164]. Mouse genetic studies have also demonstrated the importance of BMP signaling in bone formation. A critical threshold level of BMP2/4 signaling was shown to be required for trabecular bone formation in the long bones, and specifically that the BMP signal controlled the progression from Runx2- to Osterix-positive cells [62]. Contrary to BMP2 and 4, BMP3 is a negative regulator of bone mass, as BMP3-null mice contained twice as much trabecular bone volume than the control littermates at 5e6 weeks of age [60]. It is possible that BMP3 inhibits osteoblastogenesis by counteracting BMP2, as suggested by in vitro studies [60]. Besides its role in osteoblast differentiation, BMP signaling was also shown to regulate the function of mature osteoblasts. Most notably, genetic deletion of BMPR1A using Og2-Cre (osteocalcin2-Cre) decreased osteoblast function

without an obvious effect on osteoblast numbers [165]. Similarly, overexpression of noggin, a secreted inhibitor for BMPs, under the osteocalcin promoter caused a reduction in osteoblast function and a lower bone mass in postnatal mice [166]. Mouse genetic studies have also revealed important roles for FGF signaling in the osteoblast lineage. Mice lacking the mesenchymal splice form of FGFR-2 exhibited a defect in osteoblast differentiation due to a partial loss of Runx2 [167]. Conversely, mice harboring a gain-of-function mutation in FGFR-2 showed an increase in osteoblast numbers associated with an increase in Runx2 expression and osteoblast differention [168]. Likewise, a gain-of-function mutation of FGFR-1 stimulated Runx2 expression and enhanced osteoblast differentiation in the calvaria, although the status of the long bones was not reported [169]. Tissue-specific deletion of FGFR-1 revealed that FGFR-1 signaling in osteoprogenitors promotes osteoblast differentiation without affecting Runx2 expression, whereas FGFR-1 signaling in mature osteoblasts inhibits the cells’ mineralization activity [170]. In contrast, mice lacking FGFR-3 showed a decrease in bone mineral density due to defects in mineralization, even though the number of osteoblasts was increased [171]. Finally, FGF-18-null embryos exhibited defects in the formation of mature osteoblasts despite normal Runx2 expression [141,142]. Thus, Fgf signaling likely promotes osteoblast differentiation, as well as proliferation and mineralization of mature osteoblasts. In contrast to the signals discussed above, Notch normally inhibits osteoblast differentiation. Genetic removal of either the g-secretase activity, or Notch recptors in the embryonic limb mesenchyme caused excessive bone formation that began in the embryo and culminated in adolescent mice [156]. The phenotype was due to increased osteoblast differentiation from the osteogenic precursors. Consistent with the negative role of Notch in osteoblast differentiation, forced-expression of NICD in osteoblastic precursors reduced osteoblast numbers and caused osteopenia [172]. Interestingly, forced-expression of NICD at a later stage of the osteoblast lineage led to sclerosis owing to excessive proliferation of the immature osteoblasts, highlighting stagespecific functions of constitutive Notch activation in the osteoblast lineage [173,174]. In humans, Notch1 haploinsufficiency caused ectopic osteoblast differentiation and calcification in the aortic valves [175,176].

References [1] Carroll RL. Vertebrate Paleontology and Evolution. New York: Freeman; 1988. [2] Patterson C. Cartilage bones, dermal bones, and membrane bones, or the exoskeleton versus the endoskeleton. In: Andrews S, Miles R, Walker A, editors. Problems in Vertebrate Evolution. London: Academic Press; 1977. p. 77e121.

PEDIATRIC BONE

REFERENCES

[3] Gardner E. Osteogenesis in the human embryo and fetus. In: Bourne GH, editor. The Biochemistry and Physiology of Bone. New York: Academic Press; 1971. p. 77e118. [4] Thompson TJ, Owens PD, Wilson DJ. Intramembranous osteogenesis and angiogenesis in the chick embryo. J Anat 1989;166: 55e65. [5] Pritchard JJ. General histology of bone. In: Bourne GH, editor. The Biochemistry and Physiology of Bone. New York: Academic Press; 1972. p. 1e20. [6] Thorogood PV, Hinchliffe JR. An analysis of the condensation process during chondrogenesis in the embryonic chick hind limb. J Embryol Exp Morphol 1975;33:581e606. [7] Ede DA. Cellular condensations and chondrogenesis. In: Hall BK, editor. Cartilage Development, Differentiation and Growth. New York: Academic Press; 1983. p. 143e85. [8] Caplan AI, Pechak DG. The cellular and molecular embryology of bone formation. In: Peck WA, editor. Bone and Mineral Research. New York: Elsevier; 1987. p. 117e83. [9] Poole AR. The growth plate: Cellular physiology, cartilage assembly and mineralization. In: Hall BK, editor. Cartilage: Molecular Aspects. Boca Raton: CRC Press; 1991. p. 179e211. [10] Vu TH, Shipley JM, Bergers G, et al. MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 1998;93:411e22. [11] Sadler TW. Longman’s Medical Embryology. Philadelphia: Lippincott Williams & Wilkins; 2000. [12] Lemire RJ. Embryology of the skull. In: Cohen Jr MM, MacLean RE, editors. Craniosynostosis: Diagnosis, Evaluation, and Management. Oxford: Oxford University Press; 2000. p. 24e34. [13] Noback CR, Robertson GG. Sequences of appearance of ossification centers in the human skeleton during the first five prenatal months. Am J Anat 1951;89:1e28. [14] Dixon AD. Prenatal development of the facial skeleton. In: Dixon AD, Hoyte DAN, Ronning O, editors. Fundamentals of Craniofacial Growth. Boca Raton: CRC Press; 1997. p. 59e97. [15] Carlson BM. Human Embryology and Developmental Biology. St Louis: Mosby; 1999. [16] Hall BK. Embryonic bone formation with special reference to epithelialemesenchymal interactions and growth factors. In: Hall BK, editor. Bone: Mechanisms of Bone Development and Growth. Boca Raton: CRC Press; 1994. p. 137e92. [17] Hall BK, editor. Tissue interactions and chondrogenesis. In: Cartilage; Development, Differentiation, and Growth. New York: Academic Press; 1983. p. 187e222. [18] Thorogood P. Morphogenesis of cartilage. In: Hall BK, editor. Cartilage: Development, Differentiation, and Growth. New York: Academic Press; 1983. p. 223e54. [19] Kawabata M, Miyazomo K. Bone morphogenic proteins. In: Canalis E, editor. Skeletal Growth Factors. Philadelphia: Lippincott Williams & Wilkins; 2000. p. 269e90. [20] Sampath TK, Reddi AH. Dissociative extraction and reconstitution of extracellular matrix components involved in local bone differentiation. Proc Natl Acad Sci USA 1981;78:7599e603. [21] Urist MR, Mikulski A, Lietze A. Solubilized and insolubilized bone morphogenetic protein. Proc Natl Acad Sci USA 1979;76: 1828e32. [22] Rosen V, Cox K, Hattersley G. Bone morphogenic proteins. In: Bilezikian JP, Raisz LG, Rodan GA, editors. Principles of Bone Biology. New York: Academic Press; 1996. p. 661e71. [23] Reddi AH. Role of morphogenetic proteins in skeletal tissue engineering and regeneration. Nat Biotechnol 1998;16:247e52. [24] Karsenty G, Wagner EF. Reaching a genetic and molecular understanding of skeletal development. Dev Cell 2002;2: 389e406.

49

[25] Paralkar VM, Nandedkar AK, Pointer RH, Kleinman HK, Reddi AH. Interaction of osteogenin, a heparin binding bone morphogenetic protein, with type IV collagen. J Biol Chem 1990;265:17281e4. [26] Zhu Y, Oganesian A, Keene DR, Sandell LJ. Type IIA procollagen containing the cysteine-rich amino propeptide is deposited in the extracellular matrix of prechondrogenic tissue and binds to TGF-beta1 and BMP-2. J Cell Biol 1999;144:1069e80. [27] Satokata I, Maas R. Msx1 deficient mice exhibit cleft palate and abnormalities of craniofacial and tooth development. Nat Genet 1994;6:348e56. [28] Takahashi Y, Bontoux M, Le Douarin NM. Epithelioe mesenchymal interactions are critical for Quox 7 expression and membrane bone differentiation in the neural crest derived mandibular mesenchyme. Embo J 1991;10:2387e93. [29] Barlow AJ, Francis-West PH. Ectopic application of recombinant BMP-2 and BMP-4 can change patterning of developing chick facial primordia. Development 1997;124:391e8. [30] Hall BK, Miyake T. All for one and one for all: condensations and the initiation of skeletal development. Bioessays 2000;22: 138e47. [31] Coelho CN, Kosher RA. Gap junctional communication during limb cartilage differentiation. Dev Biol 1991;144:47e53. [32] Zimmermann B. Assembly and disassembly of gap junctions during mesenchymal cell condensation and early chondrogenesis in limb buds of mouse embryos. J Anat 1984;138:351e63. [33] DeLise AM, Fischer L, Tuan RS. Cellular interactions and signaling in cartilage development. Osteoarthr Cart 2000;8:309e34. [34] Haas AR, Tuan RS. Chondrogenic differentiation of murine C3H10T1/2 multipotential mesenchymal cells: II. Stimulation by bone morphogenetic protein-2 requires modulation of N-cadherin expression and function. Differentiation 1999;64: 77e89. [35] Widelitz RB, Jiang TX, Murray BA, Chuong CM. Adhesion molecules in skeletogenesis: II. Neural cell adhesion molecules mediate precartilaginous mesenchymal condensations and enhance chondrogenesis. J Cell Physiol 1993;156:399e411. [36] Flanagan JG, Vanderhaeghen P. The ephrins and Eph receptors in neural development. Annu Rev Neurosci 1998;21:309e45. [37] Davy A, Bush JO, Soriano P. Inhibition of gap junction communication at ectopic Eph/ephrin boundaries underlies craniofrontonasal syndrome. PLoS Biol 2006;4:e315. [38] Stadler HS, Higgins KM, Capecchi MR. Loss of Eph-receptor expression correlates with loss of cell adhesion and chondrogenic capacity in Hoxa13 mutant limbs. Development 2001;128: 4177e88. [39] Wada N, Kimura I, Tanaka H, Ide H, Nohno T. Glycosylphosphatidylinositol-anchored cell surface proteins regulate position-specific cell affinity in the limb bud. Dev Biol 1998;202: 244e52. [40] Goff DJ, Tabin CJ. Analysis of Hoxd-13 and Hoxd-11 misexpression in chick limb buds reveals that Hox genes affect both bone condensation and growth. Development 1997;124:627e36. [41] Yokouchi Y, Nakazato S, Yamamoto M, et al. Misexpression of Hoxa-13 induces cartilage homeotic transformation and changes cell adhesiveness in chick limb buds. Genes Dev 1995;9: 2509e22. [42] Knudson CB, Toole BP. Hyaluronate-cell interactions during differentiation of chick embryo limb mesoderm. Dev Biol 1987;124:82e90. [43] Toole BP, Jackson G, Gross J. Hyaluronate in morphogenesis: inhibition of chondrogenesis in vitro. Proc Natl Acad Sci USA 1972;69:1384e6. [44] Kosher RA. Syndecan-3 in limb skeletal development. Microsc Res Tech 1998;43:123e30.

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50

3. PRENATAL BONE DEVELOPMENT

[45] Seghatoleslami MR, Kosher RA. Inhibition of in vitro limb cartilage differentiation by syndecan-3 antibodies. Dev Dyn 1996;207:114e9. [46] Kulyk WM, Upholt WB, Kosher RA. Fibronectin gene expression during limb cartilage differentiation. Development 1989;106:449e55. [47] Frenz DA, Akiyama SK, Paulsen DF, Newman SA. Latex beads as probes of cell surface-extracellular matrix interactions during chondrogenesis: evidence for a role for amino-terminal heparinbinding domain of fibronectin. Dev Biol 1989;136:87e96. [48] Frenz DA, Jaikaria NS, Newman SA. The mechanism of precartilage mesenchymal condensation: a major role for interaction of the cell surface with the amino-terminal heparin-binding domain of fibronectin. Dev Biol 1989;136:97e103. [49] Gehris AL, Stringa E, Spina J, Desmond ME, Tuan RS, Bennett VD. The region encoded by the alternatively spliced exon IIIA in mesenchymal fibronectin appears essential for chondrogenesis at the level of cellular condensation. Dev Biol 1997;190:191e205. [50] Chimal-Monroy J, Diaz de Leon L. Expression of N-cadherin, NCAM, fibronectin and tenascin is stimulated by TGF-beta1, beta2, beta3 and beta5 during the formation of precartilage condensations. Int J Dev Biol 1999;43:59e67. [51] Leonard CM, Fuld HM, Frenz DA, Downie SA, Massague J, Newman SA. Role of transforming growth factor-beta in chondrogenic pattern formation in the embryonic limb: stimulation of mesenchymal condensation and fibronectin gene expression by exogenenous TGF-beta and evidence for endogenous TGFbeta-like activity. Dev Biol 1991;145:99e109. [52] Francis-West PH, Abdelfattah A, Chen P, et al. Mechanisms of GDF-5 action during skeletal development. Development 1999;126:1305e15. [53] Zou H, Wieser R, Massague J, Niswander L. Distinct roles of type I bone morphogenetic protein receptors in the formation and differentiation of cartilage. Genes Dev 1997;11:2191e203. [54] Tsumaki N, Nakase T, Miyaji T, et al. Bone morphogenetic protein signals are required for cartilage formation and differently regulate joint development during skeletogenesis. J Bone Miner Res 2002;17:898e906. [55] Tsumaki N, Tanaka K, Arikawa-Hirasawa E, et al. Role of CDMP-1 in skeletal morphogenesis: promotion of mesenchymal cell recruitment and chondrocyte differentiation. J Cell Biol 1999;144:161e73. [56] Merino R, Ganan Y, Macias D, Economides AN, Sampath KT, Hurle JM. Morphogenesis of digits in the avian limb is controlled by FGFs, TGFbetas, and noggin through BMP signaling. Dev Biol 1998;200:35e45. [57] Mishina Y, Suzuki A, Ueno N, Behringer RR. Bmpr encodes a type I bone morphogenetic protein receptor that is essential for gastrulation during mouse embryogenesis. Genes Dev 1995;9:3027e37. [58] Winnier G, Blessing M, Labosky PA, Hogan BL. Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse. Genes Dev 1995;9:2105e16. [59] Zhang H, Bradley A. Mice deficient for BMP2 are nonviable and have defects in amnion/chorion and cardiac development. Development 1996;122:2977e86. [60] Daluiski A, Engstrand T, Bahamonde ME, et al. Bone morphogenetic protein-3 is a negative regulator of bone density. Nat Genet 2001;27:84e8. [61] Zhao R, Lawler AM, Lee SJ. Characterization of GDF-10 expression patterns and null mice. Dev Biol 1999;212:68e79. [62] Bandyopadhyay A, Tsuji K, Cox K, Harfe BD, Rosen V, Tabin CJ. Genetic analysis of the roles of BMP2, BMP4, and BMP7 in limb patterning and skeletogenesis. PLoS Genet 2006;2:e216.

[63] Luyten FP, Kaplan FS, Shore EM. Clinical disorders associated with bone morphogenic proteins. In: Canalis E, editor. Skeletal Growth Factors. Philadelphia: Lippincott Williams & Wilkins; 2000. p. 323e34. [64] Gong Y, Krakow D, Marcelino J, et al. Heterozygous mutations in the gene encoding noggin affect human joint morphogenesis. Nat Genet 1999;21:302e4. [65] Brunet LJ, McMahon JA, McMahon AP, Harland RM. Noggin, cartilage morphogenesis, and joint formation in the mammalian skeleton. Science 1998;280:1455e7. [66] Pizette S, Niswander L. BMPs are required at two steps of limb chondrogenesis: formation of prechondrogenic condensations and their differentiation into chondrocytes. Dev Biol 2000;219: 237e49. [67] Weston AD, Rosen V, Chandraratna RA, Underhill TM. Regulation of skeletal progenitor differentiation by the BMP and retinoid signaling pathways. J Cell Biol 2000;148:679e90. [68] Barna M, Niswander L. Visualization of cartilage formation: insight into cellular properties of skeletal progenitors and chondrodysplasia syndromes. Dev Cell 2007;12:931e41. [69] Eshkar-Oren I, Viukov SV, Salameh S, et al. The forming limb skeleton serves as a signaling center for limb vasculature patterning via regulation of Vegf. Development 2009;136: 1263e72. [70] Foster JW, Dominguez-Steglich MA, Guioli S, et al. Campomelic dysplasia and autosomal sex reversal caused by mutations in an SRY-related gene. Nature 1994;372:525e30. [71] Bi W, Deng JM, Zhang Z, Behringer RR, de Crombrugghe B. Sox9 is required for cartilage formation. Nat Genet 1999;22:85e9. [72] Mori-Akiyama Y, Akiyama H, Rowitch DH, de Crombrugghe B. Sox9 is required for determination of the chondrogenic cell lineage in the cranial neural crest. Proc Natl Acad Sci USA 2003;100:9360e5. [73] Smits P, Li P, Mandel J, et al. The transcription factors L-Sox5 and Sox6 are essential for cartilage formation. Dev Cell 2001;1: 277e90. [74] Massague J, Seoane J, Wotton D. Smad transcription factors. Genes Dev 2005;19:2783e810. [75] Yoon BS, Ovchinnikov DA, Yoshii I, Mishina Y, Behringer RR, Lyons KM. Bmpr1a and Bmpr1b have overlapping functions and are essential for chondrogenesis in vivo. Proc Natl Acad Sci USA 2005;102:5062e7. [76] Retting KN, Song B, Yoon BS, Lyons KM. BMP canonical Smad signaling through Smad1 and Smad5 is required for endochondral bone formation. Development 2009;136:1093e104. [77] Zhang J, Tan X, Li W, et al. Smad4 is required for the normal organization of the cartilage growth plate. Dev Biol 2005;284: 311e22. [78] Liu F, Pouponno C, Massague J. Dual role of the Smad4/DPC4 tumor suppressor in TGFbeta-inducible transcriptional complexes. Genes Dev 1997;11:3157e67. [79] Wodarz A, Nusse R. Mechanisms of Wnt signaling in development. Annu Rev Cell Dev Biol 1998;14:59e88. [80] Wu X, Tu X, Joeng KS, Hilton MJ, Williams DA, Long F. Rac1 activation controls nuclear localization of beta-catenin during canonical Wnt signaling. Cell 2008;133:340e53. [81] Rudnicki JA, Brown AM. Inhibition of chondrogenesis by Wnt gene expression in vivo and in vitro. Dev Biol 1997;185:104e18. [82] Stott NS, Jiang TX, Chuong CM. Successive formative stages of precartilaginous mesenchymal condensations in vitro: modulation of cell adhesion by Wnt-7A and BMP-2. J Cell Physiol 1999;180:314e24. [83] Hill TP, Spater D, Taketo MM, Birchmeier W, Hartmann C. Canonical Wnt/beta-catenin signaling prevents osteoblasts from differentiating into chondrocytes. Dev Cell 2005;8:727e38.

PEDIATRIC BONE

51

REFERENCES

[84] Akiyama H, Lyons JP, Mori-Akiyama Y, et al. Interactions between Sox9 and beta-catenin control chondrocyte differentiation. Genes Dev 2004;18:1072e87. [85] Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch signaling: cell fate control and signal integration in development. Science 1999;284:770e6. [86] Chiba S. Notch signaling in stem cell systems. Stem Cells 2006;24:2437e47. [87] Kopan R, Goate A. A common enzyme connects notch signaling and Alzheimer’s disease. Genes Dev 2000;14:2799e806. [88] Kopan R, Ilagan MX. The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 2009;137:216e33. [89] Schroeter EH, Kisslinger JA, Kopan R. Notch-1 signalling requires ligand-induced proteolytic release of intracellular domain. Nature 1998;393:382e6. [90] Honjo T. The shortest path from the surface to the nucleus: RBPJ kappa/Su(H) transcription factor. Genes Cells 1996;1:1e9. [91] Dong Y, Jesse AM, Kohn A, et al. RBPjkappa-dependent Notch signaling regulates mesenchymal progenitor cell proliferation and differentiation during skeletal development. Development 2010;137:1461e71. [92] Dunwoodie SL, Clements M, Sparrow DB, Sa X, Conlon RA, Beddington RS. Axial skeletal defects caused by mutation in the spondylocostal dysplasia/pudgy gene Dll3 are associated with disruption of the segmentation clock within the presomitic mesoderm. Development 2002;129:1795e806. [93] Shen J, Bronson RT, Chen DF, Xia W, Selkoe DJ, Tonegawa S. Skeletal and CNS defects in Presenilin-1-deficient mice. Cell 1997;89:629e39. [94] Wong PC, Zheng H, Chen H, et al. Presenilin 1 is required for Notch1 and DII1 expression in the paraxial mesoderm. Nature 1997;387:288e92. [95] Zhang N, Gridley T. Defects in somite formation in lunatic fringe-deficient mice. Nature 1998;394:374e7. [96] Bulman MP, Kusumi K, Frayling TM, et al. Mutations in the human delta homologue, DLL3, cause axial skeletal defects in spondylocostal dysostosis. Nat Genet 2000;24:438e41. [97] Cash DE, Bock CB, Schughart K, Linney E, Underhill TM. Retinoic acid receptor alpha function in vertebrate limb skeletogenesis: a modulator of chondrogenesis. J Cell Biol 1997;136: 445e57. [98] Semba I, Nonaka K, Takahashi I, et al. Positionally-dependent chondrogenesis induced by BMP4 is co-regulated by Sox9 and Msx2. Dev Dyn 2000;217:401e14. [99] Takahashi K, Nuckolls GH, Takahashi I, et al. Msx2 is a repressor of chondrogenic differentiation in migratory cranial neural crest cells. Dev Dyn 2001;222:252e62. [100] Lee K, Deeds JD, Segre GV. Expression of parathyroid hormonerelated peptide and its receptor messenger ribonucleic acids during fetal development of rats. Endocrinology 1995;136: 453e63. [101] St-Jacques B, Hammerschmidt M, McMahon AP. Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev 1999;13:2072e86. [102] Vortkamp A, Lee K, Lanske B, Segre GV, Kronenberg HM, Tabin CJ. Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 1996;273:613e22. [103] Karaplis AC, Luz A, Glowacki J, et al. Lethal skeletal dysplasia from targeted disruption of the parathyroid hormone-related peptide gene. Genes Dev 1994;8:277e89. [104] Chung UI, Lanske B, Lee K, Li E, Kronenberg H. The parathyroid hormone/parathyroid hormone-related peptide receptor coordinates endochondral bone development by directly controlling

[105]

[106]

[107]

[108]

[109]

[110]

[111]

[112]

[113]

[114] [115]

[116]

[117]

[118]

[119]

[120]

[121]

[122]

chondrocyte differentiation. Proc Natl Acad Sci USA 1998;95: 13030e5. Guo J, Chung UI, Kondo H, Bringhurst FR, Kronenberg HM. The PTH/PTHrP receptor can delay chondrocyte hypertrophy in vivo without activating phospholipase C. Dev Cell 2002;3:183e94. Huang W, Chung UI, Kronenberg HM, de Crombrugghe B. The chondrogenic transcription factor Sox9 is a target of signaling by the parathyroid hormone-related peptide in the growth plate of endochondral bones. Proc Natl Acad Sci USA 2001;98:160e5. Weir EC, Philbrick WM, Amling M, Neff LA, Baron R, Broadus AE. Targeted overexpression of parathyroid hormonerelated peptide in chondrocytes causes chondrodysplasia and delayed endochondral bone formation. Proc Natl Acad Sci USA 1996;93:10240e5. Jobert AS, Zhang P, Couvineau A, et al. Absence of functional receptors for parathyroid hormone and parathyroid hormonerelated peptide in Blomstrand chondrodysplasia. J Clin Invest 1998;102:34e40. Karaplis AC, He B, Nguyen MT, et al. Inactivating mutation in the human parathyroid hormone receptor type 1 gene in Blomstrand chondrodysplasia. Endocrinology 1998;139:5255e8. Karperien M, van der Harten HJ, van Schooten R, et al. A frameshift mutation in the type I parathyroid hormone (PTH)/PTHrelated peptide receptor causing Blomstrand lethal osteochondrodysplasia. J Clin Endocrinol Metab 1999;84:3713e20. Schipani E, Kruse K, Juppner H. A constitutively active mutant PTH-PTHrP receptor in Jansen-type metaphyseal chondrodysplasia. Science 1995;268:98e100. Schipani E, Langman CB, Parfitt AM, et al. Constitutively activated receptors for parathyroid hormone and parathyroid hormone-related peptide in Jansen’s metaphyseal chondrodysplasia. N Engl J Med 1996;335:708e14. Schipani E, Lanske B, Hunzelman J, et al. Targeted expression of constitutively active receptors for parathyroid hormone and parathyroid hormone-related peptide delays endochondral bone formation and rescues mice that lack parathyroid hormonerelated peptide. Proc Natl Acad Sci USA 1997;94:13689e94. Hopyan S, Gokgoz N, Poon R, et al. A mutant PTH/PTHrP type I receptor in enchondromatosis. Nat Genet 2002;30:306e10. Rozeman LB, Sansgiorgi L, Inge H, et al. Enchondromatosis (Ollier disease, Maffucci syndrome) is not caused by the PTHR1 mutation p.R150C. Hum Mutat 2004;24:466e73. Couvineau A, Wouters V, Bertand G, et al. PTHR1 mutations associated with Ollier disease result in receptor loss of function. Hum Molec Genet 2008;17:2766e75. Huangfu D, Anderson KV. Signaling from Smo to Ci/Gli: conservation and divergence of Hedgehog pathways from Drosophila to vertebrates. Development 2006;133:3e14. Ingham PW, McMahon AP. Hedgehog signaling in animal development: paradigms and principles. Genes Dev 2001;15: 3059e87. McMahon AP, Ingham PW, Tabin CJ. Developmental roles and clinical significance of hedgehog signaling. Curr Top Dev Biol 2003;53:1e114. Long F, Zhang XM, Karp S, Yang Y, McMahon AP. Genetic manipulation of hedgehog signaling in the endochondral skeleton reveals a direct role in the regulation of chondrocyte proliferation. Development 2001;128:5099e6108. Long F, Chung UI, Ohba S, McMahon J, Kronenberg HM, McMahon AP. Ihh signaling is directly required for the osteoblast lineage in the endochondral skeleton. Development 2004;131:1309e18. Karp SJ, Schipani E, St-Jacques B, Hunzelman J, Kronenberg H, McMahon AP. Indian hedgehog coordinates endochondral bone growth and morphogenesis via parathyroid hormone

PEDIATRIC BONE

52

[123]

[124]

[125]

[126]

[127]

[128]

[129]

[130]

[131]

[132]

[133] [134]

[135]

[136]

[137]

[138]

[139]

[140]

[141]

3. PRENATAL BONE DEVELOPMENT

related-protein-dependent and -independent pathways. Development 2000;127:543e8. Hilton MJ, Tu X, Long F. Tamixofen-inducible gene deletion reveals a distinct population of osteoblast-lineage cells, and direct regulation of PTHrP expression and chondrocyte morphology by Ihh. 2006;Submitted. Kobayashi T, Soegiarto DW, Yang Y, et al. Indian hedgehog stimulates periarticular chondrocyte differentiation to regulate growth plate length independently of PTHrP. J Clin Invest 2005;115:1734e42. Hilton MJ, Tu X, Cook J, Hu H, Long F. Ihh controls cartilage development by antagonizing Gli3, but requires additional effectors to regulate osteoblast and vascular development. Development 2005;132:4339e51. Koziel L, Wuelling M, Schneider S, Vortkamp A. Gli3 acts as a repressor downstream of Ihh in regulating two distinct steps of chondrocyte differentiation. Development 2005;132:5249e60. Clemmons DR. Insulin-like growth factors. In: Canalis E, editor. Skeletal Growth Factors. Philadelphia: Lippincott Williams & Wilkins; 2000. p. 79e99. Baker J, Liu JP, Robertson EJ, Efstratiadis A. Role of insulin-like growth factors in embryonic and postnatal growth. Cell 1993;75: 73e82. DeChiara TM, Efstratiadis A, Robertson EJ. A growth-deficiency phenotype in heterozygous mice carrying an insulin-like growth factor II gene disrupted by targeting. Nature 1990;345:78e80. Liu JP, Baker J, Perkins AS, Robertson EJ, Efstratiadis A. Mice carrying null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type 1 IGF receptor (Igf1r). Cell 1993;75:59e72. Long F, Joeng KS, Xuan S, Efstratiadis A, McMahon AP. Independent regulation of skeletal growth by Ihh and IGF signaling. Dev Biol 2006;298:327e33. Ornitz DM, Marie PJ. FGF signaling pathways in endochondral and intramembranous bone development and human genetic disease. Genes Dev 2002;16:1446e65. Itoh N, Ornitz DM. Functional evolutionary history of the mouse Fgf gene family. Dev Dyn 2008;237:18e27. Eswarakumar VP, Lax I, Schlessinger J. Cellular signaling by fibroblast growth factor receptors. Cytokine Growth Factor Rev 2005;16:139e49. Rousseau F, Bonaventure J, Legeai-Mallet L, et al. Mutations in the gene encoding fibroblast growth factor receptor-3 in achondroplasia. Nature 1994;371:252e4. Shiang R, Thompson LM, Zhu YZ, et al. Mutations in the transmembrane domain of FGFR3 cause the most common genetic form of dwarfism, achondroplasia. Cell 1994;78:335e42. Chen L, Adar R, Yang X, et al. Gly369Cys mutation in mouse FGFR3 causes achondroplasia by affecting both chondrogenesis and osteogenesis. J Clin Invest 1999;104:1517e25. Naski MC, Colvin JS, Coffin JD, Ornitz DM. Repression of hedgehog signaling and BMP4 expression in growth plate cartilage by fibroblast growth factor receptor 3. Development 1998;125:4977e88. Colvin JS, Bohne BA, Harding GW, McEwen DG, Ornitz DM. Skeletal overgrowth and deafness in mice lacking fibroblast growth factor receptor 3. Nat Genet 1996;12:390e7. Deng C, Wynshaw-Boris A, Zhou F, Kuo A, Leder P. Fibroblast growth factor receptor 3 is a negative regulator of bone growth. Cell 1996;84:911e21. Liu Z, Xu J, Colvin JS, Ornitz DM. Coordination of chondrogenesis and osteogenesis by fibroblast growth factor 18. Genes Dev 2002;16:859e69.

[142] Ohbayashi N, Shibayama M, Kurotaki Y, et al. FGF18 is required for normal cell proliferation and differentiation during osteogenesis and chondrogenesis. Genes Dev 2002;16:870e9. [143] Sahni M, Ambrosetti DC, Mansukhani A, Gertner R, Levy D, Basilico C. FGF signaling inhibits chondrocyte proliferation and regulates bone development through the STAT-1 pathway. Genes Dev 1999;13:1361e6. [144] Su WC, Kitagawa M, Xue N, et al. Activation of Stat1 by mutant fibroblast growth-factor receptor in thanatophoric dysplasia type II dwarfism. Nature 1997;386:288e92. [145] Murakami S, Kan M, McKeehan WL, de Crombrugghe B. Upregulation of the chondrogenic Sox9 gene by fibroblast growth factors is mediated by the mitogen-activated protein kinase pathway. Proc Natl Acad Sci USA 2000;97:1113e8. [146] Komori T, Yagi H, Nomura S, et al. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 1997;89:755e64. [147] Otto F, Thornell AP, Crompton T, et al. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 1997;89:765e71. [148] Ducy P, Starbuck M, Priemel M, et al. A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development. Genes Dev 1999;13:1025e36. [149] Stricker S, Fundele R, Vortkamp A, Mundlos S. Role of Runx genes in chondrocyte differentiation. Dev Biol 2002;245:95e108. [150] Liu W, Toyosawa S, Furuichi T, et al. Overexpression of Cbfa1 in osteoblasts inhibits osteoblast maturation and causes osteopenia with multiple fractures. J Cell Biol 2001;155:157e66. [151] Mundlos S, Otto F, Mundlos C, et al. Mutations involving the transcription factor CBFA1 cause cleidocranial dysplasia. Cell 1997;89:773e9. [152] Nakashima K, Zhou X, Kunkel G, et al. The novel zinc fingercontaining transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 2002;108:17e29. [153] Yang X, Matsuda K, Bialek P, et al. ATF4 is a substrate of RSK2 and an essential regulator of osteoblast biology; implication for Coffin-Lowry Syndrome. Cell 2004;117:387e98. [154] Long F. Targeting intercellular signals for bone regeneration from bone marrow mesenchymal progenitors. Cell Cycle 2008;7: 2106e11. [155] Hu H, Hilton MJ, Tu X, Yu K, Ornitz DM, Long, F. Sequential roles of Hedgehog and Wnt signaling in osteoblast development. Development 2005;132:49e60. [156] Hilton MJ, Tu X, Wu X, et al. Notch signaling maintains bone marrow mesenchymal progenitors by suppressing osteoblast differentiation. Nat Med 2008;14:306e14. [157] Joeng KS, Long F. The Gli2 transcriptional activator is a crucial effector for Ihh signaling in osteoblast development and cartilage vascularization. Development 2009;136:4177e85. [158] Miao D, Liu H, Plut P, et al. Impaired endochondral bone development and osteopenia in Gli2-deficient mice. Exp Cell Res 2004;294:210e22. [159] Day TF, Guo X, Garrett-Beal L, Yang Y. Wnt/beta-catenin signaling in mesenchymal progenitors controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis. Dev Cell 2005;8:739e50. [160] Rodda SJ, McMahon AP. Distinct roles for Hedgehog and canonical Wnt signaling in specification, differentiation and maintenance of osteoblast progenitors. Development 2006;133: 3231e44. [161] Glass 2nd DA, Bialek P, Ahn JD, et al. Canonical Wnt signaling in differentiated osteoblasts controls osteoclast differentiation. Dev Cell 2005;8:751e64.

PEDIATRIC BONE

REFERENCES

[162] Holmen SL, Zylstra CR, Mukherjee A, et al. Essential role of beta-catenin in postnatal bone acquisition. J Biol Chem 2005;280: 21162e8. [163] Takada I, Mihara M, Suzawa M, et al. A histone lysine methyltransferase activated by non-canonical Wnt signalling suppresses PPAR-gamma transactivation. Nat Cell Biol 2007;9: 1273e85. [164] Tu X, Joeng KS, Nakayama KI, et al. Noncanonical Wnt signaling through G protein-linked PKCdelta activation promotes bone formation. Dev Cell 2007;12:113e27. [165] Mishina Y, Starbuck MW, Gentile MA, et al. Bone morphogenetic protein type IA receptor signaling regulates postnatal osteoblast function and bone remodeling. J Biol Chem 2004;279: 27560e6. [166] Devlin RD, Du Z, Pereira RC, et al. Skeletal overexpression of noggin results in osteopenia and reduced bone formation. Endocrinology 2003;144:1972e8. [167] Eswarakumar VP, Monsonego-Ornan E, Pines M, Antonopoulou I, Morriss-Kay GM, Lonai P. The IIIc alternative of Fgfr2 is a positive regulator of bone formation. Development 2002;129:3783e93. [168] Eswarakumar VP, Horowitz MC, Locklin R, Morriss-Kay GM, Lonai P. A gain-of-function mutation of Fgfr2c demonstrates the roles of this receptor variant in osteogenesis. Proc Natl Acad Sci USA 2004;101:12555e60.

53

[169] Zhou YX, Xu X, Chen L, Li C, Brodie SG, Deng CX. A Pro250Arg substitution in mouse Fgfr1 causes increased expression of Cbfa1 and premature fusion of calvarial sutures. Hum Mol Genet 2000;9:2001e8. [170] Jacob AL, Smith C, Partanen J, Ornitz DM. Fibroblast growth factor receptor 1 signaling in the osteo-chondrogenic cell lineage regulates sequential steps of osteoblast maturation. Dev Biol 2006;296:315e28. [171] Valverde-Franco G, Liu H, Davidson D, et al. Defective bone mineralization and osteopenia in young adult FGFR3/ mice. Hum Mol Genet 2004;13:271e84. [172] Zanotti S, Smerdel-Ramoya A, Stadmeyer L, Durant D, Radtke F, Canalis E. Notch inhibits osteoblast differentiation and causes osteopenia. Endocrinology 2008;149:3890e9. [173] Engin F, Yao Z, Yang T, et al. Dimorphic effects of Notch signaling in bone homeostasis. Nat Med 2008;14:299e305. [174] Tao J, Chen S, Yang T, et al. Osteosclerosis owing to Notch gain of function is solely Rbpj-dependent. J Bone Miner Res 2010;25: 2175e83. [175] Garg V, Muth AN, Ransom JF, et al. Mutations in NOTCH1 cause aortic valve disease. Nature 2005;437:270e4. [176] Mohamed SA, Aherrahrou Z, Liptau H, et al. Novel missense mutations (p.T596M and p.P1797H) in NOTCH1 in patients with bicuspid aortic valve. Biochem Biophys Res Commun 2006;345:1460e5.

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Postnatal Bone Growth: Growth Plate Biology, Bone Formation, and Remodeling Christa Maes 1, Henry M. Kronenberg 2 1

Assistant Professor, Katholieke Universiteit Leuven, Laboratory of Experimental Medicine and Endocrinology, Gasthuisberg O&N I, Leuven, Belgium 2 Professor of Medicine, Harvard Medical School; Chief, Endocrine Unit, Massachusetts General Hospital, Boston, Massachusetts, USA

ENDOCHONDRAL AND INTRAMEMBRANOUS BONE FORMATION

The precise arrangement of the individual anatomic elements of the skeleton involves actions and crosstalk of several morphogens including fibroblast growth factors (FGFs), sonic hedgehog (Shh), bone morphogenetic proteins (BMPs) and Wnts during fetal development [1]. These mechanisms underlying the early condensation, segmentation, differentiation and patterning events are reviewed in Chapter 3. The initial cartilage mold forms when mesenchymal cells condense and then differentiate into chondrocytes, under the direction of transcription factors including Sox-9 [2]. The chondrocytes synthesize a characteristic extracellular matrix (ECM) rich in collagens II, IX, and XI and aggrecan, among other matrix molecules. As such, a cartilaginous model or anlage is established that prefigures the future bone. The cartilage mold enlarges both through proliferation along the entire length of the mold and through matrix production. In the center of the mold, chondrocytes stop proliferating, enlarge (hypertrophy), and change the constituents of the matrix to include, for example, type X collagen. The matrix is subsequently mineralized, specialized osteoclasts called chondroclasts invade the region of hypertrophic chondrocytes along with blood vessels and preosteoblasts, and the hypertrophic chondrocytes undergo apoptosis. The invading cells come from the connective tissues surrounding the cartilage mold called the perichondrium. Perichondrial cells carry out a distinct genetic program that partly regulates the cartilage mold via the secretion of signaling molecules that regulate chondrocyte proliferation and differentiation, such as BMPs and parathyroid hormone-related peptide (PTHrP). In turn, the perichondrium is regulated by the cartilage. Perichondrial cells adjacent to prehypertrophic/

Intramembranous ossification is responsible primarily for the formation of most of the craniofacial elements, as will be discussed below. In contrast, all the long bones of the axial skeleton (vertebrae and ribs) and the appendicular skeleton (limbs) develop and grow through endochondral ossification (Fig. 4.1). This process encompasses the deposition of true bone matrix on top of scaffolding cartilaginous anlagen, and is controlled by a series of distinct cell types. The dramatic, asymmetric lengthening of the appendicular long bones is driven to a large extent by the proliferation, differentiation and matrix deposition of chondrocytes that form and make up cartilage, and of osteoblasts that build mineralized bone. Osteoclasts, giant multinuclear cells that break down (“resorb”) bone, contribute to the further modeling and remodeling of the deposited bone. In this lifelong process, packets of bone are constantly being removed and replaced to ensure an adequate shape, position, density and functionality of the mineralized bone framework. Bone growth is absolutely dependent on angiogenesis, putting vascular endothelial cells in the picture as another crucial cell type in bone. Over the last decades, intensive research has focused on the cellular and molecular control of bone development, growth and remodeling. In vitro cell systems have been extremely instructive, but insights derived from genetically altered mice and lessons from human inherited disorders have advanced the field tremendously, as reviewed in this chapter.

Pediatric Bone, Second Edition DOI: 10.1016/B978-0-12-382040-2.10004-8

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4. POSTNATAL BONE GROWTH: GROWTH PLATE BIOLOGY, BONE FORMATION, AND REMODELING

FIGURE 4.1 Stepwise schematic diagram of long bone development through endochondral ossification. Around embryonic day 12 (E12) in mice, mesenchymal progenitor cells condense and differentiate into chondrocytes to form the cartilage anlagen that prefigures future long bones. Chondrocytes in the center become hypertrophic, while cells in the surrounding perichondrium differentiate into osteoblasts forming a bone collar, the provisional cortical bone. The hypertrophic cartilage core is subsequently invaded by blood vessels, eroded and replaced by bone and marrow (“primary ossification center”, POC). In the metaphysis, hypertrophic cartilage of the growth cartilage is continually replaced with trabecular bone, a process that relies on metaphyseal vascularization and mediates longitudinal bone growth. Around postnatal day 5 (P5), epiphyseal vessels invade the avascular cartilage and initiate a secondary center of ossification (SOC). Discrete layers of residual chondrocytes form growth plates between the epiphyseal and metaphyseal bone centers to support further postnatal longitudinal bone growth. Ultimately, (in humans) the growth plates close and growth stops. A detailed view of the perinatal bone structure (boxed area) is provided in Figure 4.2.

hypertrophic chondrocytes respond to Indian hedgehog (Ihh) and other signals from the chondrocytes to become pre-osteoblasts [3]. Some of these pre-osteoblasts differentiate into mature osteoblasts that lay down and mineralize a collagen I-containing matrix, the “bone collar”, around the cartilage mold. This bone collar forms the initiation site of the cortical bone, the dense outer envelope of compact, lamellar bone that provides the long bone with most of its strength and rigidity (see Fig. 4.1). The cortical bone grows by its remodeling by endosteal osteoclastic bone resorption and periosteal new bone formation, as pre-osteoblastic cells proliferate and differentiate. In humans, the cortical bone further enlarges by the formation of Haversian systems around central blood vessels. Other pre-osteoblasts formed in the perichondrium invade the developing bone along with the blood vessels and chondroclasts that accumulated in the region just prior to the cartilage invasion and erosion, and become true osteoblasts inside the new cavity [4]. These osteoblasts lay down bone matrix dominated by collagen type I on top of the calcified 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 discrete layers of residual growth cartilage between the expanding bone in the primary and secondary ossification centers at the opposing ends of the long bone are then called the growth plates [5]. The chondrocytes of the growth plates provide the prime engine for further postnatal longitudinal bone growth. This process is typified by precise temporal and spatial regulation of chondrocyte proliferation and differentiation. In fetal life, all non-hypertrophic 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 are thought of as “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 proliferation. These proliferating cells flatten out, with their short axis parallel to the long axis of the long bone, and form

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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 large number of proliferating chondrocytes and the matrix secreted by all of the chondrocytes, and from the substantial enlargement that each chondrocyte undergoes when it hypertrophies. Because the chondrocytes and their matrix ultimately disappear, however, normal osteoblast function in the primary spongiosa and in the area adjacent to the growth plate is also essential to allow the expansion of the bone length generated by chondrocyte activity to be translated into permanent lengthening. This fact is illustrated, for example, by the short bones of mice in which the essential transcription factor Runx2 was inactivated in osteoblasts [6,7]. Another absolute requirement for endochondral bone growth to proceed is the progressive neovascularization of the region where cartilage becomes replaced by primary spongiosa. Early studies revealed that blocking of the bone’s blood supply in vivo resulted in reduced longitudinal growth [8,9]. Molecularly, this process strongly depends on vascular endothelial growth factor (VEGF) signaling, as shown first by experiments inhibiting VEGF action in juvenile mice by administration of a soluble VEGF receptor chimeric protein (sFlt-1) [10]. These mice showed impaired vascular invasion of the growth plate and, concomitantly, trabecular bone formation and bone growth were reduced while the hypertrophic cartilage zone became enlarged. Thus, endochondral bone growth involves rigorous coupling of maturation and activity of chondrocytes, osteoclasts and osteoblasts, and vascular invasion. Ultimately, at least in humans, the growth plates completely disappear or “close” during puberty, in a process that actively requires the action of estradiol acting on estrogen receptor a (ERa) in both boys and girls (see below), and longitudinal bone growth stops. Remodeling of existing bone, replacing the primary spongiosa by lamellar bone in the secondary spongiosa and renewing the cortical bone, takes place throughout adult life, ensuring optimal mechanical properties of the skeleton and contributing to mineral ion homeostasis. This continual bone turnover is accomplished through the balanced action of osteoclasts and osteoblasts (see below) and results in a dynamic organization of honeycomb plate-like structures or trabeculae in the interior of the bone that are surrounded by blood vessels and bone marrow and housed within the cortical bone. Different from the endochondral ossification process outlined above, a small number of bones, primarily the flat bones of the skull, form without a cartilage mold by a mechanism termed intramembranous bone formation [11]. The development of these bones also starts with mesenchymal progenitor cells forming

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condensations at the sites where the bones will be formed [12]. Yet, in the mesenchymal condensations of intramembranous bones, cells do not differentiate into chondrocytes but instead differentiate directly into pre-osteoblasts and then osteoblasts that deposit “osteoid” or bone matrix rich in type I collagen. As the osteoblasts mature further, the bone matrix becomes progressively mineralized. The growth of these bones occurs by waves of proliferation and differentiation of pre-osteoblasts at the perimeter of the growing bones. As the perimeters of adjacent bones approach each other, a specialized structure, the suture, forms. At the suture, proliferating mesenchymal cells differentiate into osteoblasts. Normal development of the cranium requires that the skull expands in close coordination with the growth of the underlying brain; sutures normally do not close completely until brain expansion ceases [11,13e15]. Bone growth is regulated by a large variety of transcriptional, 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. The cellular and molecular control of skeletal development and growth is first considered, followed by a discussion of some of the prime endocrine controlled regulatory systems of bone growth and remodeling.

CELLULAR AND MOLECULAR CONTROL OF SKELETAL GROWTH Spontaneous mutations in humans and mice and experimental manipulation of genes, either deletions or overexpression (causing loss- or gain-of-function) have identified many growth and transcription factors and signaling cascades involved in skeletal development and growth by regulating the coordinated differentiation and/or actions of the various cell types of bone, namely chondrocytes, vascular endothelial cells, osteoblasts and osteoclasts.

Growth Plate Biology: Chondrocyte Proliferation and Differentiation In endochondral bones, the initial conversion of progenitor cells in the mesenchymal condensations into chondrocytes is determined by the expression of the key transcription factor Sox9, whereas the combined action of Sox9, 5 and 6 is required to direct the subsequent differentiation of chondrocytes throughout all phases of the chondrocyte lineage [16,17]. These phases encompass the round upper or “reserve” chondrocytes, the flattened, columnar proliferating chondrocytes, the

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FIGURE 4.2 Schematic view of the cellular structure of developing long bones. The epiphysis is composed of chondrocytes, organized in layers of proliferation (round periarticular and flat columnar proliferating cells), progressive differentiation towards the metaphysis (prehypertrophic and hypertrophic chondrocytes), and cell death (apoptosis). The avascular cartilage is supplied by the epiphyseal blood vessel network that overlays its surface. In the metaphysis, blood vessels invading the terminal hypertrophic chondrocytes, osteoclasts resorbing the cartilage, and osteoblasts building bone on the cartilage remnants, all act coordinately to replace the cartilage anlagen with bone and marrow.

pre-hypertrophic, and the hypertrophic chondrocytes, characteristically organized as stratified layers in the growth cartilage (Fig. 4.2). The progression of the chondrocytes through these stages is the driving force of actual bone lengthening and is tightly controlled by a myriad of local signaling molecules. Some of the best-characterized ones are the pathway governed by PTHrP and Ihh, and the FGFs; their importance is reflected by the fact that mutations in their signaling receptors are causal to a number of severe human dwarfing conditions. Mutations in the PTH/PTHrP receptor cause Jansen and Blomstrand chondrodysplasia [18,19], and constitutive activation of FGF receptor (FGFR)3 leads to human achondroplasia and thanatophoric dysplasia [20e23]. We will therefore briefly review the basic mechanisms by which these signaling

systems control the pace of proliferation and differentiation of growth chondrocytes. The PTHrP/Ihh pathway provides a major regulatory system in growth chondrocytes. Ihh, a member of the conserved family of hedgehog proteins, is produced by the pre-hypertrophic and early hypertrophic chondrocytes of the growth cartilage. By as yet not fully clarified mechanisms involving signaling through the receptor Patched (Ptc), Ihh acts directly on cells expressing PTHrP to stimulate the expression of PTHrP in chondrocytes located near the periarticular ends of the bone. PTHrP in turn signals back to its receptor (the PTH/ PTHrP receptor that also responds to PTH in osteoblasts and kidney) that is expressed at low levels by proliferating chondrocytes and strongly by pre-hypertrophic cells. The result of this PTHrP signaling is that the further chondrocyte differentiation to hypertrophy is slowed down and chondrocytes are kept in the proliferative state, thereby maintaining the adequate length of columns. By preventing premature hypertrophic differentiation, the generation of cells that can produce Ihh is consequently slowed down and, therefore, the production of PTHrP is lowered. Thus, PTHrP and Ihh regulate each other via a negative feedback signaling pathway that controls the pace of chondrocyte differentiation in the growth plate [24e26]. In addition, mutant mouse models revealed that Ihh regulates endochondral bone growth through PTHrP-independent mechanisms as well. These include a direct role of Ihh signaling in the regulation of chondrocyte proliferation and accelerated conversion of round to columnar cells, and Ihhinduced direct actions on the perichondrium controlling bone collar formation [27e30]. Perichondrial cells are driven into the osteoblast lineage under the influence of Ihh stimulating the expression of Runx2, an essential transcription factor in osteoblast development (see below). Conversely, Runx2 also regulates Ihh expression and hypertrophic differentiation [24,30,31]. Also beyond development, Ihh continues to be essential in regulating and maintaining the growth plate and sustaining postnatal bone growth, as shown by inducible mutagenesis in mice [32]. The FGF pathway involves multiple ligands and receptors that regulate cell proliferation, differentiation, and/or apoptosis in several processes, including skeletal patterning and bone growth. FGFs are involved in skeletogenesis at virtually every step from the initial outgrowth of the limb to postnatal lengthening of the long bones and formation of the intramembranous bones of the skull [33]. A total of 22 different FGFs have been identified in humans and mice. 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 and -9 are both expressed by

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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 [33]. Four genes encode receptors for the FGF family (called FGF receptors 1 to 4 [FGFR1e4]), although the actual number of receptors is larger because of splice variants of these genes. 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. In the endochondral bones, FGFR3 is expressed by proliferating chondrocytes, FGFR1 is expressed by prehypertrophic and hypertrophic chondrocytes, and FGFR2 is expressed by perichondrial cells. In the sutures of the growing intramembranous bones of the skull, FGFR1 is found in the mesenchyme, whereas both FGFR1 and FGFR2 are expressed in the differentiating osteoblasts of sutures. FGFR3 is expressed late at the osteoblastic front of sutures [33]. 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. First, activating mutations in FGFR3 result in chondrodysplasias and dwarfism. Genetic analysis of the role of FGF signaling in the growth plate has, however, been complicated by the multiple roles of FGF signaling during early stages of development. Secondly, mutations in FGFR1 or 2 cause craniosynostosis, which results in skeletal dysplasias characterized by premature fusion of one or more cranial sutures, such as occur in Apert, Crouzon, and other syndromes [34]. The molecular mechanisms that contribute to these FGFR gain-of-functions mutations are complex and include constitutive (ligand-independent) or ligand-dependent FGFR activity [34]. The current insights in the FGF/FGFR signaling functions in these two settings, endochondral and intramembranous ossification, are briefly discussed here and further (see below), respectively. Humans with constitutively active FGFR3, due to point mutations such as a glycine-to-arginine mutation at residue 380 in the receptor’s transmembrane domain, have a form of dominantly inherited short-limbed dwarfism called achondroplasia. Both milder and more severe chondrodysplasias are caused by other activating mutations of FGFR3 [23]. Corresponding transgenic animal models have helped clarify the mechanisms whereby activation of FGFR3 leads to dwarfism. Activation of FGFR3 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. Conversely, mice missing the

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FGFR3 gene, normally expressed in proliferative chondrocyte columns, have elongated columns and longer bones than normal [33,35,36]. These findings were surprising since activation of FGF receptor tyrosine kinases typically leads to a positive effect on cellular proliferation in most tissues. Substantial evidence, however, suggests that FGFR3 activation in chondrocytes leads to activation of STAT1, which activates the cell cycle inhibitor p21Waf1/Cip1 and thereby decreases chondrocyte proliferation; the decrease in chondrocyte proliferation caused by FGF signaling in vivo and in vitro is blocked in chondrocytes from STAT1
/
mice [37e39]. In addition, in transgenic mice, activation of FGFR3 leads to a decrease in expression of Ihh and its receptor and downstream target Ptc, whereas in mice lacking FGFR3, Ihh and Ptc expression are upregulated [40e42]. Since Ihh stimulates chondrocyte proliferation, this provides a further, indirect mechanism supporting the decrease in chondrocyte proliferation after activation of FGFR3. Besides modulating proliferation, FGFR3 signaling in vivo also appears to affect chondrocyte differentiation, as mouse models of achondroplasia and thanatophoric dysplasia show delayed hypertrophic differentiation of chondrocytes [42e44]. During chondrocyte differentiation, the FGF and BMP pathways appear generally to antagonize each other [45]. Moreover, BMP-4 expression is altered in FGFR3 mutant mouse models, and both FGF signaling and BMP signaling regulate Ihh production. Thus, each of these pathways has multiple mechanisms for communicating with each other as they regulate chondrocyte proliferation and differentiation. Due to the plethora of FGFs expressed in the skeleton and the many actions of FGFs early in development, it remains difficult to identify the specific FGF ligands that normally activate FGFR3 in the growth plate. FGF18 [46e48] and FGF-9 [49] appear to be the most important FGF ligands in regulating chondrogenesis identified so far. These ligands activate FGFR3 expressed on proliferating chondrocytes. In this context, the observation that mice missing FGF-18 have a growth plate phenotype that closely resembles that of the FGFR3 knockout mouse is interesting, since it suggests that FGF-18, synthesized in the perichondrium surrounding the growth plate, is the primary activator of FGFR3 in chondrocytes [46e48]. This phenotype thus highlights the importance of interactions between cells in the perichondrium and chondrocytes in the growth plate [3]. Besides the abovementioned growth factors, chondrogenesis is also affected by several other growth factor and hormones, including BMPs, growth hormone (GH) and insulin-like growth factors (IGFs), thyroid hormone, estrogen, androgen, 1a,25-dihydroxyvitamin D3 (1,25(OH)2D3), glucocorticoids, as discussed below and/or in other chapters.

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Blood Vessel Invasion of Cartilage and Bone Vascularization A crucial aspect of bone growth is the vascularization of the expanding bone center. This is particularly evident during endochondral ossification: the cartilage intermediate of the long bones represents a unique mesenchymal tissue in that it is itself avascular, while fully differentiated hypertrophic cartilage undergoes angiogenic invasion, a process that is associated with the decay of the terminal cartilage and its replacement by bone. The replacement of the initially avascular cartilage template by highly vascularized bone and marrow tissue occurs through three consecutive vascularization events (see Fig. 4.1). First, the initial vascular invasion of the cartilage anlagen during embryonic development (sometimes called quiescent angiogenesis) involves endothelial cells invading from the perichondrial tissues and organizing into immature blood vessels in the primary center of ossification. Second, capillary invasion at the metaphyseal border of the growth cartilage mediates rapid bone lengthening. Third, vascularization of the cartilage ends initiates the formation of secondary ossification centers (see Fig. 4.1). Vascularization is an absolute requirement for endochondral bone development and growth to proceed, as a physical blockage of blood vessel invasion into cartilaginous fetal skeletal explants completely halted their development [50], and blocking of the bone’s blood supply in vivo resulted in reduced longitudinal growth [8,9]. In fact, as a rule, any type of bone formation occurs in close spatial and temporal association with vascularization of the ossified tissue, a concept termed angiogeniceosteogenic coupling [51,52]. The reasons that the vascular system is crucial for bone growth, homeostasis and repair obviously include its intrinsic function to supply oxygen, nutrients and growth factors/hormones to the bone cells as required for their specified activities. On top of that, the blood vessels are thought also to serve to bring in (precursors of) osteoclasts that will degrade the cartilage or bone extracellular matrix, to remove end products of the resorption processes, and to bring in progenitors of osteoblasts that will deposit bone [4,53,54]. Here, we will briefly review the basic molecular players currently known to govern the survival of the avascular cartilage, its invasion by blood vessels and the vascularization of bone. Endochondral cartilaginous condensations typically develop for a prolonged period of time as an avascular tissue. Immature chondrocytes are resistant to vascular invasion due to the production of angiogenic inhibitors [55,56]. As the fetal growth plates expand in the absence of blood vessels during development, chondrocytes in the center of the cartilage consequently face the challenge of hypoxia [57,58]. The main mediator of the

cellular responses to hypoxia is the transcription factor hypoxia-inducible factor (HIF)-1. HIF-1 is a heterodimer of two proteins, HIF-1a and HIF-1b; HIF-1b is constitutively expressed, whereas HIF-1a is the hypoxia-responsive component of the complex [59]. Particularly the stability of the HIF-1a protein is hypoxia sensitive, through oxygen-dependent hydroxylation of specific residues within its amino acid sequence. In non-hypoxic conditions, a family of HIF prolyl 4-hydroxylases is responsible for the hydroxylation of two proline residues (P402 and P564) in the oxygen dependent degradation domain (ODDD) of HIF-1a. The E3 ubiquitin ligase Von Hippel-Lindau (VHL) binds to the hydroxylated HIF-1a, and targets it to the proteasome for degradation [60,61]. In hypoxic conditions, this hydroxylation of HIF1a does not occur, and HIF-1a is stabilized and able to translocate to the nucleus, heterodimerize with the b-subunit, and initiate its transcriptional program along with other co-factors. As a result, a variety of pathways involved in the cellular adaptation to hypoxia are activated, including key regulators of glucose utilization and cell metabolism (stimulating anaerobic glycolysis), angiogenesis, and erythropoiesis [62]. In the last years, studies using genetically modified mice have shed light on how the genetic program controlled by hypoxia modulates endochondral bone development, by establishing the essential and nonredundant role of HIF-1a in chondrogenesis in vivo [57,58,63,64]. Conditional deletion of HIF-1a in limb bud mesenchyme or in chondrocytes, achieved by the use of the Cre-loxP strategy, caused massive cell death of the inner chondrocytes in the developing growth plate. This cell death region corresponds to the central hypoxic region area of the fetal growth plate [58,65]. Thus, HIF-1a is required for survival of the hypoxic chondrocytes located in the center of the growth cartilage, furthest from blood vessels. The precise mechanisms by which HIF-1a prevents chondrocyte death have yet to be fully explained. Likely, the transcriptional activity of HIF-1a is, on the one hand, required for turning on vital oxygen-sparing metabolic pathways that allow chondrocytes to survive and differentiate in their hypoxic environment. Activation of anaerobic glycolysis in mammalian hypoxic cells is HIF-1a-dependent [66]. During skeletogenesis, phosphoglycerate-kinase1 (PGK), a key enzyme of anaerobic glycolysis, was found to be strikingly stronger expressed in developing cartilage than in the surrounding tissues; HIF-1a-deficient chondrocytes, however, failed to upregulate PGK expression [58]. On the other hand, HIF-1a may additionally provide an indirect survival effect by inducing the expression of its downstream target VEGF, a principal regulator of blood vessel formation [67]. Deletion of VEGF from cartilage causes a cell death phenotype in the center of the fetal growth plate that closely mimics

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what is observed in HIF-1a deficient growth plates [65,68]. Moreover, hypoxia increases VEGF accumulation in chondrocytes in vitro in an HIF-1a-dependent manner [69,70]. Thus, HIF-1a-mediated upregulation of VEGF and induction of angiogenesis in the perichondrial tissues surrounding the avascular cartilage is likely one of the mechanisms by which chondrocytes survive hypoxia. In addition to the low level of expression of VEGF noted in the central, hypoxic immature chondrocytes of the growth cartilage [65,68], VEGF is at all times expressed abundantly by late hypertrophic chondrocytes, where it is critical for blood vessel invasion and replacement of cartilage by bone [10,71e73]. Inhibition of VEGF action in juvenile mice by administration of a soluble VEGF receptor chimeric protein (sFlt-1) impaired vascular invasion of the growth plate; concomitantly, trabecular bone formation and bone growth were reduced and the hypertrophic cartilage zone became enlarged likely due to reduced osteoclast-mediated resorption [10]. Further mouse genetic studies univocally established that VEGF is an essential physiological mediator of all three key vascularization stages of endochondral bone development (see above and Fig. 4.1). These mouse models include the Cre/LoxP-mediated conditional inactivation of the VEGF gene in chondrocytes, and genetically engineered mice expressing only one of the three major VEGF isoforms [65,68,72e75]. Altogether, these models have exposed multiple essential roles of VEGF and its isoforms in endochondral ossification, not only as a key inducer of vascularization but also as a direct modulator of bone development by affecting the various cell types involved. Perichondrial cells, osteoblasts and osteoclasts are all well documented to express several of the VEGF receptors and to respond to VEGF signaling by enhanced recruitment, differentiation, activity and/or survival (reviewed in [71,76,77]. The current model is that VEGF is produced at high levels by hypertrophic chondrocytes and is partly sequestered in the cartilage matrix upon its secretion. Trapped VEGF can be released from the matrix by proteases such as matrix metalloproteinase (MMP) 9 secreted by osteoclasts/chondroclasts during cartilage resorption. VEGF can then bind to its receptors on endothelial cells and stimulate the guided attraction of blood vessels to invade the terminal cartilage [78]. Osteoblasts and osteoclasts, or precursors thereof, associated with the newly vascularized bone region may at the same time be affected by the VEGF signaling in their recruitment, proliferation, differentiation and function. These pleiotrophic actions of VEGF on the various cells in the bone environment may contribute to the tight coordination of vascularization, ossification, and matrix resorption that is characteristically seen in endochondral bone development and growth [71,77].

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Since the loss-of-function models pointed out that VEGF is a positive regulator of bone development, skeletal growth and fracture repair [10,72,73,79,80], VEGF appears as an interesting target for stimulating fracture healing [81]. While it is being currently tested in preclinical models as a potential bone regeneration therapy, data using mice overexpressing VEGF temporally in the skeleton suggest that increasing the tightly coupled processes of angiogenesis and osteogenesis might be associated with potential side effects including disturbances in hematopoiesis [82]. In general, the actions of VEGF are highly dose dependent, and its physiological levels must be under very strict control mechanisms. Although the regulation of VEGF expression in the skeleton is still largely unresolved and may vary depending on the developmental stage and specific location and cell type, several mechanisms have been implicated to date. Hypoxia is a crucial trigger of VEGF expression in chondrocytes, osteoblasts, and possibly osteoclasts, through mechanisms that at least in part involve HIF [69,70,83e85]. During early bone development, Runx2 may be an important inducer of VEGF expression and blood vessel invasion into cartilage [86]. In addition, several hormones (including PTH, GH, 1,25(OH)2D3) and locally produced growth factors (e.g. FGFs, transforming growth factor-b [TGFb], BMPs, IGFs, plateletderived growth factor [PDGF]) have been demonstrated to be involved, at least in vitro, in the regulation of VEGF expression (see [76]).

Ossification: Osteoblast Differentiation and Activity Osteoblasts are highly specialized cells able to deposit and mineralize large amounts of matrix rich in type I collagen. Like chondrocytes, osteoblasts are cells of mesenchymal origin; they are thought to descend from mesenchymal stem cells that are pluripotent and have the capacity to differentiate into a variety of cell types, including adipocytes, chondrocytes and osteoblasts [87e89]. The prime transcription factors required to directing the cells into the osteoblastic lineage are b-catenin, Runx2 (previously termed Cbfa1), and Osterix, as discussed below [90,91]. Once committed, pre-osteoblasts further differentiate into mature osteoblasts secreting type I collagen and other bone matrix components, together called osteoid. Subsequently, osteoblasts direct the mineralization of the osteoid in a process that requires active alkaline phosphatase (ALP), expressed on the osteoblast membrane. These mature osteoblasts express characteristic genes including the gene encoding osteocalcin. Ultimately, the cells die through apoptosis, are converted to bone lining cells or become embedded within the bone matrix as osteocytes. Osteocytes are the most abundant

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cell type in postnatal bone, playing roles in the sensing of mechanical forces, the regulation of bone formation and resorption, and the control of phosphate homeostasis [92,93] (see further). Here, we will discuss a number of crucial transcription factors and extracellular signaling molecules that have been shown to control various aspects of osteoblast biology, including Wnt/b-catenin signaling, Runx2 and Osx, and actions elicited by BMPs, hedgehogs (Hh), and FGFs. At least some of these are currently known to play a role in inherited human bone diseases. Wnt/b-catenin Signaling Wnts (wingless-type MMTV integration site) are a large family of secreted growth factors (19 different members in the mouse and human genomes) that play essential roles in multiple developmental processes. Wnts are also required for adult tissue maintenance, and perturbations in Wnt signaling can lead to tumor formation and other diseases [94,95]. In the skeletal system, mutations in Wnt signaling components lead to skeletal malformations and diseases such as osteoporosis-pseudoglioma and osteoarthritis (see below). Wnt pathway modulation, particularly neutralizing inhibitors of Wnt signaling, has emerged as a promising strategy to improve bone mass. These drugs are exciting breakthroughs but are not without risks; the challenges include tissue-specific targeting and consequently, long-term safety [96e98]. Wnts can transduce their signals through several different downstream signaling pathways. The best understood pathway is the canonical or Wnt/b-catenin pathway. Central to this pathway is the regulation of the protein stability of b-catenin, which acts as a transcription factor in the canonical Wnt pathway and is also involved in cell adhesion by binding to membranous cadherins. In the absence of Wnts, cytoplasmic b-catenin is constitutively degraded through its phosphorylation by glycogen synthase kinase 3-b (GSK3-b) that targets it to the ubiquitineproteasome pathway. Upon signaling by Wnts, b-catenin is stabilized and translocates to the nucleus, where it interacts with T-cell factor (TCF)/lymphoid enhancer factor (LEF) family transcription factors to regulate the expression of its target genes. In addition, other pathways affecting GSK3-b (e.g. phosphatidyl inositol 3 [PI3]-kinase pathways) can also modulate b-catenin transcriptional activity [94,95,99,100]. The importance of b-catenin in skeletal biology was proven by conditional b-catenin loss- and gain-offunction mouse models elucidating its role as a crucial transcription factor in (i) determining osteoblast lineage commitment of early osteo-chondroprogenitors [101e104] and (ii) coupling osteoblast to osteoclast activity [105]. The first set of studies provided evidence

that, in mesenchymal progenitor cells in the perichondrium and calvarium where cells are destined to become osteoblasts, Wnt signaling is high, leading to high levels of b-catenin inducing the expression of genes that mediate osteoblast differentiation (such as Runx2) while inhibiting transcription of genes required for chondrocyte differentiation (such as Sox9). In the absence of b-catenin, these cells become chondrocytes instead of osteoblasts, as revealed via genetic modifications in mice. Conversely, in the inner region of the condensations, Wnt signaling must be low, as these cells become chondrocytes (see [100]). Secondly, inactivation of bcatenin in differentiated osteoblasts [105] or osteocytes [106] revealed that its transcriptional activity is important for stimulating osteoblastic production of osteoprotegerin (OPG or TNFRSF11B, see below), an inhibitor of osteoclast formation. As such, b-catenin plays a role in the coupling between osteoblast and osteoclast activity, the central principle of bone remodeling and maintenance of bone mass (see below). Upon Wnt stimulation, the ligands bind to two synergistically acting families of Wnt (co)-receptors, the Frizzled (Fzd) receptor family members (10 known) and low-density lipoprotein receptor-related proteins (LRP5 or LRP6). This leads to the recruitment of axin to LRP5/6 in the plasma membrane. The sequestering of axin at the plasma membrane on its turn likely leads to the disassembly of the b-catenin destruction complex, thus mediating the stabilization and transcriptional activity of b-catenin. Activation of the pathway is normally constrained by the expression of secreted Wnt inhibitors such as Dickkopfs (Dkks), sclerostin, and secreted frizzled-related proteins (Sfrps). Members of the Dkk family and sclerostin bind to LRP5/6, and thereby block the ability of Wnt ligands to interact with these co-receptors. Proteins of the Sfrp family can directly bind to Wnt ligands, offering another way to inhibit activation of the pathway [94,95,98,107]. Mutations in the Wnt receptor complexes and Wnt antagonists have been clinically associated with changes in bone mineral density and fractures. Loss-of-function mutations in the LRP5 co-receptor cause the autosomal recessive disorder osteoporosis-pseudoglioma syndrome (OPPG), which is characterized by low bone mass, ocular defects, and a predisposition to fractures [108]. These findings were recapitulated in germline LRP5 knockout mice, which developed a low bone mass phenotype similar to patients with OPPG due to decreased osteoblast proliferation [109]. Conversely, gain-of-function mutations in the same group that render LRP5 with reduced affinities for the secreted antagonists Dkk1 and sclerostin result in high bone mass phenotypes [110e113]. There is debate as to whether these actions of LRP5 are direct or indirect actions on bone; argumentation to the latter is the

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finding that activation of LRP5 in the duodenum, rather than in osteoblasts, increased osteoblast proliferation by suppressing serotonin secretion; the lower blood levels of gut-derived serotonin lead to an increase in bone mass through less serotonin binding to its osteoblast receptor, 5-hydroxytryptamine (Htr1b), and activation of the cAMP responsive element binding protein (CREB) transcription factor [114]. Mutations in SOST have also been identified in patients, namely those diagnosed with van Buchem disease and sclerosteosis, diseases associated with high bone mass [115,116]. Altogether, these findings over the last decade highlighted the important role of canonical Wnt signaling in regulating human bone mass. Extensive research is therefore being conducted in this area based on the prospect that it can possibly lead to pharmacological intervention useful in the management of osteoporosis. Among the candidate therapeutics are small molecule inhibitors of GSK3b, neutralizing antibodies to Dkk1, secreted Frizzled-related protein 1, and sclerostin. Since osteocytes are the major producer of sclerostin, inhibition of this Wnt antagonist appears as a particularly promising strategy to prevent bone loss without causing adverse side effects, such as related to the tumorigenicity and toxicity to other tissues of activated Wnt signaling [96e98]. Runx2 and Osterix: Essential Transcription Factors in Osteoblastogenesis Runx2 (runt related transcriptional factor 2, previously known as core-binding factor a1 [Cbfa1], Osf2, or AML3) and Sp7/Osterix determine the osteoblast lineage from mesenchymal stem cells along with canonical Wnt signaling. In the process of osteoblast differentiation, these factors and canonical Wnt signaling molecules inhibit mesenchymal cells from differentiating into chondrocytes and adipocytes. Runx2, a transcription factor of the ancient runt family, is the earliest known marker essential for the production of the osteoblast lineage. Functional studies have demonstrated that it is a central regulator of osteoblast differentiation and function, and absolutely essential for the induction of osteoblasts and the formation of endochondral and intramembranous bone. The dominant role of Runx2 is illustrated by the fact that Runx2-deficient mice completely lack osteoblasts and do not form bone at all e instead a completely cartilaginous skeleton develops without any true bone matrix [7,117]. In heterozygous (haploinsufficient) Runx2 mutant mice, the defect in osteoblast differentiation is limited to intramembranous bones [117]. The resulting phenotype in these mice closely resembles the cleidocranial dysplasia (CCD) syndrome in humans, a dominantly inherited developmental disorder of bone, and RUNX2 is mutated in most CCD patients [118,119]. Consistent with its function as an early transcriptional regulator of osteoblast

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differentiation, Runx2 is also an early molecular marker of the osteoblast lineage, being highly expressed in perichondrial mesenchyme and in all osteoblasts. Pre-hypertrophic chondrocytes also express Runx2, and Runx2 plays important roles in cartilage biology as well. Runx2 can be sufficient to induce differentiation of mesenchymal cells into osteoblasts: its ectopic expression in non-osteoblastic cells leads to the expression of osteoblast-specific genes in vitro and in vivo [6]. Runx2 binds to an osteoblast-specific cis-acting DNA element present in the promoter of most genes expressed in osteoblasts; as such, Runx2 mediates osteoblast differentiation by inducing ALP activity, and regulating the expression of a variety of bone matrix protein genes, including the Col1a1, osteopontin, osteonectin, bone sialoprotein, and osteocalcin genes and Runx2 itself [120,121]. The regulation of different stages of osteoblast differentiation by Runx2 is, however, very complex; Runx2 transcriptional regulation involves interactions with a myriad of transcriptional activators and repressors and other co-regulatory proteins that are under continued investigation. The current model is that Runx2 triggers the expression of major bone matrix protein genes and the acquisition of an osteoblastic phenotype at an early stage of osteoblast differentiation, while inhibiting the late osteoblast maturation stages and the transition into osteocytes. As such, Runx2 may play an important role in maintaining a supply of immature osteoblasts. Runx2 must thus be suppressed for immature osteoblasts to become fully mature osteoblasts, which form mature bone with regularly and densely packed collagen fibrils and high mineralization [120,121]. Furthermore, Runx2 regulates the expression of RANKL and OPG in osteoblasts, thus affecting osteoclast differentiation (see below). Runx2 is also required for the expression of Osterix (encoded by the Osx or Sp7 gene), an SP family transcription factor with three zinc finger motifs. Osterix is expressed in osteoblast progenitors, osteoblasts, and at a lower level also in pre-hypertrophic chondrocytes [4,122]. Like Runx2-deficient mice, mice lacking Osterix showed complete lack of osteoblasts and absence of both intramembranous and endochondral bone formation [122]. Thus, Osterix is a third transcription factor that is essential for osteoblast differentiation. Since Runx2 is expressed in the mesenchymal cells of Osx-null mice but Osterix is not expressed in Runx2-null mice, it can be concluded that Osterix acts downstream of Runx2 [122]. Furthermore, the Osx gene contains a consensus Runx2-binding site in its promoter region, suggesting that Osterix might be a direct target of Runx2 [123]. The transcriptional activity of Osterix involves its interaction with NFATc1 cooperatively forming a complex that binds to DNA and induces the expression of Col1a1 [124]. The subtleties of how Osterix regulates

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osteoblast differentiation and function, and which osteoblastic genes are directly regulated by Osterix are as yet to be elucidated. Expression of genes characteristic of mature osteoblasts (such as those encoding bone sialoprotein, osteopontin, osteonectin, osteocalcin) was absent in cells surrounding chondrocytes in Osx-null mice, and instead these cells express genes characteristic of chondrocytes (Sox9, Sox5, Col2a1) [122]. Osterix has also been reported to inhibit chondrogenesis in vitro [125,126]. Thus, Osterix may be important for directing precursor cells away from the chondrocyte lineage and toward the osteoblast lineage. Besides b-catenin, Runx2, and Osterix, various other (non-bone-specific) transcription factors are also involved in the genetic control of osteoblast differentiation and function, albeit not to a similar critical extent as their inactivation does not completely abrogate bone formation. These include ATF4, Msx1 and Msx2, Dlx5, Dlx6 and Dlx3, Twist, activator protein-1 (AP1) and its related molecules (Fos/Jun), and Schnurri-2 and -3 (for reviews and references therein, see [91,120,127,128]). Secreted Local Signaling Molecules Involved in Osteoblastogenesis A myriad of morphogens and signaling molecules control the activity of the transcription factors just described. These molecules include Wnts, as discussed before, as well as locally produced BMPs and TGF-b, Hedgehogs (particularly Ihh), FGFs, Notch ligands, IGFs, PDGF, and systemic factors like PTH, GH, prostaglandins, estrogens, androgens, 1,25(OH)2D3 and glucocorticoids. The roles of the signaling pathways involving BMPs, Ihh, and FGFs in osteoblastogenesis and bone formation are briefly reviewed here; the endocrine control of bone growth and remodeling is provided further (see below and other chapters). The family of growth factors that has received a tremendous amount of attention is that of the BMPs. These are members of the transforming growth factorb superfamily of growth factors that can, when applied locally, induce de novo bone formation; some BMPs, including BMP-2 and BMP-7, are therefore clinically used in orthopedics [129,130]. BMPs transduce signals through serine/threonine kinase receptors, composed of type I and II subtypes, activating intracellular Smads that relay the BMP signal to target genes in the nucleus [131, 132]. The analysis of their functions in vivo has relied mostly on gene deletion experiments. Although several BMPs affect skeletal patterning and joint formation, it has proved difficult using this approach to elucidate how they affect osteoblast differentiation. BMPs are crucial for regulating development in almost all the principal organs and tissues [131,133]. In the morphogenesis of the skeleton, BMPs have been

implicated in early limb patterning, mediating the correct formation, size or shape of the mesenchymal condensations, as well as converting the condensing mesenchymal cells into chondrocytes [134e136]. Their functions in osteogenesis during development, postnatal bone formation and remodeling, and fracture repair have therefore been hard to document in vivo because, in some cases, the inactivation of specific BMPs led to severe early defects and lethality (e.g. BMP-2, -4), precluding investigation of the later stages. In other cases, removal of individual family members (such as BMP-7) displayed no defects in skeletogenesis, presumably due to functional redundancy between the various BMP ligands and receptors [137]; around 20 BMP family members have been identified to date, and perichondrial cells, osteoblasts, and chondrocytes express multiple BMPs, BMP receptors, and BMP antagonists. Recent and ongoing studies therefore employ conditional (site-specific) and/or combined (multiple targets) mutagenesis strategies. As such, the double knockout of BMP-2 and BMP-4 in the limb completely disrupted osteoblast differentiation, demonstrating the crucial roles of these two BMPs in osteoblast differentiation [138]. Effects of BMP signaling in later stages of the osteoblast differentiation are suggested by studies using BMP antagonists. Targeting noggin overexpression to differentiated osteoblasts by the osteocalcin promoter results in osteopenia by 8 months of age [139]. Likewise, overexpression of gremlin, another BMP antagonist, in differentiated osteoblasts results in reduced bone mineral density and fractures [140]. On the other hand, a recent report using an osteoblast-targeted deletion of BMP receptor signaling indicated that BMP signaling in osteoblasts physiologically induces bone resorption by enhancing osteoclastogenesis via the RANKL-OPG pathway, thereby reducing bone mass [141]. Overall, signaling by BMPs plays an important role in a variety of cell types in bone such as osteoblasts, chondrocytes, and osteoclasts, and the precise mechanisms underlying their actions during bone growth have not yet been fully elucidated due to the considerable complexity and involvement of a myriad of interacting pathways. Nevertheless, particularly BMP-2 has been shown to play a critical role in osteogenic differentiation: it promotes the commitment of pluripotent mesenchymal cells to the osteoblast lineage [142,143] and has been demonstrated to induce the expression of both Runx2 and Osx during osteoblastogenesis [144e147]. The activity of BMP-2 as a potent inducer of bone formation is being applied to repair bone defects in humans [129,130,148]. Moreover, a human genetic study indicated that polymorphisms of BMP-2 gene expression are linked to a high risk for osteoporosis [149].

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The Hedgehog family member Ihh is required for endochondral but not for intramembranous bone formation, by controlling osteoblast differentiation in the perichondrium of long bones. This statement is based on the finding that Ihh-deficient mice have disorganized growth plates, as expected (see above), but also completely lack osteoblasts in bone formed through endochondral ossification; however, these do appear to be present in the skull, the mandibles, and the clavicles e bones that form by intramembranous ossification [30,150]. In these mice, Runx2 is expressed in chondrocytes but not in perichondrial cells. As well, nuclear b-catenin is absent in the perichondrial cells of Ihh-deficient mice [103]. Thus, Ihh is required for inducing the initial activity of Runx2 and b-catenin in perichondrial cells and triggering them to become endochondral osteoblasts, thereby coupling chondrocyte maturation to osteoblast differentiation during endochondral ossification. Chondrocyte-derived Ihh remains crucial for sustaining trabecular bone also in the postnatal skeleton [32]. Skeletal abnormalities have been described in mutant mice lacking some of the intracellular mediators of Ihh signaling termed Gli proteins (three related transcription factors Gli1, Gli2 and Gli3); Gli2 mediates Ihh-induced osteoblast differentiation in mesenchymal cell lines by associating with Runx2 and stimulating its expression and function, as well as inducing BMP-2 expression [151,152]. FGF signaling has also been implicated in the proliferation of immature osteoblasts and the anabolic function of mature osteoblasts in vivo [46,48,153e155]. Whether and how osteoblast differentiation per se is affected by FGFs is currently elusive, but evidence indicates that FGF signaling induces BMP-2 expression and stimulates the expression and transcriptional activities of Runx2 [156e158]. Conversely, Runx2 has been demonstrated to form a complex with Lef1 or TCF that binds to the promoter region of the gene encoding FGF18, inducing its expression [159]. These findings underscore once more how the various pathways that are essential for endochondral and intramembranous bone development physically and functionally converge. Moreover, the ultimate outcome relies on the complex integration of both stimulatory and inhibitory signaling. In any case, FGFs and their receptors have important roles in the growth of intramembranous bones and the bone adjacent to growth plates in endochondral bone growth. 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 FGFR1 or 2 in humans

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can cause craniosynostosis [34]. The ligands most crucial for activating FGF receptors in sutures and osteoblasts of the limb are uncertain. However, genetic evidence in mice established that both FGF-2 and FGF-18 are important [46e48,158]. Thus, multiple FGFs and FGF receptors are needed for normal osteoblast production and differentiation.

Bone (Re)Modeling: Osteoclastogenesis, Bone Resorption, and the Coupling Principle The Role of Osteoclasts in Bone Modeling and Remodeling Osteoclasts are giant multinucleated cells that have the unique capacity to efficiently degrade calcified tissues. During development and growth of the endochondral bones, the main tissue to be resorbed is the calcified cartilage matrix produced by hypertrophic chondrocytes. Coinciding in time and place with the vascular invasion of the terminally differentiated chondrocytes, the calcified cartilage is co-invaded by osteoclasts, or a postulated related cell type termed “chondroclasts” [160]. Osteoclasts are not strictly essential for the excavation of the marrow space and its colonization by blood vessels and bone-forming osteoblast lineage cells to replace the hypertrophic chondrocytes during endochondral bone development [4,161]. However, the degradation, digestion and removal of the septa of calcified cartilage does require osteoclasts, as mice lacking osteoclasts, such as in the case of receptor activator of nuclear factor-kB (RANK)-deficiency (see below), display poorly remodeled osteocartilaginous structures instead of normal trabecular bone [4,161]. Moreover, such mice present with disorganized growth plates and impaired longitudinal bone growth, leading to a small body size and short limbs [161]. Not surprisingly, antiresorptive drugs such as bisphosphonates can interfere with bone modeling (shaping) during growth [162,163]. Bone, like many other organs in the body, goes through a tremendous amount of destruction and growth during childhood. Bisphosphonates potently decrease the activity of osteoclasts, which can lead to the accumulation of growth plate residues within trabecular bone tissue. Calcified cartilage has a high mineral density and therefore contributes to increase densitometric results, but is less resistant to fractures than is normal bone. Low remodeling activity might also delay bone healing after injury. This is of clinical interest, for instance in the treatment of juvenile osteoporosis during growth and of osteogenesis imperfecta (OI). OI is a genetic disease characterized by increased bone fragility and low bone mass due to the synthesis and secretion of defective type I collagen

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molecules by the osteoblasts [164]. In the majority of OI patients, the disease is inherited in an autosomal dominant manner and can be linked to mutations in one of the two genes encoding collagen type I a chains (COL1A1 and COL1A2). This can be viewed as a near complete arrest of the normal osteoblast function in the most severe forms. Human OI patients exhibit characteristic short stature, which has been attributed to increased incidence of bone fractures, defective bone matrix production, and poor mineralization [165], hence illustrating the critical role played by the osteoblasts in the control of longitudinal growth. Disrupted growth plate structure was observed in severe OI patients and in various mutant mouse strains modeling the disease [165,166]. Pamidronate, a nitrogen-containing bisphosphonate that interferes with osteoclast activity, is now used worldwide to treat children and adolescents with moderate to severe forms of OI [167,168]. The hypothesis initially underlying the use of an anti-osteoclast medication in an osteoblast disorder such as OI was that a decrease in the activity of the bone resorbing system might compensate for the inadequate functioning of the bone-forming cells. This treatment has been reported to increase lumbar spine bone mineral density and metacarpal cortical width, to decrease fracture rate, and to improve mobility. These results are very encouraging, but the fact that bisphosphonates so effectively inhibit osteoclastic action raises concern that this form of treatment could further compromise longitudinal bone growth in OI children; therefore, treatment with bisphosphonates during growth is mainly reserved for patients with significant clinical problems [164,167,168]. Throughout further postnatal life, osteoclasts primarily act to degrade mineralized bone during the constant process of bone remodeling. Indeed, bone is continuously renewed via a process of self-destruction followed by a regeneration process. Its purposes are to repair damaged bone, remove old bone, and facilitate skeletal responses to changes in loading requirements and physiologic needs in ensuring mineral homeostasis. This dynamic nature of the skeleton is achieved by the coordinated actions of osteoclasts, osteoblasts, osteocytes within the bone matrix and osteoblast-derived lining cells that cover the surface of bone. In short, remodeling initiates with signals that stimulate osteoclast formation, followed by osteoclast-mediated bone resorption, a reversal period, and then a long period of bone matrix formation and mineralization mediated by osteoblasts [169,170] (Fig. 4.3). Packets of the existing trabecular and cortical mineralized bone tissue that are renewed during remodeling are called bone remodeling units (BRUs) or bone multicellular units (BMUs) [169]. The bone in an activated BRU is first removed by osteoclastic bone resorption in a process that takes a few weeks (see Fig. 4.3). During

FIGURE 4.3 Three-phase model of bone remodeling. The skeleton is a metabolically active organ that undergoes continuous remodeling throughout life. Bone remodeling involves the removal of mineralized bone by osteoclasts followed by the formation and subsequent mineralization of bone matrix by osteoblasts. Initiation starts with recruitment of hematopoietic precursors and their differentiation to osteoclasts, induced by osteoblast lineage cells that express osteoclastogenic ligands such as RANKL. Osteoclasts become multinucleated and resorb bone. Transition is marked by switching from bone resorption to formation via coupling factors, possibly including diffusible factors (e.g. hormones), membrane-bound molecules (e.g. ephrins), and factors embedded in bone matrix that become released upon osteoclastic bone resorption and can stimulate osteoblast recruitment, differentiation and/or activity. During the termination phase, the resorbed lacuna is refilled through bone formation by osteoblasts that later flatten to form a layer of lining cells on the bone surface or become osteocytes connected by canaliculi within the bone.

the time lag that occurs between the end of resorption and the beginning of formation, called the reversal phase, osteoblastic cells differentiate on the bone surface and deposit the organized matrix that then becomes mineralized. Hence, the lost bone is replaced by osteoblastic bone formation, lasting 3e4 months for one packet. This discrepancy in the kinetics of bone resorption and formation partly explains how increased resorption, even when accompanied by coupled increased formation, can cause bone loss, for example, in estrogen deficiency or hyperparathyroidism [169]. Excessive osteoclast activity can be the basis of several pathological conditions including osteoporosis, a common low bone mass disorder typically prevalent in postmenopausal women, but also periodontal disease, rheumatoid arthritis, multiple myeloma, Paget’s disease and metastatic cancer. On the other hand, impaired osteoclast differentiation and/or function leads to osteopetrosis, a rare human disease characterized by increased bone mass and obliteration of the bone marrow cavity. Many efforts have been made to dissect the regulatory pathways controlling osteoclast differentiation and function; the most important of those are reviewed here.

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Osteoclastogenesis Osteoclasts derive from hematopoietic precursor cells of the myelomonocytic lineage and share a common precursor with macrophages. The early differentiation of the bipotential macrophage/osteoclast precursor cells is regulated by PU.1, a versatile hematopoietic cellspecific transcriptional regulator of the ETS-family. Particularly, the commitment to macrophages/osteoclasts is dependent on high level/activity of PU.1; consequently, PU.1 null mice lack both macrophages and osteoclasts, and are osteopetrotic [171,172]. This is a cell autonomous defect that can be corrected by bone marrow transplantation [172]. PU.1 regulates the lineage fate decision of the early progenitors by directly controlling expression of the c-Fms gene that is a key determinant of differentiation into the macrophage/osteoclast lineage (see below) [173]. PU.1 also regulates the transcription of another key osteoclastogenesis control gene, receptor activator of nuclear factor-kB (RANK) (see below) in myeloid progenitors [174]. Although mature macrophages and osteoclasts have some cell surface markers in common, the latter express high levels of tartrate-resistant acid phosphatase (TRAP), cathepsin K, vitronectin, calcitonin receptor and avb3 integrin, that typify the osteoclast lineage (see below). PU.1 is the earliest marker of osteoclast differentiation; yet, the signals that induce its expression and that of other cell-specific transcription factors involved in osteoclastogenesis are as yet unresolved. Among the other transcription factors that control osteoclast differentiation and function is NF-kB, which appears to play a role early during osteoclast differentiation. NF-kB is formed as a dimer composed of various combinations of proteins: p50, p52, p65, c-Rel, RelA and RelB [175]. Mice deficient in both p50 and p52 harbor an osteopetrosis phenotype due to an arrest of osteoclast differentiation; the presence of a large number of macrophages in these mice places NF-kB downstream of PU.1 in the genetic pathway controlling osteoclast differentiation [176,177]. NF-kB increases the expression of another transcription factor, c-Fos, which is the cellular homolog of the v-Fos oncogene and a major component of the activator protein (AP)-1 transcriptional complex [178]. The deletion of c-Fos in mice caused an early arrest of osteoclast differentiation leading to osteopetrosis, while again macrophage numbers were enhanced [179,180]. c-Fos interacts with the master transcription factor for osteoclastogenesis, nuclear factor of activated T cells c1 (NFATc1), to induce osteoclast-specific genes such as those encoding TRAP and calcitonin receptor (see below) [181,182]. After the precursor cells have committed to the osteoclast lineage, they become subjected to a complex multistep process culminating in the generation of

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mature, multinuclear activated osteoclasts; these steps include proliferation, maturation and fusion of differentiating precursor cells, and finally activation of resorption. Several secreted molecules and signaling proteins control the process of osteoclast differentiation and function. The requirement for secreted molecules to control osteoclastogenesis was first documented with the genetic elucidation of a classical mouse mutation called op/op [183]. 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 [116,183]. The gene mutated in op/op mice encodes the growth factor macrophage colony-stimulating factor (M-CSF) [183]. The second critical cytokine essential for osteoclastogenesis is receptor activator of nuclear factor-kB ligand (RANKL) (also known previously as osteoprotegerin ligand [OPGL], osteoclast differentiation factor [ODF], or tumor necrosis factor [TNF]-related activationinduced cytokine [TRANCE]). As M-CSF, this signaling molecule is also strongly expressed by bone marrow stromal cells (i.e. osteoblast progenitors) and osteoblasts; M-CSF is produced in both a soluble and a membrane-bound form, while RANKL is made exclusively as a membrane protein by osteoblasts. The process of osteoclastogenesis requires direct cell-to-cell interaction between stromal/osteoblastic cells and osteoclast precursors presumably because of these key membrane-bound ligands. Both PTH and 1,25(OH)2vitaminD3 as well as several other osteotropic factors stimulating resorption increase the expression of RANKL on stromal/osteoblastic cells. Furthermore, not only osteoblasts but also chondrocytes and T cells synthesize and secrete RANKL and are able to support osteoclastogenesis. This may be important physiologically during osteoclast differentiation and invasion at the hypertrophic cartilage during endochondral bone development and growth, and definitely plays a major role pathologically. For instance, production of RANKL by T cells has been implicated as an activator of osteoclastic resorption in inflammatory-mediated bone and cartilage destruction such as seen in several autoimmune disorders including rheumatoid arthritis, likely working synergistically with TNF-a (for reviews, see [181,182,184e186]. M-CSF and RANKL affect several steps of the osteoclastogenesis cascade by binding to their respective receptors, c-Fms and RANK, that are expressed by osteoclast progenitors and osteoclasts at all stages of differentiation. M-CSF signaling is essential early on in the lineage for proliferation, differentiation and survival of the osteoclast/macrophage precursors. RANKL is an essential inducer of multiple aspects of osteoclastogenesis, including osteoclast differentiation, fusion, activation of

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mature osteoclasts to resorb mineralized bone, and cell survival [181,182,184e186]. Through these actions, RANKL potently stimulates bone resorption. RANKL acts via binding to its signal transducing receptor RANK, a member of the TNF receptor family present on pre-osteoclasts and mature osteoclasts. Consistent herewith, an activating mutation of the human RANK gene was found in patients with familial expansile osteolysis, a disease of excess bone resorption [187]. Recent genetic and cell biological studies have begun to elucidate the complex signaling cascade downstream of RANK/ RANKL (reviewed by [181,182,186]). Briefly stated and oversimplified, the binding of RANKL to RANK on the surface of osteoclast precursors recruits the adaptor protein TNF receptor-associated factor (TRAF) 6 to the cytoplasmic domain of RANK, leading to activation of NF-kB and its translocation to the nucleus, and the actions of c-Fos and NFATc1 as outlined above. The actions of RANKL are negatively regulated by a secreted soluble decoy receptor termed osteoprotegerin (OPG) (previously also known as osteoclastogenesis inhibitory factor [OCIF]), also a member of the TNF receptor superfamily. OPG sequesters RANKL molecules and thereby blocks their binding to RANK. As such, OPG protects bone from excessive resorption; this conclusion is supported by the finding that certain homozygous deletions of OPG in humans can cause juvenile Paget’s disease, a disorder characterized by increased bone remodeling, osteopenia, and fractures [188]. The relative concentration of RANKL and OPG in bone is thus a major determinant of bone mass and strength. OPG is widely expressed; not surprisingly, its expression by osteoblasts and stromal cells is positively regulated by bone anabolic or antiresorptive factors, such as estrogen and calcitonin (see [184,186]). The essence of the RANK/RANKL/OPG cascade was clarified by mouse genetic studies. Mice lacking RANKL or RANK and mice with increased circulating OPG by transgenic overexpression were severely osteopetrotic due to a block in osteoclastogenesis. Conversely, targeted mutagenesis of OPG, overexpression of an sRANKL transgene, and administration of RANKL in mice, all led to increased osteoclast formation, activation and/or survival and resulted in an osteoporotic phenotype (see [184,186]). In summary, RANKL and OPG act in an antagonistic fashion to regulate bone resorption and their respective expression levels are under the control of pro- and antiresorptive factors including several hormones, cytokines and growth factors. Other regulatory molecules have been implicated in the late stages of osteoclast differentiation, the fusion process and activation of resorption. Co-stimulatory molecules acting in concert with M-CSF and RANKL to complete osteoclastogenesis include proteins containing an immunoreceptor tyrosine-based activation motif

(ITAM) domain that is critical for the activation of calcium signaling and found in adapter molecules like DNAX-activating protein (DAP)12 and the Fc receptor g (FcRg) [189]. The resultant increase in intracellular calcium is required for activation of NFATc1. The fusion of mononuclear osteoclast precursor cells into mature multinucleated osteoclasts is regulated by a membrane protein called dendritic cell-specific transmembrane protein (DC-STAMP). DC-STAMP-deficient cells failed to fuse, and these mononuclear osteoclasts had reduced resorptive efficiency in vitro. Consequently, DCSTAMP-deficient mice exhibited increased bone mass [190]. Further studies on the regulatory components of osteoclastogenesis will not only expand our basic understanding of the molecular mechanisms of osteoclast differentiation during bone development and remodeling, but also increasingly offer opportunities to develop therapeutic means of intervention in osteoclast-related diseases [191,192]. OPG, as well as soluble recombinant RANK, suppresses osteoclastogenesis, while antibodies to RANK can stimulate osteoclast formation. From the clinical point of view, the RANKL signaling pathway thus holds great promise as a strategy for suppressing excessive osteoclast formation in variety of bone diseases including osteoporosis, autoimmune arthritis, periodontitis, Paget’s disease, and bone tumors/metastases. One drug that has been developed to target RANKL signaling for the treatment or prevention of bone disease is denosumab, a human monoclonal antibody that binds to RANKL and prevents RANKL interaction with RANK [193]. Bone Resorption Bone resorption involves both dissolution of bone mineral as well as degradation of organic bone matrix. Osteoclasts are highly specialized to perform both of these functions. Upon activation of the mature multinucleated osteoclasts, the cells attach themselves firmly to the bone surface, using specialized actin-rich podosomes (actin ring), through cytoskeleton reorganization and cellular polarization [194]. Within these tightly sealed zones of adhesion to the mineralized matrix, the osteoclasts form convoluted, villous-like membranes called “ruffled borders” that drastically increase the surface area of the cell membrane facing the resorption lacuna. Via these ruffled membranes the osteoclasts secrete abundant hydrochloric acid (involving the vacuolar Hþ-ATPase proton pump) mediating acidification of the compartment between the cell and the bone surface, as well as a myriad of enzymes such as lysosomal cathepsins, the phosphatase TRAP, and proteolytic MMPs. The acidity of the environment leads to dissolution of the mineral phase (crystalline hydroxyapatite), activation of the lytic enzymes, and digestion of

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organic matrix compounds. The sealing mechanism allows the localized dissolving and degrading of the mineralized bone matrix, while simultaneously protecting neighboring cells from harm. During the resorption process, dissolution of hydroxyapatite releases large amounts of soluble calcium, phosphate and bicarbonate [195,196]. These complex processes of osteoclast recruitment, polarization on the bone surface, and export of acid and enzymes are orchestrated by many factors, including RANKL, as well as integrin-mediated signaling from bone matrix itself. Particularly the avb3 integrin appeared important for osteoclast functioning based on the finding that inhibition of signaling through this avb3 integrin inhibited osteoclast-mediated bone resorption in vitro and in animal models of osteoporosis and malignant osteolysis [197]. Among the many other molecules that are important for the functional activity of osteoclasts, such as c-Src, cathepsin K, carbonic anhydrase II, TRAP, and several ion channel proteins, many have been found to cause an osteopetrotic phenotype when deleted in mice or altered in humans. The absence of these genes does not affect the differentiation into morphologically normal osteoclasts; however, the osteoclasts are not functional and fail to resorb bone effectively (reviewed in [78,195,196]. For instance, cathepsin K, the key enzyme in the digestion of bone matrix by its activity in degrading type I collagen, is highly expressed by activated osteoclasts and secreted in the resorption lacuna [198,199]. Its deletion in mice led to osteopetrosis, and mutations in the human cathepsin K gene cause pycnodysostosis. Highly selective and potent cathepsin K inhibitors have been shown useful as antiresorptive agents to treat osteoporosis, as well as being promising therapeutic tools to reduce breast cancerinduced osteolysis and skeletal tumor burden [200,201]. Besides cathepsin K, several proteolytic enzyme groups are involved in the degradation of organic components (collagens and proteoglycans) of bone and cartilage matrices after the mineral is dissolved. One of these is the MMP family, which constitutes over 25 members, including secreted collagenases, stromelysins, gelatinases and membrane-type (MT)-MMPs. Several MMPs including MMP9 and MMP14 (also known as MT1-MMP) are highly expressed in osteoclasts/chondroclasts. MMPs are synthesized as latent pro-enzymes that, upon proteolytic activation, can degrade numerous extracellular matrix components. As such, they are involved in development, growth and repair of tissues, but also in pathological conditions associated with excessive matrix degradation such as rheumatoid arthritis, osteoarthritis and tumor metastasis [202e204]. The Coupling Principle in Bone Remodeling Bone remodeling must be tightly controlled to maintain the normal bone homeostasis, but the molecular

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mechanisms that control its initiation, progression, and cessation at any given site remain poorly understood. However, failure of this normal skeletal functioning is one of the most common early manifestations of aging, for example in osteoporosis and osteoarthritis. These and a variety of other pathologic conditions affecting the skeleton (e.g. rheumatoid arthritis, periodontitis, Paget’s disease, and bone tumors) lead to perturbations in the bone remodeling process, predominantly shifting the balance towards more degradation of bone due to local and/or systemic alterations in the levels of hormones or proinflammatory cytokines that stimulate bone resorption [170,205,206]. In normal conditions of development, growth, and bone health, the osteoclast is thought to die via apoptosis after a limited period of resorptive activity [207] and the resorbed area of cartilage or bone is efficiently replaced by newly formed bone through the action of osteoblasts. Hence, skeletal homeostasis remains intact as long as the activities of both osteoclasts and osteoblasts are balanced (“coupled”) and the net bone mass is maintained. This balance implies the existence of mechanisms tightly coordinating the differentiation of osteoblasts and osteoclasts as well as their migration to locations where they function. One prime aspect of the coupling principle is provided by the direct control of osteoclastogenesis by cells of the osteoblast lineage through their expression of M-CSF and RANKL. Conversely, osteoclasts may reciprocally stimulate osteoblast differentiation and function to initiate the anabolic arm of the remodeling process. Osteoclastic bone resorption may well locally release the myriad of growth factors, like for example TGFb, that are stored in the bone matrix, which can subsequently act to attract and stimulate osteoblasts [208]. Direct signaling by osteoclasts to cells of the osteoblast lineage has recently been demonstrated and may participate in coupled osteoblastogenesis and bone formation. For instance, the ephrin/Eph receptor system allows bidirectional signaling between osteoclasts and osteoblasts; osteoclasts express ephrin B2 and osteoblasts express its EphB4 receptor, both membrane-bound proteins [209]. Signaling through EphB4 into osteoblasts (“forward signaling”) enhances osteogenic differentiation in vitro, whereas signaling through ephrin B2 into osteoclast precursors (“reverse signaling”) suppresses osteoclast differentiation [209]. The overall outcome of such interaction thus is predicted to favor bone formation [210]. It has also been reported that the v-ATPase V0 subunit D2 is not only involved in osteoclast fusion but also regulates the secretion by osteoclasts of still unidentified factors that inhibit the differentiation of osteoblast precursors into mature cells [211]. Other cytokines involved in the coupling, such as interleukin-1 (IL-1), IL-2, IL-6, and oncostatin M, transduce their signals

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through the gp130 protein and play an important role in osteoclast and osteoblast physiology [212].

Osteocytes and Mechanosensing Mechanical loading of the skeleton is essential for the development, growth, and maintenance of strong, weight-bearing bones. Altered growth and subsequent deformity resulting from abnormal mechanical loading is often referred to as mechanical modulation of bone growth. This phenomenon has key implications in the progression of infant and juvenile musculoskeletal deformities, such as adolescent idiopathic scoliosis, hyperkyphosis, genu valgum and tibia bowing. Clinical management of these deformities is often directed at modifying the mechanical environment of affected bones. However, there is limited understanding of how bone growth is regulated in response to mechanical loading with respect to growth plate chondrocytes [213]. Better characterized is the concept that mechanical loading improves bone strength particularly by inducing osteoblastic bone formation in regions of high strain energy. In order to withstand loading in the most efficient way (maximal strength for minimal material), the skeleton constantly adjusts its bone mass and architecture in response to load through bone remodeling. It has indeed long been recognized that mechanical stress induced by weight-bearing exercise increases osteoblast activity and induces bone formation. Bone tissue thus has a mechanosensing apparatus that directs osteogenesis to where it is most needed to increase bone strength. The most likely sensors of mechanical loading are the osteocytes, which sense variations in mechanical forces acting on bone and respond to this by signaling via sclerostin secretion and other mechanisms (see below), to coordinate osteogenesis [214,215]. Osteocytes represent the quantitatively dominant cell type in adult bone. Osteocytes, derived from osteoblasts, are buried within the mineralized bone matrix. They are connected to each other and to cells on the bone surface via numerous dendritic processes that run inside lacunar cannaliculi. These processes are thought to allow osteocytes to communicate with other cells including other osteocytes, and osteoblasts and osteoclasts on the bone surface, thereby playing roles in the regulation of bone formation and resorption [92,93,216]. They also allow the osteocytes to sense and respond to mechanical forces, and to secrete proteins involved in systemic phosphate homeostasis (see Chapter 7) including dentin matrix protein (DMP)-1, matrix extracellular phosphoglycoprotein (MEPE) and FGF-23 [92,217]. The importance of viable osteocytes in the maintenance of bone tissue health was shown by experimental destruction of osteocytes in murine bone: activation of the diphtheria toxin receptor, expressed

under the control of the osteocyte-specific DMP-1 promoter, quickly led to targeted ablation of osteocytes through necrosis, and was associated with a large-scale increase in bone resorption, decreased bone formation, and trabecular bone loss. Concomitantly, these mice were resistant to unloading-induced bone loss, indicating the requirement for osteocytes in the response to mechanical signals [218]. Osteocytes have been demonstrated to stimulate bone resorption in vitro and in vivo [219,220]. This modulation of bone remodeling may be elicited by osteocyte apoptosis, which can be consequential to unloading [221]. Conversely, mechanical stimulation is capable of maintaining osteocyte viability [222]. The recent deletion of b-catenin in osteocytes, using a DMP-1-Cre conditional knockout approach, indicated that b-catenin is necessary for normal osteocyte viability and function, and for the maintenance of normal bone. Indeed, these conditional knockout mice had bones with pronounced porosity and fragility due to increased osteoclast number and activity, most likely owing to reduced expression of OPG and an increase in RANKL, both found to be expressed in osteocytes [106]. The mechanisms by which a mechanical stimulus is transduced into biochemical signals in osteocytes and osteoblasts and the means whereby these cells then modulate bone remodeling have not been clearly identified. Influences that have been implicated are shearing forces produced by fluid movement (e.g. in the canaliculi surrounding the osteocytic dendrites) and a variety of membrane proteins, including integrins, connexins and stretch-sensitive ion channels [92,93]. It also has been proposed that the osteocyte senses load through cilia, single flagellar-like structures found on every cell [223]. When Pkd1, the gene encoding the large transmembrane protein polycystin-1 (PC-1) located at the primary cilia, was conditionally deleted in osteocytes using DMP-1-Cre mice, the bone anabolic response to loading was recently found to be dramatically decreased [224]. Osteocytes respond in vitro and in vivo to increased load by altering their signaling. For example, in response to loading, osteocytes upregulate nitric oxide production, release prostaglandin PGE-2 and IGF-1, and decrease glutamate transporter expression [92,93]. DMP-1 expression is also robustly increased upon mechanical stimulation [225]. DMP-1 inactivation in mice is associated with a hypomineralized phenotype linked with elevated FGF-23 expression and defective osteocyte lacuna/canalicular network formation [226]. Of great interest is the recent observation that mechanical loading suppresses the expression of SOST in osteocytes, resulting in a rapid decrease of sclerostin production [215]. As mentioned (see above), the osteocyte-specific protein sclerostin inhibits Wnt signaling

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through binding to LRP5/6. Consequently, SOST null mice have a very high bone mass [227] whereas, conversely, transgenic mice overexpressing sclerostin in osteocytes suffer severe bone loss [228]. Osteocytes thus appear to use the Wnt/beta-catenin pathway to transmit signals of mechanical loading to cells on the bone surface [92,106].

ENDOCRINE CONTROL OF POSTNATAL BONE GROWTH Several systemic factors are known to profoundly influence postnatal bone growth as well as bone remodeling. These include the principal regulators of mineral homeostasis, such as PTH and 1,25(OH)2 vitamin D that maintain calcium homeostasis by regulating osteoclastic resorption, the actions of which are discussed in detail in other chapters of this book. Here, we will briefly summarize the influences on bone growth and remodeling by the growth hormone /insulin-like growth factor-1 axis, sex steroids, thyroid hormones, and glucocorticoids.

Growth Hormone and Insulin-like Growth Factor-1 Growth hormone (GH) and insulin-like growth factor-1 (IGF-1) are central to the achievement of normal longitudinal bone growth and the acquisition of bone mass during the prepubertal period, and remain important regulators of bone homeostasis throughout life. Although GH may act directly on skeletal cells, most of its effects are mediated by IGF-1, which is present in the systemic circulation and is synthesized by peripheral tissues. Both systemic and local skeletal IGF-1 play a role in bone formation and the maintenance of bone mass [229,230]. GH is a single-chain peptide of 191 amino acids, synthesized and released from the anterior pituitary gland. Its receptor is highly expressed in the liver, adipose tissue, heart, kidneys, intestine, lung, pancreas, cartilage and skeletal muscle where it induces the synthesis of IGF-1, a member of the insulin family of growth factors [229,230]. Systemic IGF-1 is synthesized primarily in the liver and circulates bound by IGFbinding proteins (IGFBPs) that regulate its availability. The triple inactivation of IGFBP-3, -4 and -5 demonstrated that IGFBPs are necessary to maintain appropriate levels of systemic IGF-1 and adequate postnatal growth [231]. IGF-2 shares biochemical and biological properties with IGF-1; it is synthesized by skeletal cells, independent of GH, and is important in skeletal development, but its function in the adult skeleton is not proven [229,230].

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During embryonic development, IGF-1 and IGF-2 are key determinants of growth. IGF-1 deficiency retards both pre- and postnatal growth, and IGF-1 receptor (IGF-1R) null mice, exhibiting a more severe growth deficiency, die shortly after birth [232e235]. In this context, prenatally, IGF-1 signaling is considered to be GH independent, whereas postnatally, IGF-1 is partly or fully GH dependent with phosphorylation of STAT5b having an intermediary role [236]. Knockout of the IGF-2 gene leads to decreased fetal growth in analogous fashion to knockout of the IGF-1 gene [237]. Because of the additional action of IGF-2 on the insulin receptor, knockout of IGF-2 and IGF-1R results in a more severe growth abnormality than knockout of IGF-1R alone [238]. Since IGF-2 acts predominantly in fetal life and GH acts on growth postnatally, there are no expected interactions between IGF-2 and GH in mice. Postnatally and throughout puberty, GH and IGF-1 play a critical role in determining longitudinal skeletal growth. Mice missing both the GH receptor and IGF-1 are only 17% of normal size [235]. These dramatic quantitative effects show that GH and IGF-1 importantly control bone growth in mammals. 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 (GHD) who display short stature. Patients and mice with mutations in the GH receptor gene (Laron dwarfism) have a similar defect in bone growth [235,239,240]. 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 postnatal growth. In mice, the defect is detected days 10e40 after birth [235]. These mice have growth plates with short proliferative columns with fewer chondrocytes than normal, a slower rate of proliferation, and smaller hypertrophic chondrocytes than normal [235]. Thus, these mice display defects both in the proliferation and in the differentiation of chondrocytes. Clear demonstration of the physiologic importance of IGF-1 in bone growth awaited gene ablation studies [233,234,241]; subsequently, one human with IGF-1 gene mutation and a phenotype analogous to that of the knockout mice was reported [232]. 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 [235,242]. Wang et al. [242] found that proliferative columns are of normal size and that the chondrocytes proliferate at a normal rate, whereas Lupu et al. [235] found that the proliferative columns

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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 on chondrocytes 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 that the actions of GH and IGF-1 on growth are predominantly independent and additive [235]. Moreover, the traditional concept that GH primarily acts through stimulation of the IGF-1 production in the liver and increased circulating IGF-1 levels, was contested by mouse genetic studies. Indeed, when the IGF-1 gene was ablated specifically from liver in vivo using the Cre-Lox approach, circulating levels of IGF-1 decreased to approximately 20% of baseline, but the animals grew normally [243,244]. Although these studies demonstrate that a large fraction of circulating IGF-1 derives from the liver, they also suggest that IGF-1 made by the liver may not be vitally important for bone growth. The actions of IGF-1, whether produced locally or systemically, are via the IGF-1 receptor (IGF-1R) expressed on the cell surface of the chondrocytes of the growth plate. The type-2 IGF-1R is expressed equally throughout all maturational zones of the growth plate, whereas the type-1 receptor is more highly expressed by proliferating chondrocytes [245]. These data are consistent with the concept that IGF-1 has regulatory actions on all chondrocytes of the growth plate. Moreover, recently generated cartilage-specific IGF-1R knockout mice, constitutive as well as tamoxifeninducible models, indicate that the IGF-1R in chondrocytes controls cell growth, survival, and differentiation in embryonic and postnatal growth plates in part by suppression of PTHrP expression [246]. Thus, although the functional relationships between GH and IGF-1 actions on the growth plate remain unsettled, it is clear that the IGF-1 signaling pathway has a central function in modulating endochondral bone growth and regulates a number of key chondrocyte physiological processes such as chondrocyte proliferation, matrix synthesis, differentiation, hypertrophy and survival [229,230,235,246]. In addition to the effects on longitudinal growth, GH and IGF-1 are anabolic hormones and have the potential to regulate bone modeling and remodeling. During adolescence, the anabolic effects of GH and IGF-1 in

bone are important for the acquisition of bone mass. Late adolescence and early adulthood are critical periods for the acquisition of bone mass, and the achievement of peak bone mass. This is a critical determinant of future risk of osteoporosis. Adult GHD causes low bone turnover osteoporosis with high risk of vertebral and non-vertebral fractures, and the low bone mass can be partially reversed by GH replacement [230]. IGF1 mediates most of the effects of GH on skeletal metabolism. IGF-1 may reduce osteoblast apoptosis and promote osteoblastogenesis by stabilizing b-catenin, enhancing Wnt-dependent activity. This effect, associated with modest mitogenic properties, causes an increase in the number of osteoblasts, and an increase in osteoblastic function and bone formation [247,248]. Diseases affecting the GH/IGF-1 axis are frequently associated with significant alterations in bone metabolism that often lead to bone loss [229,230].

Sex Steroids: 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 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 a (ERa) [249] or defective aromatase (the enzyme that converts testosterone to estradiol) [250,251] have been identified. These patients continued to grow into adulthood owing to a lack of epiphyseal fusion in the long bones, which resulted in increased adult height. Two girls without aromatase presented with signs of androgen excess but lack of a clear pubertal growth spurt until they were treated with estrogen [252]. While estrogen therapy resulted in rapid growth plate closure in patients with aromatase deficiency, it did not in the man with a mutation in the ERa gene [249e251]. In rodents, the growth plates do not fuse directly after sexual maturation, but high-dose estradiol treatment results in a clear reduction of the growth plate height [253]. A mouse model with cartilage-specific inactivation of ERa, the main ER regulating skeletal growth, was recently generated. During sexual maturation, the skeletal growth of these mice was normal, but they continued to grow after 4 months of age, resulting in increased bone length at the age of 1 year. High-dose estradiol treatment of adult mice reduced the growth plate height as a consequence of attenuated proliferation of growth plate chondrocytes in control mice but not in cartilage-specific ERa knockout mice [254]. Sex steroids also have a pronounced influence on the process of bone modeling, remodeling and homeostasis,

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essential for maintenance of the adult skeleton. The precise time of the attainment of peak bone mass is not certain, and it is skeletal-site dependent. The increase in gonadal steroid synthesis at the time of puberty is an important hormonal regulator of bone accretion. Boys with constitutionally delayed puberty achieve lower peak bone mass than normal boys [255]. Androgens are important not only as a source of estrogen, through the action of aromatase, but also for their direct effect in stimulating bone formation [256]. Testosterone, responsible for the male phenotype characterized by a larger skeleton, has complex effects on bone metabolism. As an anabolic steroid, it stimulates bone formation in both male and females. In addition, testosterone can inhibit bone resorption directly, acting through the androgen receptor as well as through conversion by aromatase to estrogens (see below). Androgens also increase periosteal bone formation, leading to larger and therefore stronger bones. Loss of androgens in males from chemical or surgical castration or an ageassociated decline of androgen levels has the same adverse effect on the skeleton as estrogen loss in women, albeit the loss of testosterone in aging men is not as universal or as abrupt as the loss of estrogen at the time of menopause in women [256,257]. Indeed, particularly postmenopausal women suffer from osteoporosis, characterized by low bone mass and high risk of debilitating fractures of the vertebrae and long bones. This disease afflicts millions of people, and becomes increasingly prevalent with the aging of the general population. Skeletal preservation by estrogen in females may be evolutionarily related to the need of calcium stores for embryonic skeletal development. In mammalian adult males and females, including humans, estrogen has been identified as the major inhibitor of bone resorption by reducing osteoclast number [257,258]. Estrogen deficiency increases both the number of sites at which remodeling is initiated, and the extent of resorption at a given site. Increased bone resorption is accompanied by increased bone formation as a result of coupling. However, the increase in bone formation is not sufficient to maintain bone balance and prevent bone loss. The reason for this “skeletal insufficiency” in osteoporosis is not known; it could be due to lack of estrogen or other hormones, such as androgens, required for fully effective bone formation, and is partly due to the above-mentioned kinetic imbalance. In cancellous bone, the loss results in thinner trabeculae, which become rod-shaped rather than plates, and in trabecular discontinuity, which deprives them of mechanical function. In cortical bone, endosteal bone resorption causes thinning of the cortex and sometimes “trabecularization” of the endosteal surface. Enhanced resorption increases the size of haversian canals and the porosity of the cortical bone. The result of these changes is significant

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weakening of the respective bones and increased fracture risk. Treatment of women with hormone replacement therapy (either estrogen alone or estrogen plus progesterone) has been shown to prevent this bone loss [259]. Recent studies have shed light on the mechanism of estrogen action on osteoclasts: osteoclasts express estrogen receptors (ERa and ERb) and estrogen acts directly on osteoclasts to increase apoptosis [260,261]. The regulation of osteoclast life span by estrogen involves induction of the Fas/FasL system causing apoptosis, a pathway that may be involved both in osteoclast cell-autonomous effects [261], as well as in indirect mechanisms mediated via the osteoblast [262]. Furthermore, estrogen can have non-genomic effects, or rapid signaling effects, inducing the phosphorylation of components of various signaling pathways (e.g. the MAPK pathway) or calcium regulation. Such effects may contribute to the induction of osteoclast apoptosis by estrogen without the need for direct binding of ERa to DNA [262,263]. The DNA-binding function of ERa was recently shown to be dispensable also for the effects of estrogen on osteoblastogenesis and on decreasing the prevalence of mature osteoblast apoptosis [264]. In fact, Manolagas and co-workers propose that the protective effects of estrogens on bone result from their ability to attenuate oxidative stress in bone and bone marrow; estrogens diminish the generation of reactive oxygen species (ROS), stimulate the activity of glutathione reductase, and decrease the phosphorylation of p66shc, an adapter protein that serves as a key component of a signaling cascade that is activated by ROS and influences apoptosis and lifespan in invertebrates and mammals. Hence, loss of estrogens may accelerate the effects of aging on bone by decreasing the defense against oxidative stress [264,265]. A variety of other mechanisms contribute to the beneficial effects of estrogen on bone. Estrogen was reported to decrease the responsiveness of osteoclast progenitor cells to RANKL, thereby reducing osteoclastogenesis [266]. Estrogen also regulates a variety of cytokines, indirectly leading to changes in osteoclast number: estrogen suppresses the production of osteoclastogenic cytokines such as IL-1, IL-6, IL-7 and TNF-a in T cells and osteoblasts [267,268]. As well, part of the effects ascribed to estrogen deficiency were suggested to be in fact mediated by the resultant increase in pituitary gland derived follicle stimulating hormone (FSH), acting on osteoclasts [269].

Thyroid Hormones The hypothalamicepituitaryethyroid axis plays a key role in skeletal development, acquisition of peak bone mass and regulation of adult bone turnover. Euthyroid status is essential for maintenance of optimal bone mineralization and strength. In population studies,

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hypothyroidism and hyperthyroidism have both been associated with an increased risk of fracture. In children, thyrotoxicosis advances bone age and accelerates postnatal skeletal development resulting in early cessation of growth and short stature due to premature fusion of the growth plates. Conversely, children and rodents with hypothyroidism have decreased rates of bone lengthening [270]. 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 [271]. At least some of the effects of thyroid hormone are likely to be direct effects on chondrocytes that express thyroid hormone receptors [272] because both growth plates and isolated chondrocytes respond to T3 in vitro by increasing the conversion of proliferating to hypertrophic chondrocytes [273,274]. Genetically engineered mice missing all transcripts from both the thyroid hormone receptor a 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 [275]. This phenotype is not as severe as that in mice with congenital absence of thyroid glands, or as in mice missing the pax8 gene encoding an essential transcription factor required for thyroid follicular cell development [276]. These comparisons, as well as comparisons with mice missing selected transcripts from the complicated thyroid hormone receptor loci, suggest that non-ligand-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 [270,275,277,278]. 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 [279].

Glucocorticoids In contrast to sex steroids, glucocorticoid excess is catabolic to bone, as illustrated by glucocorticoidinduced osteoporosis, a devastating consequence of long-term use of glucocorticoids [280]. The mechanisms for glucocorticoid-induced bone loss are complex, including suppression of renal calcium reabsorption, reduction in intestinal calcium absorption, and hypogonadism, all of which lead to increased bone resorption and bone turnover. Perhaps more importantly, glucocorticoids also have direct effects on the skeleton [280]. Glucocorticoids impair the replication, differentiation

and function of osteoblasts and induce the apoptosis of mature osteoblasts and osteocytes, leading to a dramatic suppression of bone formation [281,282]. As well, glucocorticoids act directly on osteoclasts and prolong their life span; in addition, glucocorticoids may sensitize bone cells to regulators of bone remodeling and favor osteoclastogenesis, thus overall leading to increased bone resorption [283,284]. Glucocorticoids are widely used as anti-inflammatory and immunosuppressive drugs in children. Long-term, high-dose glucocorticoid treatment often leads to growth failure. Similarly, systemic administration of glucocorticoid in mice, rats, and rabbits decreases the rate of longitudinal bone growth, likely by inhibiting growth plate chondrocyte proliferation [285,286] and by stimulating chondrocyte apoptosis [287]. Glucocorticoid inhibits longitudinal bone growth, in part, through a direct effect on growth plate chondrocytes that exress glucocorticoid receptor [286]. In addition to its direct action on the growth plate, glucocorticoid may also suppress longitudinal bone growth through an indirect action, mediated, in part, by changes in the GH/IGF-1 axis [285,288]. Discontinuation of glucocorticoid treatment is followed by catch-up growth. Catch-up growth may occur because the decreased cell proliferation during glucocorticoid treatment conserves the proliferative capacity of the chondrocytes, thus slowing growth plate senescence. Following discontinuation of the glucocorticoid treatment, the growth plates are less senescent than normal, and hence show a greater growth rate and grow for a longer time than expected for age, resulting in catch-up growth [285]. Despite catch-up growth, prolonged glucocorticoid administration in children can result in some residual permanent growth deficit.

CONCLUDING REMARKS As outlined above, postnatal bone growth involves a complex spatiotemporal coordination of the proliferation, differentiation, and activity of multiple cell types building the multicomponent skeletal tissue. A myriad of transcription factors, local signaling molecules, endocrine, mechanical and central signals have been implicated in the regulation of bone growth and remodeling. Precise control is mandatory to build and maintain a skeleton that is fully able to fulfill its major functions, including mechanical support and mineral homeostasis. Moreover, a number of recent breakthroughs have shown that bone cells are not merely involved in the acquisition and maintenance of the bone mass, but also execute a number of critical functions that extend beyond the bone tissue proper. Among these are roles of osteoblasts and osteoclasts in the

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REFERENCES

interplay between skeletal functioning and the regulation of immunology and inflammation [289,290], hematopoiesis and stem cell biology [291e294], the central and sympathetic nervous system [295e297] and energy metabolism [296,298e300]. Further advances in the molecular and genetic understanding of skeletal biology, including these novel integrative communication pathways with other tissues and organ systems, may offer new insights as well as potential therapeutic options to treat various pediatric bone diseases presented further in this book.

References [1] Tabin C, Wolpert L. Rethinking the proximodistal axis of the vertebrate limb in the molecular era. Genes Dev 2007;21: 1433e42. [2] Bi W, Deng JM, Zhang Z, Behringer RR, de Crombrugghe B. Sox9 is required for cartilage formation. Nat Genet 1999;22: 85e9. [3] Kronenberg HM. The role of the perichondrium in fetal bone development. Ann NY Acad Sci 2007;1116:59e64. [4] Maes C, Kobayashi T, Selig MK, et al. Osteoblast precursors, but not mature osteoblasts, move into developing and fractured bones along with invading blood vessels. Dev Cell 2010;19: 329e44. [5] Kronenberg HM. Developmental regulation of the growth plate. Nature 2003;423:332e6. [6] Ducy P, Zhang R, Geoffroy V, Ridall A, Karsenty G. Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 1997;89:747e54. [7] Komori T, Yagi H, Nomura S, et al. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 1997;89:755e64. [8] Trueta J, Morgan JD. The vascular contribution to osteogenesis. I. Studies by the injection method. J Bone Joint Surg Br 1960;42-B:97e109. [9] Brashear Jr HR. Epiphyseal avascular necrosis and its relation to longitudinal bone growth. J Bone Joint Surg Am 1963;45: 1423e38. [10] Gerber HP, Vu TH, Ryan AM, Kowalski J, Werb Z, Ferrara N. VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation. Nat Med 1999;5:623e8. [11] Opperman LA. Cranial sutures as intramembranous bone growth sites. Dev Dyn 2000;219:472e85. [12] Hall BK, Miyake T. All for one and one for all: condensations and the initiation of skeletal development. Bioessays 2000;22: 138e47. [13] Morriss-Kay GM, Wilkie AO. Growth of the normal skull vault and its alteration in craniosynostosis: insights from human genetics and experimental studies. J Anat 2005;207:637e53. [14] Wilkie AO, Morriss-Kay GM. Genetics of craniofacial development and malformation. Nat Rev Genet 2001;2:458e68. [15] Helms JA, Cordero D, Tapadia MD. New insights into craniofacial morphogenesis. Development 2005;132:851e61. [16] Akiyama H, Chaboissier MC, Martin JF, Schedl A, de Crombrugghe B. The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6. Genes Dev 2002;16:2813e28.

75

[17] Smits P, Li P, Mandel J, et al. The transcription factors L-Sox5 and Sox6 are essential for cartilage formation. Dev Cell 2001;1: 277e90. [18] Karaplis AC, He B, Nguyen MT, et al. Inactivating mutation in the human parathyroid hormone receptor type 1 gene in Blomstrand chondrodysplasia. Endocrinology 1998;139:5255e8. [19] Schipani E, Kruse K, Juppner H. A constitutively active mutant PTH-PTHrP receptor in Jansen-type metaphyseal chondrodysplasia. Science 1995;268:98e100. [20] Bellus GA, McIntosh I, Smith EA, et al. A recurrent mutation in the tyrosine kinase domain of fibroblast growth factor receptor 3 causes hypochondroplasia. Nat Genet 1995;10:357e9. [21] Naski MC, Wang Q, Xu J, Ornitz DM. Graded activation of fibroblast growth factor receptor 3 by mutations causing achondroplasia and thanatophoric dysplasia. Nat Genet 1996;13: 233e7. [22] Shiang R, Thompson LM, Zhu YZ, et al. Mutations in the transmembrane domain of FGFR3 cause the most common genetic form of dwarfism, achondroplasia. Cell 1994;78:335e42. [23] Vajo Z, Francomano CA, Wilkin DJ. The molecular and genetic basis of fibroblast growth factor receptor 3 disorders: the achondroplasia family of skeletal dysplasias, Muenke craniosynostosis, and Crouzon syndrome with acanthosis nigricans. Endocr Rev 2000;21:23e39. [24] Kronenberg HM. PTHrP and skeletal development. Ann NY Acad Sci 2006;1068:1e13. [25] Lanske B, Karaplis AC, Lee K, et al. PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth. Science 1996;273:663e6. [26] Vortkamp A, Lee K, Lanske B, Segre GV, Kronenberg HM, Tabin CJ. Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 1996;273:613e22. [27] Chung UI, Schipani E, McMahon AP, Kronenberg HM. Indian hedgehog couples chondrogenesis to osteogenesis in endochondral bone development. J Clin Invest 2001;107:295e304. [28] Karp SJ, Schipani E, St Jacques B, Hunzelman J, Kronenberg H, McMahon AP. Indian hedgehog coordinates endochondral bone growth and morphogenesis via parathyroid hormone relatedprotein-dependent and -independent pathways. Development 2000;127:543e8. [29] Kobayashi T, Soegiarto DW, Yang Y, et al. Indian hedgehog stimulates periarticular chondrocyte differentiation to regulate growth plate length independently of PTHrP. J Clin Invest 2005;115:1734e42. [30] St Jacques B, Hammerschmidt M, McMahon AP. Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev 1999;13:2072e86. [31] Kim IS, Otto F, Zabel B, Mundlos S. Regulation of chondrocyte differentiation by Cbfa1. Mech Dev 1999;80:159e70. [32] Maeda Y, Nakamura E, Nguyen MT, et al. Indian Hedgehog produced by postnatal chondrocytes is essential for maintaining a growth plate and trabecular bone. Proc Natl Acad Sci USA 2007;104:6382e7. [33] Ornitz D. FGF Signaling in the developing endochondral skeleton. Cytokine Growth Factor Rev 2005;16:205e13. [34] Miraoui H, Marie PJ. Fibroblast growth factor receptor signaling crosstalk in skeletogenesis. Sci Signal 2010;3:re9. [35] Colvin JS, Bohne BA, Harding GW, McEwen DG, Ornitz DM. Skeletal overgrowth and deafness in mice lacking fibroblast growth factor receptor 3. Nat Genet 1996;12:390e7. [36] Deng C, Wynshaw-Boris A, Zhou F, Kuo A, Leder P. Fibroblast growth factor receptor 3 is a negative regulator of bone growth. Cell 1996;84:911e21.

PEDIATRIC BONE

76

4. POSTNATAL BONE GROWTH: GROWTH PLATE BIOLOGY, BONE FORMATION, AND REMODELING

[37] Legeai-Mallet L, Benoist-Lasselin C, Munnich A, Bonaventure J. Overexpression of FGFR3, Stat1, Stat5 and p21Cip1 correlates with phenotypic severity and defective chondrocyte differentiation in FGFR3-related chondrodysplasias. Bone 2004;34:26e36. [38] Sahni M, Ambrosetti DC, Mansukhani A, Gertner R, Levy D, Basilico C. FGF signaling inhibits chondrocyte proliferation and regulates bone development through the STAT-1 pathway. Genes Dev 1999;13:1361e6. [39] Su WC, Kitagawa M, Xue N, et al. Activation of Stat1 by mutant fibroblast growth-factor receptor in thanatophoric dysplasia type II dwarfism. Nature 1997;386:288e92. [40] Chen L, Li C, Qiao W, Xu X, Deng C. A Ser(365) Cys mutation of fibroblast growth factor receptor 3 in mouse downregulates Ihh/PTHrP signals and causes severe achondroplasia. Hum Mol Genet 2001;10:457e65. [41] Li C, Chen L, Iwata T, Kitagawa M, Fu XY, Deng CX. A Lys644Glu substitution in fibroblast growth factor receptor 3 (FGFR3) causes dwarfism in mice by activation of STATs and ink4 cell cycle inhibitors. Hum Mol Genet 1999;8:35e44. [42] Naski MC, Colvin JS, Coffin JD, Ornitz DM. Repression of hedgehog signaling and BMP4 expression in growth plate cartilage by fibroblast growth factor receptor 3. Development 1998;125:4977e88. [43] Iwata T, Chen L, Li C, et al. A neonatal lethal mutation in FGFR3 uncouples proliferation and differentiation of growth plate chondrocytes in embryos. Hum Mol Genet 2000;9:1603e13. [44] Segev O, Chumakov I, Nevo Z, et al. Restrained chondrocyte proliferation and maturation with abnormal growth plate vascularization and ossification in human FGFR-3(G380R) transgenic mice. Hum Mol Genet 2000;9:249e58. [45] Yoon BS, Pogue R, Ovchinnikov DA, et al. BMPs regulate multiple aspects of growth-plate chondrogenesis through opposing actions on FGF pathways. Development 2006;133: 4667e78. [46] Liu Z, Xu J, Colvin JS, Ornitz DM. Coordination of chondrogenesis and osteogenesis by fibroblast growth factor 18. Genes Dev 2002;16:859e69. [47] Liu Z, Lavine K, Hung I, Ornitz D. FGF18 is required for early chondrocyte proliferation, hypertrophy and vascular invasion of the growth plate. Dev Biol 2007;302:80e91. [48] Ohbayashi N, Shibayama M, Kurotaki Y, et al. FGF18 is required for normal cell proliferation and differentiation during osteogenesis and chondrogenesis. Genes Dev 2002;16:870e9. [49] Hung IH, Yu K, Lavine KJ, Ornitz DM. FGF9 regulates early hypertrophic chondrocyte differentiation and skeletal vascularization in the developing stylopod. Dev Biol 2007;307:300e13. [50] Colnot C, Lu C, Hu D, Helms JA. Distinguishing the contributions of the perichondrium, cartilage, and vascular endothelium to skeletal development. Dev Biol 2004;269:55e69. [51] Schipani E, Maes C, Carmeliet G, Semenza GL. Regulation of osteogenesis-angiogenesis coupling by HIFs and VEGF. J Bone Miner Res 2009;24:1347e53. [52] Wan C, Shao J, Gilbert SR, et al. Role of HIF-1alpha in skeletal development. Ann NY Acad Sci 2010;1192:322e6. [53] Karsenty G. The complexities of skeletal biology. Nature 2003;423:316e8. [54] Brandi ML, Collin-Osdoby P. Vascular biology and the skeleton. J Bone Miner Res 2006;21:183e92. [55] Hiraki Y, Shukunami C. Angiogenesis inhibitors localized in hypovascular mesenchymal tissues: chondromodulin-I and tenomodulin. Connect Tissue Res 2005;46:3e11. [56] Moses MA, Wiederschain D, Wu I, et al. Troponin I is present in human cartilage and inhibits angiogenesis. Proc Natl Acad Sci USA 1999;96:2645e50.

[57] Araldi E, Schipani E. Hypoxia, HIFs and bone development. Bone 2010;47:190e6. [58] Schipani E, Ryan HE, Didrickson S, Kobayashi T, Knight M, Johnson RS. Hypoxia in cartilage: HIF-1alpha is essential for chondrocyte growth arrest and survival. Genes Dev 2001;15: 2865e76. [59] Semenza GL. Regulation of cancer cell metabolism by hypoxiainducible factor 1. Semin Cancer Biol 2009;19:12e6. [60] Ivan M, Kondo K, Yang H, et al. HIFalpha targeted for VHLmediated destruction by proline hydroxylation: implications for O2 sensing. Science 2001;292:464e8. [61] Jaakkola P, Mole D, Tian Y, et al. Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 2001;292:468e72. [62] Simon MC, Keith B. The role of oxygen availability in embryonic development and stem cell function. Nat Rev 2008;9:285e96. [63] Amarilio R, Viukov S, Sharir A, Eshkar-Oren I, Johnson R, Zelzer E. Hif1alpha regulation of Sox9 is necessary to maintain differentiation of hypoxic prechondrogenic cells during early chondrogenesis. Development 2007;134:3917e28. [64] Provot S, Zinyk D, Gunes Y, et al. Hif-1alpha regulates differentiation of limb bud mesenchyme and joint development. J Cell Biol 2007;177:451e64. [65] Maes C, Stockmans I, Moermans K, et al. Soluble VEGF isoforms are essential for establishing epiphyseal vascularization and regulating chondrocyte development and survival. J Clin Invest 2004;113:188e99. [66] Seagroves T, Ryan H, Lu H, et al. Transcription factor HIF-1 is necessary mediator of the pasteur effect in mammalian cells. Mol Cell Biol 2001;21:3436e44. [67] Ferrara N. Vascular endothelial growth factor: basic science and clinical progress. Endocr Rev 2004;25:581e611. [68] Zelzer E, Mamluk R, Ferrara N, Johnson R, Schipani E, Olsen B. VEGFA is necessary for chondrocyte survival during bone development. Development 2004;131:2161e71. [69] Cramer T, Schipani E, Johnson RS, Swoboda B, Pfander D. Expression of VEGF isoforms by epiphyseal chondrocytes during low-oxygen tension is HIF-1 alpha dependent. Osteoarthrit Cart 2004;12:433e9. [70] Lin C, McGough R, Aswad B, Block JA, Terek R. Hypoxia induces HIF-1alpha and VEGF expression in chondrosarcoma cells and chondrocytes. J Orthop Res 2004;22:1175e81. [71] Maes C, Carmeliet G. Vascular and nonvascular roles of VEGF in bone development. In: Ruhrberg C, editor. VEGF in Development. Austin: Springer; 2008. p. 79e90. [72] Maes C, Carmeliet P, Moermans K, et al. Impaired angiogenesis and endochondral bone formation in mice lacking the vascular endothelial growth factor isoforms VEGF164 and VEGF188. Mech Dev 2002;111:61e73. [73] Zelzer E, McLean W, Ng YS, et al. Skeletal defects in VEGF(120/ 120) mice reveal multiple roles for VEGF in skeletogenesis. Development 2002;129:1893e904. [74] Haigh J, Gerber H, Ferrara N, Wagner E. Conditional inactivation of VEGF-A in areas of collagen2a1 expression results in embryonic lethality in the heterozygous state. Development 2000;127:1445e53. [75] Eshkar-Oren I, Viukov SV, Salameh S, et al. The forming limb skeleton serves as a signaling center for limb vasculature patterning via regulation of Vegf. Development 2009;136: 1263e72. [76] Dai J, Rabie AB. VEGF: an essential mediator of both angiogenesis and endochondral ossification. J Dent Res 2007;86: 937e50.

PEDIATRIC BONE

77

REFERENCES

[77] Zelzer E, Olsen B. Multiple roles of vascular endothelial growth factor (VEGF) in skeletal development, growth and repair. Curr Top Dev Biol 2005;65:169e87. [78] Karsenty G, Wagner EF. Reaching a genetic and molecular understanding of skeletal development. Dev Cell 2002;2: 389e406. [79] Jacobsen KA, Al Aql ZS, Wan C, et al. Bone formation during distraction osteogenesis is dependent on both VEGFR1 and VEGFR2 signaling. J Bone Miner Res 2008;23:596e609. [80] Street J, Bao M, deGuzman L, et al. Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover. Proc Natl Acad Sci USA 2002;99:9656e61. [81] Carano RA, Filvaroff EH. Angiogenesis and bone repair. Drug Discov Today 2003;8:980e9. [82] Maes C, Goossens S, Bartunkova S, et al. Increased skeletal VEGF enhances beta-catenin activity and results in excessively ossified bones. EMBO J 2010;29:424e41. [83] Kim HH, Lee SE, Chung WJ, et al. Stabilization of hypoxiainducible factor-1alpha is involved in the hypoxic stimuliinduced expression of vascular endothelial growth factor in osteoblastic cells. Cytokine 2002;17:14e27. [84] Knowles HJ, Athanasou NA. Hypoxia-inducible factor is expressed in giant cell tumour of bone and mediates paracrine effects of hypoxia on monocyte-osteoclast differentiation via induction of VEGF. J Pathol 2008;215:56e66. [85] Wang Y, Wan C, Deng L, et al. The hypoxia-inducible factor alpha pathway couples angiogenesis to osteogenesis during skeletal development. J Clin Invest 2007;117:1616e26. [86] Zelzer E, Glotzer D, Hartmann C, et al. Tissue specific regulation of VEGF expression during bone development requires Cbfa1/Runx2. Mech Dev 2001;106:97e106. [87] Chen Y, Shao JZ, Xiang LX, Dong XJ, Zhang GR. Mesenchymal stem cells: a promising candidate in regenerative medicine. Int J Biochem Cell Biol 2008;40:815e20. [88] Nishimura R, Hata K, Ikeda F, et al. Signal transduction and transcriptional regulation during mesenchymal cell differentiation. J Bone Miner Metab 2008;26:203e12. [89] Mendez-Ferrer S, Michurina TV, Ferraro F, et al. Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature 2010;466:829e34. [90] Harada S, Rodan GA. Control of osteoblast function and regulation of bone mass. Nature 2003;423:349e55. [91] Karsenty G. Transcriptional control of skeletogenesis. Annu Rev Genomics Hum Genet 2008;9:183e96. [92] Bonewald LF. The amazing osteocyte. J Bone Miner Res 2011;26: 229e38. [93] Noble BS. The osteocyte lineage. Arch Biochem Biophys 2008;473:106e11. [94] Clevers H. Wnt/beta-catenin signaling in development and disease. Cell 2006;127:469e80. [95] Klaus A, Birchmeier W. Wnt signalling and its impact on development and cancer. Nat Rev Cancer 2008;8:387e98. [96] Baron R, Rawadi G. Targeting the Wnt/beta-catenin pathway to regulate bone formation in the adult skeleton. Endocrinology 2007;148:2635e43. [97] Hoeppner LH, Secreto FJ, Westendorf JJ. Wnt signaling as a therapeutic target for bone diseases. Expert Opin Ther Targets 2009;13:485e96. [98] Kubota T, Michigami T, Ozono K. Wnt signaling in bone metabolism. J Bone Miner Metab 2009;27:265e71. [99] Jin T, George FI, Sun J. Wnt and beyond Wnt: multiple mechanisms control the transcriptional property of beta-catenin. Cell Signal 2008;20:1697e704. [100] Grigoryan T, Wend P, Klaus A, Birchmeier W. Deciphering the function of canonical Wnt signals in development and disease:

[101]

[102]

[103]

[104]

[105]

[106]

[107] [108]

[109]

[110]

[111]

[112]

[113]

[114]

[115]

[116]

[117]

[118]

[119]

[120]

conditional loss- and gain-of-function mutations of beta-catenin in mice. Genes Dev 2008;22:2308e41. Day T, Guo X, Garrett-Beal L, Yang Y. Wnt/beta-catenin signaling in mesenchymal progenitors controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis. Dev Cell 2005;8:739e50. Hill T, Spater D, Taketo M, Birchmeier W, Hartmann C. Canonical wnt/betacatenin signaling prevents osteoblasts from differentiating into chondrocytes. Dev Cell 2005;8:727e38. Hu H, Hilton MJ, Tu X, Yu K, Ornitz DM, Long F. Sequential roles of Hedgehog and Wnt signaling in osteoblast development. Development 2005;132:49e60. Rodda SJ, McMahon AP. Distinct roles for Hedgehog and canonical Wnt signaling in specification, differentiation and maintenance of osteoblast progenitors. Development 2006;133: 3231e44. Glass DA, Bialek P, Ahn JD, et al. Canonical Wnt signaling in differentiated osteoblasts controls osteoclast differentiation. Dev Cell 2005;8:751e64. Kramer I, Halleux C, Keller H, et al. Osteocyte Wnt/beta-catenin signaling is required for normal bone homeostasis. Mol Cell Biol 2010;30:3071e85. Baron R, Rawadi G, Roman-Roman S. Wnt signaling: a key regulator of bone mass. Curr Top Dev Biol 2006;76:103e27. Gong Y, Slee RB, Fukai N, et al. LDL receptor-related protein 5 (LRP5) affects bone accrual and eye development. Cell 2001;107: 513e23. Kato M, Patel MS, Levasseur R, et al. Cbfa1-independent decrease in osteoblast proliferation, osteopenia, and persistent embryonic eye vascularization in mice deficient in Lrp5, a Wnt coreceptor. J Cell Biol 2002;157:303e14. Little RD, Carulli JP, Del Mastro RG, et al. A mutation in the LDL receptor-related protein 5 gene results in the autosomal dominant high-bone-mass trait. Am J Hum Genet 2002;70:11e9. Balemans W, Devogelaer JP, Cleiren E, Piters E, Caussin E, Van Hul W. Novel LRP5 missense mutation in a patient with a high bone mass phenotype results in decreased DKK1-mediated inhibition of Wnt signaling. J Bone Miner Res 2007;22:708e16. Boyden LM, Mao J, Belsky J, et al. High bone density due to a mutation in LDL-receptor-related protein 5. N Engl J Med 2002;346:1513e21. Semenov MV, He X. LRP5 mutations linked to high bone mass diseases cause reduced LRP5 binding and inhibition by SOST. J Biol Chem 2006;281:38276e84. Yadav VK, Ryu JH, Suda N, et al. Lrp5 controls bone formation by inhibiting serotonin synthesis in the duodenum. Cell 2008;135:825e37. Balemans W, Patel N, Ebeling M, et al. Identification of a 52 kb deletion downstream of the SOST gene in patients with van Buchem disease. J Med Genet 2002;39:91e7. Balemans W, Van Hul W. Identification of the disease-causing gene in sclerosteosis e discovery of a novel bone anabolic target? J Musculoskelet Neuronal Interact 2004;4:139e42. Otto F, Thornell AP, Crompton T, et al. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 1997;89:765e71. Lee B, Thirunavukkarasu K, Zhou L, et al. Missense mutations abolishing DNA binding of the osteoblast-specific transcription factor OSF2/CBFA1 in cleidocranial dysplasia. Nat Genet 1997;16:307e10. Mundlos S, Otto F, Mundlos C, et al. Mutations involving the transcription factor CBFA1 cause cleidocranial dysplasia. Cell 1997;89:773e9. Franceschi RT, Ge C, Xiao G, Roca H, Jiang D. Transcriptional regulation of osteoblasts. Ann NY Acad Sci 2007;1116:196e207.

PEDIATRIC BONE

78

4. POSTNATAL BONE GROWTH: GROWTH PLATE BIOLOGY, BONE FORMATION, AND REMODELING

[121] Komori T. Regulation of bone development and extracellular matrix protein genes by RUNX2. Cell Tissue Res 2010;339: 189e95. [122] Nakashima K, Zhou X, Kunkel G, et al. The novel zinc fingercontaining transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 2002;108:17e29. [123] Nishio Y, Dong Y, Paris M, O’Keefe RJ, Schwarz EM, Drissi H. Runx2-mediated regulation of the zinc finger Osterix/Sp7 gene. Gene 2006;372:62e70. [124] Koga T, Matsui Y, Asagiri M, et al. NFAT and Osterix cooperatively regulate bone formation. Nat Med 2005;11:880e5. [125] Kaback LA, Soung dY, Naik A, et al. Osterix/Sp7 regulates mesenchymal stem cell mediated endochondral ossification. J Cell Physiol 2008;214:173e82. [126] Tominaga H, Maeda S, Miyoshi H, Miyazono K, Komiya S, Imamura T. Expression of osterix inhibits bone morphogenetic protein-induced chondrogenic differentiation of mesenchymal progenitor cells. J Bone Miner Metab 2008. [127] Komori T. Regulation of osteoblast differentiation by transcription factors. J Cell Biochem 2006;99:1233e9. [128] Marie PJ. Transcription factors controlling osteoblastogenesis. Arch Biochem Biophys 2008;473:98e105. [129] Kirker-Head CA, Boudrieau RJ, Kraus KH. Use of bone morphogenetic proteins for augmentation of bone regeneration. J Am Vet Med Assoc 2007;231:1039e55. [130] Leboy PS. Regulating bone growth and development with bone morphogenetic proteins. Ann NY Acad Sci 2006;1068:14e8. [131] Chen D, Zhao M, Mundy GR. Bone morphogenetic proteins. Growth Factors 2004;22:233e41. [132] Shi Y, Massague J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 2003;113:685e700. [133] Hogan BL. Bone morphogenetic proteins: multifunctional regulators of vertebrate development. Genes Dev 1996;10: 1580e94. [134] Pizette S, Niswander L. BMPs are required at two steps of limb chondrogenesis: formation of prechondrogenic condensations and their differentiation into chondrocytes. Dev Biol 2000;219: 237e49. [135] Tsumaki N, Nakase T, Miyaji T, et al. Bone morphogenetic protein signals are required for cartilage formation and differently regulate joint development during skeletogenesis. J Bone Miner Res 2002;17:898e906. [136] Yoon BS, Ovchinnikov DA, Yoshii I, Mishina Y, Behringer RR, Lyons KM. Bmpr1a and Bmpr1b have overlapping functions and are essential for chondrogenesis in vivo. Proc Natl Acad Sci USA 2005;102:5062e7. [137] Tsuji K, Cox K, Gamer L, Graf D, Economides A, Rosen V. Conditional deletion of BMP7 from the limb skeleton does not affect bone formation or fracture repair. J Orthop Res 2010;28: 384e9. [138] Bandyopadhyay A, Tsuji K, Cox K, Harfe BD, Rosen V, Tabin CJ. Genetic analysis of the roles of BMP2, BMP4, and BMP7 in limb patterning and skeletogenesis. PLoSGenet 2006;2:e216. [139] Wu XB, Li Y, Schneider A, et al. Impaired osteoblastic differentiation, reduced bone formation, and severe osteoporosis in noggin-overexpressing mice. J Clin Invest 2003;112:924e34. [140] Gazzerro E, Pereira RC, Jorgetti V, Olson S, Economides AN, Canalis E. Skeletal overexpression of gremlin impairs bone formation and causes osteopenia. Endocrinology 2005;146: 655e65. [141] Kamiya N, Ye L, Kobayashi T, et al. Disruption of BMP signaling in osteoblasts through type IA receptor (BMPRIA) increases bone mass. J Bone Miner Res 2008;23:2007e17. [142] Katagiri T, Yamaguchi A, Komaki M, et al. Bone morphogenetic protein-2 converts the differentiation pathway of C2C12

[143]

[144]

[145]

[146]

[147]

[148]

[149]

[150]

[151]

[152]

[153]

[154]

[155]

[156]

[157]

[158]

[159]

myoblasts into the osteoblast lineage. J Cell Biol 1994;127: 1755e66. Ryoo HM, Lee MH, Kim YJ. Critical molecular switches involved in BMP-2-induced osteogenic differentiation of mesenchymal cells. Gene 2006;366:51e7. Celil AB, Campbell PG. BMP-2 and insulin-like growth factor-I mediate Osterix (Osx) expression in human mesenchymal stem cells via the MAPK and protein kinase D signaling pathways. J Biol Chem 2005;280:31353e9. Javed A, Bae JS, Afzal F, et al. Structural coupling of Smad and Runx2 for execution of the BMP2 osteogenic signal. J Biol Chem 2008;283:8412e22. Lee KS, Kim HJ, Li QL, et al. Runx2 is a common target of transforming growth factor beta1 and bone morphogenetic protein 2, and cooperation between Runx2 and Smad5 induces osteoblast-specific gene expression in the pluripotent mesenchymal precursor cell line C2C12. Mol Cell Biol 2000;20: 8783e92. Lee MH, Kwon TG, Park HS, Wozney JM, Ryoo HM. BMP-2induced Osterix expression is mediated by Dlx5 but is independent of Runx2. Biochem Biophys Res Commun 2003;309: 689e94. Govender S, Csimma C, Genant HK, et al. Recombinant human bone morphogenetic protein-2 for treatment of open tibial fractures: a prospective, controlled, randomized study of four hundred and fifty patients. J Bone Joint Surg Am 2002;84-A: 2123e34. Styrkarsdottir U, Cazier JB, Kong A, et al. Linkage of osteoporosis to chromosome 20p12 and association to BMP2. PLoSBiol 2003;1:E69. Long F, Chung UI, Ohba S, McMahon J, Kronenberg HM, McMahon AP. Ihh signaling is directly required for the osteoblast lineage in the endochondral skeleton. Development 2004;131:1309e18. Shimoyama A, Wada M, Ikeda F, et al. Ihh/Gli2 signaling promotes osteoblast differentiation by regulating Runx2 expression and function. Mol Biol Cell 2007;18:2411e8. Zhao M, Qiao M, Harris SE, Chen D, Oyajobi BO, Mundy GR. The zinc finger transcription factor Gli2 mediates bone morphogenetic protein 2 expression in osteoblasts in response to hedgehog signaling. Mol Cell Biol 2006;26:6197e208. Eswarakumar VP, Horowitz MC, Locklin R, Morriss-Kay GM, Lonai P. A gain-of-function mutation of Fgfr2c demonstrates the roles of this receptor variant in osteogenesis. Proc Natl Acad Sci USA 2004;101:12555e60. Jacob AL, Smith C, Partanen J, Ornitz DM. Fibroblast growth factor receptor 1 signaling in the osteo-chondrogenic cell lineage regulates sequential steps of osteoblast maturation. Dev Biol 2006;296:315e28. Yu K, Xu J, Liu Z, et al. Conditional inactivation of FGF receptor 2 reveals an essential role for FGF signaling in the regulation of osteoblast function and bone growth. Development 2003;130: 3063e74. Choi KY, Kim HJ, Lee MH, et al. Runx2 regulates FGF2-induced Bmp2 expression during cranial bone development. Dev Dyn 2005;233:115e21. Kim BG, Kim HJ, Park HJ, et al. Runx2 phosphorylation induced by fibroblast growth factor-2/protein kinase C pathways. Proteomics 2006;6:1166e74. Naganawa T, Xiao L, Coffin JD, et al. Reduced expression and function of bone morphogenetic protein-2 in bones of Fgf2 null mice. J Cell Biochem 2008;103:1975e88. Reinhold MI, Naski MC. Direct interactions of Runx2 and canonical Wnt signaling induce FGF18. J Biol Chem 2007;282: 3653e63.

PEDIATRIC BONE

REFERENCES

[160] Vu TH, Shipley JM, Bergers G, et al. MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 1998;93:411e22. [161] Dougall WC, Glaccum M, Charrier K, et al. RANK is essential for osteoclast and lymph node development. Genes Dev 1999;13:2412e24. [162] Rauch F, Cornibert S, Cheung M, Glorieux FH. Long-bone changes after pamidronate discontinuation in children and adolescents with osteogenesis imperfecta. Bone 2007;40:821e7. [163] Rauch F, Plotkin H, Zeitlin L, Glorieux FH. Bone mass, size, and density in children and adolescents with osteogenesis imperfecta: effect of intravenous pamidronate therapy. J Bone Miner Res 2003;18:610e4. [164] Rauch F, Glorieux FH. Osteogenesis imperfecta, current and future medical treatment. Am J Med Genet C Semin Med Genet 2005;139C:31e7. [165] Sanguinetti C, Greco F, De Palma L, Specchia N, Falciglia F. Morphological changes in growth-plate cartilage in osteogenesis imperfecta. J Bone Joint Surg Br 1990;72:475e9. [166] Evans KD, Lau ST, Oberbauer AM, Martin RB. Alendronate affects long bone length and growth plate morphology in the oim mouse model for osteogenesis imperfecta. Bone 2003;32: 268e74. [167] Glorieux FH, Bishop NJ, Plotkin H, Chabot G, Lanoue G, Travers R. Cyclic administration of pamidronate in children with severe osteogenesis imperfecta. N Engl J Med 1998;339: 947e52. [168] Glorieux FH, Rauch F, Shapiro JR. Bisphosphonates in children with bone diseases. N Engl J Med 2003;349:2068e71. author reply 71. [169] Martin TJ, Seeman E. Bone remodelling: its local regulation and the emergence of bone fragility. Best Pract Res Clin Endocrinol Metab 2008;22:701e22. [170] Sims NA, Gooi JH. Bone remodeling: multiple cellular interactions required for coupling of bone formation and resorption. Semin Cell Dev Biol 2008;19:444e51. [171] DeKoter RP, Singh H. Regulation of B lymphocyte and macrophage development by graded expression of PU.1. Science 2000;288:1439e41. [172] Tondravi M, McKercher S, Anderson K, et al. Osteopetrosis in mice lacking haematopoietic transcription factor PU.1. Nature 1997;386:81e4. [173] Zhang DE, Hetherington CJ, Chen HM, Tenen DG. The macrophage transcription factor PU.1 directs tissue-specific expression of the macrophage colony-stimulating factor receptor. Mol Cell Biol 1994;14:373e81. [174] Kwon OH, Lee CK, Lee YI, Paik SG, Lee HJ. The hematopoietic transcription factor PU.1 regulates RANK gene expression in myeloid progenitors. Biochem Biophys Res Commun 2005;335: 437e46. [175] Boyce BF, Yao Z, Xing L. Functions of nuclear factor kappaB in bone. Ann NY Acad Sci 2010;1192:367e75. [176] Iotsova V, Caamano J, Loy J, Yang Y, Lewin A, Bravo R. Osteopetrosis in mice lacking NF-kappaB1 and NF-kappaB2. Nat Med 1997;3:1285e9. [177] Franzoso G, Carlson L, Xing L, et al. Requirement for NF-kappaB in osteoclast and B-cell development. Genes Dev 1997;11: 3482e96. [178] Wagner EF, Eferl R. Fos/AP-1 proteins in bone and the immune system. Immunol Rev 2005;208:126e40. [179] Johnson RS, Spiegelman BM, Papaioannou V. Pleiotropic effects of a null mutation in the c-fos proto-oncogene. Cell 1992;71: 577e86.

79

[180] Wang ZQ, Ovitt C, Grigoriadis AE, Mohle-Steinlein U, Ruther U, Wagner EF. Bone and haematopoietic defects in mice lacking c-fos. Nature 1992;360:741e5. [181] Asagiri M, Takayanagi H. The molecular understanding of osteoclast differentiation. Bone 2007;40:251e64. [182] Takayanagi H. Mechanistic insight into osteoclast differentiation in osteoimmunology. J Mol Med 2005;83:170e9. [183] Yoshida H, Hayashi S, Kunisada T, et al. The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature 1990;345:442e4. [184] Boyce BF, Xing L. Functions of RANKL/RANK/OPG in bone modeling and remodeling. Arch Biochem Biophys 2008;473: 139e46. [185] Leibbrandt A, Penninger JM. RANK/RANKL: regulators of immune responses and bone physiology. Ann NY Acad Sci 2008;1143:123e50. [186] Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature 2003;423:337e42. [187] Hughes A, Ralston S, Marken J, et al. Mutations in TNFRSF11A, affecting the signal peptide of RANK, cause familial expansile osteolysis. Nat Genet 2000:45e8. [188] Whyte MP, Obrecht SE, Finnegan PM, et al. Osteoprotegerin deficiency and juvenile Paget’s disease. N Engl J Med 2002;347: 175e84. [189] Koga T, Inui M, Inoue K, et al. Costimulatory signals mediated by the ITAM motif cooperate with RANKL for bone homeostasis. Nature 2004;428:758e63. [190] Yagi M, Miyamoto T, Sawatani Y, Iwamoto K, Hosogane N, Fujita N, et al. DC-STAMP is essential for cell-cell fusion in osteoclasts and foreign body giant cells. J Exp Med 2005;202: 345e51. [191] Anandarajah AP, Schwarz EM. Anti-RANKL therapy for inflammatory bone disorders: Mechanisms and potential clinical applications. J Cell Biochem 2006;97:226e32. [192] Stolina M, Kostenuik PJ, Dougall WC, Fitzpatrick LA, Zack DJ. RANKL inhibition: from mice to men (and women). Adv Exp Med Biol 2007;602:143e50. [193] Burkiewicz JS, Scarpace SL, Bruce SP. Denosumab in osteoporosis and oncology. Ann Pharmacother 2009;43:1445e55. [194] Luxenburg C, Geblinger D, Klein E, et al. The architecture of the adhesive apparatus of cultured osteoclasts: from podosome formation to sealing zone assembly. PLoSONE 2007;2:e179. [195] Teitelbaum SL. Osteoclasts: what do they do and how do they do it? Am J Pathol 2007;170:427e35. [196] Vaananen HK, Laitala-Leinonen T. Osteoclast lineage and function. Arch Biochem Biophys 2008;473:132e8. [197] Rodan GA, Martin TJ. Therapeutic approaches to bone diseases. Science 2000;289:1508e14. [198] Gowen M, Lazner F, Dodds R, et al. Cathepsin K knockout mice develop osteopetrosis due to a deficit in matrix degradation but not demineralization. J Bone Miner Res 1999;14:1654e63. [199] Johnson MR, Polymeropoulos MH, Vos HL, Ortiz de Luna RI, Francomano CA. A nonsense mutation in the cathepsin K gene observed in a family with pycnodysostosis. Genome Res 1996;6: 1050e5. [200] Le Gall C, Bonnelye E, Clezardin P. Cathepsin K inhibitors as treatment of bone metastasis. Curr Opin Support Palliat Care 2008;2:218e22. [201] Stoch SA, Wagner JA. Cathepsin K inhibitors: a novel target for osteoporosis therapy. Clin Pharmacol Ther 2008;83:172e6. [202] Delaisse JM, Andersen TL, Engsig MT, Henriksen K, Troen T, Blavier L. Matrix metalloproteinases (MMP) and cathepsin K contribute differently to osteoclastic activities. Microsc Res Tech 2003;61:504e13.

PEDIATRIC BONE

80

4. POSTNATAL BONE GROWTH: GROWTH PLATE BIOLOGY, BONE FORMATION, AND REMODELING

[203] Krane SM, Inada M. Matrix metalloproteinases and bone. Bone 2008;43:7e18. [204] Sternlicht MD, Werb Z. How matrix metalloproteinases regulate cell behavior. Annu Rev Cell Dev Biol 2001;17:463e516. [205] Martin T, Gooi JH, Sims NA. Molecular mechanisms in coupling of bone formation to resorption. Crit Rev Eukaryot Gene Express 2009;19:73e88. [206] Matsuo K, Irie N. Osteoclast-osteoblast communication. Arch Biochem Biophys 2008;473:201e9. [207] Manolagas SC. Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. Endocr Rev 2000;21:115e37. [208] Tang Y, Wu X, Lei W, et al. TGF-beta1-induced migration of bone mesenchymal stem cells couples bone resorption with formation. Nat Med 2009;15:757e65. [209] Zhao C, Irie N, Takada Y, et al. Bidirectional ephrinB2-EphB4 signaling controls bone homeostasis. Cell Metab 2006;4:111e21. [210] Edwards CM, Mundy GR. Eph receptors and ephrin signaling pathways: a role in bone homeostasis. Int J Med Sci 2008;5: 263e72. [211] Lee SH, Rho J, Jeong D, et al. v-ATPase V0 subunit d2-deficient mice exhibit impaired osteoclast fusion and increased bone formation. Nat Med 2006;12:1403e9. [212] Sims NA. gp130 signaling in bone cell biology: multiple roles revealed by analysis of genetically altered mice. Mol Cell Endocrinol 2009;310:30e9. [213] Villemure I, Stokes IA. Growth plate mechanics and mechanobiology. A survey of present understanding. J Biomech 2009;42:1793e803. [214] Bonewald LF, Johnson ML. Osteocytes, mechanosensing and Wnt signaling. Bone 2008;42:606e15. [215] Robling AG, Niziolek PJ, Baldridge LA, et al. Mechanical stimulation of bone in vivo reduces osteocyte expression of Sost/sclerostin. J Biol Chem 2008;283:5866e75. [216] Han Y, Cowin SC, Schaffler MB, Weinbaum S. Mechanotransduction and strain amplification in osteocyte cell processes. Proc Natl Acad Sci USA 2004;101:16689e94. [217] Strom TM, Juppner H. PHEX, FGF23, DMP1 and beyond. Curr Opin Nephrol Hypertens 2008;17:357e62. [218] Tatsumi S, Ishii K, Amizuka N, et al. Targeted ablation of osteocytes induces osteoporosis with defective mechanotransduction. Cell Metab 2007;5:464e75. [219] Kogianni G, Mann V, Noble BS. Apoptotic bodies convey activity capable of initiating osteoclastogenesis and localized bone destruction. J Bone Miner Res 2008;23:915e27. [220] Zhao S, Zhang YK, Harris S, Ahuja SS, Bonewald LF. MLO-Y4 osteocyte-like cells support osteoclast formation and activation. J Bone Miner Res 2002;17:2068e79. [221] Aguirre JI, Plotkin LI, Stewart SA, et al. Osteocyte apoptosis is induced by weightlessness in mice and precedes osteoclast recruitment and bone loss. J Bone Miner Res 2006;21:605e15. [222] Noble BS, Peet N, Stevens HY, et al. Mechanical loading: biphasic osteocyte survival and targeting of osteoclasts for bone destruction in rat cortical bone. Am J Physiol Cell Physiol 2003;284. C934-C43. [223] Xiao Z, Zhang S, Mahlios J, et al. Cilia-like structures and polycystin-1 in osteoblasts/osteocytes and associated abnormalities in skeletogenesis and Runx2 expression. J Biol Chem 2006;281:30884e95. [224] Xiao Z, Dallas M, Qiu N, et al. Conditional deletion of Pkd1 in osteocytes disrupts skeletal mechanosensing in mice. Faseb J 2011. [225] Yang W, Lu Y, Kalajzic I, et al. Dentin matrix protein 1 gene cisregulation: use in osteocytes to characterize local responses to

[226]

[227]

[228]

[229]

[230]

[231]

[232]

[233]

[234]

[235]

[236]

[237]

[238]

[239]

[240]

[241]

[242]

[243]

[244]

mechanical loading in vitro and in vivo. J Biol Chem 2005;280: 20680e90. Feng JQ, Ward LM, Liu S, et al. Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism. Nat Genet 2006;38:1310e5. Li X, Ominsky MS, Niu QT, et al. Targeted deletion of the sclerostin gene in mice results in increased bone formation and bone strength. J Bone Miner Res 2008;23:860e9. Winkler DG, Sutherland MK, Geoghegan JC, et al. Osteocyte control of bone formation via sclerostin, a novel BMP antagonist. EMBO J 2003;22:6267e76. Ahmed SF, Farquharson C. The effect of GH and IGF1 on linear growth and skeletal development and their modulation by SOCS proteins. J Endocrinol 2010;206:249e59. Giustina A, Mazziotti G, Canalis E. Growth hormone, insulinlike growth factors, and the skeleton. Endocr Rev 2008;29: 535e59. Ning Y, Schuller AG, Bradshaw S, et al. Diminished growth and enhanced glucose metabolism in triple knockout mice containing mutations of insulin-like growth factor binding protein-3, -4, and -5. Mol Endocrinol 2006;20:2173e86. Woods KA, Camacho-Hubner C, Savage MO, Clark AJ. Intrauterine growth retardation and postnatal growth failure associated with deletion of the insulin-like growth factor I gene. N Engl J Med 1996;335:1363e7. Baker J, Liu JP, Robertson EJ, Efstratiadis A. Role of insulin-like growth factors in embryonic and postnatal growth. Cell 1993;75: 73e82. Liu J, Baker J, Perkins A, Robertson E, Efstradiatis A. Mice carrying null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type 1 IGF receptor (Igf1r). Cell 1993;75:59e72. Lupu F, Terwilliger JD, Lee K, Segre GV, Efstratiadis A. Roles of growth hormone and insulin-like growth factor 1 in mouse postnatal growth. Dev Biol 2001;229:141e62. Woelfle J, Chia DJ, Rotwein P. Mechanisms of growth hormone (GH) action. Identification of conserved Stat5 binding sites that mediate GH-induced insulin-like growth factor-I gene activation. J Biol Chem 2003;278:51261e6. DeChiara T, Efstradiatis A, Robertson E. A growth-deficient phenotype in heterozygous mice carrying an insulin-like growth factor II gene disrupted by targeting. Nature 1990;345:78e80. Louvi A, Accili D, Efstratiadis A. Growth-promoting interaction of IGF-II with the insulin receptor during mouse embryonic development. Dev Biol 1997;189:33e48. Amselem S, Sobrier ML, Duquesnoy P, et al. Recurrent nonsense mutations in the growth hormone receptor from patients with Laron dwarfism. J Clin Invest 1991;87:1098e102. Zhou Y, Xu BC, Maheshwari HG, et al. A mammalian model for Laron syndrome produced by targeted disruption of the mouse growth hormone receptor/binding protein gene (the Laron mouse). Proc Natl Acad Sci USA 1997;94:13215e20. Powell-Braxton L, Hollingshead P, Warburton C, et al. IGF-I is required for normal embryonic growth in mice. Genes Dev 1993;7:2609e17. Wang J, Zhou J, Bondy CA. Igf1 promotes longitudinal bone growth by insulin-like actions augmenting chondrocyte hypertrophy. Faseb J 1999;13:1985e90. Sjogren K, Liu JL, Blad K, et al. Liver-derived insulin-like growth factor I (IGF-I) is the principal source of IGF-I in blood but is not required for postnatal body growth in mice. Proc Natl Acad Sci USA 1999;96:7088e92. Yakar S, Liu JL, Stannard B, et al. Normal growth and development in the absence of hepatic insulin-like growth factor I. Proc Natl Acad Sci USA 1999;96:7324e9.

PEDIATRIC BONE

81

REFERENCES

[245] Parker EA, Hegde A, Buckley M, Barnes KM, Baron J, Nilsson O. Spatial and temporal regulation of GH-IGF-related gene expression in growth plate cartilage. J Endocrinol 2007;194: 31e40. [246] Wang Y, Cheng Z, Elalieh HZ, et al. IGF-1R Signaling in chondrocytes modulates growth plate development by interacting with the PTHrP/Ihh pathway. J Bone Miner Res 2011. [247] DiGirolamo DJ, Mukherjee A, Fulzele K, et al. Mode of growth hormone action in osteoblasts. J Biol Chem 2007;282:31666e74. [248] Playford MP, Bicknell D, Bodmer WF, Macaulay VM. Insulinlike growth factor 1 regulates the location, stability, and transcriptional activity of beta-catenin. Proc Natl Acad Sci USA 2000;97:12103e8. [249] Smith EP, Boyd J, Frank GR, et al. Estrogen resistance caused by a mutation in the estrogen-receptor gene in a man. N Engl J Med 1994;331:1056e61. [250] Carani C, Qin K, Simoni M, et al. Effect of testosterone and estradiol in a man with aromatase deficiency. N Engl J Med 1997;337:91e5. [251] Bilezikian JP, Morishima A, Bell J, Grumbach MM. Increased bone mass as a result of estrogen therapy in a man with aromatase deficiency. N Engl J Med 1998;339:599e603. [252] Grumbach MM, Auchus RJ. Estrogen: consequences and implications of human mutations in synthesis and action. J Clin Endocrinol Metab 1999;84:4677e94. [253] Vanderschueren D, Boonen S, Ederveen AG, et al. Skeletal effects of estrogen deficiency as induced by an aromatase inhibitor in an aged male rat model. Bone 2000;27:611e7. [254] Borjesson AE, Lagerquist MK, Liu C, et al. The role of estrogen receptor alpha in growth plate cartilage for longitudinal bone growth. J Bone Miner Res 2010;25:2414e24. [255] Finkelstein JS, Neer RM, Biller BM, Crawford JD, Klibanski A. Osteopenia in men with a history of delayed puberty. N Engl J Med 1992;326:600e4. [256] Vanderschueren D, Gaytant J, Boonen S, Venken K. Androgens and bone. Curr Opin Endocrinol Diabetes Obes 2008;15:250e4. [257] Manolagas SC, Kousteni S, Jilka RL. Sex steroids and bone. Recent Prog Horm Res 2002;57:385e409. [258] Novack DV. Estrogen and bone: osteoclasts take center stage. Cell Metab 2007;6:254e6. [259] Rossouw JE, Anderson GL, Prentice RL, et al. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the Women’s Health Initiative randomized controlled trial. J Am Med Assoc 2002;288:321e33. [260] Kameda T, Mano H, Yuasa T, et al. Estrogen inhibits bone resorption by directly inducing apoptosis of the bone-resorbing osteoclasts. J Exp Med 1997;186:489e95. [261] Nakamura T, Imai Y, Matsumoto T, et al. Estrogen prevents bone loss via estrogen receptor alpha and induction of Fas ligand in osteoclasts 1. Cell 2007;130:811e23. [262] Krum SA, Miranda-Carboni GA, Hauschka PV, et al. Estrogen protects bone by inducing Fas ligand in osteoblasts to regulate osteoclast survival. EMBO J 2008;27:535e45. [263] Kousteni S, Chen JR, Bellido T, et al. Reversal of bone loss in mice by nongenotropic signaling of sex steroids. Science 2002;298:843e6. [264] Almeida M, Martin-Millan M, Ambrogini E, et al. Estrogens attenuate oxidative stress and the differentiation and apoptosis of osteoblasts by DNA-binding-independent actions of the ERalpha. J Bone Miner Res 2010;25:769e81. [265] Manolagas SC. From estrogen-centric to aging and oxidative stress: a revised perspective of the pathogenesis of osteoporosis. Endocr Rev 2010;31:266e300. [266] Srivastava S, Toraldo G, Weitzmann MN, Cenci S, Ross FP, Pacifici R. Estrogen decreases osteoclast formation by down-

[267] [268] [269] [270] [271]

[272]

[273]

[274]

[275]

[276]

[277]

[278]

[279]

[280]

[281]

[282]

[283]

[284]

[285]

[286]

regulating receptor activator of NF-kappa B ligand (RANKL)induced JNK activation. J Biol Chem 2001;276:8836e40. Pacifici R. Estrogen deficiency, T cells and bone loss. Cell Immunol 2008;252:68e80. Weitzmann MN, Pacifici R. Estrogen deficiency and bone loss: an inflammatory tale. J Clin Invest 2006;116:1186e94. Sun L, Peng Y, Sharrow AC, et al. FSH directly regulates bone mass. Cell 2006;125:247e60. Gogakos AI, Duncan Bassett JH, Williams GR. Thyroid and bone. Arch Biochem Biophys 2010;503:129e36. Stevens DA, Hasserjian RP, Robson H, Siebler T, Shalet SM, Williams GR. Thyroid hormones regulate hypertrophic chondrocyte differentiation and expression of parathyroid hormonerelated peptide and its receptor during endochondral bone formation. J Bone Miner Res 2000;15:2431e42. Ballock R, Mita BC, Zhou X, Chen DH, Mink LM. Expression of thyroid hormone receptor isoforms in rat growth plate cartilage in vivo. J Bone Miner Res 1999;14:1550e6. Miura M, Tanaka K, Komatsu Y, et al. Thyroid hormones promote chondrocyte differentiation in mouse ATDC5 cells and stimulate endochondral ossification in fetal mouse tibias through iodothyronine deiodinases in the growth plate. J Bone Miner Res 2002;17:443e54. Robson H, Siebler T, Stevens DA, Shalet SM, Williams GR. Thyroid hormone acts directly on growth plate chondrocytes to promote hypertrophic differentiation and inhibit clonal expansion and cell proliferation. Endocrinology 2000;141:3887e97. Gauthier K, Plateroti M, Harvey CB, et al. Genetic analysis reveals different functions for the products of the thyroid hormone receptor alpha locus. Mol Cell Biol 2001;21:4748e60. Harvey CB, O’Shea PJ, Scott AJ, et al. Molecular mechanisms of thyroid hormone effects on bone growth and function. Mol Genet Metab 2002;75:17e30. Bassett JH, O’Shea PJ, Chassande O, et al. Analysis of skeletal phenotypes in thyroid hormone receptor mutant mice. Scanning 2006;28:91e3. Bassett JH, Williams AJ, Murphy E, et al. A lack of thyroid hormones rather than excess thyrotropin causes abnormal skeletal development in hypothyroidism. Mol Endocrinol 2008;22:501e12. Weiss RE, Refetoff S. Effect of thyroid hormone on growth. Lessons from the syndrome of resistance to thyroid hormone. Endocrinol Metab Clin North Am 1996;25:719e30. Canalis E, Mazziotti G, Giustina A, Bilezikian JP. Glucocorticoid-induced osteoporosis: pathophysiology and therapy. Osteoporos Int 2007;18:1319e28. O’Brien CA, Jia D, Plotkin LI, et al. Glucocorticoids act directly on osteoblasts and osteocytes to induce their apoptosis and reduce bone formation and strength. Endocrinology 2004;145: 1835e41. Yun SI, Yoon HY, Jeong SY, Chung YS. Glucocorticoid induces apoptosis of osteoblast cells through the activation of glycogen synthase kinase 3beta. J Bone Miner Metab 2008. Jia D, O’Brien CA, Stewart SA, Manolagas SC, Weinstein RS. Glucocorticoids act directly on osteoclasts to increase their life span and reduce bone density. Endocrinology 2006;147:5592e9. Kim HJ, Zhao H, Kitaura H, et al. Glucocorticoids suppress bone formation via the osteoclast. J Clin Invest 2006;116: 2152e60. Marino R, Hegde A, Barnes KM, et al. Catch-up growth after hypothyroidism is caused by delayed growth plate senescence. Endocrinology 2008;149:1820e8. Silvestrini G, Ballanti P, Patacchioli FR, et al. Evaluation of apoptosis and the glucocorticoid receptor in the cartilage growth plate and metaphyseal bone cells of rats after high-dose treatment with corticosterone. Bone 2000;26:33e42.

PEDIATRIC BONE

82

4. POSTNATAL BONE GROWTH: GROWTH PLATE BIOLOGY, BONE FORMATION, AND REMODELING

[287] Chrysis D, Ritzen EM, Savendahl L. Growth retardation induced by dexamethasone is associated with increased apoptosis of the growth plate chondrocytes. J Endocrinol 2003;176:331e7. [288] Nilsson O, Marino R, De Luca F, Phillip M, Baron J. Endocrine regulation of the growth plate. Horm Res 2005;64:157e65. [289] Lorenzo J, Horowitz M, Choi Y. Osteoimmunology: interactions of the bone and immune system. Endocr Rev 2008;29:403e40. [290] Takayanagi H. Osteoimmunology: shared mechanisms and crosstalk between the immune and bone systems. Nat Rev Immunol 2007;7:292e304. [291] Calvi LM, Adams GB, Weibrecht KW, et al. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 2003;425: 841e6. [292] Lo Celso C, Fleming HE, Wu JW, et al. Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche. Nature 2009;457:92e6. [293] Lymperi S, Ferraro F, Scadden DT. The HSC niche concept has turned 31. Has our knowledge matured? Ann NY Acad Sci 2010;1192:12e8.

[294] Wu JY, Scadden DT, Kronenberg HM. Role of the osteoblast lineage in the bone marrow hematopoietic niches. J Bone Miner Res 2009;24:759e64. [295] Franquinho F, Liz MA, Nunes AF, Neto E, Lamghari M, Sousa MM. Neuropeptide Y and osteoblast differentiation e the balance between the neuro-osteogenic network and local control. FEBS J 2010;277:3664e74. [296] Karsenty G, Oury F. The central regulation of bone mass, the first link between bone remodeling and energy metabolism. J Clin Endocrinol Metab 2010;95:4795e801. [297] Takeda S, Karsenty G. Molecular bases of the sympathetic regulation of bone mass. Bone 2008;42:837e40. [298] Ferron M, Wei J, Yoshizawa T, et al. Insulin signaling in osteoblasts integrates bone remodeling and energy metabolism. Cell 2010;142:296e308. [299] Clemens TL, Karsenty G. The osteoblast: An insulin target cell controlling glucose homeostasis. J Bone Miner Res 2011;26: 677e80. [300] Lee NK, Karsenty G. Reciprocal regulation of bone and energy metabolism. Trends Endocrinol Metab 2008;19:161e6.

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Dental Development and Maturation, from the Dental Crypt to the Final Occlusion Jean-Marc Retrouvey 1, Michel Goldberg 2, Ste´phane Schwartz 3 1

Division of Orthodontics, McGill University, Montreal, Quebec, Canada, 2 UMR-S 747-INSERM, Universite´ Paris Descartes, 3 Dental Clinic, Montreal Children’s Hospital, MUHC (McGill University Health Center), and McGill University

The formation and maturation of dental tissues constitute an important process in craniofacial development. Genes coding the expression of growth factors, transcription factors and extracellular matrix molecules regulate this process. The interactions between the mesoderm and the ectoderm are crucial during the initial steps of tooth germ development, morphogenesis and cell differentiation, leading to crown and later to root formation. These processes are associated with the formation of alveolar bone around the dental follicle and tooth eruption in the oral cavity. Disruption of the normal developmental pattern may be due to gene mutations or to defective expression of intracellular or extracellular structural molecules. The development of the dentition is also a great contributor to the development of the lower face height by enlargement of the dento-alveolar processes. As gene mutations, epigenetic alterations or influenced by post-genomic factors, individual defects can be identified. In such cases, the mutations listed in this review are invariably associated with oral development manifestations. As a more complex phenomenon, the close association of multiple defects interacting closely may produce multifaceted syndromes.

the expression of growth factors and transcription pathways regulate these events. Any mutation in these genes is susceptible to induce morphologic and/or functional alterations in any tooth developmental process. We review here the major events that influence early tooth formation that may shed light on major tooth abnormality such as amelogenesis imperfecta and dentinogenesis imperfecta, which may or may not be associated with major craniofacial alterations or defective mineralization syndromes [2,3]. Within the first branchial arch for all the teeth, and the nasofrontal bud for the maxillary incisors, all tooth buds result from the interactions of the oral ectoderm lining the stomodeal cavity, in specific areas spatially limited as oral placodes, and the dental mesenchyme containing cells derived from the neural crest. Initial Phase In the epithelial cells expressing Otlx2/Pitx2, two families of growth factors (bone morphogenetic proteins [BMPs], fibroblast growth factors [FGFs]) are activated. The epithelial placode then becomes thicker and up to day 11 of gestation is under the influence of Left in the mouse. This leads to the formation of a dental lamina. BMP, FGF, sonic hedgehog (Shh) and Wnt are expressed in the epithelial compartment, whereas the expression of Lhx6-7, Barx1, Msx 1-2, and Pax9 is increased in the odontogenic mesenchyme. The proliferating neural crest-derived cells condense in the dental mesenchyme and express Msx1, Pax9, Left and Cbfa1 (Runx2). This implies that any alteration of the genes coding for these molecules leads to severe alteration or even the lack of tooth formation (anodontia).

THE TOOTH BUD Embryology of the Tooth Three successive steps lead to the formation of a tooth: (1) the initiation of the process, (2) the morphogenesis of the tooth, and (3) the terminal differentiation of two layers of epithelial and mesenchymal cells acquiring their terminal phenotype [1]. Several genes activating

Pediatric Bone, Second Edition DOI: 10.1016/B978-0-12-382040-2.10005-X

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Morphogenesis of the Tooth Depending on the position of the germ in the branchial arch, a different type of tooth will be produced. This is related either to a possible gradient of “morphogens”, or to the concomitant burst of cell clones predetermined to form incisors, canines, premolars and molars. The epithelial buds located along the dental lamina proliferate, become caps and later reach the bell stage. During early morphogenesis, three series of events are implicated in the shaping of the tooth crown. Initially, a semi-permeable basement membrane (BM) [4] regulates the diffusion of molecules between the enamel organ and the embryonic pulp compartments. The BM triggers the terminal differentiation of preodontoblasts into polarized secretory odontoblasts. The preameloblasts become differentiated secretory ameloblasts. Metalloproteases and other degradative enzymes trapped in the BM network are shaping the future dentino-enamel junction [5]. Following this initial step, neural crest-derived cells migrate and colonize the dental mesenchyme. Later, they slide toward the periphery of the embryonic pulp and undergo a series of cell divisions. One less division occurs in the inner enamel epithelium compared with the odontoblast layer. This induces a substantial length difference between the two compartments, hence to the folding of the cell layers and consequently to cusp formation. Finally, at an early bell stage, a niche of cells named the “enamel knot” is found in the central part of the tooth. They express the gene Msx2. This group of cells is not proliferative, in contrast with the cells located in the lateral parts of the germ. The central part stays in a fixed position whereas the rest of the enamel organ expands and, because of space limitations, contributes also to the formation of folds and consequently, cusps. The enamel knot expresses Shh, Bmp-2, Bmp-4, Bmp-7, Fgf-4 and Fgf-9 [6]. Terminal Differentiation Post-mitotic polarizing ameloblasts elongate. Intercellular junctions start to develop and the polarized young ameloblasts become functional and implicated in the synthesis and secretion of enamel proteins. Pre-odontoblasts originally bear a fibroblastic appearance. They migrate from the central part of the dental pulp to the periphery and become post-mitotic polarizing odontoblasts. Long protracted processes, characteristic of polarized odontoblasts, emerge from cell bodies associated into a palisade-like structure. They are implicated in the synthesis of collagen and non-collagenous proteins, whereas secretion and re-internalization of fragmented peptides or signal peptides occur along the cell processes, in the predentin and in dentin respectively. In the distal

part of the cell bodies, desmosomes and gap junctions form terminal junctional complexes. Root Formation/Eruption During the pre-eruptive phase, once the crown is formed, at the cervical margin of the enamel organ, epithelial cells proliferate and initiate the formation of the Hertwig’s epithelial root sheath. In the inner part of the sheath, epithelial cells promote the recruitment and cytodifferentiation of pulp progenitors. The outer layer is implicated in the recruitment of cementoblasts that cross the disaggregating intercellular Hertwig’s root sheath. The Hertwig’s root sheath is involved in the recruitment of cells of the periodontal ligament and the construction of the bony crypt [7]. The pulp progenitors migrate toward the inner surface of the Hertwig’s sheath. They become odontoblasts, organized as a palisade-like structure, forming the root dentin. The forming part of the tooth germ keeps its original position during these early stages of root formation. Gradually, the root(s) elongate(s) and the tooth erupts in the oral cavity. This is referred to as the eruptive prefunctional stage. Three possible origins have been reported for the cementoblasts: • during the early stages of formation of the root, epithelial cells of the Hertwig’s root sheath interconvert, become mesenchymal cells and ultimately acquire the cementoblast phenotype • afterwards, during root lengthening, the dissociation in the cervical area of the Hertwig’s root sheath allows some fibroblast-like cells issued from the dental follicle to migrate through these fenestrations and become cementoblasts • later, the tooth comes into contact with its antagonistic teeth, and the late steps of eruption/root formation are referred to as the functional stage. Then progenitors located in the periodontal ligament proliferate under the influence of cementum growth factor, and differentiate into cementoblasts. After stimulation of the cementum adhesion protein, cementoblast progenitors adhere to the cementum surface and contribute to the thickening of the forming cementum. Although the mechanisms implicated in the eruption are not fully elucidated, it is clear that a series of growth and transcription factors intervene in the formation of the bony socket. Colony-stimulating factor-1 (CSF-1), epithelial growth factor (EGF), transforming growth factor (TGF)1, interleukin 1a (Il-1a), receptor activator ligand of the nuclear factor-kappa B (RANKL), c-Fos, osteoprotegerin, parathyroid hormone-related protein, Cbfa1, tumor necrosis factor (TNF), vascular endothelial

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growth factor (VEGF) and bone morphogenetic protein2 (BMP-2) have all been implicated. These factors, together with a series of metalloproteases and disintegrins, contribute to the root lengthening, eruption and functional adaptation [7].

Histology of Dental Tissues Enamel, dentin and cementum constitute the three mineralized dental tissues, whereas the dental pulp is a unique non-mineralized tissue. Enamel The composition of enamel is shown in Table 5.1. ENAMEL STRUCTURE

The interacting presecretory ameloblast and odontoblast layers form distinct compartments on either side of the BM. The BM is then enzymatically degraded, and short ameloblast processes protrude toward the forming dentin, leading to the formation of a scalloped dentino-enamel junction (DEJ). Ameloblasts initiate the formation of the prismatic enamel made by rods (or prisms) and interrods (interprismatic enamel). Tightly packed hexagonal ˚ ngstro¨ms (A ˚ ) in enamel crystallites are about 700
300 A ˚ in cross-section, and vary between 2000 and 10 000 A height. Prisms (rods and interrods), Hunter-Schreger bands and Retzius lines constitute the three characteristic anatomic structures found in enamel (Fig. 5.1). Near the DEJ, in humans, between 10 and 20 prisms are seen bending alternatively to the right or to the left contributing to the formation of Hunter-Schreger’s bands. In transverse sections, Retzius lines appear as continuous lines forming concentric circles, and each enamel segment comprised between two Retzius lines, about 25 mm thick, constitutes a mineralization modulus [8]. This organization allows occlusal forces to be dissipated away from the pressure area. This type of architecture reinforces the mechanical properties of enamel.

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diffuse in the outer layer forming predentin/dentin. The first enamel layer is aprismatic. Accumulation of degraded matrix components contributes to the formation of Tomes’ process. The formation of interrod enamel then starts around the processes, developing a continuous honeycomb structure. The thickness of the forming enamel is gradually increased. In humans, the aprismatic outer enamel layer is about 30 mm. Calcium deficiencies lead to the emergence of structural defects mostly in the outer enamel (hypoplasia). Post-secretory ameloblasts are implicated in enamel maturation. Proteases such as kallikrein-4 (KLK-4) contribute to the hydrolysis of amelogenin. Amelogenin proteins represent the predominate subfamily of gene products found in developing mammalian enamel, and are implicated in the formation of the largest hydroxyapatite crystals in the vertebrate body. Maturation (or postsecretory) ameloblasts appear as ruffle-ended cells, bearing some similarities with the osteoclasts, although some segments display a smooth appearance. The organic matrix expelled from maturing enamel is pushed in deep recesses and internalized into lysosomes. Lysosomal acid phosphatase and other catalytic enzymes contribute to the degradation of this temporary organic matrix, especially amelogenin. Post-secretory ameloblasts allow calcium and phosphate ions to diffuse and contribute to the thickening and lengthening of enamel crystallites. As an enzymatic inhibitor, fluoride alters

ENAMEL FORMATION

Enamel formation results from the secretion of an extracellular matrix (ECM) by secretory ameloblasts and its eventual mineralization. Some enamel proteins TABLE 5.1

Global Composition of Enamel In weight (%)

In volume (%)

Mineral phase

96

87

Organic phase

0.6e1

2

Water

3.4e4 1% free water 2.4% bound water

FIGURE 5.1 Human dental enamel, internal zone. Groups of rods 7e11

cut longitudinally (parazones) alternate with group of rods in crosssection (diazones) at right angles from the parazones. Scanning electron microscope (SEM) preparation.

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these processes leading to mild to severe fluorosis. Defective enamel mineralization is also specifically due to the defective activity of maturation ameloblasts ENAMEL COMPOSITION

The mineral phase of enamel results in the formation of hydroxyapatite (HAp). In addition to calcium and phosphate, HAp composition also includes carbonate, magnesium, fluoride, sodium and many trace elements that are either mineral-associated or enzyme co-factors. Amelogenins constitute the bulk of the forming enamel matrix, while only a residual amount persists in the mature enamel. They are secreted as 28e25 kDa molecules, but are rapidly degraded into 21 kDa or less. They are presumably implicated in the nucleation and orientation of the crystallites. During enamel formation, proteases including enamelysine (MMP20) and other non-specific MMPs are implicated in the degradation of the molecule into small fragments that disappear during enamel maturation. KLK-4 also contributes to restriction of the organic content of mature enamel (0.4e0.6% w/v). Isoforms and spliced forms of amelogenins have been identified. Mutations of amelogenins result in different forms of amelogenesis imperfecta (AI) (Fig. 5.2). Enamelin forms about 5% of the enamel matrix, and the molecule seems to be implicated in nucleation and lengthening of crystallites and also in some forms of AI.

Ameloblastin (or amelin or shethlin) is synthesized by odontoblasts and ameloblasts, and it appears to be an adhesion molecule inhibiting cell proliferation and maintaining cell differentiation. A series of minor molecules are also present in enamel: amelotin, proteins issued from the serum, lipids and/or phospholipids, and calcium-binding proteins. Several enzymes such as MMPs, serine protease (KLK-4), acid and alkaline phosphatases are expressed during enamel formation and maturation. When these molecules are defective, due either to genetic mutations or to impaired enamel protein degradation, structural defects are detectable [9,10]. Dentin The composition of dentin is shown in Table 5.2. DIFFERENT TYPES OF DENTIN [11] OUTER PERIPHERAL DENTIN IN THE CROWN AND ROOT In the crown, odontoblasts at an early stage of

differentiation are implicated in the formation of a thin outer layer, the mantle dentin. Located beneath the dentino-enamel junction, dentin tubules are lacking or only some bended minute tubules are present. Its thickness varies between 15 and 30 mm. This layer displays specific elastic properties because it is less mineralized than the subjacent circumpulpal dentin [12] (Fig. 5.3). The proteins present in this thin layer are not phosphorylated, and a close relation has been established between

FIGURE 5.2 Human tooth displaying an amelogenesis imperfecta (AI). Left: enamel surface observed with the SEM. Hypoplastic enamel is seen, the outer part being missing. Center: longitudinal section of an AI affected tooth. Part of the outer enamel is missing. Right: the entire unsectioned tooth.

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TABLE 5.2 Global Composition of Dentin In weight (%)

In volume (%)

Mineral phase

70

50

Organic phase

20

30

Water

10e11

20

the degree of phosphorylation of the extracellular matrix molecules and their contribution to dentin or bone mineralization [14]. The same process occurs in the root where two distinct superficial layers have been identified for a total thickness of about 30 mm. The Hopewell-Smith hyaline layer and the granular Tomes’ layer are the first outer structures formed by root odontoblasts after their differentiation from the Hertwig’s root sheath. Cell interconnections and the general orientation of dentinal canaliculi along the long axis of the root suggest that these structures are implicated in the shaping and in the gradual reduction in the diameter of the root. As is

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the case in the crown, these peripheral layers are hypomineralized and there is a general increase of mineralization some distance away from the cemento-dentinal junction. CIRCUMPULPAL DENTIN Dentin is produced continuously in adults (4 mm/day) formed by regularly spaced Von Ebner lines appearing as incremental lines, and every 20e24 mm an Owen line is more prominent, implicating the dentin that includes four to six Von Ebner lines. This appearance is related to circadian and other rhythms. The primary dentin is formed until the moment the tooth becomes functional. Then the formation of secondary dentin starts and will continue for the lifetime of the tooth. Apart from the decreasing number of dentinal tubules, there is no structural or chemical difference between primary and secondary dentins. The number of tubules is about 20 000 tubules/mm2. Circumpulpal dentin includes the intertubular and peritubular dentins (Fig. 5.4). Intertubular dentin results from the transformation of predentin into dentin. It forms the bulk of dentin, whereas peritubular dentin is more variable, according to species, and location examined.

FIGURE 5.3 Upper part of the panel: normal dentin. Upper left: SEM of a tooth collected from a patient displaying an X-linked hypophosphatemia. Lower part, left: under the dentino-enamel junction, the outer mantle dentin is unaffected by the disease, whereas in the circumpulpal dentin, empty interglobular spaces are clearly seen between calcospherites. The dentino-enamel junction and the mantle dentin (md) appear to be normal, whereas the circumpulpal dentin interglobular spaces are filled with extracellular matrix partially degraded remnants accumulating between unmerged globular structures. Apparently the mantle dentin is not influenced by phosphorus homeostasis, as seen in X-linked hypophosphatemia [13].

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FIGURE 5.4

The peritubular dentin surrounds the lumen of the tubules. Intertubular dentin forms a continuous collagen-rich network. Left: horse’s dentin observed with the SEM. Right: human dentin observed with the transmission electron microscope (TEM).

Intertubular dentin is a type I collagen-rich structure. Collagen fibrils display a 100e120 nm diameter. Noncollagenous extracellular matrix proteins will be detailed in the paragraph related to the dentin composition. Some of them are phosphorylated and associated with the mineralization process either as promoters or inhibitors. Dentin crystallites appear as needle-like structures 3e4 nm thick and 60 nm long. They are located either along the surface of the collagen fibrils, in association with the so-called “holes” due to the quarter-staggered fibril structure, or filling the empty intercollagen spaces. In contrast, peritubular dentin is formed within the lumen of the tubules independently from the predentin transformation. It does not contain collagen fibrils, but is formed by a network of amorphous non-collagenous proteins, similar to those found in the intertubular dentin. In humans, the surface occupied by peritubular dentin is no more than 10% of the whole dentin volume. This distribution is related to the resistance to abrasion, intertubular dentin being elastic and easily abraded, whereas peritubular dentin is more resistant to attrition. Peritubular dentin has a higher carbonate and magnesium content than the intertubular dentin, and consequently is more soluble in acidic or in chelating solutions. The pathologic dentin defects related to specific gene mutations will be reviewed later in this chapter. They include formation of globular and interglobular

structures, defective dentin formation and altered root length. CELL AND TISSUE ORGANIZATION CELLULAR COMPARTMENT The odontoblasts are implicated in the synthesis of ECM molecules. These cells compose an individual compartment distinct from the pulp. The cells actively secrete predentin which is further transformed into mineralized dentin. THE PREDENTIN COMPARTMENT Another compartment comprises odontoblastic processes and predentin. Secretory vesicles are transferred along the processes and the larger part is secreted in the proximal predentin. Collagen fibrils start to aggregate at the site where they are secreted. The native fibrils elongate by end-to-end self-aggregation of the telopeptides and the diameter is increased by lateral aggregation of subunits. Proteoglycans such as fibromodulin and biglycan are also secreted in the proximal predentin and control collagen fibrillation. THE DENTIN COMPARTMENT The dentin compartment is a mineralized continuous structure starting at the metadentin border at the dentin edge and extending up to the dentino-enamel junction. Odontoblast processes are located within tubules (about 20 000/mm2).

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THE TOOTH BUD

A second group of non-collagenous proteins (NCPs) are secreted along the process, diffusing throughout the porous intertubular dentin and also contributing to the formation of peritubular dentin. DENTIN EXTRACELLULAR MATRIX COMPOSITION

Normal dentin extracellular matrix composition is shown in Table 5.3. Alterations of some small integrin-binding ligand N-linked glycoproteins (SIBLINGs) are related to some dentin pathologies such as dentinogenesis imperfecta or dentin dysplasia. DMP-1, PHEX and FGF-23 mutations are implicated in hyper- or hypophosphatemia. Cement/Cementogenesis [15,16] Cementum global composition in weight of mineral 65%, organic 23%, water 12%. TABLE 5.3

STRUCTURAL DIFFERENCES BETWEEN THE DIFFERENT TYPES OF CEMENT

The formation of cement, the layer that covers the root of the teeth, starts with an acellular afibrillar thin band located at the cervical junction between dentin and enamel (coronal cement). It is regulated by osteopontin [17]. Once the acellular band is completed, the formation of acellular cement begins and includes extrinsic fibrils found on the mid-root cement. The lower part of the root (apical cement) is covered by cellular and acellular cement, which includes intrinsic and/or extrinsic fibrils (Sharpey’s fibers from the periodontal ligament). Cementoblasts are either trapped in cementum, to form cellular cementum, or they slide away from the cementum surface, giving rise to acellular cementum. In the apical part, mixed cement appears as a multilayered structure that includes cellular and acellular successive layers.

Normal Dentin Extracellular Matrix Composition

Collagens 90% Non-collagenous proteins 10%

Phosphorylated proteins

Nonphosphorylated proteins

Type I collagen (89%) þ type I trimer (11%)

þ 1e3% Type III and V collagens

SIBLINGs Genes coding: in human chromosome 4 in rodent chromosome 5 Locus 4q21 Implicated in Dentinogenesis imperfecta and dentin dysplasia

DSPP (between 155 and 95 kDa) cleaved into: >DSP (N-terminal- proteoglycan forming dimers): 100e280 kDa >DPP (C-terminal) 94 kDa DMP-1: 61 kDa (proteoglycan) nucleator BSP: 95 kDa (proteoglycan) nucleation þ crystallite growth OPN: 44 kDa, glycoprotein, mineralization inhibitor MEPE: 66 kDa glycoprotein

Amelogenin

Spliced forms: Aþ 4: 8.1 kDa A-4: 6.9 kDa

Others enamel molecules

Ameloblastine

Osteocalcine DPG : dentin gla-protein (acidic g carboxyglutamic) Matrix Gla protein (MGP)

>5.7 kDa >mineralization inhibitor >14 kDa, not inhibitor of mineralization

Osteonectin- SPARC

43 kDa

Serum proteins

Albumin a2-HS glycoprotein

Small leucine-rich proteoglycans (SLRPS)

CS/DS PGs: decorine, biglycan 42 kDa KS PGs: lumican, fibromoduline, osteoadherine 50 kDa

Growth factors

FGF2, TGFb1, BMPs, ILGF I & II, PDGF

Enzymes

Alkaline and acidic phosphatase, serine proteases, MMPs: collagenases: MMP-1, 8, 13 gelatinases A (MMP-2), and B (MMP9) Stromelysine 1: MMP-3 MT1-MMP, enamelysine: MMP-20 ADAMs et ADAMTS

Proteolipids

ECM Phospholipids

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COMPOSITION OF THE ORGANIC PHASE OF CEMENTUM

(A)

The bulk of the organic fraction is formed by type I collagen (90% of the ECM) and type III collagen (z5%). The rest is formed by proteoglycans (namely decorine, biglycan, and lumican). Phosphorylated proteins such as BSP and osteopontin are implicated in cementum mineralization. Osteonectin, a nonphosphorylated glycoprotein, may also be present. Growth factors such as FGF-2, the cementum-derived growth factor (CDGF) and the cementum attachment protein (CAP) [18] play specific roles in cementum formation. CDGF is thought to be implicated in the recruitment and differentiation of cementoblasts, whereas CAP promotes the adhesion of cementoblasts on the dentin/cementum surface. Dlx-2 and Runx2/ Cbfa 1 regulate the initial cement formation and, consequently, any defective expression of these molecules induces cement alteration. Finally, alkaline phosphatase activity impairment interferes with cement formation.

(B)

MECHANISM OF SINGLE TOOTH ERUPTION Introduction The eruptive path of the dentition has been extensively studied and theories on tooth eruption have been proposed since as early as the 18th century. Teeth are unique specialized structures that develop from discrete invaginations of the oral ectoderm. These structures then invade the mesenchyme of the jaws [19]. The dental follicle, which includes the tooth bud, has to travel through alveolar bone to erupt into the oral cavity. Once the crown of the tooth is close to the gingival tissue, the oral tissues merge with the epithelium of the follicle to form the gingival and periodontal ligament. Specific steps are necessary for a specific tooth to reach and maintain its position on the occlusal plane: following intrabony development, the tooth bud migrates, and erupts in the oral cavity. Once in the oral cavity, the tooth continues its migration until contact is established with the opposing teeth and an occlusal table is established (Fig. 5.5).

Mechanism of Tooth Eruption The formation of the eruption pathway is independent of root formation. This was demonstrated by placing transmandibular wires over premolars in dogs prior to the onset of eruption. An eruption pathway caused by bone resorption formed in the alveolar bone despite the fact that the premolars were maintained stationary. As soon as the wires were removed, the teeth quickly erupted through the formed eruption pathway [7].

FIGURE 5.5 Active eruption of an upper right permanent canine. (A) The tooth is erupting through the gingival tissue slightly buccally. (B) The tooth is now in contact with the mandibular canine. All teeth in the upper quadrant have now at least one point of contact with their mandibular opposing tooth.

Role of the Dental Follicle in Tooth Eruption At the beginning of active tooth eruption (16 weeks postnatal), the dental follicle consists of a well vascularized, discrete connective tissue layer surrounding the tooth [20]. Its importance in tooth eruption was demonstrated by Cahill and Marks in 1980 [21]. They showed that tooth eruption could be interrupted if the dental follicle was removed prior to the onset of eruption. The role of the forming tooth in the formation of the eruptive pathway was also negated when they replaced the developing tooth in the dental follicle by inert (resin or metal) material. This material erupted in the same way as the tooth would have, showing that the presence of a tooth was not necessary for tooth eruption [22]. The dental follicle therefore must possess all, or the majority, of the molecules needed in the normal eruption pattern [7]. Colony stimulating factor 1 (CSF-1) is an essential factor for normal osteoclast differentiation and bone remodeling in mammals. Toothless rats show a severely reduced number of osteoclasts as may be seen in osteopetrosis [23]. Marks et al. have shown that the administration of CSF-1 to the toothless rat has a positive effect

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MECHANISM OF SINGLE TOOTH ERUPTION

91

on tooth eruption, demonstrating the importance of CSF-1 and subsequently proper osteoclast activity on normal tooth eruption pattern [24]. Speed of Intraosseous Eruption The speed of intraosseous eruption depends on several factors. Each group of teeth is programmed to erupt at a different and specific time. However, the speed of intraosseous eruption is slow in relation to intraoral eruption and requires several years to complete. Tooth buds of the majority of permanent teeth are present in the infant, but will go through the gingival tissue only in the preadolescent years. Active eruption will begin after the crown of the tooth is fully completed [25] and will continue until the root is fully formed and the tooth is in contact with the opposing tooth on the occlusal plane Mechanisms of Intraosseous Tooth Eruption Tooth eruption is a complex event that involves many types of tissues. It is the only instance where a developing organ (the tooth) must exit the confines of its bony crypt [7]. To that effect, bone remodeling via osteoblasts and osteoclasts strategically positioned around the dental follicle must take place in order to ensure a normal eruption pattern. The eruption pathway must also continuously compensate for the increase in volume of the dento-alveolar process taking place simultaneously [7]. The dental crypt must resorb bone over the dental follicle by concentrating osteoclasts on its superior border at a specific time in the process of active eruption. Osteoblasts are more concentrated and abundant at the base of the dental follicle to produce alveolar bone. The combination of bone resorption and bone apposition, if properly balanced, will result in active intraosseous eruption [26e28]. The gubernacular canal, first mentioned by Hunter in 1778 [29] and in 1909 by James, is described as an eruption pathway used by the permanent tooth to replace the primary tooth. The gubernacular canals are too small to accommodate the crowns of permanent teeth, but are considerably enlarged by osteoclastic activity during the period of intraosseous tooth eruption [30]. The primary dentition mechanism of eruption via bone resorption is slightly different (Fig. 5.6). Osteoclastic activity is high, even if the tooth is stationary [20] and takes place on the superior aspect of the dental crypt. The gubernacular canal is enlarged and the tooth uses it as a pathway of eruption [31]. Primary teeth are very close to the surface [32] and a true intraosseous eruption pattern does not take place because the tooth buds are never totally covered with dense alveolar bone [33]. The crucial role of active bone resorption by osteoclasts in tooth eruption is demonstrated in osteopetrosis [34]. The teeth of osteopetrotic patients develop to their normal length, but fail to erupt into the oral cavity due to lack of osteoclastic activity, resulting in severe dental impactions.

FIGURE 5.6 Premolar tooth buds developing under primary molars. Note that the permanent molar does not replace a primary tooth but erupts distal to the primary dentition.

Genetic Control of Tooth Eruption According to the Tooth and Craniofacial Development Group of the University of Helsinki (www.bite-it.fi), a large number of genes is involved in the mechanism of tooth development and tooth eruption. Over 300 genes have been sequenced that involve several types of tissue [35]. Pelsmaekers [36] confirmed the genetic control of dental maturation in a study involving monozygotic twins. He showed that twins developed their dentition at the same time and at the same rate, regardless of the environment. It is of no surprise that a large number of heritable syndromes have a large dento-alveolar component and result in significant facial deformities by alteration of the pattern of tooth formation and tooth eruption [37,38]. Hypodontia, a condition in which some teeth fail to develop, is often associated with several syndromes, but it can also be found in the absence of other skeletal or ectodermal anomalies, suggesting the role of specific genes in the formation of the exact number of teeth and their proper positioning on the arch [39]. Mechanisms of Intraoral Tooth Eruption Multiple factors contribute to the onset of tooth eruption. Growth factors, such as insulin-like growth factor-1 (IGF-1), VEGF, tumor necrosis factor a (TNFa), are implicated in the transformation of monocytes into osteoclasts. Colony stimulating factor 1 (CSF-1) and monocyte chemotactic protein-1 (MCF-1) act in synergy with osteoprotegerin (acting as an inhibitor) and the overexpressed RANKL to influence osteoclastogenesis [40]. Four other factors, PTH [41], retarded protein interleukin-1alpha, Cbfa1/Runx2 (transcription factor) and the presence of osteoblasts also play a role. Interactions between growth factors,

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5. DENTAL DEVELOPMENT AND MATURATION, FROM THE DENTAL CRYPT TO THE FINAL OCCLUSION

transcription factors and hormones are responsible for the differentiation of mesenchymal cells into osteoblasts at the base of the crypt and osteoclasts at the superior aspect of the dental crypt. These changes are observed during the active part of tooth eruption, and result in the tooth bud erupting through the oral mucosa. Important Factors Contributing to Lack of Tooth Eruption The best model to study proper eruption sequence is a condition that has been termed “primary failure of eruption” [42]. It is a multifactorial event where teeth develop normally but fail to erupt in the oral cavity and remain impacted. Dento-alveolar development continues around these teeth and a normal dento-alveolar height is achieved by periosteal growth of the bone formed around the adjacent teeth. This condition can affect all teeth but is more prevalent in the posterior segment. This phenomenon is also observed in osteogenesis imperfecta types III and IV patients who develop severe lateral open bites during growth (Fig. 5.7 ). The dento-alveolar process fails to develop normally in the area of the non-erupting tooth (Fig. 5.8). Severe occlusal anomalies are observed, such as posterior open bites while adjacent teeth tip into the space of the non-erupting tooth contributing to the creation of a severe malocclusion. The tooth is usually ankylosed and will not respond to orthodontic traction suggesting that the eruptive processes are absent and that the periodontal ligament’s response to mechanical traction does not result in the typical bone remodeling

FIGURE 5.8 Multiple impacted teeth of non-syndromic origin. A 16-year-old patient with only eight permanent teeth erupted, residual primary molars and 21 permanent teeth impacted. Note the lack of development of the maxillary anterior dento-alveolar process.

process observed in normal tooth eruption or orthodontic traction. Ankylosis occurs when cementum from the root of the tooth fuses with the dento-alveolar process. It may either be complete or partial ankylosis as when only some parts of the root fuse with the alveolar process. Bone remodeling stops in the affected area of ankylosis and the tooth fails to erupt. Lack or severely reduced dento-alveolar development is associated with this condition. Orthodontic traction is useless and the tooth either stays impacted or does not totally reach the occlusal plane [42]. True impaction may also occur when the tooth deviates from its normal path of eruption, totally forms in the bone and fails to erupt. Maxillary canines have the highest incidence of impaction, probably due to their complex path of eruption (Fig. 5.9). Once uncovered and provided that adequate space is created on the arch, these impacted teeth can be brought into proper position with orthodontic traction [43].

DEVELOPMENT OF THE DENTAL OCCLUSION Dental occlusion may be static or dynamic. The static phase refers to the stage when the mandibular teeth are brought into light contact with the maxillary teeth by the closing motion of the mandible. Dynamic occlusion occurs when teeth come in contact during chewing, speaking or swallowing.

Timing of Eruption FIGURE 5.7

Dentition of 12-year-old type III osteogenesis imperfecta patient showing bilateral posterior open bites. Permanent molars have erupted several years ago but have failed to come in contact, probably due to lack of growth of the dento-alveolar process. The dento-alveolar process fails to develop normally in the area of the non-erupting tooth.

Modern mammals have a diphyodont teeth arrangement, that is a deciduous and a permanent dentition in succession over their lifespan [44]. In humans, the primary dentition is made up of 20 teeth including incisors, canines and primary molars. Active intraosseous eruption of primary teeth occurs in the first and second

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93

FIGURE 5.10 Panoramic radiograph demonstrating a normal sequence of eruption in a 9-year-old patient. First permanent molars and upper and lower incisors are present in the mouth. Canines and premolars are resorbing the primary canines and molars.

FIGURE 5.9

Impacted upper right maxillary canine. The tooth has deviated from its normal path of eruption and is now resorbing the maxillary lateral incisor. The lower left second premolar is also impacted and poorly angulated as the lower left second primary molar has failed to resorb.

year of life and by 29 to 30 months, all 20 primary teeth are in place [45]. From the 30th month of life until the late teens, the permanent dentition develops, first with a path of intraosseous eruption followed by intraoral eruption. These movements are of large amplitude in relation to tooth size, occur in the three planes of space and must adjust to the continuous development of the dento-alveolar processes during growth [46]. Once teeth reach the occlusal plane, they continue to adjust with minute movements to stay in proper contact mesiodistally as well as occlusally. As the dental crypt moves toward the oral cavity, bone resorption takes place on the occlusal aspect of the crypt and bone apposition occurs at the base. At the same time, bone formation also increases the size of the dento-alveolar process. Complete remodeling of the alveolar bone occurs when deciduous teeth are replaced by succedaneous teeth. The alveolar bone associated with the primary tooth is completely resorbed together with the roots of the tooth while new alveolar bone is formed to support the newly erupted tooth [47]. The eruption sequence normally follows a path from the anterior to the posterior aspect of the mouth and is always constant during a normal eruption pattern (Fig. 5.10). Factors such as osteoprotegerin, RANKL, CSF-1 and VEGF control the path and sequence of eruption but their interactivity is still a subject of debate [24,48].

Intraosseous eruption of the lower incisors results in the eruption of the lower central incisors at 6 years of age, the first succedaneous teeth to erupt. Continuous eruption of succedaneous teeth occurs in the anterior part of the oral cavity until the age of 12e13 when the upper canines erupt [49]. The dental follicles of the permanent incisors, canines and premolars actively resorb bone and the primary tooth root structure, in order to appear in the oral cavity [50]. Odontoclasts, cells which are very close to osteoclasts, are actively involved in this process [51] and are linked to an increased expression of RANKL and to a decrease in osteoprotegerin (OPG) [52e54]. The absence of activity of odontoclasts results in non-resorption of primary teeth [55]. The succedaneous dentition continues to develop but the lack of resorption of the primary dentition creates an obstacle to eruption, resulting in an alteration of the path of eruption of the tooth buds, and severe impaction [56]. An example of this situation is the dentition of osteogenesis imperfecta types III and IV patients who show multiple impacted teeth caused by an alteration in osteoclastic activity leading to an absence of resorption of the primary dentition (Fig. 5.11) [57e59]. During the replacement of the primary dentition, intraosseous eruption takes place in the distal aspect of the primary dentition where the permanent molars

FIGURE 5.11 Impaction and malformation of premolars in an osteogenesis imperfecta type III patient. The crowns and the roots are no longer aligned as the primary tooth lack of resorption does not allow normal development of the premolar.

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erupt. A large amount of growth in the three planes of space associated with remodeling of the alveolar process and of the anterior aspect of the ramus must take place as the dental arches have to increase their size significantly to allow the eruption of these large teeth. The first molars will normally erupt between the ages of 6 and 8 years while the second ones appear around the age of 12 years. There is considerable variation in the timing of eruption between individuals. However, the sequence has to be consistent to avoid impaction and ectopic eruption of some teeth. The third molars or wisdom teeth are normally programmed to erupt around the age of 18 years but will fail to erupt and remain impacted in about 75% of the population. This is probably due to the lack of sufficient growth of the tuberosity and ramus in modern humans, resulting in a space deficiency to allow a normal eruption pattern for these teeth. The pattern of tooth eruption is an organized bilateral event where pairs of teeth from the right and left part of the jaws will erupt at about the same time [59]. Any significant change in this sequence will compromise the eruption pattern or sequence. Teeth may become impacted if primary teeth are lost prematurely and space becomes inadequate.

Pattern of Tooth Eruption Teeth are genetically designed [60] to erupt in sequence, and position themselves in an orderly manner in the plane of occlusion. The movement of eruption takes place in the three planes of space with vertical (pure eruption), labiolingual (transverse) and mesiodistal (drift) movements [31]. Tooth buds can also rotate during the intraosseous eruption phase. Each tooth shows an individualized pattern of eruption until it reaches its final position on the occlusal plane. The best example is the maxillary canine which tends to erupt in a mesial direction until it reaches the lateral incisor roots. It then turns to follow a more vertical path and may sometimes adopt a more mesial path of eruption [61]. Once in the oral cavity but still away from the occlusal plane, eruption is rapid until the tooth reaches the occlusal plane [62]. Final occlusal positioning is genetically programmed but is very dependent on the functional envelope of muscles and tissues surrounding the alveolar process. Moss [63,64] has described how the dento-alveolar process responds to alterations in the functional matrix, the development of which is guided by the patients’ vital functions such as breathing and feeding. During sleep apnea episodes, patients are unable to breathe through their nose and keep their mouth constantly open. This altered mandibular position results in a potential opening of the gonial angle (the angle formed by the junction of the ramus and the body of

the mandibular bone) and a vertical descent of the upper molars (Fig. 5.12). Constricted maxillary arches, severe crowding and anterior open bite may result from the disturbance in the proper breathing mechanism. Harvold has shown that forcing mouth breathing in monkeys resulted in the development of severe malocclusions [65]. The changes are severe enough to alter the facial appearance and modify mandibular growth [66].

Speed of Eruption While the intraosseous rate of eruption is fairly slow (1e10 mm/day) [62], once in the oral cavity, the speed of eruption is faster until teeth reach the occlusal plane. Proffitt reported a mean daily eruption rate of 25 to 75 microns for premolars out of occlusion [62]. The entire amount of eruption took place at night. Once in contact with the opposing dentition, active eruption stops but drift continues to take place via remodeling of the alveolar bone by the periodontal ligament (PDL) cells to ensure constant contacts in occlusion [67]. The speed of tooth eruption presents several characteristics. First, eruption itself has to proceed at a certain rate to ensure that the eruption sequence is respected and that the dentition develops in an orderly manner. Prolonged delays may prevent eruption and result in ankylosis of tooth to bone. Second, the eruption speed is not uniform. Erupting teeth move at different speeds at different times. Initially, eruption speed is slow within bone; it increases thereafter and becomes very slow as

FIGURE 5.12 Cephalometric radiograph of a 12-year-old male patient diagnosed with moderate to severe sleep apnea. The gonial angle of the mandible is opened and the anterior teeth are not in contact resulting in an anterior open bite.

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the tooth approaches the occlusal plane. These remarkable shifts in speed are also seen in root formation which is fast at first, slows as the apical foramen closes and is very slow thereafter. Third, this range of velocities implies that bone resorption and formation, like root growth, must occur at variable speeds depending upon the stage of eruption. Fourth, during intraosseous eruption, the rate of bone (and root) resorption determines the rate of eruption [25,68]. Continued Tooth Eruption Throughout Life Once the tooth is in occlusion, minute movements in the three dimensions of space will take place to compensate for secondary growth, and abrasion of occlusal and interproximal dental surfaces. Tooth movement takes place more as a displacement caused by dento-alveolar remodeling than tooth eruption. This stage cannot be described as active tooth eruption but as an occlusal compensation to changing conditions in the oral cavity. If an opposing tooth is lost or not present, active tooth eruption may resume with the erupting tooth elevating itself above the plane of occlusion. The dento-alveolar process will follow the erupting tooth and elongate. Tooth movement is present throughout lifetime. Changes in functional factors, such as sleep apnea, periodontal disease or tooth loss will all result in alteration of the occlusion through tooth movement and dentoalveolar remodeling [68].

MATURATION OF THE PERIODONTAL LIGAMENT The Periodontal Ligament: Role and Development of the Dento-Alveolar Complex The development of the periodontal ligament is derived from the inner layer of the dental follicle shortly after initiation of root development [69] and begins with root formation prior to tooth eruption [70]. The developing ligament contains undifferentiated cells capable of differentiating into osteoblasts, osteoclasts and fibroblasts. This combination of cells is responsible for a continuous remodeling of the alveolar bone. Dento-alveolar development from the PDL cells will compensate for the constant change in position of the tooth [68,70]. The periodontal ligament development is a highly organized process [71]. The PDL is a highly vascular and cellular connective tissue situated between the tooth and the alveolar bone. It provides supportive attachment and sensory functions [68]. Cells, vascular elements and an extracellular compartment of matrix proteins and glycosaminoglycans provide unique biophysical functions that enable mammalian teeth to adjust their position while

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remaining attached to the bony socket. Fibroblasts are the predominant cells of the periodontal ligament and have important roles in the development, structure and function of the tooth [72].

The Periodontium The periodontium, which is composed of the root cementum, the dento-alveolar process and the periodontal ligament is a unique structure with a variety of functions [73]. The main one is to provide mastication control and feedback and the second is to ensure optimum positioning of the teeth on the occlusal table. The ligament presents a high cellular turnover rate and Constant remodeling takes place as teeth being subject to masticatory forces and migrate in the dento-alveolar process. During the formation of the orofacial complex, the immature periodontal ligament around the dental follicle participates in the dental eruption and dentoalveolar process development. The maturing fibrils of the periodontal ligament shrink which may contribute to tooth eruption [74,75]. At the end of the intraosseous eruption process, the nascent periodontal ligament fuses with the oral epithelium and forms the periodontium. The ligament is responsible for the intraoral eruption stage of development and guides the tooth into its proper position on the dental arch. Once in position, the PDL will maintain the dental alignment and interdigitation position in relation to the other teeth for the life of the patient by constant remodeling of the dentoalveolar processes [68].

DENTO-ALVEOLAR GROWTH AND DISPLACEMENT OF BONE STRUCTURES Alveolar bone undergoes continuous remodeling due to tooth movement, masticatory forces and dental migration [47]. The periodontal ligament will also constantly remodel the alveolar bone, first rapidly during the period of active eruption and then more slowly once the teeth are in occlusal contact.

DENTAL DEFECTS AND SYNDROMES 1. Dental agenesis and hyperdontia 2. Dental defects • Shape defects • Enamel defects • Dentin defects

Dental Agenesis Tooth formation and morphogenesis are under strict genetic control [76]. Crucial molecules involved in tooth

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formation include RUNX2. In humans, heterozygous loss of function of MSK1 or PAX9 causes oligodontia [76,77]. Agenesis of a single tooth or teeth is a frequent anomaly in human dental development [60]. Oligodontia, hypodontia and anodontia are interchangeable names for this anomaly (Fig. 5.13). Familial tooth agenesis (FTA) is a well known condition in which the most commonly missing permanent teeth are the third molars (20% of the population), followed by the maxillary lateral incisors, the mandibular second bicuspids and the mandibular central incisors respectively [78,79]. Diseases such as scarlet fever or nutritional problems during pregnancy can result in dental agenesis. However, agenesis of several teeth is usually associated with heritable syndromes such as ectodermal dysplasias which constitute a wide and complex group of diseases. Ectodermal Dysplasia The most representative example of this group of conditions is anhydric ectodermal dysplasia with an incidence of between 1/10 000 and 1/100 000 live births. The defective gene is ED1 in humans, and Ta in the mouse [4]. It is X-linked and hemizygotes lack most of their dentition. Dental agenesis is strongly correlated to the absence or severe lack of dento-alveolar bone development as tooth eruption is largely responsible for the increase in volume of the dento-alveolar process. Thus, children affected by ectodermal dysplasia present with no or only a few teeth of conical shape and are deprived of the corresponding alveolar bone development (Fig. 5.14). Vertical face height development is stunted. The upper jaw also exhibits reduced length with a small palate and a correspondingly reduced cranial base width [80]. These patients look older than

FIGURE 5.13

Hypodontia. Very few permanent teeth are present but the ones present are normally shaped. This patient has only the upper central incisor in the maxilla and no lower incisors. Several permanent posterior teeth are also congenitally absent. The dentoalveolar process in the areas of anodontia is not severely reduced in size.

FIGURE 5.14 Ectodermal dysplasia. Hypodontia, abnormally shaped teeth such as conical crowns and short roots, and lack of development of the dento-alveolar processes are common findings in this syndrome.

their age due to sparse scalp hair, pursed lips and depressed nose base. Sofaer [81] found that 1 in 500 females with hypodontia in the permanent dentition and 1 in 50 with hypodontia in the primary dentition may be carriers for anhydric ectodermal dysplasia, suggesting a gene dose effect in ED1 mutations. Incontinentia Pigmenti-Williams syndrome Incontinentia pigmenti (BlocheSulzberger syndrome) is a syndrome similar to ectodermal dysplasia. It is also X-linked and often lethal in males. Its incidence reaches 1/40 000 in females. It affects both the primary and the permanent dentition, and 90% of patients show oral changes such as missing or conical teeth. There is a typical alopecia at the crown of the head, and the eyes are affected [82]. Ocular changes may be serious, such as severe myopia, retinal detachment or optic atrophy. Problems in mental development are found in 15% of the cases. Axelsson [83] examined 62 individuals with Williams syndrome and found that 40.5% of them lacked one or more permanent teeth while 11.6% of them lacked six or more permanent teeth. This appears to be linked to a contiguous gene deletion at chromosome 7 (7q11.23). Cherubism Cherubism is a rare neoplastic disease affecting particularly the jaw bones (Fig. 5.15). Multilocular fibrous dysplasia of both the maxilla and the mandible result in painless enlargement of these craniofacial bones. The dentition is affected by hypodontia and severe malocclusion [84]. The name cherubism comes from the fact that the face becomes deformed and almost circular due to fibrous proliferation. This condition affects mainly growing children and has been shown to regress in adulthood. Long bones are unaffected by the condition [85].

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FIGURE 5.15 Cherubism. Multilocular and radiolucent aspect of the maxillary and mandibular bones. Hypodontia, malformed teeth and severe malocclusion can be observed in this panoramic radiograph.

Cleft and Lip Palate Children born with unilateral or bilateral lip/palate clefts or palatal cleft alone present a high incidence of hypodontia (Figs 5.16 and 5.17) [86]. Lack of fusion between the maxillary and the medial nasal process explains the interruption or fragmentation of the dental lamina causing the agenesis of the lateral incisor on the cleft side. In a 2003 study, 49.8% of the cleft lip/palate children were missing this particular lateral tooth [87]. What is more intriguing is the dental agenesis of the teeth outside the cleft area, where 10.9% of patients lack the contralateral tooth [60]. The second most commonly missing permanent teeth are the maxillary second bicuspids, followed by the mandibular second bicuspids, and finally the first bicuspids [60]. Children with isolated cleft palate also have a high incidence of hypodontia (31.5%). Even in the absence of an alveolar cleft the teeth most frequently absent are the maxillary laterals

FIGURE 5.16 Occlusal view of a unilateral cleft palate. Collapse of the arch on the affected side, maxillary midline deviation and asymetry. The upper left lateral incisor is misshapen. The upper left canine is impacted.

FIGURE 5.17 Bilateral cleft lip and palate. Premaxilla is formed and includes the two central incisors. The clefts extend from the palate, the dento-alveolar process to the lip. Partial anodontia (absence of the permanent canines) and poorly shaped teeth in the area of the cleft are present.

followed by the second bicuspids. The more severe the palatal cleft, the more severe the hypodontia [88]. Down Syndrome The prevalence of tooth agenesis in Down syndrome is high. It is about 10% higher than in the general population and affects predominantly the mandibular permanent incisors [89].

Hyperdontia Hyperdontia (supernumerary teeth) is rarer than oligodontia. In the primary dentition, the maxillary lateral incisor is the only supernumerary tooth (0.10%) [90]. One to 3% of the general population will exhibit hyperdontia in the permanent dentition [91] and the supernumerary tooth is generally the maxillary lateral incisor. A Hong Kong study indicated a 2.7% rate for males and a 6.5% rate for females [104]. There is a higher incidence in Black (6%) than in Caucasian (0.64%) populations. Black children have more supernumerary teeth in the molar and bicuspid areas [92]. Some supernumerary teeth are distorted, conical and small. The most common is the mesiodens, a small conical tooth that is found between the permanent maxillary central incisors (Figs 5.18 and 5.19). If the abnormal tooth appears in the molar area, it is called a paramolar, and if located distal to the third molar, it becomes a supplemental tooth. The last category of non-syndromic hyperdontia is the odontoma. It is considered a hamartomatous

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FIGURE 5.18 The mesiodens is a small tooth usually located between the two upper central incisors and inverted (crown facing the nasal floor).

malformation. The odontoma can be complex (made of dental tissue totally disorganized) or compound (when it bears resemblance with a normal tooth). A supernumerary tooth is the primary cause of the lack of eruption of the maxillary permanent incisor [92]. The presence of multiple supernumerary teeth is often indicative of a syndrome, such as cleidocranial dysplasia [93]. Cleidocranial Dysplasia Cleidocranial dysplasia (CCD) is the best example of a syndrome associated with multiple supernumerary unerupted teeth (Fig. 5.20). Its incidence is about 1/ 1 000 000 with no preference for gender or race [94]. It is an autosomal dominant condition, characterized by delayed closure of the fontanels, brachycephalic skull, hypoplastic or aplastic clavicles and numerous dental anomalies. Multiple supernumerary teeth are part of the syndrome, as are retained primary teeth, and unerupted permanent teeth. Mutations in CBFA1 (RUNX2), mapped to chromosome 6p21, have been identified as

FIGURE 5.19 A rare view of a mesiodens that has erupted in the mouth on the labial aspect of the central incisors.

FIGURE 5.20 Cleidocranial dysplasia. A defect in the dental lamina results in a large number of supernumary teeth. These teeth usually stay impacted and are often misshapen. Primary teeth also fail to resorb especially in the posterior segments.

responsible for CCD [76,93]. CBFA1 is a transcription factor acting as a major controller of osteogenesis. Variable loss of function of CBFA1 may give rise to clinical variability, including classic CCD, mild CCD, and isolated primary dental anomalies. Ooshima et al. [95] reported genotypeephenotype associations such as “the more supernumerary teeth, the shorter the individual”. Mundlos [96] reported that lack of remodeling leads to persistence of the dental lamina, resulting in the formation of multiple supernumerary teeth at the same time that the crowns of the permanent teeth are formed, creating a third dentition [97]. Actually, it is the same type of bone dysplasia that links the supernumerary teeth to the persistence of the primary teeth in the mouth and to the delay, or non-eruption, of the permanent teeth.

Dental Defects Size and Shape Teeth can be abnormally small (microdontia), abnormally large (macrodontia), or misshaped. A study of tooth size in 43 parental pairs and their 100 offspring supported the hypothesis that tooth size is a sex-linked trait [98]. Microdontia is the most common developmental abnormality after hypodontia (Fig. 5.21). There is a significant association with hypodontia, and the more severe the hypodontia, the greater the possibility of microdontia [99]. A strong association was also found between agenesis of the second premolar and the pegged maxillary incisor [79]. Environmental conditions also contribute to variation in human tooth size [100]. In pregnant women affected with hypothyroidism, diabetes or hypertension, odontometric analyses in the offspring showed that maternal hypothyroidism and diabetes resulted in an increase in tooth size, while hypertension was associated with decreased tooth size.

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FIGURE 5.21 Non-syndromic 18-year-old female patient presenting with hypodontia, several severely misshapen and smaller than normal teeth not associated with a syndrome. Lateral and anterior open bite, anterior crossbite are also part of the malocclusion.

Chemotherapy and radiotherapy in infants, babies and young children contribute to changes in both primary and permanent dentition. The defects usually appear as hypodontia, microdontia, enamel hypoplasia and stunted roots. All these defects were present in the dentition of 52 surviving children afflicted by malignant diseases, even though none of them had received radiotherapy to their facial structures. Microdontia was present in 20% of this group, significantly above the 2.5% average for the normal population [101]. Severe and generalized microdontia is associated with several syndromes. Rieger syndrome, an autosomal dominant genetic defect, whose main features are ocular (microphthalmia), craniofacial (hypoplastic alae nasi) and hypospadia, presents with, in addition to hypodontia, an anterior mandibular tooth of smaller size and tapered in shape [102]. Identical dental findings are found in Williams syndrome, where teeth are tapered or incisors are screwdriver-shaped [83]. In Turner syndrome, there is a reduction of the dental crown’s height [103]. Several root anomalies were also reported. Trisomy 21 individuals also have a permanent dentition of reduced size [104]. The opposite was true for the deciduous dentition [104]. The authors hypothesized that, while the decreased mitotic activity of cells could logically explain the general reduction in the permanent teeth size, the phenomenon did not take place at the time of deciduous teeth mineralization (8e10 weeks of gestation) when the fetus experiences a burst of cellular activity. The result of this increased activity would be larger than normal deciduous teeth. Macrodontia is a defect rarely found in the normally developing child. However, there is a positive correlation with body stature and tooth size [100]. In subjects with a 47 XYY phenotype, the extra Y chromosome appears linked to larger teeth, probably because it

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provided a stronger and longer mitotic cell activity during the individual’s prenatal and early postnatal periods [105]. The KBG syndrome is an autosomal dominant trait characterized by short stature, facial anomalies (telecanthus, wide eyebrows, brachycephaly), mental retardation, skeletal anomalies (abnormal vertebrae, short metacarpals, short femoral necks) and macrodontia (mostly large upper incisors) [106]. Dental root anomalies are not frequent, and mostly limited to a reduction in size (short root anomaly). Normal dental roots are approximately twice the length of their corresponding crowns. A ratio of one to one or less defines a short root anomaly. Anomalies often associated with short roots are hypodontia, peg-shaped teeth, dens invaginatus and taurodontism. Short root anomalies may be limited to a few teeth (usually maxillary permanent laterals) or may affect the whole dentition. Chemotherapy, radiotherapy and total body irradiation can be devastating for the permanent dental root development in the pediatric community. A study mentioned earlier found abnormal permanent root development in long-term survivors of malignant diseases, with the root disturbance corresponding to the duration of treatment and dose of irradiation [101]. The addition of chemotherapy treatment further disturbs tooth formation. The extent of the damage seems to depend upon the stage of histodifferentiation of the tooth and the amount of rads administered. Any dose higher than 2000 rads produced serious dental defects, regardless of the child’s age. The authors described a case of a boy who had received 4050 rads to the right middle ear (rhabdomyosarcoma) and to the right cervical lymph nodes when he was 2 years old; the rads were supplemented with vincristine, actinomycin D and cyclophosphamide. At age 13, his panoramic radiograph revealed that his permanent teeth were present in the oral cavity but no root formation was noted on the first molars and several teeth such as the second premolars and molars were not developing normally (Fig. 5.22). Hultta¨ et al. reviewed the radiographs of 52 children who had received stem cell transplantation before the age of 10. The children were treated with anticancer therapy for acute lymphomatic leukemia, acute monocytic leukemia and non-Hodgkin lymphomas. A postoperative assessment took place at an average age of 7.2 years, and 945 teeth were examined. All patients presented with severe disturbances in the root development of the permanent teeth. The most severely affected teeth were found in patients who were 3.1 to 5.0 years at the time of the transplantation. Taurodontism is a dental root anomaly found in 2.5% of the Caucasian population. It is caused by the apical enlargement of the body of the tooth (or coronal part)

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FIGURE 5.22 Rhabdomyosarcoma patient having received radiotherapy at a young age. Note the total absence of development of the roots of the first molars, Conical root shape and malformed second molars and premolars. Lower left second molar is not developing as well as the third molars.

at the expense of the root length. Therefore, the roots appear much shorter. Simultaneously, the classic cemento-enamel constriction lessens or disappears. Taurodontism is often found in individuals with aneuploidy of the X chromosome [107]. Enamel Defects Enamel is a unique, highly mineralized tissue. Its protein constituents, amelogenin (80e90%), ameloblastin (5e10%), enamelin (3e5%) and enamelysin (1%) are produced by ameloblasts. They are responsible for enamel maturation. Deleterious effects of environmental and systemic factors during enamel formation are often traceable when the tooth emerges into the oral cavity. Defects in any of the above proteins can trigger enamel defects without affecting other parts of the structure [108]. Enamel defects are also part of syndromes that are hereafter mentioned. Each group of teeth has a unique anatomy and is easily recognizable. Each crown of a particular tooth may show specific alterations that may or may not endanger its integrity. For example, “dens in dente” or “dens invaginatus” is a developmental anomaly where the enamel of the maxillary incisors enfolds itself on the lingual pits and penetrates into the pulpal space. The “dens in dente” is a structure with its own inner enamel, dentin and blood supply (Fig. 5.23) [109]. As its name implies, it looks like a tooth within a tooth. If the opening is not protected and sealed in time, the invagination may communicate with the pulpal space which may become contaminated by oral bacterial flora. A pulpal infection ensues, requiring endodontic therapy. The incidence of pulpal necrosis is between 0.25 and 9.66%. “Dens evaginatus” or “tubercle shaped cusp” is the opposite of dens in dente. The enamel folds itself outwardly and resembles an extra cusp near the central

FIGURE 5.23 Dens in dente. The dental papilla is forming a second tooth inside the normally developed tooth.

groove of the tooth [110]. This event takes place in the early stage of odontogenesis. Posterior permanent teeth, notably bicuspids, most frequently present with this type of enamel defect. During active intraoral tooth eruption, the opposite tooth will eventually occlude into the central groove and fracture the little tubercle. The dentin and the minuscule pulpal tissue are then exposed, and the tooth becomes infected. This anomaly is frequent in subjects with Asian ethnic background but is also found in other races [111]. The “talon cusp”, which resembles the “dens evaginatus”, limits itself to maxillary incisors (Fig. 5.24). It favors deciduous teeth, but may be found on permanent teeth as well. It can interfere with the patient’s occlusion and presents both an esthetic and a treatment challenge [112]. Enamel defects are also associated with nutritional deficiencies. They appear as linear or irregular spots on the teeth, usually on the edge of the permanent incisors and on the superior third surfaces of the first permanent molars. These surfaces undergo their odontogenesis during the neonatal period and during the first year of life. Vitamin D, calcium and phosphorus deficiencies as well as any severe nutritional deficiency will trigger such defects. Other disturbances may also cause enamel defects. A low calcium concentration during enamel formation is a specific determinant of enamel hypoplasia [113].

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FIGURE 5.25 Hypoplastic phase of enamelogenesis imperfecta FIGURE 5.24 Talon cusp that does not allow for proper tooth positioning as it interferes with arch alignment.

Enamel mineralization defects or amelogenesis imperfecta (AI) [108] may take place at different stages of odontogenesis. When the damage occurs during the secretory time (hypoplastic defect), the enamel is of good quality, but of insufficient quantity, sometimes practically nonexistent. When the damage occurs during the mineralization time (hypomineralized defect), the enamel is of normal thickness but poor quality. When it takes place during maturation time (protein processing and crystallite maturation defect), the enamel retains its normal thickness but quality is even poorer. Most enamel defects have a dominant inheritance. Enamelin (ENAM 4q21) is the main gene involved. A classification of AI was proposed by Witkop in 1988 [108]. He stated that AI, a developmental intrinsic defect in enamel formation, can be classified into four different types: 1. Hypoplastic defect, where the enamel does not develop to its normal thickness (Fig. 5.25) 2. Hypomaturation defects, where the enamel is of normal thickness but of mottled appearance 3. Hypocalcification defects, where the enamel is of normal thickness but friable and yellowish-brown in color 4. Hypomaturationehypoplastic defects associated with taurodontism (hypoplastic and hypocalcified enamel in teeth with atypically enlarged pulp chambers). The complexity and the high intrafamilial variability, together with the difficulties in diagnosing the precise type of AI (such as hypocalcification versus hypomaturation), make it difficult to identify the precise gene defect. Teeth with AI are difficult to maintain, resulting in a high proportion of edentulism among family members. Syndromes associated with AI are rare. In

(see also Fig. 5.2). The enamel is normal but in very small quantity. Notice that the enamel is already fracturing at the incisal edges and is very translucent.

mucopolysaccharidoses (MPS) enamel can be specifically targeted. MPS is a family of metabolic disorders (lysosomal enzyme deficiencies) that share an autosomal recessive inheritance (except for MPS II which is sexlinked). They are all affected with “dysostosis multiplex”. Yet only one MPS, the Morquio syndrome (MPS IVB), is associated with enamel defects, even though the affected individuals show milder phenotypes than their counterparts with MPS IVA. The enamel is yellow, brittle and weak [114]. The tricho-dento-osseous syndrome (TDO), an autosomal dominant trait, is caused by a mutation in DLX3, mapped to 17q21. It presents with a combination of amelogenesis imperfecta (pitted thin and discolored enamel) and taurodontism. The taurodontic tooth is characterized by an abnormally large pulpal space and absence of the constriction between the crown and the root (cemento-enamel junction). TDO individuals have kinky hair (curly at birth), thick dense bones and brittle nails. The dental phenotype is present in all cases [115]. Focal dermal hypoplasia (FDH) or GoltzeGorlin syndrome presents with severe dental defects: the enamel is irregular and misshaped and covers small misaligned teeth. Hypodontia is present as well. It is transmitted as a sex-linked trait on the short arm of the X chromosome [116]. Dentine Defects In dentine, collagen (mostly collagen type I) serves as a network for the forming carbonate apatite. Dentine matrix protein (DMP1), dentin sialoprotein (DSP) and the dentin phosphoprotein (DPP) are the noncollagenous components that regulate dentinogenesis. The most important dentinal defect is dentinogenesis imperfecta (DGI), an autosomal dominant trait (Figs 5.26 and 5.27). In 1973, Shields et al. divided DGI into

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FIGURE 5.26 Dentinogenesis imperfecta. Teeth are brownish in color, fracture easily and are very difficult to restore. Enamel fractures easily as the dentineeenamel bond is weak.

three subgroups [117]. All of them are linked to chromosome 4q21, and result from mutations in DSP [108]. The first group, or DGI type I, is linked to osteogenesis imperfecta (OI); the second group, DGI type II, presents with almost the same clinical picture but with no other systemic involvement; the third type, DGI type III, is a rare isolated disorder known as the “Brandywine isolate”. Two more dentinal defects, dentin dysplasia I [101], an exceedingly rare condition and dentin dysplasia II (DDII) which bares close resemblance with DGI II, complete the list [117]. Dentinogenesis imperfecta type II is one of the most common dominantly inherited disorders. Its incidence is around 1/8000 and its penetrance is high [118].

FIGURE 5.27 Dentinogenesis imperfecta in the mixed dentition. Severe wear and attrition are visible. Permanent molars have a blueish coloration.

In 2003, Sreenath et al. [119] studied the role of the Dspp gene in tooth mineralization in Dspp-null mice and concluded that it regulated proteoglycan synthesis during dentinogenesis. Beattie et al. performed linkage and mutation analysis in studying phenotypic variation in dentinogenesis imperfecta/dentin dysplasia (DDII) linked to 4q21 in a large pedigree spread over four generations. The related individuals all displayed dentin defects associated with DGI and DDII. The pattern of inheritance was autosomal dominant with complete penetrance. They concluded that type II dentin dysplasia and DGI type II as well as DGI type III were non-syndromic heritable dentin defects and should be put in one single category and classified by their respective severities [120]. DSPP is expressed in bone as well as in dentin. However, bone is not involved in DGI type II [3]. It is thus possible that other molecules in bone exert redundancy. DGI is easily recognizable. The teeth are opalescent, with a color varying from gray to yellow. They are fragile and the enamel chips off easily because its poor adhesion to dentin and the dentin itself is defective. Dental fractures can be severe because the circumpulpal dentin (deeper dentin) is weak and does not resist stress. On radiographs, the crowns appear bulbous, the roots are slender and short and the pulp spaces are obliterated by irregular dentin. Dentinogenesis imperfecta type I is associated with the osteogenesis imperfecta syndrome (OI) [58,121] (see Chapter 18). In this heritable disorder, bone brittleness is associated with a decreased bone mass. The syndrome is associated with other anomalies, such as blue sclerae, hearing loss and joint hypermobility [57]. Most patients harbor a mutation in one of the two genes encoding type I collagen [122]. Sillence has proposed a classification into four types which is still frequently used [123]. Type I is the mildest form, type II is lethal, type III is the most severe one compatible with life (Fig. 5.28) and type IV, a heterogeneous group with variable severity. Glorieux and his group were able to describe three more types (V, VI and VII) [122]. When OI is caused by a mutation in a protein other than collagen type I, DI is not present. In the past, OI linked to collagen I defect was further classified as having DI or not. However careful examination of apparently normal dentitions may show subtle signs of DI [124]. Deciduous teeth are more affected than the permanent ones. Other problems are failure of eruption, tooth impaction and severe malocclusion. The most common type of malocclusion in the OI patient is the class III malocclusion. A lack of anteroposterior development associated with a larger mandible results in anterior crossbites. Lack of dentodevelopment, especially in the posterior region, leads to the development of lateral open bites (Figs 5.29 and 5.30) [58]. The latter may occur

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DENTAL DEFECTS AND SYNDROMES

FIGURE 5.28 Osteogenesis imperfecta type III patient presenting

103

FIGURE 5.29 Lateral cephalometric radiograph of an osteogenesis imperfecta type III patient presenting the typical lack of anterior projection of the maxilla and lateral posterior open bites.

with dentinogenesis imperfecta. Anterior crossbite is observed.

even in forms of OI not linked to type I collagen abnormalities [3,124]. X-linked hypophosphatemia (XLH), is a heritable disease caused by impaired renal phosphate transport (see Chapter 26) (Figs 5.31 and 5.32). It is linked to mutations in the PHEX gene. It results in rickets, osteomalacia, growth retardation and dentin defects [125, 126]. The latter trigger spontaneous dental abscesses on teeth that appear intact and devoid of carious lesions. Schwartz et al. examined 14 patients with XLH and found a high percentage of these particular abscesses. The teeth showed impaired dentin mineralization that allowed elongated pulp horns to reach the cementoenamel junction and, through minor enamel abrasions, be exposed to the oral bacteria [127]. Shields et al. measured the pulp profile area of the hypophosphatemic teeth on radiographs as a measure of secondary dentin development [125]. The large pulp size, the lack of secondary dentin and the globoid dentin explained

FIGURE 5.30 Intraoral pictures of an osteogenesis imperfecta type III malocclusion in the mixed dentition characterized by an anterior crossbite and lateral open bites. Dentinogenesis imperfecta is also associated with osteogenesis imperfecta in this patient.

FIGURE 5.31

Bite-wing type dental radiograph of a hypophosphatemia patient. The pulp chambers are enlarged. No protective dentin is present leading to spontaneous pulpal necrosis and dental abscesses.

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FIGURE 5.32 Periapical radiograph of a hypophosphatemia patient. Periapical radiolucency is detected on a routine radiograph. No evidence of dental decay or periodontal involvement is present.

the occurrence of dental abscesses. Chaussain-Miller et al. stated that early treatment of XLH with the association of the 1-hydroxylated form of vitamin D and phosphates was beneficial with definite improvement of the dentin tissue [121].

FIGURE 5.34 Periapical radiograph of the anterior aspect of the maxilla of an osteopetrotic patient showing lack of dental eruption, poorly formed teeth and increased density of the alveolar bone.

Osteopetrosis, also known as Albers-Scho¨nberg disease, is characterized by a defect in the differentiation and/or function of osteoclasts [128,129]. The most severe forms tend to have an autosomal recessive inheritance (1 in 250 000 births) while the milder forms have an autosomal dominant inheritance (1 in 20 000 births). Marks [130] demonstrated that tooth eruption was dependent on bone resorption in osteopetrotic rats. When teeth cannot erupt, they change their shape and become dilacerated. In the most severe form of osteopetrosis, called malignant osteopetrosis, the dental findings include delayed tooth eruption, impaction and severe root and crown malformation (Figs 5.33e5.35) [129].

CONCLUSION

FIGURE 5.33 Osteopetrosis in a 6-year-old patient. Extremely dense bone, multiple unerupted and malformed teeth are present. No osteoclastic activity is present to counterbalance the osteoblastic proliferation. Dental eruption patterns are completely disturbed.

Tooth eruption through the dental follicle is a complex phenomenon. It involves osteoblastic and osteoclastic activity that contributes to form dento-alveolar bone. This bone formation and the development of dental occlusion, first for the deciduous, then for the succedaneous dentition, will have a profound impact on the

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REFERENCES

FIGURE 5.35 Intraoral occlusal picture of an osteopetrotic patient. No bone remodeling is present. Dental eruption is incomplete. Proliferation of bone around the dental follicle without bone resorption and remodeling does not allow for normal tooth eruption.

development of the lower part of the face. If teeth are missing or fail to erupt in the proper position, dentoalveolar development will be stunted and the alveolar ridge will either not form or be severely reduced in size. In severe cases of hypodontia, very little dentoalveolar volume is present, resulting in a collapsed lower face height. The mode of tooth eruption, guided by precise genetic control, is also largely influenced by its neuromuscular environment and the response of the periodontal ligaments to the environment. Tooth positioning may be altered by functional imbalance, as the periodontal ligament will respond to forces and rapidly remodel the dento-alveolar complex in order to maintain the necessary physiological distance between the alveolar wall and the dental root. Dentofacial development is intimately linked to proper dento-alveolar development that is itself dependent on a properly developing sequence of dental eruption and PDL influence. The study of heritable conditions has helped to explain why and how dental defects take place. It has also allowed delineation of specific syndromes. As it is the case in other disciplines, a thorough oral examination together with a comprehensive medical history will continue to contribute to further our knowledge of the complex mechanisms underlying dental maturation.

References [1] Vaahtokari A, Vainio S, Jowett A. Molecular mechanisms of cell and tissue interactions during early tooth development. Anat Rec Part A 1996;245:151e61. [2] Wright J. Consequences of amelogenin mutations: implications in amelogenesis imperfecta Amelogenins: multifaceted proteins for dental and bone formation and repair. Benham 2010;88e98.

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[3] Kim J, Simmer J. Hereditary dentin defects. J Dent Res. 2007;86:392. [4] Monreal A, Ferguson BM, Headon DJ, Street SL, Overbeek PA, Zonana J. Mutations in the human homologue of mouse dl cause autosomal recessive and dominant hypohidrotic ectodermal dysplasia. Nat Genet. 1999;22:366e9. [5] Rush J-V. Determinisms of odontogenesis. Cell Biol Rev. 1987;14:86. [6] MacKenzie AM, Ferguson B, Sharpe P. Expression patterns of the homeobox gene, Hox-8, in the mouse embryo suggest a role in specifying tooth initiation and shape. Development 1992;115:403e20. [7] Wise GE, Frazier-Bowers S, D’Souza RN. Cellular, molecular, and genetic determinants of tooth eruption. Crit Rev Oral Biol Med 2002;13:323e35. [8] Sasaki ST, Goldberg M, Takuma S, Garant P. Cell biology of tooth enamel formation. Monographs in Oral Science. Karger Basel 1999. p. 1e204. [9] Snead ML, Newcomb CJ, Payne ML, et al. Cell to matrix interactions suggests a pathway for enamel regeneration using artificial matrices. Goldberg M Bentham; 2010. [10] Chun YHP, Hu JC-C, Simmer JP. The non-amelogenins: ameloblastin and enamelin in Amelogenins: multifaceted proteins for dental and bone formation and repair. M. Goldberg Bentham; 2010. p. 42e55. [11] Goldberg M, Marian KA, Young M, Boskey A. Dentin: structure, composition and mineralization. The role of dentin ECM in dentin formation and mineralization. Fronters Bioscience E3 January 1, 2011;711e35. [12] Wang R, Weiner S. Strain-structure relations in human teeth using Moire fringes. J Biomech 1997;31:135e41. [13] Boukpessi T, Septier D, Bagga S, et al. Dentin alteration of deciduous teeth in human hypophosphatemic rickets. Calcif Tissue Intern 2006;79:294e300. [14] Gericke A, Qin C, Spevak L, et al. Importance of phosphorylation for osteopontin regulation of biomineralization. Calcif Tissue Int 2005;77:45e54. [15] Schroeder H. Biological problems of regenerative cementogenesis: synthesis and attachment of collagenous matrices on growing and established root surfaces. Int Rev Cytol 1992;142:1e60. [16] Bosshardt D, Schroeder H. Cementogenesis reviewed: a comparison between human premolars and rodent molars. Anatomy 1996;245:267e92. [17] VandenBos T, Bronckers AL, Goldberg HA, Beertsen W. Blood circulation as source for osteopontin in acellular extrinsic fiber cementum and other mineralizing tissues. J Dent Res. 1999;78:1688. [18] Slomka MJ, Coward VJ, Banks J, et al. Identification of sensitive and specific avian influenza polymerase chain reaction methods through blind ring trials organized in the European Union. Avian Dis 2007;51(Suppl):227e34. [19] Marks Jr S, Schroeder H. Tooth eruption: theories and facts. Anat Rec 1996;245:374e93. [20] Wise G, Marks Jr S, Cahill D. Ultrastructural features of the dental follicle associated with formation of the tooth eruption pathway in the dog. J Oral Pathol Med 1985;14:15e26. [21] Cahill D, Marks Jr S. Tooth eruption: evidence for the central role of the dental follicle. J Oral Pathol Med 1980;9:189e200. [22] Marks Jr S, Cahill D. Experimental study in the dog of the nonactive role of the tooth in the eruptive process. Arch Oral Biol. 1984;29:311e22. [23] Schneider GBG, Marks Jr SC. The diagnosis and cure of neonatal osteopetrosis: Experimental evidence from congenitally osteopetrotic (ia) rats. Metab Bone Dis Rel Res. 1979;1:335e9.

PEDIATRIC BONE

106

5. DENTAL DEVELOPMENT AND MATURATION, FROM THE DENTAL CRYPT TO THE FINAL OCCLUSION

[24] Wesenbeeck LV, Odgrerl PR, Mackay CA, et al. The osteopetrotic mutation toothless (tl) is a loss-of-function frameshift mutation in the rat Csf1 gene: Evidence of a crucial role for CSF-1 in osteoclastogenesis and endochondral ossification. Proc Natl Acad Sci USA 2002;99:14303e98. [25] Marks S. The basic and applied biology of tooth eruption. Connect Tissue Res. 1995;32:149e57. [26] Cahill D. Eruption pathway formation in the presence of experimental tooth impaction in puppies. Anat Rec 1969;164:67e77. [27] Marks Jr S, Cahill D, Wise G. The cytology of the dental follicle and adjacent alveolar bone during tooth eruption in the dog. Am J Anat 1983;168:277e89. [28] Marks Jr S, Gorski J, Wise G. The mechanisms and mediators of tooth eruption emodels for developmental biologists. Int J Dev BioI 1995;39:223e30. [29] Hunter J. The Natural History of Human Teeth. 1778 [30] Cahill D. Histological changes in the bony crypt and gubernacular canal of erupting permanent premolars during deciduous premolar exfoliation in beagles. J Dent Res. 1974;53:786. [31] Gorski JP, Marks SC, Cahill DR, Wise GE. Developmental changes in the extracellular matrix of the dental follicle during tooth eruption. Connect Tissue Res. 1988;18:175e90. [32] Proffit W, Vig K. Primary failure of eruption: a possible cause of posterior open-bite. Am J Orthodont 1981;80:173e90. [33] McCall JO. Clinical Dental Roentgenology. W. B. Saunders Company; 1957. [34] Marks Jr S. Pathogenesis of osteopetrosis in the ia rat: Reduced bone resorption due to reduced osteoclast function. Am J Anat 1973;138:165e89. [35] Frazier-Bowers SA, Simmons D, Koehler K, Zhou J. Genetic analysis of familial non syndromic primary failure of eruption. Orthodont Craniofacial Res. 2009;12:74e81. [36] Pelsmaekers B, Loos R, Carles C, Derom C, Viletinck R. The genetic contribution to dental maturation. J Dent Res. 1997;76: 1337. [37] Wise G, Frazier-Bowers S, D’Souza R. Cellular, molecular, and genetic determinants of tooth eruption. Crit Rev Oral Biol Med 2002;13:323. [38] Lokesh Suri B, Gagari E, Vastardis H. Delayed tooth eruption: pathogenesis, diagnosis, and treatment. A literature review. Am J Orthodont Dentofacial Orthoped 2004;126:4. [39] McCollum MS. Evolution and development of teeth. J Anat. 2001;199:153e9. [40] Heinrich J, Bsoul S, Barnes J, Woodruff K, Abboud S. CSF-1, RANKL and OPG regulate osteoclastogenesis during murine tooth eruption. Arch Oral Biol. 2005;50:897e908. [41] Philbrick Willam M, Dreyer Barbara E, Nakchbandi Innaam A, Karaplis Andrew C. Parathyroid hormone-related protein is required for tooth eruption. Proc Natl Acad Sci USA 1998; 95:11846. [42] Bowers S Frazier, Koehler K, Akerman J, Proffit W. Primary failure of eruption: further characterization of a rare eruption disorder. Am J Orthodont Dentofacial Orthoped 2007;131:578. [43] Ericson S, Kurol J. Radiographic examination of ectopically erupting maxillary canines. Am J Orthodont Dentofacial Orthoped 1987;91:483e92. [44] Lee C, Proffit W. The daily rhythm of tooth eruption. Am J Orthodont Dentofacial Orthoped 1995;107:38e47. [45] Moorree C. Normal variation in dental development determined with reference to tooth eruption status. J Dent 1965;44:161. [46] Trentini C, Proffit W. High-resolution observations of human premolar eruption. Arch Oral Biol. 1996;41:63e8.

[47] Sodek J, McKee M. Molecular and cellular biology of alveolar bone. Periodontology. 200;24:99-126. [48] Dobbins DE, Sood R, Hshiramoto A, et al. Mutation of macrophage colony stimulating factor (Csf1) causes osteopetrosis in the tl rat. Biochem Biophys Res Commun 2002;294:1114e20. [49] Smith B, Garn S. Polymorphisms in eruption sequence of permanent teeth in American children. Am J Phys Anthropol 1987;74:289e303. [50] Harokopakis-Hajishengallis E. Physiologic root resorption in primary teeth: molecular and histological events. J Oral Sci. 2007;49:1e12. [51] Sahara N, Okafuji N, Toyoki A, et al. Odontoclastic resorption of the superficial nonmineralized layer of predentine in the shedding of human deciduous teeth. Cell Tissue Res. 1994;277: 19e26. [52] Fukushima H, Kajiya H, Takada K, Okamoto F. Expression and role of RANKL in periodontal ligament cells during physiological root resorption in human deciduous teeth. Eur J Oral Sci. 2003;111:346e52. [53] Bille M, Kvetny M, Kjaer I. A possible association between early apical resorption of primary teeth and ectodermal characteristics of the permanent dentition. Eur J Orthodont 2008;30:346. [54] Oshiro T, Shibasaki Y, Yoda S, et al. Immunolocalization of vacuolar type Hþ ATPase, cathepsin K, matrix metalloproteinase 9, and receptor activator of NFkB ligand in odontoclasts during physiological root resorption of human deciduous teeth. Anat Rec 2001;264:305e11. [55] Yoda S, Suda N, Kitahara Y, Komori T, Onyama K. Delayed tooth eruption and suppressed osteoclast number in the eruption pathway of heterozygous Runx2/Cbfa1 knockout mice. Arch Oral Biol 2004;49:435e42. [56] Iizuka T, Cielinski M, Aukerman SL, Marks Jr SC. The effects of colony-stimulating factor-1 on tooth eruption in the toothless (osteopetrotic) rat in relation to the critical periods for bone resorption during tooth eruption. Arch Oral Biol. 1992;37:629e36. [57] O’Connell A, Marini J. Evaluation of oral problems in an osteogenesis imperfecta population. Oral Surg Oral Med Oral Path Oral Radiol Endodontol 1999;87:189e96. [58] Schwartz S, Tsipouras P. Oral findings in osteogenesis imperfecta* 1. Oral Surg Oral Med Oral Pathol 1984;57:161e7. [59] Mossey P. The heritability of malocclusion: part 2. The influence of genetics in malocclusion. J Orthodont 1999;26:195. [60] Vastardis H. The genetics of human tooth agenesis: new discoveries for understanding dental anomalies. Am J Orthodont Dentofacial Orthoped 2000;117:650e6. [61] Ericson S, Kurol J. Resorption of maxillary lateral incisors caused by ectopic eruption of the canines: a clinical and radiographic analysis of predisposing factors. Am J Orthodont Dentofacial Orthoped 1988;94:503e13. [62] Proffit WR, Prewitt JR, Baik HS, Lee CF. Video microscope observations of human premolar eruption. J Dent Res. 1991; 70:15. [63] Moss M, Salentijn L. The primary role of functional matrices in facial growth* 1. Am J Orthodont 1969;55:566e77. [64] Moss M, Young R. A functional approach to craniology. Am J Physl Anthropol 1960;18:281e92. [65] Harvold EP, Tomer BS, Vargervik K, Chierici G. Primate experiments on oral respiration* 1. Am J Orthodont 1981;79: 359e72. [66] Vargervik K, Miller AJ, Chierici G, Harvold E, Tomer BS. Morphologic response to changes in neuromuscular patterns experimentally induced by altered modes of respiration* 1. Am J Orthodont 1984;85:115e24.

PEDIATRIC BONE

REFERENCES

[67] Saffar JL, Cherruau M. Alveolar bone and the alveolar process: the socket that is never stable. Periodontology 2000;1997(13):76e90. [68] McCulloch C, Lekic P, McKee M. Role of physical forces in regulating the form and function of the periodontal ligament. Periodontology 2000;2000(24):56e72. [69] Ten Cate A. The role of epithelium in the development, structure and function of the tissues of tooth support. Oral Dis 1996;2:55e62. [70] Cho M, Garant PR. Development and general structure of the periodontium. Periodontology 2000;2000(24):9e27. [71] Barrett A, Reade P. The relationship between degree of development of tooth isografts and the subsequent formation of bone and periodontal ligament. Periodont Res. 1981;16:456e65. [72] Lekic P, McCulloch C. Periodontal ligament cell populations: the central role of fibroblasts in creating a unique tissue. Anat Rec 1996;245:327e41. [73] Ten Cate A, Mills C. The development of the periodontium: the origin of alveolar bone. Anat Rec 1972;173:69e77. [74] Bellows C, Melcher A, Aubin J. Contraction and organization of collagen gels by cells cultured from periodontal ligament, gingiva and bone suggest functional differences between cell types. J Cell Sci. 1981;50:299. [75] Beertsen W, Everts V, Van den Hooff A. Fine structure of fibroblasts in the periodontal ligament of the rat incisor and their possible role in tooth eruption. Arch Oral Biol. 1974;19:1087e92. [76] Thesleff I. The genetic basis of tooth development and dental defects. Am J Med Genet A 2006;140:2530e5. [77] Aberg T, Cavender A, Gaikwad JS, et al. Phenotypic changes in dentition of Runx2 homozygote-null mutant mice. J Histochem Cytochem 2004;52:131. [78] Altug-Atac A, Erdem D. Prevalence and distribution of dental anomalies in orthodontic patients. Am J Orthodont Dentofacial Orthoped 2007;131:510e4. [79] Garib D, Peck S, Gomes S. Increased occurrence of dental anomalies associated with second-premolar agenesis. J Inform 2009;79:3. [80] Gorlin RG, Hennekam RCM. Syndromes of the Head and Neck. Oxford University Press; 2001. [81] Sofaer J. A dental approach to carrier screening in X linked hypohidrotic ectodermal dysplasia. J Med Genet. 1981;18:459. [82] Holmstrom G, Thoren K. Ocular manifestations of incontinentia pigmenti. Acta Ophthalmol Scand 2000;78:348e53. [83] Axelsson S. Variability of the cranial and dental phenotype in Williams syndrome. Swed Dent J Suppl. 2005;170:3. [84] Kozakiewicz M, Perczynska Partyka W, Kobos J. Cherubism e clinical picture and treatment. Oral Dis 2001;7:123e30. [85] Jones W, Gerrie J, Pritchard J. Cherubism e A familial fibrous dysplasia of the jaws* 1. Oral Surg Oral Med Oral Pathol 1952;5:292e305. [86] Lourenc¸o Ribeiro L, et al. Dental anomalies of the permanent lateral incisors and prevalence of hypodontia outside the cleft area in complete unilateral cleft lip and palate. Cleft Palate Craniofacial J 2003;40:172e5. [87] Tsai TP, Huang CS, Huang CC, See LC. Distribution patterns of primary and permanent dentition in children with unilateral complete cleft lip and palate. Cleft Palate Craniofacial J 1998; 35:154e60. [88] Ranta R, Stegars T, Rintala A. Correlations of hypodontia in children with isolated cleft palate. Cleft Palate J 1983;20:163e5. [89] Russell BG. Tooth agenesis in Down syndrome. Am J Med Genet. 1995;55:466e71. [90] Skrinjari IB- F. Anomalies of deciduous teeth and findings in permanent dentition. Acta Stomatol Croat 1991;25:151.

107

[91] Davis P. Hypodontia and hyperdontia of permanent teeth in Hong Kong schoolchildren. Commun Dent Oral Epidemiol 1987;15:218e20. [92] Harris E, Clark L. An epidemiological study of hyperdontia in American blacks and whites. J Inform 2008;78:3. [93] Garvey MT. Supernumerary teeth e an overview of classification, diagnostic and management. J Can Dent Assoc. 1999;65:612e6. [94] Baumert U, Golan I, Redlich M, Aknin JJ, Muessig D. Cleidocranial dysplasia: molecular genetic analysis and phenotypic based description of a Middle European patient group. Am J Med Genet A 2005;139:78e85. [95] Ooshima T, Mishima IR. Sobue s K. The prevalence of developmental anomalies of teeth and their association with tooth size in the primary and permanent dentitions of 1650 Japanese children. Int J Paediatr Dent 1996;6:87e94. [96] Mundlos S. Cleidocranial dysplasia: clinical and molecular genetics. J Med Genet. 1999;36:177. [97] Jensen B, Kreiborg S. Development of the dentition in cleidocranial dysplasia. J Oral Pathol Med 1990;19:89e93. [98] Lewis D, Grainger R. Sex-linked inheritance of tooth size: a family study. Arch Oral Biol. 1967;12:539e44. [99] Brook AH. A unifying aetiological explanation for anomalies of human tooth number and size. Arch Oral Biol. 1984;29:373e8. [100] Garn Sm, Osborne RH, Alvesalo L, Horowitz SL. Maternal and gestational influences on deciduous and permanent tooth size. J Dent Res. 1980;59:142e3. [101] Maguire A, Evans RG, Amineddine H, et al. The long-term effects of treatment on the dental condition of children surviving malignant disease. Cancer 1987;60:2570e5. [102] Jorgenson RJ, Levin LS, Cross HE, et al. The Rieger syndrome. Am J Med Genet. 1978;2:307e18. [103] Midtbø M, Halse A. Root length, crown height, and root morphology in Turner syndrome. Acta Odontol 1994;52:303e14. [104] Peretz B, Shapira J, Farbstein H, Arieli E, Smith P. Modification of tooth size and shape in Down’s syndrome. J Anat 1996; 188:167. [105] Alvesalo L, Osborne R, Kari M. The 47, XYY male, Y chromosome, and tooth size. Am J Hum Genet. 1975;27:53. [106] Herrmann J, Pallister PD, Tiddy W, Opitz JM. The KBG syndrome e a syndrome of short stature, characteristic facies, mental retardation, macrodontia and skeletal anomalies. Birth Defect Orig Art Ser 1975;11:7. [107] Jaspers M, Witkop Jr C. Taurodontism, an isolated trait associated with syndromes and X-chromosomal aneuploidy. Am J Hum Genet. 1980;32:396. [108] Witkop Jr C. Amelogenesis imperfecta, dentinogenesis imperfecta and dentin dysplasia revisited: problems in classification. J Oral Pathol Med 1988;17:547e53. [109] Vajrabhaya L. Nonsurgical endodontic treatment of a tooth with double dens in dente. J Endodont 1989;15:323e5. [110] Stewart R, Dixon G, Graber R. Dens evaginatus (tuberculated cusps): genetic and treatment considerations. Oral Surg Oral Med Oral Pathol 1978;46:831e6. [111] Mellor J, Ripa L. Talon cusp: a clinically significant anomaly. Oral Surg Oral Med Oral Pathol 1970;29:225e8. [112] Bailleul-Forestier I, et al. The genetic basis of inherited anomalies of the teeth. Part 2: Syndromes with significant dental involvement. Eur J Med Genet. 2008;51:383e408. [113] Nikiforuk G, Fraser D. The etiology of enamel hypoplasia: A unifying concept. J Pediatr 1981;98:888e93. [114] Gorlin RG, Hennekam RCM. Syndromes of the Head and Neck. Oxford University Press; 2001.

PEDIATRIC BONE

108

5. DENTAL DEVELOPMENT AND MATURATION, FROM THE DENTAL CRYPT TO THE FINAL OCCLUSION

[115] Price JA, Wright JT, Walker SJ, et al. Tricho-dento-osseous syndrome and amelogenesis imperfecta with taurodontism are genetically distinct conditions. Clin Genet. 1999;56:35e40. [116] Al Ghamdi K, Crawford P. Focal dermal hypoplasia e oral and dental findings. Int J Paediatr Dent 2003;13:121e6. [117] Shields E, Bixler D, El-Kafrawy A. A proposed classification for heritable human dentine defects with a description of a new entity. Arch Oral Biol. 1973;18:543e53. [118] Malmgren B, Lindskog S, Elgadi A, Norgen S. Clinical, histopathologic, and genetic investigation in two large families with dentinogenesis imperfecta type II. Hum Genet. 2004;114:491e8. [119] Sreenath T, Thyagarajan T, Hall B, et al. Dentin sialophosphoprotein knockout mouse teeth display widened predentin zone and develop defective dentin mineralization similar to human dentinogenesis imperfecta type III. J Biol Chem. 2003;278:24874. [120] Beattie ML, Kim JW, Gong SG, et al. Phenotypic variation in dentinogenesis imperfecta/dentin dysplasia linked to 4q21. J Dent Res. 2006;85:329. [121] Chaussain-Miller C, Sinding C, Septier D, et al. Dentin structure in familial hypophosphatemic rickets: benefits of vitamin D and phosphate treatment. Oral Dis 2007;13:482e9. [122] Rauch F, Glorieux F. Osteogenesis imperfecta. Lancet 2004;363:1377e85.

[123] Sillence D, Senn A, Danks D. Genetic heterogeneity in osteogenesis imperfecta. Br Med J 1979;16:101. [124] Lygidakis N, Smith R, Oulis C. Scanning electron microscopy of teeth in osteogenesis imperfecta type I. Oral Surg Oral Med Oral Pathol Oral Radiol Endodontol 1996;81:567e72. [125] Shields Ed, Scriver CR, Reade T, et al. X-linked hypophosphatemia: the mutant gene is expressed in teeth as well as in kidney. Am J Hum Genet. 1990;46:434. [126] Murayama T, Iwatsubo R, Akiyama S, Amano A, Morisaki I. Familial hypophosphatemic vitamin D-resistant rickets: dental findings and histologic study of teeth. Oral Surg Oral Med Oral Pathol Oral Radiol Endodont 2000;90:310e6. [127] Schwartz S, Scriver CR, Reade TM, Shields ED. Oral findings in patients with autosomal dominant hypophosphatemic bone disease and X-linked hypophosphatemia: further evidence that they are different diseases. Oral Surg Oral Med Oral Pathol 1988;66:310. [128] Stark Z. Osteopetrosis. Orphanet J Rare Dis 2009;4:1e10. [129] Moghadam MI, Nemati S, Johan M. Dental radiographic findings of malignant osteopetrosis: report of four cases. Iran J Radiol 2009;6:141e5. [130] Marks Jr S. Tooth eruption depends in bone resorption: experimental evidence from osteoetpotrotic (ia) rats. Metab Bone Dis Relat Res. 1981;3:107e15.

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C H A P T E R

6

Parathyroid Hormone and Calcium Homeostasis John T. Potts, Thomas J. Gardella Endocrine Unit, Department of Medicine, The Massachusetts General Hospital and Harvard Medical School Boston, MA, USA

INTRODUCTION Parathyroid hormone (PTH) and PTH-related peptide (PTHrP), along with other calciotropic hormones, play critical roles in calcium homeostasis and bone biology. In contrast to PTH, which is produced by discrete endocrine glands, PTHrP is produced as a paracrine/ autocrine factor in many different adult and fetal tissues and has, unlike PTH, multiple functions (Fig. 6.1). First discovered as a calcium-regulating hormone in the 1920s [1e3], PTH is secreted by the parathyroid glands and is the critical regulator of blood calcium concentration in all terrestrial vertebrate species. PTHrP,

FIGURE 6.1 Biology of the PTH/PTHrP receptor. The PTH/ PTHrP receptor interacts with two ligands, PTH and PTHrP, and can activate several second-messenger signaling pathways, including the cAMP/protein kinase A (PKA) and Ca2þ/inositol 1,4,5-triphosphate/ PKC pathways. The receptor is abundantly expressed in bone and kidney, where it mediates the endocrine actions of PTH, and in the metaphyseal growth plate and numerous other tissues, where it mediates the autocrine/paracrine actions of PTHrP.

Pediatric Bone, Second Edition DOI: 10.1016/B978-0-12-382040-2.10006-1

a slightly larger molecule than PTH, was discovered more recently through efforts to identify the factor that causes, when produced in excess by certain tumors, the humoral hypercalcemia of malignancy syndrome [4e6]. PTH and PTHrP most likely evolved from a common ancestral precursor, as discussed further below. The polypeptides share only limited overall amino acid sequence identity, yet at least their N-terminal regions are sufficiently homologous to enable them to bind to and activate a common G protein-coupled receptor, the PTH/PTHrP receptor (also referred to as PTH1R) [7e9]. This receptor mediates the most important biologic actions of both peptides: PTH-dependent regulation of calcium homeostasis and PTHrP-dependent regulation of endochondral bone formation [10e14]. This chapter reviews (1) the comparative chemistry of PTH and PTHrP, their genes, and their interactions with PTH1R; (2) the current molecular models of interactions between the two ligands and their common receptor; and (3) the different biologic roles of both peptides on target tissues, such as the role of PTH in calcium homeostasis and bone turnover and the role of PTHrP in bone and cartilage development, as well as the functional characteristics of two novel, closely related receptors and the pharmacologic and physiochemical evidence for several additional, still incompletely characterized receptors for PTH and PTHrP.

PHYSIOLOGICAL ROLE OF PTH To ensure a multitude of essential cellular functions, the extracellular concentration of calcium (Ca2þo) is maintained within narrow limits [15,16]. In terrestrial

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vertebrates, calcium is necessary for adequate mineralization of the skeleton, which provides mechanical support and protection for internal organs and acts as levers for the various muscle groups involved in locomotion. Because of its high calcium content, 99% of the body’s supply, the skeleton also serves as the most important reservoir from which calcium can be rapidly mobilized. Because food intake and thus the nutritional supply of calcium are usually discontinuous, intestinal calcium absorption occurs only intermittently. Maintenance of a constant blood calcium concentration thus constitutes a major homeostatic challenge which, during evolution, led to the development of highly efficient mechanisms to increase intestinal calcium absorption, reduce urinary calcium losses, and facilitate, if necessary, rapid mobilization of calcium from the skeletal reservoir [16]. In contrast to these environmental challenges of most terrestrial vertebrates, marine animals, which are usually exposed to the high environmental calcium concentration of seawater (10 mM) had to adopt mechanisms by which extracellular calcium could be reduced [17,18]. Unlike the diet of terrestrial animals, seawater provides only a very limited supply of phosphate, and this environmental deficiency resulted in the development of mechanisms to conserve phosphate. It thus, appears plausible that the efficient intestinal absorption of phosphate and the impressive capacity of the mammalian kidney to retain phosphate [15,19] are remnants of earlier evolutionary adaptations to life in the low phosphate environment of the oceans (see Chapter 7 for discussion of phosphate homeostasis). PTH and the active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), are the principal physiologic regulators of calcium homeostasis in humans and all terrestrial vertebrates [11,20,21]. Synthesis and secretion of PTH are stimulated by any decrease in blood calcium, and conversely, secretion of the hormone is inhibited by an increase in blood calcium [22,23]. This rapid negative feedback regulation of PTH production, along with the biologic actions of the hormone on different target tissues, represents the most important homeostatic mechanism for minute-to-minute control of calcium concentration in the extracellular fluid (ECF) [24e26]. In contrast to the rapid actions of PTH, 1,25(OH)2D3 is of critical importance for long-term, day-to-day, and week-to-week calcium balance. The actions of both hormones are coordinated, and each influences the synthesis and secretion of the other. At least three distinct, but coordinated, actions of PTH increase the flow of calcium into the ECF and thus increase the concentration of blood calcium [27e29]. Through its rapid actions on the kidney and bone, which are all mediated through the PTH/PTHrP receptor and subsequent secondary messages in specific

and highly specialized cells, PTH increases the release of calcium from bone, reduces the renal clearance of calcium, and stimulates the production of 1,25(OH)2D3 by activating the gene encoding 25-hydroxyvitamin D-1a-hydroxylase (1a-hydroxylase) in the kidney. The relative importance of the first two actions of PTH on the rapid, minute-to-minute regulation of calcium is not definitively resolved, but most physiologists have stressed the importance of the effects of PTH on bone in maintaining hour-to-hour calcium homeostasis in the ECF. Several lines of evidence, such as that provided by calcium kinetic analysis, indicate a transfer between ECF and bone of as much as 500 mg calcium daily, which is equivalent to one-fourth to one-half the total ECF calcium content [15]. Besides regulating this transfer of calcium from bone through direct breakdown of bone tissue (mineral and matrix), PTH influences the rates of exchange of calcium adsorbed to the surface of bone; this exchangeable calcium pool can be stimulated to provide a rapid and substantial rate of entry of calcium into blood. In addition to these actions of PTH on bone, actions of PTH on the kidney may also be extremely important in the precise hourly regulation of ECF calcium. The third action of PTH on calcium homeostasis e namely, enhancement of intestinal calcium absorption e is indirect and involves the synthesis of 1,25(OH)2D3 from the biologically inactive precursor 25(OH)D3. It is difficult, however, to analyze quantitatively or to contrast proportionately the relative physiologic importance of the direct and indirect actions of PTH on the three principal target tissues: kidney, bone, and intestine. The complexity of bone as a tissue and the many detectable rates of exchange of calcium between the skeleton and the ECF have made the action of PTH on the skeleton difficult to analyze. The state of calcium in blood is complex; much of the calcium is present as chelates or is bound to plasma proteins. Because actual filtered loads depend on the ratio of free and bound forms of calcium, it is difficult to calculate renal calcium clearance accurately. The different PTH-dependent actions to promote calcium entry into the ECF are most clearly defined in conditions of deficiency or excess of PTH, such as during experiments in animals or during controlled observations in patients with disorders of parathyroid gland function. The experimental data in these extremes abundantly affirm the crucial calcium homeostatic role of PTH. However, because of continuous and rapid adjustments in mineral ion concentration, it can be difficult to observe the consequences of hormone action under normal physiologic conditions. For example, the rate of PTH secretion changes continually and rapidly so that the controlled variable, calcium, remains constant, and it may, therefore, be difficult experimentally to detect small corrective changes.

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Teleologically, the action of PTH on the regulation of blood phosphate concentration in terrestrial species is best understood as a secondary, rather than a homeostatic, action. Phosphate is abundant in the food chain in terrestrial existence. Phosphate deficiency, unlike calcium deficiency, in the absence of specific organ dysfunction is, therefore, an unlikely environmental challenge (see Chapter 7 for detailed review of the regulation of phosphate homeostasis). To correct a deficiency in calcium, calcium phosphate stores in bone can be rapidly dissolved; such activity results, however, in the simultaneous liberation of ionic calcium and phosphate. Because a high blood phosphate level tends to lower the calcium concentration through multiple mechanisms, the rise in blood calcium that occurs after bone dissolution (desirable homeostatically) is, therefore, beneficial only if the concomitant increase in blood phosphate concentration (undesirable) can be rapidly corrected. To maximize the control of calcium homeostasis, PTH thus has divergent actions on renal tubular handling of the two mineral ions: it increases the retention of calcium and, at the same time, diminishes reabsorption of phosphate. Through these mechanisms, namely, increased renal phosphate clearance to prevent hyperphosphatemia and increased tubular calcium reabsorption, PTH guarantees that an elevation in blood calcium results from the increased release of calcium from bone. The renal action of PTH on phosphate homeostasis is biologically predominant over the increased phosphate flux from bone. Consequently, parathyroidectomy (experimentally in animals) or renal resistance to PTH, as in patients with pseudohypoparathyroidism or renal failure, leads not only to hypocalcemia but also to an increase in blood phosphate and a marked reduction in urinary phosphate excretion. This finding demonstrates the importance of the PTHdependent action on phosphate homeostasis in the kidney, which becomes particularly important in disease states when high bone turnover is the result of dietary calcium deficiency or lack of biologically active vitamin D [30,31].

Chemistry The first extracts from bovine parathyroid glands were described in 1925, and the content of biologically active PTH was assessed by their hypercalcemic and phosphaturic properties [1,2]. However, it was not until 1959, when Aurbach [32] and Rasmussen and Craig [33] developed improved extraction procedures, that it became possible to isolate and purify sufficient quantities to determine the primary structure of bovine, porcine, and human PTH through the protein sequence determination methods [34e39]. Two groups independently determined the sequences of human and bovine

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hormones [34e39]. Shown in Figure 6.2 are the sequences of the bovine, porcine, and human hormones determined by one group [35,36,38,39]. Discordant sequences for the human PTH polypeptide, and in one position for the bovine hormone, published by another group [34,37] are not shown in Figure 6.2, since nucleotide sequence analysis of genomic and complementary DNA confirmed the amino acid sequences of the first group (the only exception was residue 76 in human PTH, which was determined to be glutamine instead of glutamic acid) [35,36,39]. Based on these amino acid sequences, the PTH(1e34) fragments of the different species were synthesized, and their biologic activities were compared in vitro and in vivo with those of highly purified intact PTH from the same species. Molecular cloning techniques then led to the deduction of the amino acid sequences of rat, chicken and dog PTHs [40e43], followed more recently by the identification of PTH molecules in other mammals and fish, discussed below. The synthetic peptides used in parathyroid hormone research today are based largely on the (1e34) regions of the mammalian hormone sequences shown in Figure 6.2 [44,45]. Extensive sequence homology is present in the mammalian PTH species; these molecules consist of a single-chain polypeptide with 84 amino acids and a molecular weight of approximately 9400 Daltons (that of human PTH[1e84] is 9425 Daltons). The N-terminal region of PTH, which is necessary and sufficient for the regulation of mineral ion homeostasis, shows high sequence conservation among all the vertebrate species (see Fig. 6.2). The middle portions of the different molecules exhibit the most structural variation, which could suggest that this region of PTH is only of limited functional importance. The non-mammalian PTH homologs of chicken [41,42] and fish species Danio rerio (zebrafish) [46] and Takifugu ruberipes (puffer fish) [47,48] diverge considerably from the mammalian hormones C-terminal of amino acid residue His32. Interestingly, both fish species have two distinct genes encoding two separate PTH molecules, called PTH1 and PTH2 [46,47]. The zebrafish peptides are considerably shorter than mammalian PTH (67 and 68 residues), while fugu PTH1 is predicted to be 81 residues in length and fugu PTH2 is predicted to be 63 residues [47]. After the original work establishing that the first 34 amino acids of mammalian PTH were sufficient to produce a fully active synthetic peptide [44,45], much work has centered on defining the minimum pharmacophore essential for biologic activity. We describe below how sites of ligand interaction with the PTH/PTHrP receptor were defined by performing assays with products of various combinations of shortened and modified PTH ligands and mutagenized receptors [49]. As also discussed in the section below on hormone/receptor

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FIGURE 6.2 Sequence relationships of PTH family ligands. Comparisons of amino acid sequences of PTH and PTHrP peptides from different

species are shown in panels A and B, respectively. Note numbering indicates alignment position number, and not protein amino-acid sequence number. Comparison of TIP39 and a PTH-like peptide (PTH-L) from the pufferfish (Takifugu rubripes) with human PTH and PTHrP (the [1e84] region only) is shown in panel C. Amino acid sequences were aligned using the ClustalW software program (version 2.012; gap penalties: opening, 15; extending, 5) and further processed using the Boxshade program. Amino acid identities of 50% or more are shown in white type on black field, and similarities are shown in black type on gray field. Sequence identification or accession numbers are shown in the legend to Figure 6.4.

interactions, it has been determined that substitutions of non-naturally occurring amino acids (e.g. alpha-aminoisobutyric acid at positions 1 and 3 in the primary ligand structure) favoring formation of an alpha-helix, even in short peptides, such as PTH(1e14), produce peptides that are highly potent when tested in vitro using cellbased assays and have highly stabilized helical structure in solution (Fig. 6.3) [50e54]. The in vitro activity of the native PTH(1e14), which is quite weak, is improved about 100 000-fold by the modifications indicated in Figure 6.3. Such short-length PTH peptides have also been shown to be active in vivo, since some cause hypercalcemia and are anabolic on bone, although their potency is much less than that of PTH(1e34) due to a more rapid clearance [55]. Replacement of valine-2 in these peptides with bulky amino acids, such as tryptophan or parabenzoyl-L-phenylalanine

(Bpa), results in competitive antagonist peptides defective for AC/cAMP and PLC/IP3/Ca2þ signaling, thus confirming the critical role that this conserved valine plays in receptor activation [56]. Other longer-length PTH or PTHrP analogs having residue-1 (serine or alanine) replaced by glycine [57], Bpa [58] or tryptophan [59] exhibit signal selective properties in that they efficiently stimulate the cAMP cellular pathway but not the inositol triphosphate/intracellular Ca2þ pathway. Because of their generally demonstrated similar potencies at the PTH/PTHrP receptor, it seemed likely that both PTH(1e34) and PTHrP(1e34) ligands would adopt very similar conformations when part of the active hormone-receptor complex. However, recent data, discussed below, suggest that each ligand selectively binds to or induces a distinct receptor confirmation. Ideally, each hormone should be co-crystallized

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FIGURE 6.3 Minimized N-terminal PTH analogs. Shown is the native (rat) PTH(1e14) sequence and the locations of activity-enhancing

substitutions. The six substitutions shown above the sequence, when combined, enhance activity by as much as 100 000-fold; the Bpa2 substitution shown below confers antagonist properties to the peptide. The (1e9) region (non-shaded circles) comprises the minimum-length agonist pharmacophore. Non-encoded amino acids include a-amino-isobutyric acid (Aib), homoarginine (Har), and para-benzoyl-L-phenylalanine (Bpa).

with the PTH/PTHrP receptor to permit analysis by x-ray diffraction of those intermolecular interactions that are characteristic of the biologically active hormonereceptor complexes. G protein-coupled receptors, such as the PTH/PTHrP receptor, have multiple membraneembedded domains and are likely to have complex three-dimensional structures. Interaction with either PTH or PTHrP appears to involve several distinct receptor domains (see later discussion) that may undergo significant conformational changes after ligand binding has occurred, which makes it even more challenging to conduct x-ray or multidimensional NMR analyses. Recent advances, however, have made it possible to co-crystallize the extracellular portion of the PTH/PTHrP receptor with either the carboxyl terminal half of PTH(1e34) or that of PTHrP(1e34) [60,61].

rubripes and Tetraodon fluviatilis, reveal the duplication of the PTH gene in each case [47,65]. Both the PTH1 and PTH2 peptides derived from the zebrafish activate the PTH/PTHrP receptors from different species [48,65] and, indeed, a fugu PTH(1e34) peptide has been shown to induce bone anabolic effects in osteopenic ovariectomized rats [66]. In addition to PTH, the teleost fish also express PTHrP, again encoded by duplicate genes [65,67e70]. Furthermore, PTHrP immunoreactivity has been detected in the cartilaginous sharks and rays [71], and in a more primitive agnathan, the lamprey [72]. The

Evolution To maintain extracellular calcium and phosphate concentrations within narrow limits, the intricate regulatory system outlined above, in which PTH plays the most important role, developed in the terrestrial animals. In mammals, PTH is produced almost exclusively by the parathyroid glands (only small amounts of its messenger RNA [mRNA] have been detected elsewhere [62,63]). During evolution, these glands first appear as discrete organs in amphibians e that is, with the migration of vertebrates from an aquatic to a terrestrial existence e and their appearance most likely represents an evolutionary adaptation to an environment that is, by comparison to seawater, low in calcium [17,18,64]. Parathyroid glands have not been identified in fish or invertebrate species. However, with the rapid advances in characterization of complete genomes of multiple species we have definitive proof of the earlier evolutionary origin of both PTH and PTHrP (Fig. 6.4). Gene analyses of several teleost fish species, including the zebrafish, Danio rerio, and the puffer fishes Takifugu

FIGURE 6.4 Phylogenetic relationships of PTH family ligands. The diagram shows the separate groupings of PTH and PTHrP ligands, with duplications of each ligand in pufferfish (Takifugu ruberipes) and zebra fish (Danio rerio), as well as the relationship to an apparent ancestral, PTH-like ligand (PTH-L) in fugu. Protein data base accession numbers are as follows: human PTH, P01270; chicken PTH, A9YX65; danio PTH-1, Q6WQ25; danio PTH-2, Q6WQ24; fugu PTH-1, Q2PCS7; fugu PTH-2, Q6W9J4; human PTHrP, P12272; chicken PTHrP, Q5TLZ2; danio PTHrP-A, Q1L5E7; fugu PTHrP-A, Q9I8E9; danio PTHrP-B, Q4VVA3; fugu PTHrP-B, Q2PCS8; fugu PTH-L, Q2PCS5.

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teleost PTHrPs contain some amino acid residues characteristic of mammalian PTH; for example, fish PTHrP contains Met at position 8, Trp at position 23, and Leu at position 28, which are amino acid residues found in mammalian PTH. However, there is only one amino acid residue, Gln(Q)25, in fish PTH that is found in some mammalian PTHrP species, but not in mammalian PTH (see Fig. 6.4). This pattern suggests that the fish proteins may be phylogenetically closer to a common PTH/PTHrP precursor than are the mammalian proteins (see below). Indeed, in addition to duplicate copies of PTH and PTHrP genes, the puffer fish genome contains a fifth gene that encodes a protein containing the amino acid residues characteristic of both PTH and PTHrP. This gene, called PTH-L, is phylogenetically an intermediary to PTH and PTHrP, and may thus represent first definitive evidence for an ancestral gene from which the two divergent ligand forms evolved (see Fig. 6.4) [47,65].

The PTH Gene The human PTH gene consists of three exons located on chromosome 11p15 [73e76]. The first exon is 85 nucleotides in length and is non-coding (Fig. 6.5). Exon 2 (90 bp) encodes most amino acids of the prepropeptide sequence, whereas the third exon (612 bp) encodes the remainder of the propeptide sequence and all amino acids of the mature peptide, and it constitutes the 30 non-coding region [77]. Several frequent intragenic polymorphisms (TaqI and PstI [78], BstBI [79], DraIII [80], XmnI [81], and a tetranucleotide repeat ([AAAT]n [82]) have been identified in the human PTH gene, and some were shown to be informative in genetic linkage studies [83e85]. Two mRNAs that are 822 and 793 bp in length are derived in the human gene from the two transcriptional start sites which follow two different functional TATA boxes that are separated by 29 bp [77]. Two closely spaced TATA boxes and two distinct transcripts are also derived from the bovine PTH gene,

FIGURE 6.5 Schematic of the PTH gene. Shown is the gene along several thousand base pairs (approximate length shown by the scale marker for 500 bp). The three exons in the mRNA are represented as numbered rectangles. Control elements are identified in the 50 noncoding region (50 NC). A region responsive to vitamin D is within a few hundred base pairs of exon 1; far upstream are silencers involved in calcium regulation.

while rat and chicken PTH genes give rise to only one transcript; as a consequence of a long 30 non-coding region, the transcript from the chicken PTH gene is unusually long, and comprises 2.3 kilobases [24,86]. The genes encoding zebrafish PTH1 and PTH2 have a similar overall organization as the mammalian PTH genes [46].

Cellular Biosynthesis and Hormonal Processing During the synthesis of the preproPTH molecule, the signal sequence, which comprises the 25-amino acid containing “pre”-sequence, is cleaved off after entry of the nascent peptide chain into the intracisternal space bounded by the endoplasmic reticulum. A heterozygous mutation in this leader sequence, which changes a cysteine to an arginine at position 8 and thus impairs processing of preproPTH to proPTH, has been identified as the most plausible molecular cause of an autosomal dominant familial form of hypoparathyroidism [87,88]. The mutant hormone was found to be trapped intracellularly, predominantly in the endoplasmic reticulum (ER), leading to a marked upregulation of ER stressresponsive proteins (BiP and PERK) and the proapoptotic transcription factor CHOP, indicating that apoptosis-mediated parathyroid cell death is the likely cause of the observed hypoparathyroidism [89]. Subsequent to the removal of the pre-sequence, the pro-peptide is transported to trans-Golgi network where the pro-sequence (amino acid residues 6 through 1) is removed [90]. This latter process may involve furin (paired basic amino acid cleaving enzyme) and/or proprotein convertase-7 (PC-7), which are both expressed in parathyroid tissue; their expression levels do not appear to be regulated by either calcium or 1,25(OH)2D3 [91,92]. After removal of the basic pro-sequence, the mature polypeptide, PTH(1e84), is packaged into secretory granules. Two proteases, cathepsins B and H, are subsequently involved in the intraglandular generation of carboxyl-terminal PTH fragments from the intact hormone; no amino-terminal PTH fragments appear to be released from the gland [93e95]. Since small or intermediate size carboxyl-terminal fragments of PTH are unlikely to be involved in the regulation of calcium homeostasis, the intraglandular degradation of intact PTH is thought to represent an inactivating pathway, at least with regard to the regulation of mineral ion homeostasis. Consistent with this conclusion, hypercalcemia results in a substantial decrease in PTH secretion and, furthermore, favors the secretion of carboxylterminal PTH fragments, including a previously undetected large molecular species that is truncated at the amino-terminus (see section below) [95e98]. However, recent studies have shown that some amino-terminally truncated PTH fragments, such as PTH(7e84), have

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hypocalcemic properties in vivo and can furthermore reduce the formation of osteoclasts in vitro [99]. The pool of stored, intracellular PTH is small and the parathyroid cell must therefore have mechanisms to increase hormone synthesis and release in response to sustained hypocalcemia. One such adaptive mechanism is to reduce the intracellular degradation of the hormone, thereby increasing the net amount of intact, biologically active PTH that is available for secretion. During hypocalcemia, the bulk of the hormone that is released from the parathyroid cell is intact PTH(1e84) [93e95,97,98]. As the level of Ca2þo increases, a greater fraction of intracellular PTH is degraded, and with overt hypercalcemia, most of the secreted immunoreactive PTH consists of biologically inactive C-terminal fragments [10,24,25].

stimulation of intestinal calcium absorption and bone resorption [105e107] and may thus be attractive candidates for treating the hyperparathyroidism of chronic renal insufficiency. Adjustment of the rate of parathyroid cellular proliferation is the third adaptive mechanism contributing to changes in the overall secretory activity of the parathyroid gland. Under normal conditions, parathyroid cells have little or no proliferative activity. The parathyroid glands, however, can enlarge greatly during states of chronic hypocalcemia, particularly in the setting of renal failure, probably because of a combination of hypocalcemia, hyperphosphatemia, and low levels of 1,25(OH)2D3 in the latter condition.

Regulation of PTH Gene Expression

A large number of factors modulate PTH secretion in vitro [11,25,108] but most of these factors are not thought to control hormonal secretion in vivo in a biologically relevant manner. Therefore, we focus in this section principally on factors that are the most physiologically meaningful regulators of PTH secretion e that is, the extracellular ionized calcium concentration itself (Ca2þo), 1,25(OH)2D3, and the level of extracellular phosphate ions. Of these three, Ca2þo is most important in the minute-to-minute control of PTH secretion. Indeed, the actions of 1,25(OH)2D3 and phosphate ions on the secretion of PTH probably result, at least in part, from their effects on hormonal biosynthesis rather than secretion per se [11,25,108]. Ca2þo also modulates several other aspects of parathyroid function that indirectly affect PTH secretion, including PTH gene expression, the hormone’s intracellular degradation, and parathyroid cellular proliferation, as described previously. Recent data have shown that novel factors playing key roles in phosphate homeostasis, especially fibroblast growth factor 23 (FGF-23) and aeKlotho (a co-receptor for FGF receptors), also modulate parathyroid function, inhibiting [109,110] and enhancing [111] parathyroid function, respectively. Our rapidly improving understanding of how these factors participate in phosphate homeostasis is described in detail in Chapter 7. The relationship between PTH and Ca2þo is represented by a steep inverse sigmoidal curve that can be quantitatively described [112e114]. Parathyroid cells can readily detect reductions in Ca2þo of a few percentage points [113] and the percent coefficient of variation in Ca2þo in humans is less than 2% [115]. The set point of the parathyroid gland is the key determinant of the level at which Ca2þo is “set” in vivo [116]. Thus, the parathyroid cell is normally more than half-maximally suppressed at normal levels of Ca2þo and has a large secretory reserve for responding to hypocalcemic stress. Nevertheless, PTH levels in vivo fall dramatically

Another adaptive mechanism of the parathyroid cell to sustained reductions in Ca2þo is to increase cellular levels of PTH mRNA, a response that takes several hours. A reduction in Ca2þo increases, whereas an elevation in Ca2þo reduces the cellular levels of PTH mRNA by affecting both its stability and the transcriptional rate of its gene [11,25,100,101]. Available data suggest that phosphate ions also regulate, directly or indirectly, PTH gene expression. Hypophosphatemia and hyperphosphatemia in the rat, respectively, lower and raise the levels of mRNA for PTH through a mechanism that is independent of changes in Ca2þo or 1,25(OH)2D3. An elevated extracellular phosphate concentration could, thus, contribute importantly to the secondary hyperparathyroidism frequently encountered in patients with end-stage renal failure, who often have chronically elevated serum phosphate concentrations. Metabolites of vitamin D, principally 1,25(OH)2D3, also play an important role in the long-term regulation of parathyroid function and may act at several levels: by affecting the secretion of PTH and regulation of its gene, by regulating transcriptional activity of the genes encoding the calcium-sensing receptor (CaSR) (see below) and the vitamin D receptor (VDR), as well as by regulating parathyroid cellular proliferation [11,25,100,102]. 1,25(OH)2D3 is by far the most important vitamin D metabolite that modulates parathyroid function. It acts through a nuclear receptor, the VDR, often in concert with other such receptors (i.e. those for retinoic acid or glucocorticoids), on DNA sequences upstream from the PTH gene [103,104]. 1,25(OH)2D3induced upregulation of VDR and CaSR expression in the parathyroid could potentiate its inhibitory action(s) on PTH synthesis and secretion [11,25,100]. Non-calcemic or less calcemic analogues of 1,25(OH)2D3 inhibit PTH secretion while producing relatively little

Regulation of Secretion

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(e.g. by 80%) when Ca2þo rises to frankly hypercalcemic levels [112,113], which is thought to contribute importantly to the mineral ion homeostatic system’s defense against hypercalcemia [116]. Furthermore, elevating Ca2þo also decreases the proportion of secreted, intact PTH because of increased intraglandular degradation to inactive fragments (see the earlier section entitled PTH Biosynthesis and Intraglandular Processing and the later section entitled Metabolism of PTH) [96,117]. Even with severe hypercalcemia, however, some residual release of intact PTH(1e84) still occurs in vivo [28,98,118]. This non-suppressible basal component of PTH release may contribute to the hypercalcemia caused by hyperparathyroidism when the mass of abnormal parathyroid tissue is very great (e.g. in patients with renal failure) [114,119e121]. The parathyroid cell has a temporal hierarchy of responses to low Ca2þo that permits it to secrete progressively larger amounts of hormone during prolonged hypocalcemia [11,25,108]. To meet acute hypocalcemic challenges, PTH is released within seconds from preformed secretory vesicles by exocytosis as dictated by the sigmoidal curve. Sufficient PTH is stored in the parathyroid chief cell to sustain maximal, low Ca2þo-stimulated PTH release for about 60e90 minutes [116]. Another rapid response of the parathyroid cell to hypocalcemia that enhances its net synthetic rate of PTH is reduced intracellular hormonal degradation e the opposite of what occurs at high levels of Ca2þo e which occurs within minutes to an hour [96,117]. Hypocalcemia persisting for hours to days elicits increased PTH gene expression, whereas that lasting for days to weeks or longer stimulates parathyroid cellular proliferation [11,25,108,122]. A greater secretory capacity for PTH on a per-cell basis (e.g. as a result of enhanced PTH gene expression) increases maximal hormonal secretion in vivo, as does an increase in cell number as a result of parathyroid cellular proliferation. In severe secondary hyperparathyroidism, very large increases in parathyroid cellular mass can elevate circulating PTH levels by 100-fold or more. The molecular mechanism underlying Ca2þo-regulated PTH secretion involves a G protein-coupled, cell surface Ca2þo-sensing receptor (CaSR) [123]. The CaSR was first isolated from bovine parathyroid glands [124] and subsequently from human parathyroid and several other tissues and species [125]. The receptor exhibits the characteristic “serpentine” motif (seven membranespanning domains) of the superfamily of G proteincoupled receptors. Its long, N-terminal extracellular domain contains the major, but not all, determinants of Ca2þo binding [126e128]. Changes in Ca2þo modulate a number of second messenger systems via coupling of CaSR through its intracellular domains to the relevant G proteins regulating these signaling pathways [123].

These functions include activation of phospholipases C, A2, and D [129], stimulation of several mitogen-activated protein kinases [130] and inhibition of adenylyl cyclase [131]. Despite numerous studies conducted over the past 25 years, a full understanding of the major second messenger pathways through which changes in Ca2þo, acting via the CaSR, regulate various aspects of the function of parathyroid and other CaSR-expressing cells remains elusive. Recent evidence, however, indicates key roles for the G proteins, Gq and G11, but their downstream transduction pathways participating in the control of parathyroid function are not established [132]. In the parathyroid, the CaSR mediates the inhibitory actions of Ca2þo on PTH secretion and gene expression as well as parathyroid cellular proliferation [133,134]. The CaSR is also expressed in several additional tissues involved in systemic mineral ion homeostasis, including the calcitonin-secreting C-cells of the thyroid [135], diverse cells within the kidney [136], bone cells and/or their precursors [100], and intestinal epithelial cells. In the kidney, the CaSR in the cortical thick ascending limb of the nephron mediates direct, high Ca2þo-induced inhibition of the tubular reabsorption of Ca2þ and Mg2þ [136,137]. Therefore, raising Ca2þo both directly inhibits renal tubular reabsorption of Ca2þ via actions on the CaSR expressed in nephron segments involved in hormonal regulation of Ca2þ reabsorption (e.g. by PTH) and indirectly inhibits it by reducing PTH secretion (see Renal Calcium Reabsorption below). The identification of hypercalcemic (e.g. familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism) [138] and hypocalcemic (i.e. autosomaldominant hypocalcemia) [139] disorders caused by inactivating and activating mutations of the CaSR, respectively, illustrates the receptor’s central, nonredundant role in setting the serum calcium concentration [140,141]. Targeted disruption of the CaSR gene has also enabled the generation of mouse models of familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism via inactivation of one or both alleles of the CaSR [142], further supporting its importance in Ca2þo homeostasis. In addition to directly inhibiting PTH gene expression, 1,25(OH)2D3 also reduces PTH secretion [143,144] (for review [11,25,108]). It is not known whether this latter action is solely secondary to the effect of 1,25(OH)2D3 on biosynthesis of the hormone and/or represents a direct action on the secretory process per se. Increasing the ambient level of phosphate in vitro, independent of concomitant changes in Ca2þo, enhances parathyroid cellular proliferation, PTH gene expression and, ultimately, hormonal secretion [145e147]. Phosphate-induced changes in PTH secretion, however, take several hours and must result secondarily from

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changes in hormonal biosynthesis rather than secretion per se [147]. Finally, Mg2þo clearly functions as a CaSR agonist in vitro when tested in cells containing an endogenous CaSR [148] (e.g. parathyroid cells) or expressing the cloned CaSR [124], although it is twofold to threefold less potent than Ca2þo on a molar basis. Because levels of serum ionized Mg2þo are lower than those of Ca2þo, it is presently unclear whether Mg2þo acts as a physiologically relevant CaSR agonist at the parathyroid gland in vivo under normal circumstances. Patients with inactivating or activating CaSR mutations, however, can exhibit mild hypermagnesemia or hypomagnesemia [140], respectively, thus suggesting that the CaSR does contribute to setting Mg2þo in vivo, as previously suggested [149]. It may do so, at least in part, in the kidney, where Mg2þo in the tubular fluid of the thick ascending limb exceeds that in blood and may be sufficient to activate the CaSR that regulates tubular reabsorption of Ca2þo and Mg2þo in this nephron segment [123,137,150,151]. In addition to the inhibitory effect of elevated Mg2þo on PTH secretion, low concentrations of Mg2þo e as in patients with overt magnesium deficiency e also reduce PTH secretion [152]. The mechanism(s) underlying this effect of hypomagnesemia has recently been suggested to involve increased activity of G proteins to which the CaSR normally couples, probably Gi and Gq/11, thereby leading to increased intracellular signalling and inhibition of PTH secretion [153].

Metabolism of PTH Studies performed over more than three decades by several laboratories have focused on the heterogeneity of circulating forms of PTH, which was first identified by Berson and Yalow in 1968 [154]. From these investigations, it is now apparent that, in addition to the fulllength polypeptide PTH(1e84), which is the biologically active hormone, much of the circulating hormone lacks an intact N-terminus and most of these fragments are, thus, devoid of biologic activity, at least with regard to the PTH/PTHrP receptor-mediated regulation of mineral ion homeostasis [24]. C-terminal PTH fragments are produced in and released from the parathyroid gland, but they are also derived from circulating intact hormone by efficient, high-capacity degradative systems in the liver and kidney and most likely at other peripheral sites (Fig. 6.6). Some PTH fragments, such as PTH(7e84), appear to be generated within the gland and to have some biological activity, perhaps as inhibitors [155e157]. Direct measurement of arterial and venous differences in parathyroid effluent blood (with vigorous conditions to prevent any ex vivo cleavage of hormone after sample collection) were performed in cattle and confirmed that smaller C-terminal fragments and intact

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FIGURE 6.6 Scheme of peripheral PTH cleavage and the interaction of PTH with the PTH/PTH-related peptide receptor (PTH1R) and with other putative receptors on target cells. Within parathyroid cells, secretory vesicles (circles) and in peripheral organs various patterns of cleavages of PTH(1e84) occur, resulting in multiple circulating fragments, mostly C-terminal ones (solid bars); but not N-terminal ones (suggesting rapid degradation). The carboxyl end of intact PTH and possibly some C fragments interact with a putative C-terminal receptor and, as well, inhibit actions of PTH on the PTH1R. Cyclic adenosine monophosphate, cAMP.

hormone, but not N-terminal fragments, are secreted into the circulation. The relative concentration of these C-terminal fragments released from the gland increases under conditions of systemic hypercalcemia, when overall secretion rates of intact hormone are lower [95,158]. The C-terminal PTH fragments are similar to those generated by peripheral metabolism (see below) but were not chemically characterized. The peripheral metabolism of PTH has been analyzed by injecting intact hormone into the circulation of test animals. Such experiments have not been performed in human subjects, but it is assumed that the similar metabolism of PTH in rats, dogs, and cows is reflective of its metabolism in humans [159]. Clearance of intact PTH from plasma was found to be very rapid (halflife, 2e4 minutes) [160,161]; the major sites of clearance being the liver and kidney. Clearance by the liver predominates over clearance by the kidney; the two organs together account for virtually all clearance of intact hormone. Hepatic clearance of intact hormone has been estimated to be 40e75% and renal clearance, 20e30% [24]. In summary, current evidence indicates that intact PTH and multiple C-terminal fragments (which are derived from glandular and peripheral cleavage and may not be identical) are the principal circulating hormonal forms. In addition, recent data suggest the presence of a previously unrecognized, large,

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N-terminally truncated form of the hormone. Biologically active, N-terminal fragments of PTH, if found in the circulation at all, are likely to circulate only at extremely low concentrations (<10e13e10e14 mol/L). Because of the lack of evidence of circulating forms of biologically active, N-terminal fragments, it was feasible to introduce immunometric assays that use two different antibodies, an immobilized capturing antibody directed against the C-terminal portion of PTH(1e84) and a radiolabeled or enzyme-labeled detection antibody that is directed against an epitope within the N-terminal portion of the intact molecule [162,163]. Although these two-site assays have been clinically useful and provided a great improvement over earlier assays [162], recent studies have demonstrated the presence of circulating PTH fragments that are different in character and composition from any of the hormone fragments discussed previously [98,118,164]. High performance liquid chromatography (HPLC) analysis of PTH in blood samples from uremic patients with end-stage renal disease and from patients with other forms of hyperparathyroidism revealed two distinct immunoreactive peaks in column effluent that were detectable (although with differing sensitivity) by different commercial assays. One peak corresponded to intact PTH(1e84), whereas the other peak migrated close to the position occupied chromatographically by synthetic PTH(7e84) [98,118]. This finding suggested that the epitope of the detection antibody in these assays did not require the presence of the first six or more amino acids of PTH(1e84) [118,119]. The current view is that the molecular entity or entities detected besides PTH(1e84) are N-terminally truncated forms of the intact molecule that are similar, but not necessarily identical, to synthetic PTH(7e84) [118]. In fact, more detailed chromatographic studies performed with one of these “intact” PTH assays showed significant variation in the ratio of the N-terminally truncated PTH to PTH(1e84). Individuals with normal renal function showed a lower ratio than did patients with uremia; furthermore, the percentage of immunoreactivity representing N-terminally truncated forms of PTH (PTH[7e84]) rose in both groups when hypercalcemia was present [98]. The newer immunoradiometric assays that have N-terminal epitopes at the extreme amino-terminus of PTH (1e84) show significantly lower PTH concentrations in patients with chronic renal failure during stimulation and suppression of glandular activity with alterations in calcium, a result consistent with the conclusion that only full length and therefore biologically active PTH(1e84) is detected by these assay systems. The findings outlined previously were expected to have considerable significance for the management of patients with parathyroid dysfunction, especially in

the presence of renal failure. Treatment of patients with end-stage renal disease with large amounts of vitamin D and/or calcium (or calcimimetics) can be associated with adynamic bone disease, which can be deleterious, particularly in growing children [165e167]. Particularly during hypercalcemia, N-terminally truncated forms of PTH become a significant PTH species [168,169]. Measurement of “intact” PTH by earlier assays [162], therefore overestimated the concentrations of biologically active PTH and could result in the overtreatment of uremic patients with vitamin D-analogs and/or calcium. The resulting excessive suppression of the parathyroid gland could reduce PTH-dependent bone turnover. When secretion of PTH is suppressed and the fractional concentration of the large, N-truncated PTH increases, inhibition of the actions of native PTH on calcium or bone could also occur. Recent experiments with synthetic PTH(7e84) support the conclusion that such actions occur in vivo and in vitro. For example, PTH(7e84) was shown to have hypocalcemic properties in vivo [155,157], and it reduces, presumably via receptors specific for C-terminal PTH fragments, bone resorption that may be partly due to impaired osteoclast differentiation [99,155]. It seems useful, therefore, to monitor patients with renal failure with the newer radioimmunometric assays that detect only intact PTH [164].

PTH-DEPENDENT REGULATION OF CALCIUM HOMEOSTASIS PTH is, as outlined earlier, the most important peptide regulator of mineral ion homeostasis in mammals. Through its actions on the kidney and bone, PTH maintains blood calcium concentration within narrow limits. In bone, it stimulates the release of calcium and, in the kidney, it enhances renal tubular reabsorption of calcium (and diminishes tubular reabsorption of phosphate). Furthermore, in the kidney, it increases the synthesis of renal 1a-hydroxylase, which stimulates 1,25(OH)2D3 production and thus increases, albeit indirectly, intestinal absorption of calcium (and phosphate). These direct and indirect endocrine actions of PTH are mediated through the PTH/PTHrP receptor (PTH1R), a G protein-coupled receptor that is abundantly expressed in both major target tissues of PTH action (PTH, along with other secreted hormones such as FGF-23 are critical regulators of phosphate homeostasis as discussed in Chapter 7).

Actions of PTH on Kidney In addition to regulating renal calcium and phosphate (see Chapter 7) transport, PTH modifies the tubular

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handling of magnesium, sodium, potassium, bicarbonate, and water and, moreover, stimulates renal gluconeogenesis. Specific regions of the nephron that are involved in each of the PTH-dependent actions have been defined through in vivo micropuncture analyses and through in vitro studies with nephron segments that have been dissected from the remainder of the kidney. Furthermore, cell lines from specific regions of the kidney have also been used. However, interpretation of these results has to take into account the complexity of the organization of the kidney as an organ (e.g. the complex anatomic relationship involving different renal tubular segments spanning both the cortex and medulla and the effects of countercurrent distribution that modify solute and water transport), particularly since certain in vivo features of renal tubules are not readily imitated in vitro through the use of isolated tubules or cells. Most of the calcium in the glomerular filtrate is reabsorbed. The bulk of this reabsorption (65%) occurs via passive, paracellular mechanisms, both in the proximal tubules and, to a lesser extent, in the thick ascending limb of Henle’s loop and the distal convoluted tubule [170e174]. The calcium-sensing receptor plays an important, PTH-independent role in the adjustment of renal calcium reabsorption in the cortical thick ascending limb. The physiologically important stimulation of renal calcium reabsorption by PTH occurs almost entirely in the distal nephron. In the cortical thick ascending limb, PTH increases the magnitude of the lumen positive potential that drives the passive, paracellular reabsorption of calcium and magnesium. In the distal convoluted tubule, in contrast, PTH promotes increased transcellular Ca2þ reabsorption by coordinated upregulation of molecular components of this transcellular pathway [175]. PTH enhances uptake of Ca2þ into the tubular cells via the (luminal) plasma membrane channel, TRPV5 [176,177] as well as its active extrusion against a steep electrochemical gradient at the basolateral membrane. This latter process of extrusion involves two types of transporters. One is the plasma membrane calcium pump (Ca2þ, Mg2þ-ATPase, PMCA) and the second is an Naþ/Ca2þ exchanger (NCX1) which, in turn, is indirectly regulated by Naþ/ Kþ-ATPase(s), which maintains the transcellular Naþ gradient. Studies performed with membrane vesicles from the distal region of the kidney show increased activity of the Naþ/Ca2þ exchanger in response to PTH [178]. PTH also stimulates the translocation of preformed Ca2þ channels sequestered within the interior of certain distal tubular cells, presumably TRPV5, to the apical surface [179] (i.e. luminal) surface of the renal tubular epithelial cell to mediate increased cellular Ca2þ uptake (Fig. 6.7). Because PTH simultaneously enhances the activity of Naþ/Ca2þ exchangers in the

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FIGURE 6.7

PTH regulation of renal calcium transport. Parathyroid hormone (PTH) in distal tubular cells of kidney triggers the translocation of preformed voltage-dependent calcium channels (e.g. TRPV5) from sites of intracellular sequestration to the apical membrane, which also undergoes rapid morphologic change to greatly increase its surface area. PTH can also stimulate TRPV5 openchannel probability. Intracellular free calcium levels rise significantly, and increased net transepithelial calcium transport occurs, mainly via enhanced Naþ/Ca2þ exchange at the basolateral membrane, supported in turn by Naþ/Kþ-ATPase (see text for details).

basolateral (antiluminal) membrane, the overall process promotes an increase in transcellular Ca2þ transport from the lumen to blood e that is, from the apical to the basolateral membrane. PTH is a major inducer of the activity of proximal tubular 1a-hydroxylase, a microsomal cytochrome P-450 enzyme that synthesizes biologically active 1,25(OH)2D3 from the substrate 25(OH)D3 [180e182]. This effect of PTH on synthesis of the renal enzyme shows longer lag times than its effect on renal Ca2þ transport and is mediated, at least in part, by the protein kinase A (PKA) signaling pathway of the PTH/PTHrP receptor (see Chapter 8 for discussion of the critical role of 1,25 (OH)2D3 on intestinal calcium absorption) [183,184].

Actions of PTH on Bone PTH affects a wide variety of the highly specialized bone cells, including osteoblast/stromal cells, osteoclasts and oteocytes, the latter being the most numerous

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cell type in bone. Some of these effects reflect direct actions of PTH; both osteoblasts and osteocytes have PTH receptors. Other effects are indirect and mediated in an autocrine/paracrine manner through factors released by cells (osteoblasts) expressing PTH/PTHrP receptors that regulate the activity of yet other cells (osteoclasts) that lack these receptors [185e187]. PTH action on bone cells can be considered from at least five distinct perspectives. First is its major physiological role: the maintenance of calcium homeostasis. This action can be analyzed most clearly in experimental animals through parathyroidectomy with resultant hypocalcemia. Renal losses of calcium are significant contributors to the severe, sometimes fatal hypocalcemia, but the loss of PTH action on bone cells is the predominant cause. The traditional explanation is that the hypocalcemia is due principally to the loss of PTH stimulation of osteoclastic bone resorption, but other mechanisms may be involved (see below). A second perspective is the action of PTH when administered pharmacologically, resulting in elevation of serum calcium. It is still unclear which specific cellular actions explain the biological actions known to follow PTH administration to animals in vivo. Earlier studies had shown that the administration of PTH leads within minutes to a transient lowering of blood calcium caused by uptake of the mineral by bone cells [188], which is rapidly followed by increased mobilization of calcium from the mineral phase into the bloodstream [188,189]. Although there continue to be uncertainties regarding the cellular/ anatomic basis of these rapid responses to PTH, considerable progress has been made in elucidating the mechanisms through which bone cells respond to PTH and to other autocrine/paracrine factors, such as cytokines. A major pathway for calcium release from bone involves osteoclasts; these cells undergo multiple cellular changes involving the activation of cellular transporters and pumps, as well as the secretion of enzymes such as cathepsin K and collagenases, but other mechanisms of calcium release are proposed [188,189]. A third perspective regarding PTH action on bone cells can be appreciated through results seen when PTH is used as a therapy for osteoporosis. Chronic administration of PTH, if given intermittently, stimulates bone formation, an action that involves a complex set of cellular responses in bone affecting principally stromal cell/osteoblast proliferation, differentiation and cellular actions [190e194]. It is also clear that actions on osteocytes are involved in PTH actions on bone [194,195]; together PTH effects on the two cell types, osteoblasts and osteocytes, stimulate bone matrix formation and bone mineral deposition, as well as bone mineral mobilization.

Fourthly, pathophysiological actions of PTH are seen in hyperparathyroidism with chronic excess levels of PTH. In this situation, both bone formation and bone resorption are stimulated, the latter effect being predominant. Finally, one must consider PTH action on bone (and that of the closely related PTHrP) from the important role it plays, along with multiple other hormones and cytokines, in the complex system biology of embryonic bone formation as well as bone formation and remodeling in the adult. Numerous other hormones and autocrine- and paracrine-acting cytokines work together with PTH and PTHrP in this still incompletely understood complex cellular biology of bone. Osteoblasts express the PTH/PTHrP receptor abundantly and show vigorous responses to the hormone. PTH stimulates the generation of cAMP, inositol triphosphate, and diacylglycerol in cells expressing the cloned PTH/PTHrP receptor [7,9,196]. Many cellular activities of osteoblasts are influenced by PTH; these include ion transport, cell shape, gene transcriptional activity, and secretion of multiple proteases. Continuous PTH administration in vivo results in decreased bone mass, whereas intermittent administration of PTH leads to an increase [24,197,198]. Osteoblast synthetic activity is strongly stimulated in both situations but osteoblast/stromal cell increased production of RANK ligand and suppression of osteoprotogerin secretion [199,200] dominate through stimulation of osteoclastic activity in chronic PTH administration. PTH administration leads to multiple osteoblastic responses including reduced rates of cell apoptosis [190], increased expression and activity of Runx 2 [201,202], increased rates of osteoblastic cell differentiation [191,203] and increased resistance to oxidative stress [204]. The exact cellular mechanisms whereby intermittent PTH administration selectively enhances bone formation remain unclear at present, as are correlations between in vivo and in vitro responses. Further exploration of these mechanisms is clearly of great interest for understanding the hormone’s therapeutic potential as a bone anabolic agent [205]. It is generally agreed that osteoclasts lack PTH receptors and hence actions of PTH are indirect largely through stimulation of cytokines elaborated from osteoblasts/stromal cells. These molecules include osteoclastdifferentiating factor, now usually called RANKL [206,207], a membrane-associated protein with homology to the family of tumor necrosis factors (TNFs) that induces e upon cell-to-cell contact and in the presence of macrophage colony-stimulating factor e the differentiation of osteoclast precursors into mature bone-resorbing osteoclasts [208e210]. These effects of RANKL are likely to be mediated through

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RANK, a member of the TNF receptor family that is expressed on osteoclast precursors [207,208]. However, RANKL also interacts with osteoprotegerin, a soluble decoy receptor with homology to the TNF receptor family [211]. Transgenic expression of osteoprotegerin in mice leads to impaired osteoclastogenesis and thus to osteopetrosis [212,213], whereas ablation of the osteoprotegerin gene through homologous recombination in mice results in osteoporosis associated with arterial calcifications [214]. Ablation of the gene for RANKL results in severe osteopetrosis because of the lack of osteoclasts, a defect in tooth eruption, and a complete lack of lymph nodes, but without obvious abnormalities in mineral ion homeostasis [201]. Similarly, ablation of the RANKL receptor (RANK), leads to osteopetrotic changes similar to those observed in RANKL-ablated mice, but also to hypocalcemia and secondary hyperparathyroidism and to renal phosphate wasting [202]. It appears plausible that challenging the homeostatic control mechanisms of either of these knockout mice, in particular, through dietary calcium and vitamin D deficiency, will further aggravate the degree of hypocalcemia and urinary phosphate excretion. However, despite the absence of such experimental data, the outlined findings further illustrate the importance of osteoclastic bone resorption for maintaining blood calcium concentrations. Some of the factors previously noted to have an important role in the paracrine stimulation of osteoclast formation, such as interleukin-6, interleukin-11, prostaglandin E2, 1,25(OH)2D3, and other peptides including PTH [215e217] were shown to stimulate directly the production of RANKL by osteoblasts [218]. Other cellular responses involved in bone resorption include the development of vitronectin-mediated anchorage of osteoclasts to the bone surface, acidification of the circumscribed and sealed-off extracellular environment that is created between the osteoclast and bone and, in addition, the secretion of a variety of proteases and other enzymes. Osteocytes are the most numerous cell type in bone, and there is a growing appreciation that they play a central biological role with a myriad of functions ranging from transducing mechanical signals into bone growth and increased bone mineral density to hitherto completely unappreciated roles in phosphate homeostasis [199,200,205]. Osteocytes contain abundant PTH receptors, and there is now a clearly defined, biologically significant series of responses to PTH action that have been described [193e195,219]. The clearly defined effect of PTH to reduce sclerostin (SOST) production by osteocytes in turn enhances osteoblastic activity by blocking the suppressive effects of SOST on osteoblast activity and the Wnt signaling pathway [193e195].

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PARATHYROID HORMONE-RELATED PEPTIDE Analysis of the physiologic actions of PTH and the molecular basis of its biologic activity requires consideration of the functions of PTHrP. This peptide, discovered and characterized several decades after discovery of PTH, undoubtedly shares an evolutionary origin with PTH. PTHrP has (see Fig. 6.4), at least in mammals, both chemical and functional overlaps with PTH, but many of its biologic roles are quite different. When secreted in large concentrations, for example, by certain tumors, PTHrP has PTH-like properties. Typically, however, it functions as an autocrine/paracrine rather than an endocrine factor. PTHrP is a larger, more complex protein than PTH and is synthesized at multiple sites in different organs and tissues. The still evolving story of this protein and its proteolytic fragments is beyond the scope of this chapter (for comprehensive review see elsewhere [4,6,220]. However, selective functions in the regulation of mineral ion homeostasis and bone development are reviewed here. A substance with biologic properties similar to those of PTH was first proposed in the early 1940s, when Albright discussed a patient with malignancy-associated humoral hypercalcemia [221]. The clinical and biochemical characterization of similarly affected patients subsequently established the syndrome of humoral hypercalcemia of malignancy [222] and eventually led to the amino acid sequence analysis and molecular cloning of PTHrP from several different tumors [223e225].

The PTHrP Gene in Comparison to the PTH Gene The human PTHrP gene is located on chromosome 12p12.1-11.2 [225] which has a region analogous to that containing the human PTH gene on chromosome 11p15 [73e75]. Both the PTH and the PTHrP genes have a similar organization, including equivalent positions of the boundaries between some of the coding exons and the adjacent introns (see Fig. 6.5) [11,77, 225,226]. Like the PTH gene, the PTHrP gene contains a single exon that encodes most amino acid residues of the prepropeptide sequence, and both genes have an exon that encodes the remainder of the propeptide sequence, that is, two basic residues (Lys and Arg) that are required for endoproteolytic cleavage of the mature peptide, and either all or most of the amino acids of the secreted peptides. The similarities in their protein sequence and in the structure of their genes, as well as the overlap in some of their functional properties,

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confirmed that both peptides are derived from a common ancestor [4,6,220]. By now, both mammalian genes have diverged considerably; for example, in contrast to the less complex PTH gene, which gives rise to a single gene product, the PTHrP gene uses at least three different promoters and alternative splice patterns that lead to the synthesis of several different mRNA species encoding peptides with different C-terminal ends [6,220,225,226]. A polymorphic dinucleotide repeat sequence that has been used for genetic linkage studies is located downstream of exon 4 (which is exon 6 according to a different nomenclature) of the human PTHrP gene [227] (see Fig. 6.5); thus far, no human disorder has been discovered that is caused by mutations in the PTHrP gene. When compared with each other, chicken PTHrP and the known mammalian species of PTHrP show strong amino acid sequence homology within the first 111 residues; the degree of amino acid sequence conservation of PTHrP is considerably higher than that of the known PTH species (see Fig. 6.2). The amino acid sequence homology between both peptide families is restricted to the N-terminal portion, where six of the first 12 amino acid residues are conserved in all PTH and PTHrP species (see Fig. 6.2); the middle and Cterminal regions of both peptides share no recognizable similarity. PTHrP is likely to undergo extensive post-translational processing, resulting in peptide fragments, some of which may have biologic properties distinct from those of PTHrP(1e34) on calcium and bone metabolism. The N-terminal PTHrP fragment, functionally homologous to PTH, that is derived through cleavage at amino acid residue Arg37 [228,229], PTHrP(1e36), interacts efficiently with the PTH1R [7e9,230] and it has an in vivo efficacy similar to that of PTH(1e34) [231e233]. Longer, glycosylated fragments of N-terminal PTHrP were also described; however, these forms appear to be generated predominantly by skin-derived cells, and their biologic role, if different from that of PTHrP(1e34) or PTHrP(1e36), remains to be established [234]. In addition to the biologically active N-terminal PTHrP fragment, different C-terminal fragments are generated and accumulate in patients with end-stage renal disease [235]. A PTHrP fragment consisting of amino acids 107 to 139 and a shorter peptide, PTHrP(107e111) may be relevant to the control of bone metabolism because both fragments were shown to inhibit osteoclastic bone resorption [236,237] and to stimulate osteoblast activity and proliferation [238]. Although some investigators, using modified experimental conditions, were unable to confirm the in vitro findings with osteoclasts [239], more recent data indicate that PTHrP(107e139) reduces the number of osteoclasts and inhibits bone resorption in vivo [240]. Taken

together, these findings suggest that C-terminal PTHrP fragments may have a role in regulating bone resorption and/or formation. Cleavage at amino acid residue Arg37 also generates PTHrP(38e94) amide, a PTHrP fragment that could be of considerable importance in maintaining fetal calcium homeostasis [228,241]. This peptide is found in the circulation [242] and appears to interact with a distinct receptor that signals through changes in intracellular free calcium and is likely to be an important regulator of transplacental calcium transfer [228,241,243e245]. Studies with PTHrP(38e94) amide and PTHrP(67e86) amide have shown that these fragments increase the blood calcium concentrations of parathyroidectomized fetal lambs and PTHrP-ablated murine fetuses, respectively [228,244,245] (see below). These results confirmed earlier data that had provided the first evidence for an important role of PTHrP in the regulation of fetal calcium homeostasis [246].

Functions of PTHrP The first actions of PTHrP to be defined were the PTH-like actions associated with the humoral hypercalcemia of malignancy [4,6,220]. In this pathologic circumstance, PTHrP acts like a hormone, that is, it is secreted from the tumor into the bloodstream and then acts on bone and kidney to raise calcium levels. Whether PTHrP circulates at high enough levels in normal adults to contribute at all as a hormone to normal calcium homeostasis is an unanswered question; the levels are certainly low, and patients with congenital or acquired hypoparathyroidism are hypocalcemic despite the presence of PTHrP. Although incomplete and somewhat conflicting, growing evidence suggests, however, that PTHrP may act as a hormone in two special circumstances: during fetal life and during lactation as outlined in the next sections. During intrauterine life, fetal blood calcium is higher than maternal blood calcium, at least partly because of active transport of calcium across the placenta. In fetal life, in contrast to adulthood, PTHrP is made in easily detectable amounts in the parathyroid gland. Parathyroidectomy lowers the blood level of calcium in fetal sheep and abolishes active calcium transport across the experimentally perfused placenta. PTHrP from human tumors and synthetic PTHrP(1e84), PTHrP(1e108), PTHrP(1e141), and PTHrP(67e86) amide acutely restored placental transport of calcium in a perfused placenta preparation [247,248]. PTHrP with an intact N terminus, PTHrP extracts, or synthetic peptides such as PTHrP(1e34) or PTHrP(1e36), as well as intact PTH and PTH(1e34), had no effect on placental calcium transport. These results suggest that PTHrP secreted from the fetal parathyroids acts on the

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placenta to induce calcium transfer from the mother to the fetus. The role of PTHrP in placental calcium transport is also supported by studies in mice missing both alleles of the PTHrP gene. The blood calcium of fetal PTHrPablated mice is identical to maternal blood calcium, that is, the transport of calcium from the mother into the fetus is diminished [244]. The defect in placental calcium transport can be corrected acutely by injecting PTHrP(1e86) or PTHrP(67e86) into the fetal blood circulation, but not by injecting PTHrP(1e34) or PTH(1e84). These studies in mice and sheep suggest that a receptor distinct from the cloned PTH/PTHrP receptor mediates the action of PTHrP on placental calcium transport. Consistent with this hypothesis, placental calcium transport is actually increased in mice missing the PTH/PTHrP receptor [244]. The possible role of the fetal parathyroid gland as the crucial source of the PTHrP is suggested by the abovementioned experiments in sheep [247,248] but no measurements of PTHrP in fetal sheep blood have been made. The second possible setting for humoral actions of PTHrP is during pregnancy and lactation. During lactation, transfer of calcium from bone into milk results in a measurable decline in bone mineral content [245]. In experimental animals, PTH and 1,25(OH)2D3 have been eliminated as possible agents responsible for directing this transfer [249,250]. Furthermore, the lactating breast secretes PTHrP into the circulation [251] and urinary cAMP rises in response to suckling [252]. Postpartum lactating women have elevated levels of PTHrP in the bloodstream [253e256], and hypoparathyroid patients who are maintained normocalcemic by treatment with 1,25(OH)2D3 can become hypercalcemic during lactation. Thus, PTHrP in the bloodstream may act on bone to release calcium and on the kidney to increase reabsorption of calcium from the urine, thus retaining the calcium for transport into milk. Consistent with this hypothesis, mice missing PTHrP production specifically in breast tissue have decreased bone turnover and preservation of bone mass [257]. PTH, alone cannot effectively serve these roles because the slight elevation in ionized calcium during lactation suppresses PTH levels [253e256,258]. An exaggeration of this lactational elevation of PTHrP may explain the rare occurrence of hypercalcemia and high PTHrP levels in pregnant and lactating women [259,260]. However, PTHrP was shown to have additional roles in the breast and it has a major role in breast development, acting through the PTH1R [261,262]. PTHrP is synthesized by breast tissue and is excreted in enormous amounts into breast milk. Thus, PTHrP in the breast appears to have both paracrine and endocrine roles. These roles may be subverted in breast cancer, a setting in which PTHrP may facilitate the growth of metastases

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in bone [263] and also cause humoral hypercalcemia. The lactating mammary gland can sense extracellular calcium through the calcium-sensing receptor, thereby adjusting calcium and PTHrP secretion into milk. Further, in fetal life, PTHrP, acting through the PTH/ PTHrP receptor, is required for the normal development of breast tissue [257,262]. PTHrP has an essential role in bone development (see Chapters 1 and 2). This role is best illustrated by the phenotype of mice missing both copies of the PTHrP gene [264e266]. These mice die at birth and have diffuse abnormalities in all bones that form by the replacement of a cartilage mold with true bone (endochondral bone formation). Most of the actions of PTHrP are not thought to be hormonal, but rather to be paracrine or autocrine [267]. PTHrP is synthesized at one time or another during fetal life in virtually every tissue. This widespread expression of PTHrP in fetal life probably explains the extensive expression of PTHrP in a great variety of malignancies. Malignant tissues often revert to a fetal pattern of gene expression; synthesis of PTHrP may be part of this pattern. PTHrP is also synthesized by many adult tissues [4,220,267]. In tissues such as skin and hair, it is likely that PTHrP regulates cell proliferation and differentiation [268]. PTHrP is also synthesized widely in the smooth muscle of blood vessels and in the gastrointestinal tract, uterus, and bladder, and transgenic expression of PTHrP in smooth muscle cells leads to severe defects in cardiac development [269,270]. In these tissues, PTHrP is synthesized in response to stretch and acts on smooth muscle in an autocrine fashion to relax the muscle [271]. PTHrP is also widely expressed in neurons of the central nervous system; its function in the brain is unknown. In mice missing the PTHrP gene, widespread degeneration of neurons occurs postnatally; as explained earlier, these mice survive postnatally through expression of PTHrP only in cartilage cells [272].

RECEPTORS FOR PTH AND PTHrP Because of the diverse actions of PTH, which were shown to be either direct or indirect and to involve multiple signal transduction mechanisms, it was initially thought that several different receptors would mediate the pleiotropic actions of this peptide hormone. Although some of these actions were subsequently shown to be PTHrP- rather than PTH-dependent, it was somewhat surprising that the initial cloning approaches led to the isolation of complementary DNAs (cDNAs) encoding only a single G protein-coupled receptor, the common PTH/PTHrP receptor, also called the PTH1R (Fig. 6.8). The recombinant PTH/PTHrP receptor was shown to

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FIGURE 6.8 Schematic of the PTH/PTHrP receptor. The PTH/PTHrP receptor shows the seven-transmembrane domain architecture of the G protein-coupled receptor protein (GPCR) superfamily. The receptor large, disulfide-bonded, amino extracellular domain structure is a hallmark of the class B subgroup of GPCRs. Key residues involved in receptor structure and/or function that are illustrated in the schematic, include the extracellular cysteines (C) that form the conserved disulfide bond network [60,61]; four glycosylated extracellular asparagine (N) residues [273]; Thr33 and Gln37, which contribute interactions involving residues at or near ligand residue tryptophan-23 [274]; Phe184 and Leu187, which contribute interactions involving ligand residues at or near lysine-13 [275,276]; Ser370, Ile371, Met425, Trp437, and Gln440, which contribute interactions involving ligand residues at or near valine-2 and hence receptor activation processes [277e281]; Arg233, Asn295 and Gln451 which form a putative network of interhelical interactions (dashed connectors) [282 359] and thus contribute to PTHR activation [283] and/or internalization [284] responses; conserved proline (P) residues, including Pro132, the site of an inactivating mutation (Leu) in Blomstrand’s neonatal lethal chondrodysplasia [285]; His223, Thr410 and Ile458, the sites of activating mutations in Jansen’s chondrodysplasia [286]; Lys319 and Lys388, which contibute to interactions involving Gq or Gs and Gq coupling, respectively [287,288]; the seven serine (S) residues in the carboxy-terminal tail that are phosphorylated upon agonist activation and mediate recruitment of b-arrestins [284,289e292]; and the four C-terminal residues Glu590eMet593 that mediate interaction with the sodium-hydrogen exchanger regulating factor (NHERF) family of proteins [293,294].

interact approximately equivalently with PTH and PTHrP ligands, and to activate at least two distinct second messenger pathways, namely, the adenylyl cyclase/cAMP/PKA and phospholipase C (PLC)/IP3, Ca2þ, DAG/PKC pathways, mediated by GaS and Gaq/11 respectively [7e9,295,296]. It is now clear that, depending on cell type, the PTH/PTHrP receptor can activate various other signaling cascades, including the extracellular signal-regulated kinase (ERK) cascade [59,289,297,298] and the phospholipase D/rho A cascade

[299], the former potentially involving in addition to GaS, b-arrestins [59] or transactivation of ERK via extracellularly released epidermal growth factor [298] and the latter through activation of the Ga12/13 subtype of heterotrimeric G proteins [299]. The initial finding that both PTH and PTHrP could activate the recombinant PTH/PTHrP receptor confirmed earlier studies using different clonal cell lines or renal membrane preparations that had shown that PTH and PTHrP bind to and activate a common G

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protein-coupled receptor with similar efficiencies and efficacies [230,300e302]. Based on these and subsequent findings, such as the similar phenotypes observed in mice that are null for either PTHrP or the PTH/PTHrP receptor [12,13], it is very likely that most of the endocrine actions of PTH and most of the paracrine/ autocrine actions of PTHrP are mediated through the PTH/PTHrP receptor. The second novel PTH receptor variant isolated, the PTH3 receptor (PTH3R), has so far been found in fish [303] and birds (see NCBI chicken genome website), but not in mammals (Fig. 6.9). Functional and phylogenetic analyses indicate that the PTH3R is more closely related to the PTH1R than to the PTH2R [303]. In addition to the three identified PTH receptor subtypes, there are functional and physicochemical data suggesting that there may be additional receptors that interact with portions of either PTH or PTHrP which are C-terminal of the principal bioactive region defined by the (1e34) segment. As of yet, however, no gene or cDNA encoding such a novel receptor has been identified.

FIGURE 6.9 Phylogenetic relationships of PTH family receptors. Receptor amino acid sequences, with predicted signal peptides and exon E2-encoded segments (mammalian PTH1Rs) removed, were aligned using the ClustalW(2.012) program (gap penalties: opening, 10; extending, 0.2), and an unrooted tree in which branch distances indicate amino acid sequence divergence was generated using the Phylip(3.67) DrawTree program. The diagram illustrates the grouping of the three subtypes of PTH receptors, with the PTH1R present in all vertebrate species, the PTH2R present in humans and fish (Takifugu rubripes and Danio rerio) but apparently absent in frog (Xenopus laevis) and chicken, and the PTH3R present in chicken and fish but not higher vertebrates. A PTHR homolog (z50% identity in the TM region) is present in the genome of the tunicate Ciona intestinalis. Protein data base accession or identification numbers are as follows: human PTH1R, Q03431; pig PTH1R, P50133; rat PTH1R, P25961; opossum PTH1R, P25107; chicken PTH1R, 418507; danio PTH1R, Q9PVD3; fugu PTH1R, Q2UZQ8; chicken PTH3R, XP_425837; danio PTH3R, Q9PVD2; fugu PTH3R, Q2UZR0; ciona PTHR, ci0100139945; human PTH2R, P49190; danio PTH2R, Q9PWB7; fugu PTH2R, Q2UZQ9; xenopus PTH1R(B) from Bergwitz et al. 1998 [304].

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The PTH/PTHrP Receptor Type-1 e Evolution, Gene Structure and Protein Topology The PTH1R belongs to a distinct subgroup of G protein-coupled receptors called the class B, or secretin-family GPCRs. The first cDNAs encoding the PTH/PTHrP receptor were isolated through expression cloning techniques from two different model cell lines: OK cells, an opossum kidney derived proximal tubulelike cell, and ROS 17/2.8 cells, a rat osteosarcomaderived osteoblast-like cell [7,8]. Subsequently, cDNAs encoding the human [9,305,306], mouse [307], rat [308], chicken [13], porcine [309], dog [310], frog [304] and fish PTH/PTHrP receptors [311] were isolated through hybridization techniques from different tissue sources e i.e. kidney, osteoblast-like cells, brain, embryonic stem cells, and/or whole embryos. Northern blot and in situ studies [312e314] and data provided through available public EST (expressed sequence tag) databases confirmed that the PTH/PTHrP receptor is expressed in a wide variety of fetal and adult tissues. The tetraploid African clawed frog Xenopus laevis, expresses two nonallelic isoforms of the PTH/PTHrP receptor [304], whereas all other investigated species have only one copy of the receptor gene per haploid genome. The gene encoding the human PTH/PTHrP receptor is located on chromosome 3p (within the region 3p21.1e3p24.2). Its intron/exon structure [315e317] is largely preserved in the genes encoding the rat and mouse receptor homologs [318,319]. The gene spans at least 20 kb of genomic DNA, and consists of 14 coding exons and at least three non-coding exons. The size of the coding exons in the human gene ranges from 42 bp (exon M7) to more than 400 bp (exon T), and the introns vary in length from 81 bp (intron between exons M6/7 and M7) to more than 10 000 bp (intron between exons S and E1). Two promoters for the PTH/PTHrP receptor, P1 and P2, have been described in rodents [318e321]. The activity of P1 (also referred to as U3) is mainly restricted to the adult kidney, while that of P2 (also referred to as U1) is detected in several fetal and adult tissues, including cartilage and bone. In humans, a third promoter (P3, also referred to as S) appears to control PTH/PTHrP receptor expression in some tissues, including kidney and bone [317,322,323]. The expressed human PTH/PTHrP receptor protein is 593 amino acids in length, including the 22-amino acid N-terminal signal peptide sequence that is removed from the mature receptor during intracellular processing. The predicted topology is defined by a relatively large terminal extracellular domain, a juxtamembrane or J region concerning seven membranes spanning helices and interconnecting loops and a carboxy-terminal tail of about 110 amino acids (see Fig. 6.8).

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PTH1R, along with the receptors for secretin [324], calcitonin [325] and several other peptide hormones, form a distinct GPCR subfamily, now called the class B or secretin-family receptors [326e328]. These class B receptors share a similar overall structural topology, which is defined by the large N-terminal extracellular domain containing the conserved disulfide bond network, as well as a number of other conserved amino acids dispersed throughout the receptor protein [328]. Protein alignment analyses reveal virtually no amino acid sequence homology between the family B GPCRs and the receptors comprising the four other major GPCR subgroups, including the well-studied b2-adrenergic receptor and rhodopsin representing the family A GPCRs [326]. The structural similarity between these groups of receptors is therefore limited to the common use of a heptahelical domain organization for the membrane-spanning region [326,329]. As an evolutionary protein class, the secretin receptor family appears to have arisen during the divergence of the metazoans, as representatives of the receptor family are found in the genomes of a variety of invertebrate species, including the insect Drosophila melanogaster, and the nematode Caenorhabditsi elegans, but not in yeast or bacteria [328]. A PTH/PTHrP receptor homolog is present in the genome of the tunicate sea squirt Ciona intestinalis (see Fig. 6.9), pointing to a much earlier evolutionary origin of the PTH receptor than previously known, in fact, many millions of years before emergence of mammals [328,330]. Since in seawater calcium levels are believed then as now to be higher than the calcium content of extracellular fluid, it is intriguing to speculate what functions of the PTHR existed then. Since evolution has preserved these structures over many millions of years, the question arises: are there functions of this receptor in mammals beyond that of calcium and phosphate homeostasis? Perhaps these functions will only become apparent when and if effective hormone replacement is deployed in hypoparathyroidism.

Mechanisms of Ligand Binding and Activation at the PTH1R The mechanisms of ligand binding used by the PTH receptor have been extensively analyzed by a combination of approaches, including functional methods based on receptor site-directed mutagenesis and ligand analog design strategies [49,50,275,277,331e334] and biophysical methods based on the use of photoreactive cross-linking ligand analogs. In the cross-linking approach, which identifies intermolecular proximities, the analogs are labeled at various positions with a photoreactive group, typically benzophenone, incorporated either directly into the peptide chain, as parabenzoyl-L-phenylalanine

(BPA) [278,334e336] or attached to the epsilon amino function of lysine13 (BpLys) [276]. The combined functional and cross-linking data suggest that the ligand, as represented by the bioactive PTH(1e34) peptide, binds to the receptor via a two-step mechanism [337e340]. The C-terminal portion of PTH(1e34), representing the principal binding domain [341,342], contacts the N-terminal domain of the receptor to establish initial docking interactions [343], subsequently, the N-terminal portion of the ligand interacts with the J domain portion of the receptor to induce the conformational changes that result in G protein coupling (Fig. 6.10A). This general scheme of interaction is believed to extend to most of the class B family of peptide hormone-binding G proteinecoupled receptors [338,339,345,346]. Specific sites of intermolecular contact between the ligand binding domain and the N domain of the receptor can now be defined by the crystal structure of the isolated PTH1R N domain in complex with either the (15e34) fragment of PTH [61] or that of PTHrP [60]. The ligand binding domain thus binds as an amphiphilic a-helix, with the hydrophobic face of the helix, formed in PTH by Trp23, Leu24 and Leu28, making extensive contacts with complementary hydrophobic surfaces that line a central groove that runs through the receptor’s N domain structure. The guanidinium side-chain of the highly conserved Arg20 of the ligand makes additional, mostly polar contacts with at least five receptor residues that form a pocket at the proximal end of the groove. Binding studies, performed using either the intact receptor or the isolated N domain fragment, confirm that the contacts identified in the co-crystal contribute importantly to overall ligand binding affinity [60,61]. The ligand interactions that occur within the receptor’s J domain, containing the seven transmembrane helices (TMs) and connecting loops, are known with far less certainty, given the current absence of a crystal structure. Photoaffinity cross-linking studies suggest that the interactions involve principally the N-terminal ligand segment extending from position 1 to about position 19 [335,339,340]. This N-terminal portion of the ligand contains the activation pharmacophore, defined minimally as the PTH(1e9) segment [49] (see Fig. 6.3). A principal contact site for conserved valine-2 in the ligand, which plays a critical role in receptor activation [56,347], has been mapped by cross-linking and functional methods to Met425 at the extracellular end of TM6 [279,280], and other nearby receptor sites (e.g. Ser370 and Ile371) have also been implicated as likely contact sites for Val2 [281] (see Fig. 6.8). Cross-linking studies have also identified proximities between residue 19 in the ligand and Lys240 at the extracellular end of TM2 [348], and between residue 13 in the ligand and

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FIGURE 6.10 Mechansisms of action at the PTH1R. The proposed two site mechanism of binding at the PTH1R (A). The ligand, PTH(1e34), first interacts with the PTH1R N-domain via its C-terminal helical portion and then engages the juxtamembrane (J) region via its N-terminal segment to induce the conformational changes involved in receptor activation and G protein coupling. Kinetic analyses of ligand binding (B) and G protein coupling (C) at the PTH1R by the fluorescence resonance energy transfer (FRET) approach reveals differences in binding and coupling mechanisms for PTH(1e34) and PTHrP(1e36). In (B), the ligands are labeled on lysine13 with tetramethylrhodamine (TMR) and the PTH1R contains green-fluorescent protein (GFP) in the N domain. Upon binding, GFP fluorescence emission is reduced due to FRET-based quenching by the TMR group. The ligand was applied for z20 seconds (black bar), and then washed out. In (C), the PTH1R contains yellow-fluorescent protein (YFP) in the cytoplasmic tail, and the Gg subunit is tagged with cyan-fluorescent protein (CFP). The ratiometric FRET response (YFP emission: CFP emission upon CFP excitation) increases upon R-G coupling. The recordings show that ligand occupancy and R-G coupling are prolonged after wash-out for PTH(1e34), as compared to PTHrP(1e36). Data are adapted from Ferrandon et al. Nature Chemical Biology, 2009 [344].

Arg186 at the boundary of the N domain and TM1 [276] (see Fig. 6.8). These and other data combined suggest that the ligand’s N-terminal domain binds as an a-helix to a contact surface formed along the extracellular surface of the receptor’s juxtamembrane region [339,348,349]. Such a binding mode would allow Val2 and the other key ligand residues that form the agonist pharmacophoric domain, including Ile5 and Met8, to extend into the core of the transmembrane region and make specific intermolecular contacts that induce receptor activation [49,58,349,350]. In support of this binding mode for the juxtamembrane region of the PTH receptor, the modified

N-terminal M-PTH(1e14) fragment analogs described above (see Fig. 6.3), maintain the same high potency for cAMP generation in cells expressing a PTH1R construct in which the N-terminal domain has been deleted, as they do in cells expressing the intact receptor [49e52,56,275]. The M-PTH analogs also exhibit highly stabilized a-helical structure, in part due to the incorporation of conformationally constrained amino acid analogs, such as a-amino-isobutyric (Aib) at positions 1 and 3 [50,53,54,351]. Such a modified PTH(1e14) analog was used in a drug-screening strategy aimed at identifying small molecule ligands for the PTH1R. One compound identified, SW106, inhibits binding of

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M-PTH(1e14) tracer ligand to the PTH1R with an apparent affinity constant (IC50) of 1 mM; this compound and its analogs represent a new class of non-peptide mimetic ligand for the PTH1R that specifically targets the J domain region [352,353]. Although SW106 binds with only micromolar affinity and functions as a competitive antagonist, the findings suggest that it may be possible to develop related compounds that bind to the PTH1R J domain and function as potent agonists. Given the absence of a high-resolution, three-dimensional structure of the intact PTH1R or of the J domain region, it is not possible to describe precisely the conformational changes that occur in receptor upon agonist binding and subsequent activation and G protein coupling. Specific questions that need to be addressed include how are the N and J domains of the receptor oriented relative to each other and how does the orientation change during the binding processes; how is the PTH(1e34) ligand situated in the ligand-receptor complex, i.e. does it adopt a linear or U shaped structure [354e357]; what are the molecular movements that occur in the J domain upon agonist binding, activation, and G protein coupling, and how are the binding and activation processes related temporally? Initial clues to some of these questions come from recent studies using pharmacological and or biophysical approaches, as described below. Kinetic binding assays performed in membranes revealed that various PTH and PTHrP radioligand analogs display altered rates of receptor association and/or dissociation, as well as differential effects on binding of G protein coupling [337,358]. In particular, the studies show that PTH(1e34) can form a complex with the receptor that remains stable upon the addition of GTPgS, a reagent that is expected to dissociate the receptor-G protein heterotrimer complex and thus shift the receptor into a low-affinity state [358]. Moreover, PTH(1e34) can bind efficiently to the receptor in membranes prepared from cells lacking GaS. In contrast, the M-PTH(1e14) analog forms a complex that rapidly dissociates upon GTPgS addition and binds only poorly in the absence of GaS [358]. Thus, PTH(1e34) has the capacity to bind to, or induce, a novel, G proteinuncoupled high affinity receptor conformation, called R0 [359]; whereas M-PTH(1e14) preferentially binds mainly to a G protein-coupled high affinity conformation, called RG. The ligand PTHrP(1e36), like M-PTH(1e14), was found to bind poorly to R0 and thus more selectively to RG [360]. Analysis of the stability of the complexes using the fluorescent resonance energy transfer (FRET) approach with fluorescently-labeled ligands and proteins tagged with green fluorescent protein (GFP) confirmed a more stable binding of PTH(1e34) to the PTH1R, as compared to

PTHrP(1e36) (see Fig. 6.10B), as well as a more stable interaction of the receptor with G protein (see Fig. 6.10C). These data suggest that the binding of a PTH ligand to the R0 conformation can result in a sustained G protein coupling effect. Consistent with this possibility, the cAMP responses induced by PTH(1e34) in PTH1R-expressing cells were sustained for longer times after wash-out than were those induced by M-PTH(1e14) or PTHrP(1e36) [344,358,360].

Ligands Causing Prolonged Signaling at the PTH1R The potential biological relevance of high affinity binding to the R0 PTH1R conformation was demonstrated by studies of analogs based on the M-PTH(1e14) scaffold (see Fig. 6.3) but extended C-terminally so as to restore interaction to the receptor’s N-terminal domain [361]. The resulting analogs, M-PTH(1e28) and M-PTH(1e34), exhibit several fold higher binding affinities for R0 than does PTH(1e34), and they even induce more prolonged cAMP responses in PTH1R-expressing cells [361]. When injected into mice, M-PTH(1e28) and M-PTH(1e34) induced robust hypercalcemic responses that were sustained for several hours longer than that induced by an equivalent dose of PTH(1e34) (Fig. 6.11). Importantly, these prolonged calcemic responses could not be explained simply by a pharmacokinetic effect, as the M-PTH(1e28) and M-PTH(1e34) disappeared more rapidly from the circulation than did PTH(1e34) [361]. The subcellular mechanisms and pathways that underlie the differences in the duration of signaling responses seen for the different PTH and PTHrP analogs remain to be elucidated, but fluorescence confocal microscopy studies suggest altered modes of ligandreceptor trafficking. Thus, in transfected HEK-293 cells, the longer-acting ligands induce the formation of stable complexes that include the ligand, the receptor, G protein, and adenylyl cyclase, and these proteins remain associated as they move intracellularly and during times of continued cAMP generation [344]. These findings raise the intriguing possibility that in addition to, or as an extension of, the receptor-phosphorylation and arrestin-dependent internalization, desensitization and recycling processes that are known to contribute importantly to the regulation of PTH1R action [58,289,290,362e364], certain PTH ligands may induce a novel mechanism by which the ligand•receptor•G protein complex remains assembled and catalytically active within the endosomal domain [344]. Additional studies are needed to test this hypothesis and elucidate further the mechanisms of prolonged signaling at the PTH1R. In any case, the findings suggest potential new paths of discovery for PTH-based therapeutic

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REFERENCES

discussion), as well as modulate biologic functions in skin [243]. Likewise, actions of the C-terminal PTHrP portion on osteoclasts and osteoblasts [236e238,240] and on the central nervous system [366] appear to be mediated through a specific receptor, and other receptors have been characterized that interact equivalently with the N-terminal portions of PTH and PTHrP but signal only through changes in intracellular free calcium [367e369].

SUMMARY

FIGURE 6.11

Properties of long-acting PTH analogs in vivo. PTH analogs were injected into mice to assess acute effects on blood ionized calcium levels. The modified ligands, M-PTH(1e28) and M-PTH(1e34), which bind to the Ro PTHR conformation with higher affinity than does unmodified PTH(1e34), induce markedly more prolonged increases in blood ionized Ca2þ, as compared to PTH(1e34), when tested at the same dose. These prolonged effects were not explainable by a prolonged half-life of the modified peptides in the circulation, and they were paralleled by prolonged cAMPstimulating activity in MC3T3-E1 osteoblastic cells in culture (not shown). Thus, high-affinity binding to the Ro PTHR conformation can lead to prolonged PTHR signaling actions in vitro and in vivo. Plotted are changes in blood Ca2þ following single i.v. injection of ligands at doses of 20 nmol peptide/kg body weight (data are meansse, n ¼ 2), adapted from Okazaki et al. 2008, Proc Natl Acad Sci. [361].

ligands. For example, a modified PTH analog that induces considerably longer actions at the PTH1R than does PTH(1e34) could prove to be particularly advantageous as a therapy for hypoparathyroidism [365].

Other Receptors for C-terminal PTH or PTHrP There is evidence for the presence of additional receptors and/or binding proteins that interact with portions of PTH or PTHrP C-terminal of the PTH(1e34), but thus far no cDNA encoding such a receptor has been identified. Considerable information has accumulated that indicates that regions of PTH(1e84) other than the N-terminal residues may be responsible for novel biologic actions, although many of these effects are still largely limited to in vitro studies. There is also evidence to suggest that non-PTH1R receptors can mediate actions induced by the midregion of PTHrP, and may thus play an important role in placental calcium transport [228,244] (see earlier

The actions of the amino-terminal portion of PTH and PTHrP (or fragments of this region) are mediated through a single receptor, the PTH/PTHrP receptor, or PTH1R, which belongs to a distinct family of G protein-coupled receptors. The PTH1R mediates the actions of at least the amino-terminal regions of PTH and PTHrP, and signals through multiple second messenger pathways, including the cAMP/PKA and inositol triphosphate/Ca2þ/diacylglycerol/PKC pathways. The PTH1R gene is expressed in a large variety of fetal and adult tissues, but the receptor is most abundant in kidney and bone, where it mediates the endocrine actions of PTH in mineral ion homeostasis, and in the metaphyseal growth plate, where it mediates the autocrine/paracrine actions of locally synthesized PTHrP. Gene deletion studies demonstrate a critical role for the PTH1R and PTHrP in embryonic and postnatal development of cartilage and bone, but the biologic role of this PTHrP-PTH1R paracrine interaction system in the adult remains incompletely understood. The PTH1R is thus used by two ligands for distinct biological functions, endocrine-homeostatic versus paracrine/autocrine-developmental, and it adopts multiple conformational states to support these disparate modes of action. The biological roles of other receptors, such as the PTH-2 receptor, which binds TIP39, or the still hypothesized but uncloned receptors that specifically interact with more C-terminal regions of PTH or PTHrP, also remain undefined. New therapeutic applications of PTH analogs for hypoparathyroidism may emerge from these studies.

References [1] Albright F, Bauer W, Ropes M, Aub JC. Studies of calcium and phosphorus metabolism. IV. The effect of the parathyroid hormone. J Clin Invest 1929;7:139e81. [2] Collip JB. Extraction of a parathyroid hormone which will prevent or control parathyroid tetany and which regulates the level of blood calcium. J Biol Chem 1925;63:395e438. [3] Hansen AM. The hydrochloric X sicca: a parathyroid preparation for intramuscular injection. Milit Surg 1924;54:218e9.

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130

6. PARATHYROID HORMONE AND CALCIUM HOMEOSTASIS

[4] Broadus AE, Stewart AF. Parathyroid hormone-related protein: structure, processing, and physiological actions. In: Bilezikian JP, Levine MA, Marcus R, editors. The Parathyroids Basic and Clinical Concepts. New York: Raven Press; 1994. p. 259e94. [5] Moseley JM, Martin TJ. Parathyroid hormone-related protein: physiological actions. In: Bilezikian JP, Raisz LG, Rodan RA, editors. Principles of Bone Biology. New York: Academic Press; 1996. p. 363e76. [6] Yang KH, Stewart AF. Parathyroid hormone-related protein: the gene, its mRNA species, and protein products. In: Bilezikian JP, Raisz LG, Rodan RA, editors. Principles of Bone Biology. New York: Academic Press; 1996. p. 347e62. [7] Abou-Samra AB, Ju¨ppner H, Force T, et al. Expression cloning of a common receptor for parathyroid hormone and parathyroid hormone-related peptide from rat osteoblast-like cells: a single receptor stimulates intracellular accumulation of both cAMP and inositol triphosphates and increases intracellular free calcium. Proc Natl Acad Sci USA 1992;89:2732e6. [8] Ju¨ppner H, Abou-Samra AB, Freeman MW, et al. A G proteinlinked receptor for parathyroid hormone and parathyroid hormone-related peptide. Science 1991;254:1024e6. [9] Schipani E, Karga H, Karaplis AC, et al. Identical complementary deoxyribonucleic acids encode a human renal and bone parathyroid hormone (PTH)/PTH-related peptide receptor. Endocrinology 1993;132:2157e65. [10] Potts Jr JT, Ju¨ppner H. Parathyroid hormone and parathyroid hormone-related peptide in calcium homeostasis, bone metabolism, and bone development: the proteins, their genes, and receptors. In: Avioli L, Krane S, editors. Metabolic Bone Disease. New York: Academic Press; 1997. p. 51e94. [11] Silver J, Kronenberg HM. Parathyroid hormone e molecular biology and regulation. In: Bilezikian JP, Raisz LG, Rodan GA, editors. Principles of Bone Biology. New York: Academic Press; 1996. p. 325e46. [12] Lanske B, Karaplis AC, Luz A, et al. PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth. Science 1996;273:663e6. [13] Vortkamp A, Lee K, Lanske B, Segre GV, Kronenberg HM, Tabin CJ. Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 1996;273:613e22. [14] Kronenberg HM, Lanske B, Kovacs CS, et al. Functional analysis of the PTH/PTHrP network of ligands and receptors. Recent Prog Horm Res 1998;53:283e301. [15] Bringhurst FR. Calcium and phosphate distribution, turnover, and metabolic actions. In: DeGroot LJ, editor. Endocrinology. 2nd ed. Philadelphia: W.B. Saunders; 1989. p. 805e43. [16] Neer RM. Calcium and inorganic phosphate homeostasis. In: DeGroot LJ, editor. Endocrinology. 2nd ed. Philadelphia: W.B. Saunders; 1989. p. 927e53. [17] Wendelaar-Bonga SE, Pang PK. Control of calcium regulating hormones in the vertebrates: parathyroid hormone, calcitonin, prolactin, and stanniocalcin. Int Rev Cytol 1991;128:139e213. [18] Pang PTK, Pang RK. Hormones and calcium regulation in vertebrates: an evolutionary and overall consideration. In: Pang PKT, Schreibman MP, editors. Regulation of Calcium and Phosphate. San Diego: Academic Press; 1989. p. 343e52. [19] Drezner MK. Phosphorus homeostasis and related disorders. In: Bilezikian JP, Raisz LG, Rodan GA, editors. Principles in Bone Biology. New York: Academic Press; 1996. p. 263e76. [20] Wagner G, Dimattia G, Davie J, Copp D, Friesen H. Molecular cloning and cDNA sequence analysis of coho salmon stanniocalcin. Mol Cell Endocrinol 1992;90:7e15.

[21] Olsen H, Cepeda M, Zhang Q, Rosen C, Vozzolo B. Human stanniocalcin: a possible hormonal regulator of mineral metabolism. Proc Natl Acad Sci USA 1996;93:1792e6. [22] Ishibashi K, Miyamoto K, Taketani Y, et al. Molecular cloning of a second human stanniocalcin homologue (STC2). Biochem Biophys Res Commun 1998;250:252e8. [23] White KE, Larsson TE, Econs MJ. The roles of specific genes implicated as circulating factors involved in normal and disordered phosphate homeostasis: frizzled related protein-4, matrix extracellular phosphoglycoprotein, and fibroblast growth factor 23. Endocr Rev 2006;27:221e41. [24] Potts Jr JT, Bringhurst FR, Gardella TJ, Nussbaum SR, Segre GV, Kronenberg HM. Parathyroid hormone: physiology, chemistry, biosynthesis, secretion, metabolism, and mode of action. In: DeGroot LJ, editor. Endocrinology. Philadelphia: W.B. Saunders; 1996. p. 920e65. [25] Diaz R, El-Hajj GF, Brown E. Regulation of Parathyroid Function. New York: Oxford University Press; 1998. [26] Nordin BEC, Peacock M. Role of kidney in regulation of plasma calcium. Lancet 1969;2:1280e3. [27] Peacock M, Robertson WG, Nordin BEC. Relation between serum and urinary calcium with particular reference to parathyroid activity. Lancet 1969;1:384e6. [28] Mayer G, Habener J, Potts Jr JT. Parathyroid hormone secretion in vivo. Demonstration of a calcium-independent nonsuppressible component of secretion. J Clin Invest 1976;57: 678e83. [29] Parsons JA, Potts Jr JT, editors. Physiology and Chemistry of Parathyroid Hormone. Philadelphia: Saunders; 1972. [30] Potts Jr JT, Deftos LJ. Parathyroid hormone, calcitonin, vitamin D, bone and bone mineral metabolism. In: Bondy PK, Rosenberg LE, editors. Duncan’s Disease of Metabolism. 3rd ed. Philadelphia: W.B. Saunders; 1974. p. 1225e430. [31] Rasmussen H, Bordier P. The Physiological and Cellular Basis of Metabolic Bone Disease. Baltimore: Williams & Wilkins; 1974. [32] Aurbach GD. Isolation of parathyroid hormone after extraction with phenol. J Biol Chem 1959;234:3179. [33] Rasmussen H, Craig LC. Purification of parathyroid hormone by use of counter-current distribution. J Am Chem Soc 1959;81: 5003. [34] Brewer Jr HB, Ronan R. Bovine parathyroid hormone: amino acid sequence. Proc Natl Acad Sci USA 1970;67:1862. [35] Niall HD, Keutmann HT, Sauer RT, et al. The amino-acid sequence of bovine parathyroid hormone I. Hoppe-Seyler’s Z Physiol Chem 1970;351:1586e8. [36] Sauer RT, Niall HD, Hogan ML, et al. The amino acid sequence of porcine parathyroid hormone. Biochemistry 1974;13:1994. [37] Brewer Jr HB, Fairwell T, Ronan R, et al. Human parathyroid hormone: amino acid sequence of the amino-terminal residues 1e34. Proc Natl Acad Sci USA 1972;69:3585. [38] Niall HD, Sauer RT, Jacobs JW, et al. The amino acid sequence of the amino-terminal 37 residues of human parathyroid hormone. Proc Natl Acad Sci USA 1974;71:384. [39] Keutmann HT, Sauer MM, Hendy GN, et al. The complete amino acid sequence of human parathyroid hormone. Biochemistry 1978;17:552. [40] Heinrich G, Kronenberg HM, Potts Jr JT, Habener J. Gene encoding parathyroid hormone: nucleotide sequence of the rat gene and deduced amino acid sequence of rat preproparathyroid hormone. J Biol Chem 1984;259:3320e9. [41] Khosla S, Demay M, Pines M, Hurwitz S, Potts Jr JT, Kronenberg HM. Nucleotide sequence of cloned cDNAs encoding chicken preproparathyroid hormone. J Bone Miner Res 1988;3:689e98.

PEDIATRIC BONE

131

REFERENCES

[42] Russell J, Sherwood LM. Nucleotide sequence of the DNA complementary to avian (chicken) preproparathyroid hormone mRNA and the deduced sequence of the hormone precursor. Mol Endocrinol 1989;3:325e31. [43] Rosol TJ, Steinmeyer CL, McCauley LK, Grone A, DeWille JW, Capen CC. Sequences of the cDNAs encoding canine parathyroid hormone-related protein and parathyroid hormone. Gene 1995;160:241e3. [44] Potts Jr JT, Tregear GW, Keutmann HT, et al. Synthesis of a biologically active N-terminal tetratriacontapeptide of parathyroid hormone. Proc Natl Acad Sci USA 1971;68:63e7. [45] Tregear GW, Van Rietschoten J, Greene E, et al. Bovine parathyroid hormone: minimum chain length of synthetic peptide required for biological activity. Endocrinology 1973;93:1349e53. [46] Gensure R, Ponugoti B, Gunes Y, et al. Isolation and characterization of two PTH-like molecules in zebrafish. Endocrinology 2004;145:1634e9. [47] Guerreiro PM, Renfro JL, Power DM, Canario AV. The parathyroid hormone family of peptides: structure, tissue distribution, regulation, and potential functional roles in calcium and phosphate balance in fish. Am J Physiol Regul Integr Comp Physiol 2007;292:R679e96. [48] Danks J, Ho P, Notini A, et al. Identification of a parathyroid hormone in the fish Fugu rubripes. J Bone Miner Res 2003;18: 1326e31. [49] Shimizu M, Carter P, Gardella T. Autoactivation of type 1 parathyroid hormone receptors containing a tethered ligand. J Biol Chem 2000;275:19456e60. [50] Shimizu N, Guo J, Gardella T. Parathyroid hormone (1 14) and (1 11) analogs conformationally constrained by {alpha} aminoisobutyric acid mediate full agonist responses via the Juxtamembrane region of the PTH 1 receptor. J Biol Chem 2001;276: 49003e12. [51] Shimizu M, Carter P, Khatri A, Potts JJ, Gardella T. Enhanced activity in parathyroid hormone (1-14) and (1-11): Novel peptides for probing the ligand-receptor interaction. Endocrinology 2001;142:3068e74. [52] Shimizu M, Potts JJ, Gardella T. Minimization of parathyroid hormone: novel amino-terminal parathyroid hormone fragments with enhanced potency in activating the Type-1 parathyroid hormone receptor. J Biol Chem 2000;275:21836e43. [53] Shimizu N, Dean T, Khatri A, Gardella T. Amino-terminal parathyroid hormone fragment analogs containing a,-a-dialkyl amino acids at positions 1 and 3. J Bone Miner Res 2004;19: 2078e86. [54] Tsomaia N, Pellegrini M, Hyde K, Gardella TJ, Mierke DF. Toward parathyroid hormone minimization: conformational studies of cyclic PTH(1-14) analogues. Biochemistry 2004;43: 690e9. [55] Shimizu M, Shimizu N, Okazaki M, et al. Parathyroid hormone (1-14) fragments increase bone mass in OVX rats. Advances in Skeltal Anabolic Agents for the treatment of osteoporosismeeting abstracts 2004. M15. [56] Shimizu N, Dean T, Tsang JC, Khatri A, Potts Jr JT, Gardella TJ. Novel parathyroid hormone (PTH) antagonists that bind to the juxtamembrane portion of the PTH/PTH-related protein receptor. J Biol Chem 2005;280:1797e807. [57] Takasu H, Gardella T, Luck M, Potts Jr J, Bringhurst F. Aminoterminal modifications of human parathyroid hormone (PTH) selectively alter phospholipase C signaling via the type 1 PTH receptor: implications for design of signal-specific PTH ligands. Biochemistry 1999;38:13453e60. [58] Bisello A, Chorev M, Rosenblatt M, Monticelli L, Mierke DF, Ferrari SL. Selective ligand-induced stabilization of active and

[59]

[60]

[61]

[62]

[63]

[64]

[65]

[66]

[67]

[68]

[69]

[70]

[71]

[72]

[73]

[74]

[75]

[76]

desensitized parathyroid hormone type 1 receptor conformations. J Biol Chem 2002;277:38524e30. Gesty-Palmer D, Chen M, Reiter E, et al. Distinct beta-arrestinand G protein-dependent pathways for parathyroid hormone receptor-stimulated ERK1/2 activation. J Biol Chem 2006;281: 10856e64. Pioszak AA, Parker NR, Gardella TJ, Xu HE. Structural basis for parathyroid hormone-related protein binding to the parathyroid hormone receptor and design of conformation-selective peptides. J Biol Chem 2009;284:28382e91. Pioszak AA, Xu HE. Molecular recognition of parathyroid hormone by its G protein-coupled receptor. Proc Natl Acad Sci USA 2008;105:5034e9. Fraser RA, Kronenberg HM, Pang PK, Harvey S. Parathyroid hormone messenger ribonucleic acid in the rat hypothalamus. Endocrinology 1990;127:2517e22. Nutley MT, Parimi SA, Harvey S. Sequence analysis of hypothalamic parathyroid hormone messenger ribonucleic acid. Endocrinology 1995;136:5600e7. Roth SI, Schiller AL, editors. Comparative Anatomy of the Parathyroid Glands. Washington, DC: American Physiological Society; 1981. Canario AV, Rotllant J, Fuentes J, et al. Novel bioactive parathyroid hormone and related peptides in teleost fish. FEBS Lett 2006;580:291e9. McManus JF, Davey RA, Maclean HE, et al. Intermittent Fugu parathyroid hormone 1 (1e34) is an anabolic bone agent in young male rats and osteopenic ovariectomized rats. Bone 2008;42:1164e74. Danks JA, Devlin AJ, Ho PMW, et al. Parathyroid hormonerelated protein is a factor in normal fish pituitary. Gen Comp Endocrinol 1993;92:201e12. Devlin AJ, Danks JA, Faulkner MK, et al. Immunochemical detection of parathyroid hormone-related protein in the saccus vasculosus of a teleost fish. Gen Comp Endocrinol 1996;101: 83e90. Danks JA, McHale JC, Martin JT, Ingleton PM. Parathyroid hormone-related protein in tissues of the emerging frog (Rana temporaria): immunohistochemistry and in situ hybridization. J Anat 1997;190:229e38. Trivett M, Officer R, Clement J, et al. Parathyroid hormonerelated protein (PTHrP) in cartilaginous and bony fish tissues. J Exp Zool 1999;284:541e8. Trivett MK, Walker TI, Macmillan DL, Clement JG, Martin TJ, Danks JA. Parathyroid hormone-related protein (PTHrP) production sites in elasmobranchs. J Anat 2002;201:41e52. Trivett MK, Potter IC, Power G, et al. Parathyroid hormonerelated protein production in the lamprey Geotria australis: developmental and evolutionary perspectives. Dev Genes Evol 2005;215:553e63. Antonorakis SE, Phillips JA, Mallonee RL, et al. b-Globin locus is linked to the parathyroid hormone (PTH) locus and lies between insulin and PTH loci in man. Proc Natl Acad Sci USA 1983;80:6615e9. Naylor SL, Sakaguchi AU, Szoka P, et al. Human parathyroid hormone gene (PTH) is on short arm of chromosome 11. Somat Cell Gene 1983;9:609e16. Mayer H, Breyel E, Bostock C, Schmidtke J. Assignment of the human parathyroid hormone gene to chromosome 11. Hum Genet 1983;64:283e5. Zabel BU, Kronenberg HM, Bell GI, Shows TB. Chromosome mapping of genes on the short arm of human chromosome 11: parathyroid hormone gene is at 11p15 together with the genes for insulin, c-Harvey-ras 1, and b-hemoglobin. Cytogenet Cell Genet 1985;39:200e5.

PEDIATRIC BONE

132

6. PARATHYROID HORMONE AND CALCIUM HOMEOSTASIS

[77] Kronenberg HK, Igarashi T, Freeman MW, et al. Structure and expression of the human parathyroid hormone gene. Recent Prog Horm Res 1986;42:641e63. [78] Schmidtke J, Pape B, Krengel U, et al. Restriction fragment length polymorphism at the human parathyroid hormone gene locus. Hum Genet 1984;67:428e31. [79] Gong G, Johnson M, Barger-Lux M, Heaney R. Association of bone dimensions with a parathyroid hormone gene polymorphism in women. Osteoporos Int 1999;9:307e11. [80] Kanzawa M, Sugimoto T, Kobayashi T, Kobayashi A, Chihara K. Parathyroid hormone gene polymorphisms in primary hyperparathyroidism. Clin Endocrinol (Oxf) 1999;50:583e8. [81] Mullersman J, Shields J, Saha B. Characterization of two novel polymorphisms at the human parathyroid hormone gene locus. Hum Genet 1992;88:589e92. [82] Parkinson D, Shaw N, Himsworth R, Thakker R. Parathyroid hormone gene analysis in autosomal hypoparathyroidism using an intragenic tetranucleotide (AAAT)n polymorphism. Hum Genet 1993;91:281e4. [83] Ahn TG, Antonarakis SE, Kronenberg HM, Igarashi T, Levine MA. Familial isolated hypoparathyroidism: a molecular genetic analysis of 8 families with 23 affected persons. Medicine (Baltimore) 1986;65:73e81. [84] Miric A, Levine MA. Analysis of the preproPTH gene by denaturing gradient gel electrophoresis in familial isolated hypoparathyroidism. J Clin Endocrinol Metab 1992;74:509e16. [85] Bilous R, Murty G, Parkinson D, et al. Brief report: autosomal dominant familial hypoparathyroidism, sensorineural deafness, and renal dysplasia. N Engl J Med 1992;327:1069e74. [86] Kronenberg HM, Bringhurst FR, Nussbaum S, et al. Parathyroid hormone: biosynthesis, secretion, chemistry, and action. In: Mundy GR, Martin TJ, editors. Handbook of Experimental Pharmacology: Physiology and Pharmacology of Bone. Heidelberg, Germany: Springer-Verlag; 1993. p. 185e201. [87] Arnold A, Horst SA, Gardella TJ, Baba H, Levine MA, Kronenberg HM. Mutation of the signal peptide-encoding region of the preproparathyroid hormone gene in familial isolated hypoparathyroidism. J Clin Invest 1990;86:1084e7. [88] Karaplis AC, Lim SK, Baba H, Arnold A, Kronenberg HM. Inefficient membrane targeting, translocation, and proteolytic processing by signal peptidase of a mutant preproparathyroid hormone protein. J Biol Chem 1995;270:1629e35. [89] Datta R, Waheed A, Shah GN, Sly WS. Signal sequence mutation in autosomal dominant form of hypoparathyroidism induces apoptosis that is corrected by a chemical chaperone. Proc Natl Acad Sci USA 2007;104:19989e94. [90] Habener JF, Rosenblatt M, Potts Jr JT. Parathyroid hormone: biochemical aspects of biosynthesis, secretion, and metabolism. Physiol Rev 1984;64:985e1053. [91] Hendy GN, Bennett HP, Gibbs BF, Lazure C, Day R, Seidah NG. Proparathyroid hormone is preferentially cleaved to parathyroid hormone by the prohormone convertase furin. A mass spectrometric study. J Biol Chem 1995;270:9517e25. [92] Canaff L, Bennett HP, Hou Y, Seidah NG, Hendy GN. Proparathyroid hormone processing by the proprotein convertase-7: comparison with furin and assessment of modulation of parathyroid convertase messenger ribonucleic acid levels by calcium and 1,25-dihydroxyvitamin D3. Endocrinology 1999;140: 3633e42. [93] MacGregor RR, Hamilton JW, Kent GN. The degradation of proparathormone and parathormone by parathyroid and liver cathepsin B. J Biol Chem 1979;254:4428e33. [94] MacGregor RR, Hamilton JW, Shofstall RE, Cohn DV. Isolation and characterization of porcine parathyroid cathespin B. J Biol Chem 1979;254:4423e7.

[95] Mayer GP, Keaton JA, Hurst JG, Habener JF. Effects of plasma calcium concentration on the relative proportion of hormone and carboxyl fragments in parathyroid venous blood. Endocrinology 1979;104:1778e84. [96] Habener JF, Kemper B, Potts Jr JT. Calcium-dependent intracellular degradation of parathyroid hormone: A possible mechanism for the regulation of hormone stores. Endocrinology 1975;97:431e41. [97] D’Amour P, Palardy J, Bahsali G, Mallette LE, DeLean A, Lepage R. The modulation of circulating parathyroid hormone immunoheterogeneity in man by ionized calcium concentration. J Clin Endocrinol Metab 1992;74:525e32. [98] Brossard JH, Clouthier M, Roy L, Lepage R, Gascon-Barre M, D’Amour P. Accumulation of a non-(1e84) molecular form of parathyroid hormone (PTH) detected by intact PTH assay in renal failure: importance in the interpretation of PTH values. J Clin Endocrinol Metab 1996;81:3923e9. [99] Divieti P, John MR, Juppner H, Bringhurst FR. Human PTH-(7-84) inhibits bone resorption in vitro via actions independent of the type 1 PTH/PTHrP receptor. Endocrinology 2002;143:171e6. [100] Yamaguchi T, Chattopadhyay N, Brown EM. G Protein-coupled extracellular Ca2þ (Ca2þo)-sensing receptor (CaR): roles in cell signaling and control of diverse cellular functions. In: O’Malley B, editor. Hormones and Signalling. San Diego: Academic Press; 1998. [101] Moallem E, Kilav R, Silver J, Naveh-Many T. RNA-protein binding and post-transcriptional regulation of parathyroid hormone gene expression by calcium and phosphate. J Biol Chem 1998;273:5253e9. [102] Brown A, Zhong M, Finch J, et al. Rat calcium-sensing receptor is regulated by vitamin D but not by calcium. Am J Physiol 1996;270:F454e60. [103] Russell J, Sherwood L. The effects of 1,25-dihydroxyvitamin D3 and high calcium on transcription of the pre-proparathyroid hormone gene are direct. Trans Assoc Am Phys 1987;100: 256e62. [104] Okazaki T, Igarashi T, Kronenberg HM. 50 -Flanking region of the parathyroid hormone gene mediates negative regulation by 1,25(OH)2 vitamin D3. J Biol Chem 1989;263:2203e8. [105] Slatopolsky E, Finch J, Ritter C, et al. A new analog of calcitrol, 19-nor-1,25-(OH)2D2, suppress parathyroid hormone secretion in uremic rats in the absence of hypercalcemia. Am J Kidney Dis 1995;26:852e60. [106] Slatopolsky E. The role of calcium, phosphorus and vitamin D metabolism in the development of secondary hyperparathyroidism. Nephrol Dial Transplant 1998;13(Suppl 3):3e8. [107] Llach F, Keshav G, Goldblat M, et al. Suppression of parathyroid hormone secretion in hemodialysis patients by a novel vitamin D analogue: 19-nor-1,25-dihydroxyvitamin D2. Am J Kidney Dis 1998;32(Suppl 2):S48e54. [108] Silver J, Moallem E, Epstein E, Kilav R, Naveh-Many T. New aspects in the control of parathyroid hormone secretion. Curr Opin Nephrol Hypertens 1994;3:379e85. [109] Ben-Dov IZ, Galitzer H, Lavi-Moshayoff V, et al. The parathyroid is a target organ for FGF23 in rats. J Clin Invest 2007;117: 4003e8. [110] Krajisnik T, Bjorklund P, Marsell R, et al. Fibroblast growth factor-23 regulates parathyroid hormone and 1alpha-hydroxylase expression in cultured bovine parathyroid cells. J Endocrinol 2007;195:125e31. [111] Imura A, Tsuji Y, Murata M, et al. alpha-Klotho as a regulator of calcium homeostasis. Science 2007;316:1615e8. [112] Mayer GP, Hurst JG. Sigmoidal relationship between parathyroid hormone secretion rate and plasma calcium concentration in calves. Endocrinology 1978;102:1036e42.

PEDIATRIC BONE

133

REFERENCES

[113] Brent GA, LeBoff MS, Seely EW, Conlin PR, Brown EM. Relationship between the concentration and rate of change of calcium and serum intact parathyroid hormone levels in normal humans. J Clin Endocrinol Metab 1988;67:944e50. [114] Brown EM. Four-parameter model of the sigmoidal relationship between the parathyroid hormone release and extracellular calcium concentration in normal and abnormal parathyroid tissue. J Clin Endcrinol Metab 1983;56:572e81. [115] Parfitt AM. Bone and plasma calcium homeostasis. Bone 1987;8(Suppl. 1):1e8. [116] Brown EM. Extracellular Ca2þ sensing, regulation of parathyroid cell function, and role of Ca2þ and other ions as extracellular (first) messengers. Physiol Rev 1991;71:371e411. [117] Hanley D, Takatsuki K, Sultan J, Schneider A, Sherwood L. Direct release of parathyroid hormone fragments from functioning bovine parathyroid glands in vitro. J Clin Invest 1978;62: 1247e54. [118] Lepage R, Roy L, Brossard JH, et al. A non-(1-84) circulating parathyroid hormone (PTH) fragment interferes significantly with intact PTH commercial assay measurements in uremic samples. Clin Chem 1998;44:805e9. [119] Parfitt AM. Hypercalcemic hyperparathyroidism following renal transplantation: differential diagnosis, management, and implications for cell population control in the parathyroid gland. Min Electrol Metab 1982;8:92e112. [120] Gittes RF, Radde IC. Experimental model for hyperparathyroidism: effect of excessive numbers of transplanted isologous parathyroid glands. J Urol 1966;95:595e603. [121] Lewin E, Olgaard K. Influence of parathyroid mass on the regulation of PTH secretion. Kidney Int 2006;(Suppl):S16e21. [122] Levi R, Silver J. Pathogenesis of parathyroid dysfunction in endstage kidney disease. Pediatr Nephrol 2005;20:342e5. [123] Brown EM. The calcium-sensing receptor: physiology, pathophysiology and CaR-based therapeutics. Subcell Biochem 2007;45:139e67. [124] Brown EM, Gamba G, Riccardi D, et al. Cloning and characterization of an extracellular Ca2þ-sensing receptor from bovine parathyroid. Nature 1993;366:575e80. [125] Brown E, MacLeod R. Extracellular calcium sensing and extracellular calcium signaling. Physiol Rev 2001;81:239e97. [126] Nemeth EF. Calcium receptors as novel drug targets. In: Bilezikian JP, Raisz LG, Rodan GA, editors. Principles of Bone Biology. San Diego: Academic Press; 1996. p. 1019e35. [127] Brauner-Osborne H, Jensen AA, Sheppard PO, O’Hara P, Krogsgaard-Larsen P. The agonist-binding domain of the calcium-sensing receptor is located at the amino-terminal domain [In Process Citation]. J Biol Chem 1999;274:18382e6. [128] Hu J, Reyes-Cruz G, Chen W, Jacobson KA, Spiegel AM. Identification of acidic residues in the extracellular loops of the seven-transmembrane domain of the human Ca2þ receptor critical for response to Ca2þ and a positive allosteric modulator. J Biol Chem 2002;277:46622e31. [129] Kifor O, Diaz R, Butters R, Brown EM. The Ca2þ-sensing receptor (CaR) activates phospholipases C, A2, and D in bovine parathyroid and CaR-transfected, human embryonic kidney (HEK293) cells. J Bone Miner Res 1997;12:715e25. [130] McNeil SE, Hobson SA, Nipper V, Rodland KD. Functional calcium-sensing receptors in rat fibroblasts are required for activation of SRC kinase and mitogen-activated protein kinase in response to extracellular calcium. J Biol Chem 1998;273: 1114e20. [131] Chen CJ, Barnett JV, Congo DA, Brown EM. Divalent cations suppress 3’,5’-adenosine monophosphate accumulation by stimulating a pertussis toxin-sensitive guanine nucleotide-

[132]

[133]

[134]

[135]

[136]

[137]

[138]

[139]

[140] [141]

[142]

[143]

[144]

[145]

[146]

[147]

[148]

[149]

binding protein in cultured bovine parathyroid cells. Endocrinology 1989;124:233e40. Wettschureck N, Lee E, Libutti SK, Offermanns S, Robey PG, Spiegel AM. Parathyroid-specific double knockout of Gq and G11 alpha-subunits leads to a phenotype resembling germline knockout of the extracellular Ca2þ-sensing receptor. Mol Endocrinol 2007;21:274e80. Brown EM. Anti-parathyroid and anti-calcium sensing receptor antibodies in autoimmune hypoparathyroidism. Endocrinol Metab Clin North Am 2009;38:437e445, x. Ritter CS, Pande S, Krits I, Slatopolsky E, Brown AJ. Destabilization of parathyroid hormone mRNA by extracellular Ca2þ and the calcimimetic R-568 in parathyroid cells: role of cytosolic Ca and requirement for gene transcription. J Mol Endocrinol 2008;40:13e21. Garrett JE, Tamir H, Kifor O, et al. Calcitonin-secreting cells of the thyroid express an extracellular calcium receptor gene. Endocrinology 1995;136:5202e11. Riccardi D, Hall AE, Chattopadhyay N, Xu JZ, Brown EM, Hebert SC. Localization of the extracellular Ca2þ/polyvalent cation-sensing protein in rat kidney. Am J Physiol 1998;274: F611e22. Hebert SC, Brown EM, Harris HW. Role of the Ca(2þ)-sensing receptor in divalent mineral ion homeostasis. J Exp Biol 1997;200:295e302. Pollak MR, Brown EM, WuChou YH, et al. Mutations in the human Ca2þ-sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Cell 1993;75:1297e303. Pollak MR, Brown EM, Estep HL, et al. Autosomal dominant hypocalcaemia caused by a Ca2þ-sensing receptor gene mutation. Nat Genet 1994;8:303e7. Brown EM. Clinical lessons from the calcium-sensing receptor. Nat Clin Pract Endocrinol Metab 2007;3:122e33. Hauache OM. Extracellular calcium-sensing receptor: structural and functional features and association with diseases. Braz J Med Biol Res 2001;34:577e84. Ho C, Conner DA, Pollak M, et al. A mouse model for familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Nat Genet 1995;11:389e94. Cantley LK, Russell J, Lettieri D, Sherwood LM. 1,25-Dihydroxyvitamin D3 suppresses parathyroid hormone secretion from bovine parathyroid cells in tissue culture. Endocrinology 1985;117:2114e9. Chan YL, McKay C, Dye E, Slatopolsky E. The effect of 1,25 dihydroxycholecalciferol on parathyroid hormone secretion by monolayer cultures of bovine parathyroid cells. Calcif Tissue Int 1986;38:27e32. Naveh-Many T, Rahaminov R, Livini N, Silver J. Parathyroid cell proliferation in normal and chronic renal failure in rats. The effects of calcium, phosphate, and vitamin D. J Clin Invest 1995;96:1786e93. Almaden Y, Canalejo A, Hernandez A, et al. Direct effect of phosphorus on PTH secretion from whole rat parathyroid glands in vitro. J Bone Miner Res 1996;11:970e6. Slatopolsky E, Finch J, Denda M, et al. Phosphorus restriction prevents parathyroid gland growth. High phosphorus directly stimulates PTH secretion in vitro. J Clin Invest 1996;97:2534e40. Habener JF, Potts JTJ. Relative effectiveness of magnesium and calcium on the secretion and biosynthesis of parathyroid in vitro. Endocrinology 1976;98:197e202. Strewler GJ. Familial benign hypocalciuric hypercalcemia e from the clinic to the calcium sensor. West J Med 1994;160: 579e80.

PEDIATRIC BONE

134

6. PARATHYROID HORMONE AND CALCIUM HOMEOSTASIS

[150] Brown EM. Physiology and pathophysiology of the extracellular calcium-sensing receptor. Am J Med 1999;106:238e53. [151] Brown EM, Vassilev PM, Quinn S, Hebert SC. G-proteincoupled, extracellular Ca(2þ)-sensing receptor: a versatile regulator of diverse cellular functions. Vitam Horm 1999;55: 1e71. [152] Anast CS, Winnacker JL, Forte LF, Burns TW. Impaired release of parathyroid hormone in magnesium deficiency. J Clin Endocrinol Metab 1976;42:707e17. [153] Quitterer U, Hoffmann M, Freichel M, Lohse MJ. Paradoxical block of parathormone secretion is mediated by increased activity of G alpha subunits. J Biol Chem 2001;276:6763e9. [154] Berson SA, Yalow RS. Immunochemical heterogeneity of parathyroid hormone in plasma. J Clin Endocrinol Metab 1968;28: 1037e47. [155] Nguyen-Yamamoto L, Rousseau L, Brossard JH, Lepage R, D’Amour P. Synthetic carboxyl-terminal fragments of parathyroid hormone (PTH) decrease ionized calcium concentration in rats by acting on a receptor different from the PTH/PTHrelated peptide receptor. Endocrinology 2001;142:1386e92. [156] D’Amour P, Brossard JH, Rousseau L, et al. Structure of non(1e84) PTH fragments secreted by parathyroid glands in primary and secondary hyperparathyroidism. Kidney Int 2005;68:998e1007. [157] Slatopolsky E, Finch J, Clay P, et al. A novel mechanism for skeletal resistance in uremia. Kidney Int 2000;58:753e61. [158] Flueck JA, Dibella FB, Edis AJ, Kehrwald J, Arnaud C. Immunoheterogeneity of parathyroid hormone in venous effluent serum from hyperfunctioning parathyroid glands. J Clin Invest 1977;60:1367e75. [159] Brasier A, Wang C, Nussbaum S. Recovery of parathyroid hormone secretion after parathyroid adenomectomy. J Clin Endocrinol Metab 1988;66:495e500. [160] Bringhurst FR, Stern AM, Yotts M. Peripheral metabolism of PTH: fate of the biologically active amino-terminus in vivo. Am J Physiol 1988;255:E886e93. [161] Bringhurst FR, Stern AM, Yotts M. Peripheral metabolism of [35S]PTH in vivo: influence of alterations in calcium availability and parathyroid status. Endocrinology 1989;122:237e45. [162] Nussbaum SR, Zahradnik RJ, Lavigne JR, et al. Highly sensitive two-site immunoradiometric assay of parathyrin, and its clinical utility in evaluating patients with hypercalcemia. Clin Chem 1987;33:1364e7. [163] Blind E, Schmidt-Gayk H, Scharla S, et al. Two-site assay of intact parathyroid hormone in the investigation of primary hyperparathyroidism and other disorders of calcium metabolism compared with a midregion assay. J Clin Endocrinol Metab 1988;67:353e60. [164] John M, Goodman W, Gao P, Cantor T, Salusky I, Ju¨ppner H. A novel immunoradiometric assay detects full-length human PTH but not amino-terminally truncated fragments: implications for PTH measurements in renal failure. J Clin Endocrinol Metab 1999;84:4287e90. [165] Kuizon BD, Goodman WG, Ju¨ppner H, et al. Diminished linear growth during intermittent calcitriol therapy in children undergoing CCPD. Kidney Int 1998;53:205e11. [166] Kuizon BD, Salusky IB. Intermittent calcitriol therapy and growth in children with chronic renal failure. Miner Electrolyte Metab 1998;24:290e5. [167] Sanchez CP, Salusky IB, Kuizon BD, Abdella P, Ju¨ppner H, Goodman WG. Growth of long bones in renal failure: roles of hyperparathyroidism, growth hormone and calcitriol. Kidney Int 1998;54:1879e87. [168] Rakel A, Brossard J, Patenaude J, et al. Overproduction of an amino-terminal form of PTH distinct from human PTH(1e84) in

[169]

[170] [171] [172] [173]

[174]

[175]

[176]

[177]

[178]

[179]

[180]

[181]

[182]

[183]

[184]

[185]

[186]

[187]

a case of severe primary hyperparathyroidism: influence of medical treatment and surgery. Clin Endocrinol (Oxf) 2005;62: 721e7. Rubin MR, Silverberg SJ, D’Amour P, et al. An N-terminal molecular form of parathyroid hormone (PTH) distinct from hPTH(1 84) is overproduced in parathyroid carcinoma. Clin Chem 2007;53:1470e6. Friedman PA, Gesek FA. Calcium transport in renal epithelial cells. Am J Physiol 1993;264:F181e98. Bourdeau J. Mechanisms and regulation of calcium transport in the nephron. Semin Nephrol 1993;13:191e201. Suki WN. Calcium transport in the nephron. Am J Physiol 1979;237:F1e6. Torikai S, Wang M-S, Klein KL, Kurokawa K. Adenylate cyclase and cell cyclic AMP of rat cortical thick ascending limb of Henle. Kidney Int 1981;20:649e54. Morel F, Imbert-Teboul M, Chabardes D. Distribution of hormone-dependent adenylate cyclase in the nephron and its physiological significance. Annu Rev Physiol 1981;43:569e81. van Abel M, Hoenderop JG, van der Kemp AW, Friedlaender MM, van Leeuwen JP, Bindels RJ. Coordinated control of renal Ca(2þ) transport proteins by parathyroid hormone. Kidney Int 2005;68:1708e21. Hoenderop JG, van der Kemp AW, Hartog A, et al. Molecular identification of the apical Ca2þ channel in 1, 25-dihydroxyvitamin D3-responsive epithelia. J Biol Chem 1999;274:8375e8. Mensenkamp AR, Hoenderop JG, Bindels RJ. TRPV5, the gateway to Ca2þ homeostasis. Handb Exp Pharmacol 2007;179: 207e20. Bouhtiauy I, LaJeunesse D, Brunette MG. The mechanism of parathyroid hormone action on calcium reabsorption by the distal tubule. Endocrinology 1991;128:251e8. Bacskai BJ, Friedman PA. Activation of latent Ca2þ channels in renal epithelial cells by parathyroid hormone. Nature 1990;347: 388e91. Takeyama K, Kitanaka S, Sato T, Kobori M, Yanagisawa J, Kato S. 25-Hydroxyvitamin D3 1alpha-hydroxylase and vitamin D synthesis. Science 1997;277:1827e30. St Arnaud R, Messerlian S, Moir JM, Omdahl JL, Glorieux FH. The 25-hydroxyvitamin D 1-alpha-hydroxylase gene maps to the pseudovitamin D-deficiency rickets (PDDR) disease locus. J Bone Miner Res 1997;12:1552e9. Fu GK, Lin D, Zhang MY, et al. Cloning of human 25-hydroxyvitamin D-1 alpha-hydroxylase and mutations causing vitamin D-dependent rickets type 1. Mol Endocrinol 1997;11:1961e7.0. Henry H. Parathyroid hormone modulation of 25-hydroxyvitamin D3 metabolism by cultured chick kidney cells is mimicked and enhanced by forskolin. Endocrinology 1985;116: 503e10. Murayama A, Takeyama K, Kitanaka S, et al. Positive and negative regulations of the renal 25-hydroxyvitamin D3 1alphahydroxylase gene by parathyroid hormone, calcitonin, and 1alpha,25(OH)2D3 in intact animals. Endocrinology 1999;140: 2224e31. Chambers TJ, Athanasou NA, Fuller K. Effect of parathyroid hormone and calcitonin on the cytoplasmic spreading of isolated osteoclasts. J Endocrinol 1984;102:281e6. Wong GL. Paracrine interactions in bone-secreted products of osteoblasts permit osteoclasts to respond to parathyroid hormone. J Biol Chem 1984;259:4019e22. Perry III HM, Skogen W, Chappel J, Kahn AJ, Wilner G, Teitelbaum SL. Partial characterization of a parathyroid hormone-stimulated resorption factor(s) from osteoblast-like cells. Endocrinology 1989;125:2075e82.

PEDIATRIC BONE

REFERENCES

[188] Parsons JA, Robinson CJ. Calcium shift into bone causing transient hypocalcaemia after injection of parathyroid hormone. Nature 1971;230:581e2. [189] Talmage RV, Doppelt SH, Fondren FB. An interpretation of acute changes in plasma 45Ca following parathyroid hormone administration to thyroparathyroidectomized rats. Calcif Tissue Res 1976;22:117e28. [190] Bellido T, Ali AA, Plotkin LI, et al. Proteasomal degradation of Runx2 shortens parathyroid hormone-induced anti-apoptotic signaling in osteoblasts. A putative explanation for why intermittent administration is needed for bone anabolism. J Biol Chem 2003;278:50259e72. [191] Jilka RL. Molecular and cellular mechanisms of the anabolic effect of intermittent PTH. Bone 2007;40:1434e46. [192] Keller H, Kneissel M. SOST is a target gene for PTH in bone. Bone 2005;37:148e58. [193] Leupin O, Kramer I, Collette NM, et al. Control of the SOST bone enhancer by PTH using MEF2 transcription factors. J Bone Miner Res 2007;22:1957e67. [194] O’Brien CA, Plotkin LI, Galli C, et al. Control of bone mass and remodeling by PTH receptor signaling in osteocytes. PLoS ONE 2008;3:e2942. [195] Bellido T, Ali AA, Gubrij I, et al. Chronic elevation of parathyroid hormone in mice reduces expression of sclerostin by osteocytes: a novel mechanism for hormonal control of osteoblastogenesis. Endocrinology 2005;146:4577e83. [196] Iida-Klein A, Guo J, Xie LY, et al. Truncation of the carboxylterminal region of the parathyroid hormone (PTH)/PTH-related peptide receptor enhances PTH stimulation of adenylate cyclase but not phospholipase C. J Biol Chem 1995;270:8458e65. [197] Habener JF, Potts Jr JT. Parathyroid physiology and primary hyperparathyroidism. In: Avioli LV, Krane SM, editors. Metabolic Bone Disease. New York: Academic Press; 1978. p. 1e147. [198] Finkelstein JS. Pharmacological mechanisms of therapeutics: parathyroid hormone. In: Bilezikian JP, Raisz LG, Rodan GA, editors. Principles of Bone Biology. New York: Academic Press; 1996. p. 993e1005. [199] Noble BS. The osteocyte lineage. Arch Biochem Biophys 2008;473:106e11. [200] You L, Temiyasathit S, Lee P, et al. Osteocytes as mechanosensors in the inhibition of bone resorption due to mechanical loading. Bone 2008;42:172e9. [201] Kong Y, Yoshida H, Sarosi I, et al. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature 1999;397:315e23. [202] Li J, Sarosi I, Yan X, et al. RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism. Proc Natl Acad Sci USA 2000;15:1566e71. [203] Krishnan V, Moore TL, Ma YL, et al. Parathyroid hormone bone anabolic action requires Cbfa1/Runx2-dependent signaling. Mol Endocrinol 2003;17:423e35. [204] Jilka RL, Almeida M, Ambrogini E, et al. Decreased oxidative stress and greater bone anabolism in the aged, when compared to the young, murine skeleton with parathyroid hormone administration. Aging Cell 2010;9:851e67. [205] Martin TJ, Sims NA, Ng KW. Regulatory pathways revealing new approaches to the development of anabolic drugs for osteoporosis. Osteoporos Int 2008;19:1125e38. [206] Wong BR, Rho J, Aaron J, et al. TRANCE is a a novel ligand of the tumor necrosis factor receptor family that activates c-Jun N-terminal kinase in T cells. J Biol Chem 1997;272:25190e4. [207] Anderson DA, Maraskovsky E, Billingsley WL, et al. A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature 1997;390:175e9.

135

[208] Yasuda H, Shima N, Nakagawa N, et al. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesisinhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA 1998;95:3597e602. [209] Quinn JM, Elliott J, Gillespie MT, Martin TJ. A combination of osteoclast differentiation factor and macrophage-colony stimulating factor is sufficient for both human and mouse osteoclast formation in vitro. Endocrinology 1998;139:4424e7. [210] Fuller K, Wong B, Fox S, Choi Y, Chambers T. OC activation by TRANCE is necessary and sufficient for osteoblast-mediated activation of bone resorption in osteoclasts. J Exp Med 1998;188: 997e1001. [211] Lacey DL, Timms E, Tan HL, et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 1998;93:165e76. [212] Simonet SW, Lacey DL, Dunstan CR, et al. Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 1997;89:309e19. [213] Suda E, Goto M, Mochizuki S, et al. Isolation of a novel cytokine from human fibroblasts that specifically inhibits osteoclastogenesis. Biochem Biophys Res Commun 1997;234:137e42. [214] Bucay N, Sarosi I, Dunstan DR, et al. Osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev 1998;12:1260e8. [215] Lo¨wik CWGM, van der Pluijm G, Bloys H, et al. Parathyroid hormone (PTH) and PTH-like protein (PLP) stimulate interleukin-6 production by osteogenic cells: a possible role of interleukin-6 in osteoclastogenesis. Biochem Biophys Res Commun 1989;162:1546e52. [216] Paliwal I, Insogna K. Partial purification and characterization of the 9,000-dalton bone-resorbing activity from parathyroid hormone-related protein-treated SaOS2 cells (Abstr #245). J Bone Miner Res 1991;6(Suppl 1):S144. [217] Felix R, Fleisch H, Elford PR. Bone-resorbing cytokines enhance release of macrophage colony-stimulating activity by the osteoblastic cell MC3T3-E1. Calcif Tissue Int 1989;44:356e60. [218] Lee S, Lorenzo J. Parathyroid hormone stimulates TRANCE and inhibits osteoprotegerin messenger ribonucleic acid expression in murine bone marrow cultures: correlation with osteoclast-like cell formation. Endocrinology 1999;140:3552e61. [219] Robling AG, Niziolek PJ, Baldridge LA, et al. Mechanical stimulation of bone in vivo reduces osteocyte expression of Sost/sclerostin. J Biol Chem 2008;283:5866e75. [220] Martin JT, Moseley JM, Gillespie MT. Parathyroid hormonerelated protein: biochemistry and molecular biology. Crit Rev Biochem Mol Biol 1991;26:377e95. [221] Albright F. Case records of the Massachusetts General Hospital; case 27461. New Engl J Med 1941;255:789e91. [222] Stewart AF, Horst R, Deftos LJ, Cadman EC, Lang R, Broadus AE. Biochemical evaluation of patients with cancerassociated hypercalcemia. Evidence for humoral and nonhumoral groups. N Engl J Med 1980;303:1377e81. [223] Moseley JM, Kubota M, Diefenbach-Jagger H, et al. Parathyroid hormone-related protein purified from a human lung cancer cell line. Proc Natl Acad Sci USA 1987;84:5048e52. [224] Strewler GJ, Stern PH, Jacobs JW, et al. Parathyroid hormonelike protein from human renal carcinoma cells. Structural and functional homology with parathyroid hormone. J Clin Invest 1987;80:1803e7. [225] Mangin M, Webb AC, Dreyer BE, et al. Identification of a cDNA encoding a parathyroid hormone-like peptide from a human tumor associated with humoral hypercalcemia of malignancy. Proc Natl Acad Sci USA 1988;85:597e601.

PEDIATRIC BONE

136

6. PARATHYROID HORMONE AND CALCIUM HOMEOSTASIS

[226] Mangin M, Ikeda K, Dreyer BE, Broadus AE. Isolation and characterization of the human parathyroid hormone-like peptide gene. Proc Natl Acad Sci USA 1989;86:2408e12. [227] Pausova Z, Morgan K, Fujiwara TM, Bourdon J, Goltzman D, Hendy GN. Molecular characterization of an intragenic minisatellite (VNTR) polymorphism in the human parathyroid hormone-related peptide gene in chromsome 12p12.1-11.2. Genomics 1993;17:243e4. [228] Wu TL, Vasavada RC, Yang K, et al. Structural and physiological characterization of the mid-region secretory species of parathyroid hormone-related protein. J Biol Chem 1996;271: 24371e81. [229] Yang KH, dePapp AE, Soifer NE, et al. Parathyroid hormonerelated protein: evidence for isoform- and tissue-specific posttranslational processing. Biochemistry 1994;33:7460e9. [230] Ju¨ppner H, Abou-Samra AB, Uneno S, Gu WX, Potts Jr JT, Segre GV. The parathyroid hormone-like peptide associated with humoral hypercalcemia of malignancy and parathyroid hormone bind to the same receptor on the plasma membrane of ROS 17/2.8 cells. J Biol Chem 1988;263:8557e60. [231] Kemp BE, Moseley JM, Rodda CP, et al. Parathyroid hormonerelated protein of malignancy: active synthetic fragments. Science 1987;238:1568e70. [232] Horiuchi N, Caulfield MP, Fisher JE, et al. Similarity of synthetic peptide from human tumor to parathyroid hormone in vivo and in vitro. Science 1987;238:1566e8. [233] Everhart-Caye M, Inzucchi SE, Guinness-Henry J, Mitnick MA, Stewart AF. Parathyroid hormone (PTH)-related protein(1e36) is equipotent to PTH(1e34) in humans. J Clin Endocrinol Metab 1996;81:199e208. [234] Wu TL, Soifer NE, Burtis WJ, Milstone LM, Stewart AF. Glycosylation of parathyroid hormone-related peptide secreted by human epidermal keratinocytes. J Clin Endocrinol Metab 1991;73:1002e7. [235] Orloff JJ, Soifer NE, Fodero JP, Dann P, Burtis WJ. Accumulation of carboxy-terminal fragments of parathyroid hormone-related protein in renal failure. Kidney Int 1993;43:1371e6. [236] Fenton AJ, Kemp BE, Hammonds Jr RG, et al. A potent inhibitor of osteoclastic bone resorption within a highly conserved pentapeptide region of parathyroid hormone-related protein; PTHrP(107e111). Endocrinology 1991;129:3424e6. [237] Fenton AJ, Kemp BE, Kent GN, et al. A carboxyl-terminal peptide from the parathyroid hormone-related protein inhibits bone resorption by osteoclasts. Endocrinology 1991;129:1762e8. [238] Cornish J, Callon K, Lin C, Xiao C, Moseley J, Reid I. Stimulation of osteoblast proliferation by C-terminal fragments of parathyroid hormone-related protein. J Bone Miner Res 1999;14: 915e22. [239] Sone T, Kohno H, Kikuchi H, et al. Human parathyroid hormone-related peptide-(107e111) does not inhibit bone resorption in neonatal mouse calvariae. Endocrinology 1992;131: 2742e6. [240] Cornish J, Callon KE, Nicholson GC, Reid IR. Parathyroid hormone-related protein-(107e139) inhibits bone resorption in vivo. Endocrinology 1997;138:1299e304. [241] Soifer NE, Dee K, Insogna KL, et al. Parathyroid hormonerelated protein. Evidence for secretion of a novel mid-region fragment by three different cell types. J Biol Chem 1992;267: 18236e43. [242] Burtis WJ, Dann P, Gaich GA, Soifer NE. A high abundance midregion species of parathyroid hormone-related protein: immunological and chromatographic characterization in plasma. J Clin Endocrinol Metab 1994;78:317e22. [243] Orloff JJ, Ganz MB, Nathanson H, et al. A midregion parathyroid hormone-related peptide mobilizes cytosolic calcium

[244]

[245]

[246]

[247]

[248]

[249]

[250] [251]

[252]

[253]

[254]

[255]

[256]

[257]

[258]

[259]

[260]

[261]

and stimulates formation of inositol trisphosphate in a squamous carcinoma cell line. Endocrinology 1996;137:5376e85. Kovacs CS, Lanske B, Hunzelman JL, Guo J, Karaplis AC, Kronenberg HM. Parathyroid hormone-related peptide (PTHrP) regulates fetal placental calcium transport through a receptor distinct from the PTH/PTHrP receptor. Proc Natl Acad Sci USA 1996;93:15233e8. Kovacs CS, Kronenberg HM. Maternal-fetal calcium and bone metabolism during pregnancy, puerperium, and lactation. Endocr Rev 1997;18:832e72. Rodda CP, Kubota M, Heath JA, et al. Evidence for a novel parathyroid hormone-related protein in fetal lamb parathyroid glands and sheep placenta: comparison with a similar protein implicated in humoral hypercalcemia of malignancy. J Endocrinol 1988;117:261e71. Care A, Caple I, Abbas S, Pickard D. The effect of fetal thyroparathyroidectomy on the transport of calcium across the ovine placenta to the fetus. Placenta 1986;4:271e7. Abbas SK, Pickard DW, Rodda CP, et al. Stimulation of ovine placental calcium transport by purified natural and recombinant parathyroid hormone-related protein (PTHrP) preparations. Q J Exp Physiol 1989;74:549e52. Garner S, Boass A, Toverud S. Parathyroid hormone is not required for normal milk composition or secretion or lactationassociated bone loss in normocalcemic rats. J Bone Miner Res 1990;5:69e75. Halloran B, DeLuca H. Calcium transport in small intestine during pregnancy and lactation. Am J Physiol 1980;239:E64e8. Ratcliffe WA, Thompson GE, Care AD, Peaker M. Production of parathyroid hormone-related protein by the mammary gland of the goat. J Endocrinol 1980;133:87e93. Yamamoto M, Duong LT, Fisher JE, Thiede MA, Caulfield MP, Rosenblatt M. Suckling-mediated increases in urinary phosphate and 30 , 50 -cyclic adenosine monophosphate excretion in lactating rats: possible systemic effect of parathyroid hormonerelated protein. Endocrinology 1991;129:2614e22. Grill V, Hillary J, Ho PMW, et al. Parathyroid hormone-related protein: a possible endocrine function in lactation. Clin Endocrinol 1992;37:405e10. Dobnig H, Kainer F, Stepan V, et al. Elevated parathyroid hormone-related peptide levels after human gestation: relationship to changes in bone and mineral metabolism. J Clin Endocrinol Metab 1995;80:3699e707. Kovacs C, Chik C. Hyperprolactinemia caused by lactation and pituitary adenomas is associated with altered serum calcium, phosphate, parathyroid hormone (PTH), and PTH-related peptide levels. J Clin Endocrinol Metab 1995;80:3036e42. Sowers M, Hollis B, Shapiro B, et al. Elevated parathyroid hormone-related peptide associated with lactation and bone density loss. J Am Med Assoc 1996;276:549e54. VanHouten JN, Dann P, Stewart AF, et al. Mammary-specific deletion of parathyroid hormone-related protein preserves bone mass during lactation. J Clin Invest 2003;112:1429e36. Cross N, Hillman L, Allen S, Krause G, Vieira N. Calcium homeostasis and bone metabolism during pregnancy, lactation, and postweaning: a longitudinal study. Am J Clin Nutr 1995;61: 514e23. Reid I, Wattie D, Evans M, Budayr A. Post-pregnancy osteoporosis associated with hypercalcaemia. Clin Endocrinol (Oxf) 1992;37:298e303. Khosla S, van Heerden JA, Gharib H, et al. Parathyroid hormone-related protein and hypercalcemia secondary to massive mammary hyperplasia. N Engl J Med 1990;322:1157. Hens JR, Dann P, Zhang JP, Harris S, Robinson GW, Wysolmerski J. BMP4 and PTHrP interact to stimulate ductal

PEDIATRIC BONE

137

REFERENCES

[262]

[263]

[264]

[265]

[266]

[267]

[268]

[269]

[270]

[271]

[272]

[273]

[274]

[275]

[276]

[277]

outgrowth during embryonic mammary development and to inhibit hair follicle induction. Development 2007;134:1221e30. Wysolmerski J, Philbrick W, Dunbar M, Lanske B, Kronenberg H, Broadus A. Rescue of the parathyroid hormonerelated protein knockout mouse demonstrates that parathyroid hormone-related protein is essential for mammary gland development. Development 1998;125:1285e94. Guise TA, Yin JJ, Taylor SD, et al. Evidence for a causal role of parathyroid hormone-related protein in the pathogenesis of human breast cancer-mediated osteolysis. J Clin Invest 1996;98: 1544e9. Karaplis AC, Luz A, Glowacki J, et al. Lethal skeletal dysplasia from targeted disruption of the parathyroid hormone-related peptide gene. Genes Devel 1994;8:277e89. Amizuka N, Warshawsky H, Henderson JE, Goltzman D, Karaplis AC. Parathyroid hormone-related peptide-depleted mice show abnormal epiphyseal cartilage development and altered endochondreal bone formation. J Cell Biol 1994;126: 1611e23. Lee K, Lanske B, Karaplis AC, et al. Parathyroid hormonerelated peptide delays terminal differentiation of chondrocytes during endochondral bone development. Endocrinology 1996;137:5109e18. Philbrick WM, Wysolmerski JJ, Galbraith S, et al. Defining the roles of parathyroid hormone-related protein in normal physiology. Physiol Rev 1996;76:127e73. Wysolmerski JJ, Broadus AE, Zhou J, Fuchs E, Milstone LM, Philbrick WM. Overexpression of parathyroid hormone-related protein in the skin of transgenic mice interferes with hair follicle development. Proc Natl Acad Sci USA 1994;91:1133e7. Qian J, Lorenz J, Maeda S, et al. Reduced blood pressure and increased sensitivity of the vasculature to parathyroid hormonerelated protein (PTHrP) in transgenic mice overexpressing the PTH/PTHrP receptor in vascular smooth muscle. Endocrinology 1999;140:1826e33. Maeda S, Sutliff R, Qian J, et al. Targeted overexpression of parathyroid hormone-related protein (PTHrP) to vascular smooth muscle in transgenic mice lowers blood pressure and alters vascular contractility. Endocrinology 1999;140:1815e25. Thiede MA, Daifotis AG, Weir EC, et al. Intrauterine occupancy controls expression of the parathyroid hormone-related peptide gene in preterm rat myometrium. Proc Natl Acad Sci USA 1990;87:6969e73. Philbrick WM, Dreyer BE, Nakchbandi IA, Karaplis AC. Parathyroid hormone-related protein is required for tooth eruption. Proc Natl Acad Sci USA 1998;95:11846e51. Zhou A, Assil I, Abou Samra A. Role of asparagine linked oligosaccharides in the function of the rat PTH/PTHrP receptor. Biochemistry 2000;39:6514e20. Mannstadt M, Luck M, Gardella T, Ju¨ppner H. Evidence for a ligand interaction site at the amino-terminus of the parathyroid hormone (PTH)/PTH-related protein receptor from cross-linking and mutational studies. J Biol Chem 1998;273: 16890e6. Carter P, Shimizu M, Luck M, Gardella T. The hydrophobic residues phenylalanine 184 and leucine 187 in the type-1 parathyroid hormone (PTH) receptor functionally interact with the amino-terminal portion of PTH-(1e34). J Biol Chem 1999;274: 31955e60. Adams A, Bisello A, Chorev M, Rosenblatt M, Suva L. Arginine 186 in the extracellular N-terminal region of the human parathyroid hormone 1 receptor is essential for contact with position 13 of the hormone. Mol Endocrinol 1998;12:1673e83. Lee C, Luck MD, Ju¨ppner H, Potts Jr JT, Kronenberg HM, Gardella TJ. Homolog-scanning mutagenesis of the parathyroid

[278]

[279]

[280]

[281]

[282]

[283]

[284]

[285]

[286]

[287]

[288]

[289]

[290]

[291]

[292]

hormone (PTH) receptor reveals PTH-(1-34) binding determinants in the third extracellular loop. Mol Endocrinol 1995;9: 1269e78. Bisello A, Adams AE, Mierke DF, Pellegrini M, Rosenblatt M, Suva LJ, Chorev M. Parathyroid hormone-receptor interactions identified directly by photocross-linking and molecular modeling studies. J Biol Chem 1998;273:22498e505. Behar V, Bisello A, Bitan B, Rosenblatt M, Chorev M. Photoaffinity cross-linking identifies differences in the interactions of an agonist and an antagonist with the parathyroid hormone/ parathyroid hormone-related protein receptor. J Biol Chem 1999;275:9e17. Gensure R, Carter P, Petroni B, Juppner H, Gardella T. Identification of determinants of inverse agonism in a constitutively active parathyroid hormone/parathyroid hormone related peptide receptor by photoaffinity cross linking and mutational analysis. J Biol Chem 2001;276:42692e9. Gardella TJ, Ju¨ppner H, Wilson AK, et al. Determinants of [Arg2]PTH-(1-34) binding and signaling in the transmembrane region of the parathyroid hormone receptor. Endocrinology 1994;135:1186e94. Chugunov AO, Simms J, Poyner DR, et al. Evidence that interaction between conserved residues in transmembrane helices 2, 3, and 7 are crucial for human VPAC1 receptor activation. Mol Pharmacol 2010;78:394e401. Gardella TJ, Luck MD, Fan M-H, Lee CW. Transmembrane residues of the parathyroid hormone (PTH)/PTH-related peptide receptor that specifically affect binding and signaling by agonist ligands. J Biol Chem 1996;271:12820e5. Vilardaga J, Krasel C, Chauvin S, Bambino T, Lohse M, Nissenson R. Internalization determinants of the parathyroid hormone receptor differentially regulate beta arrestin/receptor association. J Biol Chem 2002;277:8121e9. Zhang P, Jobert AS, Couvineau A, Silve C. A homozygous inactivating mutation in the parathyroid hormone/parathyroid hormone-related peptide receptor causing Blomstrand chondrodysplasia. J Clin Endocrinol Metab 1998;83:3365e8. Schipani E, Langman CB, Hunzelman J, et al. A novel PTH/ PTHrP receptor mutation in Jansen’s metaphyseal chondrodysplasia. J Clin Endocrinol Metab 1999;84:3052e7. Iida-Klein A, Guo J, Takamura M, et al. Mutations in the second cytoplasmic loop of the rat parathyroid hormone (PTH)/PTHrelated protein receptor result in selective loss of PTH-stimulated phospholipase C activity. J Biol Chem 1997;272:6882e9. Huang Z, Chen Y, Pratt S, et al. The N-terminal region of the third intracellular loop of the parathyroid hormone (PTH)/ PTH-related peptide receptor is critical for coupling to cAMP and inositol phosphate/Ca2þþ signal transduction pathways. J Biol Chem 1996;271:33382e9. Rey A, Manen D, Rizzoli R, Caverzasio J, Ferrari SL. Proline-rich motifs in the parathyroid hormone (PTH)/PTH-related protein receptor C terminus mediate scaffolding of c-Src with betaarrestin2 for ERK1/2 activation. J Biol Chem 2006;281:38181e8. Tawfeek HA, Qian F, Abou-Samra AB. Phosphorylation of the receptor for PTH and PTHrP is required for internalization and regulates receptor signaling. Mol Endocrinol 2002;16:1e13. Malecz N, Bambino T, Bencsik M, Nissenson R. Identification of phosphorylation sites in the G protein-coupled receptor for parathyroid hormone. Receptor phosphorylation is not required for agonist-induced internalization. Mol Endocrinol 1998;12: 1846e56. Qian F, Leung A, Abou-Samra A. Agonist-dependent phosphorylation of the parathyroid hormone/parathyroid hormonerelated peptide receptor. Biochemistry 1998;37:6240e6.

PEDIATRIC BONE

138

6. PARATHYROID HORMONE AND CALCIUM HOMEOSTASIS

[293] Mahon M, Donowitz M, Yun C, Segre G. Na(þ)/H(þ) exchanger regulatory factor 2 directs parathyroid hormone 1 receptor signalling. Nature 2002;417:858e61. [294] Mahon MJ, Cole JA, Lederer ED, Segre GV. Naþ/Hþ exchanger-regulatory factor 1 mediates inhibition of phosphate transport by parathyroid hormone and second messengers by acting at multiple sites in opossum kidney cells. Mol Endocrinol 2003;17:2355e64. [295] Ju¨ppner H. Molecular cloning and characterization of a parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor: a member of an ancient family of G protein-coupled receptors. Curr Opin Nephrol Hypertens 1994;3:371e8. [296] Ju¨ppner H, Schipani E. Receptors for parathyroid hormone and parathyroid hormone-related peptide: from molecular cloning to definition of diseases. Curr Opin Nephrol Hypertens 1996;5: 300e6. [297] Sneddon WB, Yang Y, Ba J, Harinstein LM, Friedman PA. Extracellular signal-regulated kinase activation by parathyroid hormone in distal tubule cells. Am J Physiol Renal Physiol 2007;292:F1028e34. [298] Syme CA, Friedman PA, Bisello A. Parathyroid hormone receptor trafficking contributes to the activation of extracellular signal-regulated kinases but is not required for regulation of cAMP signaling. J Biol Chem 2005;280:11281e8. [299] Singh AT, Gilchrist A, Voyno-Yasenetskaya T, Radeff-Huang JM, Stern PH. G{alpha}12/G{alpha}13 subunits of heterotrimeric G proteins mediate parathyroid hormone activation of phospholipase D in UMR-106 osteoblastic cells. Endocrinology 2005;146: 2171e5. [300] Shigeno C, Yamamoto I, Kitamura N, et al. Interaction of human parathyroid hormone-related peptide with parathyroid hormone receptors in clonal rat osteosarcoma cells. J Biol Chem 1988;34:18369e77. [301] Nissenson RA, Diep D, Strewler GJ. Synthetic peptides comprising the amino-terminal sequence of a parathyroid hormone-like protein from human malignancies: binding to parathyroid hormone receptors and activation of adenylate cyclase in bone cells and kidney. J Biol Chem 1988;263:12866e71. [302] Orloff JJ, Wu TL, Heath HW, Brady TG, Brines ML, Stewart A. Characterization of canine renal receptors for the parathyroid hormone-like protein associated with humoral hypercalcemia of malignancy. J Biol Chem 1989;264:6097e103. [303] Rubin DA, Ju¨ppner H. Zebrafish express the common parathyroid hormone/parathyroid hormone-related peptide (PTH1R) and a novel receptor (PTH3R) that is preferentially activated by mammalian and fugufish parathyroid hormonerelated peptide. J Biol Chem 1999;84:28185e90. [304] Bergwitz C, Klein P, Kohno H, et al. Identification, functional characterization, and developmental expression of two nonallelic parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor isoforms in Xenopus laevis (Daudin). Endocrinology 1998;139:723e32. [305] Schneider H, Feyen JHM, Seuwen K, Movva NR. Cloning and functional expression of a human parathyroid hormone (parathormone)/parathormone-related peptide receptor. Eur J Pharmacol 1993;246:149e55. [306] Eggenberger M, Flu¨hmann B, Muff R, et al. Structure of a parathyroid hormone/parathyroid hormone-related peptide receptor of the human cerebellum and functional expression in human neuroblastoma SK-N-MC cells. Brain Res Mol Brain Res 1997;36:127e36. [307] Karperien M, van Dijk TB, Hoeijmakers T, et al. Expression pattern of parathyroid hormone/parathyroid hormone related peptide receptor mRNA in mouse postimplantation embryos

[308]

[309]

[310]

[311]

[312]

[313]

[314]

[315]

[316]

[317]

[318]

[319]

[320]

[321]

indicates involvement in multiple developmental processes. Mech Dev 1994;47:29e42. Pausova Z, Bourdon J, Clayton D, et al. Cloning of a parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR) cDNA from a rat osteosarcoma (UMR106) cell line: chromosomal assignment of the gene in the human, mouse, and rat genomes. Genomics 1994;20:20e6. Smith DP, Zang XY, Frolik CA, et al. Structure and functional expression of a complementary DNA for porcine parathyroid hormone/parathyroid hormone-related peptide receptor. Biochim Biophys Acta 1996;1307:339e47. Smock S, Vogt G, Castleberry T, Lu B, Owen T. Molecular cloning and functional characteirzation of the canine parathyroid hormone receptor 1 (PTH1). J Bone Miner Res 1999;14(Suppl. 1):S288. Rubin DA, Hellman P, Zon LI, Lobb CJ, Bergwitz C, Ju¨ppner HA. G protein-coupled receptor from zebrafish is activated by human parathyroid hormone and not by human or teleost parathyroid hormone-related peptide: Implications for the evolutionary conservation of calcium-regulating peptide hormones. J Biol Chem 1999;274:23035e42. Tian J, Smorgorzewski M, Kedes L, Massry SG. Parathyroid hormone-parathyroid hormone related protein receptor messenger RNA is present in many tissues besides the kidney. Am J Nephrol 1993;13:210e3. Urena P, Kong XF, Abou-Samra AB, et al. Parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor mRNA are widely distributed in rat tissues. Endocrinology 1993;133:617e23. van de Stolpe A, Karperien M, Lo¨wik CWGM, et al. Parathyroid hormone-related peptide as an endogenous inducer of parietal endoderm differentiation. J Cell Biol 1993;120:235e43. Schipani E, Weinstein LS, Bergwitz C, et al. Pseudohypoparathyroidism type Ib is not caused by mutations in the coding exons of the human parathyroid hormone (PTH)/PTH-related peptide receptor gene. J Clin Endocrinol Metab 1995;80:1611e21. Bettoun JD, Minagawa M, Kwan MY, et al. Cloning and characterization of the promoter regions of the human parathyroid hormone (PTH)/PTH-related peptide receptor gene: analysis of deoxyribonucleic acid from normal subjects and patients with pseudohypoparathyroidism type Ib. J Clin Endocrinol Metab 1997;82:1031e40. Manen D, Palmer G, Bonjour J, Rizzoli R. Sequence and activity of parathyroid hormone/parathyroid hormone-related protein receptor promoter region in human osteoblast-like cells. Gene 1998;218:49e56. McCuaig KA, Clarke JC, White JH. Molecular cloning of the gene encoding the mouse parathyroid hormone/parathyroid hormone-related peptide receptor. Proc Natl Acad Sci USA 1994;91:5051e5. Kong XF, Schipani E, Lanske B, Joun H, Karperien M, Defize LHK, Ju¨ppner H, Potts JT, Segre GV, Kronenberg HM, Abou-Samra AB. The rat, mouse and human genes encoding the receptor for parathyroid hormone and parathyroid hormonerelated peptide are highly homologous. Biochem Biophys Res Comm 1994;200:1290e9. McCuaig KA, Lee H, Clarke JC, Assar H, Horsford J, White JH. Parathyoid hormone/parathyroid hormone related peptide receptor gene transcripts are expressed from tissue-specific and ubiquitous promotors. Nucleic Acids Res 1995;23:1948e55. Joun H, Lanske B, Karperien M, Qian F, Defize L, AbouSamra A. Tissue-specific transcription start sites and alternative splicing of the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor gene: a new PTH/PTHrP receptor splice variant that lacks the signal peptide. Endocrinology 1997;138:1742e9.

PEDIATRIC BONE

REFERENCES

[322] Bettoun JD, Minagawa M, Hendy GN, et al. Developmental upregulation of the human parathyroid hormone (PTH)/PTHrelated peptide receptor gene expression from conserved and human-specific promoters. J Clin Invest 1998;102:958e67. [323] Giannoukos G, Williams L, Chilco P, Abou-Samra A. Characterization of an element within the rat parathyroid hormone/ parathyroid hormone-related peptide receptor gene promoter that enhances expression in osteoblastic osteosarcoma 17/2.8 cells. Biochem Biophys Res Commun 1999;258:336e40. [324] Ishihara T, Nakamura S, Kaziro Y, Takahashi T, Takahashi K, Nagata S. Molecular cloning and expression of a cDNA encoding the secretin receptor. EMBO J 1991;10:1635e41. [325] Lin HY, Harris TL, Flannery MS, et al. Expression cloning of an adenylate cyclase-coupled calcitonin receptor. Science 1991;254: 1022e4. [326] Fredriksson R, Schioth HB. The repertoire of G-protein-coupled receptors in fully sequenced genomes. Mol Pharmacol 2005;67: 1414e25. [327] Kolakowski LFJ. GCRDb: a G-protein-coupled receptor database. Receptors Channels 1994;2:1e7. [328] Cardoso JC, Pinto VC, Vieira FA, Clark MS, Power DM. Evolution of secretin family GPCR members in the metazoa. BMC Evol Biol 2006;6:108. [329] Fredriksson R, Lagerstrom MC, Schioth HB. Expansion of the superfamily of G-protein-coupled receptors in chordates. Ann NY Acad Sci 2005;1040:89e94. [330] Kamesh N, Aradhyam GK, Manoj N. The repertoire of G protein-coupled receptors in the sea squirt Ciona intestinalis. BMC Evol Biol 2008;8:129. [331] Turner PR, Bambino T, Nissenson RA. A putative selectivity filter in the G-protein-coupled receptors for parathyroid hormone and secretin. J Biol Chem 1996;271:9205e8. [332] Turner PR, Bambino T, Nissenson RA. Mutations of neighboring polar residues on the second transmembrane helix disrupt signaling by the parathyroid hormone receptor. Mol Endocrinol 1996;10:132e9. [333] Bergwitz C, Jusseaume SA, Luck MD, Ju¨ppner H, Gardella TJ. Residues in the membrane-spanning and extracellular regions of the parathyroid hormone (PTH)-2 receptor determine signaling selectivity for PTH and PTH-related peptide. J Biol Chem 1997;272:28861e8. [334] Mannstadt M, Luck M, Gardella TJ, Ju¨ppner H. Evidence for a ligand interaction site at the amino-terminus of the parathyroid hormone (PTH)/PTH-related protein receptor from cross-linking and mutational studies. J Biol Chem 1998;273: 16890e6. [335] Gensure R, Gardella T, Juppner H. Multiple sites of contact between the carboxyl terminal binding domain of PTHrP (1 36) analogs and the amino terminal extracellular domain of the PTH/PTHrP receptor identified by photoaffinity cross linking. J Biol Chem 2001;276:28650e8. [336] Greenberg Z, Bisello A, Mierke D, Rosenblatt M, Chorev M. Mapping the bimolecular interface of the parathyroid hormone (PTH) PTH1 receptor complex: spatial proximity between Lys(27) (of the hormone principal binding domain) and Leu(261) (of the first extracellular loop) of the human PTH1 receptor. Biochemistry 2000;39:8142e52. [337] Hoare S, Gardella T, Usdin T. Evaluating the signal transduction mechanism of the parathyroid hormone 1 receptor: effect of receptor-G-protein interaction on the ligand binding mechanism and receptor conformation. J Biol Chem 2001;276:7741e53. [338] Bergwitz C, Gardella TJ, Flannery MR, et al. Full activation of chimeric receptors by hybrids between parathyroid hormone and calcitonin. J Biol Chem 1996;271:26469e72.

139

[339] Gensure RC, Gardella TJ, Juppner H. Parathyroid hormone and parathyroid hormone-related peptide, and their receptors. Biochem Biophys Res Commun 2005;328:666e78. [340] Wittelsberger A, Corich M, Thomas BE, et al. The mid-region of parathyroid hormone (1e34) serves as a functional docking domain in receptor activation. Biochemistry 2006;45:2027e34. [341] Nussbaum SR, Rosenblatt M, Potts Jr JT. Parathyroid hormone/ renal receptor interactions: demonstration of two receptorbinding domains. J Biol Chem 1980;255:10183e7. [342] Rosenblatt M, Segre GV, Tyler GA, Shepard GL, Nussbaum SR, Potts Jr JT. Identification of a receptor-binding region in parathyroid hormone. Endocrinology 1980;107:545e50. [343] Ju¨ppner H, Schipani E, Bringhurst FR, et al. The extracellular, amino-terminal region of the parathyroid hormone (PTH)/ PTH-related peptide (PTHrP) receptor determines the binding affinity for carboxyl-terminal fragments of PTH(1e34). Endocrinology 1994;134:879e84. [344] Ferrandon S, Feinstein TN, Castro M, et al. Sustained cyclic AMP production by parathyroid hormone receptor endocytosis. Nat Chem Biol 2009;5:734e42. [345] Dong M, Lam PC, Pinon DI, Sexton PM, Abagyan R, Miller LJ. Spatial approximation between secretin residue five and the third extracellular loop of its receptor provides new insight into the molecular basis of natural agonist binding. Mol Pharmacol 2008;74:413e22. [346] Fortin JP, Zhu Y, Choi C, Beinborn M, Nitabach MN, Kopin AS. Membrane-tethered ligands are effective probes for exploring class B1 G protein-coupled receptor function. Proc Natl Acad Sci USA 2009;106:8049e54. [347] Gardella TJ, Axelrod D, Rubin D, et al. Mutational analysis of the receptor-activating region of human parathyroid hormone. J Biol Chem 1991;266:13141e6. [348] Gensure R, Shimizu N, Tsang J, Gardella T. Identification of a contact site for residue 19 of PTH (PTH) and PTH-related protein analogs in transmembrane domain two of the type 1 PTH receptor. Mol Endocrinol 2003;17:2642e58. [349] Monticelli L, Mammi S, Mierke D. Molecular characterization of a ligand tethered parathyroid hormone receptor. Biophys Chem 2002;95:165e72. [350] Vilardaga JP, Bunemann M, Krasel C, Castro M, Lohse MJ. Measurement of the millisecond activation switch of G proteincoupled receptors in living cells. Nat Biotechnol 2003;21:807e12. [351] Piserchio A, Shimizu N, Gardella T, Mierke D. Residue 19 of parathyroid hormone: structural consequences. Biochemistry 2002;41:13217e23. [352] Carter PH, Liu RQ, Foster WR, et al. Discovery of a small molecule antagonist of the parathyroid hormone receptor by using an N-terminal parathyroid hormone peptide probe. Proc Natl Acad Sci USA 2007;104:6846e51. [353] Gardella TJ. Mimetic ligands for the PTHR1: approaches developments, and considerations. IBMS BoneKEy e on line 2009;6:71e85. [354] Pellegrini M, Royo M, Rosenblatt M, Chorev M, Mierke D. Addressing the tertiary structure of human parathyroid hormone-(1e34). J Biol Chem 1998;273:10420e7. [355] Marx U, Adermann K, Bayer P, Forssmann W, Rosch P. Solution structures of human parathyroid hormone fragments hPTH(1 34) and hPTH(1 39) and bovine parathyroid hormone fragment bPTH(1 37). Biochem Biophys Res Commun 2000;267:213e20. [356] Chen Z, Xu P, Barbier J-R, Willick G, Ni F. Solution structure of the osteogenic 1-31 fragment of the human parathyroid hormone. Biochemistry 2000;39:12766e77. [357] Jin L, Briggs S, Chandrasekhar S, et al. Crystal structure of ˚ resolution. J Biol human parathyroid hormone 1 34 at 0.9 A Chem 2000;275:27238e44.

PEDIATRIC BONE

140

6. PARATHYROID HORMONE AND CALCIUM HOMEOSTASIS

[358] Dean T, Linglart A, Mahon MJ, et al. Mechanisms of ligand binding to the PTH/PTHrp receptor: selectivity of a modified PTH(1-15) radioligand for G{alpha}S-coupled receptor conformations. Mol Endocrinol 2006;20:931e42. [359] Hoare SR, Sullivan SK, Pahuja A, Ling N, Crowe PD, Grigoriadis DE. Conformational states of the corticotropin releasing factor 1 (CRF1) receptor: detection, and pharmacological evaluation by peptide ligands. Peptides 2003;24:1881e97. [360] Dean T, Vilardaga JP, Potts Jr JT, Gardella TJ. Altered selectivity of parathyroid hormone (PTH) and PTH-related protein (PTHrP) for distinct conformations of the PTH/PTHrP receptor. Mol Endocrinol 2008;22:156e66. [361] Okazaki M, Ferrandon S, Vilardaga JP, Bouxsein ML, Potts Jr JT, Gardella TJ. Prolonged signaling at the parathyroid hormone receptor by peptide ligands targeted to a specific receptor conformation. Proc Natl Acad Sci USA 2008;105:16525e30. [362] Tawfeek HA, Abou-Samra AB. Important role for the V-type H(þ)-ATPase and the Golgi apparatus in the recycling of PTH/ PTHrP receptor. Am J Physiol Endocrinol Metab 2004;286: E704e10. [363] Ferrari S, Bisello A. Cellular distribution of constitutively active mutant parathyroid hormone (PTH)/PTH related protein receptors and regulation of cyclic adenosine 30 ,50 monophosphate signaling by beta arrestin2. Mol Endocrinol 2001;15: 149e63.

[364] Vilardaga J, Frank M, Krasel C, Dees C, Nissenson R, Lohse M. Differential conformational requirements for activation of G proteins and regulatory proteins, arrestin and GRK in the G protein coupled receptor for parathyroid hormone (PTH)/PTH related protein. J Biol Chem 2001;31:31. [365] Winer KK, Sinaii N, Peterson D, Sainz Jr B, Cutler Jr GB. Effects of once versus twice-daily parathyroid hormone 1e34 therapy in children with hypoparathyroidism. J Clin Endocrinol Metab 2008;93:3389e95. [366] Fukayama S, Tashjian AH, Davis JN. Signaling by N- and C-terminal sequences of parathyroid hormone-related protein in hippocampal neurons. Proc Natl Acad Sci USA 1995;92: 10182e6. [367] Orloff JJ, Ganz MB, Ribaudo AE, et al. Analysis of PTHrP binding and signal transduction mechanisms in benign and malignant squamous cells. Am J Physiol 1992;262:E599e607. [368] Gaich G, Orloff JJ, Atillasoy EJ, Burtis WJ, Ganz MB, Stewart AF. Amino-terminal parathyroid hormone-related protein: specific binding and cytosolic calcium responses in rat insulinoma cells. Endocrinology 1993;132:1402e9. [369] Orloff JJ, Kats Y, Urena P, et al. Further evidence for a novel receptor for amino-terminal parathyroid hormone-related protein on keratinocytes and squamous carcinoma cell lines. Endocrinology 1995;136:3016e23.

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7

Phosphate Homeostasis Regulatory Mechanisms Clemens Bergwitz 1, Harald Ju¨ppner 1, 2 1

Endocrine Unit and Pediatric Nephrology Unit 2 Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA

INTRODUCTION Phosphate is a rare mineral that is essential for numerous biological functions. In sea and fresh water, phosphate levels are low and even for land-living animals phosphate supply is usually limited. As a result, regulatory mechanisms developed during vertebrate evolution that enhance absorption in the intestinal tract (NPT2b), minimize the loss of phosphate into the urine (NPT2a/c), and allow storage of phosphate (bone) [1e5]. These phosphate transporters in kidney and gut help maximize phosphate retention and absorption, respectively, and help maintain the extracellular concentration of inorganic phosphate (Pi) at an elevated level during growth and development when the prevailing plasma Pi concentration is considerably higher than in the adult organism. During that time, skeletal growth is particularly rapid and there is an increased need for mineralizing newly synthesized osteoid. The mechanisms responsible for maintaining higher plasma Pi levels during growth and development are not completely understood, but are likely to involve, directly or indirectly, factors that allow higher expression of Npt2c during development [6]. Two hormones, parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF-23), were adopted during evolution for the regulation of phosphate homeostasis [7e12]. PTH augments plasma phosphate levels by enhancing the release of phosphate from bone (indirectly through osteoblast-mediated stimulation of osteoclast formation and activity) and by enhancing the intestinal absorption of phosphate (indirectly through an increase of renal 1,25(OH)2D production); furthermore, it can reduce plasma phosphate levels by preventing its reabsorption in the proximal tubule thus promoting urinary phosphate excretion. FGF-23 reduces intestinal phosphate absorption by reducing the renal synthesis of 1,25(OH)2D and by

Pediatric Bone, Second Edition DOI: 10.1016/B978-0-12-382040-2.10007-3

increasing its metabolism through activation of the 24hydroxlase [13]. Like PTH, FGF-23 furthermore promotes urinary phosphate excretion and it may promote the deposition of phosphate in the skeleton. Disturbances in either of these two hormonal systems can lead to severe abnormalities in phosphate homeostasis. Hypophosphatemia is particularly problematic during growth and development, for example, in children with diminished urinary phosphate reabsorption because of secondary hyperparathyroidism due to vitamin D deficiency or because of inherited mutations in genes affecting the regulation of phosphate homeostasis, which depletes the skeletal phosphate reservoir and leads to abnormal bone mineralization and strength, impaired growth, and skeletal deformities [8,10,14e16]. Hyperphosphatemia can be the result of inappropriately reduced urinary phosphate excretion due to the lack of PTH or FGF-23, or due to resistance towards these two hormones. This can result in vascular calcifications, mineral ion deposits in skin, kidneys and other organs that impair their function and appears to decrease life expectancy [17e20]. Too little or too much phosphate is therefore associated with significant morbidity, and maintaining serum Pi concentration within normal limits is therefore of critical importance for health and longevity. This chapter will review the physiology of Pi homeostasis and the regulatory mechanisms that are involved.

PHYSIOLOGICAL ASPECTS Distribution of Phosphate in the Body The adult human body contains 15e20 moles of Pi, 80e90% of which is present in bone as hydroxyapatite [21]. The remaining 10e20% is found in soft tissues

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(primarily muscle and internal organs), extracellular fluid, and erythrocytes. In soft tissues, Pi comprises 0.1e0.3% of wet weight and is contained intracellularly as phosphorylated sugars, phospholipids, phosphoproteins, nucleic acids, and unbound inorganic Pi. In blood, Pi is present in both erythrocytes and plasma. In plasma, phospholipids comprise the main fraction (2.2e3.1 mmol/L), followed by phosphate esters (0.9e1.5 mmol/L) and unbound Pi (0.7e1.4 mmol/L). Although the concentration of Pi in plasma is not as strictly maintained at a steady-state level as the concentration of calcium, a number of homeostatic mechanisms exist that maintain Pi levels within certain limits. This normal range varies among animal species [22], with the highest levels in fish [2,3] and the lowest levels in adult humans. In the fasting human, the plasma Pi concentration varies with age: 0.71e1.36 mmol/L in adults, 1.28e2.0 mmol/L in children, and 1.39e2.67 mmol/L 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.

Intestinal Absorption of Phosphate The Pi content of different foods is variable. The concentration, expressed per 100 grams, is highest in fish, liver and other organs, eggs, meat, milk, and unprocessed cheese (30e200 mmol). Furthermore, Pi is present in bread (30e60 mmol), vegetables (10e30 mmol), and fruit (3e10 mmol). The abundance of Pi in our food explains the rarity of Pi deficiency. The average Pi intake in humans is approximately 30e50 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. Seventy percent of Pi absorption occurs by a non-saturable, paracellular pathway, which explains why increasing dietary Pi intake results in a proportional increment in net Pi absorption, with no indication of saturation in intact animals. Thirty percent of Pi absorption occurs via an active transfer of Pi across the mucosal membrane [23e26] and involves vitamin D-regulated, sodium-dependent high-affinity transporters, named NPT2b [27e29] and Pit2 [30]. Along with vitamin D, dietary Pi regulates the expression of these intestinal sodiumephosphate co-transporters

[31], which will be described later. Furthermore, there appears to be a phosphate-sensing mechanism at the luminal surface of the intestine that contributes, presumably indirectly, to the regulation of renal phosphate excretion, independent of the proximal tubular actions of PTH and FGF-23 [32].

Extrarenal Soft Tissue Handling of Phosphate In contrast to the growing knowledge of Pi transport in intestine, bone, and kidney, the molecular mechanisms mediating Pi fluxes into soft tissues remain poorly understood. For example, insulin elicits a rapid increase in Pi uptake by primary rat hepatocyte cultures [33] and in skeletal muscle [34] by increasing the Vmax of an Nadependent Pi transport system without influencing the Km. These findings suggest that insulin mediates the insertion of a preformed Pi carrier(s) 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; this process likely contributes to the hypophosphatemia observed in malnourished individuals who are given glucose infusions (“refeeding hypophosphatemia”) [35]. The type III sodiumephosphate transporter Pit-1 furthermore appears to be important for normal liver development and is upregulated after partial liver resection in mice [36], thereby providing a plausible mechanism for the FGF-23-independent hypophosphatemia which can be observed in humans after liver resection [37,38]. However, details of the regulatory mechanisms governing the rate of Pi transfer from the extracellular and intracellular compartments and their role in the maintenance of serum Pi concentration remain unclear.

Phosphate Transport and Bone A major function of bone is to provide mechanical support for the entire body. This is achieved by mineralization of an extracellular matrix, which is formed by osteogenic cells and comprises Pi as an integral component of hydroxyapatite crystals. Thus, in addition to its structural role, bone serves as a reservoir for Pi (and calcium). Pi also affects the function of chondrocytes, osteoblasts, osteocytes, and osteoclasts, thereby modifying the rate of bone growth, matrix production and bone resorption [39,40]. As mentioned above, 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

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bone elongation, either directly or indirectly, are likely to play a critical role. The importance of Pi in bone development is illustrated by abnormalities associated with hypophosphatemic disorders such as X-linked, autosomal dominant, and autosomal recessive forms of hypophosphatemia [8,41e48]. Phosphate Transport and Osteoclast Function Osteoclasts, the bone-resorbing cells, are exposed to high ambient Pi concentrations during the active resorptive process. Their precursors are derived from monocyteemacrophage lineage cells, which require macrophage colony-stimulating factor (M-CSF) and physical contact with bone-forming osteoblasts for differentiation into mature bone-resorbing osteoclasts. This process furthermore involves receptor activator of nuclear factor kappa B (NF-kappaB) (RANK) ligand (RANKL), which is a membrane-anchored protein at the surface of osteoblasts that binds to RANK, a receptor at the surface of mature osteoclasts and their precursors [49e52]. Osteoprotegerin (OPG), secreted by osteoblasts and probably more importantly by osteocytes [53], is a decoy receptor that binds to RANKL, thus limiting RANKeRANKL interaction and preventing osteoclast differentiation [50,54]. Osteoclasts comprise specific Pi transporters, including the co-transporters NPT2a and Pit1 [40,55], which are activated in vitro upon attachment of osteoclasts to bone particles in a manner that is independent of new protein synthesis [55]. On one hand, Pi is essential for osteoclast function by providing the substrate for ATP synthesis, which is required for supporting the high rates of proton transport activity involved in bone resorption. On the other hand, increased Pi inhibits both osteoclast differentiation and bone resorption [56,57], likely by enhancing osteoprotegerin expression in osteoblasts by reducing RANKLinduced JNK and Akt activation [58], and by inducting of osteoclasts apoptosis [57]. Phosphate Transport and Osteogenic Cell Function Like all eukaryotic cells, osteogenic cells (i.e. osteoblasts and chondrocytes) express the co-transporters NPT2a and Pit1 [59]. 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 (300e500 vs 50e100 mM for renal epithelial cells) and Na/Pi cotransport in osteoblasts is stimulated by an acidic extracellular pH [60], whereas renal epithelial Pi transport is stimulated by more alkaline pH. PTH, a potent osteotropic factor, stimulates Na/Pi cotransport in osteoblasts [61], but inhibits transport in renal proximal cells. Other factors that stimulate Na/Pi co-transport in osteoblasts include insulin-like growth factor-1 (IGF-1),

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platelet-derived growth factor, and fibroblast growth factor (FGF) [62e65]. Finally, fluoride, another potent activator of bone formation, also stimulates Pi transport in osteoblastic cells via the activation of a tyrosine phosphorylation process [66]. Pi affects osteogenic cell function on multiple levels as recently reviewed [67]. It induces chondrocyte apoptosis and thereby limits bone growth and permits replacement of the hypertrophic layer of the growth plate by primary spongiosa. It stimulates osteoblastic differentiation and bone formation, and it is important for matrix vesicle function, specialized extrusions of osteoblast membranes, 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 e collagen nucleation and matrix vesicles production e have been proposed to explain the mechanism involved in the initiation of calcification in skeletal tissues [68]. 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 osteogenic cells and serve as nucleation sites for mineralization of the extracellular matrix. Matrix vesicles are small organelles (100e200 nm 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 [69]. 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 [70]. Calcium enters matrix vesicles by a protease-sensitive carrier that may be related to the annexins [71]. Matrix vesicles also express Pit1 [72]. Formation of PieCaephospholipid 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 an Na/K ATP-ase dependent 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

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the time of bone matrix formation [72]. 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 co-transport 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. Pi also modulates chondrocyte differentiation and mineralization of cartilage matrix. When analyzing sections of epiphyseal growth cartilage, Pi is present at a very low concentration in pre-mineralized cartilage. At the mineralization front, however, the Pi concentration increases with mineral deposition [73]. Phosphate appears to suppress collagen type II, PTHrP and PTH/ PTHrP receptor expression, while it stimulates type X collagen in CFK2 and ATDC5 chondrogenic cell lines [74e77] and thereby promotes cartilage maturation. Terminal differentiation of growth plate chondrocytes involves a hypertrophic conversion associated with matrix mineralization and ultimately apoptosis of hypertrophic chondrocytes [78]. Several studies have shown that exposing chondrocytes to a high level of Pi leads to their terminal maturation and subsequent matrix mineralization [76,79e83]. The role of Pi in these processes is highlighted by the mineralization defects that could be observed in the case of inherited hypophosphatemias [84]. Finally, the work of Mansfield and Sabbagh et al. [82,84] has linked the elevation in environmental Pi concentration and the concomitant rise in intracellular Pi levels to rapid chondrocytic death through the apoptotic pathway. This effect of Pi is increased in the presence of calcium [85]. It has early been recognized that addition of b-glycerophosphate to the medium favors osteoblastic differentiation of several cell lines, likely by release of Pi by alkaline phosphatase leading to an increased Pi concentration in the extracellular environment [86]. The role of Pi is further emphasized by the fact that the expression level of alkaline phosphatase (ALP) increases during osteogenic differentiation. Several studies have recently implicated Pi as an important molecule capable of modulating osteoblastic proliferation. A genome-scale proteomic study in the pre-osteoblastic MC3T3-E1 cell line [87], showed that Pi modulates the expression levels of a large set of proteins within 18e24 h [88]. A pathway/function analysis of these proteins revealed wide-ranging effects from transcriptional regulators to signal transduction molecules. Several upregulated proteins suggest involvement of Pi in the regulation of cell cycle, proliferation and DNA synthesis [88]. These studies are supported by data showing that extracellular Pi dose-dependently

stimulated DNA synthesis in MC3T3-E1 cells [89] and increased the number of cells in S-phase [88,90,91]. This effect may occur at least in part via IGF-1, which is known as one of the main growth factors involved in osteoblastic cell proliferation, since neutralizing IGF1 antibodies partially inhibited Pi-induced proliferation [92]. The potential role of Pi as a signaling molecule during the course of osteoblast differentiation was first suggested by Beck et al. [93]. They showed that osteopontin expression was strongly induced in direct response to increased Pi levels in the culture medium [94]. Furthermore, Pi stimulates ERK1/2, Pit1 and Pit2 expression in the murine odontoblast-like cell line MO6-G3, which may depend at least partially on calcium/phosphate crystal formation [95]. ERK phosphorylation by Pi furthermore appears to be required for osteopontin and matrix gla protein expression [95,96]. Once mineralized, osteoblasts may become osteocytes or lining cells or die by apoptosis. The work of Adams et al. [97] has linked extracellular Pi to a decrease in the mitochondrial membrane permeability leading to the apoptosis of osteoblasts. It should be noted, however, that osteoblasts are much less sensitive to Piinduced apoptosis than chondrocytes, and that calcium is required during this process [97,98].

Renal Phosphate Transport In an adult human with a plasma Pi concentration of z1.01 mmol/L, and a glomerular filtration rate z120 ml/min the amount of Pi filtered daily z210 mmol. If 20% of the filtered load is excreted in the urine, the mass of Pi reabsorbed is z168 mmol/ day. This net renal reabsorptive flux for Pi is z10 times greater than the net intestinal absorptive flux in adults with a dietary Pi intake of 25 mmol/day and an intestinal fractional absorption of 70% [99,100]. Under normal conditions, >80% of the Pi filtered load is reabsorbed [101,102]. The proximal tubule is the major site of Pi reabsorption, with approximately 70% of the filtered load reclaimed in the proximal convoluted tubule and approximately 10% in the proximal straight tubule (Fig. 7.1). In addition, a small but variable portion (<10%) of filtered Pi is reabsorbed in the distal segments of the nephron [101,104,105]. There is evidence for interand intranephron heterogeneity in proximal tubular Pi transport capacity, with higher transport rates in early (S1/S2) vs late (S3) and in deep (juxtamedullary) vs superficial (cortical) proximal tubular segments [101,102]. Clearance studies in humans and experimental animals have shown that when the filtered load of Pi is progressively increased, Pi reabsorption increases until a maximum tubular reabsorptive rate for Pi, or TmP, is reached, after which Pi excretion increases in proportion to its filtered load. The measurement of

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appropriate compensatory change of TmP/GFR, while renal disorders of phosphate homeostasis indicate their influence on renal handling of Pi by inappropriately altering TmPi/GFR. Therefore, an understanding of the mechanisms that underlie changes in TmPi/GFR is key to our understanding of Pi homeostasis in health and disease. Cellular and Molecular Aspects

FIGURE 7.1 Intestinal and proximal tubular phosphate transport. The renal Pi-reabsorption machinery in rat and mouse proximal tubules. Naþ/Pi-co-transporters NPT2a, NPT2c and Pit-2 are localized at the brush borders of proximal tubules. NPT2a and Pit-2 proteins are detected in S1, S2 and S3 segments, whereas NPT2c co-transporter is absent in S3 segments (from [103]).

TmP varies among individuals and within the same individual, due in part to variation in glomerular filtration rate (GFR). Thus, the ratio, TmP/GFR, or the maximum tubular reabsorption of Pi per unit volume of GFR, is the most reliable quantitative estimate of the overall tubular Pi reabsorptive capacity and can be considered to reflect the quantity of sodium-dependent phosphate co-transporters (Na/Pi) available per unit of kidney mass [106]. The serum Pi concentration below which Pi reabsorption is maximal is called the “theoretical renal Pi threshold”; this value is equal to the ratio, TmP/GFR, and closely approximates the normal fasting serum Pi concentration [99,106], indicating that the capacity of the renal tubule to transport Pi is a major determinant of extrarenal Pi homeostasis. 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 [99,101,105,106]. 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 extrarenal abnormalities of phosphate homeostasis lead to an

Transepithelial Pi transport is essentially unidirectional and involves uptake across the brush border membrane, translocation across the cell and efflux at the basolateral membrane (see Fig. 7.1) as recently reviewed [107]. Pi uptake at the apical cell surface is the rate-limiting step in the overall Pi reabsorptive process and the major site of its regulation. It is mediated by Naþ-dependent Pi transporters that depend on the basolateral membrane associated Naþ/Kþ-dependent ATPase. Na/Pi co-transport is either electrogenic (NPT2a) or electroneutral (NPT2c) and sensitive to changes in pH, with 10- to 20-fold increases observed when the pH is raised from 6 to 8.5. Little is known about the translocation of Pi across the cell except that Pi anions rapidly equilibrate with intracellular inorganic and organic Pi pools. There are few data regarding the mechanisms involved in the efflux of Pi at the basolateral cell surface. Both anion exchange mechanisms and Pi leak pathways have been proposed [108e110]. In comparison, the apical mechanism of phosphate transport is better understood: three classes of Na/Pi co-transporters have been identified by expression and homology cloning, respectively. The type I Na/Pi co-transporter (NPT1, SLC17A1) is expressed predominately in brush border membranes (BBM) of proximal tubule cells [111]. The longest splice-variant of human NPT1 is 467 amino acids in length with at least seven to nine membrane spanning segments. NPT1 mediates high affinity Na/Pi co-transport, although its pH profile differs significantly from that of the pH-dependence of Na/Pi co-transport in isolated renal brush border membrane vesicles. NPT1 exhibits broad substrate specificity and mediates the transport of Cl and organic anions as well as Pi. Conditions that physiologically regulate proximal tubule phosphate transport, such as dietary phosphorus or PTH, do not alter type I Na/Pi co-transporter protein or mRNA expression. Mice that are null for NPT1 display normal serum phosphate and renal phosphate handling. Subtle changes include increased reabsorption of phosphate probably due to increased NPT2c expression [112]; defining the physiological role of NPT1 will thus require further study. The human gene encoding the type I Na/ Pi co-transporter (SLC17A1) is located on chromosome 6p21.3-p23. The type II family of Na/Pi co-transporters is comprised of three highly homologous isoforms, which

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share only 20% amino acid sequence homology with NPT1 [113,114]. The type IIa (NPT2a, SLC34A1) and the type IIc transporters (NPT2c, SLC34A3) [6,115] are expressed exclusively in the brush border membrane of the renal proximal tubule, while the type IIb transporter (NPT2b, SLC34A2) is expressed in several tissues, including small intestine and lung, but not in kidney, and it is thought to be responsible for about 30% of intestinal absorption of Pi [27,116]. The longest splice variants of human NPT2a and human NPT2c are comprised of 639 and 599 amino acids, respectively; both proteins are predicted to have eight complete and four partially membrane-spanning segments. The human genes encoding NPT2a and NPT2c are located on chromosomes 5q35 and 9q34(116), respectively. NPT2amediated Na/Pi co-transport is electrogenic and involves the inward flux of three Naþ-ions and one Pianion (preferentially divalent) [117]. The type IIb intestinal Na/Pi co-transporter is also electrogenic, whereas the Npt2c isoform mediates the electroneutral transport of two Naþ-ions with one divalent Pi-anion. In the mouse, Npt2a and Npt2c are detected exclusively in the brush border membrane of proximal tubular cells. At the mRNA level, Npt2c is approximately one order of magnitude less abundant than Npt2a. Hybrid depletion studies suggested that Npt2c accounts for approximately 30% of Na/Pi co-transport in kidneys of Pideprived adult mice [115]. The relative abundance of Npt2c protein is significantly higher in kidneys of 22-day-old rats than in those of 60-day-old rats, suggesting that Npt2c has a particularly important role during early postnatal development [6]. However, several different homozygous and compound heterozygous mutations in SLC34A3, the gene encoding human NPT2c, have been found in patients affected by hereditary hypophosphatemic rickets with hypercalciuria (HHRH) [43e45], indicating that this co-transporter has a more prominent role than initially thought. Type III Na/Pi co-transporters are cell surface retroviral receptors (gibbon ape leukemia virus [Glvr-1, Pit1, SLC20A1] and murine amphotropic virus [Ram-1, Pit-2, SLC20A2]) that mediate high affinity, electrogenic Naþ-dependent Pi transport when expressed in oocytes and in mammalian cells [30,118]. Glvr-1 and Ram-1 show no sequence similarity to NPT1 or NPT2a. Both Glvr-1 and Ram-1 proteins are widely expressed in mammalian tissues, including the basolateral membrane of the proximal tubule and are believed to serve as “housekeeping” Na/Pi co-transporters, which maintain cellular Pi homeostasis. However, recently it was suggested that the renal expression of Pit-2 is regulated by PTH and FGF-23 in a manner similar to that of NPT2a and NPT2c [13,119]. Thus, the role of type III Na/ Pi co-transporters in the regulation of phosphate homeostasis may need to be re-evaluated.

ENDOCRINE REGULATION OF PHOSPHATE HOMEOSTASIS Human Phosphate Homeostasis Over the last decade, information derived from genetic analyses of familial disorders has led to the discovery of novel genes involved in the regulation of phosphate homeostasis which, in contrast to the regulation of calcium homeostasis [120,121], has been less well understood to date. Among these novel genes is fibroblast growth factor 23 (FGF-23), named after its similarity to other fibroblast growth factors. However, different from other members of this family, it is a circulating hormone that acts at the level of the kidney to regulate phosphate reabsorption [122]. FGF-23 is produced by osteocytes and osteoblasts in bone and upregulated by an increase in dietary phosphate and by the active form of vitamin D, 1,25(OH)2D, while it is downregulated, through yet unknown mechanisms, by PHEX, DMP1, ENPP1 and probably several additional, as-of-yet unknown proteins (Fig. 7.2) [7]. FGF23 acts through one or more FGF receptors, with Klotho as co-receptor [123], to inhibit renal phosphate reabsorption [124e126], and to decrease circulating 1,25(OH)2D levels by inhibiting the 1a-hydroxylase and increasing the 24-hydroxylase [127,128]; the net effect of these actions is to lower serum phosphate levels. Furthermore, FGF-23 appears to inhibit PTH secretion by the parathyroid glands [129,130] , which leads to a reduction in bone resorption, thereby limiting the amount of phosphate that enters the blood circulation. Like FGF-23, PTH decreases renal phosphate reabsorption, thus reducing serum phosphate levels. However, it stimulates renal 1a-hydroxylase thereby increasing the serum levels of 1,25(OH)2D, which acts through VDR/RXR heterodimers to enhance the intestinal absorption of phosphate [131] and to stimulate FGF-23 synthesis and secretion by osteocytes. Phosphate feeds back to stimulate FGF-23 and PTH secretion (see below). For a detailed discussion of the receptors and signal transduction cascades for PTH, 1,25(OH)2D, and FGF-23, the reader is referred to several recent reviews [7,8,12,122,132e137].

Parathyroid Hormone The major physiological role of the parathyroid glands is to function as a “calciostat”. Consequently, PTH secretion by the parathyroid glands is tightly regulated on a transcriptional and post-transcriptional level dependent on the concentration of extracellular calcium (Cae) [138]. In addition to calcium, PTH regulates serum phosphate concentration through its actions on several organs. An elevated serum phosphate level in turn

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FIGURE 7.2 Regulation of mammalian phosphate homeostasis by PTH, FGF23, and 1,25(OH)2-D. Modified from [12], see text for an explanation of the abbreviations.

stimulates PTH-secretion, presumably by lowering extracellular calcium and increasing stability of the PTH mRNA [139,140]. PTH is synthesized as prepropeptide containing a 25-amino acid presequence (signal sequence) and a 6-amino acid prosequence [121]. Both are cleaved off in the endoplasmic reticulum and mature full-length PTH(1e84) is stored in secretory vesicles [120]. Activation of the calcium-sensor receptor (CaR) in the parathyroid cell membrane by Cae stimulates release of intracellular calcium via Gq/11/phospholipase C (PLC) and suppresses adenylate cyclase via the inhibitory G-protein Gi, respectively [141]. Mice with the parathyroid-specific ablation of Gq combined with the global ablation of G11 develop severe hyperparathyroidism similar to that present in mice homozygous for

the deletion of the CaR, thus illustrating the important role of this signal transduction pathway for calciumsensing [142]. Hydrolysis of PIP3 by PLC releases IP3, which stimulates release of intracellular calcium (Cai) and activates calcium-sensitive proteases in the secretory vesicles of the parathyroids. This results in cleavage and inactivation of PTH [120]. The activation of CaR also suppresses protein synthesis and release of PTH(1e84) into the circulation via mechanisms involving changes in the actin cytoskeleton [143]. Heterozygous activating CaR mutations cause familial forms of hypoparathyroidism [144], while heterozygous and homozygous inactivating mutations lead to familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism, respectively [145]. PTH gene expression is

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negatively regulated by 1,25(OH)2D [146]. Parathyroid gland development is regulated by Pax9 [147], GCMB [148], GATA3 [149], and possibly other, yet unknown factors. GCMB may also be involved in parathyroid cell differentiation and hormone synthesis. Phosphate modulates PTH mRNA stability [139,150] as will be reviewed in more detail below. FGF-23 suppresses PTH mRNA synthesis and secretion in vitro and in vivo in an alpha Klotho (KL)-dependent fashion [130,151e153]. End organ-resistance to FGF-23 at the level of the parathyroid may explain why one individual with hyperphosphatemic tumoral calcinosis due to a homozygous inactivating KL mutation developed parathyroid hyperplasia [154]. KL may also have an FGF-23-independent role by facilitating PTH-secretion through maintenance of membrane Na/K-ATPase activity in the setting of hypocalcemia and KL-null mice are thus desensitized to hypocalcemic stress [155]. PTH and PTH-related peptide (PTHrP) act through a common G protein-coupled receptor, the PTH/ PTHrP-receptor (PTHR1) [120]. The PTHR1 is coupled to the Gs-alpha/AC/PKA, the Gq/PLC/PKC, and the MAPK pathway, but thus far it has been difficult to attribute individual functions of PTH to selected signaling pathways. Continuous exposure to PTH induces bone resorption by activation of osteoclasts indirectly through osteoblasts, while intermittent PTH increased bone formation by activation of osteoblasts. Osteoclasts do not have receptors for PTH and thus recruitment of osteoclast precursors and activation of bone resorption occurs indirectly via osteoblasts (see above). Conversely, intermittent PTH may lead to activation of the anabolic Wnt-signaling pathway by suppressing sclerostin and dickkopf 1 expression [156]. At the level of the proximal renal tubule, PTH acts via the PTHR1 expressed at the basolateral and apical membrane to activate Gs-alpha/AC/PKA and Gq/ PLC/PKC, respectively [125,157]. Acute regulation of the sodiumephosphate co-transporters NPT2a and NPT2c by PTH appears to involve primarily the Gsalpha/AC/PKA signaling pathway of the activated PTHR1 [158], while chronic PTH-mediated regulation of phosphate appears to involve also Gq/PLC/PKC pathway downstream of the PTHR1 [159]. Besides enhancing renal phosphate excretion, PTH increases proximal tubular bicarbonate excretion by inhibiting the amiloride-sensitive Na/H-exchanger and Na/KATPase [160]. It also increases epithelial Cl efflux, thereby hyperpolarizing the distal tubular cell, which leads to increased Ca reabsorption via TRPV5 voltagesensitive Ca channels [161,162]. The net effect of the PTH actions on bone and kidneys is an increase in serum calcium level and a decrease in serum phosphorus level, and prolonged elevations can lead to (mild) hyperchloremic metabolic acidosis.

The analysis of mice lacking PTH or PTHR1, or “knock-in” mice carrying a mutant PTHR1 has provided important insights into the function of PTH and PTHrP in vivo [163e166]. For the purpose of this review, only findings relevant for phosphate homeostasis will be discussed. Mice lacking PTH are viable, yet newborns display diminished matrix mineralization, decreased neovascularization and reduced metaphyseal osteoblasts and trabecular bone [166]. PTH-null animals suffer from increased urinary calcium excretion and phosphate retention, just like humans with hypoparathyroidism [167]. Mice lacking PTHrP or the PTHR1 die at birth or shortly thereafter [168], which is similar to the findings in individuals with Blomstrand disease, a disorder caused by homozygous or compound heterozygous inactivating mutations in the PTHR1 [169,170]; no data regarding blood phosphate homeostasis have been reported for these animals. Mice in which the wild-type PTHR1 has been replaced by a receptor deficient in the activation of Gq/PLC/PKC failed to develop hypophosphatemia when secondary hyperparathyroidism is induced by a calcium-deficient diet [165], while mice expressing a PTHR1 which lacks all phosphorylation sites within the C-terminal receptor portion that are important for receptor desensitization, develop hypophosphatemia despite normal parathyroid function [171]. These findings indicate that the long-term effects of PTH on phosphate homeostasis are at least partly mediated through the Gq/PLC/PKC signaling pathway and are subject to desensitization at the level of PTHR1. Healthy individuals injected with PTH(1e34) develop hypophosphatemia and elevated 1,25(OH)2D levels, along with an increase of serum FGF-23 [172], suggesting that calcitriol is an important regulator of FGF-23 synthesis, independent of the serum phosphate concentration. Also, the parathyroid-specific knockout of the CaR leads to increased FGF-23 production along with severe hyperparathyroidism, despite presence of hypophosphatemia [173]. Increased circulating FGF-23 levels were also reported in PTH-null mice [167] as well as in some patients with hypoparathyroidism [174], indicating that the hyperphosphatemia observed under these conditions can modulate FGF-23 despite low or normal calcitriol levels. Phosphate-independent regulation of FGF-23 by 1,25(OH)2D may also explain why FGF-23 levels are not adequately adjusted in patients with hyper- or hypoparathyroidism, i.e. conditions in which 1,25(OH)2D is elevated to stimulate inappropriately, or decreased to reduce inappropriately FGF23 levels, respectively. No abnormality of phosphate homeostasis and circulating FGF-23 levels was observed in mice expressing a constitutively active PTHR1 under the control of the DMP-1 or the col1a1(3.6 kb) promoter, or in mice lacking GNAS exon 1 in osteoblasts,

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which leads to Gs-alpha deficiency in this tissue [163,164,175,176]. However, some patients with McCuneeAlbright syndrome [177e179] and one individual with Jansen metaphyseal dysplasia [180] had increased serum FGF-23 levels. Furthermore, parathyroidectomy appears to reverse an increase of FGF-23 mRNA expression and circulating FGF-23 levels in a rat model of adenine-induced renal failure [181] and thus it remains unclear to date whether there is a direct role for PTH in the regulation of FGF-23 synthesis and/or secretion.

1,25(OH)2-vitamin D Cholecalciferol is generated from its precursors 7-dehydrocholesterol and ergosterol in the skin, subjected to 25-hydroxylation in the liver and converted to the active 1,25-dihydroxycholecalciferol (1,25(OH)2D) in the kidney [182]. CYP27B1 encodes the enzyme responsible for 1a-hydroxylation in the kidney and is induced by PTH, hypocalcemia and hypophosphatemia [183]. FGF-23, hypercalcemia and hyperphosphatemia inhibit CYP27B1 expression [183,184]. Also FGF-7 and sFRP-4 appear to inhibit synthesis of 1,25(OH)2D [185,186], while MEPE lacks this inhibitory activity [187]. FGF-23 and 1,25(OH)2D also increase renal CYP24 activity [184,188,189], which converts 25-OH-vitamin D and 1,25(OH)2D into the inactive metabolites. Similar degrees of hypocalcemia and hypophosphatemia are seen in both the VDR-null mouse [190] and the CYP27B1-null mouse [191]. These findings are similar to those observed in individuals with vitamin-dependent rickets types 1 and 2 (VDDR1 and VDDR2), respectively, carrying loss-of-function mutations in either of these genes [192,193], and indicate that the effects of vitamin D on calcium and phosphate homeostasis are mediated by the liganded VDR. Upon ligand binding, the 1,25(OH)2D/VDR complex enters the nucleus, where it forms heterodimers with RXR to activate vitamin D responsive elements (VREs). 1,25(OH)2D increases expression of the transient receptor potential cation channel, subfamily V, member 6 (TRPV6) and plasma membrane Ca2þ ATPase (PMCA) in enterocytes of the intestinal tract to facilitate transcellular calcium uptake [194]. It also increases phosphate uptake from the diet [195] possibly via upregulation of Pit-2 [31], while e at least in mice e transcellular phosphate uptake via NPT2b appears to be regulated by dietary phosphate in a vitamin D-independent fashion [196,197]. VDR is also expressed in osteoblasts and 1,25(OH)2D was reported to increase bone formation and resorption. However, the complete rescue of rickets/osteomalacia of the VDR null mouse by a diet high in calcium and phosphate suggests that the major role of VDR is the delivery of calcium and phosphate to bone [198]. 1,25(OH)2D stimulates the

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synthesis and secretion of FGF-23 by osteoblasts and osteocytes [199]. At the level of the parathyroid gland, 1,25(OH)2D acts to reduce PTH synthesis and secretion directly [200] and it increases CaR expression, thereby sensitizing the parathyroid gland to inhibition by calcium [201]. At the renal distal tubules, 1,25(OH)2D increases the intracellular expression of calbindin-28kDa, expression of TRPV5 at the apical membrane, and expression of the ATP-dependent calcium transporter at the basolateral membrane, thereby enhancing PTH-dependent calcium-reabsorption from the glomerular filtrate [194]. The net effect of the actions of 1,25(OH)2D on parathyroid, gut, bone and kidneys is an increase in serum calcium and phosphate level. Intravenous injection of calcitriol increases FGF-23 levels in humans [202] and mice [199]. Mice that are null for VDR [190] or CYP27B1 [191] exhibit hypophosphatemia and have accordingly low FGF-23 levels [204]. VDR null animals on a rescue diet, which corrects secondary hyperparathyroidism and hypophosphatemia, and thus improves bone mineralization, are able to normalize circulating FGF-23 levels despite absence of 1,25(OH)2D action, suggesting a vitamin D-independent role of Pi [203]. Finally, FGF-23 levels are markedly increased in animals with selective ablation of the CaR in the parathyroid gland [173]. These animals show hypophosphatemia and increased 1,25(OH)2D because of severe hyperparathyroidism, which is known to induce CYP27B1 activity [204]. Findings from these three animal models suggest that phosphate and 1,25(OH)2D are independent regulators of the circulating FGF-23 levels. Interestingly, FGF-23 synthesis and secretion is decreased when VDR is selectively ablated in chondrocytes using VDRfl/fl mice that mated with pcol2a1-cre animals to target ablation of the VDR to proliferating chondrocytes [205]. These data suggest that a VDR-dependent regulator of FGF-23 is present in the growth plate.

Fibroblast Growth Factor 23 FGF-23 was first identified in mouse embryos by homology-based polymerase chain reaction (PCR) [206]. However, its importance for phosphate homeostasis only became apparent when FGF-23 loss-of-function mutations were shown to cause autosomal dominant hypophosphatemic rickets (ADHR) [207]. Furthermore, FGF-23 was found to be secreted by tumors causing oncogenic osteomalacia (OOM) and recombinant FGF-23 was shown to induce renal phosphate-wasting leading to hypophosphatemia and osteomalacia [208]. The human FGF-23 gene on chromosome 12p13.3 encodes for a glycoprotein comprising 251 amino acids. The main sources of FGF-23 are osteocytes and osteoblasts in the skeleton, but

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low levels of unclear significance can be detected in the ventrolateral thalamic nucleus, the thymus, small intestine and heart [206]. The phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) and dentin matrix protein 1 (DMP1) suppress expression of FGF-23 in bone most likely through indirect mechanisms [7]. Intact DMP1 is cleaved into a 35- and a 57-kDa fragment, possibly by bone morphogenic protein 1 (BMP1) [209] which, in turn, may be activated by a complex consisting of the subtilisin propeptide convertase SPC2 and the co-activator 7B2 [210]. Transgenic overexpression of the C-terminal 57kDa DMP1 fragment is both necessary and sufficient to rescue the bone phenotype (and probably the hypophosphatemia) resulting from increased FGF-23 secretion of DMP1-null mice [211]. This 57-kDa DMP1 fragment appears to have nuclear effects, which may be required for suppression and/or feedback regulation of FGF-23 gene transcription and/or FGF-23 secretion [48,211]. In contrast, dietary phosphate [212] and serum 1,25(OH)2D stimulate FGF-23 synthesis [199,202]. After cleavage of the signal sequence comprising 24 amino acids and O-glycosylation by UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyl-transferase 3 (GALNT3), the mature protein, FGF-23(25e251), is secreted into the circulation. O-glycosylation of FGF23 occurs in the 162e228 region [213] and this posttranslational modification appears to protect FGF-23 from cleavage by subtilisin-like proprotein convertases when using recombinant peptides in vitro [214]. Mutations identified in individuals affected by hyperphosphatemic familial tumoral calcinosis (HFTC2) reside in the N-terminal portion of FGF-23; these include H41Q [215], Q54K [216], S71G [217,218], M96T [219], and S129F [220]. S71G leads to an accumulation of mutant FGF-23 in the Golgi apparatus [217] and the impaired secretion of Flag-tagged versions of [G71]hFGF-23 and [F129]hFGF-23 by HEK293 cells can be rescued by lowering the culture temperature to 25 C or by compounding the mutant FGF-23 with R176Q [221]. The R176Q mutation, which was identified in patients with ADHR, appears to stabilize hFGF-23 by protecting a subtilisin-furin cleavage site [214,222]. We have recently shown that the HFTC2 mutations S129P, S71G, and S129F impair O-glycosylation of FGF-23 resulting in the intracellular accumulation of intact FGF-23 [223]. Lossof-function mutations in GALNT3 cause hyperphosphatemic familial tumoral calcinosis (HFTC1) in humans; equivalent findings were made in mice that are null for Galnt3 [224]. O-glycosylation is essential for secretion of FGF-23 by CHO cells [222] and for secretion of FGF-7 by human embryonic kidney cells (HEK293) [225]. This suggests that GALNT3 may be an important component of the regulatory mechanism of FGF-23 secretion in vivo, which is disrupted by HFTC2 mutations.

FGF-23 lacks heparan-sulfate- (HS-) binding motifs, which are characteristic for the fibroblast growth factor family. Lack of HS-binding motifs reduces matrixbinding and enable this ligand to function in an endocrine fashion [226]. Four distinct genes encode FGF receptors (FGFR; FGFR1eFGFR4). All FGFRs share a similar domain structure. The extracellular domain is made up of three immunoglobulin-like domains (D1eD3), followed by a single-pass transmembrane domain and an intracellular domain which contains tyrosine kinase activity [227]. A major tissue-specific alternative splicing event in the second half of D3 of FGFR1e3 creates epithelial lineage-specific "b" (FGFR1beFGFR3b) and mesenchymal lineage-specific "c" (FGFR1ceFGFR3c) isoforms with distinct ligand-binding specificities [229]. FGF-23 requires alpha-Klotho (KL) as a co-receptor. Immunoprecipitation experiments, surface plasmon resonance (SPR) spectroscopy, and functional assays measuring the mitogenic response of BaF3 cells or activation of the MAPK-pathway in HEK293 cells have shown that KL forms a ternary complex with FGF-23 and either FGFR1c, FGFR2c, FGFR3c, or FGFR4 [123,229e232]. Recent work using neutralizing anti-FGF-23 antibodies indicates that the N-terminal portion of FGF-23 interacts with FGFR1c, while the C-terminus binds to KL, and both interactions appear to be important for bioactivity in vitro and in vitro [233]. Deletion of FGFR3 or FGFR4 in Hyp mice, a mouse model of human X-linked hypophosphatemia (XLH), did not correct [234] and double mutants only partially rescued the hypophosphatemia in those mice [235]. Based on these data, it was concluded that neither FGFR3 nor FGFR4 are sufficient to mediate the phosphaturic activity of FGF-23, but instead act in concert with FGFR1. Indeed, mice with kidney-specific deletion of FGFR1 [236] suggest that FGFR1 mediates the phosphaturic effects of FGF-23, although lack of suppression of 1,25(OH)2D in these animals may suggest that deletion of FGFR1 in the kidney was incomplete. The site of action of FGF-23 in the kidney is still controversial. While FGF-23 decreases expression of NPT2a and NPT2c [237e239] and CYP27B1 activity in the proximal tubules [8,240,241], its co-receptor KL is expressed mainly in the distal tubules. Mice injected with FGF-23 furthermore show phosphorylation of MAPK and expression of the early growthresponse gene 1 (Egr-1) in the distal tubules [242]. These findings suggest either that FGF-23 uses a noncanonical signal-transduction pathway at the proximal tubules or that it induces the secretion of an intermediary phosphatonin in the distal tubules, which acts in a paracrine fashion on the proximal tubules. KL may furthermore have ligand-independent actions by regulating the expression of calcium channels (TRPV5) [243] and potassium channels (ROMK) at the distal tubules [244].

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The effects of FGF-23 on renal function lead to hypophosphatemia, decreased 1,25(OH)2D synthesis, increased 24-hydroxylated vitamin D analogs and suppressed or inappropriately normal parathyroid function. Regulation of PTH secretion by serum calcium independent of the effects of phosphate, FGF-23 and KL at the parathyroid gland may furthermore explain, why PTH is unable to compensate in states of FGF-23 excess or deficiency, conditions in which serum calcium is decreased to stimulate inappropriately, or increased to reduce inappropriately PTH secretion, respectively. Much has been learned about the regulation of phosphate homeostasis through the discovery of genetic causes of human hypo- and hyperphosphatemic disorders. These findings were extended by the generation of a number of animal models. Individuals suffering from familial hyperphosphatemic tumoral calcinosis (HFTC) have homozygous loss-of-function mutations in GALNT3 [245], FGF-23 [217,246], or KL [154]. Like mice that are null for FGF-23 [247,248], GALNT3 [224], or KL [249], individuals lacking these proteins suffer from hyperphosphatemia and increased 1,25(OH)2D levels due to the loss of FGF-23 activity or end-organ resistance to FGF-23, respectively. Consistent with an increased intestinal absorption of calcium from the gut, patients and mice with these genetic modifications display mild hypercalcemia, suppressed PTH levels, and hypercalciuria. The increased calciumephosphorus product in these disorders is thought to cause the characteristic tissue calcifications. Ablation of Npt2a [250], VDR [248], or CYP27B1 [251] rescues the serum biochemical abnormalities of FGF-23-null mice, and ablation of CYP27B1 was also shown to rescue the Klotho-null mice [252]. Likewise, low-phosphate/lowvitamin D diets [253] can normalize the changes in mineral ion homeostasis of FGF-23- and Klotho-null animals, although mineralization defects in the skeleton may persist [250]. Conversely, increased FGF-23 levels in most individuals affected by X-linked hypophosphatemia [46], autosomal dominant hypophosphatemic rickets [207,254], or autosomal recessive hypophosphatemia (ARHP) [47,48] leads to renal phosphate wasting. More prominent elevations in FGF-23 are observed in mice lacking PHEX (hyp mice) [255,256] or overexpressing FGF-23 [189,257e259]. Along with hypophosphatemia, these mice show low 1,25(OH)2D levels and low-normal serum calcium levels, which lead to the development of hyperparathyroidism [257,259]. Overexpression of PHEX systemically or targeted to osteoblasts of Hyp mice [260e262] rescued the skeletal phenotype, while the serum biochemical abnormalities of these animals persist (with exception of reversal of secondary hyperparathyroidism with systemic overexpression of PHEX [262]). This is in contrast to studies of Hyp mice crossed with mice carrying a reporter expressed under

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the control of the FGF-23 promoter, which show elevated FGF-23 gene transcription in bone. These findings suggest that the FGF-23 excess in mice with an abnormal PHEX function is originating from the skeleton [256,263]. Furthermore, the FGF-23-null mice and mice lacking PHEX and FGF-23 [256] as well as mice lacking DMP1 and FGF-23 [264] show indistinguishable phenotypes suggesting that the regulatory actions of PHEX and DMP1 reside upstream of FGF-23. Distinct from the FGF-23-dependent forms of renal phosphate wasting, hereditary hypophosphatemic rickets with hypercalciuria (HHRH) [45,265] leads to appropriate upregulation of CYP27B1, leading to increased 1,25(OH)2D levels and thus absorptive hypercalciuria due to loss of SLC34A3/ NPT2c in the kidney. Mice lacking SLC34A1/NPT2a also display FGF-23-independent renal phosphate wasting and absorptive hypercalciuria [266]. In contrast, mice lacking SLC34A3/NPT2c show only mild hypercalcemia and absorptive hypercalciuria at an early age only due to increased 1,25(OH)2D levels, but these animals do not develop hypophosphatemia, and thus have a milder phenotype than human individuals with HHRH [128].

Metabolic and Endocrine Effects of Phosphate Metabolic Effects of Phosphate/Activation of MAPK by Phosphate It has long been recognized that differentiation of primary or permanent osteogenic cells requires the addition of inorganic phosphate in the form of hydroxyapatite [267e269], or beta-glycerophosphate [87] along with ascorbic acid which is believed to support formation of collagen matrix [270]. Expression of the bonecell specific genes dentin matrix protein 1 (DMP1), osteopontin (OPN), and matrix-gla-protein (MGP) is stimulated by phosphate on a transcriptional level, while the expression of alkaline phosphatase (TNSALP) is suppressed [93,94,271]. Cellular uptake of phosphate by specialized transporters appears to be required since addition of phosphonoformic acid (PFA, forscarnet), an inhibitor of sodiumephosphate co-transporters, blocks these effects of Pi [94,272,273]. The downstream effects resulting in suppression of TNSALP possibly require BMP-signaling in ST2 cells [274], however, dependence on the p42/p44-MAPK/ERK pathway was shown in ATDC5 cells [275]. Likewise, activation of the p42/p44MAPK/ERK pathway is required for the induction of OPN and MGP, which can be blocked by UO126, a specific inhibitor of MAPK-kinase, MEK [276,277]. Activation of MAPK by calcium phosphate crystals in primary fibroblasts was described by Nair et al. [278]. Beck et al. subsequently showed that inorganic phosphate at concentrations between 5 and 10 mM alone is

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sufficient to activate MAPK in MC3T3 mouse fibroblast cells [276], and subsequently, activation of MAPK by inorganic phosphate was demonstrated in multiple other cell lines, including chondrogenic ATDC5 cells, MC3T3-E1 osteoblasts and ST2 murine bone marrow stromal cells [277], HEK293 human proximal tubular cells [279], and lung alveolar cells [273]. Although some cell lines, for example C2C12 or L929 cells, are less responsive than others [277], activation of MAPK by phosphate appears to be quite universal, and conserved evolutionarily, since it is likewise observed in Drosophila S2Rþ hemocyte cells [280]. Addition of PFA [82,272] or siRNA-mediated knockdown of Pit1 sodiumephosphate co-transporters blocks activation of MAPK by phosphate [279] indicating that cellular uptake of phosphate is required for the activation of MAPK. Furthermore, using cell lines expressing a Pitransport-deficient Pit1 transporter, Beck et al. recently reported that Pit1 may have transport-independent effects on cell proliferation and tumor growth in vitro and in vivo, although it remains to be shown whether these effects are dependent on phosphate binding to Pit1 [281]. Pit1 belongs, along with its mammalian paralogue Pit2, the bacterial pitA, and yeast pho89 transporters, to the NPT3 class of sodiumephosphate cotransporters [282,283]. Phosphate appears to induce phosphorylation of FRS2alpha, and siRNA mediated knockdown of FGFR1 is able to interfere with Pi-induced MAPK [275,279]. Furthermore, phosphorylation of Akt and c-Raf by phosphate was shown in human bronchial epithelial cells [273] and proliferating chondrocytes [275], respectively, suggesting that phosphate interacts with the MAPK pathway relatively close to the cell membrane. Activation of p42/p44-MAPK by phosphate is rapid and occurs within 5 min, but biphasic upregulation with a second peak after 8e12 h has been reported in some cell lines, while related kinases JNK and p38MAPK appear not to be affected by phosphate at least in those cell lines reported to date [276,277]. It is possible that binding of extracellular phosphate to Pit1 activates the MAPK pathway. However, since knockdown of members of the MFS family (i.e. NPT1), likewise blocks phosphate-induced MAPK, at least in Drosophila S2Rþ cells [280], intracellular phosphate after uptake via multiple transporter classes may be the signal for MAPK and the activation of possibly other pathways. Furthermore, block of phosphate-induced MAPK by knockdown of FGFR1 or FRS2alpha either suggests a cooperative interaction between Pit1 and FGFR1 to sense extracellular phosphate after binding to Pit1 or that intracellular phosphate acts to modulate flow through the MAPK pathway at one or multiple levels downstream of the FGFR1/FRS2alpha. Egr-1 was shown to be a downstream effector of phosphate-induced MAPK [96,284]. Pi also induces the expression of Fra-1

[96], Nrf2 [90,91], although dependence of these factors on MAPK as the intermediary has not been demonstrated [88,90]. Phosphate-induced MAPK has been implicated in proliferation of the chondrogenic cell line ATDC5, possibly by activating cyclin D1 [275]. Finally, activation of MAPK by phosphate stimulates apoptosis of hypertrophic chondrocytes [84], which also requires changes of the mitrochondrial membrane potential and activation of caspase-9 [77]. Conversely, inhibition of phosphate uptake or phosphate restriction prevents apoptosis of hypertrophic chondrocytes in vitro [76,82,285], and lead to a 2.5-fold increase in parathyroid hormone-related protein mRNA expression pointing to an important role for phosphate in regulating growth plate maturation [77]. Both effects may explain expansion of the growth plates in the growing skeleton in vivo, for example in children with hypophosphatemic rickets [286]. Regulation of Parathyroid Hormone Secretion by Phosphate In addition to calcium and 1,25(OH)2D (see above), parathyroid hormone secretion is regulated by serum phosphate levels through direct and indirect mechanisms. Elevated serum phosphate stimulates PTH-secretion presumably by lowering extracellular calcium [139,140], while hypophosphatemia suppresses PTHsecretion indirectly by upregulation of 1,25(OH)2D. Upregulation of 1,25(OH)2D also induces FGF-23 synthesis, which acts at the parathyroids to suppress PTH mRNA synthesis and secretion in vitro and in vivo in a KL-dependent fashion [130,151,153,287]. Finally, KL may also have an FGF-23-independent role by facilitating PTH-secretion through maintenance of membrane Na/K-ATPase activity in the setting of hypocalcemia and KL-null mice are thus desensitized to hypocalcemic stress [155]. In addition to these indirect effects, phosphate directly acts on the parathyroids to stimulate PTH secretion through post-transcriptional mechanisms, which have been elucidated in recent years. Using Northwestern blot analysis, RNA electrophoretic mobility shift and cross-linking analysis of parathyroid extracts, cytosolic trans-acting factors were identified which bind to a defined 63 bp cis-acting instability element in the PTH mRNA 30 -untranslated region (UTR) [139,150]. It appears that there is a balanced interaction of the PTH mRNA with stabilizing proteins AUF1 (AU-rich element binding protein, isoforms p37, p40, p42 and p45) and Unr (upstream of N-ras) [288] and the destabilizing protein KSRP (K-homology splicing regulator protein) in vivo [289]. In the setting of hypocalcemia or chronic kidney disease, the peptidyl-prolyl isomerase Pin1 is inactive [290], resulting in KSRP phosphorylation and hence its inactivation. This presumably

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allows AUF1 and Unr to bind the PTH mRNA 30 UTR ARE with a greater affinity, leading to increased PTH mRNA stability. Conversely, hypercalcemia and hypophosphatemia lead to destabilization of the PTH mRNA, which then is targeted for degradation by the exosome. Since studies to show direct regulation of mRNA stability by phosphate in transfected cells using a reporter containing the PTH 30 -UTR are missing, the nature of the upstream phosphate sensing mechanism continues to remain unclear. Regulation of 1a-hydroxylase by Phosphate Cholecalciferol is generated from its precursors 7-dehydrocholesterol and ergosterol in the skin, subjected to 25-hydroxylation in the liver and converted to the active 1,25-dihydroxycholecalciferol (1,25(OH)2D) in the kidney [182,291]. 1a-hydroxylation in the kidney is tightly regulated and induced by PTH, hypocalcemia and hypophosphatemia, which appear to induce gene expression of CYP27B1, the gene encoding 1ahydroxylase [183]. FGF-23, hypercalcemia and hyperphosphatemia, on the other hand, inhibit CYP27B1 expression [183,184]. Also, FGF-7 and sFRP-4 appear to inhibit synthesis of 1,25(OH)2D [185,186]. FGF-23 and 1,25(OH)2D also increase renal CYP24 activity [184,188,189], which converts 25-OH-vitamin D and 1,25(OH)2D into inactive metabolites. The upstream sensing mechanism and whether phosphate regulates CYP27B1 on the transcriptional or post-transcriptional level remain unclear. Regulation of FGF-23 Secretion by Phosphate The main sources of FGF-23 are osteocytes and osteoblasts in the skeleton [207]. As mentioned above, dietary phosphate and 1,25(OH)2D stimulate FGF-23 secretion [199,202,212], which appear to occur independently from each other. For example, injection of 1,25-dihydroxyvitamin D3 increases serum FGF-23 levels within hours in rodents [292] without significant increase in serum phosphate levels, indicating that vitamin D can increase FGF-23 independently of phosphate [292]. The effect of 1,25-dihydroxyvitamin D3 is via its nuclear vitamin D receptor (VDR) which heterodimerizes with another nuclear receptor RXR. The VDReRXR heterodimer in turn binds to the promoter region of the FGF-23 gene and transactivates its expression [133]. Similarly, phosphate positively regulates FGF-23 expression, although the exact mechanisms need to be defined. VDR-deficient mice (VDR/ mice) have low serum levels of phosphate and FGF-23, but when placed on a “rescue” diet (rich in calcium and phosphate) serum FGF-23 levels are restored, indicating that phosphate can increase FGF-23 independently of vitamin D [203]. Due to lack of suitable in vitro culture models which show regulation of FGF-23 secretion by phosphate, it remains

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unclear whether the in vivo effects of phosphate on FGF-23 transcription and/or secretion are cell autonomous or mediated via intermediary factors. Insights from cell lines transfected with plasmids encoding for human FGF-23 indicate that FGF-23 is subjected to post-translational modification, and that this modification may be regulated by phosphate. After cleavage of the signal sequence comprising 24 amino acids is removed, FGF-23(25e251) is O-glycosylated by GALNT3. O-glycosylation is essential for secretion of FGF-23 by CHO cells [222] and for secretion of FGF-7 by human embryonic kidney cells (HEK293) [225], and mutations impairing O-glycosylation of FGF-23 lead to hyperphosphatemic familial tumoral calcinosis in humans [221,223] and equivalent findings in mice [224] due to lack of bioactive FGF-23. Expression of GALNT3 appears to be stimulated by extracellular phosphate and is suppressed by extracellular calcium and 1,25(OH)2D in HEK293 cells [225]. It also appears to be stimulated by AMP medicated mechanisms in vitro [294] and by fibrous dysplastic lesions caused by activating GNAS mutations in vivo [178]. This suggests that GALNT3 may be an important component of the regulatory mechanism of FGF-23 secretion by phosphate and cAMP. Recently, ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) was found to be a negative regulator of FGF-23 secretion. ENPP1 is a membrane-bound ecto-enzyme responsible for the generation of the mineralization inhibitor pyrophosphate (PPi) [294]. Loss-of function mutations in this enzyme are the cause of generalized arterial calcifications of infancy (GACI) [295,296], and hypophosphatemic rickets due to FGF-23-dependent renal phosphate-wasting [297,298]. The cause of FGF-23 excess may be directly related to lack of PPi production, or may be due to accumulation of precursors, such as ATP, in the extracellular space. However, the presence of mild hyperphosphatemia in individuals and carriers suffering from hypophosphatasia, which is caused by loss-of-function mutations in the PPi-degrading enzyme, namely tissue non-specific alkaline phosphatase (TNALP) [299], suggests that PPi is the intermediary suppressing FGF-23 production.

References [1] Coloso RM, King K, Fletcher JW, Weis P, Werner A, Ferraris RP. Dietary P regulates phosphate transporter expression, phosphatase activity, and effluent P partitioning in trout culture. J Comp Physiol B 2003;173:519e30. [2] Sugiura SH, Ferraris RP. Contributions of different NaPi cotransporter isoforms to dietary regulation of P transport in the pyloric caeca and intestine of rainbow trout. J Exp Biol 2004; 207:2055e64. [3] Sugiura SH, Hardy RW, Roberts RJ. The pathology of phosphorus deficiency in fish e a review. J Fish Dis 2004;27:255e65.

PEDIATRIC BONE

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7. PHOSPHATE HOMEOSTASIS REGULATORY MECHANISMS

[4] Beyenbach KW. Kidneys sans glomeruli. Am J Physiol Renal Physiol 2004;286:F811e27. [5] Bijvoet OL, Reitsma PH. Phylogeny of renal phosphate transport in the vertebrates. Adv Exp Med Biol 1977;81:41e53. [6] Segawa H, Kaneko I, Takahashi A, et al. Growth-related renal type II Na/Pi cotransporter. J Biol Chem 2002;277:19665e72. [7] Quarles LD. Endocrine functions of bone in mineral metabolism regulation. J Clin Invest 2008;118:3820e8. [8] Strom TM, Ju¨ppner H. PHEX, FGF23, DMP1 and beyond. Curr Opin Nephrol Hypertens 2008;17:357e62. [9] Razzaque MS. The FGF23-Klotho axis: endocrine regulation of phosphate homeostasis. Nat Rev Endocrinol 2009;5:611e9. [10] Ju¨ppner H, Wolf M, Salusky IB. FGF23: more than a regulator of renal phosphate handling? J Bone Miner Res 2010;25:2091e7. [11] Kuro-o M. Overview of the FGF23-Klotho axis. Pediatr Nephrol 2010;25:583e90. [12] Bergwitz C, Ju¨ppner H. Regulation of phosphate homeostasis by PTH, vitamin D, and FGF23. Annu Rev Med 2010;61:91e104. [13] Tomoe Y, Segawa H, Shiozawa K, et al. Phosphaturic action of fibroblast growth factor 23 in Npt2 null mice. Am J Physiol Renal Physiol 2010;298:F1341e50. [14] Yu X, White KE. FGF23 and disorders of phosphate homeostasis. Cytokine Growth Factor Rev 2005;16:221e32. [15] Gattineni J, Baum M. Regulation of phosphate transport by fibroblast growth factor 23 (FGF23): implications for disorders of phosphate metabolism. Pediatr Nephrol 2010;25:591e601. [16] Bergwitz C, Ju¨ppner H. Disorders of phosphate homeostasis and tissue mineralisation. Endocr Dev 2009;16:133e56. [17] Parker BD, Schurgers LJ, Brandenburg VM, et al. The associations of fibroblast growth factor 23 and uncarboxylated matrix Gla protein with mortality in coronary artery disease: the Heart and Soul Study. Ann Intern Med 2010;152:640e8. [18] Kanbay M, Goldsmith D, Akcay A, Covic A. Phosphate e the silent stealthy cardiorenal culprit in all stages of chronic kidney disease: a systematic review. Blood Purif 2009;27:220e30. [19] Hruska KA, Mathew S, Lund R, Qiu P, Pratt R. Hyperphosphatemia of chronic kidney disease. Kidney Int 2008; 74:148e57. [20] Kuro-o M. A potential link between phosphate and aging e lessons from Klotho-deficient mice. Mech Ageing Dev 2010; 131:270e5. [21] Bringhurst FR, Leder BZ. Regulation of calcium and phosphate homeostasis. In: DeGroot LJ, Jameson JL, editors. Endocrinology. 5th ed. Philadelphia: W.B. Saunders Co.; 2006. p. 805e43. [22] Kuro-o M, Klotho. Pflu¨gers Arch 2010;459:333e43. [23] Berner W, Kinne R, Murer H. Phosphate transport into brushborder membrane vesicles isolated from rat small intestine. Biochem J 1976;160:467e74. [24] Danisi G, Bonjour JP, Straub RW. Regulation of Na-dependent phosphate influx across the mucosal border of duodenum by 1,25-dihydroxycholecalciferol. Pflu¨gers Arch 1980;388:227e32. [25] Lee DB, Walling MW, Corry DB. Phosphate transport across rat jejunum: influence of sodium, pH, and 1,25-dihydroxyvitamin D3. Am J Physiol 1986;251:G90e5. [26] Peterlik M, Wasserman RH. Effect of vitamin D on transepithelial phosphate transport in chick intestine. Am J Physiol 1978;234:E379e88. [27] Sabbagh Y, O’Brien SP, Song W, et al. Intestinal npt2b plays a major role in phosphate absorption and homeostasis. J Am Soc Nephrol 2009;20:2348e58. [28] Radanovic T, Wagner CA, Murer H, Biber J. Regulation of intestinal phosphate transport. I. Segmental expression and adaptation to low-P(i) diet of the type IIb Na(þ)-P(i)

[29]

[30]

[31]

[32]

[33]

[34]

[35] [36]

[37]

[38]

[39]

[40]

[41]

[42] [43]

[44]

[45]

[46]

[47]

cotransporter in mouse small intestine. Am J Physiol Gastrointest Liver Physiol 2005;288:G496e500. Stauber A, Radanovic T, Stange G, Murer H, Wagner CA, Biber J. Regulation of intestinal phosphate transport. II. Metabolic acidosis stimulates Na(þ)-dependent phosphate absorption and expression of the Na(þ)-P(i) cotransporter NaPi-IIb in small intestine. Am J Physiol Gastrointest Liver Physiol 2005; 288:G501e6. Collins JF, Bai L, Ghishan FK. The SLC20 family of proteins: dual functions as sodium-phosphate cotransporters and viral receptors. Pflu¨gers Arch 2004;447:647e52. Katai K, Miyamoto K, Kishida S, et al. Regulation of intestinal Naþ-dependent phosphate co-transporters by a low-phosphate diet and 1,25-dihydroxyvitamin D3. Biochem J 1999;343:705e12. Berndt T, Thomas LF, Craig TA, et al. Evidence for a signaling axis by which intestinal phosphate rapidly modulates renal phosphate reabsorption. Proc Natl Acad Sci USA 2007;104: 11085e90. Butterworth PJ, Younus MJ. Uptake of phosphate by rat hepatocytes in primary culture: a sodium-dependent system that is stimulated by insulin. Biochim Biophys Acta 1993;1148:117e22. Polgreen KE, Kemp GJ, Leighton B, Radda GK. Modulation of Pi transport in skeletal muscle by insulin and IGF-1. Biochim Biophys Acta 1994;1223:279e84. Marinella MA. The refeeding syndrome and hypophosphatemia. Nutr Rev 2003;61:320e3. Beck L, Leroy C, Beck-Cormier S, et al. The phosphate transporter PiT1 (Slc20a1) revealed as a new essential gene for mouse liver development. PLoS One 2010;5:e9148. Nafidi O, Lepage R, Lapointe RW, D’Amour P. Hepatic resection-related hypophosphatemia is of renal origin as manifested by isolated hyperphosphaturia. Ann Surg 2007;245:1000e2. Salem RR, Tray K. Hepatic resection-related hypophosphatemia is of renal origin as manifested by isolated hyperphosphaturia. Ann Surg 2005;241:343e8. Caverzasio J, Bonjour JP. Characteristics and regulation of Pi transport in osteogenic cells for bone metabolism. Kidney Int 1996;49:975e80. Gupta A, Miyauchi A, Fujimori A, Hruska KA. Phosphate transport in osteoclasts: a functional and immunochemical characterization. Kidney Int 1996;49:968e74. Baylink D, Wergedal J, Stauffer M. Formation, mineralization, and resorption of bone in hypophosphatemic rats. J Clin Invest 1971;50:2519e30. Albright F, Butler AM, Bloomberg E. Rickets resistant to vitamin D therapy. Am J Dis Child 1937;54:529e47. Bergwitz C, Roslin NM, Tieder M, et al. SLC34A3 mutations in patients with hereditary hypophosphatemic rickets with hypercalciuria predict a key role for the sodium-phosphate cotransporter NaPi-IIc in maintaining phosphate homeostasis. Am J Hum Genet 2006;78:179e92. Ichikawa S, Sorenson AH, Imel EA, Friedman NE, Gertner JM, Econs MJ. Intronic deletions in the SLC34A3 gene cause hereditary hypophosphatemic rickets with hypercalciuria. J Clin Endocrinol Metab 2006;91:4022e7. Lorenz-Depiereux B, Benet-Pages A, Eckstein G, et al. Hereditary hypophosphatemic rickets with hypercalciuria is caused by mutations in the sodium-phosphate cotransporter gene SLC34A3. Am J Hum Genet 2006;78:193e201. HYP-Consortium. A. gene (PEX) with homologies to endopeptidases is mutated in patients with X-linked hypophosphatemic rickets. Nat Genet 1995;11:130e6. Feng JQ, Ward LM, Liu S, et al. Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism. Nat Genet 2006;38:1310e5.

PEDIATRIC BONE

155

REFERENCES

[48] Lorenz-Depiereux B, Bastepe M, Benet-Pages A, et al. DMP1 mutations in autosomal recessive hypophosphatemia implicate a bone matrix protein in the regulation of phosphate homeostasis. Nat Genet 2006;38:1248e50. [49] Anderson DM, Maraskovsky E, Billingsley WL, et al. A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature 1997;390:175e9. [50] Lacey DL, Timms E, Tan HL, et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 1998;93:165e76. [51] Wong BR, Rho J, Arron J, et al. TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c-Jun N-terminal kinase in T cells. J Biol Chem 1997;272:25190e4. [52] Yasuda H, Shima N, Nakagawa N, et al. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesisinhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA 1998;95:3597e602. [53] Kramer I, Halleux C, Keller H, et al. Osteocyte Wnt/beta-catenin signaling is required for normal bone homeostasis. Mol Cell Biol 2010;30:3071e85. [54] Simonet WS, Lacey DL, Dunstan CR, et al. Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 1997;89:309e19. [55] Gupta A, Guo XL, Alvarez UM, Hruska KA. Regulation of sodium-dependent phosphate transport in osteoclasts. J Clin Invest 1997;100:538e49. [56] Yates AJ, Oreffo RO, Mayor K, Mundy GR. Inhibition of bone resorption by inorganic phosphate is mediated by both reduced osteoclast formation and decreased activity of mature osteoclasts. J Bone Miner Res 1991;6:473e8. [57] Kanatani M, Sugimoto T, Kano J, Kanzawa M, Chihara K. Effect of high phosphate concentration on osteoclast differentiation as well as bone-resorbing activity. J Cell Physiol 2003;196:180e9. [58] Mozar A, Haren N, Chasseraud M, et al. High extracellular inorganic phosphate concentration inhibits RANK-RANKL signaling in osteoclast-like cells. J Cell Physiol 2008;215:47e54. [59] Zoidis E, Ghirlanda-Keller C, Gosteli-Peter M, Zapf J, Schmid C. Regulation of phosphate (Pi) transport and NaPi-III transporter (Pit-1) mRNA in rat osteoblasts. J Endocrinol 2004;181:531e40. [60] Caverzasio J, Selz T, Bonjour JP. Characteristics of phosphate transport in osteoblastlike cells. Calcif Tissue Int 1988;43:83e7. [61] Selz T, Caverzasio J, Bonjour JP. Regulation of Na-dependent Pi transport by parathyroid hormone in osteoblast-like cells. Am J Physiol 1989;256:E93e100. [62] Montessuit C, Bonjour JP, Caverzasio J. Pi transport regulation by chicken growth plate chondrocytes. Am J Physiol 1994; 267:E24e31. [63] Palmer G, Bonjour JP, Caverzasio J. Expression of a newly identified phosphate transporter/retrovirus receptor in human SaOS-2 osteoblast-like cells and its regulation by insulin-like growth factor I. Endocrinology 1997;138:5202e9. [64] Suzuki A, Palmer G, Bonjour JP, Caverzasio J. 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 2000;15:95e102. [65] Zhen X, Bonjour JP, Caverzasio J. Platelet-derived growth factor stimulates sodium-dependent Pi transport in osteoblastic cells via phospholipase Cgamma and phosphatidylinositol 30 -kinase. J Bone Miner Res 1997;12:36e44. [66] Caverzasio J, Palmer G, Suzuki A, Bonjour JP. Mechanism of the mitogenic effect of fluoride on osteoblast-like cells: evidences for a G protein-dependent tyrosine phosphorylation process. J Bone Miner Res 1997;12:1975e83. [67] Khoshniat S, Bourgine A, Julien M, Weiss P, Guicheux J, Beck L. The emergence of phosphate as a specific signaling molecule

[68]

[69] [70]

[71]

[72]

[73]

[74]

[75]

[76]

[77]

[78]

[79]

[80]

[81]

[82]

[83]

[84]

[85]

in bone and other cell types in mammals. Cell Mol Life Sci 2010;68:205e18. Williams DC, Frolik CA. Physiological and pharmacological regulation of biological calcification. Int Rev Cytol 1991;126:195e292. Anderson HC. Matrix vesicle calcification. Fed Proc 1976;35:104. Felix R, Herrmann W, Fleisch H. Stimulation of precipitation of calcium phosphate by matrix vesicles. Biochem J 1978;170:681e91. Genge BR, Cao X, Wu LN, et al. Establishment of the primary structure of the major lipid-dependent Ca2þ binding proteins of chicken growth plate cartilage matrix vesicles: identity with anchorin CII (annexin V) and annexin II. J Bone Miner Res 1992;7:807e19. Montessuit C, Caverzasio J, Bonjour JP. Characterization of a Pi transport system in cartilage matrix vesicles. Potential role in the calcification process. J Biol Chem 1991;266:17791e7. Kakuta S, Golub EE, Shapiro IM. Morphochemical analysis of phosphorus pools in calcifying cartilage. Calcif Tissue Int 1985;37:293e9. Wang D, Canaff L, Davidson D, et al. Alterations in the sensing and transport of phosphate and calcium by differentiating chondrocytes. J Biol Chem 2001;276:33995e4005. Fujita T, Meguro T, Izumo N, et al. Phosphate stimulates differentiation and mineralization of the chondroprogenitor clone ATDC5. Jpn J Pharmacol 2001;85:278e81. Magne D, Bluteau G, Faucheux C, et al. Phosphate is a specific signal for ATDC5 chondrocyte maturation and apoptosisassociated mineralization: possible implication of apoptosis in the regulation of endochondral ossification. J Bone Miner Res 2003;18:1430e42. Miedlich SU, Zalutskaya A, Zhu ED, Demay MB. Phosphateinduced apoptosis of hypertrophic chondrocytes is associated with a decrease in mitochondrial membrane potential and is dependent upon Erk1/2 phosphorylation. J Biol Chem 2010; 285:18270e5. Magne D, Julien M, Vinatier C, Merhi-Soussi F, Weiss P, Guicheux J. Cartilage formation in growth plate and arteries: from physiology to pathology. Bioessays 2005;27:708e16. Cecil DL, Rose DM, Terkeltaub R, Liu-Bryan R. Role of interleukin-8 in PiT-1 expression and CXCR1-mediated inorganic phosphate uptake in chondrocytes. Arthritis Rheum 2005; 52:144e54. Alini M, Carey D, Hirata S, Grynpas MD, Pidoux I, Poole AR. Cellular and matrix changes before and at the time of calcification in the growth plate studied in vitro: arrest of type X collagen synthesis and net loss of collagen when calcification is initiated. J Bone Miner Res 1994;9:1077e87. Mansfield K, Rajpurohit R, Shapiro IM. Extracellular phosphate ions cause apoptosis of terminally differentiated epiphyseal chondrocytes. J Cell Physiol 1999;179:276e86. Mansfield K, Teixeira CC, Adams CS, Shapiro IM. Phosphate ions mediate chondrocyte apoptosis through a plasma membrane transporter mechanism. Bone 2001;28:1e8. Teixeira CC, Mansfield K, Hertkorn C, Ischiropoulos H, Shapiro IM. Phosphate-induced chondrocyte apoptosis is linked to nitric oxide generation. Am J Physiol Cell Physiol 2001;281: C833e9. Sabbagh Y, Carpenter TO, Demay MB. Hypophosphatemia leads to rickets by impairing caspase-mediated apoptosis of hypertrophic chondrocytes. Proc Natl Acad Sci USA 2005; 102:9637e42. Mansfield K, Pucci B, Adams CS, Shapiro IM. Induction of apoptosis in skeletal tissues: phosphate-mediated chick

PEDIATRIC BONE

156

[86]

[87]

[88]

[89]

[90]

[91]

[92] [93]

[94]

[95]

[96]

[97]

[98]

[99]

[100]

[101]

[102]

[103]

[104] [105]

7. PHOSPHATE HOMEOSTASIS REGULATORY MECHANISMS

chondrocyte apoptosis is calcium dependent. Calcif Tissue Int 2003;73:161e72. Bellows CG, Heersche JN, Aubin JE. Inorganic phosphate added exogenously or released from beta-glycerophosphate initiates mineralization of osteoid nodules in vitro. Bone Miner 1992; 17:15e29. Quarles LD, Yohay DA, Lever LW, Caton R, Wenstrup RJ. Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: an in vitro model of osteoblast development. J Bone Miner Res 1992;7:683e92. Conrads KA, Yi M, Simpson KA, et al. A combined proteome and microarray investigation of inorganic phosphate-induced pre-osteoblast cells. Mol Cell Proteomics 2005;4:1284e96. Kanatani M, Sugimoto T, Kano J, Chihara K. IGF-I mediates the stimulatory effect of high phosphate concentration on osteoblastic cell proliferation. J Cell Physiol 2002;190:306e12. Conrads KA, Yu LR, Lucas DA, et al. Quantitative proteomic analysis of inorganic phosphate-induced murine MC3T3-E1 osteoblast cells. Electrophoresis 2004;25:1342e52. Beck Jr GR, Moran E, Knecht N. Inorganic phosphate regulates multiple genes during osteoblast differentiation, including Nrf2. Exp Cell Res 2003;288:288e300. Baylink DJ, Finkelman RD, Mohan S. Growth factors to stimulate bone formation. J Bone Miner Res 1993;8(Suppl. 2):S565e72. Beck Jr GR, Sullivan EC, Moran E, Zerler B. Relationship between alkaline phosphatase levels, osteopontin expression, and mineralization in differentiating MC3T3-E1 osteoblasts. J Cell Biochem 1998;68:269e80. Beck Jr GR, Zerler B, Moran E. Phosphate is a specific signal for induction of osteopontin gene expression. Proc Natl Acad Sci USA 2000;97:8352e7. Wittrant Y, Bourgine A, Khoshniat S, et al. Inorganic phosphate regulates Glvr-1 and -2 expression: role of calcium and ERK1/2. Biochem Biophys Res Commun 2009;381:259e63. Julien M, Khoshniat S, Lacreusette A, et al. Phosphate-dependent regulation of MGP in osteoblasts: role of ERK1/2 and Fra-1. J Bone Miner Res 2009;24:1856e68. Adams CS, Mansfield K, Perlot RL, Shapiro IM. Matrix regulation of skeletal cell apoptosis. Role of calcium and phosphate ions. J Biol Chem 2001;276:20316e22. Meleti Z, Shapiro IM, Adams CS. Inorganic phosphate induces apoptosis of osteoblast-like cells in culture. Bone 2000;27:359e66. Bonjour JP, Caverzasio J, Fleisch H, Muhlbauer R, Troehler U. The adaptive system of the tubular transport of phosphate. Adv Exp Med Biol 1982;151:1e11. Bonjour JP, Fleisch H. Tubular adaption to the supply and requirement of phosphate. In: Massry SG, Fleisch H, editors. Renal Handling of Phosphate. New York: Plenum; 1980. p. 243e64. Berndt J, Knox FG. Renal regulation of phosphate excretion. In: Seldin DW, Giebisch G, editors. The Kidney: Physiology and Pathophysiology. New York: Raven Press; 1992. p. 2511e32. Silve C, Friedlander G. Regulation of phosphate excretion. In: Seldin DW, Giebisch G, editors. The Kidney: Physiology and Pathophysiology. Philadelphia: Lippincott Williams&Wilkins; 2000. p. 1885e904. Biber J, Hernando N, Forster I, Murer H. Regulation of phosphate transport in proximal tubules. Pflu¨gers Arch 2009;458: 39e52. Suki W, Lederer E, Rouse D. Renal Transport of Calcium, Magnesium, and Phosphate. Philadelphia: W.B. Saunders; 2000. Bonjour JP, Caverzasio J. Phosphate transport in the kidney. Rev Physiol Biochem Pharmacol 1984;100:161e214.

[106] Bijvoet OLM. The importance of the kidneys in phosphate homeostasis. In: Avioli LV, editor. Phosphate Metabolism, Kidney and Bone. Paris: Armour Montagu; 1976. p. 421e74. [107] Ju¨ppner H, Portale A. Endocrine regulation of phosphate homeostasis. In: Singh AK, Williams GH, editors. Textbook of Nephro-Endocrinology. New York: Academic Press; 2009. [108] Murer H, Hernando N, Forster I, Biber J. Proximal tubular phosphate reabsorption: molecular mechanisms. Physiol Rev 2000;80:1373e409. [109] Murer H, Kaissling B, Forster I, Biber J, editors. Cellular mechanisms in proximal tubular handling. 3rd ed. New York: Lippincott Williams & Wilkins; 2000. [110] Barac-Nieto M, Alfred M, Spitzer A. Basolateral phosphate transport in renal proximal-tubule-like OK cells. Exp Biol Med (Maywood) 2002;227:626e31. [111] Custer M, Meier F, Schlatter E, et al. Localization of NaPi-1, a Na-Pi cotransporter, in rabbit kidney proximal tubules. I. mRNA localization by reverse transcription/polymerase chain reaction. Pflu¨gers Arch 1993;424:203e9. [112] Segawa H, Sawai Y, Tominaga R et al. Inorganic phosphate homeostasis in the type I Na/Pi co-transporter (NPT1) null mice. Renal Week 2009. San Diego; abstract F-FC255. [113] Werner A, Moore ML, Mantei N, Biber J, Semenza G, Murer H. Cloning and expression of cDNA for a Na/Pi cotransport system of kidney cortex. Proc Natl Acad Sci USA 1991;88: 9608e12. [114] Magagnin S, Werner A, Markovich D, et al. Expression cloning of human and rat renal cortex Na/Pi cotransport. Proc Natl Acad Sci USA 1993;90:5979e83. [115] Ohkido I, Segawa H, Yanagida R, Nakamura M, Miyamoto K. Cloning, gene structure and dietary regulation of the type-IIc Na/Pi cotransporter in the mouse kidney. Pflu¨gers Arch 2003; 446:106e15. [116] Hilfiker H, Hattenhauer O, Traebert M, Forster I, Murer H, Biber J. Characterization of a murine type II sodium-phosphate cotransporter expressed in mammalian small intestine. Proc Natl Acad Sci USA 1998;95:14564e9. [117] Forster IC, Loo DD, Eskandari S. Stoichiometry and Naþ binding cooperativity of rat and flounder renal type II Naþ-Pi cotransporters. Am J Physiol 1999;276:F644e9. [118] Kavanaugh MP, Kabat D. Identification and characterization of a widely expressed phosphate transporter/retrovirus receptor family. Kidney Int 1996;49:959e63. [119] Breusegem SY, Takahashi H, Giral-Arnal H, et al. Differential regulation of the renal sodium-phosphate cotransporters NaPiIIa, NaPi-IIc, and PiT-2 in dietary potassium deficiency. Am J Physiol Renal Physiol 2009;297:F350e61. [120] Ju¨ppner H, Gardella TJ, Brown EM, Kronenberg HM, Potts Jr JT. Parathyroid hormone and parathyroid hormone-related peptide in the regulation of calcium homeostasis and bone development. In: DeGroot LJ, Jameson JL, editors. Endocrinology. Philadelphia: Elsevier Saunders; 2006. p. 1377e417. [121] Potts Jr JT, Ju¨ppner H. Parathyroid hormone and parathyroid hormone-related peptide in calcium homeostasis, bone metabolism, and bone development: the proteins, their genes, and receptors. In: Avioli L, Krane S, editors. Metabolic Bone Disease. New York: Academic Press; 1997. p. 51e94. [122] White KE, Larsson TE, Econs MJ. The roles of specific genes implicated as circulating factors involved in normal and disordered phosphate homeostasis: frizzled related protein-4, matrix extracellular phosphoglycoprotein, and fibroblast growth factor 23. Endocr Rev 2006;27:221e41. [123] Urakawa I, Yamazaki Y, Shimada T, et al. Klotho converts canonical FGF receptor into a specific receptor for FGF23. Nature 2006;444:770e4.

PEDIATRIC BONE

REFERENCES

[124] Miyamoto KI, Ito M, Tatsumi S, Kuwahata M, Segawa H. New aspect of renal phosphate reabsorption: the type IIc sodiumdependent phosphate transporter. Am J Nephrol 2007;27: 503e15. [125] Forster IC, Hernando N, Biber J, Murer H. Proximal tubular handling of phosphate: A molecular perspective. Kidney Int 2006;70:1548e59. [126] Murer H, Forster I, Biber J. The sodium phosphate cotransporter family SLC34. Pflu¨gers Arch 2004;447:763e7. [127] Stubbs J, Liu S, Quarles LD. Role of fibroblast growth factor 23 in phosphate homeostasis and pathogenesis of disordered mineral metabolism in chronic kidney disease. Semin Dial 2007;20:302e8. [128] Segawa H, Onitsuka A, Kuwahata M, et al. Type IIc sodiumdependent phosphate transporter regulates calcium metabolism. J Am Soc Nephrol 2009;20:104e13. [129] Silver J, Naveh-Many T. FGF23 and the parathyroid glands. Pediatr Nephrol 2010;25:2241e5. [130] Krajisnik T, Bjorklund P, Marsell R, et al. Fibroblast growth factor23 regulates parathyroid hormone and 1alpha-hydroxylase expression in cultured bovine parathyroid cells. J Endocrinol 2007;195:125e31. [131] Wilz DR, Gray RW, Dominguez JH, Lemann Jr J. Plasma 1,25(OH)2-vitamin D concentrations and net intestinal calcium, phosphate, and magnesium absorption in humans. Am J Clin Nutr 1979;32:2052e60. [132] Nabeshima Y. Discovery of alpha-klotho unveiled new insights into calcium and phosphate homeostasis. Proc Jpn Acad Ser B Phys Biol Sci 2009;85:125e41. [133] Kuro-o M. Endocrine FGFs and klothos: emerging concepts. Trends Endocrinol Metab 2008;19:239e45. [134] Bikle D. Nonclassic actions of vitamin D. J Clin Endocrinol Metab 2009;94:26e34. [135] Silver J, Levi R. Regulation of PTH synthesis and secretion relevant to the management of secondary hyperparathyroidism in chronic kidney disease. Kidney Int Suppl 2005;95:S8e12. [136] Gardella TJ. Mimetic ligands for the PTHR1: approaches developments, and considerations. IBMS BoneKey 2009:71e85. [137] Kronenberg HM. PTH regulates the hematopoietic stem cell niche in bone. Adv Exp Med Biol 2007;602:57e60. [138] Kemper B, Habener JF, Rich A, Potts Jr JT. Parathyroid secretion: discovery of a major calcium-dependent protein. Science 1974;184:167e9. [139] Moallem E, Kilav R, Silver J, Naveh-Many T. RNA-protein binding and post-transcriptional regulation of parathyroid hormone gene expression by calcium and phosphate. J Biol Chem 1998;273:5253e9. [140] Naveh-Many T, Rahaminov R, Livini N, Silver J. Parathyroid cell proliferation in normal and chronic renal failure in rats. The effects of calcium, phosphate, and vitamin D. J Clin Invest 1995; 96:1786e93. [141] Diaz R, Brown EM. Familial hypocalciuric hypercalcemia and other disorders due to calcium-sensing receptor mutations. In: DeGroot LJ, Jameson JL, editors. Endocrinology. Philadelphia: Elsevier Saunders; 2006. p. 1595e609. [142] Wettschureck N, Lee E, Libutti SK, Offermanns S, Robey PG, Spiegel AM. Parathyroid-specific double knockout of Gq and G11 alpha-subunits leads to a phenotype resembling germline knockout of the extracellular Ca2þ-sensing receptor. Mol Endocrinol 2007;21:274e80. [143] Quinn SJ, Kifor O, Kifor I, Butters Jr RR, Brown EM. Role of the cytoskeleton in extracellular calcium-regulated PTH release. Biochem Biophys Res Commun 2007;354:8e13.

157

[144] Pollak MR, Brown EM, Estep HL, et al. Autosomal dominant hypocalcaemia caused by a Ca2þ-sensing receptor gene mutation. Nat Genet 1994;8:303e7. [145] Pollak MR, Brown EM, WuChou YH, et al. Mutations in the human Ca2þ-sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Cell 1993;75:1297e303. [146] Silver J, Russell J, Sherwood LM. Regulation by vitamin D metabolites of messenger ribronucleic acid for preproparathyroid hormone in isolated bovine parathyroid cells. Proc Natl Acad Sci USA 1985;82:4270e3. [147] Peters H, Neubuser A, Kratochwil K, Balling R. Pax9-deficient mice lack pharyngeal pouch derivatives and teeth and exhibit craniofacial and limb abnormalities. Genes Dev 1998;12: 2735e47. [148] Ding C, Buckingham B, Levine MA. Familial isolated hypoparathyroidism caused by a mutation in the gene for the transcription factor GCMB. J Clin Invest 2001;108:1215e20. [149] Van Esch H, Groenen P, Nesbit MA, et al. GATA3 haploinsufficiency causes human HDR syndrome. Nature 2000;406: 419e22. [150] Kilav R, Silver J, Naveh-Many T. Parathyroid hormone gene expression in hypophosphatemic rats. J Clin Invest 1995; 96:327e33. [151] Galitzer H, Ben-Dov I, Lavi-Moshayoff V, Naveh-Many T, Silver J. Fibroblast growth factor 23 acts on the parathyroid to decrease parathyroid hormone secretion. Curr Opin Nephrol Hypertens 2008;17:363e7. [152] Galitzer H, Lavi-Moshayoff V, Nechama M, Meir T, Silver J, Naveh-Many T. The calcium-sensing receptor regulates parathyroid hormone gene expression in transfected HEK293 cells. BMC Biol 2009;7:17. [153] Ben-Dov IZ, Galitzer H, Lavi-Moshayoff V, et al. The parathyroid is a target organ for FGF23 in rats. J Clin Invest 2007; 117:4003e8. [154] Ichikawa S, Imel EA, Kreiter ML, et al. A homozygous missense mutation in human KLOTHO causes severe tumoral calcinosis. J Clin Invest 2007;117:2684e91. [155] Imura A, Tsuji Y, Murata M, et al. alpha-Klotho as a regulator of calcium homeostasis. Science 2007;316:1615e8. [156] Kousteni S, Bilezikian JP. The cell biology of parathyroid hormone in osteoblasts. Curr Osteoporos Rep 2008;6:72e6. [157] Mahon MJ. Ezrin promotes functional expression and parathyroid hormone-mediated regulation of the sodium-phosphate cotransporter 2a in LLC-PK1 cells. Am J Physiol Renal Physiol 2008;294:F667e75. [158] Nagai S, Okazaki M, Segawa H, et al. Acute down-regulation of sodium-dependent phosphate transporter NPT2a involves predominantly the cAMP/PKA pathway as revealed by signaling-selective parathyroid hormone analogs. J Biol Chem 2010;286:1618e26. [159] Guo J, Liu M, Yang D, et al. Phospholipase C signaling via the parathyroid hormone (PTH)/PTH-related peptide receptor is essential for normal bone responses to PTH. Endocrinology 2010;151:3502e13. [160] Alpern RJ. Cell mechanisms of proximal tubule acidification. Physiol Rev 1990;70:79e114. [161] Mensenkamp AR, Hoenderop JG, Bindels RJ. TRPV5, the gateway to Ca2þ homeostasis. Handb Exp Pharmacol 2007; 179:207e20. [162] Friedman PA, Gesek FA. Calcium transport in renal epithelial cells. Am J Physiol 1993;264:F181e98. [163] Calvi L, Sims N, Hunzelman J, et al. Activation of the PTH/ PTHrP receptor in osteoblastic cells has differential effects on cortical and trabecular bone. J Clin Invest 2001;107:277e86.

PEDIATRIC BONE

158

7. PHOSPHATE HOMEOSTASIS REGULATORY MECHANISMS

[164] Schipani E, Lanske B, Hunzelman J, et al. Targeted expression of constitutively active PTH/PTHrP receptors delays endochondral bone formation and rescues PTHrP-less mice. Proc Natl Acad Sci USA 1997;94:13689e94. [165] Guo J, Chung U-i, Kondo H, Brighurst FR, Kronenberg HM. The PTH/PTHrP receptor can delay chondrocyte hypertrophy in vivo without activating phospholipase C. Mol Cell 2002;3: 183e94. [166] Miao D, He B, Karaplis A, Goltzman D. Parathyroid hormone is essential for normal fetal bone formation. J Clin Invest 2002;109:1173e82. [167] Bai X, Miao D, Goltzman D, Karaplis AC. Early lethality in Hyp mice with targeted deletion of Pth gene. Endocrinology 2007; 148:4974e83. [168] Lanske B, Karaplis AC, Luz A, et al. PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth. Science 1996;273:663e6. [169] Zhang P, Jobert AS, Couvineau A, Silve C. A homozygous inactivating mutation in the parathyroid hormone/parathyroid hormone-related peptide receptor causing Blomstrand chondrodysplasia. J Clin Endocrinol Metab 1998;83:3365e8. [170] Karperien MC, van der Harten HJ, van Schooten R, et al. A frame-shift mutation in the type I parathyroid hormone/ parathyroid hormone-related peptide receptor causing Blomstrand lethal osteochondrodysplasia. J Clin Endocrinol Metab 1999;84:3713e20. [171] Bounoutas GS, Tawfeek H, Frohlich LF, Chung UI, AbouSamra AB. Impact of impaired receptor internalization on calcium homeostasis in knock-in mice expressing a phosphorylation-deficient parathyroid hormone (PTH)/PTH-related peptide receptor. Endocrinology 2006;147:4674e9. [172] Burnett-Bowie SM, Henao MP, Dere ME, Lee H, Leder BZ. Effects of hPTH(1-34) infusion on circulating serum phosphate, 1,25-dihydroxyvitamin D, and FGF23 levels in healthy men. J Bone Miner Res 2009;24:1681e5. [173] Chang W, Tu C, Chen TH, Bikle D, Shoback D. The extracellular calcium-sensing receptor (CaSR) is a critical modulator of skeletal development. Sci Signal 2008;1. ra1. [174] Gupta A, Winer K, Econs M, Marx S, Collins M. FGF-23 is elevated by chronic hyperphosphatemia. J Clin Endocrinol Metab 2004;89:4489e92. [175] O’Brien CA, Plotkin LI, Galli C, et al. Control of bone mass and remodeling by PTH receptor signaling in osteocytes. PLoS One 2008;3:e2942. [176] Wu JY, Purton LE, Rodda SJ, et al. Osteoblastic regulation of B lymphopoiesis is mediated by Gs{alpha}-dependent signaling pathways. Proc Natl Acad Sci USA 2008;105:16976e81. [177] Yamamoto T, Miyamoto KI, Ozono K, et al. Hypophosphatemic rickets accompanying McCune-Albright syndrome: evidence that a humoral factor causes hypophosphatemia. J Bone Miner Metab 2001;19:287e95. [178] Riminucci M, Collins MT, Fedarko NS, et al. FGF-23 in fibrous dysplasia of bone and its relationship to renal phosphate wasting. J Clin Invest 2003;112:683e92. [179] Collins MT, Chebli C, Jones J, et al. Renal phosphate wasting in fibrous dysplasia of bone is part of a generalized renal tubular dysfunction similar to that seen in tumor-induced osteomalacia. J Bone Miner Res 2001;16:806e13. [180] Brown AJ, Slatopolsky E. Vitamin D analogs: therapeutic applications and mechanisms for selectivity. Mol Aspects Med 2008;29:433e52. [181] Lavi-Moshayoff V, Wasserman G, Meir T, Silver J, NavehMany T. PTH increases FGF23 gene expression and mediates the high-FGF23 levels of experimental kidney failure: a bone

[182]

[183] [184]

[185]

[186]

[187]

[188] [189]

[190]

[191]

[192]

[193]

[194]

[195] [196]

[197]

[198]

[199]

[200]

parathyroid feedback loop. Am J Physiol Renal Physiol 2010; 299:F882e9. Boullion R. Vitamin D: from photosynthesis, metabolism, and action to clinical application. In: DeGroot LJ, Jameson JL, editors. Endocrinology. Philadelphia: Elsevier Saunders; 2006. p. 1435e63. Sakaki T, Kagawa N, Yamamoto K, Inouye K. Metabolism of vitamin D3 by cytochromes P450. Front Biosci 2005;10:119e34. Bai XY, Miao D, Goltzman D, Karaplis AC. The autosomal dominant hypophosphatemic rickets R176Q mutation in fibroblast growth factor 23 resists proteolytic cleavage and enhances in vivo biological potency. J Biol Chem 2003;278:9843e9. Carpenter TO, Ellis BK, Insogna KL, Philbrick WM, Sterpka J, Shimkets R. Fibroblast growth factor 7: an inhibitor of phosphate transport derived from oncogenic osteomalacia-causing tumors. J Clin Endocrinol Metab 2005;90:1012e20. Berndt T, Craig T, Bowe A, et al. Secreted frizzled-related protein 4 is a potent tumor-derived phosphaturic agent. J Clin Invest 2003;112:785e94. Sommer S, Berndt T, Craig T, Kumar R. The phosphatonins and the regulation of phosphate transport and vitamin D metabolism. J Steroid Biochem Mol Biol 2007;103:497e503. DeLuca HF. The metabolism and functions of vitamin D. Adv Exp Med Biol 1986;196:361e75. Inoue Y, Segawa H, Kaneko I, et al. Role of the vitamin D receptor in FGF23 action on phosphate metabolism. Biochem J 2005;390:325e31. Li YC, Pirro AE, Amling M, et al. Targeted ablation of the vitamin D receptor: an animal model of vitamin D-dependent rickets type II with alopecia. Proc Natl Acad Sci USA 1997;94:9831e5. Panda DK, Miao D, Tremblay ML, et al. Targeted ablation of the 25-hydroxyvitamin D 1alpha-hydroxylase enzyme: evidence for skeletal, reproductive, and immune dysfunction. Proc Natl Acad Sci USA 2001;98:7498e503. Kitanaka S, Takeyama K, Murayama A, et al. Inactivating mutations in the 25-hydroxyvitamin D3 1alpha-hydroxylase gene in patients with pseudovitamin D-deficiency rickets. N Engl J Med 1998;338:653e61. Hughes MR, Malloy PJ, Kieback DG, et al. Point mutations in the human vitamin D receptor gene associated with hypocalcemic rickets. Science 1988;242:1702e5. Suzuki Y, Landowski CP, Hediger MA. Mechanisms and regulation of epithelial Ca2þ absorption in health and disease. Annu Rev Physiol 2008;70:257e71. DeLuca HF. New developments in the vitamin D endocrine system. J Am Diet Assoc 1982;80:231e7. Capuano P, Radanovic T, Wagner CA, et al. Intestinal and renal adaptation to a low-Pi diet of type II NaPi cotransporters in vitamin D receptor- and 1alphaOHase-deficient mice. Am J Physiol Cell Physiol 2005;288:C429e34. Segawa H, Kaneko I, Yamanaka S, et al. Intestinal Na-P(i) cotransporter adaptation to dietary P(i) content in vitamin D receptor null mice. Am J Physiol Renal Physiol 2004;287:F39e47. Amling M, Priemel M, Holzmann T, et al. Rescue of the skeletal phenotype of vitamin D receptor-ablated mice in the setting of normal mineral ion homeostasis: formal histomorphometric and biomechanical analyses. Endocrinology 1999;140:4982e7. Kolek OI, Hines ER, Jones MD, et al. 1alpha,25-Dihydroxyvitamin D3 upregulates FGF23 gene expression in bone: the final link in a renal-gastrointestinal-skeletal axis that controls phosphate transport. Am J Physiol Gastrointest Liver Physiol 2005;289:G1036e42. Demay MB, Kiernan MS, DeLuca HF, Kronenberg HM. Sequences in the human parathyroid hormone gene that bind the 1,25-dihydroxyvitamin D3 receptor and mediate

PEDIATRIC BONE

REFERENCES

[201]

[202]

[203]

[204]

[205]

[206]

[207]

[208]

[209]

[210]

[211]

[212]

[213]

[214]

[215]

[216]

[217]

transcriptional repression in response to 1,25-dihydroxyvitamin D3. Proc Natl Acad Sci USA 1992;89:8097e101. Canaff L, Hendy GN. Human calcium-sensing receptor gene. Vitamin D response elements in promoters P1 and P2 confer transcriptional responsiveness to 1,25-dihydroxyvitamin D. J Biol Chem 2002;277:30337e50. Nishi H, Nii-Kono T, Nakanishi S, et al. Intravenous calcitriol therapy increases serum concentrations of fibroblast growth factor-23 in dialysis patients with secondary hyperparathyroidism. Nephron Clin Pract 2005;101:c94e9. Yu X, Sabbagh Y, Davis SI, Demay MB, White KE. Genetic dissection of phosphate- and vitamin D-mediated regulation of circulating Fgf23 concentrations. Bone 2005;36:971e7. Murayama A, Takeyama K, Kitanaka S, et al. Positive and negative regulations of the renal 25-hydroxyvitamin D3 1alphahydroxylase gene by parathyroid hormone, calcitonin, and 1alpha,25(OH)2D3 in intact animals. Endocrinology 1999; 140:2224e31. Masuyama R, Stockmans I, Torrekens S, et al. Vitamin D receptor in chondrocytes promotes osteoclastogenesis and regulates FGF23 production in osteoblasts. J Clin Invest 2006; 116:3150e9. Yamashita T, Yoshioka M, Itoh N. Identification of a novel fibroblast growth factor, FGF-23, preferentially expressed in the ventrolateral thalamic nucleus of the brain. Biochem Biophys Res Commun 2000;277:494e8. ADHR Consortium T, White KE, Evans WE, O’Riordan JLH, et al. Autosomal dominant hypophosphataemic rickets is associated with mutations in FGF23. Nat Genet 2000;26:345e8. Shimada T, Mizutani S, Muto T, et al. Cloning and characterization of FGF23 as a causative factor of tumor-induced osteomalacia. Proc Natl Acad Sci USA 2001;98:6500e5. von Marschall Z, Fisher LW. Dentin matrix protein-1 isoforms promote differential cell attachment and migration. J Biol Chem 2008;283:32730e40. Yuan B, Meudt J, Feng JO, Drezner MK. 7B2 protein mediated inhibition of DMP1 cleavage in osteoblasts enhances FGF-23 production in hyp-mice. J Bone Miner Res 2008;23:s16 (abstract 1053). Lu Y, Qin C, Xie Y, Bonewald LF, Feng JQ. Studies of the DMP1 57-kDa functional domain both in vivo and in vitro. Cells Tissues Organs 2009;189:175e85. Burnett SM, Gunawardene SC, Bringhurst FR, Ju¨ppner H, Lee H, Finkelstein JS. Regulation of C-terminal and intact FGF23 by dietary phosphate in men and women. J Bone Miner Res 2006;21:1187e96. Shimada T, Muto T, Urakawa I, et al. Mutant FGF-23 responsible for autosomal dominant hypophosphatemic rickets is resistant to proteolytic cleavage and causes hypophosphatemia in vivo. Endocrinology 2002;143:3179e82. Kato K, Jeanneau C, Tarp MA, et al. Polypeptide GalNActransferase T3 and familial tumoral calcinosis. Secretion of fibroblast growth factor 23 requires O-glycosylation. J Biol Chem 2006;281:18370e7. Masi L, Gozzini A, Carbonell S, et al. A novel recessive mutation in fibroblast growth factor-23 (FGF23) causes a tumoral calcinosis. J Bone Miner Res 2005;(Suppl):S128. Garringer HJ, Malekpour M, Esteghamat F, et al. Molecular genetic and biochemical analyses of FGF23 mutations in familial tumoral calcinosis. Am J Physiol Endocrinol Metab 2008; 295:E929e37. Benet-Pages A, Orlik P, Strom TM, Lorenz-Depiereux B. An FGF23 missense mutation causes familial tumoral calcinosis with hyperphosphatemia. Hum Mol Genet 2005;14:385e90.

159

[218] Larsson T, Yu X, Davis SI, et al. A novel recessive mutation in fibroblast growth factor-23 causes familial tumoral calcinosis. J Clin Endocrinol Metab 2005;90:2424e7. [219] Chefetz I, Heller R, Galli-Tsinopoulou A, et al. A novel homozygous missense mutation in FGF23 causes familial tumoral calcinosis associated with disseminated visceral calcification. Hum Genet 2005;118:261e6. [220] Araya K, Fukumoto S, Backenroth R, et al. A novel mutation in fibroblast growth factor 23 gene as a cause of tumoral calcinosis. J Clin Endocrinol Metab 2005;90:5523e7. [221] Larsson T, Davis SI, Garringer HJ, et al. Fibroblast growth factor-23 mutants causing familial tumoral calcinosis are differentially processed. Endocrinology 2005;146:3883e91. [222] Frishberg Y, Ito N, Rinat C, et al. Hyperostosis-hyperphosphatemia syndrome: a congenital disorder of O-glycosylation associated with augmented processing of fibroblast growth factor 23. J Bone Miner Res 2007;22:235e42. [223] Bergwitz C, Banerjee S, Abu-Zahra H, et al. Defective O-glycosylation due to a novel homozygous S129P mutation is associated with lack of fibroblast growth factor 23 secretion and tumoral calcinosis. J Clin Endocrinol Metab 2009;94:4267e74. [224] Ichikawa S, Sorenson AH, Austin AM, et al. Ablation of the Galnt3 gene leads to low-circulating intact fibroblast growth factor 23 (Fgf23) concentrations and hyperphosphatemia despite increased Fgf23 expression. Endocrinology 2009;150:2543e50. [225] Chefetz I, Kohno K, Izumi H, Uitto J, Richard G, Sprecher E. GALNT3, a gene associated with hyperphosphatemic familial tumoral calcinosis, is transcriptionally regulated by extracellular phosphate and modulates matrix metalloproteinase activity. Biochim Biophys Acta 2009;1792:61e7. [226] Mohammadi M, Olsen SK, Ibrahimi OA. Structural basis for fibroblast growth factor receptor activation. Cytokine Growth Factor Rev 2005;16:107e37. [227] Powers CJ, McLeskey SW, Wellstein A. Fibroblast growth factors, their receptors and signaling. Endocr Relat Cancer 2000; 7:165e97. [228] Ornitz DM, Itoh N. Fibroblast growth factors. Genome Biol 2001;2:3005. [229] Zhang X, Ibrahimi OA, Olsen SK, Umemori H, Mohammadi M, Ornitz DM. Receptor specificity of the fibroblast growth factor family. The complete mammalian FGF family. J Biol Chem 2006; 281:15694e700. [230] Yu X, White KE. Fibroblast growth factor 23 and its receptors. Ther Apher Dial 2005;9:308e12. [231] Yamashita T, Konishi M, Miyake A, Inui K, Itoh N. Fibroblast growth factor (FGF)-23 inhibits renal phosphate reabsorption by activation of the mitogen-activated protein kinase pathway. J Biol Chem 2002;277:28265e70. [232] Kurosu H, Yamamoto M, Clark JD, et al. Suppression of aging in mice by the hormone Klotho. Science 2005;309:1829e33. [233] Yamazaki Y, Tamada T, Kasai N, et al. Anti-FGF23 neutralizing antibodies show the physiological role and structural features of FGF23. J Bone Miner Res 2008;23:1509e18. [234] Liu S, Vierthaler L, Tang W, Zhou J, Quarles LD. FGFR3 and FGFR4 do not mediate renal effects of FGF23. J Am Soc Nephrol 2008;19:2342e50. [235] Li H, Martin AC, David V, Quarles LD. Compound deletion of FGFR3 and FGFR4 partially rescues the Hyp mouse phenotype. Am J Physiol Endocrinol Metab 2010;300:E508e17. [236] Gattineni J, Bates C, Twombley K, et al. FGF23 decreases renal NaPi-2a and NaPi-2c expression and induces hypophosphatemia in vivo predominantly via FGF receptor 1. Am J Physiol Renal Physiol 2009;297:F282e91.

PEDIATRIC BONE

160

7. PHOSPHATE HOMEOSTASIS REGULATORY MECHANISMS

[237] Segawa H, Yamanaka S, Ohno Y, et al. Correlation between hyperphosphatemia and type II Na-Pi cotransporter activity in klotho mice. Am J Physiol Renal Physiol 2007;292:F769e79. [238] Baum M, Schiavi S, Dwarakanath V, Quigley R. Effect of fibroblast growth factor-23 on phosphate transport in proximal tubules. Kidney Int 2005;68:1148e53. [239] Segawa H, Onitsuka A, Aranami F, et al. Npt2a and Npt2c in mice play distinct and synergistic roles in inorganic phosphate metabolism and skeletal development. Renal Week 2006. San Francisco: JASN; 2007. 2007. p. abstract SA-FC101. [240] Liu S, Gupta A, Quarles LD. Emerging role of fibroblast growth factor 23 in a bone-kidney axis regulating systemic phosphate homeostasis and extracellular matrix mineralization. Curr Opin Nephrol Hypertens 2007;16:329e35. [241] Fukumoto S, Yamashita T. FGF23 is a hormone-regulating phosphate metabolism e unique biological characteristics of FGF23. Bone 2007;40:1190e5. [242] Farrow EG, Davis SI, Summers LJ, White KE. Initial FGF23mediated signaling occurs in the distal convoluted tubule. J Am Soc Nephrol 2009;20:955e60. [243] Chang Q, Hoefs S, van der Kemp AW, Topala CN, Bindels RJ, Hoenderop JG. The beta-glucuronidase klotho hydrolyzes and activates the TRPV5 channel. Science 2005;310:490e3. [244] Cha SK, Hu MC, Kurosu H, Kuro-o M, Moe O, Huang CL. Regulation of renal outer medullary potassium channel and renal K(þ) excretion by Klotho. Mol Pharmacol 2009;76:38e46. [245] Frishberg Y, Araya K, Rinat C, et al. Hyperostosis-hyperphosphatemia syndrome caused by mutations in GALNT3 and associated with augmented processing of FGF-23. Philadelphia: American Society of Nephrology; 2004. 2004. p. F-P0937. [246] Ichikawa S, Lyles KW, Econs MJ. A novel GALNT3 mutation in a pseudoautosomal dominant form of tumoral calcinosis: evidence that the disorder is autosomal recessive. J Clin Endocrinol Metab 2005;90:2420e3. [247] Shimada T, Kakitani M, Yamazaki Y, et al. Targeted ablation of Fgf23 demonstrates an essential physiological role of FGF23 in phosphate and vitamin D metabolism. J Clin Invest 2004;113: 561e8. [248] Hesse M, Frohlich LF, Zeitz U, Lanske B, Erben RG. Ablation of vitamin D signaling rescues bone, mineral, and glucose homeostasis in Fgf-23 deficient mice. Matrix Biol 2007;26:75e84. [249] Kuro-o M, Matsumura Y, Aizawa H, et al. Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature 1997;390:45e51. [250] Sitara D, Kim S, Razzaque MS, et al. Genetic evidence of serum phosphate-independent functions of FGF-23 on bone. PLoS Genet 2008;4:e1000154. [251] Razzaque MS, Sitara D, Taguchi T, St-Arnaud R, Lanske B. Premature aging-like phenotype in fibroblast growth factor 23 null mice is a vitamin D-mediated process. Faseb J 2006;20: 720e2. [252] Ohnishi M, Nakatani T, Lanske B, Razzaque MS. Reversal of mineral ion homeostasis and soft-tissue calcification of klotho knockout mice by deletion of vitamin D 1alpha-hydroxylase. Kidney Int 2009;75:1166e72. [253] Stubbs JR, Liu S, Tang W, et al. Role of hyperphosphatemia and 1,25-dihydroxyvitamin d in vascular calcification and mortality in fibroblastic growth factor 23 null mice. J Am Soc Nephrol 2007;18:2116e24. [254] Brownstein CA, Adler F, Nelson-Williams C, et al. A translocation causing increased alpha-klotho level results in hypophosphatemic rickets and hyperparathyroidism. Proc Natl Acad Sci USA 2008;105:3455e60.

[255] Eicher E, Southard J, Scriver C, Glorieux F. Hypophosphatemia: mouse model for human familial hypophosphatemic (vitamin D-resistant) rickets. Proc Natl Acad Sci USA 1976;73:4667e71. [256] Liu S, Zhou J, Tang W, Jiang X, Rowe DW, Quarles LD. Pathogenic role of Fgf23 in Hyp mice. Am J Physiol Endocrinol Metab 2006;291:E38e49. [257] Bai X, Miao D, Li J, Goltzman D, Karaplis AC. Transgenic mice overexpressing human fibroblast growth factor 23 (R176Q) delineate a putative role for parathyroid hormone in renal phosphate wasting disorders. Endocrinology 2004;145:5269e79. [258] DeLuca S, Sitara D, Kang K, et al. Amelioration of the premature ageing-like features of Fgf-23 knockout mice by genetically restoring the systemic actions of FGF-23. J Pathol 2008;216: 345e55. [259] Larsson T, Marsell R, Schipani E, et al. Transgenic mice expressing fibroblast growth factor 23 under the control of the alpha1(I) collagen promoter exhibit growth retardation, osteomalacia, and disturbed phosphate homeostasis. Endocrinology 2004;145:3087e94. [260] Bai X, Miao D, Panda D, et al. Partial rescue of the Hyp phenotype by osteoblast-targeted PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) expression. Mol Endocrinol 2002;16:2913e25. [261] Liu S, Guo R, Tu Q, Quarles LD. Overexpression of Phex in osteoblasts fails to rescue the Hyp mouse phenotype. J Biol Chem 2002;277:3686e97. [262] Erben RG, Mayer D, Weber K, Jonsson K, Ju¨ppner H, Lanske B. Overexpression of human PHEX under the human beta-actin promoter does not fully rescue the Hyp mouse phenotype. J Bone Miner Res 2005;20:1149e60. [263] Sitara D, Razzaque MS, Hesse M, et al. Homozygous ablation of fibroblast growth factor-23 results in hyperphosphatemia and impaired skeletogenesis, and reverses hypophosphatemia in Phex-deficient mice. Matrix Biol 2004;23:421e32. [264] Liu S, Zhou J, Tang W, Menard R, Feng JQ, Quarles LD. Pathogenic role of Fgf23 in Dmp1-null mice. Am J Physiol Endocrinol Metab 2008;295:E254e61. [265] Bergwitz C, Kremke B, Bounoutas G, Hiort O, Insogna K, Ju¨ppner H. Hypophosphatemic rickets with hypercalciuria (HHRH) can be associated with renal stones. ASBMR. Philadelphia. J Bone Miner Res 2006. p. SU419. [266] Beck L, Karaplis AC, Amizuka N, Hewson AS, Ozawa H, Tenenhouse HS. Targeted inactivation of Npt2 in mice leads to severe renal phosphate wasting, hypercalciuria, and skeletal abnormalities. Proc Natl Acad Sci USA 1998;95:5372e7. [267] Beck Jr GR. Inorganic phosphate as a signaling molecule in osteoblast differentiation. J Cell Biochem 2003;90:234e43. [268] Schoeters GE, de Saint-Georges L, Van den Heuvel R, Vanderborght O. Mineralization of adult mouse bone marrow in vitro. Cell Tissue Kinet 1988;21:363e74. [269] Xie J, Baumann MJ, McCabe LR. Osteoblasts respond to hydroxyapatite surfaces with immediate changes in gene expression. J Biomed Mater Res A 2004;71:108e17. [270] Otsuka E, Yamaguchi A, Hirose S, Hagiwara H. Characterization of osteoblastic differentiation of stromal cell line ST2 that is induced by ascorbic acid. Am J Physiol 1999;277:C132e8. [271] Berendsen AD, Smit TH, Schoenmaker T, et al. Inorganic phosphate stimulates DMP1 expression in human periodontal ligament fibroblasts embedded in three-dimensional collagen gels. Cells Tissues Organs 2010;192:116e24. [272] Yoshiko Y, Candeliere GA, Maeda N, Aubin JE. Osteoblast autonomous Pi regulation via Pit1 plays a role in bone mineralization. Mol Cell Biol 2007;27:4465e74.

PEDIATRIC BONE

REFERENCES

[273] Chang SH, Yu KN, Lee YS, et al. Elevated inorganic phosphate stimulates Akt-ERK1/2-Mnk1 signaling in human lung cells. Am J Respir Cell Mol Biol 2006;35:528e39. [274] Goseki-Sone M, Yamada A, Hamatani R, Mizoi L, Iimura T, Ezawa I. Phosphate depletion enhances bone morphogenetic protein-4 gene expression in a cultured mouse marrow stromal cell line ST2. Biochem Biophys Res Commun 2002;299:395e9. [275] Kimata M, Michigami T, Tachikawa K, et al. Signaling of extracellular inorganic phosphate up-regulates cyclin D1 expression in proliferating chondrocytes via the Na(þ)/Pi cotransporter Pit-1 and Raf/MEK/ERK pathway. Bone 2010;47:938e47. [276] Beck Jr GR, Knecht N. Osteopontin regulation by inorganic phosphate is ERK1/2-, protein kinase C-, and proteasomedependent. J Biol Chem 2003;278:41921e9. [277] Julien M, Magne D, Masson M, et al. Phosphate stimulates matrix Gla protein expression in chondrocytes through the extracellular signal regulated kinase signaling pathway. Endocrinology 2007;148:530e7. [278] Nair D, Misra RP, Sallis JD, Cheung HS. Phosphocitrate inhibits a basic calcium phosphate and calcium pyrophosphate dihydrate crystal-induced mitogen-activated protein kinase cascade signal transduction pathway. J Biol Chem 1997;272:18920e5. [279] Yamazaki M, Ozonoa K, Okada T, et al. Both FGF23 and extracellular phosphate activate Raf/MEK/ERK pathway via FGF receptors in HEK293 cells. J Cell Biochem 2010;111:1210e21. [280] Bergwitz C, DeRobertis C, Chen H, Friedman A, Ju¨ppner H, Perrimon N. Role of sodium-phosphate co-transporters in the activation of the MAPK pathway by phosphate. J Am Soc Nephrol 2009. abstract F-FC258. [281] Beck L, Leroy C, Salaun C, Margall-Ducos G, Desdouets C, Friedlander G. Identification of a novel function of PiT1 critical for cell proliferation and independent of its phosphate transport activity. J Biol Chem 2009;284:31363e74. [282] Werner A, Kinne RK. Evolution of the Na-P(i) cotransport systems. Am J Physiol Regul Integr Comp Physiol 2001;280: R301e12. [283] Hubbard TJ, Aken BL, Beal K, et al. Ensembl 2007. Nucleic Acids Res 2007 Jan;35(Database issue):D610e7. [284] Meng Z, Camalier CE, Lucas DA, Veenstra TD, Beck Jr GR, Conrads TP. Probing early growth response 1 interacting proteins at the active promoter in osteoblast cells using oligoprecipitation and mass spectrometry. J Proteome Res 2006;5:1931e9. [285] Oshima Y, Akiyama T, Hikita A, et al. Pivotal role of Bcl-2 family proteins in the regulation of chondrocyte apoptosis. J Biol Chem 2008;283:26499e508. [286] Donohue MM, Demay MB. Rickets in VDR null mice is secondary to decreased apoptosis of hypertrophic chondrocytes. Endocrinology 2002;143:3691e4.

161

[287] Lavi-Moshayoff V, Silver J, Naveh-Many T. Human PTH gene regulation in vivo using transgenic mice. Am J Physiol Renal Physiol 2009;297:F713e9. [288] Dinur M, Kilav R, Sela-Brown A, Jacquemin-Sablon H, NavehMany T. In vitro evidence that upstream of N-ras participates in the regulation of parathyroid hormone messenger ribonucleic acid stability. Mol Endocrinol 2006;20:1652e60. [289] Nechama M, Ben-Dov IZ, Briata P, Gherzi R, Naveh-Many T. The mRNA decay promoting factor K-homology splicing regulator protein post-transcriptionally determines parathyroid hormone mRNA levels. Faseb J 2008;22:3458e68. [290] Nechama M, Uchida T, Mor Yosef-Levi I, Silver J, NavehMany T. The peptidyl-prolyl isomerase Pin1 determines parathyroid hormone mRNA levels and stability in rat models of secondary hyperparathyroidism. J Clin Invest 2009;119:3102e14. [291] Bikle D, Adams J, Christakos C. Vitamin D: production, metabolism, mechanism of action, and clinical requirements. In: Rosen C, editor. Primer on Metabolic Bone Disease and Disorders of Mineral Metabolism. Philadelphia, PA: Lippincott-Raven; 2008. p. 141e9. [292] Shimada T, Hasegawa H, Yamazaki Y, et al. FGF-23 is a potent regulator of vitamin D metabolism and phosphate homeostasis. J Bone Miner Res 2004;19:429e35. [293] Bhattacharyya N, Andreopoulou P, Dumitrescu C, Miwa H, Gafni R, Collins M. Cyclic AMP-mediated processing of FGF23: Regulation by O-glycosylation. J Bone Miner Res 2009. abstract A09003028. [294] Terkeltaub R. Physiologic and pathologic functions of the NPP nucleotide pyrophosphatase/phosphodiesterase family focusing on NPP1 in calcification. Purinergic Signal 2006;2: 371e7. [295] Rutsch F, Vaingankar S, Johnson K, et al. PC-1 nucleoside triphosphate pyrophosphohydrolase deficiency in idiopathic infantile arterial calcification. Am J Pathol 2001;158:543e54. [296] Rutsch F, Ruf N, Vaingankar S, et al. Mutations in ENPP1 are associated with ’idiopathic’ infantile arterial calcification. Nat Genet 2003;34:379e81. [297] Levy-Litan V, Hershkovitz E, Avizov L, et al. Autosomal-recessive hypophosphatemic rickets is associated with an inactivation mutation in the ENPP1 gene. Am J Hum Genet 2010;86:273e8. [298] Lorenz-Depiereux B, Schnabel D, Tiosano D, Hausler G, Strom TM. Loss-of-function ENPP1 mutations cause both generalized arterial calcification of infancy and autosomalrecessive hypophosphatemic rickets. Am J Hum Genet 2010; 86:267e72. [299] Chodirker BN, Evans JA, Seargeant LE, Cheang MS, Greenberg CR. Hyperphosphatemia in infantile hypophosphatasia: implications for carrier diagnosis and screening. Am J Hum Genet 1990;46:280e5.

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Vitamin D Biology Rene´ St-Arnaud 1, Marie B. Demay 2 1

Genetics Unit, Shriners Hospital for Children, Montre´al, Que´bec and Departments of Surgery and Human Genetics, McGill University, Montre´al, Que´bec, Canada 2 Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

METABOLIC ACTIVATION OF VITAMIN D Overview 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 which 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 (CYP2R1) 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 1a by the enzyme 25-hydroxyvitamin D-1a-hydroxylase (CYP27B1) converts 25(OH)D to 1a,25-dihydroxyvitamin D (1,25(OH)2D), the active, hormonal form of vitamin D. Upon reaching target tissues, 1,25(OH)2D 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

Pediatric Bone, Second Edition DOI: 10.1016/B978-0-12-382040-2.10008-5

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 D3 (cholecalciferol) is produced in fish and mammals. The slight differences in the chemical structure of the two compounds (Fig. 8.1A) do not affect function or metabolism in mammals. The generic term of vitamin D (without subscript) will be used hereafter. This chapter will review several aspects of vitamin D biology. Vitamin D metabolism will be 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 will be presented. The reader is referred to Chapter 25 for a discussion of the pediatric disorders involving the vitamin D endocrine system.

Hepatic 25-Hydroxylation In humans, a large percentage (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 7-dehydrocholesterol into previtamin D which rapidly becomes a more thermodynamically stable molecule, vitamin D [3]. Vitamin D then exits the keratinocyte cells and enters the dermal capillary bed where it is bound to the vitamin D binding protein, DBP. Once associated with DBP in the circulation, vitamin D is transported to the liver where the cytochrome P450 enzyme vitamin D 25-hydroxylase (CYP2R1) adds a hydroxyl group on carbon 25 to produce 25-hydroxyvitamin D (25(OH)D).

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FIGURE 8.1 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 cyclopentanoperhydrophenantren core identifies vitamin D metabolites as seco-steroids 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.

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 (see Fig. 8.1B). Early studies using perfused rat liver revealed kinetics of vitamin D metabolism that supported two 25-hydroxylase activities: a microsomal (endoplasmic reticulum) high-affinity, low-capacity enzyme and a mitochondrial low-affinity, high capacity form [4]. The microsomal, high-affinity vitamin D 25-hydroxylase was recently identified as CYP2R1 using an elegant expression-based screening strategy [5]. Several criteria suggest that CYP2R1 is the physiologically relevant 25-hydroxylase in vivo. CYP2R1 is a microsomal cytochrome P450 expressed primarily in liver and testis [5]. It can hydroxylate vitamin D2 and vitamin D3 at physiologically-relevant substrate concentrations [5]. It exhibits specificity towards vitamin D as a substrate and does not metabolize cholesterol or 7-dehydrocholesterol [6]. Finally, a mutation in CYP2R1 was reported in a patient with vitamin D-dependent rickets, type 1A [7e9]. Another cytochrome shown to hydroxylate vitamin D at position 25 is the bifunctional CYP27A1 that derives

its name for its ability to 27-hydroxylate the side-chains of cholesterol-derived intermediates involved in bile acid biosynthesis [10]. The physiological relevance of CYP27A1 as a vitamin D 25-hydroxylase was always considered debatable. Indeed, mice with a disrupted Cyp27a1 gene have reduced bile acid synthesis, but normal vitamin D metabolite levels [11]. Also, patients with the inherited disease cerebrotendinous xanthomatosis, caused by mutations in CYP27A1 [12,13], exhibit normal vitamin D metabolism and no obvious 25(OH)D or 1,25(OH)2D deficiency [14]. Finally, CYP27A1 is a mitochondrial enzyme [15,16]. It is likely that CYP2R1 is the physiologically relevant enzyme at normal, nanomolar vitamin D concentrations, but that it is backed up by CYP27A1 when substrate concentrations rise into the pharmacological range (high nanomolar to low micromolar).

Renal 1a-Hydroxylation 25(OH)D must be further hydroxylated to gain hormonal bioactivity. This takes place in the convoluted and straight portions of the proximal kidney tubule. Hydroxylation at position 1a by the mitochondrial cytochrome P450 enzyme 25-hydroxyvitamin D1a-hydroxylase (CYP27B1, hereafter referred to as 1a-hydroxylase) converts 25(OH)D to 1a,25-dihydroxyvitamin D (1,25(OH)2D), the active, hormonal form of vitamin D.

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The 1a-hydroxylase also belongs to the class of the mixed-function oxidases. The 1a-hydroxylase cDNA was cloned from rat and mouse kidney, as well as from human keratinocytes and kidney [17e20]. 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 1a-hydroxylase at both the nucleotide and the amino acid levels. The human 1a-hydroxylase cDNA is 2469 bp in length, and codes for a deduced protein of 508 amino acids containing a ferredoxinbinding domain and a heme-binding domain. The deduced amino acid sequence has substantial homology to members of the mitochondrial P450 family [21], particularly CYP27A1 (40%) [16], CYP24A1 (32%) [22], P450scc, the cholesterol side-chain cleavage enzyme (CYP11A, 33%) [23], and P450c11b, the steroid 11b-hydroxylase (CYP11B1, 30%) [24]. Every laboratory that isolated the 1a-hydroxylase cDNA rapidly obtained the sequence of the 1a-hydroxylase gene in various species. The gene exists as a single copy in the human genome and contains nine exons spanning 5 kb of sequence. The ferredoxin-binding domain is encoded by sequences contained in exons 6 and 7, while the heme-binding domain is contained in exon 8 [25,26]. The human gene has been localized to chromosome 12 using somatic cell hybrids [26], then mapped to 12q13.1e13.3 by fluorescence in situ hybridization [18,27,28]. The chromosomal location provided further circumstantial evidence that mutations in the gene were responsible for the hereditary disease, pseudo vitamin D deficiency rickets (PDDR), also termed vitamin D dependency rickets type I (see Chapter 25), since the disease had been previously mapped to this locus by linkage analysis [29,30]. Incontrovertible proof that mutations of the 1a-hydroxylase caused PDDR was provided by the characterization of CYP27B1 mutations in patients with the disease (reviewed in [31]). Animal models of the disease have recently been engineered by gene targeting technology (see below) [32,33]. The main site for the 1a-hydroxylation of 25(OH)D is the proximal tubule of the renal cortex [34]. The expression of the enzyme has also been reported in cells from other tissues: osteoblasts [35], keratinocytes [17], and cells of the lympho-hematopoeitic system [36]. The identification of these extrarenal sites of expression of the 1a-hydroxylase has led investigators to hypothesize that local production of 1,25(OH)2D could play an important autocrine or paracrine role in the differentiation or function of these cells. In the kidney, the expression of the 1a-hydroxylase gene is subject to complex regulation by parathyroid hormone, calcitonin, fibroblast growth factor 23, calcium, phosphorus, and 1,25(OH)2D itself.

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Parathyroid hormone (PTH) has long been known to stimulate 1a-hydroxylase enzymatic activity in kidney [37] and this has now been demonstrated to occur at the level of gene transcription [38,39]. The PTH response involves signal transduction through cyclic AMP and protein kinase A [38e41]. Putative cyclic AMP response elements have been identified within the proximal 1a-hydroxylase promoter [39], but individual mutation of these sites did not significantly affect transcriptional responses [42]. Recently, regulated epigenetic modification was demonstrated to contribute to the PTH control of CYP27B1 expression. Treatment with PTH induces active demethylation of CpG sites within the CYP27B1 promoter [43]. This mechanism involves the DNA glycosylase, MBD4 [44], which is phosphorylated through protein kinase C upon PTH treatment. This promotes incision of methylated DNA through glycosylase activity, and a base-excision repair process leads to DNA demethylation and transcriptional derepression [43]. Conversely, treatment with 1,25(OH)2D induces methylation through the DNA methyltransferases, DNMT1 and DNMT3B [43]. The identification of this methylation switching at the DNA level constitutes a novel mechanism in the hormonal control of gene transcription. Calcitonin is also a positive regulator of 1a-hydroxylase activity [45,46], and this also occurs at the transcriptional level [38]. Results obtained in a rat model demonstrated that while PTH is mainly responsible for Cyp27b1 induction in hypocalcemic animals, calcitonin appears to be the major regulator of the expression of the 1a-hydroxylase gene in normocalcemic rats [47]. Dietary calcium and phosphate intake are critical in the control of 1a-hydroxylase enzymatic activity [48]. Decreases in blood calcium stimulate the synthesis and secretion of PTH which, in turn, increases expression of the 1a-hydroxylase gene as mentioned above [49], and thus regulation by calcium is indirect. Hyperphosphatemia inhibits CYP27B1 expression. This regulation involves the phosphaturic hormone fibroblast growth factor 23 (FGF-23) [50]. Dietary phosphate stimulates FGF-23 synthesis [51]. In turn, FGF-23 inhibits CYP27B1 expression [52]. The regulation of phosphate homeostasis by PTH, FGF-23, and vitamin D remains a current research focus and has been the subject of recent reviews [50,53]. PTH induces the expression of the 1a-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 1a-hydroxylase gene expression [54e58]. The characterization of this transcriptional repression feedback has highlighted a novel mechanism of action for the VDR [59e62] which is detailed below.

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24-Hydroxylation Hypocalcemia results in increased production of 1,25(OH)2D via PTH. As discussed above, the expression of the 1a-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 hormone induces the expression of the gene encoding the key effector of its catabolic breakdown: 25-hydroxyvitamin D-24-hydroxylase (CYP24A1). The CYP24A1 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)2D); 1,25(OH)2D, to produce 1,24,25-trihydroxyvitamin D (1,24,25(OH)3D), and even 1a-hydroxyvitamin D (1a (OH)D), to generate 1a,24-dihydroxyvitamin D (1,24(OH)2D). 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 24-hydroxylase enzyme, long before 1,25(OH)2D and the 1a-hydroxylase were identified. The enzyme was purified to homogeneity from rat kidney mitochondria [63] and the purified protein was used to raise antibodies [64] permitting cloning of the cDNA [22]. This then allowed cloning of the gene from various species [65e67], 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)2D itself [68,69]. Since the product of that reaction, 1,24,25(OH)3D, was 10 times less active than the 1,25(OH)2D substrate [68], investigators began to reason 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)2D and 1,24,25(OH)3D, could be further converted to different metabolic products sporting 24-oxo and/or 23-hydroxy groups [70e72]. Studies using perfused rat kidney then led to the identification of additional metabolites: a 23-alcohol [73] and a 23-acid [74,75]. Those metabolites had not been previously identified in vitro. Calcitroic acid, the 1a-hydroxylated 23-acid, was shown to be the main biliary excretory form of 1,25(OH)2D [76]. With most metabolites identified, investigators deduced that 24-hydroxylation initiates the C-24 oxidation pathway that leads to 1,25(OH)2D degradation [74,75]. This pathway comprises five enzymatic steps which involve successive hydroxylation/oxidation reactions at carbons 24 and 23, followed by cleavage of the secosteroid at the C-23/C-24 bond and subsequent oxidation of the

cleaved product to calcitroic acid [74,75]. Under basal conditions, low levels of Cyp24a1 mRNA [34], protein [77], and enzymatic activity [78] can be detected in kidney, but treatment with 1,25(OH)2D induces expression in a wide range of tissues, such as bone, heart, intestine, kidney, lung, pancreas, skin, spleen, testis and thymus [79]. The 1,25(OH)2D-inducible 24-hydroxylation and calcitroic acid production is also observed in cell lines from kidney, bone, intestine, skin, and breast [80e82], demonstrating that the C-24 oxidation catabolic pathway can be induced in most vitamin D target cells. Interestingly, the CYP24A1 enzyme is able to hydroxylate both the C23 or the C24 side-chain carbons of 25(OH)D or 1,25(OH)2D [83]. The relative level of C23and C24-hydroxylase activity appears species-specific and the structural basis of this altered specificity has recently been examined using sequence alignment and site-directed mutagenesis [84]. C24 hydroxylation leads to side-chain cleavage and oxidation to a carboxylic acid (C24 oxidation pathway), while hydroxylation at carbon 23 results in side-chain lactone formation (C23 hydroxylation pathway). The recombinant CYP24A1 protein, when associated with its electron-transport co-factors, NADPH-ferredoxin reductase and ferredoxin, has been shown to perform multiple steps of the C-24 oxidation pathway. This includes 23-hydroxylation, dehydrogenation of the 24-hydroxyl group, and side-chain cleavage [85,86]. The hypothesis that the main role of the C-24 oxidation pathway is attenuation of the 1,25(OH)2D 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)2D accumulation and extended hormone action [87]. This hypothesis was also tested and confirmed in vivo by engineering Cyp24a1-deficient mice. Animals homozygous for the Cyp24a1 mutation cannot effectively clear 1,25(OH)2D from their circulation [88]. The phenotype of the Cyp24a1 knockout mice will be described in detail below. There has been significant interest in performing structureefunction analyses and generating homology models of the CYP24A1 structure while awaiting crystal structure resolution, which was recently reported [89]. Efforts stemmed from two different perspectives: on the one hand, investigators were intrigued by the speciesspecific bias for C23- or C24-hydroxylation [83,85,86, 90,91]. On the other hand, there has been steady pharmacological interest in the development of CYP24A1 inhibitors to prolong the bioactivity of 1,25(OH)2D or its relevant analogs [92e95]. Information on the tertiary structure of the substrate-binding pocket was perceived as relevant to develop specific inhibitors. Recombinant CYP24A1 crystals were obtained following ferredoxinsepharose affinity chromatography purification of

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bacterially expressed rat Cyp24a1 sequence deleted of its mitochondrial import signal (D2e32) [96] and mutated at residue 57 (S57D) to stabilize the recombinant protein [89,97]. The CYP24A1 crystal structure displays the canonical P450 fold, including the 12 a-helices (AeL) and four b-sheet systems (b1eb4). Five additional short helices (A’, B’, G’, K’ and K”) were also identified [89]. The substrate-binding cavity is defined by the b1 and b4 sheets, the BeC loop, and helices E, F, G, I, and K surrounding the heme. Alignment of helices A’ and G’ with other mitochondrial P450 sequences identifies them as membrane insertion sequences (MIS) and computational modeling suggests that residues in the hydrophobic surfaces of helices A’ and G’ can penetrate ˚ into the mitochondrial inner approximately 7 A membrane to serve as anchors flanking the substrate access channel. The CYP24A1 crystal structure thus provides a template for understanding membrane insertion, substrate binding, and electron chain partner interactions. This molecular understanding of the structuree function relationships of the CYP24A1 protein will greatly facilitate biorational drug development.

MECHANISM OF ACTION Overview The vitamin D receptor (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 1,25(OH)2D by binding this ligand, heterodimerizing with the retinoid-X receptor (RXR) and interacting with DNA sequences on target genes, thereby regulating their transcription. Nuclear receptor co-activators have been shown to contribute to the transcriptional effects of the liganded VDReRXR heterodimer. The VDR has also been shown to bind non-traditional ligands including lithocholic acid [98] and curcumin [99], as well as to have actions which are not thought to be dependent upon ligand binding [100].

Vitamin D Receptor The biological effects of 1,25(OH)2D are thought to be mediated by a nuclear receptor, the VDR (reviewed in [101]). The VDR belongs to the subfamily of nuclear hormone receptors, which also includes the retinoic acid receptors, retinoid X receptors and thyroid hormone receptors. Unlike these other members of the subfamily, which each has more than one characterized isoform (a, b and g), only one nuclear VDR has been

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FIGURE 8.2

Evolutionary conservation of functional domains in the vitamin D receptor (VDR). A schematic representation of the VDR is shown with its principal domains: AF1 and AF2 activation domains, DNA binding domain (DBD), heterodimerization domain (HD) and hormone binding domain (HBD). The evolutionary conservation of the amino acid sequence of the various domains.

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. 8.2). These domains are highly evolutionarily conserved among vertebrates, with 75% identity between the human [102] and Xenopus [103] VDRs. The DNA binding domain is more highly conserved with greater than 90% conservation of amino acid sequence across vertebrate species [103] and 84% between the human and invertebrate lamprey VDR [104]. 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 24e90) are responsible for DNA binding as well as for nuclear localization and also have 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 co-activators (reviewed in [101]). The affinity of the VDR for 1,25(OH)2D is approximately three orders of magnitude higher than that for 25(OH)D. Hormone binding by the receptor results in phosphorylation, promotes heterodimerization, high affinity binding to DNA response elements and the recruitment of nuclear receptor co-activators. An amphipathic alpha-helical region at the C terminus (residues 416e422), referred to as the AF2 region, has been shown to be critical for transactivation and for recruitment of nuclear receptor co-activators. The transcriptional activity of the VDR is modulated by post-translational modifications as well as by association with nuclear receptor co-activators. Like other members of the nuclear receptor superfamily, the VDR undergoes rapid phosphorylation. 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

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fact, purified casein kinase II phosphorylates the human VDR at this residue [105]. Phosphorylation at serine 208 has been shown to be the principal site of hormonedependent VDR phosphorylation [106]. Although phosphorylation of this residue does not alter ligand binding [105], nuclear localization or binding to vitamin D response elements, it results in a dose-dependent enhancement of 1,25(OH)2D-mediated transactivation of reporter genes [107] due to enhanced interaction with the DRIP nuclear receptor co-activator complex [108]. In contrast, the rapid protein kinase A-mediated phosphorylation of the VDR is neither mediated by, nor dependent upon ligand. Protein kinase A-dependent phosphorylation of serine 182 impairs induction of target genes by impairing RXR heterodimerization [109]. Attenuation of ligand-mediated transactivation is also seen with protein kinase C-mediated VDR phosphorylation at serine 51 [110], correlating with impaired binding of the VDR to its DNA response elements [111]. The carboxy-terminal region of the vitamin D receptor interacts 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 proteineprotein interactions that ultimately leads to the modulation of gene expression by the VDR.

Nuclear Receptor Co-activators Nuclear receptor co-activators provide a critical link between ligand-activated nuclear receptors and the basal transcription machinery. The best characterized nuclear receptor co-activators belong to the SRC/p160 (steroid receptor coactivator) family [113,114]. The members of this family interact with nuclear receptors via an alpha-helical LXXLL motif that binds to an alpha helical region in the distal carboxy terminal region (416e422) of the VDR. It is postulated that ligand binding by the VDR results in a conformational change, exposing residues of the carboxy terminal to the DNA binding domain (224e246, 255e278 and 416e422) 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 (TGF) beta signaling pathway, and the liganded VDR, suggesting that nuclear co-activators play a key role in modulating the VDR-mediated transcriptional effects of growth factors [116]. SRC-1 knockout mice

exhibit abnormal response to several nuclear receptors but have not been shown to have impaired 1,25-dihydroxyvitamin D action in vivo. 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/p160 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 co-activator 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 [120]. However, sequences in helix 10 of the VDR which interact with RXR, also interact with NCoA62 and TFIIB [121]. A mulimeric complex, isolated based on its ability to interact with the hormone binding domain of the VDR, DRIP (D receptor interacting proteins), plays a crucial role in direct recruitment of the transcriptional machinery. This complex is capable of enhancing VDRmediated transactivation in a cell free system [122] and is essential for ligand-dependent transactivation [123]. 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 [124]. However, several components of the DRIP complex are also present in the activation complexes purified using non-nuclear receptor transactivators, such as Sp1, suggesting that they may be universally required for transcriptional activation. Studies in epidermal keratinocytes have demonstrated that different co-activatoreVDR interactions are found in proliferating versus differentiating keratinocytes [125]. Thus, the finding that DRIPeVDR complexes predominate in proliferative cells, whereas SRC-1 predominates in differentiating cells provides a paradigm for how a transcription factor can regulate different subsets of genes during differentiation of a single cell type. Co-repressors (NcoR and SMRT) also interact with nuclear hormone receptors and their actions are thought to be responsible for the transcription repressing activities of unliganded receptors for triiodothyronine and

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retinoic acid. These co-repressors stabilize chromatin by deacetylation of histones. In contrast, 1,25(OH)2D recruits SMRT and NCoR to the promoters of target genes. The ligand-bound VDR promotes binding of these co-repressors to RXR, whereas, the RXR ligand results in co-repressor release from the heterodimer [126]. Hairless, which was first characterized as a thyroid hormone receptor co-repressor, binds the VDR and represses VDR-mediated transcription in both the presence and absence of ligand [127]. While the in vivo consequences of this interaction have not been established, the absence of functional VDR or Hairless protein is associated with alopecia in humans and mice [128,129], suggesting that their cooperative effects are critical for maintenance of hair follicle homeostasis.

Vitamin D Response Elements and Target Genes 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(OH)2D [130,131]. These response elements have been identified in several genes, including the osteocalcin [132,133], osteopontin [134], aVb3 integrin [135,136] and vitamin D 24-hydroxylase (Cyp24a1) genes [137,138]. Vitamin D responsiveness has also been demonstrated for the calbindin-D9K and 28K genes. Although vitamin D response elements have been characterized in both of these genes [139,140], 1,25(OH)2D does not induce the expression of calbindin-D9K in all cells that express this transcript [141,142]. Interestingly, transgenic experiments have demonstrated that the 1,25(OH)2D responsiveness of the 50 regulatory region of calbindin-D9K is

not dependent upon the characterized vitamin D response element, but rather requires other DNA sequences, including the consensus binding motif for the homeodomain gene Cdx2 [143]. 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. In the case of the human c-fos promoter, nuclear factor 1 has been shown to be involved in the VDReRXR complex that interacts with a DNA sequence consisting of three direct repeats, separated by seven bases [144]. In an analogous fashion, perhaps binding of the VDR to “non-traditional” response elements requires stabilization of the DNA protein interactions by recruitment of other nuclear proteins. With the advent of chromatin immunoprecipitation and analyses of DNA sequences that bind the VDR in the context of intact chromatin, a complex regulatory network is being unveiled. Studies of the 24 hydroxylase (CYP24A1) gene, which is induced by 1,25(OH)2D, demonstrate that, in addition to the two VDREs located near the proximal promoter, the VDReRXR heterodimer also binds a cluster of potential enhancers in the intergenic region, 50e69 kilobase pairs downstream from the human gene and 35e45 kilobase pairs downstream from the mouse gene. These sequences contribute to transcriptional activation by 1,25(OH)2D as well as to co-regulator recruitment [145]. The VDR also suppresses the expression of several genes (Table 8.1). The VDR interacts with DNA sequences in the parathyroid hormone gene that are homologous to the motifs required for transcriptional induction by this receptor [56,146]. The mechanism by which these sequences lead to 1,25(OH)2D-mediated

TABLE 8.1 Activating and Repressing Vitamin D Response Elements (VDREs) Activating VDREs

RXR

VDR

Rat osteocalcin

GGGTGA

atg AGGACA

Human osteocalcin

GGGTGA

acg GGGGCA

Mouse osteopontin

AGGTTC

acg AGGTTC

Avian aVb3 Integrin

GAGGCA

gaa GGGAGA

Rat 24 hydroxylase (i)

AGGTGA

gtg AGGGCG

(ii)

GGTTCA

gcg GGTGCG

Repressing VDREs 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.

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transcriptional repression is thought to be due to the use of alternate heterodimerization partners [147] and to additional sequences which bind VDR interacting proteins [148]. The human parathyroid hormone-related peptide (PTHrP) negative VDRE also binds the VDR but not RXR. Transcriptional repression mediated by this negative VDRE is dependent on interactions of the VDR with the Ku antigen [149]. The sequences that are responsible for 1,25(OH)2D-mediated transcriptional repression of the vitamin D 1a-hydroxylase gene, bind a basic helix-loop helix transcription factor known as the VDR interacting repressor (VDIR) [62]. The VDIR binds this response element resulting in transactivation. The liganded VDReRXR heterodimer interacts with VDIR, leading to the dissociation of the histone acetyl transferase p300/CBP and recruitment of the histone deacetylase nuclear receptor co-repressor HDAC. This results in transcriptional repression of the vitamin D 1a-hydroxylase gene. Similar interactions of the VDIR with sequences in the PTH and PTHrP promoters have been implicated in the transcriptional repression of these genes by the liganded VDR [148]. In vitro data have suggested that the unliganded VDR can repress basal transcription and that the unliganded VDReRXR heterodimer causes VDR-mediated repression of basal transcription on classic vitamin D response elements [150]. The in vivo significance of these observations has not been established, but the existence of ligand-independent VDR actions are supported by the observation that mice and humans lacking functional VDRs develop alopecia [129], whereas absence of ligand does not result in this phenotype [151].

Chromatin and DNA Methylation In addition to binding histone acetyl transferases and deacetylases, the VDR interacts with chromatin remodeling complexes that rearrange nucleosomes in an energy-dependent fashion, rendering chromosomal DNA accessible to transcription factors. The VDR binds a multimeric complex with ATP-dependent chromatin remodeling activity known as WINAC (WSTF including nucleosome assembly complex). The complex derives its name, in part from the Williams syndrome transcription factor (WSTF), a protein that is mutated in a congenital disorder characterized by abnormal vitamin D metabolism, vascular lesions, developmental delay and atypical facies. The VDR binds directly to WSTF which, in turn, binds to ATP-dependent chromatin remodeling enzymes, including Brg1 and Brm [152]. In addition to promoting ligand-dependent transactivation by the VDR, the WINAC complex is also required for transrepression of the 1a-hydroxylase gene by the liganded VDR. This transrepression requires the bromodomain of WSTF that interacts with acetylated histones.

As previously mentioned, the VDR also promotes DNA methylation of CpG islands in the 1a-hydroxylase promoter, revealing another level at which the liganded VDR represses expression of this gene [43]. The upregulation of the 1a-hydroxylase by parathyroid hormone involves demethylation of the methylated CpG sites. The complex that associates with the VDR to regulate methylation involves two DNA methyl transferases as well as a DNA glycosylase, MBD4. This latter protein is phosphorylated by PKC in response to PTH stimulation, leading to incision of methylated DNA promoting a base-excision repair process that results in complete DNA demethylation. Thus, the VDR regulates transcription of target genes by direct binding to DNA response elements, recruiting nuclear receptor co-modulators, as well as by modifying the chromatin environment in association with the WINAC complex and regulating DNA methylation.

Non-classical Effects Several actions of 1,25(OH)2D occur too rapidly to be mediated by transcriptional mechanisms. These effects involve rapid activation of second messengers [153,154] and activation of voltage-dependent calcium channels [153e156]. As well, 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 24-hydroxylated metabolites on growth plate chondrocyte maturation [157,158]. Studies in mice lacking the VDR DNA binding and AF1 domains have demonstrated absence of rapid increases in intracellular calcium in response to 1,25dihydroxyvitamin D, demonstrating that this response requires an intact nuclear VDR [159]. ERp57/GRp58 is also known as the membrane-associated rapid response steroid binding (MARRS) protein that binds vitamin D metabolites. Its biological role in mediating effects of 1,25(OH)2D and other vitamin D metabolites will require investigations in mice lacking this protein [160].

ROLE OF VITAMIN D IN CALCIUM HOMEOSTASIS Physiological Actions of 1a,25-Dihydroxyvitamin D Animal models and studies in humans with dietary vitamin D deficiency have contributed significantly to our understanding of the role of 1,25(OH)2D in mineral ion homeostasis [161e164]. These studies have demonstrated that 1,25(OH)2D plays a critical role in intestinal calcium absorption. Vitamin D deficient animals develop hypocalcemia, hypophosphatemia, hyperparathyroidism, rickets and osteomalacia [161]. In vitro

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studies have demonstrated that 1,25(OH)2D has an antiproliferative effect on parathyroid cells [165]. Furthermore, 1,25(OH)2D has been shown both in vitro [166,167] and in vivo [58] 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 [168]. However, investigations in mice lacking functional 1ahydroxylase or VDR genes have demonstrated that normalizing mineral ion levels can prevent hyperparathyroidism, thus, in the setting of normocalcemia, the effects of 1,25(OH)2D on parathyroid function are redundant [169,170]. Numerous studies have examined the skeletal actions of 1,25(OH)2D. 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 of the hypertrophic chondrocyte layer [161]. 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 [171e174]. Murine studies directed at clarifying the cellular and molecular basis for the rachitic changes demonstrated that expansion of the growth plate was due to impaired hypertrophic chondrocyte apoptosis [175]. This impaired apoptosis was a result of hypophosphatemia, and was not prevented by suppression of PTH levels or hypercalcemia [176]. Osteoblasts express vitamin D receptors and 1,25(OH)2D plays a critical role in the regulation of several bone matrix proteins. 1,25(OH)2D has been shown to downregulate the transcription of the alpha1(I) collagen gene in osteoblasts [177] and is a potent transcriptional activator of the genes encoding osteocalcin and osteopontin [134]. However, experiments performed in vitamin D deficient rats have failed to show significant differences in the bone content of numerous matrix proteins, including osteocalcin [178]. Studies performed in vitamin D deficient rats [179] and in humans [173] with vitamin D receptor mutations have demonstrated that calcium and phosphorus repletion can mineralize osteomalacic lesions in the presence of impaired 1,25(OH)2D action, suggesting that this steroid hormone is not required for mineralization. Similarly, preventing hypocalcemia can prevent the osteomalacia observed in mice with 1a-hydroxylase and VDR mutations [169,172]. 1,25(OH)2D also stimulates the differentiation of osteoclasts from monocyteemacrophage stem cell precursors. However, monocyteemacrophage precursors isolated from vitamin D receptor null mice are capable of differentiating into multinucleated osteoclasts [180]. This is consistent with the effect of 1,25(OH)2D and

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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 which, in the presence of M-CSF, leads to their differentiation into mature osteoclasts. Because RANK ligand synthesis is also induced by PTH, in the absence of the genomic actions of 1,25(OH)2D, PTH-stimulated production of this factor is sufficient to sustain osteoclast differentiation. One of the most important in vivo roles of 1,25(OH)2D is to promote intestinal calcium absorption. Several proteins present in the brush border of the intestinal cell membrane, including the Ca/Mg ATPase, brush border alkaline phosphatase and intestinal membrane calcium binding protein, are induced by 1,25(OH)2D. In addition, two calcium channels, expressed in the intestine, TRPV5 and TRPV6 [181,182] have been shown to promote entry of calcium into the enterocyte. These transient receptor potential vanilloid receptor (TRPV) subfamily members are both induced by 1,25(OH)2D. However, TRPV5 is expressed primarily in the kidney and mice lacking TRPV5 exhibit renal calcium wasting with enhanced intestinal calcium absorption, due to a compensatory increase in 1,25(OH)2D [183]. 1,25(OH)2D is also a potent inducer of the gene encoding calbindin-D9K, 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-D9K levels are increased early and dramatically after injection of vitamin D metabolites and correlate with an increase in active transport of calcium across the intestine [184]. However, mice lacking both TRPV6, the predominant intestinal calcium channel, and calbindin-D9K exhibit enhancement of calcium transport in response to 1,25(OH)2D [185], suggesting that additional proteins or pathways are involved in 1,25(OH)2D regulated intestinal calcium absorption. Two proteins thought to form paracellular calcium channels, claudin 2 and claudin 12, are also induced by 1,25(OH)2D [186], suggesting that this steroid hormone regulates paracellular as well as transcellular calcium transport. Vitamin D has been shown to have effects not related to mineral ion homeostasis. Consistent with these observations, the VDR is expressed in several tissues including skin, breast, prostate, pancreas, colon, muscle and immune cells. 1,25(OH)2D induces the differentiation and attenuates the proliferation of hematopoietic tumor cells [187], breast cancer cells [188,189], prostate cancer cells [190] and keratinocytes [191e193]. While the biological implications of VDR expression in many of these non-traditional tissues is an active area of investigation, 1,25(OH)2D analogs have been used for the treatment of psoriasis [194] and clinical studies have

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demonstrated that vitamin D deficiency leads to impaired muscle function and an increase in falls [195]. The molecular basis for this latter effect of vitamin D metabolites has yet to be elucidated. Studies of the immunomodulatory effects of vitamin D metabolites have demonstrated that activation of Toll-like receptors (TLRs) induces the 1a-hydroxylase in cells of the macrophage lineage. Coupled with the increase in VDR expression which is also observed, this leads to a dramatic enhancement in local autocrine signaling by 1,25(OH)2D which, in turn, induces the synthesis of antimicrobial peptides that play a role in host defense to microbes, including mycobacteria [196]. In addition to demonstrating the importance of the VDR in immune function, this model system identifies a critical biological role for the local activation of vitamin D to 1,25(OH)2D in target tissues. Similarly, the molecular basis for the effects of 1,25(OH)2D on colon cancer have been extensively studied. Mutations in APC occur in colonic polyps in the early stages of carcinogenesis [197]. These mutations result in constitutive activation of the canonical Wnt signaling pathway that plays a critical role in subsequent neoplastic transformation. The liganded VDR attenuates this pathway by causing redistribution of b-catenin, a key transcriptional effector of this pathway, from the nucleus to the cell membrane, promoting the formation of adherens junctions and attenuating canonical Wnt signaling [198].

Animal Models Cyp27a1 Knockout Currently, there are no published reports of mice deficient for CYP2R1, the microsomal vitamin D-25-hydroxylase. A strain deficient for Cyp27a1, the bifunctional mitochondrial P450 able to hydroxylate vitamin D at position 25 [199,200] as well as 27-hydroxylate the side chains of cholesterol-derived intermediates involved in bile acid biosynthesis [10,15,16], has been described [11]. Mutations in the gene cause cerebrotendinous xanthomatosis (CTX), a lipid storage disorder leading to premature atherosclerosis and progressive neurological dysfunction [12,13]. Homozygous Cyp27a1/ animals have larger livers and larger adrenals [201], decreased synthesis and excretion of bile acids [11], and hypertriglyceridemia [201]. The increased formation of 25-hydroxylated bile alcohols and cholestarol observed in CTX patients was not observed in Cyp27a1/ mice, and no CTX-related pathological abnormalities were evident [11]. At present, it remains unclear whether the Cyp27a1-deficient mouse is a valid animal model for CTX. Interestingly, Cyp27a1-null mice had normal serum concentrations of 1,25(OH)2D and slightly

elevated levels of 25(OH)D in their blood [11]. These results support the hypothesis that CYP27A1 is not the sole vitamin D-25-hydroxylase and that CYP2R1 is physiologically relevant in vitamin D metabolism. DBP Knockout The bulk of 25(OH)D circulates bound to the carrier protein, vitamin D binding protein (DBP), also known as the group-specific component of serum (Gc-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 [202]. 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 [203]. The effect of the mutation was more evident when the mice were challenged by feeding them a vitamin D-deficient diet. Under those conditions, the DBP-deficient mice became hypocalcemic, hypophosphatemic, and 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 [203]. Thus, 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 [203]. Urinary excretion of 25(OH)D was higher in DBP-null mice [203]. DBP-deficient animals were less susceptible to the toxic effects of hypervitaminosis D, exhibiting reduced nephrocalcinosis and reduced hypercalcemia when compared with wild-type littermates [203]. 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 [204,205]. It binds numerous molecules including vitamin-binding proteins, hormones, lipoproteins, proteases, and protease inhibitors [204,205]. Megalin is expressed in the neuroepithelium, in proximal tubular cells of the kidney and in the parathyroids; mice deficient for megalin die soon after birth from a developmental defect of the forebrain [206]. The

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severity of the phenotype differs between animals, however, and one in 50 of the megalin/ mice make it to adulthood. Analysis of the phenotype of these survivors identified a key endocytic pathway involved in renal uptake and activation of 25(OH)D [207]. Analysis of the urine from surviving adult megalin/ mice revealed several excreted low-molecular-weight proteins that are normally reabsorbed in normal mice. Amino acid sequence analysis of the major protein band from knockout urine matched the sequence of DBP [207]. 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)D-DBP complexes [207]. Moreover, perfused rat kidney experiments showed that blocking megalin activity in the tubules with a specific antagonist prevented conversion of 25(OH)D into 1,25(OH)2D [207]. These results suggest that filtered and reabsorbed DBP-bound 25(OH)D serves as a substrate for the renal 1a-hydroxylase enzyme and that loss of megalin activity prevents delivery of the substrate to tubular cells. Since close to 90% of circulating 25(OH)D is bound to DBP [208], the massive excretion of DBP in the urine of megalin/ mice leads to vitamin D deficiency and bone defects in megalin-null mice [207]. Megalin requires co-receptors to achieve ligand specificity [209], and one such co-receptor is the plasmamembrane anchored protein cubilin [210]. It was shown that cubilin binds DBP [211]. Interestingly, dogs with an uncharacterized mutation affecting cubilin biosynthesis excrete DBP and 25(OH)D in the urine and show reduced levels of blood 25(OH)D and 1,25(OH)2D [211], supporting a role for cubilin in the delivery of DBP-bound 25(OH)D to tubular cells. A model for the role of cubilin and megalin in vitamin D homeostasis was proposed [207,211]. Cubilin binds the excreted 25(OH)D-DBP complexes on the cell surface of proximal tubular kidney cells. This facilitates the endocytotic process by sequestering the vitamin Dcarrier complex before internalization via megalin. Delivery of the endocytosed 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. Cyp24a1 Knockout The CYP24A1 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 [74, 75]. The role of the CYP24A1 enzyme in the catabolism of 1,25(OH)2D had already been

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demonstrated in tissue culture [87]. The Cyp24a1 gene has been inactivated in mice to examine the role of the CYP24A1 enzyme in vitamin D homeostasis in vivo [88]. Fifty percent of Cyp24a1/ mice die before 3 weeks of age [88,212]. 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 Cyp24a1 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 [88]. Chronic 1,25(OH)2D administration in Cyp24a1/ 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 [88]. Since half of the mutant progeny appear unaffected by the Cyp24a1 deficiency, these animals most likely use alternate means of regulating vitamin D homeostasis. Clearance and metabolism of labeled 1,25(OH)2D was measured in Cyp24a1/ survivors and heterozygote controls. These experiments have shown that Cyp24a1-null mice have impaired clearance of 1,25(OH)2D [213,214]. Surprisingly, Cyp24a1/ mice appear to lack not only 24-hydroxylated metabolites but also 1,25(OH)2D-26,23-lactone, supporting the view that the CYP24A1 enzyme is also responsible for hydroxylating 1,25(OH)2D at C-23 and C-26 [86,215]. These surprising findings suggest that the Cyp24a1null survivors adapt to the impaired vitamin D catabolism not by using an alternative catabolic route, but by limiting the synthesis of the active compound. Indeed, reduced expression of the 1a-hydroxylase gene, Cyp27b1, was measured in Cyp24a1/ mice [214]. The survival of some Cyp24a1/ 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 of homozygous females [88,216]. Histological examination of the bones from these animals revealed an accumulation of osteoid at sites of intramembranous ossification [88,216]. 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 Cyp24a1-deficient embryos from Cyp24a1 mutant dams: (1) perturbation of 1,25(OH)2D catabolism through the inactivation of the

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C-24 oxidation pathway affects bone development; (2) metabolites of vitamin D hydroxylated at position 24 are essential for bone development. To differentiate between these possibilities, the Cyp24a1-deficient animals were bred to mice carrying an inactivating mutation of the vitamin D receptor gene [217]. 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 Cyp24a1 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 Cyp24a1-deficient animals: double mutant homozygotes (Cyp24a1/ and VDR/) showed normal intramembranous bone formation at all sites examined [88]. This demonstrates that elevated 1,25(OH)2D levels during gestation affect mineralization and suggest that impaired vitamin D metabolism during development perturbs bone formation. 1a-hydroxylase (Cyp27B1) Knockout The 1a-hydroxylase enzyme (CYP27B1) converts 25(OH)D to 1,25(OH)2D, the biologically active metabolite of vitamin D. Mutations in the CYP27B1 gene cause pseudo vitamin D deficiency rickets (PDDR), also named vitamin D dependent rickets type I, a rare autosomal disease characterized by growth retardation, failure to thrive, rickets and osteomalacia [218,219] (see also Chapter 25). Biochemical analysis of serum from affected patients reveals hypocalcemia, secondary hyperparathyroidism, and undetectable levels of 1,25(OH)2D3. An animal model of PDDR was generated by targeted inactivation of the 1a-hydroxylase gene in mice [32]. The strategy, based on the Cre/lox methodology, allows tissue-specific inactivation of the targeted gene [220]. 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)2D in various cell types such as osteoblasts, chondrocytes, macrophages, or keratinocytes. Exon 8 of the 1a-hydroxylase gene, encoding the heme binding domain [25], was flanked by loxP recognition sites. Homologous recombination at the 1a-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 [32]. Homozygous mutant animals were phenotypically normal at birth but exhibited retarded growth as measured by weight gain from 3 to 8 weeks of age (not shown) and femur length at 8 weeks (Fig. 8.3). Serum analysis of homozygous mutant animals confirmed that they were hypocalcemic (Fig. 8.4),

FIGURE 8.3 Reduced growth of 25-hydroxyvitamin D-1ahydroxylase (Cyp27b1; 1a-hydroxylase) mouse mutants. Animals from all genotypes (þ/þ, þ/, /) and þ/ 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 1a-hydroxylase/ animals (/) exhibited reduced growth rates and had significantly shorter femurs at 8 weeks when compared to heterozygote (þ/) littermate controls, *** P<0.001.

FIGURE 8.4 Serum concentrations of total calcium in 25-hydroxyvitamin D-1a-hydroxylase (Cyp27b1; 1a-hydroxylase) 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 (þ/þ), heterozygotes (þ/), and homozygote mutant (/) animals using an automated analyzer. The horizontal bars indicate the mean for each population. ** P<0.01 by analysis of variance (ANOVA). The mean value for / mice is at the lower end of the normal range for this strain.

hypophosphatemic, suffered from secondary hyperparathyroidism and exhibited undetectable circulating levels of 1,25(OH)2D3 [32]. From a biochemical standpoint, the only difference between the 1a-hydroxylase/ mice and patients with PDDR is that patients with the disease have normal serum levels of 25(OH)D [221e223] and 24,25(OH)2D [224], whereas elevated levels of 25(OH)D and very low levels of 24,25(OH)2D

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are seen in 1a-hydroxylase mutant mice [32]. These discrepancies with the human disease remain to be explained but could result from species differences. Histological analysis of the bones from 3 week-old mutant animals confirmed the evidence of rickets [32]. At the age of 8 weeks, femurs from 1a-OHase-ablated mice present a severe disorganization in the architecture of the growth plate and marked osteomalacia [32]. The x-ray features of the bones from 1a-hydroxylase/ animals also matched the clinical manifestations of PDDR. Contact radiography of femurs from mutant animals revealed diffuse osteopenia (hypomineralization) and rachitic metaphyseal changes [225]. Interestingly, the expression of vitamin D-dependent genes such as osteopontin and osteocalcin was not changed in bone tissue from 1a-hydroxylase mutant mice [32]. The treatment of choice for PDDR is long-term replacement therapy with 1,25(OH)2D3 [219,221,226]. Treatment of 1a-hydroxylase-deficient mice with 1,25(OH)2D for 5 weeks corrects the hypocalcemia and secondary hyperparathyroidism. Bone histology and histomorphometry confirmed that the rickets and osteomalacia were cured. The biomechanical properties of the bone tissue (load bearing, deformation, and stiffness) were also normalized by the rescue treatment [227]. Rescuing mineral homeostasis in 1a-hydroxylasedeficient mice by feeding of a high calcium, high lactose diet corrected all aspects of the phenotype, except long bone growth [169]. Since the 1a-hydroxylase enzyme that synthesizes 1,25(OH)2D is expressed in chondrocytes, it was suggested that local production of 1,25(OH)2D could play an autocrine or paracrine role in the differentiation of these cells and explain the partial rescue of the phenotype when mineral homeostasis is corrected. To test this hypothesis, mutant mice that do not express the 1a-hydroxylase gene in chondrocytes were generated. This led to increased width of the hypertrophic zone of the growth plate at E15.5, increased bone mass in neonatal long bones, and increased expression of the chondrocytic differentiation markers Indian Hedgehog and PTH/PTHrP receptor. VEGF mRNA levels were decreased, accompanied by decreased PECAM-1 immunostaining, suggesting a delay in vascularization [228]. These results agree with the phenotype observed in chondrocyte-specific VDR-ablated mice [229] and support an autocrine/paracrine role of 1,25(OH)2D in endochondral ossification and chondrocyte development in vivo. VDR Knockout Although studies in vitamin D deficient animals and in vitro studies using cell culture models clarified many actions of vitamin D metabolites, there remained several unanswered questions concerning the principal actions of the VDR in vivo. To clarify further the role of the

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VDR in vivo, mice with targeted ablation of the vitamin D receptor were generated. As anticipated, these mice demonstrate several of the phenotypic abnormalities present in humans with vitamin D receptor mutations [217,230]. The pups are phenotypically indistinguishable from their wild-type and heterozygous littermates at birth. At the end of the third week of life, the mice develop hyperparathyroidism, presumably secondary to impaired intestinal calcium absorption [231]. 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 demonstrate that intestinal calcium absorption is 1,25(OH)2D-independent the first 3 weeks of life [232]. Thereafter, it gradually becomes hormone dependent and, by 7 weeks of life, a 1,25(OH)2D-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 [170]. By 70 days of age, the circulating immunoreactive PTH levels in the VDR null mice are 16-fold elevated 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 [159,170]. Thus, in the presence of normocalcemia, the genomic actions of 1,25(OH)2D 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, rather than lack of hormone, is the pathophysiological basis for hyperparathyroidism when 1,25(OH)2D action is impaired. Analogous to humans with VDR mutations and to conditions associated with 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 suggested that these rachitic changes were a direct consequence of hypocalcemia, hypophosphatemia or secondary hyperparathyroidism, rather than to impaired genomic actions of 1,25(OH)2D. Confirming this hypothesis is the observation that prevention of

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impaired mineral ion homeostasis prevents the growth plate abnormalities in the VDR null mice [170]. Although chondrocyte proliferation and differentiation was not altered in the VDR null mice, there was a marked impairment of hypertrophic chondrocyte apoptosis [175]. Investigations in additional mouse models, including the murine model of X-linked hypophosphatemia, demonstrated that hypophosphatemia led to impaired apoptosis, regardless of circulating calcium and PTH levels [176,233]. Studies in cellular models and in growing mice demonstrated that phosphate-induction of chondrocyte apoptosis is differentiation-stage specific and dependent upon Erk1/2 phosphorylation [234]. Impaired mineralization of newly synthesized osteoid (osteomalacia) is a prominent feature in the VDR null mice [172]. By 70 days of age, there is a 30fold increase in osteoid volume which leads to a fivefold increase in bone volume. In spite of this increase in bone volume, biomechanical analyses demonstrate a marked decrease in the strength of the bones in the VDR null mice, consistent with the increased fracture risk of patients with osteomalacia [235e237]. Analysis of the cellular content of the bones of the VDR null mice demonstrates a twofold increase in osteoblast number, presumably due to acceleration of bone turnover in response to profound secondary hyperparathyroidism. Paradoxically, the number of osteoclasts is not significantly altered, despite the elevation of immunoreactive PTH levels. In vitro studies demonstrated that the absence of the VDR in osteoclast precursors does not impair differentiation of these cells into mature osteoclasts. However, absence of the VDR in marrow stromal cells (osteoblast precursors), leads to impaired osteoclastogenesis in response to 1,25(OH)2D, but not to PTH [180]. These in vitro data suggest that there should be a compensatory increase in osteoclast number in response to the secondary hyperparathyroidism observed in the VDR null mice. Two additional variables may explain the inappropriately normal osteoclast number observed in these mice. First, the osteoclast avb3 integrin gene is highly induced by 1,25(OH)2D [135,136], thus cell attachment may be impaired; and secondly, osteoclasts are unable to resorb osteoid. Prevention of abnormal mineral ion homeostasis prevents the development of osteomalacia [172]. The increased bone volume observed 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 increase in bone formation and 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 [172]. The

biomechanical properties of the bones of the normocalcemic VDR null mice are indistinguishable from those of control littermates, as is the cellular content of the bone [172]. These data demonstrate that the genomic actions of the VDR are not essential 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 in vivo targets of the liganded VDR. The VDR null mice exhibit a dramatic impairment in intestinal calcium absorption, associated with a reduction in the mRNA levels encoding two calcium channels, TRPV5 and TRPV6, the latter of which is thought to play a critical role in the regulation of calcium entry into the enterocyte [231]. Levels of calbindin-D9K mRNA were also dramatically reduced in the duodenum of the VDR null mice [170,231], however, the level of the plasma membrane ATPase was not affected by VDR status [231]. These data suggest that expression of the channels that regulate calcium entry into the enterocyte and that of calbindin-D9K, 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. However, mice lacking TRPV5, the major renal calcium transporter, or lacking both TRPV6 and calbindinD9K exhibit 1,25(OH)2D-dependent intestinal calcium absorption [183,185]. Thus, 1,25(OH)2D-dependent calcium absorption involves other modulators. Notable in this respect is the induction of claudin 2 and claudin 12 by 1,25(OH)2D. These proteins are thought to form paracellular calcium channels, thereby providing an alternative to transcellular calcium absorption and do not require the expression of membrane calcium channels for their action [186]. 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 [151,238], however, there have been no cases of vitamin D deficiency reported with undetectable circulating levels of 1,25(OH)2D. Both mice and humans with VDR mutations have normal hair neonatally, suggesting that hair follicle morphogenesis does not require a functional VDR. However, clinically, the VDR null mice begin to develop alopecia by the fourth week of life [217,230]. This process is not prevented by normalization of mineral ion levels, suggesting that it is truly VDR dependent [170]. Because 1,25(OH)2D has been shown to play an important role in inhibiting proliferation and promoting differentiation of neonatal keratinocytes [191], studies were undertaken to determine whether absence of the VDR led to increased keratinocyte proliferation and impaired differentiation. Studies using neonatal keratinocytes isolated from VDR null mice demonstrated that the proliferation rate and the

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acquisition of selected markers of keratinocyte differentiation were indistinguishable from those of keratinocytes isolated from control littermates [239]. However, in vivo studies demonstrated impaired keratinocyte differentiation in the VDR null mice, a phenotype that was reversed by maintenance of normal calcium levels [240]. Thus, similar to the parathyroid gland, the effects of calcium and 1,25(OH)2D on keratinocyte proliferation and differentiation are redundant. Hair follicle morphogenesis in mice begins at embryonic day 14.5 and ends day 14 of life, the end of the first hair cycle. While there is no apparent defect in hair follicle morphogenesis, the VDR null mice are unable to initiate post-morphogenic hair cycles [239]. Hair reconstitution assays [151] and studies in VDR knockout mice expressing a VDR transgene in keratinocytes demonstrated that the presence of the VDR in the keratinocyte compartment of the hair follicle is both necessary and sufficient to restore post-morphogenic hair cycling. This was somewhat at odds with the apparently redundant effects of calcium and the VDR on keratinocyte proliferation and differentiation, since maintaining normal calcium levels cannot restore post-morphogenic hair cycles [170]. This suggested there was a unique population of keratinocytes in which normocalcemia cannot compensate for the lack of VDR. The bulge region of the hair follicle, which forms below the sebaceous gland at the end of the morphogenic period (Fig. 8.5), contains keratinocyte stem cells that give rise to the hair follicle, the sebaceous glands and play a role in epidermal regeneration postinjury. While this stem cell niche forms normally in

FIGURE 8.5 Schematic representation of the hair follicle. Keratinocyte stem cells reside in the bulge of the hair follicle. These cells give rise to the sebaceous gland and the keratinocytes in the lower part of the hair follicle. They also contribute to epidermal regeneration during wound repair. (Figure reproduced from Vitamin D. Pike JW, Adams JS and Feldman D, eds.)

177

VDR null mice, the number of keratinocyte stem cells decreased with age [241,242]. However, at a time when keratinocyte stem cell number is normal, these cells are unable to regenerate a hair follicle. This observation, combined with the expanded sebocyte compartment observed in VDR null mice, and lipid laden dermal cysts suggests a defect in lineage progression of these keratinocyte stem cells [241]. The observation that wild-type mice with undetectable circulating levels of 25(OH)D and 1,25(OH)2D do not develop alopecia [151] suggested that the actions of the VDR required to maintain post-morphogenic hair cycles are ligand independent. This was confirmed by studies in VDR knockout mice with keratinocytespecific expression of a VDR transgene containing a mutation that abolishes ligand binding and liganddependent transactivation [100]. Mice with keratinocyte specific ablation of RXRa also demonstrate impairment of post-morphogenetic hair cycling, suggesting that a functional VDReRXR heterodimer is required for normal skin homeostasis. The keratinocyte ablated RXRa mice have a more profound skin phenotype than the VDR null mice, including keratinocyte hyperproliferation and impaired keratinocyte differentiation [243], suggesting that RXRa interacts with alternative nuclear receptors to mediate these latter homeostatic effects. Humans and mice lacking the nuclear co-repressor Hairless also develop alopecia [128]. Hairless mice develop lipid-laden dermal cysts, analogous to those observed in the VDR null mice. Hairless binds the VDR and represses its activity in both the presence and absence of ligand [127]. Hairless also suppresses the expression of Soggy and WISE [244], two inhibitors of the canonical Wnt signaling pathway, a pathway that is essential for post-morphogenic hair cycling [245] and which is dependent upon the VDR for activation of a reporter gene in keratinocytes [241]. Thus, maintenance of keratinocyte stem cell function by Hairless and the VDR may involve modulation of the canonical Wnt signaling pathway. The immune system has also been the focus of numerous investigations in the VDR null mice. The VDR is expressed in T lymphocytes, dendritic cells and macrophages. The 1a-hydroxylase expressed in cells of the immune system is identical to the renal enzyme, however, it is induced by cytokines, including interferon (IFN) g and by activation of Toll-like receptors (TLRs), rather than by PTH, growth hormone (GH) and hypophosphatemia. In addition, 1,25(OH)2D does not exert negative feedback regulation in these cells, thus local hormone production in cells of the immune system may contribute to circulating levels of 1,25(OH)2D, resulting in the hypercalcemia seen with granulomatous disease and some hematologic malignancies.

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1,25(OH)2D has been shown to prevent the development of autoimmune diseases [246] and delay graft rejection in experimental models. Furthermore, 1,25(OH)2D regulates the expression of several key genes involved in immunomodulation, including interleukin-1, interleukin-12, IFNg and granulocyte macrophage colonystimulating factor (GM-CSF). Vitamin D deficient animals exhibit defects in macrophage and neutrophil chemotaxis as well as impaired cell-mediated immunity. However, the contribution of impaired genomic actions of 1,25(OH)2D to the immune defects in vitamin D deficiency is not clear. Specific studies of dendritic cell function in VDR null mice demonstrate an increase in mature dendritic cells, consistent with the known effect of 1,25(OH)2D on inhibition of dendritic cell maturation [247]. Interestingly, the increase in mature dendritic cells in the VDR knockout animals was seen only in lymph nodes, not in the spleen. Other studies in the VDR null mice demonstrated normal leukocyte and lymphocyte subset composition [248]. Characterization of splenocyte proliferation in response to numerous stimuli was notable only for a minor decrease in anti-CD3 stimulation. In spite of mildly impaired macrophage chemotaxis, phagocytosis and killing by these cells was normal as was in vivo graft rejection. These minor abnormalities in T cells and macrophages were not observed in cells isolated from normocalcemic VDR null mice, thus are not a direct reflection of impaired VDR action. 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 [248], thus demonstrating specific effects of hypocalcemia rather than impaired VDR activity. In contrast, normocalcemic VDR null mice were less susceptible to experimentally-induced autoimmune encephalomyelitis, the murine model of multiple sclerosis [249]. Studies in murine models demonstrated increased severity of inflammatory bowel disease in the absence of the VDR, associated with an increase in inflammatory and Th1-related cytokines [250]. In contrast, a decrease in experimentally-induced asthma and airway inflammation was observed, perhaps reflecting the absence of 1,25(OH)2D-mediated migration of immune cells [251]. These data suggest that many of 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 Metabolites 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 to date [252]; while most of them are catabolic 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 Cyp24a1 is expressed in growth plate chondrocytes and that cells from the growth plate respond to 24,25(OH)2D in a cell maturation-dependent manner [253]. Most of these studies were performed using the in vitro rat costochondral primary culture system. Dissection of the tissue allows culture of the 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 [254]. The more mature cells of the growth zone, comprising the prehypertrophic and hypertrophic compartments, respond primarily to 1,25(OH)2D [255]. Interestingly, treatment of resting zone chondrocytes with 24,25(OH)2D induces a change in maturation state [256], 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 non-genomic effects [257]. Contrasting with the tissue culture results is the observation that the growth plates from Cyp24a1/ mice do not show major defects [88,258]. These observations suggest that the absence of CYP24A1 activity does not affect growth plate development and that 24,25(OH)2D is not required for chondrocyte maturation in vivo [88]. It remains possible, however, that a redundant endocrine system is able to compensate for the function of 24,25(OH)2D in animals. It has also been proposed that 24,25(OH)2D might play a role in fracture repair, but there is limited information available on this putative function of the metabolite. The circulating levels of 24,25(OH)2D increase during fracture repair in chickens due to an increase in CYP24A1 activity [259]. When the effect of various vitamin D metabolites on the mechanical properties of healed bones was tested, treatment with 24,25(OH)2D3 was necessary to achieve maximal healing [260]. The Cyp24a1-deficient mouse strain [88] represents an invaluable tool to examine the putative role of 24,25(OH)2D in mammalian fracture repair. Fracture repair was compared between Cyp24a1/- mice and wild-type controls. A delay in the mineralization of the cartilaginous matrix of the soft callus was observed in Cyp24a1/ mutant animals [261]. The repair delay and the aberrant pattern of gene expression could be rescued by treatment with 24,25(OH)2D3 [261]. These results strongly support a role for 24,25(OH)2D in mammalian fracture repair.

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These studies could open up exciting perspectives for treatment following fracture or orthopedic surgery using 24-hydroxylated vitamin D metabolites or analogs. The CYP24 enzyme acts on 1,25(OH)2D to produce 1,24,25-trihydroxyvitamin D, the initial reactant in the C-24 oxidation pathway that leads to metabolite inactivation [74,75]. Treatment of ovariectomized rats with 1,24,25-trihydroxyvitamin D increased bone mass, serum calcium, and reduced bone resorption [262]. The second enzymatic step in the C-24 oxidation pathway is the oxidation of 1,24,25-trihydroxyvitamin D to 1,25(OH)224-oxo-D, a metabolite which showed activity in bone resorption assays and antiproliferation assays [263,264]. Hydroxylation of 1,25(OH)2D on carbon 26 eventually leads to formation of 1,25(OH)2D-26,23-lactone [265]. This metabolite affects intestinal calcium transport and can modulate bone resorption [266,267]. There have also been claims that 1,25(OH)2D-26,23-lactone could act as a VDR antagonist [268,269]. 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 carbon C-3 from the b to the a position. The enzyme responsible for this epimerization remains unknown. The 3-epimerization pathway is only detected in differentiated Caco-2 cells, but not in proliferating, undifferentiated cells [270]. This observation suggests that 3-epimerization is a tightly regulated metabolic pathway and supports the hypothesis that 3-epi1,25(OH)2D 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 [271], human keratinocytes [272], rat osteosarcoma cells [273], but not in perfused rat kidney or human promyelocytic leukemia cells [274]. The 3-epi1,25(OH)2D compound was also detected as a circulating metabolite in rats [275]. Biological activity associated with the 3-epi-1,25(OH)2D metabolite includes suppression of parathyroid hormone (PTH) secretion in bovine parathyroid cells [271] and induction of keratinocyte differentiation [276]. Despite lower affinity for the VDR [271,277], all the experimental evidence accumulated so far indicates that any bioactivity of the 3-epi1,25(OH)2D metabolite is mediated through the VDR and not a distinct receptor [278,279]. In summary, while some vitamin D metabolites have been shown to exhibit bioactivity, they are less active than 1,25(OH)2D and seem to act through the classical VDR, raising doubts as to their importance under physiological conditions. The identification of putative specific receptors [280,281] 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 dramatic progress in our understanding of vitamin D metabolism and function. For example, the engineering of VDR-deficient 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. The VDR-ablated mice also served as a useful tool to allow the cloning of the 1a-hydroxylase cDNA. The analysis of the negative VDRE from the 1a-hydroxylase gene characterized new mechanisms of vitamin D-controlled gene expression. Mice deficient in 1a-hydroxylase activity were engineered and represent a valid animal model for pseudo vitamin D deficiency rickets. The conditional ablation strategy used to engineer these animals has identified a role for locally synthesized 1,25(OH)2D3 in the neovascularization of the growth plate and will help elucidate the hypothesized autocrine/paracrine roles of 1,25(OH)2D in other target cells such as osteoblasts, keratinocytes, 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. 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 the non-nuclear 1,25(OH)2D receptor is involved in vitamin D biology, or whether other receptors with high affinity for alternative vitamin D metabolites play physiological roles in vitamin D action. It was recently shown that some forms of idiopathic infantile hypercalcemia (IIH), a genetic condition characterized by hypercalcemia and nephrocalcinosis, were due to mutations in the vitamin D inactivating enzyme, CYP24A1. (Schlingmann et al. 2011. New Engl J M 10.1056/NEJMoa1103864). The mutations occcur in key parts (C-or I-Helices) of the CYP24A1 protein which affect enzyme function.

References [1] McCollum EV, Simmonds N, Becker JE, Shipley PG. An experimental demonstration of the existence of a vitamin which promotes calcium deposition. J Biol Chem 1922;53:293e8. [2] Steenbock H, Black A. Fat-soluble vitamins. XVII. The induction of growth-promoting and calcifying properties in a ration by exposure to ultraviolet light. J Biol Chem 1924;61:405e22. [3] Holick MF. Photobiology of vitamin D. In: Feldman D, Glorieux FH, Pike JW, editors. Vitamin D. San Diego: Academic Press; 1997. p. 33e9.

PEDIATRIC BONE

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8. VITAMIN D BIOLOGY

[4] Fukushima M, Suzuki Y, Tohira Y, Nishii Y, Suzuki M. 25-Hydroxylation of 1alpha-hydroxyvitamin D3 in vivo and in perfused rat liver. FEBS Lett 1976;65:211e4. [5] Cheng JB, Motola DL, Mangelsdorf DJ, Russell DW. De-orphanization of cytochrome P450 2R1: a microsomal vitamin D 25-hydroxylase. J Biol Chem 2003;278:38084e93. [6] Strushkevich N, Usanov SA, Plotnikov AN, Jones G, Park HW. Structural analysis of CYP2R1 in complex with vitamin D3. J Mol Biol 2008;380:95e106. [7] Casella SJ, Reiner BJ, Chen TC, Holick MF, Harrison HE. A possible genetic defect in 25-hydroxylation as a cause of rickets. J Pediatr 1994;124:929e32. [8] Cheng JB, Levine MA, Bell NH, Mangelsdorf DJ, Russell DW. Genetic evidence that the human CYP2R1 enzyme is a key vitamin D 25-hydroxylase. Proc Natl Acad Sci USA 2004;101: 7711e5. [9] Fraser D, Kooh SW, Kind HP, Holick MF, Tanaka Y, DeLuca HF. Pathogenesis of hereditary vitamin-D-dependent rickets. An inborn error of vitamin D metabolism involving defective conversion of 25-hydroxyvitamin D to 1 alpha,25-dihydroxyvitamin D. N Engl J Med 1973;289:817e22. [10] Okuda KI, Usui E, Ohyama Y. Recent progress in enzymology and molecular biology of enzymes involved in vitamin D metabolism. J Lipid Res 1995;36:1641e52. [11] Rosen H, Reshef A, Maeda N, et al. Markedly reduced bile acid synthesis but maintained levels of cholesterol and vitamin D metabolites in mice with disrupted sterol 27-hydroxylase gene. J Biol Chem 1998;273:14805e12. [12] Salen G, Shefer S, Cheng FW, Dayal B, Batta AK, Tint GS. Cholic acid biosynthesis: the enzymatic defect in cerebrotendinous xanthomatosis. J Clin Invest 1979;63:38e44. [13] Cali JJ, Hsieh CL, Francke U, Russell DW. Mutations in the bile acid biosynthetic enzyme sterol 27-hydroxylase underlie cerebrotendinous xanthomatosis. J Biol Chem 1991;266:7779e83. [14] Kuriyama M, Fujiyama J, Kubota R, Nakagawa M, Osame M. Osteoporosis and increased bone fractures in cerebrotendinous xanthomatosis. Metabolism 1993;42:1497e8. [15] Cali JJ, Russell DW. Characterization of human sterol 27hydroxylase. A mitochondrial cytochrome P-450 that catalyzes multiple oxidation reactions in bile acid biosynthesis. J Biol Chem 1991;266:7774e8. [16] Andersson D, Davis DL, Dahlback H, Jornvall H. Cloning, structure and expression of the mitochondrial cytochrome P-450 sterol 26-hydroxylase, a bile acid biosynthetic enzyme. J Biol Chem 1989;264:8222e9. [17] Fu GK, Lin D, Zhang MY, et al. Cloning of human 25-hydroxyvitamin D-1 alpha-hydroxylase and mutations causing vitamin D-dependent rickets type 1. Mol Endocrinol 1997;11:1961e70. [18] St-Arnaud R, Messerlian S, Moir JM, Omdahl JL, Glorieux FH. The 25-hydroxyvitamin D 1-alpha-hydroxylase gene maps to the pseudovitamin D-deficiency rickets (PDDR) disease locus. J Bone Miner Res 1997;12:1552e9. [19] Shinki T, Shimada H, Wakino S, et al. Cloning and expression of rat 25-hydroxyvitamin D3-1alpha-hydroxylase cDNA. Proc Natl Acad Sci USA 1997;94:12920e5. [20] Takeyama K, Kitanaka S, Sato T, Kobori M, Yanagisawa J, Kato S. 25-Hydroxyvitamin D3 1alpha-hydroxylase and vitamin D synthesis. Science 1997;277:1827e30. [21] Hasemann CA, Kurumbail RG, Boddupalli SS, Peterson JA, Deisenhofer J. Structure and function of cytochromes P450: a comparative analysis of three crystal structures. Structure 1995;3:41e62. [22] Ohyama Y, Noshiro M, Okuda K. Cloning and expression of cDNA encoding 25-hydroxyvitamin D3 24-hydroxylase. FEBS Lett 1991;278:195e8.

[23] Chung BC, Matteson KJ, Voutilainen R, Mohandas TK, Miller WL. Human cholesterol side-chain cleavage enzyme, P450scc: cDNA cloning, assignment of the gene to chromosome 15, and expression in the placenta. Proc Natl Acad Sci USA 1986;83:8962e6. [24] Mornet E, Dupont J, Vitek A, White PC. Characterization of two genes encoding human steroid 11 beta-hydroxylase (P-450(11) beta). J Biol Chem 1989;264:20961e7. [25] Panda DK, Al Kawas S, Seldin MF, Hendy GN, Goltzman D. 25-hydroxyvitamin D 1alpha-hydroxylase: structure of the mouse gene, chromosomal assignment, and developmental expression. J Bone Miner Res 2001;16:46e56. [26] Fu GK, Portale AA, Miller WL. Complete structure of the human gene for the vitamin D 1alpha- hydroxylase, P450c1alpha. DNA Cell Biol 1997;16:1499e507. [27] Kitanaka S, Takeyama K, Murayama A, et al. Inactivating mutations in the 25-hydroxyvitamin D3 1alpha-hydroxylase gene in patients with pseudovitamin D-deficiency rickets. N Engl J Med 1998;338:653e61. [28] Yoshida T, Monkawa T, Tenenhouse HS, et al. Two novel 1alphahydroxylase mutations in French-Canadians with vitamin D dependency rickets type I. Kidney Int 1998;54:1437e43. [29] Labuda M, Morgan K, Glorieux FH. Mapping autosomal recessive vitamin D dependency type I to chromosome 12q14 by linkage analysis. Am J Hum Genet 1990;47:28e36. [30] Labuda M, Labuda D, Korab-Laskowska M, et al. Linkage disequilibrium analysis in young populations: pseudo-vitamin D- deficiency rickets and the founder effect in French Canadians. Am J Hum Genet 1996;59:633e43. [31] Portale AA, Miller WL. Human 25-hydroxyvitamin D-1alphahydroxylase: cloning, mutations, and gene expression. Pediatr Nephrol 2000;14:620e5. [32] Dardenne O, Prud’homme J, Arabian A, Glorieux FH, St-Arnaud R. Targeted inactivation of the 25-hydroxyvitamin D(3)-1(alpha)- hydroxylase gene (CYP27B1) creates an animal model of pseudovitamin D-deficiency rickets. Endocrinology 2001;142:3135e41. [33] Panda DK, Miao D, Tremblay ML, et al. Targeted ablation of the 25-hydroxyvitamin D 1alpha-hydroxylase enzyme: evidence for skeletal, reproductive, and immune dysfunction. Proc Natl Acad Sci USA 2001;98:7498e503. [34] Yamagata M, Kimoto A, Michigami T, Nakayama M, Ozono K. Hydroxylases involved in vitamin D metabolism are differentially expressed in murine embryonic kidney: application of whole mount in situ hybridization. Endocrinology 2001;142: 3223e30. [35] Puzas JE, Turner RT, Howard GA, Brand JS, Baylink DJ. Synthesis of 1,25-dihydroxycholecalciferol and 24,25-dihydroxycholecalciferol by calvarial cells. Characterization of the enzyme systems. Biochem J 1987;245:333e8. [36] Reichel H, Bishop JE, Koeffler HP, Norman AW. Evidence for 1,25-dihydroxyvitamin D3 production by cultured porcine alveolar macrophages. Mol Cell Endocrinol 1991;75:163e7. [37] Fraser DR, Kodicek E. Regulation of 25-hydroxycholecalciferol1-hydroxylase activity in kidney by parathyroid hormone. Nat New Biol 1973;241:163e6. [38] Murayama A, Takeyama K, Kitanaka S, Kodera Y, Hosoya T, Kato S. The promoter of the human 25-hydroxyvitamin D3 1 alpha-hydroxylase gene confers positive and negative responsiveness to PTH, calcitonin, and 1 alpha,25(OH)2D3. Biochem Biophys Res Commun 1998;249:11e6. [39] Brenza HL, Kimmel-Jehan C, Jehan F, et al. Parathyroid hormone activation of the 25-hydroxyvitamin D3-1alphahydroxylase gene promoter. Proc Natl Acad Sci USA 1998;95: 1387e91.

PEDIATRIC BONE

181

REFERENCES

[40] Horiuchi N, Suda T, Takahashi H, Shimazawa E, Ogata E. In vivo evidence for the intermediary role of 30 ,50 -cyclic AMP in parathyroid hormone-induced stimulation of 1alpha,25-dihydroxyvitamin D3 synthesis in rats. Endocrinology 1977;101:969e74. [41] Henry HL. Parathyroid hormone modulation of 25-hydroxyvitamin D3 metabolism by cultured chick kidney cells is mimicked and enhanced by forskolin. Endocrinology 1985;116: 503e10. [42] Gao XH, Dwivedi PP, Choe S, et al. Basal and parathyroid hormone induced expression of the human 25-hydroxyvitamin D 1alpha-hydroxylase gene promoter in kidney AOK-B50 cells: role of Sp1, Ets and CCAAT box protein binding sites. Int J Biochem Cell Biol 2002;34:921e30. [43] Kim MS, Kondo T, Takada I, et al. DNA demethylation in hormone-induced transcriptional derepression. Nature 2009;461: 1007e12. [44] Hendrich B, Hardeland U, Ng HH, Jiricny J, Bird A. The thymine glycosylase MBD4 can bind to the product of deamination at methylated CpG sites. Nature 1999;401:301e4. [45] Horiuchi N, Takahashi H, Matsumoto T, et al. Salmon calcitonininduced stimulation of 1 alpha,25-dihydroxycholecalciferol synthesis in rats involving a mechanism independent of adenosine 30 :50 -cyclic monophosphate. Biochem J 1979;184:269e75. [46] Galante L, Colston KW, MacAuley SJ, MacIntyre I. Effect of calcitonin on vitamin D metabolism. Nature 1972;238:271e3. [47] Shinki T, Ueno Y, DeLuca HF, Suda T. Calcitonin is a major regulator for the expression of renal 25-hydroxyvitamin D31alpha-hydroxylase gene in normocalcemic rats. Proc Natl Acad Sci USA 1999;96:8253e8. [48] Friedlander EJ, Henry HL, Norman AW. Studies on the mode of action of calciferol. Effects of dietary calcium and phosphorus on the relationship between the 25-hydroxyvitamin D3-1alphahydroxylase and production of chick intestinal calcium binding protein. J Biol Chem 1977;252:8677e83. [49] Heaney RP. Vitamin D: role in the calcium economy. In: Feldman D, Glorieux FH, Pike JW, editors. Vitamin D. SanDiego: Academic Press; 1997. p. 485e98. [50] Quarles LD. Endocrine functions of bone in mineral metabolism regulation. J Clin Invest 2008;118:3820e8. [51] Burnett SM, Gunawardene SC, Bringhurst FR, Juppner H, Lee H, Finkelstein JS. Regulation of C-terminal and intact FGF23 by dietary phosphate in men and women. J Bone Miner Res 2006;21:1187e96. [52] Bai XY, Miao D, Goltzman D, Karaplis AC. The autosomal dominant hypophosphatemic rickets R176Q mutation in fibroblast growth factor 23 resists proteolytic cleavage and enhances in vivo biological potency. J Biol Chem 2003;278:9843e9. [53] Bergwitz C, Juppner H. Regulation of phosphate homeostasis by PTH, vitamin D, and FGF23. Annu Rev Med 2010;61:91e104. [54] Henry HL. Regulation of the hydroxylation of 25-hydroxyvitamin D3 in vivo and in primary cultures of chick kidney cells. J Biol Chem 1979;254:2722e9. [55] Booth BE, Tsai HC, Morris Jr RC. Vitamin D status regulates 25hydroxyvitamin D3-1 alpha-hydroxylase and its responsiveness to parathyroid hormone in the chick. J Clin Invest 1985;75: 155e61. [56] Demay MB, Kiernan MS, DeLuca HF, Kronenberg HM. Sequences in the human parathyroid hormone gene that bind the 1,25-dihydroxyvitamin D3 receptor and mediate transcriptional repression in response to 1,25-dihydroxyvitamin D3. Proc Natl Acad Sci USA 1992;89:8097e101. [57] Murayama A, Takeyama K, Kitanaka S, et al. Positive and negative regulations of the renal 25-hydroxyvitamin D3 1alphahydroxylase gene by parathyroid hormone, calcitonin, and

[58]

[59]

[60]

[61]

[62]

[63]

[64]

[65]

[66]

[67]

[68] [69]

[70]

[71]

[72]

[73]

[74]

1alpha,25(OH)2D3 in intact animals. Endocrinology 1999;140: 2224e31. Silver J, Naveh-Many T, Mayer H, Schmelzer HJ, Popovtzer MM. Regulation by vitamin D metabolites of parathyroid hormone gene transcription in vivo in the rat. J Clin Invest 1986;78:1296e301. Kato S, Kim MS, Yamaoka K, Fujiki R. Mechanisms of transcriptional repression by 1,25(OH)2 vitamin D. Curr Opin Nephrol Hypertens 2007;16:297e304. Kim MS, Fujiki R, Kitagawa H, Kato S. 1alpha,25(OH)2D3induced DNA methylation suppresses the human CYP27B1 gene. Mol Cell Endocrinol 2007;265e266:168e73. Murayama A, Takeyama K, Asahina T, Kitanaka S, Kato S. Cloning of a novel transcription factor mediating the negative vitamin D responsiveness through the human 25-hydroxyvitamin D-1alpha-hydroxylase nVDRE. J Bone Miner Res 2000;15:S199. Murayama A, Kim MS, Yanagisawa J, Takeyama K, Kato S. Transrepression by a liganded nuclear receptor via a bHLH activator through co-regulator switching. Embo J 2004;23: 1598e608. Ohyama Y, Hayashi S, Okuda K. Purification of 25-hydroxyvitamin D3 24-hydroxylase from rat kidney mitochondria. FEBS Lett 1989;255:405e8. Ohyama Y, Okuda K. Isolation and characterization of a cytochrome P-450 from rat kidney mitochondria that catalyzes the 24-hydroxylation of 25-hydroxyvitamin D3. J Biol Chem 1991;266:8690e5. Chen KS, DeLuca HF. 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 1995;1263:1e9. Hahn CN, Kerry DM, Omdahl JL, May BK. Identification of a vitamin D responsive element in the promoter of the rat cytochrome P450(24) gene. Nucleic Acids Res 1994;22:2410e6. Ohyama Y, Noshiro M, Eggertsen G, et al. Structural characterization of the gene encoding rat 25-hydroxyvitamin D3 24-hydroxylase. Biochemistry 1993;32:76e82. Tanaka Y, Castillo L, DeLuca HF. The 24-hydroxylation of 1,25dihydroxyvitamin D3. J Biol Chem 1977;252:1421e4. Tanaka Y, DeLuca HF. Stimulation of 24,25-dihydroxyvitamin D3 production by 1,25-dihydroxyvitamin D3. Science 1974;183: 1198e200. Napoli JL, Horst RL. C(24)- and C(23)-oxidation, converging pathways of intestinal 1,25-dihydroxyvitamin D3 metabolism: identification of 24-keto-1,23,25-trihydroxyvitamin D3. Biochemistry 1983;22:5848e53. Ohnuma N, Norman AW. Identification of a new C-23 oxidation pathway of metabolism for 1,25-dihydroxyvitamin D3 present in intestine and kidney. J Biol Chem 1982;257:8261e71. Takasaki Y, Suda T, Yamada S, Takayama H, Nishii Y. Isolation, identification, and biological activity of 25-hydroxy-24-oxovitamin D3: a new metabolite of vitamin D3 generated by in vitro incubations with kidney homogenates. Biochemistry 1981;20:1681e6. Jones G, Kano K, Yamada S, Furusawa T, Takayama H, Suda T. Identification of 24,25,26,27-tetranor-23-hydroxyvitamin D3 as a product of the renal metabolism of 24,25-dihydroxyvitamin D3. Biochemistry 1984;23:3749e54. Makin G, Lohnes D, Byford V, Ray R, Jones G. Target cell metabolism of 1,25-dihydroxyvitamin D3 to calcitroic acid. Evidence for a pathway in kidney and bone involving 24oxidation. Biochem J 1989;262:173e80.

PEDIATRIC BONE

182

8. VITAMIN D BIOLOGY

[75] Reddy GS, Tserng KY. Calcitroic acid, end product of renal metabolism of 1,25-dihydroxyvitamin D3 through C-24 oxidation pathway. Biochemistry 1989;28:1763e9. [76] Esvelt RP, Schnoes HK, DeLuca HF. Isolation and characterization of 1 alpha-hydroxy-23-carboxytetranorvitamin D: a major metabolite of 1,25-dihydroxyvitamin D3. Biochemistry 1979;18: 3977e83. [77] Kumar R, Schaefer J, Grande JP, Roche PC. Immunolocalization of calcitriol receptor, 24-hydroxylase cytochrome P-450, and calbindin D28k in human kidney. Am J Physiol 1994;266: F477e85. [78] Kawashima H, Torikai S, Kurokawa K. Localization of 25hydroxyvitamin D3 1 alpha-hydroxylase and 24-hydroxylase along the rat nephron. Proc Natl Acad Sci USA 1981;78: 1199e203. [79] Akeno N, Saikatsu S, Kawane T, Horiuchi N. Mouse vitamin D24-hydroxylase: molecular cloning, tissue distribution, and transcriptional regulation by 1alpha,25-dihydroxyvitamin D3. Endocrinology 1997;138:2233e40. [80] Masuda S, Strugnell S, Calverley MJ, Makin HL, Kremer R, Jones G. In vitro metabolism of the anti-psoriatic vitamin D analog, calcipotriol, in two cultured human keratinocyte models. J Biol Chem 1994;269:4794e803. [81] Tomon M, Tenenhouse HS, Jones G. Expression of 25-hydroxyvitamin D3-24-hydroxylase activity in Caco-2 cells. An in vitro model of intestinal vitamin D catabolism. Endocrinology 1990;126:2868e75. [82] Reinhardt TA, Horst RL. Self-induction of 1,25-dihydroxyvitamin D3 metabolism limits receptor occupancy and target tissue responsiveness. J Biol Chem 1989;264:15917e21. [83] Sakaki T, Sawada N, Komai K, et al. Dual metabolic pathway of 25-hydroxyvitamin D3 catalyzed by human CYP24. Eur J Biochem/FEBS 2000;267:6158e65. [84] Prosser DE, Kaufmann M, O’Leary B, Byford V, Jones G. Single A326G mutation converts human CYP24A1 from 25-OH-D3-24hydroxylase into -23-hydroxylase, generating 1alpha,25-(OH) 2D3-26,23-lactone. Proc Natl Acad Sci USA 2007;104:12673e8. [85] Akiyoshi-Shibata M, Sakaki T, Ohyama Y, Noshiro M, Okuda K, Yabusaki Y. Further oxidation of hydroxycalcidiol by calcidiol 24-hydroxylase. A study with the mature enzyme expressed in Escherichia coli. Eur J Biochem/FEBS 1994;224:335e43. [86] Beckman MJ, Tadikonda P, Werner E, Prahl J, Yamada S, DeLuca HF. Human 25-hydroxyvitamin D3-24-hydroxylase, a multicatalytic enzyme. Biochemistry 1996;35:8465e72. [87] Reinhardt TA, Horst RL. Ketoconazole inhibits self-induced metabolism of 1,25-dihydroxyvitamin D3 and amplifies 1,25dihydroxyvitamin D3 receptor up-regulation in rat osteosarcoma cells. Arch Biochem Biophys 1989;272:459e65. [88] St-Arnaud R, Arabian A, Travers R, et al. Deficient mineralization of intramembranous bone in vitamin D-24- hydroxylaseablated mice is due to elevated 1,25-dihydroxyvitamin D and not to the absence of 24,25-dihydroxyvitamin D. Endocrinology 2000;141:2658e66. [89] Annalora AJ, Goodin DB, Hong WX, Zhang Q, Johnson EF, Stout CD. Crystal structure of CYP24A1, a mitochondrial cytochrome P450 involved in vitamin D metabolism. J Mol Biol 2010;396:441e51. [90] Engstrom GW, Reinhardt TA, Horst RL. 25-Hydroxyvitamin D323-hydroxylase, a renal enzyme in several animal species. Arch Biochem Biophys 1986;250:86e93. [91] Sakaki T, Sawada N, Nonaka Y, Ohyama Y, Inouye K. Metabolic studies using recombinant Escherichia coli cells producing rat mitochondrial CYP24. CYP24 can convert 1alpha,25-dihydroxyvitamin D3 to calcitroic acid. Eur J Biochem/FEBS 1999;262:43e8.

[92] Kahraman M, Sinishtaj S, Dolan PM, et al. Potent, selective and low-calcemic inhibitors of CYP24 hydroxylase: 24-sulfoximine analogues of the hormone 1alpha,25-dihydroxyvitamin D(3). J Med Chem 2004;47:6854e63. [93] Schuster I, Egger H, Nussbaumer P, Kroemer RT. Inhibitors of vitamin D hydroxylases: structureeactivity relationships. J Cell Biochem 2003;88:372e80. [94] Schuster I, Egger H, Reddy GS, Vorisek G. Combination of vitamin D metabolites with selective inhibitors of vitamin D metabolism. Recent Results Cancer Res 2003;164:169e88. [95] Schuster I, Egger H, Herzig G, et al. Selective inhibitors of vitamin D metabolism e new concepts and perspectives. Anticancer Res 2006;26:2653e68. [96] Annalora A, Bobrovnikova-Marjon E, Serda R, et al. Rat cytochrome P450C24 (CYP24A1) and the role of F249 in substrate binding and catalytic activity. Arch Biochem Biophys 2004; 425:133e46. [97] Omdahl JL, Swamy N, Serda R, Annalora A, Berne M, Rayb R. Affinity labeling of rat cytochrome P450C24 (CYP24) and identification of Ser57 as an active site residue. J Steroid Biochem Mol Biol 2004;89e90:159e62. [98] Makishima M, Lu TT, Xie W, et al. Vitamin D receptor as an intestinal bile acid sensor. Science 2002;296:1313e6. [99] Bartik L, Whitfield GK, Kaczmarska M, et al. Curcumin: a novel nutritionally derived ligand of the vitamin D receptor with implications for colon cancer chemoprevention. J Nutr Biochem 2010. [100] Skorija K, Cox M, Sisk JM, et al. Ligand-independent actions of the vitamin D receptor maintain hair follicle homeostasis. Mol Endocrinol 2005;19:855e62. [101] Haussler MR, Whitfield GK, Haussler CA, et al. The nuclear vitamin D receptor: biological and molecular regulatory properties revealed. J Bone Miner Res 1998;13:325e49. [102] Baker AR, McDonnell DP, Hughes M, et al. Cloning and expression of full-length cDNA encoding human vitamin D receptor. Proc Natl Acad Sci USA 1988;85:3294e8. [103] Li YC, Bergwitz C, Juppner H, Demay M. Cloning and characterization of the vitamin D receptor from Xenopus laevis. Endocrinology 1997;138:2347e53. [104] Whitfield GK, Dang HT, Schluter SF, et al. Cloning of a functional vitamin D receptor from the lamprey (Petromyzon marinus), an ancient vertebrate lacking a calcified skeleton and teeth. Endocrinology 2003;144:2704e16. [105] Jurutka PW, Hsieh JC, MacDonald PN, et al. Phosphorylation of serine 208 in the human vitamin D receptor. The predominant amino acid phosphorylated by casein kinase II, in vitro, and identification as a significant phosphorylation site in intact cells. J Biol Chem 1993;268:6791e9. [106] Hilliard GMT, Cook RG, Weigel NL, Pike JW. 1,25-dihydroxyvitamin D3 modulates phosphorylation of serine 205 in the human vitamin D receptor: site-directed mutagenesis of this residue promotes alternative phosphorylation. Biochemistry 1994;33:4300e11. [107] Jurutka PW, Hseih J-C, Nakajima S, Haussler CA, Haussler M. Human vitamin D receptor phosphorylation by casein kinase II at Ser-208 potentiates transcriptional activation. Proc Natl Acad Sci USA 1996;93:3519e24. [108] Arriagada G, Paredes R, Olate J, et al. Phosphorylation at serine 208 of the 1alpha,25-dihydroxy vitamin D3 receptor modulates the interaction with transcriptional coactivators. J Steroid Biochem Mol Biol 2007;103:4259. [109] Hsieh JC, Dang HT, Galligan MA, et al. Phosphorylation of human vitamin D receptor serine-182 by PKA suppresses 1,25(OH)2D3-dependent transactivation. Biochem Biophys Res Commun 2004;324:801e9.

PEDIATRIC BONE

REFERENCES

[110] Hsieh JC, Jurutka PW, Galligan MA, et al. Human vitamin D receptor is selectively phosphorylated by protein kinase C on serine 51, a residue crucial to its trans-activation function. Proc Natl Acad Sci USA 1991;88:9315e9. [111] Hsieh JC, Jurutka PW, Nakajima S, et al. Phosphorylation of the human vitamin D receptor by protein kinase C. Biochemical and functional evaluation of the serine 51 recognition site. J Biol Chem 1993;268:15118e26. [112] MacDonald PN, Sherman DR, Dowd DR, Jefcoat Jr SC, DeLisle RK. The vitamin D receptor interacts with general transcription factor IIB. J Biol Chem 1995;270:4748e52. [113] McKenna N, Xu J, Nawaz Z, Tsai S, Tsai M, O’Malley B. Nuclear receptor coactivators: multiple enzymes, multiple complexes, multiple functions. J Steroid Biochem Molec Biol 1999;69:3e12. [114] Onate S, Tsai S, Tsai M, O’Malley B. Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science 1995;270:1354e7. [115] Kraichely D, Collins J, DeLisle R, MacDonald PN. The autonomous transactivation domain in helix H3 of the vitamin D receptor is required for transactivation and coactivator interaction. J Biol Chem 1999;274:14352e8. [116] Yanagisawa J, Yanagi Y, Masuhiro Y, et al. Convergence of transforming growth factor beta and vitamin D signaling pathways on SMAD transcriptional coactivators. Science 1999; 283:1317e21. [117] Takeshita A, Yen P, Misiti S, Cardona G, Liu Y, Chin W. Molecular cloning and properties of a full-length putative thyroid hormone receptor coactivator. Endocrinology 1996; 137:3594e7. [118] Spencer T, Jenster G, Burcin M, et al. Steroid receptor coactivator-1 is a histone acetyltransferase. Nature 1997;389:194e8. [119] Bannister A, Kouzarides T. The CBP coactivator is a histone acetyl transferease. Nature 1996;384:641e3. [120] Baudino TA, Kraichely DM, Jefcoat Jr SC, et al. Isolation and characterization of a novel coactivator protein, NCoA-62, involved in vitamin D-mediated transcription. J Biol Chem 1998;273:16434e41. [121] Barry JB, Leong GM, Church WB, Issa LL, Eisman JA, Gardiner EM. Interactions of SKIP/NCoA-62, TFIIB, and retinoid X receptor with vitamin D receptor helix H10 residues. J Biol Chem 2003;278:8224e8. [122] Rachez C, Suldan Z, Ward J, et al. A novel protein complex that interacts with the vitamin D3 receptor in a ligand-dependent manner and enhances VDR transactivation in a cell-free system. Genes Dev 1998;12:1787e800. [123] Rachez C, Lemon B, Suldan Z, et al. Ligand-dependent transcription activation by nuclear receptors requires the DRIP complex. Nature 1999;398:824e8. [124] Chiba N, Suldan Z, Freedman LP, Parvin J. Binding of liganded vitamin D receptor to the vitamin D receptor interacting protein coactivator complex induces interaction with RNA polymerase II holoenzyme. J Biol Chem 2000;275:10719e22. [125] Oda Y, Ishikawa MH, Hawker NP, Yun QC, Bikle DD. Differential role of two VDR coactivators, DRIP205 and SRC-3, in keratinocyte proliferation and differentiation. J Steroid Biochem Mol Biol 2007;103:776e80. [126] Sanchez-Martinez R, Zambrano A, Castillo AI, Aranda A. Vitamin D-dependent recruitment of corepressors to vitamin D/ retinoid X receptor heterodimers. Mol Cell Biol 2008; 28:3817e29. [127] Hsieh JC, Sisk JM, Jurutka PW, et al. Physical and functional interaction between the vitamin D receptor and hairless corepressor, two proteins required for hair cycling. J Biol Chem 2003;278:38665e74.

183

[128] Ahmad W, Faiyaz ul Haque M, Brancolini V, et al. Alopecia universalis associated with a mutation in the human hairless gene. Science 1998;279:720e4. [129] Marx SJ, Bliziotes MM, Nanes M. Analysis of the relation between alopecia and resistance to 1,25-dihydroxyvitamin D. Clin Endocrinol 1986;25:373e81. [130] Demay MB, Kiernan MS, DeLuca HF, Kronenberg HM. Characterization of 1,25-dihydroxyvitamin D3 receptor interactions with target sequences in the rat osteocalcin gene. Mol Endocrinol 1992;6:557e62. [131] Umesono K, Murakami KK, Thompson CC, Evans RM. Direct repeats as selective response elements for the thyroid hormone, retinoic acid and vitamin D3 receptors. Cell 1991;65:1255e66. [132] Demay MB, Gerardi JM, DeLuca HF, Kronenberg HM. DNA sequences in the rat osteocalcin gene that bind the 1,25-dihydroxyvitamin D3 receptor and confer responsiveness to 1,25dihydroxyvitamin D3. Proc Natl Acad Sci USA 1990;87:369e73. [133] Ozono K, Liao J, Kerner SA, Scott RA, Pike JW. The vitamin Dresponsive element in the human osteocalcin gene: association with a nuclear proto-oncogene enhancer. J Biol Chem 1990; 265:21881e8. [134] Noda M, Vogel RL, Craig AM, Prahl J, DeLuca HF, Denhardt DT. Identification of a DNA sequence responsible for binding of the 1,25-dihydroxyvitamin D3 receptor and 1,25dihydroxyvitamin D3 enhancement of mouse secreted phosphoprotein 1 (Spp-1 or osteopontin) gene expression. Proc Natl Acad Sci USA 1990;87:9995e9. [135] Cao X, Ross FP, Zhang L, MacDonald PN, Chappel J, Teitelbaum SL. Cloning of the promoter for the avian integrin b3 subunit gene and its regulation by 1,25-dihydroxyvitamin D3. J Biol Chem 1993;268:27371e80. [136] Medhora MM, Teitelbaum S, Chappel J, Alvarez J, Mimura H, Ross FP, Hruska K. 1a,25-Dihydroxyvitamin D3 up-regulates expression of the osteoclast integrin avb3. J Biol Chem 1993; 268:1456e61. [137] Kerry DM, Dwivedi PP, Hahn CN, Morris HA, Omdahl JL, May BK. Transcriptional synergism between vitamin Dresponsive elements in the rat 25-hydroxyvitamin D3 24hydroxylase (CYP24) promoter. J Biol Chem 1996;271:29715e21. [138] Ohyama Y, Ozono K, Uchida M, et al. Identification of a vitamin D-responsive element in the 50 -flanking region of the rat 25hydroxyvitamin D3 24-hydroxylase gene. J Biol Chem 1994;269:10545e50. [139] Darwish HM, DeLuca HF. Identification of a 1,25-dihydroxyvitamin D3-response element in the 50 -flanking region of the rat calbindin D-9k gene. Proc Natl Acad Sci USA 1992;89:603e7. [140] Gill RK, Christakos S. Identification of sequence elements in mouse calbindin-D28k gene that confer 1,25-dihydroxyvitamin D3- and butyrate-inducible responses. Proc Natl Aca Sci USA 1993;90:2984e8. [141] Dupret JM, L’Horset F, Perret C, Bernaudin J-F, Thomasset M. Calbindin-D9K gene expression in the lung of the rat. Absence of regulation by 1,25-dihydroxyvitamin D3 and estrogen. Endocrinology 1992;131:2643e8. [142] Glazier JD, Mawer EB, Sibley CP. Calbindin-D9k gene expression in rat chorioallantoic placenta is not regulated by 1,25dihydroxyvitamin D3. Pediatr Res 1995;37:720e5. [143] Colnot S, Ovejero C, Romagnolo B, et al. Transgenic analysis of the response of the rat calbindin-D 9k gene to vitamin D. Endoocrinology 2000;141:2301e8. [144] Candeliere GA, Jurutka PW, Haussler MR, St-Arnaud R. A composite element binding the vitamin D receptor, retinoid X receptor alpha, and a member of the CTF/NF-1 family of transcription factors mediates the vitamin D responsiveness of the c-fos promoter. Mol Cell Biol 1996;16:584e92.

PEDIATRIC BONE

184

8. VITAMIN D BIOLOGY

[145] Meyer MB, Goetsch PD, Pike JW. A downstream intergenic cluster of regulatory enhancers contributes to the induction of CYP24A1 expression by 1alpha,25-dihydroxyvitamin D3. J Biol Chem 2010;285:15599e610. [146] Liu SM, Koszewski N, Lupez M, Malluche HH, Olivera A, Russell J. Characterization of a response element in the 50 -flanking region of the avian (chicken) PTH gene that mediates negative regulation of gene transcription by 1,25-dihydroxyvitamin D3 and binds the vitamin D3 receptor. Mol Endocrinol 1996;10:206e15. [147] Mackey SL, Heymont JL, Kronenberg HM, Demay MB. Vitamin D receptor binding to the negative human parathyroid hormone vitamin D response element does not require the retinoid x receptor. Mol Endocrinol 1996;10:298e305. [148] Kim MS, Fujiki R, Murayama A, et al. 1Alpha,25(OH)2D3induced transrepression by vitamin D receptor through E-boxtype elements in the human parathyroid hormone gene promoter. Mol Endocrinol 2007;21:334e42. [149] Nishishita T, Okazaki T, Ishikawa T, et al. A negative vitamin D response DNA element in the human parathyroid hormonerelated peptide gene binds to vitamin D receptor along with Ku antigen to mediate negative gene regulation by vitamin D. J Biol Chem 1998;273:10901e7. [150] Yen P, Liu Y, Sugawara A, Chin W. Vitamin D receptors repress basal transcription and exert dominant negative activity on triiodothyronine-mediated transcriptional activity. J Biol Chem 1996;271:10910e6. [151] Sakai Y, Kishimoto J, Demay MB. Metabolic and cellular analysis of alopecia in vitamin D receptor knockout mice. J Clin Invest 2001;107:961e6. [152] Kitagawa H, Fujiki R, Yoshimura K, et al. The chromatinremodeling complex WINAC targets a nuclear receptor to promoters and is impaired in Williams syndrome. Cell 2003; 113:905e17. [153] Barsony J, Marx S. Rapid accumulation of cyclic GMP near activated vitamin D receptors. Proc Natl Acad Sci USA 1991; 88:1436e40. [154] Civitelli R, Kim YS, Gunsten SL, et al. Nongenomic activation of the calcium message system by vitamin D metabolites in osteoblast-like cells. Endocrinology 1990;127:2253e62. [155] de Boland AR, Nemere I, Norman AW. Ca2þ-Channel agonist bay K8644 mimics 1,25(OH)2-vitamin D3 rapid enhancement of Ca2þ transport in chick perfused duodenum. Biochem Biophys Res Commun 1990;166:217e22. [156] Farach-Carson MC, Sergeev I, Norman AW. Nongenomic actions of 1,25-dihydroxyvitamin D3 in rat osteosarcoma cells: structure-function studies using ligand analogs. Endocrinology 1991;129:1876e84. [157] Schwartz Z, Brooks B, Swain L, Del Toro F, Norman A, Boyan B. 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 1992;130:2495e504. [158] Schwartz Z, Sylvia VL, Luna MH, et al. The effect of 24R,25(OH)(2)D(3) on protein kinase C activity in chondrocytes is mediated by phospholipase D whereas the effect of 1alpha,25(OH)(2)D(3) is mediated by phospholipase C. Steroids 2001;66:683e94. [159] Erben RG, Soegiarto DW, Weber K, et al. Deletion of deoxyribonucleic acid binding domain of the vitamin D receptor abrogates genomic and nongenomic functions of vitamin D. Mol Endocrinol 2002;16:1524e37. [160] Khanal RC, Nemere I. The ERp57/GRp58/1,25D3-MARRS receptor: multiple functional roles in diverse cell systems. Curr Med Chem 2007;14:1087e93.

[161] Halloran BP, DeLuca HF. Effect of vitamin D deficiency on skeletal development during early growth in the rat. Arch Biochem Biophys 1981;209:7e14. [162] Peacock M, Selby PL, Francis RM, Brown WB, Hordon L. Vitamin D deficiency, insufficiency, sufficiency, and intoxication: what do they mean? In: Norman AW, Schaefer K, Grigoleit H-G, von Herrath D, editors. Vitamin D: Chemical, Biochemical, and Clinical Update: Sixth Workshop on Vitamin D. New York: Walter de Gruyter; 1985. p. 569e70. [163] Pettifor MP, Daniels ED. Vitamin D Deficiency and Nutritional Rickets in Children. San Diego, CA: Academic Press; 1997. [164] Thomas MK, Demay MB. Vitamin D deficiency and disorders of vitamin D metabolism. Endocrinol Metab Clin North Am 2000;29:611e27. viii. [165] Ishimi Y, Russell J, Sherwood LM. Regulation by calcium and 1,25-(OH)2D3 of cell proliferation and function of bovine parathyroid cells in culture. J Bone Miner Res 1990;5:755e60. [166] Russell J, Lettieri D, Sherwood LM. Suppression by 1,25(OH)2D3 of transcription of the pre-proparathyroid hormone gene. Endocrinology 1986;119:2864e6. [167] Silver J, Russell J, Sherwood LM. Regulation by vitamin D metabolites of messenger ribonucleic acid for preproparathyroid hormone in isolated bovine parathyroid cells. Proc Natl Acad Sci USA 1985;82:4270e3. [168] Szabo A, Merke J, Beier E, Mall G, Ritz E. 1,25(OH)2 vitamin D3 inhibits parathyroid cell proliferation in experimental uremia. Kidney Int 1989;35:1049e56. [169] Dardenne O, Prud’homme J, Hacking SA, Glorieux FH, StArnaud R. Correction of the abnormal mineral ion homeostasis with a high-calcium, high-phosphorus, high-lactose diet rescues the PDDR phenotype of mice deficient for the 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1). Bone 2003;32: 332e40. [170] Li YC, Amling M, Pirro AE, et al. Normalization of mineral ion homeostasis by dietary means prevents hyperparathyroidism, rickets, and osteomalacia, but not alopecia in vitamin D receptor-ablated mice. Endocrinology 1998;139:4391e6. [171] Al-Aqeel A, Ozand P, Sobki S, Sewairi W, Marx S. The combined use of intravenous and oral calcium for the treatment of vitamin D dependent rickets type II (VDDRII). Clin Endocrinol 1993; 39:229e37. [172] Amling M, Priemel M, Holzmann T, et al. Rescue of the skeletal phenotype of vitamin D receptor-ablated mice in the setting of normal mineral ion homeostasis: formal histomorphometric and biomechanical analyses. Endocrinology 1999;140:4982e7. [173] Balsan S, Garabedian M, Larchet M, et al. Long-term nocturnal calcium infusions can cure rickets and promote normal mineralization in hereditary resistance to 1,25-dihydroxyvitamin D. J Clin Invest 1986;77:1661e7. [174] Weinstein RS, Underwood JL, Hutson MS, DeLuca HF. Bone histomorphometry in vitamin D-deficient rats infused with calcium and phosphorus. Am J Physiol 1984;246:E499e506. [175] Donohue MM, Demay MB. Rickets in VDR null mice is secondary to decreased apoptosis of hypertrophic chondrocytes. Endocrinology 2002;143:3691e4. [176] Sabbagh Y, Carpenter TO, Demay MB. Hypophosphatemia leads to rickets by impairing caspase-mediated apoptosis of hypertrophic chondrocytes. Proc Natl Acad Sci USA 2005; 102:9637e42. [177] Harrison JR, Petersen DN, Lichtler AC, Mador AT, Rowe DW, Kream BE. 1,25-dihydroxyvitamin D3 inhibits transcription of type I collagen genes in the rat osteosarcoma line ROS 17/2.8. Endocrinology 1989;125:327e33.

PEDIATRIC BONE

185

REFERENCES

[178] Wientroub S, Fisher L, Reddi A, Termine J. Noncollagenous bone proteins in experimental rickets in the rat. Mol Cell Biochem 1987;74:157e62. [179] Underwood JL, DeLuca HF. Vitamin D is not directly necessary for bone growth and mineralization. Am J Physiol 1984; 246:E493e8. [180] Takeda S, Yoshizawa T, Nagai Y, et al. Stimulation of osteoclast formation by 1,25-dihydroxyvitamin D requires its binding to vitamin D receptor (VDR) in osteoblastic cells: studies using VDR knockout mice. Endocrinology 1999;140:1005e8. [181] Hoenderop J, VanDerKemp A, Hartog A, et al. Molecular identification of the apical Ca2þ channel in 1,25-dihydroxyvitamin D-responsive epithelia. J Biol Chem 1999;274:8375e8. [182] Peng J, Chen X, Berger U, et al. Molecular cloning and characterization of a channel-like transporter mediating intestinal calcium absorption. J Biol Chem 1999;274:22739e46. [183] Hoenderop JG, van Leeuwen JP, van der Eerden BC, et al. Renal Ca2þ wasting, hyperabsorption, and reduced bone thickness in mice lacking TRPV5. J Clin Invest 2003;112:1906e14. [184] Wasserman RH, Fullmer CS. Calcium transport proteins, calcium absorption, and vitamin D. Ann Rev Physiol 1983; 45:375e90. [185] Benn BS, Ajibade D, Porta A, et al. Active intestinal calcium transport in the absence of transient receptor potential vanilloid type 6 and calbindin-D9k. Endocrinology 2008;149:3196e205. [186] Fujita H, Sugimoto K, Inatomi S, et al. Tight junction proteins claudin-2 and -12 are critical for vitamin D-dependent Ca2þ absorption between enterocytes. Mol Biol Cell 2008;19:1912e21. [187] Manolagas SC, Yu X-P, Girasole G, Bellido T. Vitamin D and the hematolymphopoietic tissue: a 1994 update. Semin Nephrol 1994;14:129e43. [188] Colston KW, Mackay AG, James SY, Binderup L, Chanders S, Coombes RC. EB 1089: a new vitamin D analogue that inhibits the growth of breast cancer cells in vivo and in vitro. Biochem Pharmacol 1992;44:2273e80. [189] Colston KW, Perks CM, Xie SP, Holly JM. 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 1998;20:157e62. [190] Feldman D, Skowronski RJ, Peehl DM. Vitamin D and prostate cancer. Adv Exp Med Biol 1996;375:53e63. [191] Bikle DD, Pillai S. Vitamin D, calcium, and epidermal differentiation. Endocr Rev 1993;14:3e19. [192] Sorensen S, Solvsten H, Politi Y, Kragballe K. Effects of vitamin D3 on keratinocyte proliferation and differentiation in vitro: modulation by ligands for retinoic acid and retinoid X receptors. Skin Pharmacol 1997;10:144e52. [193] Su MJ, Bikle DD, Mancianti ML, Pillai S. 1,25-Dihydroxyvitamin D3 potentiates the keratinocyte response to calcium. J Biol Chem 1994;269:14723e9. [194] Holick MF, Smith E, Pincus S. Skin as the site of vitamin D synthesis and target tissue for 1,25-dihydroxyvitamin D3. Use of calcitriol (1,25-dihydroxyvitamin D3) for treatment of psoriasis. Arch Dermatol 1987;123:1677e83. [195] Bischoff-Ferrari HA. Optimal serum 25-hydroxyvitamin D levels for multiple health outcomes. Adv Exp Med Biol 2008; 624:55e71. [196] Liu PT, Stenger S, Li H, et al. Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response. Science 2006;311:1770e3. [197] Powell SM, Zilz N, Beazer-Barclay Y, et al. APC mutations occur early during colorectal tumorigenesis. Nature 1992;359:235e7. [198] Palmer HG, Sanchez-Carbayo M, Ordonez-Moran P, Larriba MJ, Cordon-Cardo C, Munoz A. Genetic signatures of

[199]

[200]

[201]

[202] [203]

[204]

[205]

[206]

[207]

[208]

[209]

[210]

[211]

[212] [213]

[214]

[215]

differentiation induced by 1alpha,25-dihydroxyvitamin D3 in human colon cancer cells. Cancer Res 2003;63:7799e806. Usui E, Noshiro M, Okuda K. Molecular cloning of cDNA for vitamin D3 25-hydroxylase from rat liver mitochondria. FEBS Lett 1990;262:135e8. Su P, Rennert H, Shayiq RM, et al. 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 1990;9:657e67. Repa JJ, Lund EG, Horton JD, et al. Disruption of the sterol 27-hydroxylase gene in mice results in hepatomegaly and hypertriglyceridemia. Reversal by cholic acid feeding. J Biol Chem 2000;275:39685e92. Cooke NE, Haddad JG. Vitamin D binding protein (Gc-globulin). Endocr Rev 1989;10:294e307. Safadi FF, Thornton P, Magiera H, et al. Osteopathy and resistance to vitamin D toxicity in mice null for vitamin D binding protein. J Clin Invest 1999;103:239e51. Verroust PJ, Kozyraki R. The roles of cubilin and megalin, two multiligand receptors, in proximal tubule function: possible implication in the progression of renal disease. Curr Opin Nephrol Hypertens 2001;10:33e8. Barth JL, Argraves WS. Cubilin and megalin: partners in lipoprotein and vitamin metabolism. Trends Cardiovasc Med 2001;11:26e31. Willnow TE, Hilpert J, Armstrong SA, et al. Defective forebrain development in mice lacking gp330/megalin. Proc Natl Acad Sci USA 1996;93:8460e4. Nykjaer A, Dragun D, Walther D, et al. An endocytic pathway essential for renal uptake and activation of the steroid 25-(OH) vitamin D3. Cell 1999;96:507e15. Bikle DD, Gee E, Halloran B, Kowalski MA, Ryzen E, Haddad JG. Assessment of the free fraction of 25-hydroxyvitamin D in serum and its regulation by albumin and the vitamin D-binding protein. J Clin Endocrinol Metab 1986; 63:954e9. Gliemann J. Receptors of the low density lipoprotein (LDL) receptor family in man. Multiple functions of the large family members via interaction with complex ligands. Biol Chem 1998;379:951e64. Moestrup SK, Kozyraki R, Kristiansen M, et al. The intrinsic factor-vitamin B12 receptor and target of teratogenic antibodies is a megalin-binding peripheral membrane protein with homology to developmental proteins. J Biol Chem 1998;273: 5235e42. Nykjaer A, Fyfe JC, Kozyraki R, et al. Cubilin dysfunction causes abnormal metabolism of the steroid hormone 25(OH) vitamin D(3). Proc Natl Acad Sci USA 2001;98:13895e900. St-Arnaud R. Targeted inactivation of vitamin D hydroxylases in mice. Bone 1999;25:127e9. Masuda S, Kaufmann M, Byford V, et al. Insights into Vitamin D metabolism using cyp24 over-expression and knockout systems in conjunction with liquid chromatography/mass spectrometry (LC/MS). J Steroid Biochem Mol Biol 2004;89e90:149e53. Masuda S, Byford V, Arabian A, et al. Altered pharmacokinetics of 1alpha,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 in the blood and tissues of the 25-hydroxyvitamin D-24hydroxylase (Cyp24a1) null mouse. Endocrinology 2005;146: 825e34. Miyamoto Y, Shinki T, Yamamoto K, et al. 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 1alpha,25-dihydroxyvitamin D3. J Biol Chem 1997;272: 14115e9.

PEDIATRIC BONE

186

8. VITAMIN D BIOLOGY

[216] St-Arnaud R, Glorieux FH. Vitamin D and bone development. In: Feldman D, Glorieux FH, Pike JW, editors. Vitamin D. San Diego: Academic Press; 1997. p. 293e303. [217] Li YC, Pirro AE, Amling M, et al. Targeted ablation of the vitamin D receptor: an animal model of vitamin D-dependent rickets type II with alopecia. Proc Natl Acad Sci USA 1997;94: 9831e5. [218] Miller WL, Portale AA. Genetic causes of rickets. Curr Opin Pediatr 1999;11:333e9. [219] St-Arnaud R, Glorieux FH. In: DeGroot LJ, Jameson JL, editors. Endocrinology. Hereditary defects in vitamin D metabolism and action. 4th ed, Vol. 2. Philadelphia: W.B. Saunders Company; 2000. p. 1154e68. [220] Schipani E. Conditional gene inactivation using cre recombinase. Calcif Tissue Int 2002;71:100e2. [221] Delvin EE, Glorieux FH, Marie PJ, Pettifor JM. Vitamin D dependency: replacement therapy with calcitriol. J Pediatr 1981; 99:26e34. [222] Rosen JF, Finberg L. Vitamin D-dependent rickets: actions of parathyroid hormone and 25-hydroxycholecalciferol. Pediatr Res 1972;6:552e62. [223] Scriver CR, Reade TM, DeLuca HF, Hamstra AJ. Serum 1,25dihydroxyvitamin D levels in normal subjects and in patients with hereditary rickets or bone disease. N Engl J Med 1978; 299:976e9. [224] Mandla S, Jones G, Tenenhouse HS. Normal 24-hydroxylation of vitamin D metabolites in patients with vitamin D-dependency rickets type I. Structural implications for the vitamin D hydroxylases. J Clin Endocrinol Metab 1992;74:814e20. [225] St-Arnaud R, Dardenne O, Glorieux FH. Etiologie mole´culaire des rachitismes vitamino-de´pendents he´re´ditaires. Me´d/Sci 2001;17:1289e96. [226] Glorieux FH, St-Arnaud R. Vitamin D pseudodeficiency. In: Feldman D, Glorieux FH, Pike JW, editors. Vitamin D. SanDiego: Academic Press; 1997. p. 755e64. [227] Dardenne O, Prudhomme J, Hacking SA, Glorieux FH, StArnaud R. Rescue of the pseudo-vitamin D deficiency rickets phenotype of CYP27B1-deficient mice by treatment with 1,25dihydroxyvitamin D3: biochemical, histomorphometric, and biomechanical analyses. J Bone Miner Res 2003;18:637e43. [228] Naja RP, Dardenne O, Arabian A. St Arnaud R. Chondrocytespecific modulation of Cyp27b1 expression supports a role for local synthesis of 1,25-dihydroxyvitamin D3 in growth plate development. Endocrinology 2009;150:4024e32. [229] Masuyama R, Stockmans I, Torrekens S, et al. Vitamin D receptor in chondrocytes promotes osteoclastogenesis and regulates FGF23 production in osteoblasts. J Clin Invest 2006; 116:3150e9. [230] Yoshizawa T, Handa Y, Uematsu Y, et al. Mice lacking the vitamin D receptor exhibit impaired bone formation, uterine hypoplasia and growth retardation after weaning. Nat Genet 1997;16:391e6. [231] Van Cromphaut SJ, Dewerchin M, Hoenderop JG, et al. Duodenal calcium absorption in vitamin D receptor-knockout mice: functional and molecular aspects. Proc Natl Acad Sci USA 2001;98:13324e9. [232] Dostal LA, Toverud SU. Effect of vitamin D3 on duodenal calcium absorption in vivo during early development. Am J Physiol 1984;246:G528e34. [233] Tu Q, Pi M, Karsenty G, Simpson L, Liu S, Quarles LD. Rescue of the skeletal phenotype in CasR-deficient mice by transfer onto the Gcm2 null background. J Clin Invest 2003;111:1029e37. [234] Miedlich SU, Zalutskaya A, Zhu ED, Demay MB. Phosphateinduced apoptosis of hypertrophic chondrocytes is associated with a decrease in mitochondrial membrane potential and is

[235]

[236]

[237]

[238]

[239]

[240]

[241]

[242]

[243]

[244]

[245]

[246]

[247]

[248]

[249]

[250]

[251]

[252]

[253]

dependent upon Erk1/2 phosphorylation. J Biol Chem 2010; 285:18270e5. Baker MR, McDonnell H, Peacock M, Nordin BE. Plasma 25-hydroxy vitamin D concentrations in patients with fractures of the femoral neck. Br Med J 1979;1:589. Chapuy MC, Arlot ME, Duboeuf F, et al. Vitamin D3 and calcium to prevent hip fractures in elderly women. N Engl J Med 1992;327:1637e42. Lips P, Graafmans WC, Ooms ME, Bezemer PD, Bouter LM. Vitamin D supplementation and fracture incidence in elderly persons. Ann Intern Med 1996;124:400e6. Pavlovitch JH, Galoppin L, Rizk M, Didierjean L, Balsan S. Alterations in rat epidermis provoked by chronic vitamin D deficiency. Am J Physiol 1984;247:E228e33. Sakai Y, Demay MB. Evaluation of keratinocyte proliferation and differentiation in vitamin D receptor knockout mice. Endocrinology 2000;141:2043e9. Xie Z, Komuves L, Yu QC, et al. Lack of the vitamin D receptor is associated with reduced epidermal differentiation and hair follicle growth. J Invest Dermatol 2002;118:11e6. Cianferotti L, Cox M, Skorija K, Demay MB. Vitamin D receptor is essential for normal keratinocyte stem cell function. Proc Natl Acad Sci USA 2007;104:9428e33. Palmer HG, Anjos-Afonso F, Carmeliet G, Takeda H, Watt FM. The vitamin D receptor is a Wnt effector that controls hair follicle differentiation and specifies tumor type in adult epidermis. PLoS One 2008;3:e1483. Li M, Chiba H, Warot X, et al. RXR-alpha ablation in skin keratinocytes results in alopecia and epidermal alterations. Development 2001;128:675e88. Beaudoin 3rd GM, Sisk JM, Coulombe PA, Thompson CC. Hairless triggers reactivation of hair growth by promoting Wnt signaling. Proc Natl Acad Sci USA 2005;102:14653e8. Huelsken J, Vogel R, Erdmann B, Cotsarelis G, Birchmeier W. beta-Catenin controls hair follicle morphogenesis and stem cell differentiation in the skin. Cell 2001;105:533e45. Mathieu C, Waer M, Laureys J, Rutgeerts O, Bouillon R. Prevention of autoimmune diabetes in NOD mice by 1,25 dihydroxyvitamin D3. Diabetologia 1994;37:552e8. Griffin M, Lutz W, Phan V, et al. Dendritic cell modulation by 1,25-dihydroxyvitamin D and its analogs: A vitamin D receptordependent pathway that promotes a persistent state of immaturity in vivo and in vitro. Proc Natl Acad Sci USA 2001; 98:6800e5. Mathieu C, VanEtten E, Gysemans C, et al. In vitro and in vivo analysis of the immune system of vitamin D receptor knockout mice. J Bone Miner Res 2001;16:2057e65. Meehan TF, DeLuca HF. The vitamin D receptor is necessary for 1alpha,25-dihydroxyvitamin D(3) to suppress experimental autoimmune encephalomyelitis in mice. Arch Biochem Biophys 2002;408:200e4. Froicu M, Cantorna MT. Vitamin D and the vitamin D receptor are critical for control of the innate immune response to colonic injury. BMC Immunol 2007;8:5. Wittke A, Chang A, Froicu M, et al. Vitamin D receptor expression by the lung micro-environment is required for maximal induction of lung inflammation. Arch Biochem Biophys 2007;460:306e13. Bouillon R, Okamura WH, Norman AW. Structureefunction relationships in the vitamin D endocrine system. Endocr Rev 1995;16:200e57. Boyan BD, Sylvia VL, Dean DD, Schwartz Z. 24,25-(OH)(2)D(3) regulates cartilage and bone via autocrine and endocrine mechanisms. Steroids 2001;66:363e74.

PEDIATRIC BONE

187

REFERENCES

[254] Boyan BD, Sylvia VL, Dean DD, Del Toro F, Schwartz Z. Differential regulation of growth plate chondrocytes by 1alpha,25-(OH)2D3 and 24R,25-(OH)2D3 involves cell-maturation-specific membrane-receptor-activated phospholipid metabolism. Crit Rev Oral Biol Med 2002;13:143e54. [255] Boyan BD, Jennings EG, Wang L, Schwartz Z. Mechanisms regulating differential activation of membrane-mediated signaling by 1alpha,25(OH)2D3 and 24R,25(OH)2D3. J Steroid Biochem Mol Biol 2004;89e90:309e15. [256] Schwartz Z, Dean D, Walton J, Brooks B, Boyan B. Treatment of resting zone chondrocytes with 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] induces differentiation into a 125-(OH)2D3responsive phenotype characteristic of growth zone chondrocytes. Endocrinology 1995;136:402e11. [257] Boyan BD, Schwartz Z. Cartilage and vitamin D: genomic and nongenomic regulation by 1,25(OH)2D3 and 24,25(OH)2D3. In: Feldman D, Pike JW, Glorieux FH, editors. Vitamin D. 2nd ed, Vol. 1. San Diego: Elsevier Academic Press; 2005. p. 575e97. [258] St-Arnaud R. In: Feldman D, Pike JW, Glorieux FH, editors. Vitamin D. Mutant mouse models of vitamin D metabolic enzymes. 2nd ed. Vol. 1. San Diego: Elsevier Academic Press; 2005. p. 105e16. [259] Seo EG, Norman AW. Three-fold induction of renal 25-hydroxyvitamin D3-24-hydroxylase activity and increased serum 24,25dihydroxyvitamin D3 levels are correlated with the healing process after chick tibial fracture. J Bone Miner Res 1997; 12:598e606. [260] Seo EG, Einhorn TA, Norman AW. 24R,25-dihydroxyvitamin D3: an essential vitamin D3 metabolite for both normal bone integrity and healing of tibial fracture in chicks. Endocrinology 1997;138:3864e72. [261] Naja RP. The role of vitamin D metabolic enzymes in bone development and repair. Ph.D. Thesis, Department of Human Genetics. Montreal: McGill; 2009. [262] Erben RG, Bante U, Birner H, Stangassinger M. Prophylactic effects of 1,24,25-trihydroxyvitamin D3 on ovariectomy-induced cancellous bone loss in the rat. Calcif Tissue Int 1997;60:434e40. [263] Campbell MJ, Reddy GS, Koeffler HP. 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 1997;66:413e25. [264] Stern PH, Rappaport MS, Mayer E, Norman AW. 24-Oxo and 26,23-lactone metabolites of 1,25-dihydroxyvitamin D3 have direct bone-resorbing activity. Arch Biochem Biophys 1984; 230:424e9. [265] Jones G. Analog metabolism. In: Feldman D, Glorieux F, Pike J, editors. Vitamin D. San Diego, CA: Academic Press; 1997. p. 973e94. [266] Ishizuka S, Ishimoto S, Norman AW. Biological activity assessment of 1 alpha,25-dihydroxyvitamin D3-26,23-lactone in the rat. J Steroid Biochem 1984;20:611e5. [267] Ishizuka S, Norman AW. The difference of biological activity among four diastereoisomers of 1 alpha,25-dihydroxycholecalciferol-26,23-lactone. J Steroid Biochem 1986;25:505e10. [268] Toell A, Gonzalez MM, Ruf D, Steinmeyer A, Ishizuka S, Carlberg C. Different molecular mechanisms of vitamin D(3) receptor antagonists. Mol Pharmacol 2001;59:1478e85. [269] Ishizuka S, Kurihara N, Reddy SV, Cornish J, Cundy T, Roodman GD. (23S)-25-Dehydro-1{alpha}-hydroxyvitamin

[270]

[271]

[272]

[273]

[274]

[275]

[276]

[277]

[278]

[279]

[280]

[281]

D3-26,23-lactone, a vitamin D receptor antagonist that inhibits osteoclast formation and bone resorption in bone marrow cultures from patients with Paget’s disease. Endocrinology 2005;146:2023e30. Bischof MG, Siu-Caldera ML, Weiskopf A, et al. Differentiationrelated 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 1998;241:194e201. Brown AJ, Ritter C, Slatopolsky E, Muralidharan KR, Okamura WH, Reddy GS. 1Alpha,25-dihydroxy-3-epi-vitamin D3, a natural metabolite of 1alpha,25-dihydroxyvitamin D3, is a potent suppressor of parathyroid hormone secretion. J Cell Biochem 1999;73:106e13. Astecker N, Reddy GS, Herzig G, Vorisek G, Schuster I. 1alpha,25-Dihydroxy-3-epi-vitamin D3 a physiological metabolite of 1alpha,25-dihydroxyvitamin D3: its production and metabolism in primary human keratinocytes. Mol Cell Endocrinol 2000;170:91e101. Siu-Caldera ML, Sekimoto H, Weiskopf A, et al. Production of 1alpha,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 1999;24:457e63. Rao DS, Campbell MJ, Koeffler HP, et al. Metabolism of 1alpha,25-dihydroxyvitamin D(3) in human promyelocytic leukemia (HL-60) cells: in vitro biological activities of the natural metabolites of 1alpha,25-dihydroxyvitamin D(3) produced in HL-60 cells. Steroids 2001;66:423e31. Sekimoto H, Siu-Caldera ML, Weiskopf A, et al. 1alpha,25dihydroxy-3-epi-vitamin D3: in vivo metabolite of 1alpha,25dihydroxyvitamin D3 in rats. FEBS Lett 1999;448:278e82. Schuster I, Astecker N, Egger H, et al. Vitamin D-metabolism in human keratinocytes and biological role of products. In: Norman A, Bouillon R, Thomasset M, editors. Vitamin D: Chemistry, Biology and Clinical Applications of the Steroid Hormone. Riverside, CA: University of California Press; 1997. p. 551e8. Norman A, Bouillon R, Farach-Carson M, et al. Demonstration that 1b,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 1993;268:20022e30. Harant H, Spinner D, Reddy GS, Lindley IJ. Natural metabolites of 1alpha,25-dihydroxyvitamin D(3) retain biologic activity mediated through the vitamin D receptor. J Cell Biochem 2000; 78:112e20. Messerlian S, Gao X, St-Arnaud R. The 3-epi- and 24-oxoderivatives of 1alpha,25 dihydroxyvitamin D(3) stimulate transcription through the vitamin D receptor. J Steroid Biochem Mol Biol 2000;72:29e34. Seo EG, Kato A, Norman AW. Evidence for a 24R,25(OH)2vitamin D3 receptor/binding protein in a membrane fraction isolated from a chick tibial fracture-healing callus. Biochem Biophys Res Commun 1996;225:203e8. Kato A, Seo EG, Einhorn TA, Bishop JE, Norman AW. Studies on 24R,25-dihydroxyvitamin D3: evidence for a nonnuclear membrane receptor in the chick tibial fracture-healing callus. Bone 1998;23:141e6.

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9

Peak Bone Mass and Its Regulation Jean-Philippe Bonjour, Thierry Chevalley, Serge Ferrari, Rene Rizzoli Division of Bone Diseases*, University Hospitals and Faculty of Medicine of Geneva, Switzerland

DEFINITION AND IMPORTANCE OF PEAK BONE MASS The concept of “peak bone mass” became familiar about 25 years ago with the frequent use of non-invasive quantitative technology enabling the measurement of the amount of mineral contained at several sites of the skeleton. Interestingly, the first quoted papers referring to “peak bone mass” (PBM) dealt with the influence of disease (anorexia nervosa), medication (glucocorticoids in rheumatoid arthritis) or environmental factors (physical activity) on bone health in young adults [1e3]. It is still convenient to refer to this concept even though “stricto sensu” its actual meaning is not limited to the maximal bone “mass” attained by the end of the period of growth. In the clinical setting, areal (a) bone mineral density (BMD) measured by dual x-ray energy absorptiometry (DXA) in young healthy adults is used as the reference value for evaluating the risk of osteoporotic fracture. It is based on the inverse relationship found between areal BMD values determined at the forearm, spine and hip and the risk of fragility fracture at these three skeletal sites. The relative contribution of peak bone mass to fracture risk has been explored by examining the variability of areal bone mineral density (aBMD) values in relation with age. If peak bone mass is relatively unimportant to aBMD and fracture risk in later life, then the range of aBMD values would become wider as a function of age during adult life. However, several observations are not consistent with such an increased range in aBMD values in relation to age. In untreated postmenopausal women, the standard deviation (SD) of bone mineral mass measured at both the proximal and distal radius was no greater in women aged 70e75 than in women aged 55e59 years [4]. Similar findings were reported at two other clinically relevant skeletal sites at risk of osteoporotic fractures. At both the lumbar spine and femoral

neck, the range of aBMD values was no wider in women aged 70e90 than in women aged 20e30 years [5]. This constant range of individual aBMD values was observed despite the marked reduction in spine and femoral neck aBMD values in the older women (see for review [6]). In agreement with these cross-sectional findings, a longitudinal study of women ranging in age from 20 to 94 years (median age 60 years), with follow-up periods of 16 to 22 years, showed that the average annual rate of bone loss was relatively constant and tracked well within an individual [7,8]. High correlations were observed between the baseline aBMD values and those obtained after 16 (r ¼ 0.83) and 22 (r ¼ 0.80) years of follow up [7,8]. This tracking pattern of aBMD, which was already observed during growth, appears to be maintained over six decades of adult life. This notion of “tracking” has two important implications. First, the prediction of fracture risk based on one single measurement of femoral neck aBMD remains reliable in the long term [8]. Second, within the large range of femoral neck aBMD values little variation occurs during adult life in individual Z-scores or percentiles. From these two implications, it can be inferred that bone mass acquired at the end of the growth period appears to be more important than bone loss occurring during adult life. In a mathematical model using several experimental variables to predict the relative influences of PBM, menopause and age-related bone loss on the development of osteoporosis [9], it was calculated that an increase in peak bone mass of 10% would delay the onset of osteoporosis by 13 years [10]. In comparison, a 10% increase in the age of menopause, or a 10% reduction in age-related (non-menopausal) bone loss would only delay the onset of osteoporosis by 2 years [9]. Thus, this theoretical analysis indicates that peak bone mass might be the single most important factor for the prevention of osteoporosis later in life [9].

*World Health Organization Collaborating Center for Osteoporosis Prevention

Pediatric Bone, Second Edition DOI: 10.1016/B978-0-12-382040-2.10009-7

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There is also evidence that the risk of fracture after the sixth decade may be related to bone structural and biomechanical properties acquired during the first few decades of life. Duan et al. [11,12] calculated the fracture risk index (FRI) of the vertebral bodies based on the ratio of the compressive load and strength in young and older adults (z30e70 years of age). Load was determined by upper body weight, height and the muscle moment arm, whereas bone strength was estimated from the bone cross-sectional area (CSA) and volumetric (v) BMD [11]. From young to older adulthood, this index increased more in women (Chinese and Caucasian) than in men of the same ethnicity [12]. However, the dispersion of CSA, vBMD and FRI values around the mean did not increase with age within a given sex in either the Chinese or the Caucasian ethnic groups [12], suggesting an important role of bone acquired prior to the age of 30. The importance of maximizing peak bone mass has also been estimated from the determination of the risk of experiencing an osteoporotic fracture in adulthood. Epidemiological studies allow one to predict that a 10% increase (about 1 SD) in peak bone mass could reduce the risk of fracture by 50% in women after the menopause [10,13e15]. Altogether, these findings provide strong evidence for the notion that maximizing bone health during growth may represent an important strategy to prevent osteoporosis and fractures during aging. As a result, there has been considerable interest in exploring whether environmental factors can modify the genetically predetermined bone mineral mass trajectory during growth. Before discussing this possibility, the role of heredity and putative candidate genes that might be implicated in the determination of peak bone mass are presented.

CHARACTERISTICS OF BONE MASS AND STRENGTH ACQUISITION Assessment Several structural elements determine the mechanical strength of bone. The size of the bone, the amount of bony tissue within the periosteal envelope and its spatial distribution, i.e. the micro- and macroarchitecture, and the degree of mineralization and structural organization of the organic matrix are the most important elements that determine the resistance to mechanical loading. In each individual, these components as a whole follow a trajectory from intrauterine life to completion of the skeletal growth process, i.e. at the attainment of peak bone mass. To date, aBMD (in g/cm2), as assessed by DXA, is the most common variable studied during infancy, childhood and adolescence. There are several explanations for the widespread use of aBMD

measurement to study bone acquisition during growth in relation to the risk of osteoporosis in adulthood. Determination of aBMD is particularly convenient in terms of availability of equipment, low exposure to irradiation, reproducibility of the measurement at several sites of the skeleton and, last but not least, its relationship with adult osteoporosis fracture risk as adequately documented in large cohorts of women and men. Because of this last characteristic, aBMD was recognized by several national and international institutions, including the World Health Organization (WHO) [10], as the variable to be measured for establishing the diagnosis of adult osteoporosis. More recent practical guides and recommendations for the use of DXA scans in children and adolescents have been presented by national and international bone specialist societies [16,17]. The positive aspects of DXA scanning in clinical use do not mean that aBMD measurement integrates all the determinants of bone strength. Structural and functional components contribute to the degree of bone fragility and therefore to the risk of experiencing fragility fractures. In clinical research, the recent increasing use of high-resolution peripheral quantitative computed tomography (HR-pQCT) and finite element analysis (FEA) can provide additional information on more subtle bone structural mechanical resistance components [18e25]. This technical approach is expected to improve the prediction of bone strength as compared to the current use of the variables that can easily be captured by DXA: aBMD, bone mineral content (BMC), and the bone size of the region of interest. In addition, at some skeletal sites, an estimate of volumetric BMD, cortical thickness, cross-sectional area and moment of inertia can also be computed. Quantitative ultrasonography (QUS) has been compared to DXA for identifying adults with osteoporosis and fragility fractures. Although QUS parameters determined in some but not all tested devices can predict the osteoporotic fracture risk, their use is still not recommended for the diagnosis or treatment monitoring of adult osteoporosis [26]. The application of QUS technology in pediatric populations is attractive because of several characteristics including absence of ionizing radiation, portability and low cost. Calcaneous QUS measurements can detect low bone mass during childhood and adolescence. However, the clinical utility of QUS in children is yet to be determined, and thus this technique remains a research tool in the pediatric population [16,27,28].

Structural Development During growth, aBMD increment is essentially due to an increase in bone size [29,30], which is closely linked to a virtually commensurate increment in the amount of

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Lumbar Spine Increment (%) from Prepuberty to Postpuberty

260 240 220 200 180 160 140 120 BM C (g)

AR E (cm A )

aB (g/ MD cm 2 )

Tr ab . (g/ vBM cm 3 D )

100

FIGURE 9.1 Change in bone traits at the lumbar spine from prepuberty to postpuberty in white females. The results are expressed as the percent increment from prepubertal values. The trabecular volumetric density (Trab.vBMD) change is taken from Gilsanz et al. [32]. The areal bone mineral density (aBMD), bone size (AREA) and bone mineral content (BMC) are from Bonjour et al. [29] and Theintz et al. [33]. They were measured by dual-energy x-ray absorptiometry (DXA) in the anteroposterior view. The much greater change in BMC (þ151%) as compared to Trab.vBMD (þ14%), demonstrates that pubertal bone growth is essentially due to an increase in bone size including an increment in cortical thickness, with very little gain in volumetric trabecular density. (See text for further details.)

Δ L2-L4 aBMD

9–10

g/cm2 per year ± SEM

mineralized tissue contained within the periosteal envelope. Consequently, as compared to bone size, vBMD increases very little from infancy to the end of the growth period [31]. Changes in bone traits from prepuberty to postpuberty also indicate the much greater increment in bone size and BMC, including increased cortical thickness, than in trabecular vBMD as illustrated in the lumbar spine (Fig. 9.1). In healthy girls, longitudinal examination of lumbar spine development indicates that the standard deviation scores (Z-scores) of aBMD, BMC, apparent vBMD, as well as vertebral body width and height are highly correlated, with correlation coefficients (R) ranging from 0.83 to 0.92, as compared to 0.91 for standing height [34]. Before puberty, no substantial sex difference has been reported in the bone mineral mass of the axial (lumbar spine) or appendicular (e.g. radius and femur) skeleton when adjusted for age, nutrition and physical activity. There is no evidence of a sex difference in bone mass at birth; the vBMD appears to be similar in female and male newborns. This absence of substantial sex differences in bone mass is maintained until the onset of pubertal maturation. The gender dimorphism in bone mass is expressed during puberty. It appears to be mainly due to a longer period of bone maturation in males than in females, resulting in a larger increment in bone size and cortical thickness (Figs 9.2 and 9.3).

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Age range (years) FIGURE 9.2 Bone mass gain at the lumbar spine during the years of pubertal maturation. The approximately fourfold increment in bone mass gain, as estimated by aBMD measurement by DXA, is particularly pronounced during 3 and 4 years in females (from 11e14) and males (from 13e17), respectively. (Adapted from Theintz et al. [33].)

As mentioned above, pubertal maturation affects bone size much more than it does vBMD [31]. There is no significant sex difference in trabecular vBMD at the end of pubertal maturation [30,32,35,36]. Bone mass accumulation rate at both lumbar spine (Fig. 9.2) and femoral neck regions increases four- to sixfold over a 3- and 4-year period in females and males, respectively. The increment in bone mass gain is less marked in long bone diaphyses (Fig. 9.3) [33]. As first described by Garn [37] by measuring metacarpal bones during sexual maturation, cortical thickness increases more by periosteal development in males, but more by endosteal deposition in females. This concept is illustrated in Figure 9.3 depicting the gain in femoral shaft cortical thickness from 9 to 20 years in both female and male healthy subjects. In the lumbar spine, the gender difference observed when PBM is attained consists essentially of a greater vertebral body diameter in the frontal plane of males as compared to females [38,39] (Fig. 9.4A and B). This gender-related structural dimorphism does not attenuate with aging. It certainly represents an important macroarchitectural characteristic accounting for the greater incidence of vertebral fragility fractures recorded in female than male subjects in later life. Within each gender, this structural property also plays an important role in vertebral fracture risk. In postmenopausal women, a smaller cross-sectional area of vertebral bodies was measured in those with than without vertebral fractures despite the fact that the two groups displayed equally low trabecular vBMD as determined by spinal QCT [40]. The gender difference in either

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Femoral Shaft aBMD g/cm2 per year ± SEM

Males

Females 9–10

–

Age Range (years)

FIGURE 9.3 Bone mass gain in the midfemoral shaft during the years of pubertal maturation. The increment in bone mass gain, as estimated by aBMD measurement by DXA, takes place during 3 and 4 years in females (from 11e14) and males (from 13e17), respectively. (Adapted from Theintz et al. [33].) On the right side is schematized the sex difference occurring during pubertal maturation in the cross-section of a long bone. Before pubertal maturation (age 9e10 years), both periosteal and endosteal circumferences (dotted circles) are similar in girls and boys. At the end of pubertal maturation (age 17e20 years), cortical thickness has increased by more periosteal deposition in males than in females (continuous circles) in whom the increment in cortical width is also due to increased endosteal formation. (See text for further details.)

(A)

Morphometric lumbar spine gender differences

Lumbar vertebral endplate Measured diameters Sagittal: small arrows Diagonal: mid-sized arrows Transverse: bold arrows

Lumbar vertebral body Measured heights Posterior: thin arrows Anterior: bold arrows

Male

Female

Transverse diameter

56.1 ± 5.5

50.6 ± 8.6

1.11*

Diagonale diameter

49.4 ± 5.6

45.4 ± 5.1

1.09*

Sagittal diameter

36.1 ± 3.9

35.4 ± 3.6

1.02

Vertebral height ant.

35.5 ± 4.2

27.0 ± 4 .1

1.32*

Vertebral height post.

27.4 ± 5.4

25.9 ± 4.2

1.06

Measurements are in millimeters ± SD

Male/Female Ratio

*P<0.05

FIGURE 9.4 (A) Sex-related morphometric differences in lumbar vertebrae. Measurements were made on 68 macerated lumbar vertebrae. Note the significantly wider transversal diameter in males than in females, whereas there is no difference in the sagittal diameter. These direct morphometric measurements from Aharinejad et al. [38] are relevant to the sex differences in PBM as recorded by DXA and illustrated in (B).

aBMD or BMC observed in the radial or femoral diaphysis once PBM is attained also appears to be essentially due to a greater gain in bone size in males than in females during pubertal maturation. A study comparing bone variables (BMC, aBMD and vBMD) in opposite-sex twins corroborates this notion [41].

Peripubertal Transient Fragility There is an asynchrony between the gain in standing height and the growth of bone mineral mass during pubertal maturation [33,42]. This phenomenon may be responsible for the occurrence of a transient bone

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Lumbar spine osteodensitometric gender differences

(B)

C

V

B

V

A

C BM

R A B V

BM

-l

-la EA

D M aB B V

at

t

t -la

p -a C BM

R A

aB

M

EA

D

-a

-a

p

p

L3 BMC =

FIGURE 9.4 (B) Sex-related osteodensitometric differences in lumbar vertebrae. Measurements of aBMD, AREA and BMC were made by both anteroposterior (ap) and lateral (lat) DXA scans in 65 male and female subjects with ages ranging from 20 to 35 years. Vertebral BMC of L2eL3 or L3 alone, was quite similar in anteroposterior and lateral scans, as schematized in the upper part of the diagram. Male to female ratios were not different from 1.0 for aBMD in ap view, but significantly higher for aBMD in lat view as shown in the lower part of the diagram. VB, vertebral body; VA, vetebral arch. ***P < 0.001 as compared with a sex ratio of 1.0. Note that the most prominent difference between male and female osteodensitometric values was for VB BMC in lat view, as explained, at least in part, by the greater transverse (frontal) than sagittal dimension of the vertebral body in males than in females as shown in Figure 9.4A. (Adapted from Fournier et al. [39].)

fragility in adolescence that may contribute to the higher incidence of fractures that occurs when the dissociation is maximal between the rates of standing height and mineral mass accrual [43,44]. In healthy girls and boys aged 6 to 21 years, bone structure measurement by HR-pQCT associated with FEA at the distal radius suggests that the proportion of load borne by cortical bone decreased transiently during mid- to late puberty [23]. This change could correspond to an increase in cortical porosity during the pubertal growth spurt and may contribute to the adolescent peak in forearm fractures [23]. This possibility would be consistent with the hypothesis that cortical porosity would develop in response to the increased calcium demand related to the rapid peripubertal skeletal growth [45]. Nevertheless, some trauma fractures observed during childhood or adolescence may be unrelated to transient bone

fragility that occurs at peak height velocity during the peripubertal growth phase. It is possible that some of these fractures may also be determined by tracking, from infancy to the end of skeletal maturation, along a relatively low bone mass percentile (Z-score) [46]. Thus, the deficit would be permanent and expressed by a relatively low PBM and increased risk of fragility fracture in later adult life [47].

Time of Peak Bone Mass Attainment In adolescent females, gain in bone mass declines rapidly after menarche (Fig. 9.5) [33]. No further statistical significant gains can be monitored within 2e4 years postmenarche, at least in sites such as the lumbar spine or femoral neck (Fig. 9.5). In adolescent males, the gain

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in 16-year-old and 30-year-old subjects [48]. This observation supports the notion that the modest increase in vertebral trabecular vBMD is achieved soon after menarche. This is in keeping with numerous observations indicating that, at most skeletal sites, total bone mineral mass does not significantly increase from the third to the fifth decade. Nevertheless, a few crosssectional studies suggest that bone mass acquisition may still be substantial during the third and fourth decades. In any case, the balance of published data does not sustain the concept that bone mass at any skeletal site, in either gender and in any ethnic and/or geographic population group, continues to accumulate through the fourth decade [30].

FIGURE 9.5 Relationship between spinal and femoral aBMD gains and years after menarche in healthy girls. Results are means  SEM. Time after menarche: P5a, less than 1.0 yr (age: 13.8  0.4 yearrs); P5b, 1e2 years (age 14.5  0.2 years); P5c, 2e4 years (age: 16.6  0.3 years); P5d, more than 4 years (age: 17.4  0.3 years). Statistical significance of aBMD gain: *P < 0.05; **P < 0.01; ***P < 0.001. The increment in aBMD dramatically fell after 16 years (see Figure 9.2) and/or 2 years after menarche. The mean aBMD gains in healthy girls at the level of lumbar spine, femoral neck and femoral shaft were not statistically significant between 17 and 20 years. Data from Theintz et al. [33].

Peak Bone Mass Variability At the beginning of the third decade, there is a large variability (CV ¼ SD/mean100) in the normal values of aBMD or BMC in the axial and appendicular skeleton [30]. This large variability, 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 increase substantially during adult life (Fig. 9.6) [39]. The height-independent broad variability 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 [29,33]. Note that in young healthy adults the biological variability in lumbar spine BMC is four or five times higher than that in standing height. Of note, the variability in standing height does not increase during pubertal maturation [42].

Bone Biochemical Markers During Puberty in BMD or BMC that is accelerated particularly from 13 to 17 years declines markedly thereafter, although it remains significant between 17 and 20 years in both lumbar spine BMD and BMC and in mid-femoral shaft BMD; in contrast, no significant increase is observed in femoral neck BMD [33]. In subjects who reached pubertal stage P5 and grew less than 1 cm/year, a significant bone mass gain persisted in males but not in females [33]. This suggests the existence of an important sex difference in the magnitude and/or duration of the so-called “consolidation” phenomenon that contributes to PBM. As described above, the change in trabecular vBMD during growth is very modest as compared to the increment in bone size (see Fig. 9.1). Furthermore, the increased vBMD, as measured by QCT, has been detected in vertebral cancellous bone but not in appendicular cortical tissue [35]. In the lumbar vertebral body, no difference is observed between the mean values

Taking into account the different bone growth processes which encompass endochondral formation, modeling and remodeling, it is no wonder that 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 [49e53]. 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 cross-links, 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) [49e53]. This probably reflects the decrease in the resorption rate associated with the reduction and eventually the arrest in longitudinal bone growth. In a prospective study of pubertal girls, bone turnover markers (osteolcalcin, bone-specific

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L2 - L4 BMC (g)

FEMALES

MALES

70

70

60

60

50

50

40

40

30

30

20

20

10 0 120

P1 130

140

195

10

P2 P3 P4 P5 150

160

170

180

P1

P2 P3 P4 P5

0 120 130 140 150 160 170 180 190 200

STANDING HEIGHT (cm)

FIGURE 9.6

Relationship between lumbar spine bone mineral content (BMC) and standing height in healthy females and males from prepuberty to completion of pubertal maturation. Data are from 207 healthy Caucasian subjects aged 9e18 years. BMC was measured by DXA. Pubertal stages (P1 to P5) are marked on the standing height horizontal axis. Vertical lines illustrate the marked variability of bone mineral mass at the end of pubertal maturation for a standing height corresponding to the mean value of the young adult population of reference. A twofold difference in the amount of bone mineral contained within the three lumbar vertebrae is observed for the same standing height in both female and male subjects. The mean L2eL4 BMC at the end of pubertal maturation as indicated by the horizontal solid lines was 10% greater in males than in females. Data from Bonjour et al. [29].

alkaline phosphatase, and collagen pyridium crosslinks) were modestly related to standing height gain, but they were not predictive of gains in either total body BMC or BMD as assessed by DXA [54]. The reader should refer to Chapter 15 for more detailed discussion on the clinical use of biochemical markers of bone metabolism.

DETERMINANTS OF PEAK BONE MASS AND STRENGTH Several interconnected factors influence bone mass accumulation during growth. These physiological determinants classically include heredity, vitamin D and bonetropic nutrients, endocrine factors, and mechanical forces (Fig. 9.7). Quantitatively, the most prominent determinant appears to be genetically related.

Genetic Factors Parent(s)eoffspring comparison studies reveal a significant relationship in the risk of osteoporosis within families, with apparent transmission from either mothers or fathers to their children [34,55e58]. The

familial resemblance for bone mineral mass in mothers and daughters is expressed before the onset of pubertal maturation [34]. Comparison in the degree of correlation between pairs of monozygotic versus dizygotic twins allows one to estimate more precisely the contribution of heritability to the variance of bone mineral mass [59,60]. This computation suggests that heritability, i.e. the additive effects of genes, explains 60e80% in the variance of adult bone mineral mass. This “genetic effect” appears to be greater in skeletal sites such as the lumbar spine compared to the femoral neck [61]. It is possible that mechanical factors (e.g. physical activity, body weight, muscle force) exert a greater influence on the cortical component of the bony structure of the femur, thus explaining the relatively low heritability at that site. Despite the strong impact of heritability on aBMD, environmental factors still play an important role since they may account for up to 20e40% of PBM variance. Two main approaches have dominated the search for genetic factors that influence bone acquisition and thereby modify the susceptibility to osteoporosis in later life. One approach is to search by genome-wide screening for loci flanked by DNA microsatellite markers that would co-segregate with the phenotype of interest in a population of related individuals. The

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FIGURE 9.7 Physiologic determinants of peak bone mass and

Genetics

Endocrine Sex Hormones IGFIGF-I

strength. The black arrows and dashed lines illustrate the interdependency of the four categories of factors influencing the mass and strength acquired by the end of skeletal development in healthy female and male subjects. IGF-I: Insulin-like growth factor-I. The major importance of heredity in peak bone mass and strength is symbolized by the wider white arrow from the “Genetics” box. (See text for further details on the determinants and their interactions as schematized on this diagram.)

pedigrees investigated to date consist mainly of families with a member at either extreme of the skeletal phenotype spectrum; particularly those exhibiting either very high or very low bone mineral mass or areal density [62e64]. Genome screening for quantitative trait loci (QTLs) has also been used to detect within the “normal” population families and/or siblings with marked difference in bone mass, size [65e67], or geometry [64,68,69]. The second frequently used approach is to search for an association between allelic variants or polymorphisms of genes coding for products that are implicated in bone acquisition or loss. The most studied phenotype has been aBMD or bone mineral content (BMC) because of both the ease of access and reliability of its measurement, as well as the relatively good predictability for osteoporotic fracture risk [10,13e15]. Association between polymorphic candidate genes and fracture has also been reported [70e73]. The fracture phenotype is certainly attractive since it is the most convincing evidence of osteoporosis-induced bone fragility. However, fragility fracture is a very complex phenotype that depends not only on bone quantity and quality, but also on other endogenous factors, such as the propensity to fall, protective responses, soft tissue padding, and exogenous elements present in the living environment [74e76]. Numerous studies have reported an association between bone phenotype and polymorphic candidate genes coding for hormones, hormonal receptors or enzymes involved in their biochemical pathways; local regulators of bone metabolism; structural molecules of the bone matrix [59,60,77e79]. Meta-analyses have been reported for the most studied polymorphisms, which include: vitamin D receptor (VDR) [80,81], estrogen receptor alpha (ESR1) [72] and type I collagen A1 chain (CollA1) [71]. The polymorphisms considered in these three genes were significantly associated with aBMD, BMC and/or fracture risk [82]. However, none of these polymorphisms appears to be responsible for

a substantial proportion, i.e. more than 1e3%, of PBM variance. Furthermore, significant associations appear to depend upon several factors including the skeletal sites measured, age, gender, ethnicity, genetic homogeneity of the investigated population, and the interaction between genes and environmental factors (refer to the next sections of this chapter). Only a few studies have explored the contribution of these candidate genes using bone geometry or strength as an outcome. Findings from these studies are contradictory which, given the small sample size, are largely explained by a lack of statistical power. One of the most interesting aspects concerning heritability of bone mass and strength is the implication of the gene coding for low-density lipoprotein receptorrelated protein-5 (LRP5). LRP5 is a member of the lowdensity lipoprotein (LDL) receptor-related family coding for a transmembrane co-receptor for Wnt signaling [83]. Several lines of evidence point to LRP5 as a candidate gene for osteoporosis. Mutation in the LRP5 gene has been found in patients with the human osteoporosis pseudoglioma syndrome (OPPG), an autosomal recessive disorder characterized by low bone mass, spontaneous fractures and blindness [84,85]. Interestingly LRP5-deficient mice develop osteoporosis and sustain fractures due to reduced osteoblast proliferation and function [86]. In sharp contrast to LRP5 mutations reducing the functional osteogenic capacity, other mutations in the same gene can lead to increased bone formation. Such a gain-of-function mutation in LRP5 is associated with an autosomal dominant high bone mass (HBM) and sclerosing bone dysplasias [87e89]. Most importantly, a QTL for aBMD in the general population was mapped at 11q12-13, the LRP5 locus [65,69,90]. A population-based study of five LRP5 polymorphisms with allele frequencies > 2% found that a missense substitution in exon 9 (c.2047G > A, p.V667M) and haplotypes based on exon 9 and exon 18 (c.4037 C > T, p.A1330V) alleles were significantly

Nutrition Vitamin D Calcium Proteins

Peak Bone Mass/Strength

Mechanics Physical Activity Body Weight

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associated with lumbar spine bone mass and projected area in adult males, but not females. These polymorphisms accounted for up to 15% of the population variance for these traits in men [91]. Consistent with the presence of a QTL for stature at 11q12-13 [92,93], the exon 9 variant was also significantly associated with standing height in both genders [91]. Moreover, 1-year changes in lumbar spine bone mass and size in prepubertal boys were also significantly associated with these LRP5 variants, suggesting that LRP5 polymorphisms could contribute to the risk of spine osteoporosis in men by influencing vertebral bone growth during childhood [91]. These observations in young healthy males led to investigating LRP5 polymorphisms in men with idiopathic osteoporosis [79]. This rather uncommon form of osteoporosis affects middle-aged men and is characterized by low PBM and an increased incidence of vertebral fractures in the absence of any secondary causes, and by a clear heritable component [58,94]. In keeping with the previous association study, exon 9A and exon 18T alleles were twice as common in men with idiopathic osteoporosis as in age-matched controls (mean age: 50.4 years, range 23e70). The odds were greater than two for idiopathic male osteoporosis and fractures among carriers of the 9A-18T haplotype [79]. In a large prospective population-based study, a variant (1330valine) of the LRP5 gene was associated with decreased aBMD and reduced bone size (vertebral body size and femoral neck width) at both the lumbar spine and femoral neck in elderly white men [95]. In summary, these different studies suggest that some LRP5 gene variants would increase the osteoporosis risk profile in men, possibly by influencing bone size during growth and thereby affecting an important component of peak bone strength. In a recent meta-analysis of several studies, nine out of the 150 candidate genes were associated with bone mineral density at the lumbar spine and only three at both lumbar spine and femoral sites [96]. In another large scale meta-analysis of genome-wide association studies (GWAS), only nine out of 20 gene loci were associated with bone mineral density at both lumbar and femoral neck sites [97]. The contribution of these genes to the inter-individual wide range of bone mineral density was very weak, explaining only z3% and z2% of the variance, respectively [97]. This very small variance contribution markedly contrasts with the large genetic effects on bone mineral density reported in twin studies [61,98,99]. Possible limitations regarding large-scale studies to identify important genetic determinants have been explicitly considered, including ethnic heterogeneity, population sampling, use of genetic markers instead of functional variants, lack of geneegene or geneeenvironment interaction analysis

197

[100]. The disappointing outcome of large scale metaanalysis of either candidate gene or GWAS studies suggests that other methodology approaches should be explored, particularly regarding geneeenvironment interactions, in order better to identify the main factors explaining the large PBM variance, and thereby the individual osteoporosis risk in later adulthood [100,101].

Endocrine Factors Among endocrine factors, both sex hormones [36,102,103) and the growth hormoneeinsulin-like growth factor-1 (GH-IGF-1) system [104,105] exert a specific impact on bone and play an important role, particularly during the phase of pubertal maturation. Furthermore, these two hormonal systems interact to stimulate the longitudinal and cross-sectional growth of the skeleton. Sex Hormones As already discussed, the development of bone mineral mass during the whole growth period, including pubertal maturation, is due essentially to an increase in bone size, with a very small change in the unit amount of mineralized tissue within the bone envelope [30,36,103,106]. In other words, the volumetric bone mineral density (vBMD) remains virtually constant from birth to the end of the growth period. Similarly, once pubertal maturation is achieved, the gender difference in bone mass results essentially from a greater bone size in male subjects [30,36,103,106]. In boys, the onset of puberty occurs later than in girls and the period of accelerated bone growth lasts 4 years, as compared to 3 years in girls [33]. These two characteristics probably account, to a large extent, for the gender difference in mean PBM observed in healthy young adults. Androgen receptors have been localized in growth plate chondrocytes in humans during pubertal maturation [107,108]. However, there is no evidence that androgens stimulate longitudinal bone growth by a direct action on the skeleton. At adult age, patients affected by the androgen insensitivity syndrome and who have an XY genotype associated with a markedly female phenotype, are taller than the average standing height of the corresponding female population [109]. In contrast, it is well documented that estrogens play an essential role in longitudinal bone growth. Estrogens exert biphasic effects, accelerating bone growth at the beginning of puberty and playing a key role in the closing of growth plates in both genders [36,102,103]. During pubertal maturation, cross-sectional analysis of appendicular bone, at least in the upper limb, reveals distinct gender dimorphisms. In female subjects, bone mineral mass increases more by endosteal than

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periosteal accrual [37]. In male subjects, the opposite structural modifications are observed, with a greater increase in periosteal than endosteal deposition, resulting in an increment in both external and internal perimeters of the cortex [37]. As mentioned above, at the end of pubertal maturation, cortical thickness is greater in male than in female subjects. In vertebral bodies, the gender structural dimorphism is mainly expressed in the frontal axis, which is 10e15% larger in males than in females [39]. These morphological differences and mineral mass distribution of both axial and appendicular bones confer to male skeleton a greater resistance to mechanical loading. The increased deposition of bone mineral at the level of the endosteal surface during puberty in female subjects may represent, according to a teleological explanation, a biological adaptation allowing the rapid mobilization of bone mineral in response to increased need during pregnancy and lactation. Experimental evidence indicates that alterations in maternal estrogen levels during pregnancy can not only influence the early phases of fetal bone tissue development, but can also exert long-term imprinting effects on bone cellular activity and eventually on adult skeletal mass [110]. The Growth Hormone-IGF-I System From birth to the end of adolescence, the GH-IGF-I system is essential for the harmonious development of the skeleton [111]. During puberty, the plasma level of IGF-I transiently rises according to a pattern similar to the curve of the gain in bone mass and size [30]. IGF-I positively influences the growth of the skeletal pieces in both length and width. IGF-I exerts a direct action on growth plate chondrocytes as well as on osteogenic cells responsible for building both cortical and trabecular bone tissue constituents [111]. This activity is also expressed by parallel changes in the circulating biochemical markers of bone formation, osteocalcin and alkaline phosphatase. In addition, IGF-I exerts an important impact on renal endocrine and transport functions that are essential for bone mineral economy. IGF-I receptors are localized in renal tubular cells. They are connected to both the production machinery of the hormonal form of vitamin D, namely 1,25dihydroxyvitamin D (1,25(OH)2D) [112,113], and to the transport system of inorganic phosphate (Pi) [114] localized in the luminal membrane of tubular cells. By enhancing the production and circulating level of 1,25(OH)2D [115], IGF-I indirectly stimulates the intestinal absorption of Ca and Pi. Coupled with the stimulation of the tubular capacity to reabsorb Pi [115], the extracellular CaePi product is increased by IGF-I which, through this dual renal action, favors bone matrix mineralization. Furthermore, at the bone level, IGF-I directly enhances the osteoblastic formation of the extracellular

matrix [116]. Growth plate chondrocytes as well as their plasma membrane derived extracellular matrix vesicles are equipped with a Pi transport system that plays a key role in the process of primary mineralization and, thereby, in bone development [117e119]. This Pi transport system is also present in other osteogenic cells [120] and is regulated by IGF-I [121]. The hepatic production of IGF-I, which is the main source of circulating IGF-I, is influenced not only by GH, but also by other factors, particularly by amino acids from dietary proteins (see below). During pubertal maturation, there is an interaction between sex steroids and the GH-IGF-1 system. The modalities of this interaction remain to be delineated in humans. In animal studies, relatively low concentrations of estrogens stimulate the hepatic production of IGF-I, whereas large concentrations exert an inhibitory effect [103]. Androgens act mainly at the pituitary level, but only after being converted into estrogens by the enzymatic activity of aromatase [103].

Importance of Pubertal Timing Physiologically, there are large variations in the onset of pubertal maturation which ranges from 8 to 12 and from 9 to 13 years of age in girls and boys respectively [122]. In affluent populations, the coefficient of variation (CV) is around 10%. It may even be larger in developing countries [123]. Pubertal timing is much easier to determine in females than in males. Indeed, the first menstruation represents a relatively precise milestone of sexual development, remaining a memorable event for most subjects. The occurrence of the first menstruation is a relatively late marker of pubertal maturation [122]. Nevertheless, it is a quite reliable milestone of pubertal maturation onset, since menarcheal age is highly correlated with the thelarcheal age, the time of first appearance of breast bud development [124]. The notion that pubertal timing within the physiological range is related to the risk of osteoporosis during adult life has been mostly documented in female subjects. Thus, in postmenopausal women, the risk of osteoporotic (fragility) fractures occurring at several skeletal sites, including forearm, spine and proximal femur, is associated with later age at menarche [125e128]. That these observations were related to an intrinsic bone fragility component was documented by other observations reporting low aBMD at these three skeletal sites in postmenopausal women with later menarche [129e132]. Taking into account the tracking phenomenon described above, it is no wonder that low aBMD was also recorded in premenopausal women with later menarche [133e135]. An inverse relationship between menarcheal age and osteoporosis risk is not limited to some regional distinctive characteristics, since

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DETERMINANTS OF PEAK BONE MASS AND STRENGTH

it was observed in adult women living in Asia, Europe and/or North America. More recently, prospective studies from prepuberty to time of PBM attainment in healthy subjects indicate that menarcheal age is inversely related to aBMD at several sites of the skeleton, including forearm, lumbar spine and proximal femur [21,22,136]. As a tentative explanation, this inverse relationship was supposed to be the expression of an uneven duration of estrogen exposure from sexual maturation to menopause [137e139]. However, recent data do not sustain this concept. Indeed, in a prospective study, the mean aBMD gain from prepuberty (mean age 8.9 years) to PBM (mean age 20.4 years) of six skeletal sites including radial metaphysis and diaphysis, femoral neck and shaft, trochanter and lumber spine, was not different in subjects having experienced a relatively earlier or later menarche [136]. In fact, the difference in PBM between those who experienced a relatively later vs earlier pubertal timing was already fully expressed before any sign of pubertal maturation [136]. Menarcheal age is under the strong influence of heritable factors as reported many years ago [140]. By the mid1930s, mean differences in menarcheal age between identical twins, non-identical twins, sisters and unrelated women had been found to be 2.2, 12.0, 12.9 and 18.6 months, respectively [140]. Further reports on menarcheal age corroborated these early findings by documenting a greater correlation coefficient within monozygotic (MZ, R ¼ 0.75) than dizygotic (DZ, R ¼ 0.31) twin pairs, as shown for instance, in a Finnish study [141]. From a mathematical analysis that included the contribution of body mass index (BMI) to pubertal onset, 74% and 26% of the variance in menarcheal age could be attributed to genetic and environmental factors, respectively [141]. The genetic regulation of pubertal timing in females is also supported by significant correlations between ages at which mothers and daughters experience their first menstruation [142]. Thus, like PBM, pubertal timing is under strong genetic influence. From twin models, it can be estimated that heredity accounts for about 75% of the variance of either menarcheal age [141] or PBM [59]. In the general population, both pubertal timing and PBM with its mechanical strength components are traits characterized by large variance and Gaussian distribution. Both variables are under the strong influence of heredity and moderately affected by environmental factors. Therefore, it was postulated that pubertal timing, PBM and consecutive osteoporosis risk later in life may well be part of common programming in which both genetic factors and in uteri influences are important determinants [47]. Both variables probably arise from the additive influence of multiple genes. Several variants in relation to either pubertal timing [143,144] or PBM [59,96,97,100,101,145] have been identified. However,

199

none of them can explain more than a few percent of the wide physiological variance in these two traits. By the end of the growth period in healthy females, the negative influence of later menarcheal age was expressed not only in lower aBMD but also in decreased cortical thickness without reduced cortical surface area (CSA) [21], a finding compatible with less endocortical accrual. Such structural characteristics could explain how later menarcheal age can increase the risk of forearm osteoporotic fractures in postmenopausal women [127]. Likewise, regarding the increased risk of hip fracture with later menarche [146] in young healthy women with later pubertal timing, lower femoral neck aBMD was associated with reduced cortical thickness and vBMD in the distal tibia [22]. Of note, the deleterious influence of later menarcheal age at weight-bearing sites was of similar magnitude in premenopausal middle-aged as compared to adult females in their early twenties [22]. Furthermore, this effect was independent of age-related premenopausal bone loss (Fig. 9.8) [22]. In growing males, pubertal timing was prospectively estimated by determining the age at peak height velocity (PHV), an anthropometric characteristic which occurred at a mean age of 13.6 years, ranging from 10.9 to 16.9 years [147]. Age at PHV was a negative predictor of several bone variables measured a few years later at an age close to PBM attainment [147]. In addition, an association was found between age at PHV and fracture incidence during growth [147]. A one year increment in age at PHV increased by 40% the risk of upper limb fracture occurring during growth [147]. Thus, in both healthy females and males, later pubertal timing is associated with low PBM, and increases the risk of fragility fractures in later life, taking account, as discussed above, that bone traits track during adulthood. In addition, the deficit in bone acquisition that is detectable before the first clinical expression of sexual maturation [136], may also explain the influence of pubertal timing on the risk of fracture during growth [46,147e149].

Mechanical Factors Mechanical forces impinge on the skeleton by enhancing osteoblastic bone formation, while inhibiting osteoclastic bone resorption [150]. The effect on osteoblast number and activity probably involve several local factors. Some appear to be produced by the osteocytes. The density, distribution and extensive communication network of osteocytes make them particularly well structured to function as detectors of mechanical strain by sensing fluid movement within the bone canaliculi. They can direct the formation of new bone by activating lining cells to differentiate into preosteoblasts [150]. A key molecule implicated in this

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9. PEAK BONE MASS AND ITS REGULATION

EARLIER LATER 0.6

Menarche

Femoral Neck aBMD (g/cm 2)

0.4

0.4

0.3 0.2

T-score

0.2

0.1

0

0 –0.1

–0.2

–0.2

–0.4

–0.3 –0.4

–0.6 –0.8 Age at menarche Age at examination

Distal Tibia vBMD (mg/cm3)

–0.5 –0.6

12.1 14.0 11.8 14.4 20.4 20.4 45.6 46.0 YAD

PREMENO

*P = 0.0001 #P

= 0.004

12.1 14.0

11.8 14.4

yrs

20.4 20.4

45.6 46.0

yrs

YAD

*Difference between YAD and PREMENO # Difference between EARLIER and LATER

PREMENO *P = 0.024 #P

= 0.0005

FIGURE 9.8 Deleterious impact of later pubertal timing on bone traits in healthy women. Two cohorts of young adult (YAD, 20.4 years, n ¼ 124) and middle-aged premenopausal (PREMENO, 45.8 years, n ¼ 120) women. Menarcheal age (MENA) represents a reliable marker of pubertal timing. Each cohort was dichotomized by the median of MENA into EARLIER and LATER. Values are T-scores (  SEM) of femoral neck aBMD and trabecular density (vBMD) of distal tibia measured by DXA and HR-pQCT, respectively. Age at MENA and bone trait measurements are indicated below the histogram. The data show the significant negative impact of later pubertal timing on peak femoral neck aBMD and tibial vBMD. The deleterious effect of later MENA remains unabated 25 years later, and adds to the significant age-dependent bone loss that occurs at these weight-bearing skeletal sites during the premenopausal years. Data are from Chevalley et al. [22].

mechanotransduction process appears to be sclerostin, the product of the SOST gene [151] (see later).

Age and Optimal Response to Loading Growing bones are usually more responsive to mechanical loading than adult bones. Physical activity increases bone mineral mass accumulation in both children and adolescents. However, the impact appears to be stronger before than during or after the period of pubertal maturation [152]. Children and adolescents involved in various competitive sports such as gymnastics, freestyle skiing, figure or speed skating, soccer, and therefore undergoing intense training, display increased bone mineral mass gain. The greater gain in aBMD or BMC in young athletes compared with less active controls is preferentially localized in weight-bearing bones, such as the proximal femur. Studies in adult elite 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 (see next section).

Exercise During Growth and Fracture Prevention in Adulthood The question whether the increased PBM induced by physical exercise will be maintained into old age and confer a reduction in fracture rate remains uncertain. A cross-sectional study of retired Australian elite soccer players suggested that this might not be the case [153]. However, in another study, benefits were attenuated but not lost [154]. Thus, in ice hockey and soccer players, although exercise-induced BMD benefits during growth are partially reduced after retirement from sports, higher PBM may contribute to the lower incidence of fragility fractures observed in retired athletes beyond 60 years of age compared to matched controls [154]. Some of the bone structural change can be retained after training discontinuation. This can be explainable when exercise produces an increased bone size as observed in adult former tennis players [155]. Higher humeral shaft side-to-side differences in crosssectional area, cortical area, bone strength index were recorded, consistent with the persistence of exerciseinduced periosteal expansion [155]. Nevertheless,

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DETERMINANTS OF PEAK BONE MASS AND STRENGTH

residual benefits to bone structural properties conferring increased resistance to loading may not be the only factor explaining the reduced fragility fracture risk recorded in former athletes [156,157]. A positive impact on skeletal muscle strength and improved movement coordination and balance may also contribute to the residual benefits [156].

Moderate Exercise for Public Health Program In the perspective of public health programs aimed at increasing bone mineral mass gain in children and adolescents, it is obvious that only physical exercise of moderate intensity, duration and frequency, but which would still be effective, can be taken into consideration. In children, prepubertal individuals or those at an early stage of sexual maturation, several interventions implemented within the school curriculum indicate that physical exercise can positively impact on bone development (Fig. 9.9) [158e165]. Nevertheless, it remains uncertain to what extent the greater bone mineral accrual in response to moderate and readily accessible weightbearing exercise is associated with a commensurate increase in bone strength. The magnitude of benefit in terms of bone strength will depend upon the nature of the structural change. The structural change underlying the beneficial effects of weight-bearing exercises on aBMD and BMC remains to be better determined. Surface-specific effects on long bones appear to be

pubertal maturation and sex specific [166]. Prior to puberty, exercise leads to increased periosteal apposition in both sexes, although weight-bearing bones could be more responsive to physical exercise in prepubertal or early pubertal boys than in girls [167]. During pubertal maturation, this specific surface effect persists in boys, whereas exercise in girls leads to endocortical contraction [166]. Basic concepts of biomechanics predict that an effect consisting primarily of an increased periosteal apposition should confer greater resistance than a response limited to the endosteal apposition rate leading essentially to a reduction in the endocortical “diameter” [157]. Skeletal Site Specificity Recent studies suggest site-specific differences in how the prepubertal skeleton develops in response to repetitive loading [168]. At some sites, such as the tibia diaphysis, loading will result in geometrical changes with larger bone and greater cortical area, whereas at sites consisting predominantly of trabecular tissue, such as the distal radius and tibia, physical activity may increase the volumetric mineral density [168]. Quantitative bone structural analysis in children and adolescents [27] will provide a clearer assessment of the actual effects of mechanical loading components, such as intensity, duration and frequency of various types of exercises on the size, geometry and mineral density of cortical and trabecular bones in children and adolescents.

Controls Jumpers Percent Change BMC 12

p < 0.01

p < 0.05

10 8 6 4 2 0

Femoral Neck

Lumbar Spine

FIGURE 9.9 Impact of increased physical activity on bone acquisition. Prepubescent children with an age range of 5.9 to 9.8 years were randomized into a jumping (25 boys and 20 girls) and a control group (26 boys and 18 girls). The jumping group performed 100 two-footed jumps off 61-cm boxes during the school day, three times a week, during 7 months, as shown by the pictorial representation on the left side of the figure. The control group performed during the same time non-impact stretching exercises. Seven months of jumping significantly improves gains in both femoral neck and lumbar spine BMC (g) as shown by the diagram on the right side of the figure. Data adapted are from Fuchs et al. [158].

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9. PEAK BONE MASS AND ITS REGULATION

Role of Energy Intake and Muscle Mass Development In healthy subjects, the energy intake is adjusted to increased physical activity. Hence it is difficult to ascribe the additional gain in bone mass to mechanical loading alone. Indeed, nutrients such as calcium and proteins that are usually consumed in various amounts in relation to physical activity could substantially contribute to the positive effect on bone mass acquisition. The independent mechanical contribution can be measured by the differential effect observed according to the skeletal sites solicited. However, the best evidence of the distinct effect of mechanical loading from concomitant increase in nutritional intakes is provided by studies on the use of rackets, as determined by measuring the difference between loaded and unloaded arms. It has been suggested that the exercise-induced gain in bone mass, size and strength essentially results from an adaptation secondary to the increase in muscle mass and strength [169]. However, this model has been challenged by several observations demonstrating that bone growth can be dissociated from muscle development [170,171].

GeneeEnvironment Interaction in the Bone Response to Physical Activity There is evidence from several studies that suggests geneeenvironment interactions in the skeletal responses to exercise in children and young adults. The findings from a cross-sectional study in pre- and early pubertal girls revealed that PvuII polymorphism in the estrogen receptor-alpha (ER-alpha) gene may modulate the effect of exercise on aBMD at loaded sites [172]. Girls with heterozygote ER-alpha genotype (Pp) and high physical activity had higher total body, lumbar spine and femur BMC and aBMD, in addition to greater tibia cortical thickness, than their low physical activity counterparts. Interestingly, no differences were found between the groups in bone properties at the distal radius, a nonweight bearing site. Consistent with these findings, Remes et al. [173] reported that changes in lumbar spine aBMD following an exercise intervention were associated with the ER-a genotype in men who completed the exercise training. Other retrospective [174] and cross-sectional studies [175] of the VDR genotype have also reported variances in the osteogenic responses to loading in young adults. The VDR genotype was also attributed to variance in the bone metabolic response following strenuous resistance training in young adult males [176]. There is also preliminary evidence for an association between a functional polymorphism in the interleukin-6 (IL-6) gene and the response of femoral cortical bone area to 10 weeks of training in male army recruits [177]. Together, these studies provide some

evidence that genetic polymorphisms may influence an individual’s response to mechanical loading. These data are important because they open a way to study inter-individual differences in the bone response to physical activity. Furthermore, they provide new information on how the human genome can affect the relationship between mechanical loading and bone health (or vice versa). Along this line, one may also speculate on the interaction of mechanical strain and bone formation in relation to osteocyte function [178e180]. Osteocyctes are terminally differentiated osteoblasts that become embedded within the newly mineralized matrix during bone formation. Each osteocyte has long cell processes or canaliculi that connect other osteocytes and surface lining cells [181]. The density, distribution and extensive communication network of osteocytes make them particularly well structured to function as detectors of mechanical strain by sensing fluid movement within the bone canaliculi [178e180]. They can direct the formation of new bone by activating lining cells to differentiate into pre-osteoblasts. A key molecule implicated in this mechanotransduction process appears to be sclerostin, the product of the SOST gene [182e184]. Patients with sclerosteosis and high bone mass can have mutations in either the LRP5 or SOST gene [87e89,182e184]. Sclerostin can bind and antagonize LRP5 [185], a Wnt coreceptor that is required for bone formation in response to a mechanical load [186]. Mechanical loading can induce a marked reduction of sclerostin in both osteocytes and in the canaliculi network [151]. Furthermore, evidence for a key role of this molecular pathway has been reported by demonstrating that administration of sclerostin monoclonal antibodies to primates leads to dramatic increases in bone formation, trabecular thickness, radial, femoral and vertebral BMD as well as bone strength [187]. Therefore, genes coding for the LRP5-Wnt co-receptor and sclerostin are implicated in the bone anabolic response to increased mechanical strain. Polymorphisms in the SOST gene region that may modulate its expression have been shown to be associated with aBMD in elderly white subjects [188]. Thus, it will be of interest to explore in the future whether polymorphisms already observed in both LRP5 [79,91,95] and SOST [188,189] genes may be associated with variability in the bone anabolic response to mechanical loading during infancy, childhood and adolescence.

Negative Impact of Intensive Physical Exercise Impaired bone mass acquisition can occur when intensive physical activity leads to hypogonadism and low body mass [190]. Both nutritional and hormonal factors probably contribute to this impairment.

PEDIATRIC BONE

DETERMINANTS OF PEAK BONE MASS AND STRENGTH

Disordered eating in combination with high physical activity expenditure can lead to a negative energy balance and menstrual cycle disturbance [191]. Intake of energy, protein and calcium may be inadequate as athletes go on diets to maintain an idealized physique for their sport. Intensive training during childhood may contribute to a later onset and completion of puberty. Hypogonadism, as expressed by the occurrence of oligomenorrhea or amenorrhea, can lead to bone loss in females who begin training intensively after menarche [190,192]. Reduced IGF-1, presumably mainly due to low protein intake, can explain the decreased bone formation, contributing to reduce bone acquisition during adolescence and bone loss in young adulthood in combination with estrogen deprivation.

Nutritional Factors The extent to which variations in the intake of certain nutrients by healthy, apparently well-nourished children and adolescents affect bone mass accumulation, particularly at sites susceptible to osteoporotic fractures in late adulthood, has received increasing attention over the last two decades. Most studies have focused on the intake of calcium. In a tight relationship with calcium, vitamin D is also presented in this section, because of its essential role in calciumephosphate economy, although its supply does not only depend on dietary sources. Moreover, several studies have reported on the relationship between the vitamin D status and bone acquisition, and/or on the effects of vitamin D supplementation. Proteins, the intake of which is essential for adequate bone accrual, not only as brick builders but also as a stimulators of the bone tropic growth factor IGF-1, are also considered in relation to bone acquisition.

Vitamin D Vitamin D plays a pivotal role in calcium homeostasis and bone mineral mass gain during growth. Nutritional sources of vitamin D, whether ergocalciferol (vitamin D2) or cholecalciferol (vitamin D3) are limited. Oily fishes represent the main dietary supply. Nevertheless, adequate sunshine exposure through the activity of the UVB radiation provides vitamin D3, by the conversion of dehydrocholecalciferol to cholecalciferol in the skin. (See Chapter 8 for detailed discussion on vitamin D metabolism and action.) The circulating level of 25-hydroxycholecalciferol (25OHD), the enzymatic conversion product of vitamin D in the liver, is a reliable marker of vitamin D status. Serum level of 25OHD < 15 nmol/L ( < 6 ng/mL) indicates a state of severe vitamin D deficiency that leads to rickets, bone deformities and increased risk of fracture due to impaired mineralization of osteoid and

203

cartilage growth plates, and secondary hyperparathyroidism. This last anomaly can be observed in less severe vitamin D deficiency associated with reduced bone mass. Observational Studies Lower forearm and/or tibia aBMD was associated with serum 25OHD level  25e40 mol/L, concentrations found in up to 62% of healthy females aged 10e16 years and living in Finland or Ireland [177,193,194]. In other epidemiological studies, a serum level of 25OHD < 30 nmol/L was recorded during winter and spring in 30e50% and in 17e35% of non-immigrant children living in European countries located in relatively higher [195,196] and lower latitudes [197,198], respectively. In several other regions of the world, vitamin D insufficiency and/or deficiency was also documented not only in elderly but also in children and adolescents [199,200]. The important notion that vitamin D is usually very low in human milk (z20 IU, or z0.5 mg/L), and is thus far from meeting the needs (400 IU ¼ 10 mg/day) of breastfed infants, is not fully appreciated, not only in the general population, but also among heath-care providers [201]. Inadequate supplementation during the first year of life can have not only short-term severe consequences [202] but also longer-term negative effects on bone health [203]. Furthermore, as recently reported, maternal vitamin D status appears to influence tibia mineral content and size during intrauterine life, as assessed by pQCT a few days postpartum [204]. In late pubertal girls (11 to 16.9 years), adverse interactions were seen between relatively low 25OHD (40 nmol/L) and calcium intake below 600 mg/day, as expressed by a significantly reduced lumbar spine aBMD or BMC as compared to subjects with higher dietary calcium and/or vitamin D status [205]. Interventional Studies Scarce supplemental vitamin D randomized control trials (RCTs) have so far been reported in children and adolescents. In girls living in Finland with mean age 11.4 years, vitamin D supplementation of 5 (200 IU) and 10 mg/day (400 IU) during one year, increased femoral BMC by 14.3 and 17.2%, respectively [206]. Only the 10 mg dose induced a positive effect on lumbar spine BMC [206]. In another randomized placebo controlled trial in girls aged 10e17 years living in Lebanon, total hip BMC measured by DXA significantly increased at an equivalent daily dose of 2000 IU ( ¼ 50 mg) taken during one year [207]. Dietary Vitamin D Recommendations Taking into account: (i) the relatively high prevalence of low vitamin D status, particularly in winter time, which seems to have negative effects on bone

PEDIATRIC BONE

204

9. PEAK BONE MASS AND ITS REGULATION

acquisition; (ii) the limited natural dietary sources of vitamin D and adequate sunshine exposure; (iii) the positive effect of supplemental vitamin D observed in interventional trials, it is recommended that not only infants, but also children and adolescents have a minimum intake of 400 IU (10 mg) per day [202]. To meet this intake requirement, recent specific recommendations from the American Academy of Pediatrics have been made for breastfed and non-breastfed infants, children with increased risk of vitamin D deficiency, and adolescents who do not obtain 400 IU (10 mg) per day through the consumption of fortified foods [202]. In November 2010, the US Institute of Medicine (IOM) released a report on “Dietary Reference Intakes for Calcium and Vitamin D” that proposes new reference values. Regarding vitamin D, for the life stage groups from 1 to 30 years, the estimated average requirement (EAR) and the recommended dietary allowance (RDA) are set at 400 and 600 IU/day, respectively (www.iom. edu/vitamind). In European children and adolescents, based on relatively high prevalence of lower limb deformities in relation to low serum 25OHD level combined with low calcium/dairy product intake, dietary fortification or supplementation with vitamin D was also recently recommended at least during winter/spring time [208].

Calcium In most regions of the world, the supply of calcium is sufficient to avoid the occurrence of clinically manifest bone disorders during growth. Nevertheless, by securing adequate calcium intake, provided the skin and food supply of vitamin D is adequate, it is expected

that bone mass gain can be increased during infancy, childhood and adolescence and thereby optimal PBM can be achieved. The prevention of adult osteoporotic fractures is the main reason for this widespread preoccupation. Observational Studies Retrospective epidemiological data obtained in women aged 20e49 years indicated that milk consumption during childhood and adolescence can be positively correlated to bone mineral mass [209]. In some but not all observational studies carried out during childhood and adolescence, a positive correlation between dietary calcium and bone mineral mass was reported [210,211]. In our own longitudinal prospective observational study, results were analyzed by taking into account the influence of age and pubertal maturation [211]. A significant positive relationship between total calcium intake, as determined by two 5-day diaries, and bone mineral mass accrual was found in the pubertal subgroup P1, but not in the P2eP4 or P5 subgroups (Fig. 9.10). These results suggested that dietary calcium might be more important before than during pubertal maturation [211]. Interventional Studies Several calcium intervention studies have been carried out in children and adolescents. (See the following for reviews [210,213].) Overall, these studies indicated a greater bone mineral mass gain in children and adolescents receiving calcium supplementation over periods varying from 12 to 36 months (Table 9.1). Nevertheless, the response appears to vary markedly according to several factors including the skeletal sites

Yearly Z-score Gain

Lumbar spine BMC

Calcium intake (mg/day)

FIGURE 9.10 Relation between calcium intakes and changes in lumbar BMC in pre-, peri- and postpubertal female and male adolescents. Mean calcium intakes from dairy, vegetable and mineral sources were recorded in two five-day diet diaries at one year interval in 193 healthy adolescents (96 females and 97 males) aged 9e19 years. Each dot corresponds to individual change in BMC adjusted for age and gender (Z score). A positive correlation was found in prepubertal (P1), but not in either peripubertal (P2eP4) or postpubertal (P5) subjects. The methods and corresponding macronutrient intakes are described in Clavien et al. [212]. The BMC data are from Theintz et al. [33].

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DETERMINANTS OF PEAK BONE MASS AND STRENGTH

TABLE 9.1 Calcium Intervention Trials. Annualized % Differences in aBMD or BMC Between Calcium Supplemented and Placebo Groups Triala (Calcium salt e mg/day)

Age (years)

Johnston et al. 1992 (Citrate malate e 1000)

Spontaneous Ca intake (mg/day)

Midradius aBMD/ BMC

LS aBMD/ BMC

FN aBMD/ BMC

FS aBMD/ BMC

Sex

n

Duration (months)

6e14

Twins (all) Prepubertal

90

36

900

0.8 1.7*

0.2 0.9*

0.1 0.4

NA

Lloyd et al. 1993 (Citrate malate e 350)

11e13

F

94

18

960

NA

2.0*/1.6#

NA

NA

Lee et al. 1994 (Calcium carbonate e 300)

6e8

F/M

162

18

280

1.1#

1.2

0.4

NA

Lee et al. 1995 (Calcium carbonate e 300)

6e8

F/M

84

18

565

NA/0.7b

0.6/2.7*

NA

NA

Bonjour et al. 1997 (Milk extracted Ca-Pi salt e 850)

6e9

F

144

12

900

1.7*

0.4

1.0

1.2*

Nowson et al. 1997 (Lactate gluconate e 1000)

10e17

F, twins

74

18

730

NA

1.5*

0.9

NA

Dibba et al. 2000 (Ca carbonate e 700)

8e12

F/M

160

12

340

3.9***/2.1

NA

NA

NA

Cameron et al. 2004 (Ca carbonate e 1200)

8e17

F, twins

48

24

715

NA

0.7

0.3

NA

Prentice et al. 2005 (Ca carbonate e 1000)

16e18

M

143

13

1200

0.3/0.8

1.0/2.5**

1.5*/2.4*

NA

Chevalley et al. 2005 (Milk extracted Ca-Pi salt e 850)

6e8

M

235

12

750

0.7/0.7

0.3/0.0

0.0/0.1

1.3**/1.3

Iuliano-Burns et al. 2006 (Milk minerals or Ca carbonate e 800)

5e11

F/M

99

10

800

NA

NA/0.1

NA

NA/0.2

Lambert et al. 2008 (Citrate malate e 792)

11e12

F

96

18

636

NA

2.1/4.0*

2.2*/2.3

NA

a

Analysis in Intention-to-Treat except in trials from Johnston et al., 1992, Lloyd et al., 1993, Nowson et al., 1997 and Cameron et al., 2004. P < 0.10 * P < 0.05 ** P < 0.01 *** P < 0.001. b By single photon absorptiometry. F ¼ females; M ¼ males; NA: not available; LS ¼ lumbar spine; FN ¼ femoral neck; FS ¼ femoral shaft. References: Johnston CC et al. N Engl J Med 1992;327:82e7; Lloyd T et al. J Am Med Assoc 1993;270:841e4; Lee WT et al. Am J Clin Nutr 1994;60:744e50 and Br J Nutr 1995;74:125e39; Bonjour JP et al. J Clin Invest 1997;99:1287e94; Nowson CA et al. Osteoporos Int 1997;7:219e25; Dibba B et al. Am J Clin Nutr 2000;71:544e9; Cameron MA et al. J Clin Endocrinol Metab 2004;89:4916e22; Prentice A et al. J Clin Endocrinol Metab 2005;90:3153e61; Chevalley T et al. J Clin Endocrinol Metab 2005;90:3342e9. Iuliano-Burns S et al. Osteoporos Int 2006;17:1794e800. Lambert HL et al. Am J Clin Nutr 2008;87:455e62. #

examined, the stage of pubertal maturation, the basal nutritional conditions, i.e. spontaneous calcium and protein intakes, the level of physical activity and the genetic background (Fig. 9.11). DIFFERENTIAL SKELETAL SITE RESPONSIVENESS

The benefit of supplemental calcium was usually greater in the appendicular than in the axial skeleton [210,213]. 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 in the proximal femur [210,213]. INFLUENCE OF PUBERTAL MATURATION

In agreement with our longitudinal observation in healthy subjects aged 8 to 19 years (see Fig. 9.10), the skeleton appears to be more responsive to calcium supplementation before the onset of pubertal maturation than during the peripubertal period [210,213]. Randomized placebo-controlled trials in twins are particularly informative in this regard [214]. Indeed,

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9. PEAK BONE MASS AND ITS REGULATION

Genetics Gender

Other Nutrients

Physical Activity

4

Gain induced by Ca Supplement

Skeletal Sites

Mean aBMD Difference (%) within Twin Pairs between Calcium Suppplemented and Placebo Groups

3

2 Spontaneous Ca Intake Vitamin D Status

Pubertal Stage 1

FIGURE 9.11

Potential factors influencing the bone response to calcium supplementation in healthy children and adolescents. As presented in Table 9.1, analysis of 12 randomized controlled trials reveals variability in aBMD or BMC response to calcium supplementation. Differences in annualized percent gains between calcium supplemented and placebo groups vary from less than 1.0% to 2.1, 2.2 and 3.9%, in lumbar spine, femoral neck and midradius, respectively. The impact of increasing calcium intake during growth appears to be better in boys than girls; in predominantly cortical than trabecular bone; before pubertal maturation; with relatively low spontaneous calcium consumption; and with increased physical activity, this factor possibly explaining the gender difference in the calcium response. Vitamin D status below a certain threshold may be expected to influence the calcium response. Interactions with some genotypes/ haplotypes, and pubertal timing have also been suggested. (See text for further details.)

the co-twin design to test the effect of calcium supplementation confers a substantial advantage in statistical power, compared with interventional studies in unrelated individuals. Co-twin studies strongly suggest that increasing calcium intake after the onset of pubertal maturation above a daily spontaneous intake of about 800e900 mg does not exert a significant positive effect on bone mineral mass acquisition (Fig. 9.12) [215,216]. This contrasts with the widespread intuitive belief that the period of pubertal maturation, with its acceleration of bone mineral mass accrual, would be the most attractive time for enhancing calcium intake well above the prepubertal requirements. As described above, efficient adaptive mechanisms secure an adequate bone mineral economy in response to the increased demand of the peripubertal growth spurt. Thanks to these adaptive processes, one can infer that the dependency on environmental mineral supply to secure bone growth demand is not necessarily much increased during the peripubertal period as compared to the years preceding the onset of sexual maturation [214]. SPONTANEOUS CALCIUM INTAKE

As intuitively expected, the benefit observed at the end of intervention is particularly substantial in children with a relatively low calcium intake [210,213]. In 8-year-old prepubertal girls with relatively low spontaneous calcium intake, increasing the calcium intake resulted in an additional gain by about 0.25 SD as

0 Prepubertal Twin Pairs

Pubertal or Postpubertal Twin Pairs

FIGURE 9.12 Influence of pubertal status on the response to calcium supplementation in a twin study. Mean percent differences in aBMD measured at several skeletal sites, including midshaft and distal radius, lumbar spine and hip, after 3 years of intervention. The average difference in aBMD within twin pairs between the calciumsupplemented and the placebo groups was significant in prepubertal twins (þ2.9%), but not in peripubertal and postpubertal twins (þ0.3%). Data adapted from Johnston et al. [215].

compared to the placebo group after one year of supplementation. In contrast, the additional gain was minimal in those girls with a relatively high calcium intake (Fig. 9.13) [217]. Therefore, below a certain threshold in the spontaneous supply, it is quite likely that increasing the calcium intake can “push upward” the individual’s bone growth trajectory and thereby positively influence the value of PBM. According to the “programming” concept, environmental stimuli during critical periods of early development can provoke long-lasting modifications in structure and function of various biological systems [218]. Interventions limited to the first period of life may modify the trajectory of bone mass accrual [219]. This concept received some support in relation to calcium economy since, as discussed above, vitamin D given in physiological doses (400 IU ¼ 10 mg/day) to female infants for an average of one year was associated with a significant increase in aBMD measured at the age of 7e9 years [203]. In this study, the aBMD difference between the vitamin D-supplemented and non-supplemented infants was most significant at the femoral neck, trochanter and radial metaphysis [203]. CALCIUM INTAKE AND PHYSICAL ACTIVITY INTERACTION

The possibility that physical activity could modulate the bone response to dietary calcium supplementation during growth has been considered in infants, children and adolescents. Overall, the results suggest an interaction: the higher the calcium intake, the more positive the

PEDIATRIC BONE

DETERMINANTS OF PEAK BONE MASS AND STRENGTH

Spontaneous Ca intake (mg/day)

<

Median 855 > Placebo Ca Suppl.

6 aBMD Gain (% per year)

*** 5 4 3 2 1 0 Total Ca Intake (mg/day±SEM)

694 1238 ±16 ±56

1175 1805 ±64 ±54

FIGURE 9.13 Influence of spontaneous calcium intake on change in aBMD in response to calcium-supplementation in prepubertal girls. Bars are means (  SEM) of aBMD change after 12 months of intervention. Average of six skeletal sites, including lumbar spine, radial diaphysis and radial metaphysis, femoral neck, femoral diaphysis and trochanter. The median spontaneous calcium intake is indicated above the bars. The total calcium intake of each group is indicated below the bars. ***P < 0.001 for the calcium supplemented (Ca Suppl.) as compared to the corresponding placebo group with spontaneous calcium intake below the median. Intention-to-Treat cohort (n ¼ 144) with mean age at baseline of 7.9 years. Data adapted from Bonjour et al. [217].

207

effect that increased physical activity exerts on bone growth. At moderately low calcium intake, the effect may not be positive. Thus, in a longitudinal study in infants 6e18 months of age, i.e. during rapid bone growth, loading of the skeleton was associated with a reduced increase in total body BMC in the presence of a moderately low calcium intake [220]. In young children aged 3e5 years, the bone response to calcium supplement was greater in children with gross than fine motor activity (Fig. 9.14) [221,222]. Furthermore, in another study in 8e9-year-old girls, greater gains in bone mass at weight-bearing skeletal sites were observed when moderate exercise was combined with calcium supplementation [223]. Likewise, in premenarcheal girls, calcium supplementation significantly enhanced the positive impact of physical activity in the femoral neck but not in the forearm [224]. Thus, the positive interaction of calcium intake and physical activity appears to be region specific. This regional specificity suggests that the effect of physical activity alone or combined with relatively high calcium supply is not merely due to an indirect influence on the energy intake, which in turn would positively affect bone mass acquisition.

FIGURE 9.14

Interaction between calcium intake and physical activity. Results from a randomized trial, enrolling 239 children aged 3e5 years, designed to test of interaction between physical exercise and calcium intake [221]. Half the children were randomized to participate in a gross motor activity program involving bone-loading exercises, 30 min per day, 5 days per week for 1 year. The other half of children were assigned to a fine motor activity program that was designed to keep them sitting quietly. On the right part, the change in leg BMC measured by DXA shows a significant interaction of exercise-by-calcium intake. The images above the bars illustrate the pQCT findings of the cross-section analyzed at 20% of distal tibia. In the left upper part is schematically illustrated the periosteal and endosteal circumference and thickness of the cortical shell (gray-shaded area) in which cortical volumetric (v)BMD can be measured by pQCT. These morphometric changes suggest that physical activity increases periosteal circumference, whereas calcium supplementation appears to decrease endosteal expansion. Data adapted from Specker and Binkley [222].

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9. PEAK BONE MASS AND ITS REGULATION

INDIVIDUAL HETEROGENEITY IN BONE RESPONSE TO CALCIUM SUPPLEMENTATION

This notion is illustrated in two randomized doubleblind placebo-controlled trials in which the effect of calcium supplementation (850 mg/day for one year) was examined in prepubertal girls and boys (Fig. 9.15A and B) [217,225]. The greatest response in terms of gain in aBMD was observed at the femoral

Analysis of calcium intervention trials indicate that there is a marked interindividual variability in bone mineral accrual, despite recruitment of homogeneous cohorts with respect to age and pubertal maturation.

Yearly Gain in FS aBMD (mg/cm2 ±SEM)

(A)

Mean Gain

6.4%

80 70

6.3%

5.3%

Ca suppl. (+850 mg/d)

60 50

53

88

55

86

N

40

Placebo

30 20 10 0

Baseline Age (yrs) Height (cm) Weight (kg) Ca Intake (mg/d)

(B)

Yearly Gain in FS aBMD (mg/cm2)

P < 0.001 8.1%

P < 0.01

90

Girls

Boys

7.9 128

7.4 126

27 890

25 760

Individual Gain Range

180

P < 0.001

160

*** P < 0.01

140

100

Ca-suppl. (+850 mg/d)

**

120 53

88

55

86

80

+ 1.8%

+ 1.1%

60

N

Placebo 40 20 0 –20 –40

Girls

Boys

FIGURE 9.15 Gain of femoral shaft aBMD in prepubertal girls and boys: response to calcium supplementation. Data are from two randomized, placebo controlled trials [217,225]. The calcium supplement (Ca suppl.) was given during one year. All subjects remained prepubertal at the end of the intervention. Femoral shaft was the most responsive site to the calcium supplementation among the six sites examined that also included: lumbar spine, radial metaphysis and diaphysis, femoral neck and trochanter. The calcium supplementation was given as phosphate salt extracted from milk. This salt was used to enrich several foods: cakes, biscuits, fruit juices, powdered drinking chocolate or chocolate bars. It is important to underscore that the same foods were consumed by the placebo groups, so that the ingested amounts of energy, proteins, lipids, and glucides were identical in the two groups. Mid femoral shaft (FS) was measured by DXA. (A) Mean gain. The baseline characteristics are indicated under the histogram. In the placebo groups, the mean gain (  SEM) in FS aBMD was slightly greater in boys than in girls. Likewise, the response to calcium supplementation, þ1.8% as compared to þ1.1% in girls. (B) Individual gain range. In both genders, there was a wide range of FS aBMD gain. In girls, the effect of calcium supplementation was to increase the lower limit of the range without affecting the upper limit. In contrast, in boys the effect of calcium supplementation appeared to shift upward the whole range of FS aBMD gain. (See text for further details.)

PEDIATRIC BONE

DETERMINANTS OF PEAK BONE MASS AND STRENGTH

shaft. In both trials, the differences between the placebo and the calcium-supplemented group (Ca) were significant (P < 0.01) in both the intention-to-treat and the active-treatment cohorts [217,225]. In the active treatment cohort of girls (mean age at entry: 7.9 years, range 6.6e9.4), the yearly gain in femoral shaft aBMD was 54  4 and 66  3 mg/cm2 (mean  SEM, P < 0.01) in the placebo and Ca supplemented group, respectively (Fig. 9.15A) [217]. This represented a gain of 5.3% and 6.4% in the placebo and Ca supplemented groups, respectively. In the active treatment boy cohort (mean age at entry: 7.4 years, range 6.5e8.5), the yearly gain in femoral shaft aBMD was 64.3  4 and 76.3  3 mg/ cm2 (P < 0.01) in the placebo and Ca supplemented group, respectively [225]. This represented a gain of 6.3% and 8.1% in the placebo and Ca supplemented group, respectively (Fig. 9.15A). In both studies, there was a wide range of individual bone mineral mass accrual [217,225]. In girls, the yearly gains in femoral shaft aBMD ranged from 19 to þ127 mg/cm2 and from þ10 to þ122 mg/cm2 in the placebo and Ca supplemented groups, respectively [217] (see Fig. 9.15B). In boys, the yearly gains ranged from 12 to þ140 mg/cm2 and from þ2 to þ160 mg/ cm2 in the placebo and Ca supplemented groups, respectively (see Fig. 9.15B) [225]. Similar ranges of aBMD gains were recorded at the other five skeletal sites measured: radial metaphysis and diaphysis, femoral neck and trochanter, and lumbar spine. However, in neither study could the marked variability in bone mineral accrual be explained, even to a small extent, by interindividual differences in the spontaneous (baseline) dietary calcium consumed during the intervention year. Furthermore, in boys, the large variance in aBMD gains was not significantly reduced after adjustment for both physical activity and dietary protein intake [225]. GENEeENVIRONMENT INTERACTION IN RESPONSE TO DIETARY CALCIUM

As discussed above in relation to the impact of physical activity on prepuberty, compared to peri- or postpuberty, may represent an opportune time for environmental factors to modify the genetically predetermined bone growth trajectory [214]. The relatively modest effect of calcium supplementation, even at the femoral shaft, a weight-bearing cortical bone site, contrasts with the large variability in bone mass accrual observed in healthy prepubertal children. The strong influence of heritability in peak bone mass variance does not exclude the possibility that environmental factors, either mechanical or nutritional in nature, modulate the expression of genetic susceptibility to osteoporosis. Variability in the bone response to environmental factors can have several origins in relation to

209

individual genetic profiles. With regard to nutrition, food ingredients may modulate the induction or expression of genes. They can also modify, by quantitative or qualitative alterations, the activity of protein gene products and their metabolites. Nutrients may also interact with variants in the coding or promoter sequences of specific genes, thus modulating the level of expression, or the number copies or even the function of protein products. These kinds of interaction responsible for interindividual variability belong to the field termed “nutrigenetics” [226e229]. Whether some of these genetic polymorphisms may alter bone mineral accretion in response to specific nutrients with or without physical activity is a vast research domain which, to date, has received little attention. However, such an interaction has been explored in relation to calcium intake and VDR polymorphisms [230]. Geneeenvironment interactions may explain the inconsistent relationships reported between bone mineral mass and vitamin D receptor (VDR)-30 and 50 -genotypes [80e82,231,232]. Significant aBMD differences between VDR-30 BsmI genotypes (BB, Bb and bb) were detected in children [230,233], but were absent in premenopausal women with the same genetic background [231]. Moreover, the latter study found that aBMD gain in prepubertal girls increased at several skeletal sites in Bb and BB girls in response to calcium supplements. In contrast, calcium supplementation had no apparent effect in bb girls, who had a trend for greater aBMD gain than other genotypes on their usual calcium diet [230,231]. Accordingly, a model taking into account the early influence of VDR-30 polymorphisms, calcium intake and puberty on aBMD gain has been proposed to explain the relation between these genotypes and peak bone mass [230,231]. The theory that VDR-30 alleles together with environmental calcium might exert an indirect and complex influence on peak bone mass by regulating skeletal growth remains speculative [59,231]. Therefore, the possibility that calcium supplementation could be associated with a greater bone mass response in carriers of the VDR allele B needs to be investigated in prospective, calcium-dosing trials stratifying the cohort by VDR genotypes [59,230,231]. This type of investigation will require a large investment and intricate study design. OTHER UNCERTAINTIES

It has not been established whether the type of calcium salt used to supplement diets may modulate the nature of the bone response. The observation that calcium supplementation can increase bone size, at least transiently, has been observed using either milk extracted calcium phosphate or calcium carbonate salt [210,211]. It is interesting to note that an effect on bone size has been observed in response to whole milk supplementation [234]. In this type of intervention, the

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9. PEAK BONE MASS AND ITS REGULATION

effect could be due to other nutrients, or their association with calcium, such as milk proteins. The associated increase in circulating IGF-1 supports a key role of milk proteins on bone size [234]. Another uncertainty is the question of whether gains observed by the end of the intervention are maintained or lost after discontinuation of calcium supplementation. A clear answer to this question requires long-term follow up, since sustained gain even on bone mass and size may be transient, possibly resulting from some indirect influence of calcium supplementation on the tempo of pubertal and thereby bone maturation [235,236].

(IOM) as indicated in its report released on November 2010 (www.iom.edu/calcium) (Table 9.2). For female adolescents aged 11e14 years, a similar calcium intake difference of 500 mg/day (800 vs 1300 mg/day) separates the UK from the US recommendations (Table 9.2). Such a variability in calcium intake recommendations can partly be explained by the discrepant results obtained in observational and interventional studies (see Table 9.1). As discussed above, numerous factors can modulate the bone response to calcium intake, hence, the difficulty to reach a scientifically based worldwide consensus on dietary allowance recommendation for children and adolescents.

Dietary Calcium Recommendations International and national agencies have adopted recommendations for calcium intake from infancy to the last decades of life. Decisions from these recommending bodies can be based on either calcium balance e enabling the estimations of maximal retention e or on the factorial calculation that takes into account calcium accretion for growth, and the amount needed to cover endogenous losses, adjusted for fractional absorption to determined required intake. Observational and interventional studies are also taken into consideration. The recommendations vary widely among regional agencies (Table 9.2) [237e240]. Thus, for children aged 4e10 years, the recommended daily calcium intakes can vary from 450 to 550 mg/day for the UK Reference Nutrient Intake (RNI) to 1000 mg/day for the US Institute of Medicine

Proteins Effect of Protein Intake on CalciumePhosphate Economy and Bone Metabolism Protein supply from foods is required to promote bone formation. As for any other organs of the body, amino acids are required for the synthesis of intracellular and extracellular bone proteins, and other nitrogen-containing compounds. Besides this role as “brick supplier”, proteins, through some of their amino acids, can influence calciumephosphate economy and bone metabolism. Thus, dietary proteins stimulate the formation of IGF-1 from hepatic cells, which are the main source of this circulating growth factor (Fig. 9.16).

TABLE 9.2 Dietary Calcium Reference Values for Toddlers, Children and Adolescents Country Age group (years)

A/G/S RNI

UK RNI

UK LRNI

EU PRI

French ANC

Nordic NR

USA RDA

Canadian RNI

USA/ Canadian AI

Aust/NZ RDI

1e3

600

350

200

400

600

600

700

500e550

500

700

4e6

700

450

275

400

700

600

600

800

800

7-10

900

550

325

550

700 (7e9)

700

1000 (4e8)

700 (7e9)

800 (7e8) 1300 (9e10)

900 F (8e11) 800 M (8e11)

11e14 M

1100e1200

1000

325

1000

1000 (10e12) 1200 (13e14)

900

900 (10e12) 1100 (13e15)

1300

1200 (12e15)

11e14 F

1100e1200

800

325

800

1000 (10e12) 1200 (13e14)

900

1100 (10e12) 1000 (13e15)

1300

1000 (12e15)

15e18 M

1200

1000

480

1000

1200

900

900 (16e18)

1300

1000 (16e18)

15e18 F

1200

800

450

800

1200

900

700 (16e18)

1300

800 (16e18)

1300 (9e13)

1300 (14e18)

Values are in mg per day. F ¼ females; M ¼ males. Subgroup year ranges are indicated in parenthesis. Adapted from: (a) Valeurs de re´fe´rence pour les apports nutritionnels. Socie´te´ Suisse de Nutrition 2002. (b) Departement of Health Nutrition and Bone Health: with particular reference to calcium and vitamin D. Report on Health and Social Subjects 1998; 49. (c) World Health Organization Prevention and management of osteoporosis. Report of a scientific group. WHO Technical Report Series 2003; 921. (d) Dietary Reference intakes for calcium and vitamin D. Institute of Medicine of the National Academy. Report Released November 2010. (www.iom.edu/calcium). (e) Apports Nutritonnels Conseille´s pour la Population Franc¸aise. 3rd edn. TEC & DOC. 2001. A/G/S RN ¼ Austrian-German & Swiss Reference Nutrient Intake; UK RNI ¼ UK Reference Nutrient Intake; UK LRNI ¼ UK Lower Reference Nutrient Intake; *EU PRI ¼ European Union Population Reference Intake; ANC ¼ Apports Nutrionnels Conseille´s; NR ¼ Nutrition Recommendations; RDA ¼ Recommended Dietary Allowance; RNI ¼ Recommended Nutrient Intake; AI ¼ Adequate Intake; Aust/NZ RDI ¼ Australian & New Zealand Recommended Dietary Intake; *A nutrient intake level notionally representing 2 SD below the estimated average requirement (EAR). People habitually having intakes less than the LRNI will almost certainly be deficient.

PEDIATRIC BONE

DETERMINANTS OF PEAK BONE MASS AND STRENGTH

Skin

Dietary Supply

Liver

Vit D

Vit D, Ca, Pi, Proteins IGF-I

GH

+

Bone

a.a Gut G t

Ca Pi

Ca Pi

+

TmPi

Blood IGF-I

Heredity Sex Hormones Mechanical Forces

+ 1,25D

Kidney

+

211

FIGURE 9.16 Relation between essential nutrients for bone mass accrual, insulin-like growth factor-1 (IGF-1), and calcium phosphate metabolism during growth. The hepatic production of IGF-1 is under the positive influence of growth hormone (GH) and essential amino acids (a.a.). IGF-1 exerts a direct action on bone growth. In addition, at the kidney level, IGF-1 increases both the 1,25-dihydroxyvitamin D (1,25D) conversion from 25-hydroxyvitamin D (25D) and the maximal tubular reabsorption of Pi (TmPi). By this dual renal action IGF-1 favors a positive calcium and phosphate balance as required for the increased bone mineral accrual. The effects of essential nutrients for bone growth can interact with factors such as heredity, mechanical forces, sex hormones and risk factors. (See text for further details.)

+

25D

Ca Pi

An increment in the circulating level of IGF-1 can be observed in response to increased protein intake. This effect can be observed in the absence of any difference in dietary energy supply [241,242]. As decribed above, by stimulating IGF-1, food proteins can also exert a favorable impact on bone mineral economy by a dual renal action (see Fig. 9.16). The indirect positive effect of proteins on intestinal calcium absorption, via the IGF-1-1,25(OH)2D link, is associated with a direct stimulatory effect of amino acids such as arginine and lysine on calcium translocation from the luminal to the contraluminal side of the intestinal mucosa (see below). The overall effect of increased protein intake is enhanced intestinal calcium absorption, and this accounts for the associated increased calciuria. In fact, the increased urinary calcium excretion which can be associated with a high protein diet does not result in negative skeletal calcium balance that would reflect bone loss [243]. Another effect of dietary proteins on mineral metabolism could include an inhibitory activity of L-amino acids on Ca2þ-sensing receptors (CaR), leading to a decrease in PTH secretion [244]. Furthermore, broadspectrum amino acid-sensing receptors from class 3 of the G-protein coupled receptors superfamily that also includes CaR, could be involved in the positive impact of protein on bone acquisition [245]. The stimulatory effect of arginine on the osteoblatic production of both IGF-1 and collagen synthesis [246] could be mediated by these amino acid sensing receptors [245]. Protein Intake and Bone Acquisition Both animal and human studies indicate that low protein intake per se could be particularly detrimental

to bone acquisition [242]. Malnutrition, including inadequate supplies of energy and protein during growth, can severely impair bone development [247]. An inadequate protein supply appears to play a central role in the pathogenesis of the delayed skeletal growth and reduced bone mass that is observed in undernourished children. Low protein intake could be detrimental to skeletal integrity by lowering the production of IGF-1 [248]. 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 dietary protein intake. Indeed, the plasma level of IGF-1 is closely related to the growth rate of the organism. In humans, circulating IGF-1 rises progressively from 1 year of age to reach peak values during puberty. As mentioned above, this factor appears to play a key role in calciumephosphate metabolism during growth. Furthermore, the positive effect of dietary proteins on bone longitudinal growth, by stimulating the proliferation and differentiation of chondrocytes in the epiphyseal plate, appears to be mediated, at least in part, by IGF-1. Likewise, the physiological link between protein intake and IGF-1 production exerts positive effects on trabecular and cortical bone formation. During growth, this nutritionaleendocrine link influences bone mass and size by increasing the external diameter of long bone, probably by enhancing the process of periosteal apposition. Therefore, during childhood and adolescence, a relative deficiency in IGF-1 or a resistance to its action due to an inadequate supply in protein [242] may result in a reduction in the skeletal longitudinal growth, and impaired width- or cross-sectional bone development.

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9. PEAK BONE MASS AND ITS REGULATION

FN BMC

FN aBMD

0.4

Physical activity (kcal.d–-1) Protein intake (g. d–-1)

0.2 0.1 0

– 0.2 – 0.3

>

< > < >

< < > >

< < > >

< > <

P =0.006 0.6 0.5 0.4 0.3 0.2 0.1 0 – 0.1 – 0.2 – 0.3 – 0.4

Z-score ± SEM

0.3

– 0.1

–0.1 -0.1 –-0.2 –0.3 -0.3 –0.4 -0.4

FN AREA or FN Width

P =0.040

0.5

0.6 0.5 0.4 0.3 0.2 0.1 0

Z-score ± SEM

Z-score ± SEM

P =0.001

< > < > < < > >

Median

FIGURE 9.17 Influence of protein intake on the impact of increased physical activity on BMC, aBMD and AREA or width of femoral neck (FN) in prepubertal boys. Data, as expressed in Z-scores, are from 237 healthy boys, with mean age 7.4 years. They are distributed in four subgroups according to the median of both physical activity and protein intake. Increased physical activity from 168  40 to 303  54 (SD) kcal/ day was not associated with a significant increment in Z-score of FN variables, when protein intake was below the median (38.0  6.9 g/day). In contrast, increased physical activity of a similar magnitude, i.e. from 167  33 to 324  80 kcal/day was associated with a marked increment in FN variables, when protein intake was above the median (56.2  9.5 g/day). In this situation of relatively high protein intake, the greater FN Zscore associated with more sustained physical activity was þ0.66 and þ0.59 for BMC and AREA. The morphometric difference reflected a larger width of the femoral neck, since for all DXA scans the height of the region of interest parallel to the femoral axis was constant. (Adapted data from Chevalley et al. [262]. See text for further details.)

In “well-nourished” children and adolescents, the question arises whether or not variations in the protein intake within the “normal” range can influence skeletal growth and thereby modulate the influence of genetic determinants on peak bone mass attainment [6]. In the relationship between protein intake and bone mass gain, it is not surprising to find a positive correlation between these two variables [242,249,250]. Similar to calcium intake, the association appears to be particularly significant in prepubertal children [242]. These observations suggest that relatively high protein intakes could favor bone mass accrual during childhood. Positive relationships were reported between increased calcium intake from dairy foods and bone mineral acquisition during childhood and adolescence [234,251e254]. The associated increase in protein intake from dairy foods may have substantially contributed to such positive relationships. Likewise, in the often-quoted study carried out in two Yugoslav populations, the difference in young adult bone mass has usually been ascribed to calcium intake [255]. However, both protein intake and physical activity were also associated with peak bone mass [255]. The reasons why protein intake was not considered early on as an essential nutrient for calcium economy and bone health appears to stem from longterm prejudice or “belief sytems in the conduct of nutritional science” as thoughtfully analyzed by Heaney [256]. Claims against dietary proteins, particularly against those from animal food sources were mainly

based on a questionable dietary acid load hypothesis which has been refuted by several analyses or metaanalyses of experimental data [257e261]. In order to determine the quantitative relationship between protein intake and bone mass acquisition during childhood and adolescence, interventional studies testing different levels of protein intakes in otherwise isocaloric diets remain to be conducted. Furthermore, calcium requirement for optimal bone mass accrual could vary according to the level of protein intake. Thus, the possible positive interaction between protein and calcium intake deserves to be investigated in the perspective of increasing peak bone mass by modifying bone trophic nutrients. Interaction of Protein Intake and Physical Activity As already underscored, growing bones are usually more responsive to mechanical loading than adult bones. Increased physical activity was shown to increase bone mineral mass accumulation in children and adolescents. The positive impact of increased physical activity on bone acquisition might be greater before than during or after the period of pubertal maturation [6], although this pubertal maturation modulation may depend upon the skeletal site (axial vs appendicular) and/or structural (cortical vs trabecular) components examined [166]. Adequate nutritional supply can be expected to sustain the anabolic effect of mechanical loading on bone tissue as it does on skeletal muscle development.

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CONCLUSIONS

Among nutrients, high calcium intake was shown to enhance the response to physical activity in healthy children aged 3e5 years [221]. In healthy children and adolescents aged 6e18 years, long-term protein consumption exerts a stronger impact than calcium intake on bone mass and strength acquisition [249]. That high protein intake may enhance the bone response to increased physical activity has been recently reported in 8-year-old prepubertal boys [262]. At the femoral neck level, the increased aBMD and BMC was associated with a wider external perimeter (Fig. 9.17) [262]. Such an effect on the macroarchitecture of the femoral neck would be compatible with a greater resistance to mechanical load [166,263]. Potential of Increased Protein Intake in the Management of a Genetic Bone Disorder An interesting observation has been reported regarding the effect of a specific nutritional intervention to correct a defect in skeletal development. The CoffineLowry syndrome is a rare X-linked disorder in which males show severe mental retardation associated with several skeletal defects including short stature, kyphosis and/or scoliosis [264]. The skeletal manifestations worsen over time. The genetic defect is caused by loss-of-function mutations in a gene (RPS6KA3) coding for a growth factor-regulated protein kinase hRSK2 [265]. One mechanism whereby RSK2 favors skeletal development and bone formation appears to be by phosphorylating ATF4. This transcriptional factor itself regulates osteoblast differentiation during development and favors bone formation postnatally [266]. ATF4 exerts a stimulatory effect on amino acid import in eukaryotic cells [267]. Likewise, in osteoblasts, this transcription factor stimulates the amino acid import and regulates the synthesis of type I collagen, the main constituent of the bone matrix [266]. ATF4 deficiency in mice results in delayed bone formation during embryonic development and low bone mass throughout postnatal life [266]. Interestingly, a high protein diet in ATF4 deficient mice normalizes osteoblast differentiation and collagen synthesis in bone [268]. Furthermore, both bone formation and bone mass are enhanced. These observations suggest that the severe expression of genetic defect could be alleviated by simple dietary manipulation [269]. Dietary Protein Recommendations According to several national and international agencies, the daily recommended protein allowance for children and adolescents declines slightly from age 5 (1.05 g/kg b.w.) to 17 years (0.80 g/kg b.w.) [270e273]. In sharp contrast to a recommended amount of approximately 0.9e1.0 kg/b.w. per day for children aged 7e12 years, surveys made from several countries

in Europe, as well as in North America and Australia have recorded much higher daily protein intake of about 1.9e2.0 g/kg b.w. [24,212,249,250,262,273,274]. It has been underscored that the literature is essentially void of studies specifically directed at quantifying protein needs of healthy children between 7 and 12 years of age [275]. Current recommendations for protein intake in children remain speculative, since they correspond to estimates derived from interpolation of very shortterm nitrogen balance studies carried out in infants and young adults [272,275,276]. As described above in prepubertal boys aged 7.4 years (see Fig. 9.17), increased physical activity from about 170 to 315 Kcal/day in the presence of a spontaneous protein intake of z2.0 instead of 1.5 g/kg b.w. per day was associated with higher mean BMC at six skeletal sites of þ 0.64 vs þ 0.15 Z-score. This difference of z0.5 Z-score could well be expressed by a substantial shift of the BMC trajectory, resulting in a higher PBM and possibly in a reduction of fragility fractures in adulthood [15]. Likewise, such an upward shift in bone mass acquisition could also reduce the incidence of fractures during growth [46,147].

CONCLUSIONS Bone mass and strength achieved at the end of the growth period, simply designated as “peak bone mass (PBM)”, play an essential role in the risk of osteoporotic fractures occurring in adulthood. It is considered that an increase of PBM by 1 standard deviation would reduce the fracture risk by 50%. As estimated from twin studies, genetics is the major determinant of PBM, accounting for about 60e80% of its variance. Numerous polymorphisms of “candidate” genes have been found to be associated with the areal bone mineral density (aBMD), so far the most convenient measurable surrogate of bone mass and strength. The studied genes code for molecules implicated in bone function and structure such as circulating endocrine factors, hormone receptors, local regulators of bone modeling and remodeling or matrix molecules. None of these genes appears to account for more than a few percent of PBM variance. Before puberty, there is no substantial gender difference in aBMD when adjusted for age, nutritional factors and physical activity. During pubertal maturation, the size of the bone increases whereas the volumetric bone mineral density remains constant in both genders. At the end of puberty, the sex difference is essentially due to a greater bone size in male than female subjects. This is achieved by larger periosteal deposition in boys, thus conferring at PBM a better resistance to mechanical forces in men than in women. Sex hormones and the IGF-1 system are implicated in the bone sexual dimorphism occurring

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during pubertal maturation. The genetically determined trajectory of bone mass development can be modulated to a certain extent by modifiable environmental factors. Interventions aimed at increasing the physical activity of children have been shown to exert a positive impact on cortical bones of the weight-bearing appendicular skeleton. Likewise, interventional studies using either calcium salt or milk supplementation have documented an increased bone mass gain, particularly at weightbearing cortical bones. Some studies suggest a positive interaction between calcium supplementation and increased physical activity on bone development. In healthy prepubertal boys, the impact of increased physical activity affecting bone acquisition is enhanced by protein intake within limits above the usual recommended allowance. Prepuberty appears to be an opportune time to modify environmental factors that impinge on bone mineral mass acquisition and eventually peak bone mass and strength.

References [1] Block JE, Genant HK, Black D. Greater vertebral bone mineral mass in exercising young men. West J Med 1986;145:39e42. [2] de Deuxchaisnes CN, Devogelaer JP, Esselinckx W, et al. The effect of low dosage glucocorticoids on bone mass in rheumatoid arthritis: a cross-sectional and a longitudinal study using single photon absorptiometry. Adv Exp Med Biol 1984;171: 209e39. [3] Crosby LO, Kaplan FS, Pertschuk MJ, Mullen JL. The effect of anorexia nervosa on bone morphometry in young women. Clin Orthop Relat Res 1985;201:271e7. [4] Davis JW, Grove JS, Ross PD, Vogel JM, Wasnich RD. Relationship between bone mass and rates of bone change at appendicular measurement sites. J Bone Miner Res 1992;7:719e25. [5] Buchs B, Rizzoli R, Slosman D, Nydegger V, Bonjour JP. Densite´ mine´rale osseuse de la colonne lombaire, du col et de la diaphyse fe´moraux d’un e´chantillon de la population genevoise. Schweiz Med Wochenschr 1992;122:1129e36. [6] Bonjour JP, Chevalley T, Rizzoli R, Ferrari S. Gene-environment interactions in the skeletal response to nutrition and exercise during growth. Med Sport Sci 2007;51:64e80. [7] Melton 3rd LJ, Atkinson EJ, O’Connor MK, O’Fallon WM, Riggs BL. Determinants of bone loss from the femoral neck in women of different ages. J Bone Miner Res 2000;15:24e31. [8] Melton 3rd LJ, Atkinson EJ, Khosla S, Oberg AL, Riggs BL. Evaluation of a prediction model for long-term fracture risk. J Bone Miner Res 2005;20:551e6. [9] Hernandez CJ, Beaupre GS, Carter DR. A theoretical analysis of the relative influences of peak BMD, age-related bone loss and menopause on the development of osteoporosis. Osteoporos Int 2003;14:843e7. [10] World Health Organization. Assessment of fracture risk and its application to screening for postmenopausal osteoporosis. Report of a WHO study group. WHO Technical Report Series. 1994;No 843. [11] Duan Y, Seeman E, Turner CH. The biomechanical basis of vertebral body fragility in men and women. J Bone Miner Res 2001;16:2276e83.

[12] Duan Y, Wang XF, Evans A, Seeman E. Structural and biomechanical basis of racial and sex differences in vertebral fragility in Chinese and Caucasians. Bone 2005;36:987e98. [13] Cummings SR, Black DM, Nevitt MC, et al. Bone density at various sites for prediction of hip fractures. The Study of Osteoporotic Fractures Research Group. Lancet 1993;341:72e5. [14] Melton 3rd LJ, Atkinson EJ, O’Fallon WM, Wahner HW, Riggs BL. Long-term fracture prediction by bone mineral assessed at different skeletal sites. J Bone Miner Res 1993;8:1227e33. [15] Marshall D, Johnell O, Wedel H. Meta-analysis of how well measures of bone mineral density predict occurrence of osteoporotic fractures. Br Med J 1996;312:1254e9. [16] Adams JE, Shaw N. A Practical Guide to Bone Densitometry in Children. National Osteoporosis Society, Camerton, Bath, UK; 2004. [17] Baim S, Leonard MB, Bianchi ML, et al. Official Positions of the International Society for Clinical Densitometry and executive summary of the 2007 ISCD Pediatric Position Development Conference. J Clin Densitom 2008;11:6e21. [18] Boutroy S, Bouxsein ML, Munoz F, Delmas PD. In vivo assessment of trabecular bone microarchitecture by high-resolution peripheral quantitative computed tomography. J Clin Endocrinol Metab 2005;90:6508e15. [19] Sornay-Rendu E, Boutroy S, Munoz F, Delmas PD. Alterations of cortical and trabecular architecture are associated with fractures in postmenopausal women, partially independent of decreased BMD measured by DXA: the OFELY study. J Bone Miner Res 2007;22:425e33. [20] Boutroy S, Van Rietbergen B, Sornay-Rendu E, Munoz F, Bouxsein ML, Delmas PD. Finite element analysis based on in vivo HR-pQCT images of the distal radius is associated with wrist fracture in postmenopausal women. J Bone Miner Res 2008;23:392e9. [21] Chevalley T, Bonjour JP, Ferrari S, Rizzoli R. Influence of age at menarche on forearm bone microstructure in healthy young women. J Clin Endocrinol Metab 2008;93:2594e601. [22] Chevalley T, Bonjour JP, Ferrari S, Rizzoli R. Deleterious effect of late menarche on distal tibia microstructure in healthy 20-yearold and premenopausal middle-aged women. J Bone Miner Res 2009;24:144e52. [23] Kirmani S, Christen D, van Lenthe GH, et al. Bone structure at the distal radius during adolescent growth. J Bone Miner Res 2009;24:1033e42. [24] Bacchetta J, Boutroy S, Vilayphiou N, et al. Early impairment of trabecular microarchitecture assessed with HR-pQCT in patients with stage IIeIV chronic kidney disease. J Bone Miner Res 2010;25:849e57. [25] Burghardt AJ, Issever AS, Schwartz AV, et al. High-resolution peripheral quantitative computed tomographic imaging of cortical and trabecular bone microarchitecture in patients with type 2 diabetes mellitus. J Clin Endocrinol Metab 2010;95: 5045e55. [26] Briot K, Roux C. What is the role of DXA, QUS and bone markers in fracture prediction, treatment allocation and monitoring? Best Pract Res Clin Rheumatol 2005;19:951e64. [27] Specker BL, Schoenau E. Quantitative bone analysis in children: current methods and recommendations. J Pediatr 2005;146: 726e31. [28] Binkley TL, Berry R, Specker BL. Methods for measurement of pediatric bone. Rev Endocr Metab Disord 2008;9:95e106. [29] Bonjour JP, Theintz G, Buchs B, Slosman D, Rizzoli R. Critical years and stages of puberty for spinal and femoral bone mass accumulation during adolescence. J Clin Endocrinol Metab 1991;73:555e63.

PEDIATRIC BONE

REFERENCES

[30] Bonjour JP, Theintz G, Law F, Slosman D, Rizzoli R. Peak bone mass. Osteoporos Int 1994;4(Suppl. 1):7e13. [31] Rizzoli R, Bonjour JP. Determinants of peak bone mass and mechanisms of bone loss. Osteoporos Int 1999;9(Suppl. 2): S17e23. [32] Gilsanz V, Roe TF, Mora S, Costin G, Goodman WG. Changes in vertebral bone density in black girls and white girls during childhood and puberty. N Engl J Med 1991;325:1597e600. [33] Theintz G, Buchs B, Rizzoli R, et al. Longitudinal monitoring of bone mass accumulation in healthy adolescents: evidence for a marked reduction after 16 years of age at the levels of lumbar spine and femoral neck in female subjects. J Clin Endocrinol Metab 1992;75:1060e5. [34] Ferrari S, Rizzoli R, Slosman D, Bonjour JP. Familial resemblance for bone mineral mass is expressed before puberty. J Clin Endocrinol Metab 1998;83:358e61. [35] Gilsanz V, Skaggs DL, Kovanlikaya A, et al. Differential effect of race on the axial and appendicular skeletons of children. J Clin Endocrinol Metab 1998;83:1420e7. [36] Seeman E. Pathogenesis of bone fragility in women and men. Lancet 2002;359:1841e50. [37] Garn SM. The earlier gain and later loss of cortical bone. In: nutritional perspective: Charles C Thomas, Springfield IL; 1970. p 3e120. [38] Aharinejad S, Bertagnoli R, Wicke K, Firbas W, Schneider B. Morphometric analysis of vertebrae and intervertebral discs as a basis of disc replacement. Am J Anat 1990;189:69e76. [39] Fournier PE, Rizzoli R, Slosman DO, Buchs B, Bonjour JP. Relative contribution of vertebral body and posterior arch in female and male lumbar spine peak bone mass. Osteoporos Int 1994;4:264e72. [40] Gilsanz V, Loro ML, Roe TF, Sayre J, Gilsanz R, Schulz EE. Vertebral size in elderly women with osteoporosis. Mechanical implications and relationship to fractures. J Clin Invest 1995;95:2332e7. [41] Naganathan V, Sambrook P. Gender differences in volumetric bone density: a study of opposite-sex twins. Osteoporos Int 2003;14:564e9. [42] Fournier PE, Rizzoli R, Slosman DO, Theintz G, Bonjour JP. Asynchrony between the rates of standing height gain and bone mass accumulation during puberty. Osteoporos Int 1997;7: 525e32. [43] Bailey DA, Wedge JH, McCulloch RG, Martin AD, Bernhardson SC. Epidemiology of fractures of the distal end of the radius in children as associated with growth. J Bone Joint Surg Am 1989;71:1225e31. [44] Landin LA. Fracture patterns in children. Analysis of 8,682 fractures with special reference to incidence, etiology and secular changes in a Swedish urban population 1950e1979. Acta Orthop Scand Suppl 1983;202:1e109. [45] Parfitt AM. The two faces of growth: benefits and risks to bone integrity. Osteoporos Int 1994;4:382e98. [46] Ferrari SL, Chevalley T, Bonjour JP, Rizzoli R. Childhood fractures are associated with decreased bone mass gain during puberty: an early marker of persistent bone fragility? J Bone Miner Res 2006;21:501e7. [47] Bonjour JP, Chevalley T. Pubertal timing, peak bone mass and fragility fracture risk. BoneKey-Osteovision 2007;4:30e48. http:// www.bonekey-ibms.org/cgi/content/full/ibmske 4/2/30. [48] Gilsanz V, Gibbens DT, Roe TF, et al. Vertebral bone density in children: effect of puberty. Radiology 1988;166:847e50. [49] Szulc P, Seeman E, Delmas PD. Biochemical measurements of bone turnover in children and adolescents. Osteoporos Int 2000;11:281e94.

215

[50] Rauch F, Georg M, Stabrey A, et al. Collagen markers deoxypyridinoline and hydroxylysine glycosides: pediatric reference data and use for growth prediction in growth hormone-deficient children. Clin Chem 2002;48:315e22. [51] Yang L, Grey V. Pediatric reference intervals for bone markers. Clin Biochem 2006;39:561e8. [52] Jurimae J. Interpretation and application of bone turnover markers in children and adolescents. Curr Opin Pediatr 2010;22:494e500. [53] Jurimae J, Maestu J, Jurimae T. Bone turnover markers during pubertal development: relationships with growth factors and adipocytokines. Med Sport Sci 2010;55:114e27. [54] Cadogan J, Blumsohn A, Barker ME, Eastell R. A longitudinal study of bone gain in pubertal girls: anthropometric and biochemical correlates. J Bone Miner Res 1998;13(10):1602e12. [55] Seeman E, Hopper JL, Bach LA, et al. Reduced bone mass in daughters of women with osteoporosis. N Engl J Med 1989;320:554e8. [56] Soroko SB, Barrett-Connor E, Edelstein SL, Kritz-Silverstein D. Family history of osteoporosis and bone mineral density at the axial skeleton: the Rancho Bernardo Study. J Bone Miner Res 1994;9:761e9. [57] Barthe N, Basse-Cathalinat B, Meunier PJ, et al. Measurement of bone mineral density in mother-daughter pairs for evaluating the family influence on bone mass acquisition: a GRIO survey. Osteoporos Int 1998;8:379e84. [58] Cohen-Solal ME, Baudoin C, Omouri M, Kuntz D, De Vernejoul MC. Bone mass in middle-aged osteoporotic men and their relatives: familial effect. J Bone Miner Res 1998;13:1909e14. [59] Eisman JA. Genetics of osteoporosis. Endocr Rev 1999;20(6):788e804. [60] Peacock M, Turner CH, Econs MJ, Foroud T. Genetics of osteoporosis. Endocr Rev 2002;23:303e26. [61] Pocock NA, Eisman JA, Hopper JL, Yeates MG, Sambrook PN, Eberl S. Genetic determinants of bone mass in adults. A twin study. J Clin Invest 1987;80:706e10. [62] Johnson ML, Gong G, Kimberling W, Recker SM, Kimmel DB, Recker RB. Linkage of a gene causing high bone mass to human chromosome 11 (11q12-13). Am J Hum Genet 1997;60:1326e32. [63] Devoto M, Shimoya K, Caminis J, et al. First-stage autosomal genome screen in extended pedigrees suggests genes predisposing to low bone mineral density on chromosomes 1p, 2p and 4q. Eur J Hum Genet 1998;6:151e7. [64] Deng HW, Shen H, Xu FH, et al. Several genomic regions potentially containing QTLs for bone size variation were identified in a whole-genome linkage scan. Am J Med Genet A 2003;119:121e31. [65] Koller DL, Rodriguez LA, Christian JC, et al. Linkage of a QTL contributing to normal variation in bone mineral density to chromosome 11q12-13. J Bone Miner Res 1998;13:1903e8. [66] Koller DL, Econs MJ, Morin PA, et al. Genome screen for QTLs contributing to normal variation in bone mineral density and osteoporosis. J Clin Endocrinol Metab 2000;85:3116e20. [67] Karasik D, Myers RH, Cupples LA, et al. Genome screen for quantitative trait loci contributing to normal variation in bone mineral density: the Framingham Study. J Bone Miner Res 2002;17:1718e27. [68] Koller DL, Liu G, Econs MJ, et al. Genome screen for quantitative trait loci underlying normal variation in femoral structure. J Bone Miner Res 2001;16:985e91. [69] Carn G, Koller DL, Peacock M, et al. Sibling pair linkage and association studies between peak bone mineral density and the gene locus for the osteoclast-specific subunit (OC116) of the vacuolar proton pump on chromosome 11p12-13. J Clin Endocrinol Metab 2002;87:3819e24.

PEDIATRIC BONE

216

9. PEAK BONE MASS AND ITS REGULATION

[70] Langdahl BL, Ralston SH, Grant SF, Eriksen EF. An Sp1 binding site polymorphism in the COLIA1 gene predicts osteoporotic fractures in both men and women. J Bone Miner Res 1998;13:1384e9. [71] Mann V, Hobson EE, Li B, et al. A COL1A1 Sp1 binding site polymorphism predisposes to osteoporotic fracture by affecting bone density and quality. J Clin Invest 2001;107:899e907. [72] Ioannidis JP, Stavrou I, Trikalinos TA, et al. Association of polymorphisms of the estrogen receptor alpha gene with bone mineral density and fracture risk in women: a meta-analysis. J Bone Miner Res 2002;17:2048e60. [73] Garnero P, Munoz F, Borel O, Sornay-Rendu E, Delmas PD. Vitamin D receptor gene polymorphisms are associated with the risk of fractures in postmenopausal women, independently of bone mineral density. J Clin Endocrinol Metab 2005;90:4829e35. [74] Cummings SR, Nevitt MC. Non-skeletal determinants of fractures: the potential importance of the mechanics of falls. Study of Osteoporotic Fractures Research Group. Osteoporos Int 1994;4(Suppl. 1):67e70. [75] Cummings SR. Treatable and untreatable risk factors for hip fracture. Bone 1996;18(Suppl):165Se7S. [76] Schwartz A, Capezuti E, Grisso JA. Risk factors for falls as a cause of hip fracture in women. In: Marcus R, Feldman D, Kelsey J, editors. Osteoporosis. San Diego: Academic Press; 2001. p. 795e808. [77] Rizzoli R, Bonjour JP, Ferrari SL. Osteoporosis, genetics and hormones. J Mol Endocrinol 2001;26:79e94. [78] Liu YZ, Liu YJ, Recker RR, Deng HW. Molecular studies of identification of genes for osteoporosis: the 2002 update. J Endocrinol 2003;177:147e96. [79] Ferrari SL, Deutsch S, Baudoin C, et al. LRP5 gene polymorphisms and idiopathic osteoporosis in men. Bone 2005;37: 770e5. [80] Thakkinstian A, D’Este C, Attia J. Haplotype analysis of VDR gene polymorphisms: a meta-analysis. Osteoporos Int 2004;15: 729e34. [81] Thakkinstian A, D’Este C, Eisman J, Nguyen T, Attia J. Metaanalysis of molecular association studies: vitamin D receptor gene polymorphisms and BMD as a case study. J Bone Miner Res 2004;19:419e28. [82] Ferrari SL. Osteoporosis: a complex disorder of aging with multiple genetic and environmental determinants. World Rev Nutr Diet 2005;95:35e51. [83] Hey PJ, Twells RC, Phillips MS, et al. Cloning of a novel member of the low-density lipoprotein receptor family. Gene 1998;216:103e11. [84] Gong Y, Slee RB, Fukai N, et al. LDL receptor-related protein 5 (LRP5) affects bone accrual and eye development. Cell 2001;107:513e23. [85] Ai M, Heeger S, Bartels CF, Schelling DK. Clinical and molecular findings in osteoporosis-pseudoglioma syndrome. Am J Hum Genet 2005;77:741e53. [86] Kato M, Patel MS, Levasseur R, et al. Cbfa1-independent decrease in osteoblast proliferation, osteopenia, and persistent embryonic eye vascularization in mice deficient in Lrp5, a Wnt coreceptor. J Cell Biol 2002;157:303e14. [87] Boyden LM, Mao J, Belsky J, et al. High bone density due to a mutation in LDL-receptor-related protein 5. N Engl J Med 2002;346:1513e21. [88] Little RD, Carulli JP, Del Mastro RG, et al. A mutation in the LDL receptor-related protein 5 gene results in the autosomal dominant high-bone-mass trait. Am J Hum Genet 2002;70:11e9. [89] Van Wesenbeeck L, Cleiren E, Gram J, et al. Six novel missense mutations in the LDL receptor-related protein 5 (LRP5) gene in

[90]

[91]

[92]

[93]

[94]

[95]

[96]

[97]

[98]

[99]

[100]

[101] [102]

[103]

[104]

[105] [106]

[107]

[108]

different conditions with an increased bone density. Am J Hum Genet 2003;72:763e71. Livshits G, Trofimov S, Malkin I, Kobyliansky E. Transmission disequilibrium test for hand bone mineral density and 11q12-13 chromosomal segment. Osteoporos Int 2002;13:461e7. Ferrari SL, Deutsch S, Choudhury U, et al. Polymorphisms in the low-density lipoprotein receptor-related protein 5 (LRP5) gene are associated with variation in vertebral bone mass, vertebral bone size, and stature in whites. Am J Hum Genet 2004;74:866e75. Perola M, Ohman M, Hiekkalinna T, et al. Quantitative-traitlocus analysis of body-mass index and of stature, by combined analysis of genome scans of five Finnish study groups. Am J Hum Genet 2001;69:117e23. Hirschhorn JN, Lindgren CM, Daly MJ, et al. Genomewide linkage analysis of stature in multiple populations reveals several regions with evidence of linkage to adult height. Am J Hum Genet 2001;69:106e16. Van Pottelbergh I, Goemaere S, Zmierczak H, De Bacquer D, Kaufman JM. Deficient acquisition of bone during maturation underlies idiopathic osteoporosis in men: evidence from a threegeneration family study. J Bone Miner Res 2003;18:303e11. van Meurs JB, Rivadeneira F, Jhamai M, et al. Common genetic variation of the low-density lipoprotein receptor-related protein 5 and 6 genes determines fracture risk in elderly white men. J Bone Miner Res 2006;21:141e50. Richards JB, Kavvoura FK, Rivadeneira F, et al. Collaborative meta-analysis: associations of 150 candidate genes with osteoporosis and osteoporotic fracture. Ann Intern Med 2009;151: 528e37. Rivadeneira F, Styrkarsdottir U, Estrada K, et al. Twenty bonemineral-density loci identified by large-scale meta-analysis of genome-wide association studies. Nat Genet 2009;41:1199e206. Dequeker J, Nijs J, Verstraeten A, Geusens P, Gevers G. Genetic determinants of bone mineral content at the spine and radius: a twin study. Bone 1987;8:207e9. Slemenda CW, Turner CH, Peacock M, et al. The genetics of proximal femur geometry, distribution of bone mass and bone mineral density. Osteoporos Int 1996;6:178e82. Eisman J. Is the genomics glass half empty or completely empty? BoneKey IBMS 2010:27e31. http://www.bonekey-ibms. org/cgi/content/full/ibmske 7/1/27. Ferrari S. Human genetics of osteoporosis. Best Pract Res Clin Endocrinol Metab 2008;22:723e35. Riggs BL, Khosla S, Melton 3rd LJ. Sex steroids and the construction and conservation of the adult skeleton. Endocr Rev 2002;23:279e302. Vanderschueren D, Vandenput L, Boonen S. Reversing sex steroid deficiency and optimizing skeletal development in the adolescent with gonadal failure. Endocr Dev 2005;8:150e65. Bonjour JP, Rizzoli R. Bone acquisition in adolescence. In: Marcus R, Feldman D, Kelsey J, editors. Osteoporosis. San Diego: Academic Press; 2001. p. 621e38. Niu T, Rosen CJ. The insulin-like growth factor-I gene and osteoporosis: a critical appraisal. Gene 2005;361:38e56. Vanderschueren D, Vandenput L, Boonen S, Lindberg MK, Bouillon R, Ohlsson C. Androgens and bone. Endocr Rev 2004;25:389e425. Abu EO, Horner A, Kusec V, Triffitt JT, Compston JE. The localization of androgen receptors in human bone. J Clin Endocrinol Metab 1997;82:3493e7. Nilsson O, Chrysis D, Pajulo O, et al. Localization of estrogen receptors-alpha and -beta and androgen receptor in the human growth plate at different pubertal stages. J Endocrinol 2003;177: 319e26.

PEDIATRIC BONE

REFERENCES

[109] Marcus R, Leary D, Schneider DL, Shane E, Favus M, Quigley CA. The contribution of testosterone to skeletal development and maintenance: lessons from the androgen insensitivity syndrome. J Clin Endocrinol Metab 2000;85:1032e7. [110] Migliaccio S, Newbold RR, Bullock BC, et al. Alterations of maternal estrogen levels during gestation affect the skeleton of female offspring. Endocrinology 1996;137:2118e25. [111] Yakar S, Rosen CJ. From mouse to man: redefining the role of insulin-like growth factor-I in the acquisition of bone mass. Exp Biol Med (Maywood) 2003;228:245e52. [112] Condamine L, Menaa C, Vrtovsnik F, Friedlander G, Garabedian M. Local action of phosphate depletion and insulinlike growth factor 1 on in vitro production of 1,25-dihydroxyvitamin D by cultured mammalian kidney cells. J Clin Invest 1994;94:1673e9. [113] Menaa C, Vrtovsnik F, Friedlander G, Corvol M, Garabedian M. Insulin-like growth factor I, a unique calcium-dependent stimulator of 1,25-dihydroxyvitamin D3 production. Studies in cultured mouse kidney cells. J Biol Chem 1995;270:25461e7. [114] Palmer G, Bonjour JP, Caverzasio J. Stimulation of inorganic phosphate transport by insulin-like growth factor I and vanadate in opossum kidney cells is mediated by distinct protein tyrosine phosphorylation processes. Endocrinology 1996;137: 4699e705. [115] Caverzasio J, Montessuit C, Bonjour JP. Stimulatory effect of insulin-like growth factor-1 on renal Pi transport and plasma 1,25-dihydroxyvitamin D3. Endocrinology 1990;127:453e9. [116] Spelsberg TC, Subramaniam M, Riggs BL, Khosla S. The actions and interactions of sex steroids and growth factors/cytokines on the skeleton. Mol Endocrinol 1999;13:819e28. [117] Montessuit C, Bonjour JP, Caverzasio J. Pi transport regulation by chicken growth plate chondrocytes. Am J Physiol 1994;267:E24e31. [118] Montessuit C, Caverzasio J, Bonjour JP. Characterization of a Pi transport system in cartilage matrix vesicles. Potential role in the calcification process. J Biol Chem 1991;266:17791e7. [119] Palmer G, Zhao J, Bonjour J, Hofstetter W, Caverzasio J. In vivo expression of transcripts encoding the Glvr-1 phosphate transporter/retrovirus receptor during bone development. Bone 1999;24:1e7. [120] Caverzasio J, Bonjour JP. Characteristics and regulation of Pi transport in osteogenic cells for bone metabolism. Kidney Int 1996;49:975e80. [121] Palmer G, Bonjour JP, Caverzasio J. Expression of a newly identified phosphate transporter/retrovirus receptor in human SaOS-2 osteoblast-like cells and its regulation by insulin-like growth factor I. Endocrinology 1997;138:5202e9. [122] Ducharme JR, Forest MG. Normal pubertal development. In: Betrand J, Rappaport R, Sizonenko PC, editors. Pediatric Endocrinology Physiology, Pathophysiology, and Clinical Aspects. Baltimore: Williams & Wilkins; 1993. p. 372e86. [123] Parent AS, Teilmann G, Juul A, Skakkebaek NE, Toppari J, Bourguignon JP. The timing of normal puberty and the age limits of sexual precocity: variations around the world, secular trends, and changes after migration. Endocr Rev 2003;24: 668e93. [124] Tanner JM. Growth at Adolescence. 2nd ed. Oxford, UK: Blackwell; 1962. [125] Johnell O, Gullberg B, Kanis JA, et al. Risk factors for hip fracture in European women: the MEDOS Study. Mediterranean Osteoporosis Study. J Bone Miner Res 1995;10:1802e15. [126] Melton 3rd LJ. Epidemiology of spinal osteoporosis. Spine 1997;22(Suppl):2Se11S.

217

[127] Silman AJ. Risk factors for Colles’ fracture in men and women: results from the European Prospective Osteoporosis Study. Osteoporos Int 2003;14:213e8. [128] Paganini-Hill A, Atchison KA, Gornbein JA, Nattiv A, Service SK, White SC. Menstrual and reproductive factors and fracture risk: the Leisure World Cohort Study. J Womens Health (Larchmt) 2005;14:808e19. [129] Ribot C, Pouilles JM, Bonneu M, Tremollieres F. Assessment of the risk of post-menopausal osteoporosis using clinical factors. Clin Endocrinol (Oxf) 1992;36:225e8. [130] Fox KM, Magaziner J, Sherwin R, et al. Reproductive correlates of bone mass in elderly women. Study of Osteoporotic Fractures Research Group. J Bone Miner Res 1993;8:901e8. [131] Tuppurainen M, Kroger H, Saarikoski S, Honkanen R, Alhava E. The effect of gynecological risk factors on lumbar and femoral bone mineral density in peri- and postmenopausal women. Maturitas 1995;21:137e45. [132] Varenna M, Binelli L, Zucchi F, Ghiringhelli D, Gallazzi M, Sinigaglia L. Prevalence of osteoporosis by educational level in a cohort of postmenopausal women. Osteoporos Int 1999;9: 236e41. [133] Rosenthal DI, Mayo-Smith W, Hayes CW, et al. Age and bone mass in premenopausal women. J Bone Miner Res 1989;4:533e8. [134] Ito M, Yamada M, Hayashi K, Ohki M, Uetani M, Nakamura T. Relation of early menarche to high bone mineral density. Calcif Tissue Int 1995;57:11e4. [135] Fujita Y, Katsumata K, Unno A, Tawa T, Tokita A. Factors affecting peak bone density in Japanese women. Calcif Tissue Int 1999;64:107e11. [136] Chevalley T, Bonjour JP, Ferrari S, Rizzoli R. The influence of pubertal timing on bone mass acquisition: a predetermined trajectory detectable five years before menarche. J Clin Endocrinol Metab 2009;94:3424e31. [137] Armamento-Villareal R, Villareal DT, Avioli LV, Civitelli R. Estrogen status and heredity are major determinants of premenopausal bone mass. J Clin Invest 1992;90:2464e71. [138] Nguyen TV, Jones G, Sambrook PN, White CP, Kelly PJ, Eisman JA. Effects of estrogen exposure and reproductive factors on bone mineral density and osteoporotic fractures. J Clin Endocrinol Metab 1995;80:2709e14. [139] Galuska DA, Sowers MR. Menstrual history and bone density in young women. J Womens Health Gend Based Med 1999;8: 647e56. [140] Petri E. Untersuchungen zur Erbbedingheit der Menarche. Z Morphol Anthropol 1935;33:43e8. [141] Kaprio J, Rimpela A, Winter T, Viken RJ, Rimpela M, Rose RJ. Common genetic influences on BMI and age at menarche. Hum Biol 1995;67:739e53. [142] Salces I, Rebato EM, Susanne C, San Martin L, Rosique J. Familial resemblance for the age at menarche in Basque population. Ann Hum Biol 2001;28:143e56. [143] Gajdos ZK, Butler JL, Henderson KD, et al. Association studies of common variants in 10 hypogonadotropic hypogonadism genes with age at menarche. J Clin Endocrinol Metab 2008;93: 4290e8. [144] Seminara SB. Lack of association of hypogonadotropic genes with age at menarche: prospects for the future. J Clin Endocrinol Metab 2008;93:4224e5. [145] Ralston SH, Uitterlinden AG. Genetics of osteoporosis. Endocr Rev 2010;31:629e62. [146] Johnell O, Kanis JA, Oden A, et al. Predictive value of BMD for hip and other fractures. J Bone Miner Res 2005;20:1185e94. [147] Kindblom JM, Lorentzon M, Norjavaara E, et al. Pubertal timing predicts previous fractures and BMD in young adult men: the GOOD study. J Bone Miner Res 2006;21:790e5.

PEDIATRIC BONE

218

9. PEAK BONE MASS AND ITS REGULATION

[148] Ferrari S, Chevalley T, Van Rietbergen B, Bonjour J-P, Rizzoli R. Reduced bone strength in young adult women with clinical fractures during childhood and adolescence. J Bone Miner Res 2010;25(Suppl. 1):S40. [149] Chevalley T, Bonjour J-P, Van Rietbergen B, Ferrari S, Rizzoli R. Fractures in healthy adolescent boys are associated with reduced bone strength as assessed by finite element analysis at weightbearing skeletal sites. J Bone Miner Res 2010;25(Suppl. 1):S64e5. [150] Robling AG, Castillo AB, Turner CH. Biomechanical and molecular regulation of bone remodeling. Annu Rev Biomed Eng 2006;8:455e98. [151] Robling AG, Bellido TM, Turner CH. Mechanical loading reduced osteocyte expression of sclerostin protein. J Bone Miner Res 2006;21(Suppl. 1):S72. [152] Bass SL, Saxon L, Daly RM, et al. The effect of mechanical loading on the size and shape of bone in pre-, peri-, and postpubertal girls: a study in tennis players. J Bone Miner Res 2002;17:2274e80. [153] Karlsson MK, Linden C, Karlsson C, Johnell O, Obrant K, Seeman E. Exercise during growth and bone mineral density and fractures in old age. Lancet 2000;355:469e70. [154] Nordstrom A, Karlsson C, Nyquist F, Olsson T, Nordstrom P, Karlsson M. Bone loss and fracture risk after reduced physical activity. J Bone Miner Res 2005;20:202e7. [155] Haapasalo H, Kontulainen S, Sievanen H, Kannus P, Jarvinen M, Vuori I. Exercise-induced bone gain is due to enlargement in bone size without a change in volumetric bone density: a peripheral quantitative computed tomography study of the upper arms of male tennis players. Bone 2000;27:351e7. [156] Karlsson MK. Does exercise during growth prevent fractures in later life? Med Sport Sci 2007;51:121e36. [157] Kontulainen SA, Hughes JM, Macdonald HM, Johnston JD. The biomechanical basis of bone strength development during growth. Med Sport Sci 2007;51:13e32. [158] Fuchs RK, Bauer JJ, Snow CM. Jumping improves hip and lumbar spine bone mass in prepubescent children: a randomized controlled trial. J Bone Miner Res 2001;16:148e56. [159] MacKelvie KJ, Khan KM, McKay HA. Is there a critical period for bone response to weight-bearing exercise in children and adolescents? a systematic review. Br J Sports Med 2002;36: 250e7. discussion 7. [160] McKay HA, MacLean L, Petit M, et al. “Bounce at the Bell”: a novel program of short bouts of exercise improves proximal femur bone mass in early pubertal children. Br J Sports Med 2005;39:521e6. [161] Hind K, Burrows M. Weight-bearing exercise and bone mineral accrual in children and adolescents: a review of controlled trials. Bone 2007;40:14e27. [162] Hughes JM, Novotny SA, Wetzsteon RJ, Petit MA. Lessons learned from school-based skeletal loading intervention trials: putting research into practice. Med Sport Sci 2007;51:137e58. [163] Gunter K, Baxter-Jones AD, Mirwald RL, et al. Impact exercise increases BMC during growth: an 8-year longitudinal study. J Bone Miner Res 2008;23:986e93. [164] Gunter K, Baxter-Jones AD, Mirwald RL, et al. Jump starting skeletal health: a 4-year longitudinal study assessing the effects of jumping on skeletal development in pre and circum pubertal children. Bone 2008;42:710e8. [165] Macdonald HM, Kontulainen SA, Petit MA, Beck TJ, Khan KM, McKay HA. Does a novel school-based physical activity model benefit femoral neck bone strength in pre- and early pubertal children? Osteoporos Int 2008;19:1445e56. [166] Daly RM. The effect of exercise on bone mass and structural geometry during growth. Med Sport Sci 2007;51:33e49.

[167] Kriemler S, Zahner L, Puder JJ, et al. Weight-bearing bones are more sensitive to physical exercise in boys than in girls during pre- and early puberty: a cross-sectional study. Osteoporos Int 2008;19:1749e58. [168] Ward KA, Roberts SA, Adams JE, Mughal MZ. Bone geometry and density in the skeleton of pre-pubertal gymnasts and school children. Bone 2005;36:1012e8. [169] Schoenau E, Frost HM. The ”muscle-bone unit“ in children and adolescents. Calcif Tissue Int 2002;70:405e7. [170] Daly RM, Saxon L, Turner CH, Robling AG, Bass SL. The relationship between muscle size and bone geometry during growth and in response to exercise. Bone 2004;34:281e7. [171] Ducher G, Courteix D, Meme S, Magni C, Viala JF, Benhamou CL. Bone geometry in response to long-term tennis playing and its relationship with muscle volume: a quantitative magnetic resonance imaging study in tennis players. Bone 2005; 37:457e66. [172] Suuriniemi M, Mahonen A, Kovanen V, et al. Association between exercise and pubertal BMD is modulated by estrogen receptor alpha genotype. J Bone Miner Res 2004;19:1758e65. [173] Remes T, Vaisanen SB, Mahonen A, et al. Aerobic exercise and bone mineral density in middle-aged Finnish men: a controlled randomized trial with reference to androgen receptor, aromatase, and estrogen receptor alpha gene polymorphisms small star, filled. Bone 2003;32:412e20. [174] Omasu F, Kitagawa J, Koyama K, et al. The influence of VDR genotype and exercise on ultrasound parameters in young adult Japanese women. J Physiol Anthropol Appl Human Sci 2004;23:49e55. [175] Nakamura O, Ishii T, Ando Y, et al. Potential role of vitamin D receptor gene polymorphism in determining bone phenotype in young male athletes. J Appl Physiol 2002;93:1973e9. [176] Tajima O, Ashizawa N, Ishii T, et al. Interaction of the effects between vitamin D receptor polymorphism and exercise training on bone metabolism. J Appl Physiol 2000;88:1271e6. [177] Dhamrait SS, James L, Brull DJ, et al. Cortical bone resorption during exercise is interleukin-6 genotype-dependent. Eur J Appl Physiol 2003;89:21e5. [178] Klein-Nulend J, Bacabac RG, Mullender MG. Mechanobiology of bone tissue. Pathol Biol (Paris) 2005;53:576e80. [179] Suva LJ, Gaddy D, Perrien DS, Thomas RL, Findlay DM. Regulation of bone mass by mechanical loading: microarchitecture and genetics. Curr Osteoporos Rep 2005;3:46e51. [180] Rubin J, Rubin C, Jacobs CR. Molecular pathways mediating mechanical signaling in bone. Gene 2006;367:1e16. [181] Klein-Nulend J, Nijweide PJ, Burger EH. Osteocyte and bone structure. Curr Osteoporos Rep 2003;1:5e10. [182] Balemans W, Ebeling M, Patel N, et al. Increased bone density in sclerosteosis is due to the deficiency of a novel secreted protein (SOST). Hum Mol Genet 2001;10:537e43. [183] Balemans W, Van Hul W. Identification of the disease-causing gene in sclerosteosis e discovery of a novel bone anabolic target? J Musculoskelet Neuronal Interact 2004;4:139e42. [184] Balemans W, Cleiren E, Siebers U, Horst J, Van Hul W. A generalized skeletal hyperostosis in two siblings caused by a novel mutation in the SOST gene. Bone 2005;36:943e7. [185] Li X, Zhang Y, Kang H, et al. Sclerostin binds to LRP5/6 and antagonizes canonical Wnt signaling. J Biol Chem 2005;280: 19883e7. [186] Sawakami K, Robling AG, Ai M, et al. The Wnt co-receptor LRP5 is essential for skeletal mechanotransduction but not for the anabolic bone response to parathyroid hormone treatment. J Biol Chem 2006;281:23698e711. [187] Ominsky M, Stouch B, Doellgast G, et al. Administration of sclerostin antibodies to female cynomolgus monkeys results in

PEDIATRIC BONE

219

REFERENCES

[188]

[189]

[190] [191]

[192]

[193]

[194]

[195]

[196]

[197]

[198]

[199]

[200]

[201]

[202]

[203]

[204]

[205]

[206]

increased bone formation, bone mineral density and bone strength. J Bone Miner Res 2006;21(Suppl. 1):S44. Uitterlinden AG, Arp PP, Paeper BW, et al. Polymorphisms in the sclerosteosis/van Buchem disease gene (SOST) region are associated with bone-mineral density in elderly whites. Am J Hum Genet 2004;75:1032e45. Husted LB, Carstens M, Stenkir L, Langdahl BL. A polymorphism in the Van Buchem deletion region of the SOST gene is associated with increased bone mass. J Bone Miner Res 2006;21(Suppl. 1):S423. Warren MP, Goodman LR. Exercise-induced endocrine pathologies. J Endocrinol Invest 2003;26:873e8. Zanker C, Hind K. The effect of energy balance on endocrine function and bone health in youth. Med Sport Sci 2007;51: 81e101. Gremion G, Rizzoli R, Slosman D, Theintz G, Bonjour JP. Oligoamenorrheic long-distance runners may lose more bone in spine than in femur. Med Sci Sports Exerc 2001;33:15e21. Outila TA, Karkkainen MU, Lamberg-Allardt CJ. Vitamin D status affects serum parathyroid hormone concentrations during winter in female adolescents: associations with forearm bone mineral density. Am J Clin Nutr 2001;74:206e10. Cashman KD, Hill TR, Cotter AA, et al. Low vitamin D status adversely affects bone health parameters in adolescents. Am J Clin Nutr 2008;87:1039e44. Cheng S, Tylavsky F, Kroger H, et al. Association of low 25hydroxyvitamin D concentrations with elevated parathyroid hormone concentrations and low cortical bone density in early pubertal and prepubertal Finnish girls. Am J Clin Nutr 2003;78:485e92. Andersen R, Molgaard C, Skovgaard LT, et al. Teenage girls and elderly women living in northern Europe have low winter vitamin D status. Eur J Clin Nutr 2005;59:533e41. Lapatsanis D, Moulas A, Cholevas V, Soukakos P, Papadopoulou ZL, Challa A. Vitamin D, a necessity for children and adolescents in Greece. Calcif Tissue Int 2005;77:348e55. Ginty F, Cavadini C, Michaud PA, et al. Effects of usual nutrient intake and vitamin D status on markers of bone turnover in Swiss adolescents. Eur J Clin Nutr 2004;58:1257e65. Mithal A, Wahl DA, Bonjour JP, et al. Global vitamin D status and determinants of hypovitaminosis D. Osteoporos Int 2009;20:1807e20. Arabi A, El Rassi R, El-Hajj Fuleihan G. Hypovitaminosis D in developing countries e prevalence, risk factors and outcomes. Nat Rev Endocrinol 2010;6:550e61. Taylor JA, Geyer LJ, Feldman KW. Use of supplemental vitamin d among infants breastfed for prolonged periods. Pediatrics 2010;125:105e11. Wagner CL, Greer FR. Prevention of rickets and vitamin D deficiency in infants, children, and adolescents. Pediatrics 2008;122:1142e52. Zamora SA, Rizzoli R, Belli DC, Slosman DO, Bonjour JP. Vitamin D supplementation during infancy is associated with higher bone mineral mass in prepubertal girls. J Clin Endocrinol Metab 1999;84:4541e4. Viljakainen HT, Saarnio E, Hytinantti T, et al. Maternal vitamin D status determines bone variables in the newborn. J Clin Endocrinol Metab 2010;95:1749e57. Esterle L, Nguyen M, Walrant-Debray O, Sabatier JP, Garabedian M. Adverse interaction of low-calcium diet and low 25(OH)D levels on lumbar spine mineralization in late-pubertal girls. J Bone Miner Res 2010;25:2392e8. Viljakainen HT, Natri AM, Karkkainen M, et al. A positive doseresponse effect of vitamin D supplementation on site-specific bone mineral augmentation in adolescent girls: a double-

[207]

[208]

[209]

[210] [211]

[212]

[213]

[214]

[215]

[216]

[217]

[218] [219]

[220]

[221]

[222]

[223]

[224]

[225]

blinded randomized placebo-controlled 1-year intervention. J Bone Miner Res 2006;21:836e44. El-Hajj Fuleihan G, Nabulsi M, et al. Effect of vitamin D replacement on musculoskeletal parameters in school children: a randomized controlled trial. J Clin Endocrinol Metab 2006;91:405e12. Voloc A, Esterle L, Nguyen TM, et al. High prevalence of genu varum/valgum in European children with low vitamin D status and insufficient dairy products/calcium intakes. Eur J Endocrinol 2010;163:811e7. Kalkwarf HJ, Khoury JC, Lanphear BP. Milk intake during childhood and adolescence, adult bone density, and osteoporotic fractures in US women. Am J Clin Nutr 2003;77:257e65. Wosje KS, Specker BL. Role of calcium in bone health during childhood. Nutr Rev 2000;58:253e68. Bonjour JP, Ammann P, Chevalley T, Ferrari S, Rizzoli R. Nutritional aspects of bone growth: an overview. In: New S, Bonjour JP, editors. Nutritional Aspects of Bone Health. Cambridge, UK: The Royal Society of Chemistry; 2003. p. 111e27. Clavien H, Theintz G, Rizzoli R, Bonjour JP. Does puberty alter dietary habits in adolescents living in a western society? J Adolesc Health 1996;19:68e75. Bonjour JP, Chevalley T, Ferrari S, Rizzoli R. The importance and relevance of peak bone mass in the prevalence of osteoporosis. Salud Publica Mex 2009;51(Suppl. 1):S5e17. Bonjour JP. Is peripuberty the most opportune time to increase calcium intake in healthy girls? BoneKEy-Osteovision 2005 March;2(3):6e11. http://wwwbonekey-ibmsorg/cgi/content/ full/ibmske 2/3/6. Johnston Jr CC, Miller JZ, Slemenda CW, et al. Calcium supplementation and increases in bone mineral density in children. N Engl J Med 1992;327:82e7. Nowson CA, Green RM, Hopper JL, et al. A co-twin study of the effect of calcium supplementation on bone density during adolescence. Osteoporos Int 1997;7:219e25. Bonjour JP, Carrie AL, Ferrari S, et al. Calcium-enriched foods and bone mass growth in prepubertal girls: a randomized, double-blind, placebo-controlled trial. J Clin Invest 1997;99:1287e94. Barker DJ. Intrauterine programming of adult disease. Mol Med Today 1995;1:418e23. Cooper C, Westlake S, Harvey N, Javaid K, Dennison E, Hanson M. Review: developmental origins of osteoporotic fracture. Osteoporos Int 2006;17:337e47. Specker BL, Mulligan L, Ho M. Longitudinal study of calcium intake, physical activity, and bone mineral content in infants 6e18 months of age. J Bone Miner Res 1999;14:569e76. Specker B, Binkley T. Randomized trial of physical activity and calcium supplementation on bone mineral content in 3- to 5year-old children. J Bone Miner Res 2003;18:885e92. Specker B, Vukovich M. Evidence for an interaction between exercise and nutrition for improved bone health during growth. Med Sport Sci 2007;51:50e63. Iuliano-Burns S, Saxon L, Naughton G, Gibbons K, Bass SL. Regional specificity of exercise and calcium during skeletal growth in girls: a randomized controlled trial. J Bone Miner Res 2003;18:156e62. Courteix D, Jaffre C, Lespessailles E, Benhamou L. Cumulative effects of calcium supplementation and physical activity on bone accretion in premenarchal children: a double-blind randomised placebo-controlled trial. Int J Sports Med 2005;26:332e8. Chevalley T, Bonjour JP, Ferrari S, Hans D, Rizzoli R. Skeletal site selectivity in the effects of calcium supplementation on areal bone mineral density gain: a randomized, double-blind,

PEDIATRIC BONE

220

[226] [227] [228]

[229]

[230]

[231] [232]

[233]

[234]

[235]

[236]

[237]

[238]

[239] [240]

[241]

[242] [243]

[244]

[245]

9. PEAK BONE MASS AND ITS REGULATION

placebo-controlled trial in prepubertal boys. J Clin Endocrinol Metab 2005;90:3342e9. Ordovas JM, Mooser V. Nutrigenomics and nutrigenetics. Curr Opin Lipidol 2004;15:101e8. van Ommen B. Nutrigenomics: exploiting systems biology in the nutrition and health arenas. Nutrition 2004;20:4e8. Mutch DM, Wahli W, Williamson G. Nutrigenomics and nutrigenetics: the emerging faces of nutrition. Faseb J 2005;19: 1602e16. Mariman EC. Nutrigenomics and nutrigenetics: the ’omics’ revolution in nutritional science. Biotechnol Appl Biochem 2006;44:119e28. Ferrari SL, Rizzoli R, Slosman DO, Bonjour JP. Do dietary calcium and age explain the controversy surrounding the relationship between bone mineral density and vitamin D receptor gene polymorphisms? J Bone Miner Res 1998;13:363e70. Ferrari S, Bonjour JP, Rizzoli R. The vitamin D receptor gene and calcium metabolism. Trends Endocrinol Metab 1998;9:259e65. Gong G, Stern HS, Cheng SC, et al. The association of bone mineral density with vitamin D receptor gene polymorphisms. Osteoporos Int 1999;9:55e64. Sainz J, Van Tornout JM, Loro ML, Sayre J, Roe TF, Gilsanz V. Vitamin D-receptor gene polymorphisms and bone density in prepubertal American girls of Mexican descent. N Engl J Med 1997;337:77e82. Cadogan J, Eastell R, Jones N, Barker ME. Milk intake and bone mineral acquisition in adolescent girls: randomised, controlled intervention trial. Br Med J 1997;315:1255e60. Chevalley T, Rizzoli R, Hans D, Ferrari S, Bonjour JP. Interaction between calcium intake and menarcheal age on bone mass gain: an eight-year follow-up study from prepuberty to postmenarche. J Clin Endocrinol Metab 2005;90:44e51. Prentice A, Ginty F, Stear SJ, Jones SC, Laskey MA, Cole TJ. Calcium supplementation increases stature and bone mineral mass of 16- to 18-year-old boys. J Clin Endocrinol Metab 2005;90:3153e61. Institute of Medicine Food and Nutrition Board. Dietary References Intakes for Calcium, Phosphorus, Magnesium, Vitamin D and Fluoride. Washington, DC: National Academy Press; 1997. Committee on Medical Aspects of Food and Nutrition Policy (COMA). Nutrition and Bone Health: with particular reference to calcium and vitamin D. London, UK: The Stationery Office; 1998. Valeur de re´fe´rences pour les apports nutrtritionnels. Socie´te´ Suisse de Nutrition; 2002. World Health Organization. Prevention and management of osteoporosis. Report of a scientific group. WHO Technical Report Series. 2003;No 921. Schurch MA, Rizzoli R, Slosman D, Vadas L, Vergnaud P, Bonjour JP. Protein supplements increase serum insulin-like growth factor-I levels and attenuate proximal femur bone loss in patients with recent hip fracture. A randomized, double-blind, placebo-controlled trial. Ann Intern Med 1998;128:801e9. Bonjour JP, Ammann P, Chevalley T, Rizzoli R. Protein intake and bone growth. Can J Appl Physiol 2001;26(Suppl):S153e66. Kerstetter JE, O’Brien KO, Insogna KL. Dietary protein, calcium metabolism, and skeletal homeostasis revisited. Am J Clin Nutr 2003;78(Suppl):584Se92S. Conigrave AD, Mun HC, Delbridge L, Quinn SJ, Wilkinson M, Brown EM. L-amino acids regulate parathyroid hormone secretion. J Biol Chem 2004;279:38151e9. Conigrave AD, Brown EM, Rizzoli R. Dietary protein and bone health: roles of amino acid-sensing receptors in the control of

[246]

[247]

[248]

[249]

[250]

[251]

[252]

[253]

[254]

[255]

[256]

[257] [258] [259]

[260]

[261]

[262]

[263]

[264] [265]

calcium metabolism and bone homeostasis. Annu Rev Nutr 2008;28:131e55. Chevalley T, Rizzoli R, Manen D, Caverzasio J, Bonjour JP. Arginine increases insulin-like growth factor-I production and collagen synthesis in osteoblast-like cells. Bone 1998;23:103e9. Garn SM, Rohmann CG, Behar M, Viteri F, Guzman MA. Compact bone deficiency in protein-calorie malnutrition. Science 1964;145:1444e5. Thissen JP, Triest S, Maes M, Underwood LE, Ketelslegers JM. The decreased plasma concentration of insulin-like growth factor-I in protein-restricted rats is not due to decreased numbers of growth hormone receptors on isolated hepatocytes. J Endocrinol 1990;124:159e65. Alexy U, Remer T, Manz F, Neu CM, Schoenau E. Long-term protein intake and dietary potential renal acid load are associated with bone modeling and remodeling at the proximal radius in healthy children. Am J Clin Nutr 2005;82:1107e14. Iuliano-Burns S, Stone J, Hopper JL, Seeman E. Diet and exercise during growth have site-specific skeletal effects: a co-twin control study. Osteoporos Int 2005;16:1225e32. Cheng S, Lyytikainen A, Kroger H, et al. Effects of calcium, dairy product, and vitamin D supplementation on bone mass accrual and body composition in 10-12-y-old girls: a 2-y randomized trial. Am J Clin Nutr 2005;82:1115e26. quiz 47e8. Du X, Zhu K, Trube A, et al. School-milk intervention trial enhances growth and bone mineral accretion in Chinese girls aged 10e12 years in Beijing. Br J Nutr 2004;92:159e68. Merrilees MJ, Smart EJ, Gilchrist NL, et al. Effects of diary food supplements on bone mineral density in teenage girls. Eur J Nutr 2000;39:256e62. Teegarden D, Lyle RM, Proulx WR, Johnston CC, Weaver CM. Previous milk consumption is associated with greater bone density in young women. Am J Clin Nutr 1999;69:1014e7. Matkovic V, Kostial K, Simonovic I, Buzina R, Brodarec A, Nordin BE. Bone status and fracture rates in two regions of Yugoslavia. Am J Clin Nutr 1979;32:540e9. Heaney RP. Protein intake and bone health: the influence of belief systems on the conduct of nutritional science. Am J Clin Nutr 2001;73:5e6. Rizzoli R, Bonjour JP. Dietary protein and bone health. J Bone Miner Res 2004;19:527e31. Bonjour JP. Dietary protein: an essential nutrient for bone health. J Am Coll Nutr 2005;24(Suppl):526Se36S. Fenton TR, Lyon AW, Eliasziw M, Tough SC, Hanley DA. Metaanalysis of the effect of the acid-ash hypothesis of osteoporosis on calcium balance. J Bone Miner Res 2009;24:1835e40. Darling AL, Millward DJ, Torgerson DJ, Hewitt CE, LanhamNew SA. Dietary protein and bone health: a systematic review and meta-analysis. Am J Clin Nutr 2009;90:1674e92. Fenton TR, Eliasziw M, Tough SC, Lyon AW, Brown JP, Hanley DA. Low urine pH and acid excretion do not predict bone fractures or the loss of bone mineral density: a prospective cohort study. BMC Musculoskelet Disord 2010;11:88. Chevalley T, Bonjour JP, Ferrari S, Rizzoli R. High-protein intake enhances the positive impact of physical activity on BMC in prepubertal boys. J Bone Miner Res 2008;23:131e42. Bouxsein ML. Biomechanics of age-related fractures. In: Marcus R, Feldman D, Kelsey J, editors. Osteoporosis. San Diego: Academic Press; 2001. p. 509e34. Hanauer A, Young ID. CoffineLowry syndrome: clinical and molecular features. J Med Genet 2002;39:705e13. Pereira PM, Schneider A, Pannetier S, Heron D, Hanauer A. CoffineLowry syndrome. Eur J Hum Genet 2010;18:627e33.

PEDIATRIC BONE

REFERENCES

[266] Yang X, Matsuda K, Bialek P, et al. ATF4 is a substrate of RSK2 and an essential regulator of osteoblast biology; implication for CoffineLowry syndrome. Cell 2004;117:387e98. [267] Harding HP, Zhang Y, Zeng H, et al. An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. Mol Cell 2003;11:619e33. [268] Elefteriou F, Benson MD, Sowa H, et al. ATF4 mediation of NF1 functions in osteoblast reveals a nutritional basis for congenital skeletal dysplasiae. Cell Metab 2006;4:441e51. [269] Martin TJ. Protein nutrition as therapy for a genetic disorder of bone? Cell Metab 2006;4:419e20. [270] World Health Organization. Energy and protein requirements. Report of a joint FAO/WHO/UNO expert consultation. Geneva, Switzerland; 1985.

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[271] National Research Council. Recommended Dietary Allowances. 10th ed. Washington, DC: National Academy Press; 1989. [272] Dewey KG, Beaton G, Fjeld C, Lonnerdal B, Reeds P. Protein requirements of infants and children. Eur J Clin Nutr 1996;50(Suppl. 1):S119e47. discussion S47e50. [273] Martin AD. Apports nutritionnels conseille´s pour la population franc¸aise. 3rd ed. Paris: TEC&DOC; 2001. [274] Bounds W, Skinner J, Carruth BR, Ziegler P. The relationship of dietary and lifestyle factors to bone mineral indexes in children. J Am Diet Assoc 2005;105:735e41. [275] Rodriguez NR. Optimal quantity and composition of protein for growing children. J Am Coll Nutr 2005;24:150Se4S. [276] Young VR, Borgonha S. Nitrogen and amino acid requirements: the Massachusetts Institute of Technology amino acid requirement pattern. J Nutr 2000;130:1841Se9S.

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Pregnancy and Lactation Ann Prentice Medical Research Council Human Nutrition Research, Elsie Widdowson Laboratory, Cambridge, UK

INTRODUCTION 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 MOTHER TO OFFSPRING At birth, the skeleton contains approximately 20e30 g calcium [1e3]. This represents 98e99% of the total body 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 mid-gestation

Pediatric Bone, Second Edition DOI: 10.1016/B978-0-12-382040-2.10010-3

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 highest 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 minerals e phosphorus, 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 750 mg, of which 60% is in the skeleton, and a typical whole-body accretion rate in infancy is 3 mg/day. For zinc, typically the whole-body content is around 50 mg at birth, of which 30% is in the skeleton, and the accretion rate in infancy is around 0.4 mg/day [7,8]. Breast milk secretion during full lactation averages approximately 120, 25, and 1.6 mg/day for phosphorus, magnesium, and zinc, respectively, but the ranges of values between individuals are wide. These mineral fluxes from mother to child represent a significant proportion of the mineral intakes of the mother, especially for calcium [9]. Intakes of these minerals vary in different areas of the world and range

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widely between individuals, but average intakes for women are of the order of 300e1500 mg/day calcium, 1000 mg/day phosphorus, 250 mg/day magnesium, and 10 mg/day zinc [10e13]. 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 Metabolism and Bone Biochemistry Human pregnancy is associated with major changes in mineral and bone metabolism (Table 10.1). 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 non-pregnant state (Fig. 10.1) [14e20]. The elevation in calcium absorption is associated with increased intestinal expression of calbindinD9K, a vitamin D-dependent calcium-binding protein [21,22]. The increased expression of calbindin-D9K is a result of increased transcription and reduced posttranslational degradation mediated by 1,25-dihydroxyvitamin D, which is elevated in pregnancy, and by other factors [21]. 1,25-Dihydroxyvitamin D also acts at the enterocyte brush border to open voltage-gated calcium channels (primarily TRPV6). However, there is evidence TABLE 10.1

from animal studies to suggest that this mechanism may not be fully responsible for the upregulation of intestinal calcium absorption in pregnancy, which has been shown to occur in the absence of 1,25-dihydroxyvitamin D and its receptor [23]. 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 [16,24e26]. Measured calcium balance in the later stages of pregnancy is generally positive and retention approximates to that required for fetal growth [27]. 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 throughout [28,29]. 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 largely reflects the change in serum albumin concentration associated with the increased intravascular fluid volume of pregnancy and the resulting hemodilution [29,30]. Calcium transport across the placenta is predominantly through an active transcellular pathway involving TRPV6 expressed on the apical membrane of the trophoblast, intracellular calcium-binding proteins (primarily calbindin-D9K) and active transport into the fetal circulation at the basolateral membrane through the calcium pump PMCA3 [31,32]. The fetal calcium concentration is maintained at a higher level than the maternal concentration through the actions of fetal parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) on the expression of the intracellular calbindins [32,33]. Other regulators of calbindin expression include progesterone and estrogen, but studies in animals do not support an essential role for the vitamin D system in placental calcium transfer, although it may have a role as a negative modulator [31e33].

Calcium and Bone Metabolic Changes in Human Pregnancy, Compared to the Non-Pregnant, Non-Lactating State

Calcium absorption

[

Serum 1,25 (OH)2 vitamin D (free and bound)

[

Urinary calcium excretion, daily

[

Serum parathyroid hormone (intact)

4Y

Fasting urinary calcium, creatinine corrected

4Y

Serum parathyroid hormone-related protein

[

Serum calcium (ionized)

4(Y)

Nephrogenous cyclic AMP

4

Serum calcium (total)

Y

Serum calcitonin

[4

Tubular phosphate reabsorption

4Y

Bone resorption histology and markers*

[

Urinary phosphate excretion

[4

Bone formation markers (except Oc)**

[

Serum phosphate

4Y

Osteocalcin (intact)

Y

* Urinary collagen cross-links, telopeptides, hydroxyproline, serum tartrate-resistant acid phosphatase. ** Serum bone alkaline phosphatase and procollagen peptides

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↑ 1,25 (OH)2D ↑ PTHrP ↑ Prolactin ↑ Placental lactogen

pregnancy to term. This has been attributed to hemodilution, decreased concentrations of the zinc binding protein, hormonal changes in pregnancy and active transport of zinc to the fetus [8]. There is evidence that zinc absorption and urinary zinc excretion are increased, although individual responses are highly variable [13,41e43].

↑ intestinal Ca++ absorption ↑ Ca-release from bone ↑ Ca-uptake into bone

↑ urinary Ca excretion

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Bone Turnover

Ca transport to fetus

FIGURE 10.1 Schematic representation of the changes in calcium and bone metabolism that occur during pregnancy.

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 [14], and net absorption is higher in pregnant women compared with non-pregnant women [34]. Some studies have indicated a decrease in serum phosphorus concentration [16] and the renal phosphate threshold in the second and third trimesters of pregnancy, with a corresponding increase in urinary phosphate excretion [16,18]. The data are inconsistent, and other studies have shown no changes in phosphate metabolism [18,24,35]. Little is known about the mechanisms of transporting phosphate to the fetus [32]. As with calcium, fetal phosphate concentrations are maintained at a higher level than in the maternal circulation which suggests that there is active transport across the placenta, although the mechanisms involved have yet to be fully elucidated [32]. 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 [36e39] and are lower in pregnant women than preconception [18] or compared to non-pregnant controls [40]. These effects parallel the changes in serum proteins due to hemodilution and are not considered to represent alterations in maternal magnesium status [10]. Lymphocyte magnesium concentration is unchanged or decreased in pregnancy [38,39]. Urinary magnesium excretion is elevated in late pregnancy [18]. Serum zinc concentration decreases in pregnancy by around 35% from early

Bone resorption increases during pregnancy. This has been indicated both histologically [44] and biochemically by urinary markers, such as collagen cross-links, telopeptides, and hydroxyproline, serum markers such as tartrate-resistant acid phosphatase [17e19,45,46] and stable isotope kinetic studies [47]. 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 [17e19,45,46,48e50]. However, serum osteocalcin concentration, a commonly used marker of bone formation, is decreased relative to preconception levels [17,18], although its concentration in late gestation is higher than in early pregnancy [17e19,51]. Increases in bone turnover indices are observed in early gestation. Their levels increase by 50e200% during pregnancy [17e19,46,48,52]. The increases in markers of bone resorption occur before those of bone formation [19,53], suggestive of bone mineral mobilization. In support of this, a stable isotope study of Brazilian women at 10e12 weeks of pregnancy recorded a net deficit in bone calcium turnover [47]. 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 procollagen (PICP), an index of bone formation [45]. 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. Decreases in bone resorption markers have been noted in pregnant women following calcium supplementation [54,55], indicating that the physiological response to an increased calcium load remains intact during pregnancy. 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 [22]. The disparity between the different indices of bone formation, for example, may be due to the degradation or uptake of osteocalcin by the placenta [49,56]. It has been suggested that the reduction in osteocalcin

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concentration may facilitate bone formation [28]. Measurements of an osteocalcin metabolite e octeocalcin N terminal fragment, Ocf e 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 [19]. Total alkaline phosphatase is not a useful index of bone formation in pregnancy because of the contribution from the placental isoform [22]. The increase in resorption markers may partly reflect a contribution from the turnover of the fetal skeleton. However, a study of the ratio of a to b 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 a-CTX and only 2% of b-CTX [19]. Calciotropic Hormones The concentration of serum 1,25-dihydroxyvitamin D is increased substantially throughout pregnancy [16e18,57], while observational studies suggest there is no change or a modest decline in 25-hydroxyvitamin D concentration [23]. The elevation in 1,25-dihydroxyvitamin D concentration occurs in both the free and protein-bound forms [24,58]. The increase is apparent in the first trimester and continues to rise during gestation by several-fold. Until late in pregnancy, the increase in 1,25-dihydroxyvitamin D parallels an increase in the concentration of vitamin D-binding protein, such that the proportion of the free hormone in the circulation is only elevated in the last trimester [23,59,60]. It is unlikely, therefore, that the early rise in 1,25-dihydroxyvitamin D is fully responsible for the increase in intestinal absorption at that time, and there is animal evidence to suggest that neither this hormone nor its receptor are necessary for the higher calcium absorption efficiency of pregnancy [23]. The mechanisms underlying the increase in 1,25dihydroxyvitamin D are unclear but may involve placental or fetal synthesis of the hormone from maternal 25-hydroxyvitamin D, upregulation of maternal renal 1a-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 [20,22,61e64]. It is likely that the production of 1,25-dihydroxyvitamin 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 [22]. Fetal cord blood concentrations of 25-hydroxyvitamin D are similar to or up to 20% lower than maternal concentrations [23], while fetal 1,25-dihydroxyvitamin D concentrations are low. 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 [10,65]. Furthermore,

although vitamin D and its metabolites are essential for mineral ion homeostasis in adults, there is growing evidence from animal studies that fetal mineral ion homeostasis appears to be mostly independent of this hormonal system [32], or that it plays only a minor modulating role in placental calcium transfer [33]. Although serum 1,25-dihydroxyvitamin D is raised, there is no evidence of an increase in intact PTH concentration in pregnancy [16,20,24,35,52,66], and it may be decreased [17,18,53,67]. There may be a nadir in PTH concentration in early gestation followed by an increase in later pregnancy [28]. 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 [20]. 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 [20,68]. This is supported by normal nephrogenous cyclic adenosine monophosphate (NcAMP) production, a marker of PTH-like bioactivity [16,35,66]. Consequently, the view of pregnancy as a period of physiological hyperparathyroidism driven by the fetal demand for calcium [69] is no longer regarded as tenable [16]. However, the homeostatic response to a calcium load by an increase in PTH appears to remain intact [70]. Fetal serum concentrations of PTH, which originates from the fetal parathyroids and thymus, are low and decrease further towards the end of gestation, although fetal serum ionized calcium remains stable [20]. Increased concentrations of PTHrP are detected in the maternal circulation during pregnancy [71], probably originating from fetal, placental, or mammary tissues [20]. PTHrP, or more specifically its amino-terminal fragments, has close homology with the N-terminal 1e34 amino acid sequence of PTH. It has the ability to activate the PTH/PTHrP receptor [72] and, consequently, has PTH-like characteristics. It stimulates renal 1a-hydoxylase activity and NcAMP production, thereby promoting 1,25-dihydroxyvitamin D synthesis and calcium reabsorption [21]. In addition, N-terminal PTHrP promotes bone resorption via the classical PTH/PTHrP receptor, although the C-terminal fragment PTHrP (107e139) inhibits osteoclastic bone resorption through a different receptor [72]. 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. Non-pregnant women administered PTHrP (1e36) subcutaneously have elevated 1,25-dihydroxyvitamin D levels, urinary calcium excretion, and

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NcAMP production with no alteration in serum PTH or calcium concentrations [73] e a response that resembles some of the biochemical changes of pregnancy. The response of calcitonin (CT) to pregnancy appears to be highly variable [74], with some studies reporting elevations [20,40,75] and others reporting no changes [17,28,61,74]. Increases in circulating CT have been observed in thyroidectomized women during pregnancy, probably as a result of CT synthesis by mammary and placental tissues [20]. The physiological function of CT is not fully understood, although a role in protecting the maternal skeleton from resorption during pregnancy has been proposed [76], and it may promote renal calcium excretion [22]. Calcitonin is secreted by the fetal thyroid and concentrations in the fetal circulation are higher than maternal concentrations [32]. Nevertheless, animal studies do not suggest a major role for calcitonin in fetal mineral homeostasis or bone metabolism [32]. 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, insulin-like growth factor-1 (IGF-1), components of the OPG/RANKL/RANK system and the ratio of osteoprotegerin to the other components [19,20,50,77,78]. Their relative contributions to mineral and bone metabolism in human pregnancy, and the interactions between their effects, have yet to be established. Positive correlations with bone turnover markers and kinetically derived measures of bone mineral mobilization have been observed in pregnant women for maternal IGF-1, estrogen and placental lactogen concentrations [47,79e81)] but not for components of the OPG/ RANKL/RANK system [78].

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 sufficient to result in an overall increase or decrease in bone mineral content is not clear. 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 dualand single-energy x-ray absorptiometry, and quantitative computed tomography. Although the radiation doses incurred with modern instruments are generally

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low and similar to that received from background radiation, these techniques are not useful for measurements of the axial skeleton in pregnant women because of the involvement of fetal tissues. As a consequence, investigations of the axial skeleton and whole body are limited to estimating the integrated skeletal response over the whole of pregnancy by measuring bone mineral status before conception and after delivery. Pregnancy is accompanied by changes in weight and advances in age, both of which can have independent effects on the bone mineral content of the skeleton, and these should be factored into any conclusions about bone mineral changes during pregnancy [82]. In addition, the bone mineral content of the skeleton postpartum is affected by lactation (discussed later) and, consequently, the timing of the postpartum measurement can confound the interpretation of the bone changes during pregnancy in women who breastfeed. To date, of the prospective studies that have been undertaken, most involved relatively small numbers of individuals [17e19,46,52,53,82e89], and only a few have compared the results with a group of non-pregnant women, measured over the same time period, to consider age and weight changes [82,89]. Changes in bone mineral content have been reported in many of these studies, but the observed response differs widely between studies, between individual women and between skeletal sites. The average effect of pregnancy on the axial skeleton reported in these studies ranges from no change in bone mineral content to a decrease of around 4e5%. Nevertheless, a pattern is emerging for pregnancy to be associated with a reduction in bone mineral content at one or more axial sites, reflected in a decrease in whole-body bone mineral content of around 2%. In a longitudinal study of pregnant women in the UK, the decrease in bone mineral content at the spine and whole body was largely independent of concomitant changes in weight and age [82], but the decrease at the femoral neck was similar to that experienced by non-pregnant women studied over the same time period, and therefore could not be ascribed to pregnancy [82]. The calculated release of calcium from the maternal skeleton over the course of pregnancy was estimated to be have been sufficient to cover most of the calcium required for fetal bone mineral accretion [82]. In the case of women entering pregnancy during or after a period of extended lactation, substantial increases in bone mineral content have been reported [83,85], suggesting that maternal adaptative processes are sufficient to supply mineral for both maternal and fetal mineral accretion. Measurements at peripheral skeletal sites, using single- or dual-energy absorptiometry (DXA), peripheral quantitative computed tomography or bone ultrasonography, have also been used to investigate the pattern

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of skeletal response during pregnancy [18,24,28,48, 52,82,86,90e94]. 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 [44]. 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 [28,52,86,91,92], but generally not at more proximal appendicular sites and not in all studies [18,24,28,82]. Ultrasound studies of the os calcis and phalanges have reported decreases in the measured variables in the later stages of pregnancy [48,90,93,94]. Speed of sound and broadband ultrasound attenuation, measured by bone ultrasonography are regarded as indices of bone mineral density for fracture risk prediction in older people. However, the validity of this assumption for quantifying changes in bone mineral content during pregnancy is not known, particularly in the presence of peripheral edema, and has been called into question by a longitudinal study of lactating women in which ultrasound measurements of the os calcis were not concordant with the skeletal bone mineral changes observed by DXA [95]. 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 [83,85]. 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. Slim pregnant women (body mass index <22 kg/m2) exhibited significant increases in bone mineral content at the femoral neck that were independent of weight gain in pregnancy and were not observed in larger pregnant women or size-matched controls [89,96]. Weight changes in pregnancy have been shown to be positively associated with similar changes in bone mineral content to nonpregnant women who gain weight, which are superimposed on the skeletal response to pregnancy [82]. In addition, there may be pregnancy changes in the size, internal architecture and orientation of the skeleton that may influence the in vivo measurement of bone mineral content and bone mineral density and alter their interpretation in terms of bone strength [82,97]. A study of older women reported a positive correlation between bone area of the whole body or femoral neck with parity,

without changes in bone mineral content (implying an increase in bone strength despite a decrease in bone mineral density), which supported studies suggesting that higher parity (i.e. greater number of pregnancies) is associated with lower fracture risk in later life [97]. However, any change in tissue depth, bone mineral content, soft tissue composition or bone orientation can affect bone measurements using the currently available techniques because of alterations in bone edge detection and calibration, and, therefore, the possibility of technical artifacts also needs to be considered [82]. Osteoporosis of Pregnancy Fragility fractures due to osteoporosis can occur in pregnancy, although the incidence is rare [26,98e100]. The condition often involves the spine or hip, is more common in the first pregnancy, and usually resolves spontaneously a few months postpartum [26]. Osteoporosis of pregnancy is generally either idiopathic or secondary to treatment with corticosteroids, magnesium, or warfarin [99e101]. 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 [26,102]. However, fractures associated with pregnancy can occur in the absence of low bone mineral density [103]. There are no data to suggest that osteoporosis of pregnancy is either an exaggerated metabolic response to pregnancy or a consequence of dietary deficiencies or other environmental factors [104,105]. 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 on osteoporosis risk have produced conflicting results [9,106]. The disparities may be due to the difficulties of defining reproductive history adequately, of separating the effects of pregnancy and lactation, and of 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 increasing parity is either associated

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with higher bone mineral content, greater bone area and/or reduced fracture risk [97,107e114] or has no effect [114e120]. There is no evidence that women who have become pregnant but miscarried have altered bone mineral status [121]. 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 osteoporotic fracture in later life [106,114]. The potential influence of individual differences in hormonal milieu has been suggested by a report of an interaction between parity and oral contraceptive use on hip fracture risk [111]. 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 [111].

Influence of Maternal Diet and Nutritional Status on Mineral Metabolism of the Pregnant Mother and Skeletal Development of the 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 non-pregnant controls [16,70] but experience similar increases in urinary excretion and decreases in bone resorption [70]. These data provide further evidence that pregnancy is a state of physiological hyperabsorption [16]. 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 [18,122]. Few investigations have been conducted on women with a low calcium intake. Cross-sectional studies of Malay and Brazilian women reported lower urinary calcium excretion [47,123] and higher serum concentrations of intact PTH in late pregnancy compared to early pregnancy [123]. 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. Similarly,

229

studies in Brazil and Mexico (where mean calcium intakes are around 450e500 mg/day) reported larger increments in bone turnover markers from the second to the third trimester in pregnant women with the lower calcium intake. 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 [27,124]. However, a stable isotope study in 10 Brazilian women reported smaller deficits in net calcium balance in early and late pregnancy associated with higher calcium intake [47]. There is also little evidence to suggest that the plane of maternal calcium nutrition modifies the integrated skeletal response to pregnancy. In absorptiometric studies of Western women consuming an average of 1100e1200 mg/day of calcium, the decreases in bone mineral content observed across pregnancy were not correlated with calcium intake [82,87]. This contrasts with the report of larger decreases in phalangeal ultrasonographic bone propagation velocity in pregnant women with a calcium intake less than 1000 mg/day compared with those with a higher intake [90]. The effects of calcium supplementation in pregnancy have also been little studied. 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 [125], whereas a small, nonblinded study of Chinese women given milk powder (350 mg/day of calcium) with and without calcium supplements (600 mg/day of calcium) reported greater bone mineral density, measured by absorptiometry, at the whole body and spine in those consuming milk powder [54]. Conversely, a randomized, placebocontrolled study of Gambian women (mean intake 355 mg/day) supplemented with 1500 mg/day of calcium from 20 weeks’ pregnancy to term demonstrated no significant differences in these absorptiometric measures at 2 weeks postpartum but lower bone mineral at the hip in those who had received the calcium supplement [126]. 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 [127e129]. 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 [130]. Taken together, the limited data indicate that physiological adaptations occur during human pregnancy that provide an adequate supply of calcium to meet fetal

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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 300 mg/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 [125]. A possible effect of maternal calcium intake on fetal mineralization has also been suggested from a study of US women taking part in a calcium supplementation trial during pregnancy (2 g/day calcium or placebo from 22 weeks’ gestation) [131]. For women with a low customary calcium intake (<600 mg/day), calcium supplementation was associated with greater total bone mineral content of the neonate, but there was no significant difference in infant size. No supplement effect was noted for women with higher calcium intakes. However, a trial of Gambian women (mean dietary intake 350 mg/day of calcium) supplemented with 1500 mg/day of calcium or placebo from 20 weeks’ pregnancy to term reported no significant effects on birth weight or length, or on infant size and bone mineral content at 2, 13 or 52 weeks of age [132] or on stature at age 5e8 years [133]. 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 pre-eclampsia. 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 pre-eclampsia has been associated with neonatal hypermagnesemia, elevated alkaline phosphatase and bone abnormalities [134e136]. Some observational studies suggest that poor maternal zinc status is associated with fetal growth retardation, but the data are conflicting [137]. Meta-analyses and systematic reviews of zinc supplementation trials in pregnant mothers have not shown an effect on infant birth weight or other indicators of fetal growth such as crowneheel length, fetal femur diaphysis length and head circumference [8,137,138]. There is some evidence of a positive effect in a subset of studies involving underweight or zinc-deficient women, but the data are limited [8]. There is evidence of greater fractional zinc absorption and intestinal conservation of endogenous fecal zinc in pregnant mothers with low zinc intakes or plasma zinc concentrations [139,140]. It is probable, therefore, that, in healthy women, metabolic adaptations during

pregnancy ensure an adequate transfer of zinc to the fetus even when dietary zinc intakes are marginal [12,139]. Vitamin D Status Vitamin D deficiency during pregnancy is associated with congenital rickets and craniotabes in the newborn and with the development of rickets in infancy, especially when the child is exclusively breastfed [10,141]. 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 UVB exposure are at risk unless their diet provides sufficient vitamin D [10,141]. Osteomalacia and hypovitaminosis D are common among subgroups of pregnant women from many parts of the world [142] but there is little to suggest that this problem is specific to or is worsened by pregnancy [23]. The effect of the seasonal variations in vitamin D status experienced in temperate countries on maternal bone outcomes has been little investigated. One observational study of British women reported greater reductions in calcaneal ultrasound bone measures during pregnancy among those pregnant in the winter than in the summer [94]. Supplementation of pregnant women with vitamin D increases circulating 25-hydroxyvitamin D concentrations but there is no evidence that other maternal outcomes are affected [23]. Poor maternal vitamin D status in pregnancy can affect fetal and infant skeletal growth and ossification, tooth enamel formation, and calcium handling [65,143,144], although there have been no systematic studies [23]. 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 low cord serum 25-hydroxyvitamin D concentrations [145]. Lower 25-hydroxyvitamin D concentrations in pregnant women are associated with greater ‘splaying’ of the fetal femoral metaphysis assessed with high-resolution ultrasound [146] and with shorter kneeeheel length at birth [147]. In Japan, seasonal variations in the rate of neonatal craniotabes parallel the seasonal variations in maternal vitamin D status [148] while in America birth length is greater among neonates born in winter/spring than those born in summer/autumn [149]. Seasonal variations in maternal vitamin D status are reflected in neonatal bone mineral content [150e152], though the patterns observed in different populations are not consistent. In 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 [150]. Conversely, in American studies, neonates born in the winter had higher bone mineral

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content and lower bone turnover than those born in the summer [151,152]. The differences in these findings may indicate that there is a vulnerable period to vitamin D insufficiency in early gestation [65] or that there is greater use of vitamin D supplements by American women in winter [150] or that they reflect other seasonal factors unrelated to vitamin D. Newborns of mothers at risk of vitamin D deficiency given a vitamin D supplement during pregnancy have higher serum calcium concentrations than those of mothers who have not been supplemented [143,145,153]. Vitamin D supplementation of British Asian mothers, who are at risk of vitamin D deficiency, resulted in smaller fontanels and a trend to higher birth weight in their newborns compared to controls [153]. The influence of season and maternal vitamin D status on infant growth appears 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 [154]. Lower vitamin D status in pregnant British women was associated with lower whole body and lumbar spine bone mineral content in their children at 9 years of age [155], and winter/spring born American infants were heavier, taller and had larger head circumference at seven years of age than those born in summer/autumn [149].

nutritional environment in utero and in early life, in addition to influencing the risk of adult chronic diseases [165], may be an important modulating factor of skeletal growth in childhood and of bone health in old age.

LACTATION Mineral Metabolism and Bone Biochemistry Major changes in mineral and bone metabolism occur for several months postpartum (Figs 10.2 and 10.3). The pattern of change is dependent on whether the mother chooses to breastfeed and, if lactation is initiated, on the pattern of breastfeeding adopted by the mother. Likely lactation-associated variables include the duration of exclusive breastfeeding, the timing of introduction of other foods to the infant (as a complement to breastfeeding), breastfeed frequency (which will vary with stage of lactation), the initiation of replacement of breastfeeding by other meals (when breastfeeding frequency is reduced), and the timing of cessation of breastfeeding. 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.

General Nutrition

Suckling

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 [156]. Poor nutrition during pregnancy may reduce neonatal bone density as well as size [156,157]. 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 been subjected to systematic analysis and review [158,159]. The intakes of specific foods in pregnancy, such as milk, milk products, pulses and green-leafy vegetables have been related to high bone mineral status in the infant [160] and child [161]. Deficiencies of specific nutrients, such as folic acid and other micronutrients, and their replenishment through supplementation, can also influence fetal bone growth and development, and are associated with increases in weight, size, bone mineral content and indices of calcium and bone metabolism at birth [159,162] and later in childhood [161]. Size at birth and in infancy predicts adult bone mineral mass, probably through tracking of skeletal size through childhood [163,164]. This suggests that the

↑ prolactin

↓ GnRH

↑ PTHrP

↓ estrogen

↑ bone resorption

↑ blood Ca++

↓ PTH

↑ Ca++uptake by mammary tissue

milk calcium

FIGURE 10.2 Schematic representation 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 [166]).

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Estrogen repletion

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. Also, because non-lactating women may experience changes as a result of not initiating breastfeeding or stopping breastfeeding early, in addition to no longer being pregnant, comparisons between lactating and non-lactating postpartum women can obscure or exaggerate the response to lactation.

↑ osteoblast activity

↑ Ca-uptake into bone

↓ serum Ca++

Calcium

↑ PTH

↑ 1,25 (OH)2D

↑ renal Ca++ reabsorption

↑ intestinal Ca++ absorption

FIGURE 10.3 Schematic representation of the proposed mechanism that increases bone mineral after weaning permission from Kalkwarf [166]).

(reproduced with

The main metabolic and biochemical differences for breastfeeding mothers during full lactation (first 3e6 months) and during the periods before and after cessation of breastfeeding are summarized in Tables 10.2 and 10.3. 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 10.2 and 10.3. It should be appreciated that because non-lactating women may experience TABLE 10.2

Calcium absorption and urinary calcium excretion decline from the high levels of pregnancy to prepregnancy levels after delivery [15,17,19,81,167,168]. The decrease in urinary calcium output partly reflects the reduction in glomerular filtration rate after parturition [22]. Further decreases in total urinary calcium output, and reductions in fasting calcium excretion, have been reported for breast-feeding women [17,20,40,42,65,169e171]. This finding has not been observed in all studies, particularly those that have made comparisons with non-lactating mothers at the same stage postpartum [18,172e175]. Serum ionized and total calcium concentrations are raised in lactation compared to late pregnancy and normal controls [17,20,122,126,171], whereas studies comparing lactating and non-lactating mothers at the same stage postpartum have reported no differences [172,174]. Studies comparing serum calcium in lactation and prepregnancy show either an elevation or no difference [17,18]. Taken together, the data suggest that the first 3e6 months of breastfeeding are, or can be, associated with increased renal conservation of calcium but not with increased calcium absorption. The period immediately following the cessation of breastfeeding and, for women who breastfeed for 6e12 months or more, the later months of lactation may be

Calcium and Bone Metabolic Changes in Human Lactation During 3e6 Months of Full Breastfeeding NP

NL

NP

NL

Calcium absorption

4

4

Serum 1,25 (OH)2 vitamin D (free and bound)

4Y

[4

Urinary calcium excretion, daily

4Y

4

Serum parathyroid hormone (intact)

4Y

4Y

Fasting urinary calcium, creatinine corrected

Y

nk

Serum parathyroid hormone-related protein

[

[

Serum calcium (ionized)

[4

4

Nephrogenous cyclic AMP

[4

[

Serum calcium (total)

[4

4

Serum calcitonin

[4

4

Tubular phosphate reabsorption

[

4Y

Bone resorption markers*

[

[

Urinary phosphate excretion

Y

[

Bone formation markers (not osteocalcin)**

[

[

Serum phosphate

[

[4

Osteocalcin (intact)

4

[

* Urinary collagen cross-links, telopeptides, hydroxyproline, serum tartrate-resistant acid phosphatase. ** Serum bone alkaline phosphatase and procollagen peptides. nk¼not known Data for lactating women compared to not-recently pregnant women (NP) and to post-pregnant mothers who did not lactate (NL).

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TABLE 10.3

Calcium and Bone Metabolic Changes in Human Lactation During the Later Stages or After the Cessation of Breastfeeding NP

NL

NP

NL

Calcium absorption

4

[

Serum 1,25 (OH)2 vitamin D

[

[4

Urinary calcium excretion, daily

4Y

4

Serum parathyroid hormone (intact)

[4

[4

Fasting urinary calcium, creatinine corrected

4Y

nk

Serum parathyroid hormone-related protein

nd

nd

Serum calcium (ionized)

4

nk

Serum calcitonin

4

4

Serum calcium (total)

4

4

Nephrogenous cyclic AMP

4

4

Tubular phosphate reabsorption

[4

4

Bone resorption markers*

4

[4

Urinary phosphate excretion

4Y

4

Bone formation markers (except Oc)**

4

[4

Serum phosphate

4Y

4

Osteocalcin

4

[4

* Urinary collagen cross-links, telopeptides, hydroxyproline, serum tartrate-resistant acid phosphatase. ** Serum bone alkaline phosphatase and procollagen peptides. nd¼not detectable, nk¼not known Data for lactating women compared to not-recently pregnant women (NP) and to post-pregnant mothers who did not lactate (NL).

a time of recovery when the changes in calcium and bone metabolism are relaxed and calcium retention is increased. Some studies have reported decreases in calcium excretion in women after breastfeeding has stopped [18,171]. Others indicate that urinary calcium output increases during long lactation and after the cessation of breastfeeding to prepregnancy levels or to levels typical of women of the same age who have not recently been pregnant [17,169,176]. A study comparing women in the period after breastfeeding to mothers at the same stage postpartum but who had not breastfed found no differences in urinary calcium excretion or serum calcium concentration [174]. Increased calcium absorption efficiency has been observed 2 or 3 months after the cessation of breastfeeding [168] but not in all studies [17,18] and not 6 months or longer after breastfeeding has stopped [167]. The return of ovarian function may complicate the findings, although the picture is not clear-cut [166]. The resumption of menstruation has been associated with decreased urinary calcium excretion [17,171] and higher calcium absorption efficiency [166] but not in all studies [17,174]. Use of oral contraceptives containing ethinyl estradiol by lactating women has been associated with reduced urinary calcium excretion [169]. 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 [171,173,176]. Compared to non-lactating mothers at the same stage postpartum, breastfeeding women have similar or raised serum

phosphate concentrations [10,172,174] and increased urinary phosphate excretion [172]. Tubular phosphate reabsorption is decreased [174] or unchanged [172]. In the later stages of lactation and after the cessation of breastfeeding, serum and urinary phosphate levels normalize [171,173,176] and may be reduced compared to prepregnancy, with an increase in tubular phosphate reabsorption [18]. 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 [177] or unaffected by lactation [18,169]. Lactating women have been reported to excrete less zinc than non-lactating mothers and women who have not recently been pregnant [169], but not in a longitudinal study comparing zinc excretion in lactation with that in preconception [41,42]. The potential for conserving endogenous zinc by reducing renal excretion is regarded as limited [139]. However, in contrast to calcium and the other minerals, zinc absorption efficiency has been shown to increase markedly in early lactation compared to late pregnancy and to preconception levels [42,139,140] and for there to be greater intestinal conservation of endogenous fecal zinc in lactating mothers with a low zinc intake [139]. 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 [171,175,178,179]. Indices of bone resorption (e.g. cross-linked amino-terminal telopeptide of procollagen 1 (NTX), cross-linked carboxy-terminal telopeptide of type 1 collagen (CTX), carboxy-terminal telopeptide of type 1 collagen (1CTP), deoxypridinoline

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and hydroxyproline) and of bone formation (e.g. osteocalcin, amino-terminal telopeptide of procollagen 1 (P1NP), carboxy-terminal propeptide of type 1 collagen (P1CP), and bone alkaline phosphatase) have been studied. Some changes are evident after delivery even in women who do not breastfeed [172,174,179]. 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 [17,18,52]. Measured osteocalcin concentration is at variance with other markers because, although it is elevated in lactation compared to non-lactating controls, this represents an increase from the low concentrations observed in pregnancy to levels similar to those prepregnancy [17,18,48,51]. The length of lactation and the duration of postpartum amenorrhea influence the patterns of changes of these markers, which occur for longer and are more pronounced in those who breastfeed for a longer time and in those whose menses return later [48,178,180]. The concentrations of bone turnover markers decline after 6e12 months, even in women who continue to breastfeed for 18 months or longer [176]. The levels also decline when lactation stops, but differences have been observed for several weeks after the cessation of breastfeeding between mothers who lactated and those at the same stage postpartum who did not [174,178]. 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 [52,174,176,180,181]. Such a pattern would allow for the release of mineral from bone, followed by its restitution after a period of time (as described later) [176]. However, other explanations have been proposed for bone loss in the face of these increases in both bone resorption and formation, a pattern that distinguishes lactation from pathological bone loss where marked decoupling of resorption and formation occurs [179]. These include the possibility that completion of osteoblast differentation and osteoid mineralization does not occur during lactation in spite of accelerated initiation of the osteoblast program in the bone remodeling unit, and that the accumulated pre-osteoblasts rapidly proceed to complete the differentiation cycle after lactation stops [179]. 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 [17,18,58,167,172,176,182,183]. Similar or higher concentrations of 1,25-dihydroxyvitamin D have been reported in lactating women compared to non-

lactating mothers [168,172,182e185], but similar or slightly decreased compared to prepregnancy or nonpregnant, non-lactating women [17,172,176,183]. PTH is similar or lower in lactating women than in nonlactating women [174,183] and in women before pregnancy or who have not been pregnant recently [17,172,176,182,183,186]. Raised serum CT concentrations in early lactation have been reported in some studies [40,176], but not in others [17,172,187]. Elevated or normal NcAMP production indicates enhanced PTH-like activity during lactation in the face of normal or lowered PTH concentrations [171,174]. Mothers who are breastfeeding twins have elevated concentrations of PTH, 1,25-dihydroxyvitamin D, and calcitonin compared to women nursing singletons [188]. 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 [172,176,183,187]. The later stages of lactation and the period following the cessation of breastfeeding have been associated with increased serum PTH and 1,25-dihydroxyvitamin D concentrations [18,171,176], although this finding is not consistent [182,183] and no changes are seen in serum CT concentration [17,172]. Increases in PTH and 1,25dihydroxyvitamin D have been reported by 6 months of lactation, with 1,25-dihydroxyvitamin D concentrations exceeding those of non-lactating mothers by this stage postpartum [174]. Other studies show no difference by breastfeeding status [183]. No difference in NcAMP production has been noted between women who have recently stopped breastfeeding and either non-lactating controls or women who have not recently been pregnant [171,174]. It therefore appears that the changes in calcium and bone metabolism observed in lactation are not driven by the classical PTHevitamin 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 in response to a change in calcium load [174]. 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 breastfeeding 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 early concept that human lactation is a period of physiological hyperparathyroidism [189]. PTHrP is regarded as a prime candidate for the role of principal regulator of calcium and bone metabolism in lactation [173,190,191]. It is produced by the lactating mammary gland, possibly under the influence of

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progresses [183], 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 [174], but this has not been observed in all studies [168]. In addition, estrogen deficiency in non-pregnant, non-lactating women of reproductive age resulting from GnRH agonist therapy causes increased calcium excretion with suppression of PTH and 1,25-dihydroxyvitamin D, a pattern that does not resemble the metabolic response to lactation [20]. It is likely that the observed changes seen in lactation are due to the combined effects of many different factors.

Effect on the Maternal Skeleton Physiological Changes Human lactation is accompanied by significant decreases in maternal bone mineral content during the first 3e6 months, as shown by prospective, longitudinal studies [52,88,91,170e172,175,195e203]. The reductions are particularly pronounced in the axial skeleton, where average decreases of 3e5% have been observed at the spine and hip (Fig. 10.4). Changes in appendicular sites are less marked or absent. Whole-body bone mineral content is generally decreased by approximately 0.5e1%. For a typical woman, this represents Effect of lactation on bone mineral content 4

Cambridge Mean±SE, n=38

Lumbar spine

2 Change in BMC (%)

prolactin, and is secreted into breast milk and released in significant amounts into the maternal bloodstream [173,192,193]. PTHrP has PTH-like activity in the kidney, where it stimulates renal calcium conservation, increases urinary phosphorus excretion, and elevates NcAMP production [174]. Serum PTHrP concentration is higher in lactating women than in non-lactating postpartum women [183,190,191] and in those who are postlactation [173]. 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 [194]. 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 [22]. However, evidence regarding the importance of PTHrP in lactation is inconsistent. Subcutaneous administration of PTHrP(1e36) to non-pregnant, non-lactating women elevates serum 1,25-dihydroxyvitamin D concentration, increases urinary phosphate and calcium excretion, and decreases serum phosphate [73]. This pattern of changes does not resemble the metabolic response to 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 [183,190]. 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 breastfeed [183]. In addition, no correlations have been demonstrated between PTHrP and other biochemical indices or bone mineral changes during established lactation [181,190], although an inverse correlation was observed with PTH during the initiation of lactation (2 or 3 days postpartum) [181]. 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 [72]. 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 estradiol tend to normalize as lactation

Whole body

0

Femoral neck

–2

–4

–6

0

13

26

39

52

65

W eeks post-partum

FIGURE 10.4 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 women who breastfed exclusively for at least 2 months (reproduced with permission from Prentice et al. [6]).

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a mobilization of approximately 5e10 g of calcium which, when averaged over a 3-month period, equates to approximately 50e100 mg/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 2e4 g overall and 20e40 mg/day averaged over 3 months. These rates of change are remarkably rapid, given that menopausal bone loss is typically 1e3% per year. The magnitude and duration of the skeletal response are greater in women who breastfeed longer [190,196,203] and who produce larger volumes of breast milk [204]. The effects are attenuated or do not occur in mothers who do not breastfeed [52,196,198], indeed, increases in bone mineral content have been reported [196,205]. On an individual basis, the bone changes associated with lactation 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 breastfeeding 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 [204], age, and parity [203]. Lactation-associated bone loss is recovered during late lactation and after breastfeeding stops [170,195,196,199]. For women who conceive during lactation, increases in bone mineral are observed during the following pregnancy [83]. At most skeletal sites, women have a higher bone mineral status after breastfeeding has stopped for at least 2 or 3 months than immediately after delivery [196,199]. Exceptions occur at the femoral neck and wrist, where bone mineral status tends to be lower later in lactation [91,196,201] and after the cessation of breastfeeding [196] than immediately postpartum. However, similar changes at these sites are observed in mothers who do not breastfeed, and there is no evidence that duration of lactation, or lactation itself, is a determinant of a mother’s bone mineral status after lactation [196,199]. Furthermore, breastfeeding among adolescent mothers is not associated with decreased hip bone mineral density compared with adolescent mothers who did not breastfeed [206]. There has been considerable debate about whether the trigger for the recovery of bone mineral is cessation of breastfeeding or the return of ovarian function. However, the strong interrelation between length of lactation and duration of amenorrhea 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 [196]. 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 breastfeeding, the onset of regular menstruation, or delivery, with no control of the other

variables [17,86,99,195,196,200,201,207]. In a study in which all women fully breastfed for 6 months and stopped breastfeeding 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 [199]. 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 breastfeeding [208]. In one study, more than half the patients had onset of symptoms after delivery and approximately threefourths breastfed [208]. A combination of increased plasma PTHrP concentration and a low bone mineral density has been implicated in postpartum-onset osteoporosis of pregnancy [209], but case reports have not addressed whether the patients had an exaggerated response compared to healthy mothers who had breastfed 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 [98,104,210]. 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 have associated lactation history and duration of breastfeeding with increased bone mineral [107,211,212], with decreased bone mineral [213e215], or with no effect [107,110,201,216,217]. No association has been observed between lactation history and risk of spinal deformity [118]. Postmenopausal women who breastfed when younger appear to be at lower risk of hip fracture than women who had children but did not breastfeed, with the protective effect increasing with duration of lactation [115,119,218]. The lack of consistent definitions and the failure adequately to control for confounding factors, such as obesity and estrogen use, make interpretation of these studies difficult [6,106]. The term lactation encompasses a range of breastfeeding behaviors that differ in the duration of exclusive and partial breastfeeding, the number of breastfeeds 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 breastfeeding incidence and in body size that may confound the associations between bone mineral measurements and lactation history

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[6,117,219]. 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 [220]. 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 [221e223].

Secretion of Minerals in Breast Milk Calcium is present in breast milk predominantly (60e70%) in a diffusible form, either as ionized calcium or associated with small inorganic ions such as citrate and phosphate [224,225]. The remaining 30e40% is associated with casein and whey proteins, such as a-lactalbumin and serum albumin. This contrasts with bovine milk, in which 60e70% of calcium is associated with casein. This is due in part to the fact that the caseins of human milk (which are 75% b- and 25% k-casein) bind less calcium per molecule than the caseins of bovine milk (which are 50% a-, 37.5% b-, 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 [224]. The compartmentation of zinc in human milk is unclear, but at least 25% occurs in the fat layer. Because some breast milk calcium, phosphorus, 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 [226] 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 6e12 weeks of lactation but declines progressively thereafter [6,227,228]. There are geographical variations in breast milk calcium concentration. Average values at 2 or 3 months of lactation range from 300 mg/L in regions of the USA and Europe to 200 mg/L in areas of Africa and Asia [6,229]. 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. Associations

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have been reported between breast milk calcium concentration and the concentrations of PTHrP in breast milk [230,231] and plasma [232], but the major determinants of an individual’s breast milk calcium concentration are likely to be those that regulate the casein, phosphate and citrate contents of breast milk [225]. The total amount of calcium transferred from the mother to her breastfed infant depends on both the calcium concentration and the quantity of breast milk consumed; there is no relationship between the two [6,233,234]. 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 breastfed infants from the UK and The Gambia resulted in a range of 79e450 mg/day [6]. Breast milk concentrations of phosphorus, magnesium, and zinc also decline as lactation progresses [13,235e238]. The following are typical concentrations at 3 months of lactation: phosphorus, 150 mg/L; magnesium, 30 mg/L; and zinc, 2 mg/L [13,229,238]. 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 [236]. Studies in Africa suggest that women from developing countries may have a lower ratio of calcium to phosphorus, which declines further during lactation [236,237].

Other Modulators 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 [239]. In addition, breast milk contains hormones and growth factors that may promote bone development. Examples include PTH, calcitonin, thyroid hormones, IGF, and transforming growth factor [240]. Breastfed infants have higher circulating concentrations of osteocalcin than formula-fed infants, which suggests that they have higher rates of bone turnover [241].

Influence of Maternal Diet and Nutritional Status on Mineral Metabolism and Breast Milk Production of the Lactating Mother Calcium The evidence to date suggests that maternal calcium intake does not modify the biochemical response to human lactation. Lactating women have an exaggerated

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reduction in urinary hydroxyproline excretion and decreased calciuric response to an acute oral calcium load but similar calcemic response compared to nonpregnant, non-lactating control women [70]. 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 [168,170,174,176,197,242]. Calcium supplementation produced a similar decrease in PTH and 1,25-dihydroxyvitamin D concentrations in lactating and non-lactating American mothers [174], whereas no significant effect of supplementation on calciotropic hormone levels or intestinal calcium absorption efficiency was seen in Gambian women [176,242]. In a small study, breast milk PTHrP concentration was not altered by calcium supplementation, suggesting that maternal calcium intake does not influence the production of this hormone in the mammary gland [193]. The pattern of bone loss and gain that accompanies lactation is also independent of the current calcium intake of the mother [6,243,244]. Lactating women with high customary calcium intakes exhibit the typical changes in bone mineral, as do those who consume calcium supplements [91,197,199,200,204]. Randomized, placebo-controlled studies have demonstrated little effect of calcium supplementation on the magnitude of bone changes during and after lactation [170,197,198,245]. Calcium supplementation postpartum produces a small increase in bone mineral status [198,199], but this is seen in both lactating and nonlactating women [198] and is likely a bone remodeling transient effect [199,246]. Conversely, calcium supplementation in the preceding pregnancy among Gambian women with a very low customary intake has been associated with more accentuated lactational bone changes in the mother, especially at the spine and distal radius [126]. Most observational studies have found no correlation between lactational bone changes and maternal calcium intake [91,195,196,200,204]. Where associations have been reported [171,247], these have been in bone mineral status and not in the magnitude of the lactational bone response, and they probably reflect the interrelationships between bone mineral density, body size, and dietary intake [219]. 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 [248]. 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 [170,176].

Similarly, the amount of calcium transferred into breast milk is not dependent on the calcium intake of the breastfeeding 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, direct experimental evidence has demonstrated that calcium intake during lactation does not influence breast milk calcium secretion [6,243]. In particular, no changes in breast milk calcium concentration were observed in two randomized, controlled calcium supplementation studies of lactating mothers (Figs 10.5 and 10.6) [170,198,249], even in women with a very low calcium intake (300 mg/day) [170]. It was considered possible that the calcium intake of the mother during pregnancy may predetermine breast milk calcium concentration in the subsequent lactation [250,251], thus providing a link between the observational data and the results of intervention studies. However, formal testing of this hypothesis in a calcium supplementation trial among pregnant Gambian women has discounted this possibility [132]. In addition, on theoretical physicochemical grounds, it is unlikely that there is a mechanism whereby breast milk calcium concentration can be modulated by maternal calcium intake [225]. 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, Effect on breast-milk calcium concentration Breast-milk calcium concentration (mmol/l)

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6

Supplement n=30 Placebo n=30

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39

52

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FIGURE 10.5 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 (n¼30 per group). There were no significant differences (reproduced with permission from Prentice et al. [170]).

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FIGURE 10.6 Effect of calcium supplements on breast milk calcium concentration. (A) Gambian women consuming 283 mg calcium/day supplemented with either 714 mg calcium/day (n¼30) or placebo (n¼30) from 0.5 to 12 months postpartum [170]. Milk samples were obtained from all participants at each time point. (B) American women consuming 720 mg calcium/day supplemented with either 1000 mg 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, S¼8 and P¼9; 3 months, S¼9 and P¼11; 6 months, S¼16 and P¼16. Data are mean  SE. Solid bars, calcium group; open bars, placebo group (reproduced with permission from Prentice [249]).

and zinc. However, there is little evidence that dietary intakes of these minerals influence breast milk composition or mineral outputs [8,139,238,252e254] or affect the changes in maternal bone metabolism that accompany lactation. Vitamin D There is no evidence that maternal vitamin D requirements are greater during lactation than at any other period [23,65]. It is possible that maternal vitamin D deficiency could impact on the breast milk concentrations of calcium and vitamin D. Breast milk, however, contains only 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 [10,65,141]. In addition, neither maternal vitamin D status in the normal range [255] nor high dose vitamin D supplementation influence breast milk calcium concentration [256]. 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 than by maternal nutritional status during lactation [10,65,141]. Whether this applies when the infant has limited exposure to sunlight of the correct wavelengths is not known. Breastfed black and Asian infants born in temperate climates are at risk of vitamin D-deficiency rickets [65,257,258]. 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 [259]. General Nutrition 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 the evidence which demonstrates that human lactation is robust in the face of poor maternal nutrition, unless there is severe malnutrition [240,260,261]. 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 [240]. 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 high-dose supplements can result in elevated breast milk concentrations, potentially to toxic levels [252]. It is well recognized that breastfed children have different rates of growth during infancy than those fed formula milk, and that the pattern of growth depends on the approach adopted to infant feeding [239,262e264]. Different patterns of growth in childhood may have longterm effects on skeletal size and bone health in adult life [163,265]. 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.

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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 EM, Dickerson JWT. In: Comar CL, Bronner F, editors. Mineral Metabolism. An Advanced Treatise. Chemical Composition of the Body. New York: Academic Press; 1964. p. 2e247. [2] Givens MH. The chemical composition of the human fetus. J Biol Chem 1933;102:7e17. [3] Prentice A, Bates CJ. Adequacy of dietary mineral supply for human bone growth and mineralisation. Eur J Clin Nutr 1994;48S:161e77. [4] Forbes GB. Calcium accumulation by the human fetus. Pediatrics 1976;57:976e7. [5] Fomon SJ. Infant Nutrition. Philadelphia: W.B. Saunders; 1974. [6] Prentice A, Laskey MA, Jarjou LMA. In: Tsang RC, Bonjour J-P, editors. Lactation and Bone Development: Implications for the Calcium Requirements of Infants and Lactating Mothers. Nutrition and Bone Development. New York: Vevey/Lippincott-Raven; 1999. p. 127e45. [7] Krebs NF, Hambidge KM. Zinc requirements and zinc intakes of breast-fed infants. Am J Clin Nutr 1986;45:288e92. [8] Hess YH, King JC. Effects of maternal zinc supplementation on pregnancy and lactation outcomes. Food Nutr Bull 2009;30:S60e78. [9] Prentice A. Maternal calcium requirements during pregnancy and lactation. Am J Clin Nutr 1994;59S:477e83. [10] Institute of Medicine Food and Nutrition Board. Dietary Reference Intakes for Calcium, Phosphorus, Magnesium, Vitamin D, and Fluoride. Washington, DC: National Academy Press; 1997. [11] Department of Health. Nutrition and Bone Health: with particular reference to Calcium and Vitamin D. London: The Stationery Office; 1998. [12] Department of Health. Dietary Reference Values for Food Energy and Nutrients for the United Kingdom. London: Her Majesty’s Stationery Office; 1991. [13] Institute of Medicine Food and Nutrition Board. Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium and Zinc. Washington, DC: National Academy Press; 2001.

[14] Heaney RP, Skillman TG. Calcium metabolism in normal human pregnancy. J Clin Endocrinol Metab 1971;33:661e70. [15] Kent GN, Price RI, Gutteridge DH. The efficiency of intestinal calcium absorption is increased in late pregnancy but not in established lactation. Calcif Tissue Int 1991;48:293e5. [16] Gertner JM, Coustan DR, Kliger AS, Mallette LE, Ravin N. Pregnancy as a state of physiologic absorptive hypercalciuria. Am J Med 1986;81:451e6. [17] Ritchie LD, Fung EB, Halloran BP, et al. A longitudinal study of calcium homeostasis during human pregnancy and lactation and after resumption of menses. Am J Clin Nutr 1998;67:693e701. [18] Cross NA, Hillman LS, Allen AH, Krause GF, Vieira NE. Calcium homeostasis and bone metabolism during pregnancy, lactation. and postweaning: a longitudinal study. Am J Clin Nutr 1995;61:514e23. [19] Naylor KE, Iqbal P, Fraser RB, Eastell R. The effect of pregnancy on bone density and bone turnover. J Bone Miner Res 2000;15:129e37. [20] Kovacs CS, Kronenberg HM. Maternal-fetal calcium and bone metabolism during pregnancy, puerperium, and lactation. Endocr Rev 1997;18:832e72. [21] Hosking DJ. Calcium homeostasis in pregnancy. Clin Endocrinol 1996;45:1e6. [22] Kovacs CS. Calcium and bone metabolism in pregnancy and lactation. J Clin Endocrinol Metab 2001;86:2344e8. [23] Kovacs CS. Vitamin D in pregnancy and lactation: maternal, fetal, and neonatal outcomes from human and animal studies. Am J Clin Nutr 2008;88:520Se8S. [24] Kent GN, Price RI, Gutteridge DH, et al. Effect of pregnancy and lactation on maternal bone mass and calcium metabolism. Osteoporos Int 1993;(Suppl. 1):S44e7. [25] Howarth AT, Morgan DB, Payne RB. Urinary excretion of calcium in late pregnancy and its relation to creatinine clearance. Am J Obstet Gynecol 1977;129:499e502. [26] Mestman JH. Parathyroid disorders of pregnancy. Semin Perinatol 1998;22:485e96. [27] Paterson CR. Calcium requirements in man: a critical review. Postgrad Med J 1978;54:244e8. [28] Wisser J, Florio I, Neff M, et al. Changes in bone density and metabolism in pregnancy. Acta Obstet Gynecol Scand 2005;84:349e54. [29] Kovacs CS. Calcium and bone metabolism during pregnancy and lactation. J Mammary Gland Biol Neoplas 2005;10:105e18. [30] Pitkin RM, Gebhardt MP. Serum calcium concentrations in human pregnancy. Am J Obstet Gynecol 1977;127:775e8. [31] Belkacemi L, Bedard I, Simoneau L, Lafond J. Calcium channels, transporters and exchangers in placenta: a review. Cell Calcium 2005;37:1e8. [32] Mitchell DM, Juppner H. Regulation of calcium homeostasis and bone metabolism in the fetus and neonate. Curr Opin Endocrinol Diabetes Obes 2010;17:25e30. [33] Simmonds CS, Karsenty G, Karaplis AC, Kovacs CS. Parathyroid hormone regulates fetal-placental mineral homeostasis. J Bone Miner Res 2010;25:594e605. [34] Wilkinson R. In: Nordin BEC, editor. Calcium, Phosphate and Magnesium Metabolism. Edinburgh: Churchill Livingstone; 1976. p. 36e112. [35] Gillette ME, Insogna KL, Lewis AM, Baran DT. Influence of pregnancy on immunoreactive parathyroid hormone levels. Calcif Tissue Int 1982;34:9e12. [36] Standley CA, Whitty JE, Mason BA, Cotton DB. Serum ionized magnesium levels in normal and preeclamptic gestation. Obstet Gynecol 1997;89:24e7.

PEDIATRIC BONE

REFERENCES

[37] Handwerker SM, Altura BT, Altura BM. Serum ionized magnesium and other electrolytes in the antenatal period of human pregnancy. J Am Coll Nutr 1996;15:36e43. [38] Bardicef M, Bardicef O, Sorokin Y, et al. Extracellular and intracellular magnesium depletion in pregnancy and gestational diabetes. Am J Obstet Gynecol 1995;172:1009e13. [39] Seydoux J, Girardin E, Paunier L, Beguin F. Serum and intracellular magnesium during normal pregnancy and in patients with pre-eclampsia. Br J Obstet Gynaecol 1992;99:207e11. [40] Dahlman T, Sjoberg HE, Bucht E. Calcium homeostasis in normal pregnancy and puerperium. Acta Obstet Gynecol Scand 1994;73:393e8. [41] Fung EB, Ritchie LD, Woodhouse LR, Roehl R, King JC. Zinc absorption in women during pregnancy and lactation: a longitudinal study. Am J Clin Nutr 1997;66:80e8. [42] King JC. Effect of reproduction on the bioavailability of calcium, zinc and selenium. J Nutr 2001;131:1355Se8S. [43] King JC. Determinants of maternal zinc status during pregnancy. Am J Clin Nutr 2000;71:1334Se43S. [44] Purdie DW, Aaron JE, Selby P. Bone histology and mineral homeostasis in human pregnancy. Br J Obstet Gynaecol 1988;95:849e54. [45] Ogueh O, Khastgir G, Abbas A, et al. The feto-placental unit stimulates the pregnancy-associated increase in maternal bone metabolism. Human Reprod 2000;15:1834e7. [46] Ulrich U, Miller PB, Eyre DR, Chesnut 3rd CH, Schlebusch H, Soules MR. Bone remodeling and bone mineral density during pregnancy. Arch Gynecol Obstet 2003;268:309e16. [47] O’Brien KO, Donangelo CM, Zapata CL, Abrams SA, Spencer EM, King JC. Bone calcium turnover during pregnancy and lactation in women with low calcium diets is associated with calcium intake and circulating insulin-like growth factor 1 concentrations. Am J Clin Nutr 2006;83:317e23. [48] Yamaga A, Taga M, Minaguchi H, Sato K. 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 1996;81:752e6. [49] Rodin A, Duncan A, Quartero HWP, Pistofidis G, et al. Serum concentrations of alkaline phosphatase isoenzymes and osteocalcin in normal pregnancy. J Clin Endocrinol Metab 1989;68:1123e7. [50] Haruna M, Fukuoka H. Metabolic turnover of bone during pregnancy and puerperium. Bull Physical Fitness Res Instit 1996;91:109e15. [51] Cole DEC, Gundberg CM, Stirk LJ, et al. Changing osteocalcin concentrations during pregnancy and lactation: implications for maternal mineral metabolism. J Clin Endocrinol Metab 1987;65:290e4. [52] More C, Bhattoa HP, Bettembuk P, Balogh A. The effects of pregnancy and lactation on hormonal status and biochemical markers of bone turnover. Eur J Obstet Gynecol Reprod 2003;106:209e13. [53] Black AJ, Topping J, Durham B, Farquharson RG, Fraser WD. A detailed assessment of alterations in bone turnover, calcium homeostasis, and bone density in normal pregnancy. J Bone Miner Res 2000;15:557e63. [54] Liu Z, Qiu L, Chen YM, Su YX. Effect of milk and calcium supplementation on bone density and bone turnover in pregnant Chinese women: a randomized controlled trial. Arch Gynecol Obstet 2010. Jan 1, epub ahead of print. [55] Janakiraman V, Ettinger A, Mercado-Garcia A, Hu H, Hernandez-Avila M. Calcium supplements and bone resorption in pregnancy: a randomized crossover trial. Am J Prevent Med 2003;24:260e4.

241

[56] Salle BL, Delvin EE, Lapillonne A, Bishop NJ, Glorieux FH. Perinatal metabolism of vitamin D. Am J Clin Nutr 2000;71:1317Se24S. [57] Kumar R, Cohen WR, Silva P, Epstein FH. Elevated 1,25-dihydroxyvitamin D plasma levels in normal human pregnancy and lactation. J Clin Invest 1979;63:342e4. [58] Wilson SG, Retallack RW, Kent JC, Worth GK, Gutteridge DH. 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 1990;32:613e22. [59] Bouillon R, Van Assche FA, Van Baelen H, Heyns W, De Moor P. 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 1981;67:589e96. [60] Bilke DD, Gee E, Halloran B, Haddad JG. Free 1,25dihydroxyvitamin D levels in serum from normal subjects, pregnant subjects, and subjects with liver disease. J Clin Invest 1984;74:1966e71. [61] Hillman LS, Slatopolsky E, Haddad JG. Perinatal vitamin D metabolism. IV. Maternal and cord serum 24,25-dihydroxyvitamin D concentrations. J Clin Endocrinol Metab 1978;47:1073e7. [62] Evans KN, Bulmer JN, Kilby MD, Hewison M. Vitamin D and placental-decidual function. J Soc Gynecol Invest 2004;11: 263e71. [63] Zerwekh JE, Breslau NA. Human placental production of 1a,25dihydroxyvitamin D3: biochemical characterization and production in normal subjects and patients with pseudohypoparathyroidism. J Clin Endocrinol Metab 1986;62:192e6. [64] Reiter EO, Braunstein GD, Vargas A, Root AW. Changes in 25hydroxyvitamin D and 24,25-dihydroxyvitamin D during pregnancy. Am J Obstet Gynecol 1979;135:227e9. [65] Specker BL. Do North American women need supplemental vitamin D during pregnancy or lactation? Am J Clin Nutr 1994;59:484Se91S. [66] Gallacher SJ, Fraser WD, Owens OJ, et al. Changes in calciotrophic hormones and biochemical markers of bone turnover in normal human pregnancy. Eur J Endocrinol 1994;131:369e74. [67] Seely EW, Brown EM, DeMaggio DM, Weldon DK, Graves SW. 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 1997;176:214e7. [68] Davis OK, Hawkins DS, Rubin LP, Posillico JT, Brown EM, Schiff I. Serum parathyroid hormone (PTH) in pregnant women determined by an immunoradiometric assay for intact PTH. J Clin Endocrinol Metab 1988;67:850e2. [69] Cushard WG, Creditor MA, Canterbury JM, Reiss E. Physiologic hyperthyroidism in pregnancy. J Clin Endocrinol Metab 1972;34:767e71. [70] Kent GN, Price RI, Gutteridge DH, et al. Acute effects of an oral calcium load in pregnancy and lactation: findings on renal calcium conservation and biochemical indices of bone turnover. Miner Electrolyte Metab 1991;17:1e7. [71] Ardawi MSM, Nasrat HAN, BA’Aqueel HS. Calcium-regulating hormones and parathyroid hormone-related peptide in normal human pregnancy and postpartum: a longitudinal study. Eur J Endocrinol 1997;137:402e9. [72] Wysolmerski JJ, Stewart AF. The physiology of parathyroid hormone-related protein: an emerging role as a developmental factor. Annu Rev Physiol 1998;60:431e60. [73] Henry JG, Mitnick M, Dann PR, Stewart AF. Parathyroid hormone-related protein-(1e36) is biologically active when

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[74]

[75]

[76]

[77]

[78]

[79]

[80]

[81]

[82]

[83] [84]

[85]

[86]

[87]

[88]

[89]

[90]

[91]

[92]

10. PREGNANCY AND LACTATION

administered subcutaneously to humans. J Clin Endocrinol Metab 1997;82:900e6. Pitkin RM, Reynolds WA, Williams GA, Hargis GK. Calcium metabolism in normal pregnancy: a longitudinal study. Am J Obstet Gynecol 1979;133:781e90. Whitehead M, Lane G, Osyth Y, et al. Interrelations of calciumregulating hormones during normal pregnancy. Br Med J 1981;283:10e2. Stevenson JC, Hillyard CJ, MacIntyre I. A physiological role for calcitonin: protection of the maternal skeleton. Lancet 1979:769e71. ii. Naylor KE, Rogers A, Fraser RB, Hall V, Eastell R, Blumsohn A. Serum osteoprotegerin as a determinant of bone metabolism in a longitudinal study of human pregnancy and lactation. J Clin Endocrinol Metab 2003;88:5361e5. Uemura H, Yasui T, Kiyokawa M, et al. Serum osteoprotegerin/ osteoclastogenesis-inhibitory factor during pregnancy and lactation and the relationship with calcium-regulating hormones and bone turnover markers. J Endocrinol 2002;174:353e9. Paoletti AM, Orru M, Floris L, et al. Pattern of bone markers during pregnancy and their changes after delivery. Hormone Res 2003;59:21e9. Naylor KE, Iqbal P, Fledelius C, Fraser RB, Eastell R. The effect of pregnancy on bone density and bone turnover. J Bone Miner Res 2000;15:129e37. Vargas Zapata CL, Donangelo CM, Woodhouse LR, Abrams SA, Spencer EM, King JC. Calcium homeostasis during pregnancy and lactation in Brazilian women with low calcium intakes: a longitudinal study. Am J Clin Nutr 2004;80:417e42. Olausson H, Laskey MA, Goldberg G, Prentice A. Changes in bone mineral status and bone size during pregnancy, and the influences of body weight and calcium intake. Am J Clin Nutr 2008;88:1032e9. Laskey MA, Prentice A. Effect of pregnancy on recovery of lactational bone loss. Lancet 1997;349:1518e9. Drinkwater BL, Chesnut CH. Bone density changes during pregnancy and lactation in active women: a longitudinal study. Bone Miner 1993;14:153e60. Sowers MF, Randolph J, Shapiro B, Jannausch M. A prospective study of bone density and pregnancy after an extended period of lactation with bone loss. Obstet Gynecol 1995;85:285e98. Holmberg-Marttila D, Sievanen H, Tuimala R. Changes in bone mineral density during pregnancy and postpartum: prospective data on five women. Osteoporos Int 1999;10:41e6. Sowers M, Crutchfield M, Jannausch M, Updike S, Corton G. A prospective evaluation of bone mineral change in pregnancy. Obstet Gynecol 1991;77:841e5. Pearson D, Kaur M, San P, Lawson N, Baker P, Hosking D. Recovery of pregnancy mediated bone loss during lactation. Bone 2004;34:570e8. Butte NF, Ellis KJ, Wong WW, Hopkinson JM, Smith EO. Composition of gestational weight gain impacts maternal fat retention and infant birth weight. Am J Obstet Gynecol 2003;189:1423e32. Aguado F, Revilla M, Hernandez ER, et al. Ultrasonographic bone velocity in pregnancy: a longitudinal study. Am J Obstet Gynecol 1998;178:1016e21. Kolthoff N, Eiken P, Kristensen B, Nielsen SP. Bone mineral changes during pregnancy and lactation: a longitudinal cohort study. Clin Sci 1998;94:405e12. Lamke B, Brundin J, Moberg P. Changes in bone mineral content during pregnancy and lactation. Acta Obstet Gynecol Scand 1977;56:217e9.

[93] Gambacciani M, Spinetti A, Gallo R, Cappagli B, Teti GC, Facchini V. Ultrasonographic bone characteristics during normal pregnancy: longitudinal and cross-sectional evaluation. Am J Obstet Gynecol 1995;173:890e3. [94] Javaid MK, Crozier SR, Harvey NC, et al. Maternal and seasonal predictors of change in calcaneal quantitative ultrasound during pregnancy. J Clin Endocrinol Metab 2005;90:5182e7. [95] Laskey MA, Prentice A. Can bone ultrasound measurements detect bone mineral changes during lactation? Osteoporos Int 1997;7:296. [96] Sowers M, Crutchfield M, Jannausch M, Updike S, Corton G. A prospective evaluation of bone mineral change in pregnancy. Obstet Gynecol 1991;77:841e5. [97] Specker B, Binkley T. High parity is associated with increased bone size and strength. Osteoporos Int 2005;16:1969e74. [98] O’Sullivan SM, Grey AB, Singh R, Reid IR. Bisphosphonates in pregnancy and lactation-associated osteoporosis. Osteoporos Int 2006;17:1008e12. [99] Smith R, Athanasou NA, Ostlere SJ, Vipond SE. Pregnancyassociated osteoporosis. Q J Med 1995;88:865e78. [100] Dunne F, Walters B, Marshall T, Heath DA. Pregnancy associated osteoporosis. Clin Endocrinol 1993;39:487e90. [101] Levav AL, Chan L, Wapner RJ. Long-term magnesium sulfate tocolysis and maternal osteoporosis in a triplet pregnancy: a case report. Am J Perinatol 1998;15:43e6. [102] Rizzoli R, Bonjour JP. Pregnancy-associated osteoporosis. Lancet 1996;347:1274e7. [103] Rousiere M, Kahan A, Job-Deslandre C. Postpartal sacral fracture without osteoporosis. Joint Bone Spine 2001;68:71e3. [104] Smith R, Phillips AJ. Osteoporosis during pregnancy and its management. Scand J Rheumatol 1998;27:66e7. [105] Gruber HE, Gutteridge DH, Baylink DJ. Osteoporosis associated with pregnancy and lactation: bone biopsy and skeletal features in three patients. Metab Bone Dis Relat Res 1984;5:159e65. [106] Sowers MF. Pregnancy and lactation as risk factors for subsequent bone loss and osteoporosis. J Bone Min Res 1996;11:1052e60. [107] Melton LJ, Bryant SC, Wahner HW, et al. Influence of breastfeeding and other reproductive factors on bone mass later in life. Osteoporos Int 1993;3:76e83. [108] Laitenen K, Valimaki M, Keto P. Bone mineral density measured by dual-energy x-ray absorptiometry in healthy Finnish women. Calcif Tiss Int 1991;48:224e31. [109] Murphy S, Khaw K-T, May H, Compston JE. Parity and bone mineral density in middle-aged women. Osteoporos Int 1994;4:162e6. [110] Fox KM, Magaziner J, Sherwin R, et al. Reproductive correlates of bone mass in elderly women. J Bone Miner Res 1993;8:901e8. [111] Michaelsson K, Baron JA, Farahmand BY, Ljunghall S. Influence of parity and lactation on hip fracture risk. Am J Epidemiol 2001;153:1166e72. [112] Paganini-Hill A, Chao A, Ross RK, Henderson BE. Exercise and other factors in the prevention of hip fracture: The Leisure World Study. Epidemiology 1991;2:16e25. [113] Hoffman S, Grisso JA, Kelsey JL, Gammon MD, O’Brien LA. Parity, lactation and hip fracture. Osteoporos Int 1993;3:171e6. [114] Hillier TA, Rizzo JH, Pedula KL, et al. Nulliparity and fracture risk in older women: the study of osteoporotic fractures. J Bone Miner Res 2003;18:893e9. [115] Alderman BW, Weiss NS, Daling JR, Ure CL, Ballard JH. Reproductive history and postmenopausal risk of hip and forearm fracture. Am J Epidemiol 1986;124:262e7. [116] Cox M, Khan SA, Gau DW, Cox SAL, Hodkinson HM. Determinants of forearm bone density on premenopausal women: a study in one general practice. Br J Gen Pract 1991;41:194e6.

PEDIATRIC BONE

REFERENCES

[117] Kritz-Silverstein D, Barrett-Connor E, Hollenbach KA. Pregnancy and lactation as determinants of bone mineral density in postmenopausal women. Am J Epidemiol 1992;136:1052e9. [118] O’Neill TW, Silman AJ, Naves Diaz M, et al. The European Vertebral Osteoporosis Study Group. Influence of hormonal and reproductive factors on the risk of vertebral deformity in European women. Osteoporos Int 1997;7:72e8. [119] Cumming RG, Klineberg RJ. Breastfeeding and other reproductive factors and the risk of hip fractures in elderly women. Int J Epidemiol 1993;22:684e91. [120] Ribot C, Tremollieres F, Pouilles JM, et al. Risk factors for hip fracture. MEDOS study: results of the Toulouse centre. Bone 1993;14:S77e80. [121] Hamed MH, Purdie DW, Steel SA, Howey S. The relation between bone mineral density and early pregnancy loss. Br J Obstet Gynaecol 1992;99:946e9. [122] King JC, Halloran BP, Huq N, Diamond T, Buckendahl PE. In: Picciano MF, Lonnerdal B, editors. Mechanisms Regulating Lactation and Infant Nutrient Utilization. New York: Wiley-Liss; 1992. p. 129e46. [123] Singh HJ, Mohammed NH, Nila A. Serum calcium and parathormone during normal pregnancy in Malay women. J Maternal-Fetal Med 1999;8:95e100. [124] Shenolikar IS. Absorption of dietary calcium in pregnancy. Am J Clin Nutr 1970;23:63e7. [125] Raman L, Rajalakshmi K, Krishnamachari KAVR, Sastry KG. Effect of calcium supplementation on undernourished mothers during pregnancy on the bone density of the neonates. Am J Clin Nutr 1978;21:466e9. [126] Jarjou LM, Laskey MA, Sawo Y, Goldberg GR, Cole TJ, Prentice A. Effect of calcium supplementation in pregnancy on maternal bone outcomes in women with a low calcium intake. Am J Clin Nutr 2010;92:450e7. [127] Gulson BL, Jameson CW, Mahaffey KR, Mizon KJ, Korsch MJ, Vimpani G. Pregnancy increases mobilization of lead from maternal skeleton. J Lab Clin Med 1997;130:51e62. [128] Gulson BL, Mahaffey KR, Jameson CW, et al. Mobilization of lead from the skeleton during the postnatal period is larger than during pregnancy. J Lab Clin Med 1998;131:324e9. [129] Lagerkvist BJ, Ekesrydh S, Englyst V, Hordberg G, Soderberg H-A, Wiklund D-E. Increased blood lead and decreased calcium levels during pregnancy: a prospective study of Swedish women living near a smelter. Am J Publ Hlth 1996;86:1247e52. [130] Mushak P. Lead’s toxic legacy for human reproduction: new studies establish significant bone lead release during pregnancy and nursing. J Lab Clin Med 1998;131:295e7. [131] Koo WW, Walters JC, Esterlitz J, Levine J, Bush AJ, Sibai B. Maternal calcium supplementation and fetal bone mineralization. Obstet Gynecol 1999;94:577e82. [132] Jarjou L, Prentice A, Sawo Y, et al. Randomized, placebocontrolled, calcium supplementation study in pregnant Gambian woman: effects on breastmilk calcium concentrations and infant birth weight, growth, and bone mineral accretion in the first year of life. Am J Clin Nutr 2006;83:657e66. [133] Hawkesworth S, Sawo Y, Fulford AJ, et al. Effect of maternal calcium supplementation on offspring blood pressure in 5- to 10-y-old rural Gambian children. Am J Clin Nutr 2010;92:741e7. [134] Matsuda Y, Maeda Y, Ito M, et al. Effect of magnesium sulfate treatment on neonatal bone abnormalities. Gynecol Obstet Invest 1997;44:82e8. [135] Edwards DK. Skeletal growth lines seen on radiographs of newborn infants: prevalence and possible association with obstetric abnormalities. Am J Roentgenol 1993;161:141e5.

243

[136] Yokoyama K, Takahashi N, Yada Y, et al. Prolonged maternal magnesium administration and bone metabolism in neonates. Early Hum Dev 2010;86:187e9. [137] Shah D, Sachdev HPS. Zinc deficiency in pregnancy and fetal outcome. Nutr Rev 2006;64:15e30. [138] Mahomed K. Zinc supplementation in pregnancy. Cochrane Databse Syst Rev 2000;2:CD000230. [139] Sian L, Krebs N, Westcott JE, et al. Zinc homeostasis during lactation with a low zinc intake. Am J Clin Nutr 2002;75:99e103. [140] Donangelo CM, Vargas Zapata CL, Woodhouse LR, Shames DM, Mukherjea R, King JC. Zinc absorption and kinetics during pregnancy and lactation in Brazilian women. Am J Clin Nutr 2005;82:118e24. [141] Scientific Advisory Committee on Nutrition Update on Vitamin D. London: The Stationery Office; 2007. [142] Prentice A. Vitamin D deficiency: a global perspective. Nutr Rev 2008;66:S153e64. [143] Delvin EE, Salle BL, Glorieux F, Adeleine P, David LS. Vitamin D supplementation during pregnancy: effect on neonatal calcium homeostasis. J Pediatr 1986;109:328e34. [144] Cockburn F, Belton NR, Purvis RJ, et al. Maternal vitamin D intake and mineral metabolism in mothers and their newborn infants. Br Med J 1970;281:11e4. [145] Specker B, Ho M, Oestreich A, et al. Prospective study of vitamin D supplementation and rickets in China. J Pediatr 1992;120:733e9. [146] Mahon P, Harvey N, Crozier S, et al. the SWS Study Group. Low maternal vitamin D status and fetal bone development: cohort study. J Bone Miner Res 2010;25:14e9. [147] Morley R, Carlin JB, Pasco JA, Wark JD. Maternal 25-hydroxyvitamin D and parathyroid hormone concentrations and offspring birth size. J Clin Endocrinol Metab 2006;91:906e12. [148] Yorifuji J, Yorifuji T, Tachibana K, et al. Craniotabes in normal newborns: the earliest sign of subclinical vitamin D deficiency. J Clin Endocrinol Metab 2008;93:1784e8. [149] McGrath JJ, Saha S, Lieberman DE, Buka S. Season of birth is associated with anthropometric and neurocognitive outcomes during infancy and childhood in a general population birth cohort. Schizophrenia Res 2006;81:91e100. [150] Namgung R, Tsang RC, Lee C, Han D-G, Ho ML, Sierra RI. Low total body bone mineral content and high bone resportion in Korean winter-born versus summer-born newborn infants. J Pediatr 1998;132:421e5. [151] Namgung R, Mimouni F, Campaigne BN, Ho ML, Tsang RC. Low bone mineral content in summer-born compared with winter-born infants. J Pediatr Gastroenterol Nutr 1992;15:285e8. [152] Namgung R, Tsang RC, Specker BL, Sierra RI, Ho ML. Low bone mineral content and high serum osteocalcin and 1,25-dihydroxyvitamin D in summer- versus winter-born newborn infants: an early fetal effect? J Pediatr Gastroenterol Nutr 1994;19:220e7. [153] Brooke OG, Brown IRF, Bone CDM et al. Vitamin D supplements in pregnant Asian women: effects on calcium status and fetal growth. Br Med J. 198;280:751-4. [154] Brooke OG, Butters F, Wood C. Intrauterine vitamin D nutrition and postnatal growth in Asian infants. Br Med J 1981;283:1024. [155] Javaid MK, Crozier SR, Harvey NC, et al. Maternal vitamin D status during pregnancy and childhood bone mass at age 9 years: a longitudinal study. Lancet 2006;367:36e43. [156] Rigo J, De Curtis M, Nyamugabo K, Pieltain C, Gerard P, Senterre J. In: Bonjour J-P, Tsang RC, editors. Nutrition and Bone Development. Philadelphia: Vevey/Lippincott-Raven; 1999. p. 83e93.

PEDIATRIC BONE

244

10. PREGNANCY AND LACTATION

[157] Krishnamachari KAVR, Iyengar L. Effect of maternal malnutrition on the bone density of the neonates. Am J Clin Nutr 1975;28:482e6. [158] Gulmezoglu M, de Onis M, Villar J. Effectiveness of interventions to prevent or treat impaired fetal growth. Obstet Gynecol Survey 1997;52:139e49. [159] Bhutta ZA, Ahmed T, Black RE, et al. The Maternal and Child Undernutrition Study Group. What works? Interventions for maternal and child undernutrition and survival. Lancet 2008;371:417e40. [160] Chan GM, McElligott K, McNaught T, Gill G. Effects of dietary calcium intervention on adolescent mothers and newborns: a randomized controlled trial. Obstet Gynecol 2006;108:565e71. [161] Ganpule A, Yajnik CS, Fall CH, et al. Bone mass in Indian children e relationships to maternal nutritional status and diet during pregnancy: the Pune Maternal Nutrition Study. J Clin Endocrinol Metab 2006;91:2994e3001. [162] Hossein-Nezhad A, Mirzaei K, Maghbooli Z, Najmafshar A, Larijani B. The influence of folic acid supplementation on maternal and fetal bone turnover. J Bone Miner Metab 2010. Jul 3, epub ahead of print. [163] Cooper C, Fall C, Egger P, Hobbs R, Eastell R, Barker D. Growth in infancy and bone mass in later life. Ann Rheum Dis 1997;56:17e21. [164] Gale CR, Martyn CN, Kellingray S, Eastell R, Cooper C. Intrauterine programming of adult body composition. J Clin Endocrinol Metab 2001;86:267e72. [165] Godfrey KM, Barker DJP. Fetal nutrition and adult disease. Am J Clin Nutr 2000;71:1344Se52S. [166] Kalkwarf HJ. Hormonal and dietary regulation of changes in bone density during lactation and after weaning in women. J Mammary Gland Biol Neoplas 1999;4:319e29. [167] Specker BL, Vieira NE, O’Brien KO, et al. Calcium kinetics in lactating women with high and low calcium intakes. Am J Clin Nutr 1994;59:593e9. [168] Kalkwarf HJ, Specker BL, Heubi JE, Vieira NE, Yergey AL. Intestinal calcium absorption of women during lactation and after weaning. Am J Clin Nutr 1996;63:526e31. [169] Klein CJ, Moser-Veillon B, Douglass LW, Ruben KA, Trocki O. A longitudinal study of urinary calcium, magnesium, and zinc excretion in lactating and nonlactating postpartum women. Am J Clin Nutr 1995;61:779e86. [170] Prentice A, Jarjou LMA, Cole TJ, Stirling DM, Dibba B, Fairweather-Tait S. Calcium requirements of lactating Gambian mothers: effects of a calcium supplement on breastmilk calcium concentration, maternal bone mineral content, and urinary calcium excretion. Am J Clin Nutr 1995;62:58e67. [171] Kent GN, Price RI, Gutteridge DH, et al. Human lactation: forearm trabecular bone loss, increased bone turnover, and renal conservation of calcium and inorganic phosphate with recovery of bone mass following weaning. J Bone Miner Res 1990;5:361e9. [172] Krebs NF, Reidinger CJ, Robertson AD, Brenner M. Bone mineral density changes during lactation: maternal, dietary, and biochemical correlates. Am J Clin Nutr 1997;65:1738e46. [173] Lippuner K, Zehnder H-K, Casez J-P, Takkinen R, Jaeger P. PTHrelated protein is released into the mother’s bloodstream during lactation: evidence for beneficial effects on maternal calciumphosphate metabolism. J Bone Miner Res 1996;11:1394e9. [174] Kalkwarf HJ, Specker BL, Ho M. Effects of calcium supplementation on calcium homeostasis and bone turnover in lactating women. J Clin Endocrinol Metab 1999;84:464e70. [175] Affinito P, Tommaselli GA, Di Carlo C, Guido F, Nappi C. Changes in bone mineral density and calcium metabolism in

[176]

[177]

[178]

[179]

[180]

[181]

[182]

[183]

[184]

[185]

[186]

[187]

[188]

[189]

[190]

[191]

[192]

breastfeeding women: a one year follow-up study. J Clin Endocrinol Metab 1996;81:2314e8. Prentice A, Jarjou LMA, Stirling DMS, Buffenstein R, Fairweather-Tait S. Biochemical markers of calcium and bone metabolism during 18 months of lactation in Gambian women accustomed to a low calcium intake and in those consuming a calcium supplement. J Clin Endocrinol Metab 1998;83:1059e66. Dengel JL, Mangels AR, Moser-Veillon PB. Magnesium homeostasis: conservation mechanism in lactating women consuming a controlled-magnesium diet. Am J Clin Nutr 1994;59:990e4. Sowers M, Eyre D, Hollis BW, et al. Biochemical markers of bone turnover in lactating and nonlactating postpartum women. J Clin Endocrinol Metab 1995;80:2210e6. Carneiro RM, Prebehalla L, Tedesco MB, et al. Lactation and bone turnover: a conundrum of marked bone loss in the setting of coupled bone turnover. J Clin Endocrinol Metab 2010;95:1767e76. Holmberg-Marttila D, Leino A, Sievanen H. Bone turnover markers during lactation, postpartum amenorrhea and resumption of menses. Osteoporos Int 2003;14:103e9. Dobnig H, Kainer F, Stepan V, et al. Elevated parathyroid hormone-related peptide levels after human gestation: relationship to changes in bone and mineral metabolism. J Clin Endocrinol Metab 1995;80:3699e707. Specker BL, Tsang RC, Ho ML. Changes in calcium homeostasis over the first year postpartum: effect of lactation and weaning. Obstet Gynecol 1991;78:56e62. Sowers MF, Zhang D, Hollis BW, et al. Role of calciotrophic hormones in calcium mobilization of lactation. Am J Clin Nutr 1998;67:284e91. Hillman L, Sateesha S, Haussler M, Wiest W, Stapolsky E, Haddad J. Control of mineral homeostasis during lactation: interrelationships of 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D, 1,25-dihydroxyvitamin D, parathyroid hormone, calcitonin, prolactin and estradiol. Am J Obstet Gynecol 1981;139:471e6. Specker BL, Tsang RC, Ho M, Miller D. Effect of vegetarian diet on serum 1,25-dihydroxyvitamin D concentrations during lactation. Obstet Gynecol 1987;70:870e4. Chan SM, Nelson EA, Leung SS, Cheng JC. Bone mineral density and calcium metabolism of Hong Kong Chinese postpartum women e a 1-y longitudinal study. Eur J Clin Nutr 2005;59:868e76. Greer FR, Tsang RC, Searcy JE, Levin RS, Steichen JJ. Mineral homeostasis during lactation e relationship to serum 1,25dihydroxyvitamin D, 25-hydroxyvitamin D, parathyroid hormone and calcitonin. Am J Clin Nutr 1982;36:431e7. Greer FR, Lane J, Ho M. Elevated serum parathyroid hormone, calcitonin and 1,25-dihydroxyvitamin D in lactating women nursing twins. Am J Clin Nutr 1984;40:562e8. Retallack RW, Jeffries M, Kent GN, Hitchcock NE, Gutteridge DH, Smith M. Physiological hyperparathyroidism in human lactation. Calcif Tissues Res 1977;22(Suppl):142e6. Sowers MF, Hollis BW, Shapiro B, et al. Elevated parathyroid hormone-related peptide associated with lactation and bone density loss. J Am Med Assoc 1996;276:549e54. Grill V, Hillary J, Ho PWH, et al. Parathyroid hormone-related protein: a possible endocrine function in lactation. Clin Endocrinol 1992;37:405e10. Budayr AA, Halloran BP, King JC, Diep D, Nissenson RA, Strewler GJ. High levels of parathyroid hormone-like protein in milk. Proc Natl Acad Sci 1989;86:7183e5.

PEDIATRIC BONE

REFERENCES

[193] Cross NA, Hillman LS, Forte LR. The effects of calcium supplementation, duration of lactation, and time of day on concentrations of parathyroid hormone-related protein in human milk: a pilot study. J Hum Lact 1998;14:111e7. [194] Mather KJ, Chik CL, Corneblum B. Maintenance of serum calcium by parathyroid hormone-related peptide during lactation in a hypoparathyroid patient. J Clin Endocrinol Metab 199;84:424-7. [195] Sowers MF, Corton G, Shapiro B, et al. Changes in bone density with lactation. J Am Med Assoc 1993;269:3130e5. [196] Laskey MA, Prentice A. Bone mineral changes during and after lactation. Obstet Gynecol 1999;94:608e15. [197] Cross NA, Hillman LS, Allen SH, Krause GF. Changes in bone mineral density and markers of bone remodeling during lactation and postweaning in women consuming high amounts of calcium. J Bone Miner Res 1995;10:1312e20. [198] Kalkwarf HJ, Specker BL, Bianchi DC, Ranz J, Ho M. The effect of calcium supplementation on bone density during lactation and after weaning. New Engl J Med 1997;337:523e8. [199] Polatti F, Capuzzo E, Viazzo F, Colleoni R, Klersy C. Bone mineral changes during and after lactation. Obstet Gynecol 1999;94:52e6. [200] Lopez JM, Gonzalez G, Reyes V, Campino C, Diaz S. Bone turnover and density in healthy women during breastfeeding and after weaning. Osteoporos Int 1996;6:153e9. [201] Karlsson C, Obrant KJ, Karlsson M. Pregnancy and lactation confer reversible bone loss in humans. Osteoporos Int 2001;12:828e34. [202] Akesson A, Vahter M, Berglund M, Eklof T, Bremme K, Bjellerup P. Bone turnover from early pregnancy to postweaning. Acta Obstet Gynecol Scand 2004;83:1049e55. [203] Hopkinson JM, Butte NF, Ellis K, O’Brian Smith E. Lactation delays postpartum bone mineral accretion and temporarily alters its regional distribution in women. J Nutr 2000;130:777e83. [204] Laskey MA, Prentice A, Hanratty LA, et al. Bone changes after 3 months of lactation: influence of calcium intake, breast-milk output and vitamin-D receptor genotype. Am J Clin Nutr 1998;67:685e92. [205] Kalkwarf HJ, Specker BL. Bone mineral loss during lactation and recovery after weaning. Obstet Gynecol 1995;86:26e32. [206] Chantry CJ, Auinger P, Byrd RS. Lactation among adolescent mothers and subsequent bone mineral density. Arch Pediatr Adolesc Med 2004;158:650e6. [207] Holmberg-Marttila D, Harri S. Prevalence of bone mineral changes during postpartum amenorrhea and after resumption of menstruation. Am J Obstet Gynecol 1999;180:537e8. [208] Khovidhunkit W, Epstein S. Review article: Osteoporosis in pregnancy. Osteoporos Int 1996;6:345e54. [209] Anai T, Tomiyasu T, Arima K, Miyakawa I. Pregnancy-associated osteoporosis with elevated levels of circulating parathyroid hormone-related protein: a report of two cases. J Obstet Gynaecol 1999;25:63e7. [210] Gruber HE, Gutteridge DH, Baylink DJ. Osteoporosis associated with pregnancy and lactation: bone biopsy and skeletal features in three patients. Metab Bone Dis Rel Disorders 1984;5:159e65. [211] Feldblum PJ, Zhang J, Rich LE, Fortney JA, Talmage RV. Lactation history and bone mineral density among perimenopausal women. Epidemiology 1992;3:527e31. [212] Hreshchyshyn MM, Hopkins A, Zylstra S, Anbar M. Associations of parity, breast-feeding, and birth control pills with lumbar spine and femoral neck bone densities. Am J Obstet Gynecol 1988;159:318e22.

245

[213] Goldsmith NF, Johnston JO. Bone mineral: effects of oral contraceptives, pregnancy, and lactation. J Bone Joint Surg 1975;57A:657e68. [214] Lopez-Jaramillo P, Delgado F, Jacome P, Teran E, Ruano C, Rivera J. Calcium supplementation and the risk of preeclampsia in Ecuadorian pregnant teenagers. Obstet Gynecol 1997;90:162e7. [215] Wardlaw GM, Pike AM. The effect of lactation on peak adult shaft and ultra-distal forearm bone mass in women. Am J Clin Nutr 1986;44:283e6. [216] Koetting CA, Wardlaw GM. Wrist, spine, and hip bone density in women with variable histories of lactation. Am J Clin Nutr 1988;48:1479e81. [217] Sinigaglia L, Varenna M, Binelli L, Gallazzi M, Calori G, Ranza R. Effect of lactation on postmenopausal bone mineral density of the lumbar spine. J Reprod Med 1996;41:439e43. [218] Kreiger N, Kelsey JL, Holford TR, O’Connor T. An epidemiologic study of hip fracture in postmenopausal women. Am J Epidemiol 1982;116:141e8. [219] Prentice A, Parsons TJ, Cole TJ. Uncritical use of bone mineral density in absorptiometry may lead to size-related artifacts in the identification of bone mineral determinants. Am J Clin Nutr 1994;60:837e42. [220] Kleerekoper M, Peterson E, Nelson D, et al. Identification of women at risk for developing postmenopausal osteoporosis with vertebral fractures: role of history and single photon absorptiometry. Bone Min 1989;7:171e86. [221] Bererhi H, Kolhoff N, Constable A, Nielsen SP. Multiparity and bone mass. Br J Obstet Gynaecol 1996;103:818e21. [222] Walker ARP, Richardson B, Walker F. The influence of numerous pregnancies and lactations on bone dimensions in South African Bantu and Caucasian mothers. Clin Sci 1972;42:189e96. [223] Aspray TJ, Prentice A, Cole TJ, Sawo Y, Reeve J, Francis RM. Low bone mineral content is common but osteoporotic fractures are rare in elderly rural Gambian women. J Bone Miner Res 1996;11:1018e24. [224] Neville MC, Zhang P, Allen JC. In: Jensen RG, editor. Handbook of Milk Composition. San Diego: Academic Press; 1995. p. 577e92. [225] Kent JC, Arthur PG, Mitoulas LR, Hartmann PE. Why calcium in breastmilk is independent of maternal dietary calcium and vitamin D. Breastfeeding Rev 2009;17:5e11. [226] Laskey MA, Dibba B, Prentice A. A semi-automated micromethod for the determination of calcium and phosphorus in human milk. Ann Clin Biochem 1991;28:49e54. [227] Vaughan LA, Weber CW, Kemberling SR. Longitudinal changes in the mineral content of human milk. Am J Clin Nutr 1979;32:2301e6. [228] Prentice A, Laskey MA, Shaw J, Cole TJ, Fraser DR. Bone mineral content of Gambian and British children aged 0e36 months. Bone Miner 1990;10:211e24. [229] Prentice A. In: Jensen RG, editor. Handbook of Milk Composition. San Diego: Academic Press; 1995. p. 115e221. [230] Uemura H, Yasui T, Yoneda N, Irahara M, Aono T. Measurement of N- and C-terminal-region fragments of parathyroid hormone-related peptide in milk from lactating women and investigation of the relationship of their concentrations to calcium in milk. J Endocrinol 1997;153:445e51. [231] Seki K, Kato T, Sekiya S, et al. Parathyroid-hormone-related protein in human milk and its relation to milk calcium. Gynecol Obstet Invest 1997;44:102e6. [232] DeSantiago S, Alonso L, Halhali A, Larrea F, Isoard F, Bourges H. Negative calcium balance during lactation in rural Mexican women. Am J Clin Nutr 2002;76:845e51.

PEDIATRIC BONE

246

10. PREGNANCY AND LACTATION

[233] Laskey MA, Prentice A, Shaw J, Zachou T, Ceesay SM. Breastmilk calcium concentrations during prolonged lactation in British and rural Gambian mothers. Acta Paediatr Scand 1990;79:507e12. [234] Laskey MA, Jarjou L, Dibba B, Prentice A. Does maternal calcium intake influence the calcium nutrition of the breast-fed baby? Proc Nutr Soc 1997;56:1Ae6A. [235] Casey CE, Smith A, Zhang P. In: Jensen RG, editor. Handbook of Milk Composition. San Diego: Academic Press; 1995. p. 622e74. [236] Laskey MA, Dibba B, Prentice A. Low ratios of calcium to phosphorus in the breast-milk of rural Gambian mothers. Acta Paediatr Scand 199;80:250-1. [237] Prentice A, Barclay DV. Breast-milk calcium and phosphorus concentrations of mothers in rural Zaire. Eur J Clin Nutr 1991;45:611e7. [238] Brown KH, Engle-Stone R, Krebs NF, Peerson JM. Dietary intervention strategies to enhance zinc nutrition: Promotion and support of breastfeeding fo infants and young children. Food Nutr Bull 2009;30:S144e71. [239] Dewey KG. Nutrition, growth, and complementary feeding of the breastfed infant. Pediatr Clin North Am 2001;48:87e104. [240] Prentice A. The constituents of human milk. Food Nutr Bull 1997;17:305e15. [241] Michaelsen KF, Johansen JS, Samuelson G, Price PA, Christiansen C, Skakkebaek NE. In: Picciano MF, Lonnerdal B, editors. Mechanisms Regulating Lactation and Infant Nutrient Utilization. New York: Wiley-Liss; 1992. p. 419e23. [242] Fairweather-Tait SJ, Prentice A, Heumann KG, et al. 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 1995;62:1188e92. [243] Prentice A. Calcium in pregnancy and lactation. Annu Rev Nutr 2000;20:249e72. [244] Prentice A. Calcium supplementation during breast-feeding. N Engl J Med 1997;337:558e9. [245] Kent GN, Price RI, Gutteridge DH, et al. Site-specific reduction in bone loss by calcium supplements in normal lactation. Osteoporos Int 1995;5:315(A). [246] Parfitt AM. Morphologic basis of bone mineral measurements: transient and steady state effects of treatment in osteoporosis. Miner Electrolyte Metab 1980;4:273e87. [247] Morales A, Tud-Tud Hans L, Herber M, Taylor AK, Baylink DJ. Lactation is associated with an increase in spinal bone density. J Bone Miner Res 1995;8:S156. [248] Chan GM, McMurry M, Westover K, Engelbert-Fenton K, Thomas MR. Effects of increased dietary calcium intake upon the calcium and bone mineral status of lactating adolescent and adult women. Am J Clin Nutr 1987;46:319e23. [249] Prentice A. Calcium requirements of breast-feeding mothers. Nutr Rev 1998;56:124e7.

[250] Prentice A, Dibba B, Jarjou LMA, Laskey MA, Paul AA. Is breast-milk calcium concentration influenced by calcium intake during pregnancy? Lancet 1994;344:411e2. [251] Ortega RM, Martinez RM, Quintas ME, Lopez-Sobaler AM, Andrews P. Calcium levels in maternal milk: relationships with calcium intake during the third trimester of pregnancy. Br J Nutr 1998;79:501e7. [252] Bates CJ, Prentice A. In: Bennett P, editor. Drugs and Human Lactation. 2nd ed. Amsterdam: Elsevier; 1996. p. 533e607. [253] Krebs NF, Reidinger CJ, Hartley S, Robertson AD, Hambidge KM. Zinc supplementation during lactation: effects on maternal status and milk zinc concentrations. Am J Clin Nutr 1995;61:1030e6. [254] Moser-Veillon PB, Reynolds RD. A longitudinal study of pyridoxine and zinc supplementation of lactating women. Am J Clin Nutr 1990;52:135e41. [255] Prentice A, Yan L, Jarjou LMA, Dibba B, Laskey MA, Fairweather-Tait S. Vitamin D status does not influence the breastmilk calcium concentration of lactating mothers accustomed to a low calcium intake. Acta Paediatr 1997;86:1006e8. [256] Basile LA, Taylor SN, Wagner CL, Horst RL, Hollis BW. The effect of high-dose vitamin D supplementation on serum vitamin D levels and milk calcium concentration in lactating women and their infants. Breastfeeding Med 2006;1:27e35. [257] Mughal MZ, Salama H, Greenaway T, Laing I, Mawer EB. Lesson of the week: florid rickets associated with prolonged breast-feeding without vitamin D supplementation. Br Med J 1999;318:1417. [258] Kreiter SR, Schwartz RP, Kirkman HN, Charlton PA, Calikoglu AS, Davenport ML. Nutritional rickets in African American breast-fed infants. J Pediatr 2000;137:153e7. [259] Specker BL, Tsang RC, Hollis BW. Effect of race and diet on human milk vitamin D and 25-hydroxyvitamin D. Am J Dis Chld 1985;139:1134e7. [260] Prentice AM, Paul AA, Prentice A, Black AE, Cole TJ, Whitehead RG. In: Hamosh M, Goldman AS, editors. Human Lactation 2: Maternal and Environmental Factors. New York: Plenum Press; 1986. p. 13e44. [261] Prentice AM, Prentice A. Evolutionary and environmental influences on human lactation. Proc Nutr Soc 1995;54:391e400. [262] Whitehead RG, Paul AA. Growth charts and the assessment of infant feeding practices in the western world and in developing countries. Early Hum Dev 1984;9:187e207. [263] Michaelsen KF, Larsen PS, Thomsen BL, Samuelson G. The Copenhagen Cohort Study on Infant Nutrition and Growth: breast milk intake, human milk macronturient content and influencing factors. Am J Clin Nutr 1994;59:600e11. [264] Prentice A. Breast feeding and the older infant. Acta Paediatr Scand 1991;374(Suppl):78e88. [265] Cooper C, Eriksson JG, Forsen T, Osmond C. Maternal height, childhood growth and risk of hip fracture in later life: a longitudinal study. Osteoporos Int 2001;12:623e9.

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Fetal Mineral Homeostasis Christopher S. Kovacs Faculty of Medicine e Endocrinology, Memorial University of Newfoundland, St John’s, Newfoundland, Canada

FETAL ADAPTIVE GOALS Much of normal calcium and bone homeostasis in the adult can be explained by the interactions of parathyroid hormone (PTH), 1,25-dihydroxyvitamin D (calcitriol), fibroblast growth factor-23 (FGF-23), calcitonin, and the sex steroids. Deficiency of PTH causes potentially fatal hypocalcemia as well as hyperphosphatemia, hypercalciuria, low bone turnover, and basal ganglia calcifications. Vitamin D deficiency causes more modest hypocalcemia as well as hypophosphatemia and rickets or osteomalacia. Estrogen deficiency causes increased skeletal resorption and osteoporosis. Many of our current treatments for osteoporosis and other metabolic bone disorders have been extrapolated from the roles that these hormones play, and utilize pharmacological doses of these hormones or their synthetic equivalents. In contrast to the adult, many of these hormones are not required to regulate fetal calcium and bone metabolism. The needs of this developmental period include to actively to pump calcium (and other minerals) across the placenta against a concentration gradient, to maintain high extracellular levels of calcium (and other minerals) compared to the mother, and to mineralize fully the skeleton by the time of birth. A human fetus typically accumulates 21 g of calcium by term, and 80% of this is obtained in the third trimester with 200e300 mg transported daily across the placenta [1]. In order to regulate the extracellular level of calcium, the fetus makes use of the placenta, kidneys, skeleton, and intestines. Maternal hypo- or hypercalcemia can disrupt mineralization of the fetal skeleton and parathyroid development. This chapter will also show that PTHrelated protein (PTHrP) is a major regulator of placental calcium transport, while PTHrP and PTH act in concert to regulate the blood calcium and control skeletal mineralization. Data from human fetuses are quite limited and so regulation of fetal mineral homeostasis must be largely

Pediatric Bone, Second Edition DOI: 10.1016/B978-0-12-382040-2.10011-5

inferred from animal data, including sheep and rodents. The first insights into the regulation of fetal mineral homeostasis were gleaned from studying the effects of fetal or maternal thyroparathyroidectomy, experimental vitamin D deficiency, and pharmacological doses of hormones. More recently, genetically engineered mice have enabled the study of models that cannot be created by surgical or pharmacological techniques, such as fetal mice that are completely devoid of PTHrP. The key mouse models are briefly described in the Appendix.

MINERALS Calcium The fetal serum calcium is maintained 0.30e 0.50 mmol/L higher than the maternal level in all mammals that have been studied, including humans [2e6], rhesus monkeys [7,8], sheep [6,9], cattle [10], rodents [9,11,12], pigs [13], and horses [13]. The ionized calcium is 0.25e0.45 mmol/L higher than the mother’s value and represents about 80% of the total calcium value, in contrast to the adult where about 45e50% of calcium is ionized [6,11,14]. This fetal hypercalcemia has been found at the earliest time points in which it has been technically possible to measure it. In humans, this is 15e20 weeks of gestation (by fetoscopy) [15] and at delivery of preterm singleton and twin pregnancies (mean gestational age 33 weeks) [16]. Fetal hypercalcemia has also been demonstrated from the 35th day of gestation in sheep [17,18], during the last 7 days in rats [19e21], and the last 4 days in mice [22]. This blood calcium level exceeds that which the calcium sensing receptor (CaR) sets in the adult through its regulation of PTH secretion; consequently, fetal hypercalcemia causes the CaR to suppress fetal PTH (see below, Calcium Sensing Receptor). The roles of

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PTH and PTHrP in regulating the blood calcium are discussed in later sections. The high level of blood calcium is set and maintained independently of the maternal blood calcium [13]. Fetal rats were normocalcemic despite maternal hypocalcemia due to a calcium-restricted diet [23], vitamin D deficiency [24e26], or thyroparathyroidectomy [27,28]. Use of both Car [29] and Vdr [30] mutants has shown that wild-type fetuses will maintain the same blood calcium regardless of whether the mother is normocalcemic, hypercalcemic, or hypocalcemic (Figs 11.1 and 11.2) [12,31]. Acute alterations in the maternal blood calcium of rodents and primates (by infusions of calcium, calcitriol, calcitonin, PTH, or EDTA) also have minimal or no effect on the fetal blood calcium level [32e36]. However, other investigators have found that following maternal parathyroidectomy, rat fetuses were normocalcemic between the 12th and 17th day of gestation but became hypocalcemic during the last few days of gestation when the skeleton is rapidly accreting mineral [37e39]. The robust maintenance of fetal hypercalcemia across all species studied suggests that it must have physiological importance. However, it is not necessary for fetal viability because survival to the end of gestation is unaffected by significant hypocalcemia in Pthrp null, Pthr1 null, Hoxa3 null, Trpv6 null, and Hoxa3/Pthrp double mutant fetuses [13,40e42]. But survival after birth may be aided by a high blood calcium level in utero. Normally, the postnatal onset of breathing and obligatory rise in pH cause a rapid fall in calcium within the first 12 hours after birth, followed by an increase to the adult value during the succeeding day [13]. The magnitude of decline is approximately 20e30% in humans [2,3,43] and 40% in rodents [9,20]. Conceivably, a lower fetal blood calcium in utero will predispose to

an even lower trough level being reached after birth, thereby increasing the risk of tetany and death. The early postnatal mortality of Pthrp null, Pthr1 null, and Hoxa3 null fetuses is consistent with this possibility [11,41,44,45]. Finally, maintaining the fetal calcium concentration above the maternal level is required to achieve normal mineralization of the fetal skeleton (see Fetal Skeleton, below). In summary, the total and ionized calcium level is higher in the fetus than in the mother from early in gestation. This level is set independently from the mother and can be robustly maintained despite significant maternal hypocalcemia of various causes, although hypocalcemia may develop during the time of rapid accretion of calcium by the fetal skeleton. Fetal hypercalcemia may help avoid neonatal hypocalcemia in the first 12e36 hours after birth, and it is also required to enable full mineralization of the skeleton.

Phosphorus Serum phosphorus is set about 0.5 mmol/L higher in the fetus than in the mother as observed in humans [2,3,5,46], rats [21,47,48], mice [49], sheep [23,41], and pigs [50,51]. Combined with fetal hypercalcemia, this means a high calcium
phosphorus product that causes soft tissue calcifications in the adult but seems necessary (and non-hazardous) in utero to mineralize the fetal skeleton. Phosphorus drops sharply in the first few hours after birth, at least in rats [9].

Magnesium Serum magnesium is also set independently of the maternal value and most human studies show there to be modest hypermagnesemia (0.05 mmol/L higher

Fetal ionized calcium is unaffected by maternal hypercalcemia. Ionized calcium was measured in fetuses from (A) Carþ/ females, and (B) wild-type females, which had been mated with the same Carþ/ male mice. The fetal blood calcium of wild-type (WT) and Carþ/ fetuses is the same regardless of the mother’s genotype and blood calcium level. A dashed line in each graph indicates the respective mean maternal ionized calcium for Carþ/ (A) and WT females (B). The number of observations for each genotype is indicated within parentheses. Reproduced with permission from [12] Ó 1998 American Society of Clinical Investigation.

FIGURE 11.1

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Fetal ionized calcium is unaffected by maternal hypocalcemia. Ionized calcium was measured in fetuses obtained from Vdrþ/ mothers (A) and Vdr null mothers (B). No difference in ionized calcium level was noted among the fetal genotypes, and the mother’s genotype or ionized calcium level did not affect the fetal ionized calcium level. The maternalefetal calcium gradient (double-headed arrows) is the difference between the fetal ionized calcium and the corresponding mean maternal ionized calcium level (line). The SE on the maternal values is indicated by errors bars on the far right of each line. The maternalefetal gradient was strikingly increased in fetuses of Vdr null mothers as compared to fetuses of Vdrþ/ mothers (P<0.001). The number of observations is indicated in parentheses. Reproduced with permission from Kovacs et al. [31] Ó2005 American Physiological Society.

FIGURE 11.2

than maternal) [2,3,5,46,52]. Within animal models, variable differences have been seen, including small increases [22,53,54] and decreases in fetal sheep [23], modest increases [55] or no change in fetal rats [9], and small but statistically significant increases in fetal mice (including unpublished data) [49,56].

CALCIOTROPIC HORMONES Parathyroid Hormone In humans [2,46,57e69) and other mammals [10,19,70,71), PTH typically circulates at very low (<0.5 pmol/L) to undetectable concentrations in the fetal circulation, and lower than the simultaneous maternal value. The earliest available measurements in preterm infants are from 19 weeks of gestation and have shown suppressed PTH compared to the maternal value [57,63,64,67], although one study found unsuppressed PTH values in cord blood of infants aged 31e36 weeks [16]. The suppression of fetal PTH is especially marked when it is considered that the maternal PTH value is usually at the lower end of the adult normal range or below it during pregnancy [13]. Intact PTH does not cross the placenta of non-human primates, sheep and rodents [33,36,41,72,73] and the low levels maintained in human fetuses indicate that it probably does not cross the human placenta either. PTH in fetal blood therefore derives from fetal sources alone, including parathyroids and possibly placenta [49]. PTH immunoreactivity is present in fetal parathyroid glands as early as 10 weeks of gestation in humans

[74], and PTH mRNA is present during early fetal development in parathyroids of rats and sheep [75,76]. But despite its low levels, PTH remains important for maintaining the fetal serum calcium since hypocalcemia occurs in Pth null, Gcm2 null, Hoxa3 null, and Pthr1 null fetuses [41,49]. The fetal PTH level appears to be suppressed by a normally functioning CaR in response to the high fetal blood calcium. When CaR is ablated, PTH increases significantly and the fetal blood calcium rises further (see Figs 11.1 and 11.3) [12]. In the absence of PTHrP (Pthrp null fetuses), the circulating PTH level rises but is constrained by the CaR to maintain the blood calcium at the adult level and no higher [11,40], but when CaR is simultaneously ablated in Pthrp null fetuses, the blood calcium rises well above the maternal level [12]. Maternal hypercalcemia suppresses the fetal PTH level as observed in the offspring of wt and Carþ/ mice (Fig. 11.3) [12]; conversely, maternal hypocalcemia due to parathyroidectomy or calcitonin infusion induces secondary hyperparathyroidism, bone resorption, and reduced mineralization in rat fetuses [77e81]. PTH also regulates serum phosphorus since hyperphosphatemia occurs in thyroparathyroidectomized fetal lambs [82,83], and in Hoxa3 null, Gcm2 null, and Pth null mouse fetuses, all of which are deficient in PTH [41,49]. In the adult, loss of PTH causes hyperphosphatemia through reduced renal phosphorus excretion and low bone turnover. Fetal hyperphosphatemia can be explained in part by reduced renal phosphorus excretion which was observed in thyroparathyroidectomized fetal lambs [82,83], but renal phosphorus excretion has not been measured in any of these PTH-deficient mouse

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FIGURE 11.3

Fetal CaR and maternal blood calcium determine the fetal PTH level. In the Car knockout model, ablation of the Car results in a step-wise increase in PTH concentrations in the fetal circulation (A). Fetuses obtained from hypercalcemic Carþ/ mothers (A) have lower PTH levels than in 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 [12] Ó 1998 American Society of Clinical Investigation.

models. However, reduced calcium and magnesium excretion into amniotic fluid of Hoxa3 nulls, Gcm2 nulls, and Pth nulls predict that phosphorus excretion is likely reduced as well [41,49]. Skeletal mineralization is reduced in all PTH-deficient models and so decreased utilization of phosphorus for mineralization could also contribute to hyperphosphatemia. Placental phosphorus transfer has not been assayed in these mouse models but data from fetal sheep suggest that PTH does not regulate it [84]. PTH also regulates serum magnesium because its absence leads to lower serum magnesium levels, most notably a 20% decline in aparathyroid Hoxa3 null fetuses [41] while Gcm2 null and Pth nulls had a 5% but statistically significant decrease [49]. Conversely, excess PTH does not cause hypermagnesemia; Carþ/ and Car null fetuses had normal magnesium levels despite significant hyperparathyroidism [12] (unpublished data). Overall, despite low circulating levels, PTH is an important regulator of serum calcium, phosphorus, and magnesium; in its absence, these mineral concentrations are reduced and skeletal mineralization is impaired.

Calcitriol and Vitamin D Calcitriol circulates at low levels in fetal humans [64,69,85,86], rodents [31], and pigs [87], but it may exceed the maternal value in fetal sheep [87]. Calcitriol does not cross the rat placenta [88] and so the low fetal values must derive from fetal sources. Fetal kidneys and placenta possess the 1a-hydroxylase (CYP27b1) which converts 25-hydroxyvitamin D to calcitriol [89,90]. The contribution of the fetal kidneys is significant because fetal nephrectomy reduced the fetal

calcitriol levels in sheep and rats [91,92]. Moreover, in humans the umbilical artery levels of calcitriol are higher than umbilical venous levels, indicating contribution from the fetal kidneys [69]. The low levels of calcitriol are not due to inadequate supply of its precursor because 25-hydroxyvitamin D readily crosses the placenta [93,94] and achieves cord blood levels that are typically 75e100% of the maternal value at term [64,69,85,86,95]. Instead, the renal 1a-hydroxylase is suppressed by the ambient high calcium, high phosphorus, and low PTH concentrations in fetal serum. Also, although PTHrP circulates at high levels (see below), it is much less potent than PTH at stimulating the 1a-hydroxylase [96e99]. Only when the VDR is ablated (Vdr null fetuses) do fetal calcitriol levels reach normal adult values (Fig. 11.4) [100]. The 1a-hydroxylase is responsive to higher levels of PTH in utero because Carþ/ and Car null fetuses demonstrated a step-wise increase in both PTH and calcitriol levels [12]. Even so, the high calcitriol levels attained in Car null fetuses remained lower than the normally observed values in adult wild-type mice [12,31]. Maternal vitamin D deficiency in rats and absence of the vitamin D receptor in mice (Vdr null) reduces fertility and litter size [24,31,101]. In contrast to these maternal effects, several animal models indicate that calcitriol 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 phosphorus levels and had fully mineralized skeletons at term, as determined by total weight, ash weight, and calcium content of femurs [23e26,102].

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FIGURE 11.4 Fetal serum calcitriol increases with loss of VDR. Serum calcitriol was increased only in Vdr null fetuses as compared to wild-type (WT) and Vdrþ/ fetuses. For comparison, mean values of calcitriol in pregnant adult WT, Vdrþ/, and Vdr null mice are represented by horizontal lines with the SE indicated on the far right of each line. The SE for WT is too small to be represented. The maternal values were significantly different from each other (P<0.001). The number of observations is indicated in parentheses. Reproduced with permission from Kovacs et al. [31] Ó2005 American Physiological Society.

Definitive evidence that calcitriol is not needed for normal fetal calcium and bone homeostasis comes from 1a-hydroxylase-deficient Hannover pigs [50] and Vdr null mice [30,31]. In Hannover pigs, fetuses of homozygous calcitriol-deficient sows had normal blood calcium and phosphorus levels and fully mineralized skeletons [50]. Similarly, Vdr null fetuses had normal calcium, magnesium and phosphorus levels, and fully mineralized skeletons [31]. It is only after weaning that Vdr null neonates develop hypocalcemia and rickets [30], indicating that the VDR is also not required in the early neonatal period before intestinal calcium absorption becomes an active process. Additional evidence that calcitriol is not required is that fetal nephrectomy in rats did not affect fetal blood calcium or phosphorus levels when measured 48 hours later, even though fetal calcitriol levels had fallen [92]. Although the intestinal and renal expression of the vitamin D-dependent calcium binding proteins (calbindin-D9K and calbindin-D28K) are critically dependent on the presence of calcitriol 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, intestines, and other tissues [31,102e104]. Some data from humans lend support to the observation that calcitriol is not needed for normal fetal calcium and bone metabolism (discussed in greater detail in [105]). At term, cord blood calcium and skeletal mineralization are normal in the offspring of severely vitamin D-deficient mothers [106e108]. In a clinical trial that

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resulted in 25-hydroxyvitamin D levels of 138 nmol/L in fetuses of vitamin D-treated mothers versus the rachitic range of 10 nmol/L in fetuses of placebo-treated, severely vitamin D-deficient mothers, there was no difference in cord blood calcium or phosphorus between groups and no radiologic evidence of rickets [109]. In the only attempt at a systematic examination of the fetal skeleton, investigators in China examined the ash weight and mineral content of 15 fetuses that had died due to obstetrical accidents, including seven born of mothers known to have severe osteomalacia due to vitamin D deficiency and malnutrition, and eight born of healthy well-nourished mothers [110]. There were no significant differences in ash weight or calcium, phosphorus, and magnesium content of the ash between the two groups. The authors also described that centers of ossification were normal and that there were no radiographic signs of rickets [110]. A few rare, isolated case reports have indicated that craniotabes and other suggestive skeletal changes of rickets can be detected at birth [111e114], whereas in other reports that described craniotabes or rickets being present “at birth”, the diagnosis was actually made within the first or second week [114e119]. In one such case, convincing radiographic findings were not present at day 2 after birth but had developed by day 16 [119]. More recent clinical experience is that vitamin D deficient rickets usually does not develop (or become recognized) until weeks to months after birth with a peak incidence between 6 and 18 months, even in regions where severe vitamin D deficiency during pregnancy is endemic [106e108,120]. The clinical courses of children born with genetic disorders of vitamin D physiology have also been reported and the findings are consistent with Hannover pigs and Vdr null mice. Babies with 1a-hydroxylase deficiency (vitamin D dependent rickets type I; VDDR-I) and those lacking the vitamin D receptor (vitamin D dependent rickets type II or hereditary vitamin D resistant rickets; VDDR-II) are normal at birth [121e126]. In both conditions, the child inevitably develops hypocalcemia, hypophosphatemia, and rickets. VDDR-I presents early in infancy to within the first year of life; VDDR-II can also present in infancy but more often is diagnosed during the second year of life [121e126]. Overall, the animal and human data indicate that hypocalcemia and rickets are not present at birth but develop postnatally. These observations do not mean that calcitriol is inactive or has no role in fetal life. Widespread expression of VDR early in fetal skeletal development suggests a role for its ligand in fetal bone development [127], but the fetal skeleton forms normally without it [30,128]. Other investigators have manipulated the fetal calcitriol level by other means and found evidence of possible calcitriol-mediated

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effects. Infusion of an antibody to calcitriol decreased the ovine fetal blood calcium level [91]. Pharmacological doses of calcitriol given to pregnant guinea pigs increased the fetal calcium and phosphorus levels [129]. Bilateral nephrectomy in fetal sheep lowered the ionized and total calcium, and increased the phosphorus and PTH levels; these changes could be reversed by administration of calcitriol to the fetus and did not seem explainable by uremia alone [130]. Thus, at least in the absence of normal renal function, the low levels of calcitriol may still influence fetal mineral ion homeostasis. In summary, data from humans and several animal models indicate that loss of calcitriol and absence of VDR do not impair fetal skeletal formation and mineralization, nor the ability of the fetus to maintain normal blood levels of calcium and phosphorus. Pharmacological doses of calcitriol have been demonstrated to affect fetal calcium metabolism but this may not be physiologically relevant.

FGF-23 Fibroblast growth factor-23 (FGF-23) is a recently recognized hormone relevant to adult mineral homeostasis; excess levels lead to hypophosphatemia [131]. The role of FGF-23 in fetal mineral and bone homeostasis has not been studied, although it has been noted that FGF-23 null mice have normal length, weight, appearance, and serum calcium and phosphorus at birth [132]. Recent human data indicate that intact FGF-23 levels are low in cord blood but reach adult values by 5 days after birth [133].

Calcitonin Calcitonin can be detected in human fetal thyroid glands from as early as the 15th week of gestation [134], and fetal calcitonin levels are elevated compared to the simultaneous maternal value in term and preterm humans [5,58,64,69,135e137), rodents [9,56], and sheep [9,138]. Maternal calcitonin cannot cross the placenta [139] and so the high fetal levels derive from the fetal C-cells, stimulated by a high fetal serum calcium level. Several acute experimental perturbations suggest a role for calcitonin in fetal calcium homeostasis. Infusion of calcitonin antiserum to near-term fetal rats caused a slight increase in the fetal blood calcium after 1 hour [140], while fetal injection of calcitonin caused hypocalcemia and hypophosphatemia [47]. However, the bulk of evidence suggests that physiological levels of calcitonin play no significant role in regulating fetal calcium homeostasis. 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 [141]. In calcitonin/calcitonin gene-related peptide-alpha gene ablation model (Ctcgrp null fetuses) [142], fetal ionized calcium levels and the calcium content of their skeletons were normal [56]. On the other hand, calcitonin may play a role in regulating magnesium because, in Ctcgrp null fetuses, the serum magnesium and skeletal mineral content of magnesium were both significantly reduced [56].

Parathyroid Hormone-Related Protein Although immunoreactive PTH levels are low in cord blood, in vitro cytochemical bioassays have found high PTH-like bioactivity [59,63,143] which is attributable to high circulating levels of PTHrP [60,68,76,144,145]. When measured simultaneously and expressed in equivalent units (pmol/L), human cord blood PTHrP levels are 2 to 4 pmol/L and up to 15-fold higher than the levels of PTH (0.2 to 0.5 pmol/L) [60e62]. PTHrP is a prohormone that is processed into separate circulating fragments, each of which may have different functional roles and receptors [146e148]. The structures of these fragments have been deduced from studies of tumor cell lines transfected with the PTHrP gene, but it has yet to be determined which of them normally circulate in fetal life. Full length PTHrP has twice the molecular weight of PTH, and since PTH does not cross the placenta, full length PTHrP is unlikely to do so. A shorter fragment, PTHrP 1e86, did not cross the placentas of sheep and goats [149] but whether smaller fragments cross the placenta has not been determined. PTHrP is produced in many tissues throughout the developing embryo and fetus, including the skeletal growth plates [150,151], cardiac and vascular smooth muscle [152], trophoblast cells of the placenta [40,75,153], intraplacental yolk sac [40], amnion [154,155], chorion [155], umbilical cord [156], and many other organs. The expression of PTHrP in many of these locations has physiological importance as shown by Pthrp null fetuses, which have multiple defects that include short-limbed chondrodysplasia and abnormal rib cage [44], absence of mammary bud and nipple development [157], delayed differentiation of type II alveolar cells and surfactant deficiency [158], etc. Which of these tissues contribute to the circulating level of PTHrP and are important for regulation of fetal calcium and bone metabolism is less clear. Since venous umbilical PTHrP levels are higher than umbilical arterial levels in pigs, the placenta may be an important source of circulating PTHrP in the fetus [144]. Due to local production of PTHrP by the umbilical cord [156], the level of PTHrP in cord blood might not accurately reflect the systemic level of PTHrP. The importance of the placenta as a source of PTHrP in the fetal circulation is further supported by

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the finding that Pthr1 null fetuses have increased placental expression of PTHrP (mRNA and protein) and an 11-fold increase in circulating PTHrP [41]. Whether PTHrP is produced by the fetal parathyroid glands remains unresolved, and this is discussed below in the parathyroid section. PTHrP is an important regulator of the fetal blood calcium, as shown by Pthrp null fetuses in which loss of PTHrP results in a fetal blood calcium reduced to the maternal level (Fig. 11.5), the value normally set by the CaR in adults [11]. In sheep, fetal parathyroidectomy causes hypocalcemia that can be reversed by PTH or PTHrP infusion [141,159,160]. Since PTH normally circulates at low or undetectable levels, it is possible that the hypocalcemic effect of fetal parathyroidectomy is due in part to the loss of PTHrP produced by the parathyroids. Consistent with this, loss of parathyroids in Hoxa3 null fetuses causes more profound hypocalcemia than that due to loss of PTH alone, as observed in Pth null fetuses [40,41,49]. However, since aparathyroid Hoxa3 null fetuses had no change in circulating PTHrP levels or in the expression of PTHrP within the neck region [41], it remains unclear whether hypocalcemia due to loss of fetal parathyroids is at all attributable to loss of PTHrP. PTHrP must also regulate phosphate metabolism because Pthrp null fetuses have a 20% increase in serum phosphorus [41]. However, the explanation for this hyperphosphatemia is not clear. Although renal phosphorus excretion has not been measured, amniotic fluid calcium excretion was normal in Pthrp null fetuses and so phosphorus excretion may also be normal. Impaired skeletal mineralization also cannot explain the hyperphosphatemia since the calcium and phosphorus content of the Pthrp null skeleton is normal. Data from fetal sheep also suggest that PTHrP does not regulate placental phosphorus transfer [84]. PTHrP may not be an important regulator of magnesium since the serum magnesium and skeletal mineral content of magnesium were normal in Pthrp null fetuses [40 and unpublished data]. However, Pthrp nulls also

have hyperparathyroidism which may have a confounding effect on the serum magnesium. In summary, PTHrP is produced by diverse fetal tissues and circulates in fetal blood at levels up to 15fold higher than PTH, and higher than in the mother. PTHrP appears to regulate the fetal blood calcium and phosphorus. It also regulates fetaleplacental calcium transport, discussed below.

Other Hormones and Factors The sex steroids (estradiol and testosterone) play important roles in regulating calcium and bone homeostasis in the adult. In the fetus, these hormones circulate at low levels and may be unimportant for calcium and bone homeostasis. Mice lacking estrogen receptor alpha or beta, or the aromatase, have normal skeletal lengths at birth and do not develop altered skeletal metabolism until several weeks after birth [161e165]. However, in none of these models has calcium homeostasis in the fetus been investigated to date. NF-kappaB (RANK), RANK ligand (RANKL), and osteoprotegerin (OPG) play key roles in regulating the function of osteoclasts and bone turnover in the adult. Surprisingly, this system may be relatively unimportant in the development of the fetal skeleton. Deletion of RANKL leads to mice that show osteopetrotic changes by 2 days after birth; however, growth and serum levels of calcium, phosphorus and alkaline phosphatase are normal until weaning [166]. Mice in which RANK is deleted also appear normal at birth, but by 3 weeks of age develop stunted growth, osteopetrosis, lack of tooth eruption, hypocalcemia, hypophosphatemia and secondary hyperparathyroidism [167]. Deletion of OPG causes the opposite phenotype of early, severe osteoporosis and arterial calcifications in adult mice [168,169]; however, it has not been determined if fetal skeletal development and calcium metabolism are affected. The serum ionized calcium concentration in fetuses and adults differs significantly among different FIGURE 11.5

Loss of PTHrP causes hypocalcemia. Pthrp null fetuses (HOM) have lower blood calcium (A) and zero maternalefetal calcium gradient (B) as 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 [11] Ó 1996 National Academy of Sciences, USA.

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commercially available strains of normal mice [11 and unpublished data]. Normal peak bone density or skeletal calcium content also differs markedly among strains [170]. It seems likely that unidentified genes must contribute to the regulation of calcium and bone metabolism during fetal development and adults in order to explain this variability.

FETAL PARATHYROIDS AND THYMUS Parathyroids and thymus share a common developmental origin within the third pharyngeal pouch in both mice and humans [171e175]. In humans, a second pair of parathyroids originates from the fourth pouch but, in mice, the second set of parathyroids does not form [171e175]. Parathyroids are friable and so parathyroid cells within the thymus and adjacent tissues conceivably represent fragments that detached during migration [176e179]; analysis of parathyroid organogenesis in the mouse supports this possibility [174]. The primordium derived from the third pharyngeal pouch intensely expresses Gcm2 and so deletion of this gene should ablate parathyroids and PTH in mice, in the same way that absence of Hoxa3 ablates the parathyroids and PTH [40,41,176,180]. Indeed, neither PTH mRNA nor protein was present in the common parathyroid/thymus primordium in the Gcm2 null mice on embryonic days 11.5e12.5, while both were clearly present in the wild-type mice [177]. However, circulating PTH has been detected in Gcm2 null fetuses and adults even though the parathyroids (and parathyroid remnants in the thymus) have been eliminated [49]. Retained expression of circulating PTH in Gcm2 null fetuses may be the result of recently confirmed placental expression of the gene [49], whereas expression in adult Gcm2 null mice is not readily explainable. It may indicate that other Gcm2-independent sources of PTH activate in the absence of normal parathyroids [181]. As noted above, fetal parathyroids may contribute to calcium homeostasis by secretion of both PTH and PTHrP, unlike in the adult, where the parathyroids secrete PTH alone. However, the evidence that fetal parathyroids make PTHrP is inconclusive. PTHrP was originally detected by immunocytochemical methods with polyclonal antibodies in parathyroids of fetal sheep [76,153]. However, in two subsequent studies, PTHrP mRNA was not detected in fetal or adult rat parathyroids by in situ hybridization or reverse transcriptionepolymerase chain reaction (RTePCR) [182,183], whereas it was detected by RT-PCR in a third study of adult rat parathyroids [184]. Although murine parathyroids have not been studied in isolation, loss of parathyroids in Hoxa3 null and Gcm2 null fetuses did not alter the plasma PTHrP level [41,49], while an 11-fold

upregulation in plasma PTHrP in Pthr1 null mice was not associated with any detectable increase in expression of PTHrP mRNA in the neck region [41]. Human fetal parathyroids have not been examined but PTHrP expression has been detected in adult normal, hyperplastic, and adenomatous parathyroids by Northern, immunohistochemical staining, immunoblot, and both radioimmunoand radioimmunometric assays [185e189]. The evidence is consistent across species that intact parathyroid glands are required for maintenance of a normal fetal calcium level as demonstrated in the studies of fetal lambs (thyroparathyroidectomy), rats (decapitation), and mice (loss of parathyroids through ablation of Hoxa3 or Gcm2, or loss of PTH alone in Pth null fetuses). Recent studies in genetically engineered mice have demonstrated that PTH and PTHrP have an additive effect in regulating the blood calcium [40,41]. In a double-mutant colony, Pthrp null fetuses had a modestly reduced blood calcium (equal to the maternal calcium concentration), aparathyroid 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. 11.6) [40]. However, in a similar genetic background, aparathyroid Gcm2 null and also Pth null fetuses had a blood calcium equal to the mother and not as low as in aparathyroid Hoxa3 null fetuses

FIGURE 11.6 PTH and PTHrP additively regulate the fetal blood calcium. Serum ionized calcium in wild-type, single mutant Hoxa3 null, single mutant Pthrp null, and Hoxa3/Pthrp double mutant fetuses. For clarity, the other five possible genotypes have been omitted from the figure. The upper dashed line indicates the mean maternal ionized calcium level in this genetic background (equal to Pthrp null), while the lower dashed line indicates the mean ionized calcium level in Pthr1 null fetuses in a similar genetic background. Reproduced with permission from [40] Ó 2001 The Endocrine Society.

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(Fig. 11.7). The explanation for this discrepancy is not clear, but it may relate in part to the presence of hyperplastic parathyroids (possibly producing PTHrP) in Pth null fetuses [190], and to the upregulation of placental PTH expression in Gcm2 null fetuses [49]. As noted earlier, fetal parathyroids are also required for regulation of serum magnesium and phosphorus concentrations, since thyroparathyroidectomized fetal lambs, and Hoxa3 null, Gcm2 null, and Pth null fetal mice have hypomagnesemia and hyperphosphatemia [18,41,49,82,191]. Absence of PTHrP alone also causes hyperphosphatemia, but the serum magnesium concentration is unaltered [41 and unpublished data]. As discussed below, there is conflicting evidence between sheep and mouse models that parathyroids are required to regulate placental calcium transfer.

CALCIUM SENSING RECEPTOR Ablation of CaR (in Carþ/ and Car null fetuses) increases both the fetal blood calcium (see Fig. 11.1) and the circulating PTH level (see Fig. 11.3) above the corresponding wild-type values [12]. This hypercalcemia is dependent upon PTH because simultaneous ablation of the PTH1 receptor (PTH1R) (Car/Pthr1 double mutant) prevents the hypercalcemia [12]. Postnatally, homozygous ablation of CaR results in severe hyperparathyroidism and hypercalcemia [29], analogous to the human condition of neonatal severe

FIGURE 11.7

Loss of PTH causes only modest hypocalcemia. Pth null fetuses (/) have a blood calcium that is reduced to the maternal calcium concentration (line). Not shown is that the blood calcium of Gcm2 null fetuses was also reduced to the maternal level. The number of observations is indicated in parentheses. Adapted with permission from [49] Ó2010 American Society for Bones and Mineral Research.

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primary hyperparathyroidism. However, in fetal life, the serum calcium of Car null fetuses is no higher than the heterozygous siblings (see Fig. 11.1) [12], indicating that some aspect of the intrauterine environment prevents Car null fetuses from achieving a higher blood calcium level. Although CaR functions in fetal life (as it does in the adult) to regulate PTH secretion, it does not regulate PTHrP. The fetal serum calcium is maintained at a higher level than in the mother partly through the action of PTHrP; CaR responds to this fetal hypercalcemia by suppressing PTH (Fig. 11.8A). With loss of PTHrP (Pthrp null), the blood calcium falls but is then maintained at the adult level through the actions of the CaR to stimulate PTH (Fig. 11.8B) [11,40]. In the absence of PTH (Hoxa null), the serum calcium falls sharply because PTH is absent and CaR has no ability to stimulate PTHrP; plasma PTHrP levels remain normal in these fetuses. CaR is also expressed in the murine placenta [192] and Car null fetuses have reduced transfer of calcium across the placenta [12]. This reduction in placental calcium transfer may be a consequence of the loss of calcium sensing within the placenta, and the lower plasma PTHrP levels observed in these fetuses are consistent with this possibility. Alternatively, placental calcium transfer might be downregulated in response to the elevated serum calcium or hyperparathyroidism that occur in these null mice. Ablation of CaR would be expected to decrease renal calcium clearance as it does in the adult [29]. However, Carþ/ and Car null fetal mice have increased amniotic fluid calcium, suggesting that the renal filtered load and excretion of calcium are increased by the higher serum calcium [12]. The discrepancy between adult and fetal effects of CaR ablation on renal calcium handling may be explained by the observation that the

FIGURE 11.8 Fetal blood calcium regulation. (A) Normal high fetal calcium level, which is dependent upon PTHrP, activates the parathyroid CaR, and PTH is suppressed. (B) In the absence of PTHrP, the fetal calcium level falls to a level that is now set by the parathyroid CaR; PTH is stimulated to maintain the ionized calcium at the normal adult level (¼ maternal).

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kidneys express very low levels of CaR mRNA until the first postnatal day [193].

PLACENTAL CALCIUM TRANSPORT Overview Human babies obtain 80% of the required 20e30 g of calcium during the third trimester [1,194], while rats accrete 95% of the required 12.5 mg of calcium during the last 5 days of a 3-week gestational period [195]. Active transport of calcium across the placenta is required because the diffusional flow is insufficient and the calcium exchange occurs against a substantial electrochemical gradient [196,197]. Calcium presumably enters calcium-transporting cells in the placenta via gated calcium channels (such as transient receptor potential vanilloid 6 [TRPV6]) in the maternal-facing basement membranes, shuttles to the opposite basement membrane bound to calcium binding proteins, and is extruded at the fetal-facing basement membranes by Ca2þ-ATPase [42,196]. The calcium transporting cells appear to be syncytiotrophoblasts in human placenta, and trophoblasts and intraplacental yolk sac cells in rodents [192,198,199]. Study of perfused human and sheep placentas estimated that active transport of calcium comprises one to two-thirds of the forward flow from mother to fetus [22,200]. Backflux of calcium from fetus to mother has been estimated to be 1% (sheep) to 80% (primates) of the forward flow [201,202]. Calbindin-D9K shuttles calcium across kidney and intestinal cells and likely plays a similar role in trophoblasts and yolk sac cells [203]. Its expression within placenta begins as early as day 10 of gestation in rodents [204] and both the mRNA and protein increase manyfold over the last 7 days of gestation in rats [205e207] and mice [42,208]. Expression of the Ca2þ-ATPase doubles over the same interval [205,209]. TRPV6 expression increases 14-fold during the last 4 days of gestation in the mouse [42]. All three calciotropic genes co-localize within trophoblasts but, in rodent placentas, the most intense expression is within the intraplacental yolk sac, a structure that also expresses other calciotropic genes at the highest intensity compared to the surrounding trophoblasts (Fig. 11.9) [42]. Whether calbindin-D9K, Ca2þ-ATPase, and TRPV6 are essential for placental calcium transport is unclear. A murine knockout of calbindin-D9K has normal serum calcium and intestinal calcium absorption as a young adult, and evidently compensates by upregulating other calcium-binding and transporter genes; however, placental calcium transfer has not been measured in this model [210e212]. Calbindin-D9K expression was

reduced within the intraplacental yolk sac of two mouse models that have reduced placental calcium transfer (Pthrp null and Trpv6 null) [42,192], but whether the reduced expression was a cause or consequence of a lower rate of placental calcium transfer is unknown. Knockout of Pmca1, the gene encoding the dominant placental Ca2þ-ATPase, results in embryonic death at the preimplantation stage but placental calcium transfer and skeletal mineralization has not been assessed in the

FIGURE 11.9 Anatomy of the intraplacental yolk sac. By the time the mature placenta has formed (A), the compressed yolk sac lines the uterine cavity (decidua) that is not in contact with the placenta, and it overlies the dome of the placenta. The parietal layer and Reichert’s membrane are in contact with the decidua and the dome of the placenta, while the columnar or visceral yolk sac layer is in apposition to this layer, separated by the yolk sac cavity. The yolk sac bilayer that overlies the dome of the placenta forms finger-like projections into the placenta near the insertion of the fetal vessels (boxed area in [B], the intraplacental yolk sac). The intraplacental yolk sac is positioned between maternal and fetal blood spaces, as visualized in detail in (B). Within this structure, the parietal layer and Reichert’s membrane overlie maternal blood spaces and vessels, while the columnar layer overlies fetal blood vessels. Between these layers is the sinus of Duval, which also communicates with the yolk sac cavity and the uterine lumen. Reproduced with permission from Kovacs et al. [192] Ó2002 American Physiological Society.

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heterozygotes [213,214]. Knockout of the isoforms Pmca2 or Pmca4 (which are also expressed in placenta) [215,216] each result in mild phenotypes such as deafness, impaired sperm motility, and infertility, but possible effects on fetal skeleton and placenta have not been assessed [213,214]. On the other hand, although knockout of Trpv6 causes no impairment of intestinal calcium absorption in adult mice [211], placental calcium transfer was reduced 40%, skeletal ash weight at term was reduced 50%, and the Trpv6 null fetuses were severely hypocalcemic [42]. Therefore, TRPV6 plays an important role in placental calcium transfer and fetal calcium homeostasis, while the effect of absence of the Pmca1 isoform of Ca2þ-ATPase or of calbindin-D9K remains unknown.

Placental Structure and Gene Expression There are significant structural and functional differences among placentas of the various species that have been studied [217e222]. Placentas of sheep, goats, and pigs have a cotyledonary structure of 60e70 individual units (cotyledons) which are spread over the entire uterine wall. The microscopic structure is epitheliochorial, meaning that the maternal and fetal circulations are separated by full thicknesses of maternal and fetal tissues [217,218,221]. Calbindin-D9K expression is most concentrated in the interplacentomal region (i.e. the flat intercotyledonary trophoblasts) [223e225]. This structure makes up about 2% of the volume of the mature placenta and has been postulated to be an important site of calcium transfer between mother and fetus in ruminants [226]. The placentome and interplacentomal region also contain the highest expression of Ca2þATPase [224]. The placentas of humans, monkeys, and rodents are discoid, in which the placental tissue is confined to a single plate [217,218]. The microscopic structure is hemochorial, meaning that fetal trophoblasts have fully invaded the uterine tissue such that maternal blood comes in direct contact with the fetal chorion. The barriers to exchange of nutrients and waste are reduced as compared to epitheliochorial placentas [217,218]. Although hemochorial placentas of humans, mice, and rats share similar structure and function [219e222], they do exhibit other significant differences. First, the human placenta is hemomonochorial, meaning that a single trophoblast cell layer separates maternal and fetal blood. Rodent placentas are hemotrichorial since three layers of trophoblasts are interposed between the maternal and fetal circulations [218,221,227]. Second, in human placentas, finger-like projections (villi) protrude and float freely into a large intervillous maternal bloodfilled space. In rodent placentas, the villi are surrounded by maternal blood in a labyrinthine series of small

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channels created by trophoblasts [228]. Third, rodent placentas contain the intraplacental yolk sac, a bilayered membrane interposed between fetal and maternal blood vessels (see Fig. 11.9) [229e231]. Many calciotropic genes are intensely expressed within this structure, including calbindin-D9K, Ca2þ-ATPase, PTHrP, PTH1R, TRPV6, vitamin D receptor, CaR, etc. [42,192,232e234]. Due to its positioning between the maternal and fetal vessels and its intense expression of calciotropic factors, it has been postulated to be a site of maternalefetal calcium exchange [22,42,192]. Such an alternate route for active calcium transport may enable rodents to meet proportionately larger calcium demands created by larger litters and a short gestation period [22]. How much the intraplacental yolk sac contributes to maternalefetal calcium exchange remains speculative. Absence of platelet-derived growth factor alpha, which encodes the intraplacental yolk sac, leads to embryonic lethality [235]. However, support for a role of the intraplacental yolk sac in calcium transfer comes from Pthrp null and Trpv6 null fetuses in which placental calcium transfer was reduced (see below) [11,42]. In the Pthrp null, calbindin-D9K mRNA and protein expression were substantially reduced within the intraplacental yolk sac, whereas there was no difference in expression noted by Northern or Western blots of the trophoblasts which make up the bulk of the placenta [11,192]. The Trpv6 null also had reduced expression of calbindinD9K in the intraplacental yolk sac [42]. In human placentas, trophoblasts contain the highest expression of calbindin-D9K, Ca2þ-ATPase, and TRPV6, as compared to the intraplacental yolk sac in rodent placentas [42,192,196,199,206e208,236]. PTHrP mRNA and protein are widely expressed in human syncytiotrophoblasts, cytotrophoblasts, amnion, decidua and myometrium; the highest levels may be in the amnion [154,237,238]. In the rodent, PTHrP is expressed in giant trophoblasts between embryonic days (ED) 7.5 and 13.5 [75,239]; after that, it is most intensely expressed in columnar cells of the intraplacental yolk sac, giant trophoblasts, and spongiotrophoblasts [192]. The PTH1R is most intensely expressed in chorionedecidua of the human placenta, with lower levels in the amnion, smooth muscle cells and endothelium of the chorionic plate vessels, syncytiotrophoblasts and capillary endothelium of the chorionic villi, and myometrium [238,240]. In mice, PTH1R is expressed in the preimplantation embryo [241], and later in parietal and visceral extraembryonic endoderm of the postimplantation embryo from ED 7.5 through 10.5 [242]. PTH1R mRNA and protein expression continues on intensely in that part of the parietal and visceral extraembryonic endoderm that becomes the intraplacental yolk sac (especially the parietal cells); PTH1R was not detected in murine trophoblasts [192].

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CaR mRNA is present in cytotrophoblasts of the human term placenta [243]. In the mouse, CaR mRNA and protein are expressed in trophoblasts and intraplacental yolk sac, with the most intense expression in the parietal cells of the intraplacental yolk sac [12,192]. VDR is expressed in human placenta but its localization has not been described [244]. Within rodent placentas, VDR is expressed by trophoblasts but its highest expression is in the columnar cells of the intraplacental yolk sac [192,234,245]. Calcitonin is expressed by trophoblasts in the human placenta and by trophoblasts and intraplacental yolk sac in rodent placentas [192,246,247). The calcitonin receptor has been localized to both fetus-facing and maternalfacing plasma membranes of syncytiotrophoblasts in humans [248,249], and to trophoblasts and intraplacental yolk sac in murine placentas [192]. Overall, there are substantial structural and possibly functional differences between epitheliochorial placentas of sheep and the hemochorial placentas of humans and rodents, but also between human and rodent placentas. Notably all in vivo data about placental function come from study of non-human placentas.

Techniques for Measuring Placental Calcium Transport Over the years, several techniques have been utilized to measure placental calcium transport. These differ significantly from one another and offer varying benefits and limitations which, in turn, affect the conclusions that can be drawn from the data. An in situ placental perfusion technique has been utilized in sheep, goats, and rats (Fig. 11.10) [141,250e252]. The fetus is discarded and the placenta is connected via the umbilical vessels to a semi-closed circuit. The placenta is then perfused in situ with either autologous fetal blood or a blood substitute, and the flow rate and perfusion pressure are maintained at arbitrary set levels. Both 45Ca and 51Cr-EDTA are

administered into the maternal circulation, and repeated sampling from the maternal circulation and umbilical vein enables the radioisotope clearance rates to be calculated. Since 51Cr-EDTA has been shown to diffuse across the placenta and not be actively transported, the rate of disappearance of 45Ca from the maternal circulation, and the appearance of 45Ca relative to 51Cr-EDTA in the placental outflow, are used to calculate accurately the rate of active calcium transfer [251]. Peptides or pharmacologic agents can be infused directly into the umbilical arteries (placental in-flow) or into the maternal circulation, while continuous sampling of the calcium concentration in the umbilical vein portion of the circuit (placental out-flow) is used to determine the effects of the intervention on calcium transport [250]. Limitations of this technique are that it is done in the absence of fetal control, a single placenta is measured with no matched control, and the perfusate pressure and rate are at artificially set levels. On the other hand, continuous sampling from the maternal circulation and from the placental circuit enables the experimental conditions (concentration of 45 Ca and 51Cr-EDTA in the maternal circulation, for example) to be kept nearly the same from one pregnant dam to the next. A second technique keeps the fetus intact and in control of placental function, and has been utilized in mice (Fig. 11.11). The pregnant mother receives an intracardiac injection containing 45Ca and 51Cr-EDTA during brief (30 second) anesthesia. As early as 5 minutes after the maternal injection, the fetuses are removed, and the amount of isotope accumulated within each fetus is measured. Because 51Cr-EDTA crosses the placenta only by passive diffusion, it serves as a control for differences in flow rate between the individual placentas in one litter. The relative rate of placental calcium transfer for each fetus in the litter can then be determined by expressing the accumulation of 45Ca relative to 51CrEDTA within each fetus [11]. The importance of the use of 51Cr-EDTA is illustrated by the observation that 51 Cr accumulation commonly varies two- to threefold FIGURE 11.10

Placental perfusion studies in fetal lambs and rats. The fetus is removed from the uterus, and the umbilical vessels are connected to a semi-closed circuit to enable perfusion of the placenta in situ. Test peptides are administered on the arterial side of the circuit, and changes in maternalefetal calcium transfer are detected by measurements on the venous side of the circuit. The reservoir contains autologous fetal blood or a blood substitute.

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259 FIGURE 11.11 Placental calcium transfer studies in fetal mice. The pregnant mother receives an intracardiac injection of isotope [45Ca and 51Cr-EDTA] (A). After 5 minutes, 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 intra-abdominal injection of the test substance or diluent (B). Following a predetermined interval of 60 minutes or longer, the placental calcium transfer experiment proceeds with the intracardiac injection of isotope administered to the mother as in (A).

between placentas within the same uterus while the 45 Ca/51Cr ratio remains unchanged [unpublished data]. This indicates that blood flow to any one murine placenta is not equal to all the others in the same uterus at any point in time, but whether these differences in blood flow are permanent or changing from minute to minute is unknown. Strengths of this procedure include that the fetuses are not disturbed and remain in control of placental function, placental perfusion pressure has not been altered, the mother receives only brief anesthesia, the isotopes are rapidly and uniformly presented to the placental circulations following a single maternal intracardiac injection, and the eight to ten normal and heterozygous fetuses within each litter serve as controls for the null fetuses and each other. Limitations of this procedure include that a relative rate of calcium transfer among fetuses is obtained in order to be able to compare results from one experiment to the next; with an intracardiac injection, some isotope may leak into the pericardial space or the lungs, and so the actual amount of isotope entering the maternal circulation may differ from one experiment to the next. Most recently an adaptation of the in situ placental perfusion technique has been performed in mice. The small (<1 cm) diameter of murine placentas creates substantial technical challenges, and in order to catheterize successfully the umbilical vessels, it is necessary first to maximally dilate the vasculature with nitroglycerin [253,254]. 45Ca is administered without 51Cr-EDTA

or another control for diffusion of isotope. In the original technique used in sheep, goats, and rats, serial blood samples collected throughout the experiment enabled maternalefetal 45Ca clearance to be formally and accurately calculated. However, due to the small blood volumes of adult and fetal mice, numerous maternal or umbilical blood samples cannot be collected over the course of the experiment, and maternalefetal clearance of 45Ca cannot be directly calculated. Instead, the declining maternal radioisotope concentration was determined by sampling from different mothers at single time points and creating a maternal radioisotope disappearance curve from the aggregate results [253,254]. This curve was then used to estimate maternalefetal 45Ca clearance. Strengths of this procedure are that it attempts to quantify the rate of placental calcium transfer in mice under conditions in which the effects of different hormones or pharmacological agents could be evaluated. Weaknesses include those mentioned earlier for use of the procedure in placentas of sheep and rats. But there are additional significant limitations. The use of nitroglycerin to dilate maximally the placental vessels before cannulation may create an artifactual result, especially if the wild-type and mutant placentas differ in their ambient blood pressure or responsiveness to vasodilators. The lack of monitoring of maternal clearance of radioisotope within the same experiment, and substitution of a clearance curve from multiple mice assessed at single time points, means that the amount of isotope

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presented to the individual placenta at successive time points is assumed rather than directly measured, and so it is less certain that the clearance formula is correct for each experiment. As noted above, blood flow from one wild-type placenta to the next differs two- to threefold as assessed by 51Cr administered to the mother, but this experiment utilizes a single, arbitrary rate and pressure of perfusate. In addition to these techniques, some investigators have inferred a rate of placental calcium transfer by injecting 45Ca into the circulation of pregnant ewes and then measuring the amount of 45Ca in the fetal skeleton several days later [255]. This technique is less than optimal. 45Ca that enters the fetal circulation can cycle within several compartments including blood, amniotic fluid, and skeleton, and then return to the maternal circulation. Thus, at best this technique serves as an indirect measure of placental calcium transfer [22].

Maternal Regulation Maternal hormones can influence fetaleplacental calcium transport by acting directly on the placenta or by raising or lowering the maternal blood calcium level. However, studies in animals indicate that the normal maternal-to-fetal calcium transfer rate can usually be maintained despite maternal hypocalcemia or hormone deficiencies [13,256]. For example, maternal hypocalcemia due to parathyroidectomy or a severely calciumdeficient diet did not alter the rate of calcium transfer across perfused placentas of sheep [250,257]. Similarly, in fetal mice, severe hypocalcemia in Vdr null mothers did not adversely affect placental calcium transfer [31]. It is likely that the fetaleplacental unit works harder in response to maternal hypocalcemia in order to extract the required calcium. Indeed, the following observation from intact fetal rats confirmed that the rate of placental calcium transfer must be upregulated in response to maternal hypocalcemia due to parathyroidectomy. A maternal calcium infusion caused a marked, acute rise in the blood calcium of fetuses from parathyroidectomized, hypocalcemic rats, but had no effect on the fetuses of normal rats [37]. Maternal loss of calcitonin in Ctcgrp null mice did not adversely affect placental calcium transfer or the expected accretion of mineral by the fetal skeletons [56]. Additional studies have indirectly assessed the effect of other maternal hormone deficiencies by determining the net fetal accumulation of calcium at term in vitamin D-deficient rats [102], thyroidectomized, thyroxine-supplemented (“calcitonin-deficient”) sheep [255,258], and in sheep that received daily administration of prolactin and/or bromocriptine [259]. 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. However, the apparent lack of effect of maternal vitamin D or calcitonin deficiency on placental calcium transfer have since been confirmed by studies of Vdr null and Ctcgrp null mice [31,56]. Although rodents show that the normal amount of mineral may be extracted by term despite severe maternal hypocalcemia, this may not be true for humans. Maternal hypocalcemia due to hypoparathyroidism in humans has been associated with intrauterine fetal hyperparathyroidism, the consequences of which include skeletal demineralization, intrauterine fractures, and bowing of the long bones [260,261].

Fetal Regulation The Role of PTHrP That PTH or PTHrP might regulate placental calcium transfer became apparent upon studying the effects of fetal parathyroidectomy. In thyroparathyroidectomized fetal sheep supplemented with thyroid hormone [141,250], and in decapitated fetal rats (thought to approximate a thyroparathyroidectomy) [251], placental calcium transfer was reduced as assessed by in situ placental perfusion [141,250,251]. When autologous blood from intact fetal sheep was infused into the placental circuit of thyroparathyroidectomized fetal sheep, calcium transfer was restored [141]. These findings led to the conclusion that the fetal parathyroids regulate active exchange of calcium between mother and fetus. Since the parathyroids produce PTH, it was thought that loss and restoration of PTH explained the experimental results. However, in subsequent studies in thyroparathyroidectomized fetal sheep, PTH did not restore calcium transport, and it was concluded that the fetal parathyroids must regulate placental calcium transfer through the production of other factor(s) [160]. Subsequently PTHrP (1e141), PTHrP (1e86) and PTHrP (67e86) all stimulated placental calcium transfer in thyroparathyroidectomized fetal sheep, whereas amino-terminal fragments of PTH or PTHrP had no effect [160,262,263]. A mid-molecular fragment of PTHrP, specifically PTHrP (67e86), appeared to contain the relevant bioactivity. More recent studies in fetal lambs demonstrated that PTHrP (38e94), considered to be the true mid-molecular form of PTHrP, also stimulated placental calcium transfer [148]. These results in sheep are consistent with the notion that PTHrP, originating from the fetal parathyroids, is an important regulator of placental calcium transfer, while PTH is not. Circulating levels of PTHrP were not measured, and the evidence (discussed earlier) that fetal parathyroids produce PTHrP is mixed, and so it remains unknown

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whether fetal thyroparathyroidectomy lowers the rate of placental calcium transfer by reducing fetal PTHrP. Subsequent studies utilized the placental calcium transfer technique in intact fetal mice and found that, depending upon the time point studied, Pthrp null fetal mice had a 25e40% reduction in the rate of placental calcium transfer. When treated with PTHrP (1e86) or PTHrP (67e86), the isotope accumulation increased to normal [11]. Treatment with PTHrP (1e34) or PTH (1e86) had no effect, confirming that the mid-molecule of PTHrP must contain the bioactivity needed to stimulate placental calcium transfer [11]. Notably, PTH was subsequently found to be upregulated threefold in these fetuses, and so any effect of PTH to stimulate calcium transfer may have already reached its maximum, thus rendering the Pthrp nulls unable to respond to exogenous PTH. Additional studies done in other knockout mouse models support the notion that PTHrP regulates placental calcium transfer, and that PTH may not. Hoxa3 null fetuses completely lack PTH [180] but have a normal rate of placental calcium transfer and unaltered plasma PTHrP levels [41]. Pthr1 null fetuses (which lack the PTH1R) had a 50% increased rate of placental calcium transfer relative to the diffusion of 51Cr-EDTA [11,41]. The absolute amount of 45Ca transferred to Pthr1 null fetuses was lower than in their wild-type and Pthr1þ/ littermates, due to the much smaller size of the Pthr1 null fetuses and placentas [11, unpublished data]. It was deduced and later confirmed that Pthr1 null fetuses have markedly upregulated circulating levels of PTH and PTHrP [11,41]. Since the PTHrP mid-molecule contains the bioactivity to stimulate placental calcium transfer and does not activate the PTH1R, it was concluded that the high circulating levels of PTHrP were stimulating placental calcium transfer by acting on an as-yet-uncloned mid-molecular receptor [11,41]. The placental calcium transfer technique in intact mice has also been validated in other models. Pth nulls have hypocalcemia but no alteration in circulating PTHrP and a normal rate of placental calcium transfer (discussed below) [49]; Gp130 null mice have hypocalcemia, bone abnormalities, and a compensatory increased rate of placental calcium transfer [264]; Vdr null fetuses have increased placental calcium transfer accompanied by upregulation in TRPV6 [31]; Trpv6 null mice have severe hypocalcemia and decreased placental calcium transfer, accompanied by reduced expression of calbindin-D9K in the intraplacental yolk sac [42]; Car null mice have reduced placental calcium transfer and significantly lower plasma PTHrP levels [12, unpublished data]; and Ctcgrp null fetuses which lack calcitonin show no alteration in calcium homeostasis and have a normal rate of placental calcium transfer [56].

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Pthrp null fetuses do not produce any PTHrP and so this model does not address whether fetal parathyroids produce PTHrP or even if such parathyroid-derived PTHrP regulates placental calcium transfer. Studies in Hoxa3 and Pthr1 null fetuses support the hypothesis that the placenta may be the relevant source of PTHrP. Hoxa3 nulls are aparathyroid but have normal circulating PTHrP levels and a normal rate of placental calcium transfer [41]. On the other hand, Pthr1 nulls have an increased rate of placental calcium transfer, elevated circulating PTHrP levels, and an increase in PTHrP mRNA in both placenta and liver, but not in the neck region that contains the parathyroids [11,41]. Thus, the studies in fetal sheep and mice agree that a mid-molecular form of PTHrP stimulates placental calcium transfer. But whereas the fetal sheep studies concluded that it was parathyroid-derived PTHrP which stimulated placental calcium transfer, the studies in fetal mice are consistent with a placental source of PTHrP. Species differences might also explain the apparently discrepant results of the sheep and mouse studies; there are no equivalent human data to shed light on this. Most recently, contrary evidence about the role of PTHrP comes from the in situ placental perfusion technique adapted for use in mice as described above. Perfused placentas from Pthrp null fetuses had an increased rate of 45Ca transfer across the placenta as compared to parallel studies done in wild-type and Pthrpþ/ fetuses [254]. This surprising result may be explainable by the experimental methodology described earlier, most notably the use of nitroglycerin to dilate the placental and umbilical vasculature prior to catheterization of the umbilical vessels and initiation of the experiment. PTHrP is expressed in the smooth muscle vasculature, functions as a smooth muscle relaxant and vasodilator, and exerts effects on vascular development and physiology [147,265]. In the absence of PTHrP, the Pthrp null placental vasculature likely differs from wild-type in its ambient pressure, structure, and responsiveness to vasodilators [265]. Therefore, dilating the vasculature with nitroglycerin prior to measuring placental calcium transfer may lead to a greater response in the Pthrp null and an artifactual result. Furthermore, the ambient blood flow rate from the maternal circulation to the Pthrp null fetus may differ from wild-type, and this is suggested by a trend for higher 51Cr-EDTA accumulation observed in the Pthrp null mice in the original placental calcium transfer experiments [11, unpublished data]. If maternal/placental/fetal blood flow dynamics differ between Pthrp null and wild-type in vivo, the use of a vasodilator followed by a set perfusion pressure and flow rate would obliterate any effect that the absence of PTHrP had caused. Additional limitations are that the fetus is absent in this experimental method and therefore the placenta

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has been released from fetal control, leaving it subjected only to regulation by maternal factors and substances added to the perfusate. Furthermore, with only one placenta examined per pregnancy there are no littermate controls which would allow for the effect of the vasodilator and variations in experimental conditions from dam to dam to be taken into account. Overall, the results from this adapted placental perfusion technique in Pthrp null placentas remain puzzling but possibly explainable by the experimental methodology as described. Overall, the in situ placental perfusion technique in sheep and the intact placental calcium transfer method in mice led to consistent results suggesting that the mid-molecule of PTHrP stimulates placental calcium transfer. Pthr1 null mice support this conclusion by demonstrating an increase in placental calcium transfer under conditions in which PTHrP is upregulated, while Car null mice show a reduction in placental calcium transfer with reduced PTHrP [11,12, unpublished data]. The intact placental calcium transfer method has been validated in other mouse models such as Vdr, Trpv6, Hoxa3, Ctcgrp, Gp130, Car, Pth, and Gcm2 [31,41,42,49,56,264]. Thus, the aggregate results are consistent with the conclusion that PTHrP is an important stimulator of placental calcium transfer, although its relevant source remains uncertain (Fig. 11.12). The Role of PTH PTH circulates at low levels in the fetus and most studies suggest that it does not regulate placental calcium transfer. However, other studies have indicated that PTH can stimulate this process. Among the earliest were the rat decapitation studies mentioned earlier in which perfused placentas showed a reduced rate of calcium transfer. When PTH was administered to the decapitated fetal rats, placental calcium transfer was stimulated [251]. More recently, PTH (1e34) increased calcium accumulation in vitro within vesicles created from human syncytiotrophoblast basal membranes [266]. Additional supportive evidence for a role of PTH is that the PTH1R is highly expressed in the placenta, particularly in the intraplacental yolk sac which is believed to be a site of active maternalefetal calcium exchange [192]. And, as noted earlier, Pthrp null fetuses have a threefold increase in endogenous circulating PTH which may have made exogenous administration of PTH to Pthrp null fetuses ineffective at raising placental calcium transfer further [11,40]. Indeed, in retrospect a non-significant 5.9% increase in placental calcium transfer occurred in response to PTH (1e84) administration to Pthrp null fetuses [11]. That PTH has a role in regulating placental calcium transfer was recently elucidated in studies of Pth null fetal mice, which completely lack PTH, and Gcm2 null

fetal mice, which have very low levels of circulating PTH [49]. Placental calcium transfer was not decreased in Pth nulls and the circulating PTHrP level was unchanged. However, microarray and real-time RTPCR analysis of placental gene expression revealed reduced expression of several calciotropic genes (Trpv6, Cabp9k, Vdr) as well as other genes involved in cation and solute transport. To confirm that these changes in gene expression were due to absence of PTH, and to determine if PTH could stimulate placental calcium transfer, Pth null fetuses were treated in utero with either saline or PTH (1e84) before performing the placental calcium transfer experiment. Pth null fetuses treated with PTH (1e84) had a 30% higher rate of 45Ca/51Cr-EDTA accumulation as compared to Pth

FIGURE 11.12 Placental calcium transfer is regulated by PTHrP and PTH. While the exact tissue source(s) of PTHrP and PTH that are relevant for placental calcium transfer remain uncertain, the placenta is a source of both PTHrP and PTH from which location both might regulate placental calcium transfer. Whether the parathyroids produce PTHrP or not is uncertain.

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null fetuses treated with saline, confirming that PTH can stimulate placental calcium transfer in vivo [49]. Furthermore, during the 90-minute experiment, PTH treatment increased the expression of several genes including Vdr, vitamin D binding protein, and other solute carriers [49]. In contrast to the Pth null phenotype, Gcm2 nulls showed a significant baseline increase in placental calcium transfer, possibly in response to fetal hypocalcemia [49]. Further investigation showed no upregulation in PTHrP (in the circulation, neck, or placenta) that could explain this increase in placental calcium transfer. Gcm2 nulls had significantly increased expression of placental Trpv6, Cabp9k, and Vdr as compared to Pth null placentas, and these changes may account for the increase in placental calcium transfer. Pth nulls and Gcm2 nulls each display a hypoparathyroid phenotype, but since the Gcm2 nulls showed upregulation of placental calcium transfer and several genes known to be involved in calcium transport, the authors speculated that PTH is expressed in the placenta and acts locally to regulate placental calcium transfer. Placental expression of PTH mRNA was subsequently confirmed and found to be increased in Gcm2 null relative to Pth null placentas [49]. Within Pth null placentas, the expression was near the limit of detectability and may have represented expression in maternal blood vessels or a false-positive detection. Overall, the placental expression of PTH and the PTH1R, the stimulation of placental calcium transfer and gene expression in response to PTH injection, and the downregulation of placental genes in the absence of PTH, are all consistent with the conclusion that PTH acts locally in the placenta to regulate the transfer of calcium and other solutes (see Fig. 11.12). The low circulating PTH level in the normal fetus, and the much lower circulating level of PTH in the Gcm2 nulls, may be irrelevant to the regulation of placental calcium transfer because PTH is locally expressed in the placenta. PTH’s ability to stimulate placental calcium transfer must be weaker or secondary to that of PTHrP since, in the absence of PTHrP, and despite a threefold elevation in endogenous PTH, placental calcium transfer was reduced in Pthrp null fetuses. The Role of Calcitriol and VDR A possible role for calcitriol in fetaleplacental calcium transport has been implied by the observation that VDRs are present in the placentas of humans, rats, mice and sheep [40,234,244,245,267]. Prior nephrectomy of fetal sheep reduced calcium transfer in the in situ placental perfusion model, and this effect could be partly restored by administering calcitriol [196]. Also, pharmacological doses of calcitriol or 1a-hydroxycholecalciferol increased calcium transfer using in situ

263

placental perfusion models in rats, guinea pigs and sheep [129,268,269]; however, those findings do not necessarily mean that calcitriol is important at its physiologically low levels that are normally present in the fetus. Evidence against a role for calcitriol in regulating placental calcium transfer comes from Vdr null fetal mice, which have a non-significantly increased rate of placental calcium transfer, increased placental expression of TRPV6, and no alteration in other calciotropic hormones [31]. Since absence of VDR does not reduce placental calcium transfer, it is likely that calcitriol is not required to stimulate this process. Further, Vdr null placentas have normal expression of calbindin-D9K [31], as do placentas obtained from rat models of vitamin D deficiency [102,104]. Therefore, although the expression of calbindin-D9K in the adult intestine is critically dependent on sufficiency of vitamin D and calcitriol, the expression of placental calbindin-D9k is independent of vitamin D and calcitriol. Overall, it appears doubtful that calcitriol is an important stimulator of placental calcium transfer. The Role of Calcitonin A role for fetal calcitonin in regulating placental calcium transfer is also unlikely. Pharmacological doses of calcitonin were found to reduce the PTHrP-mediated increases in the apparent rate of calcium transfer in sheep [255,270]. However, in those 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 [141]. More recently, studies in Ctcgrp null fetal mice found that absence of calcitonin and calcitonin gene-related peptide-alpha does not impair placental calcium transfer or net calcium accretion by the skeleton [56].

PLACENTAL TRANSPORT OF MAGNESIUM AND PHOSPHORUS Magnesium and phosphorus are also actively transported across the placenta against a concentration gradient but the regulators of this process are uncertain [6,263]. Mid-molecular fragments of PTHrP stimulated magnesium transport across in situ perfused placentas of fetal lambs [191,263] but neither N-terminal or midmolecular forms of PTHrP stimulated magnesium transfer across in situ perfused rat placentas [271]. Neither PTHrP or PTH stimulated placental transport of phosphorus in sheep and no other candidates are known [84,272].

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FETAL KIDNEYS AND AMNIOTIC FLUID Fetal kidneys partly regulate fetal calcium homeostasis by adjusting the relative reabsorption and excretion of minerals in response to the filtered load and other factors such as PTHrP and/or PTH. Thyroparathyroidectomy in fetal lambs caused increased fractional excretion of calcium and reduced phosphorus excretion [82,273], and these were reversed by treatment with amino-terminal fragments of either PTH or PTHrP [82,273]. Thus, the hypocalcemia of thyroparathyroidectomized fetal lambs may be a consequence not only of reduced placental calcium transfer but also loss of the effects of parathyroid-derived PTH and PTHrP on the renal tubules and skeleton. In contrast to these studies in fetal lambs, absence of parathyroids in Hoxa3 null fetuses, loss of PTH1R in Pthr1 null fetuses, and absence of PTH in Pth null fetuses, were each accompanied by reduced amniotic fluid calcium, an indirect measure of the renal calcium excretion [41,49, unpublished data]. Since blood calcium is low in Hoxa3 null, Pthr1 null, and Pth null fetuses, the renal filtered load of calcium is likely to be low and may explain the low amniotic fluid calcium content. The fetal kidneys synthesize calcitriol which may have relevance for fetal mineral homeostasis. However, given that the levels of calcitriol in the fetus are normally low (except in sheep, as discussed earlier), and that absence of VDR in fetal mice does not impair fetal calcium homeostasis or placental calcium transfer [31], it appears likely that renal production of calcitriol is relatively unimportant for fetal calcium homeostasis in most mammals. Renal calcium handling in fetal life may also be less important as compared to the adult because calcium excreted by the kidneys is made available again to the fetus when amniotic fluid is swallowed [274].

FETAL SKELETON Apart from the vault of the skull and several other bones that form by intramembranous ossification, most of the skeleton is formed by endochondral bone formation [275,276]. Detailed discussion of this process is beyond the scope of this chapter, but it should be noted that PTHrP plays a crucial rule through its local expression within the perichondrium and proliferating chondrocytes, and by its actions on the PTH1R which is expressed within pre-hypertrophic chondrocytes [150,276,277]. PTHrP delays terminal differentiation and hypertrophy of chondrocytes and, in its absence (Pthrp null), hypertrophy occurs prematurely and bone formation begins before the cartilaginous template has reached its intended length [44,45,276,278]. Loss of the

PTH1R (Pthr1 null) leads to a similar phenotype of accelerated endochondral ossification and dysplasia in humans and mice [45,279,280]. Conversely, an activating mutation of the PTH1R [281] or overexpression of PTHrP within the developing skeleton [282] exaggerate the normal actions of PTHrP to delay chondrocyte hypertrophy, resulting in a skeleton that is largely cartilaginous at birth. It is unclear if PTH plays a similar role in endochondral bone development of the fetus. Careful analysis of aparathyroid Hoxa3 null fetuses showed no abnormality in endochondral bone formation, including skeletal lengths, growth plate morphology, trabecular bone volume, and the developmental expression of growth plate genes known to be disrupted by absence of PTHrP or the PTH1R [40,41]. The only abnormality was that the trabecular bone compartment was undermineralized [40,41]. Hoxa3/Pthrp double mutants displayed the Pthrp null chondrodysplasia except that the mouse was globally smaller and the skeleton was now undermineralized [40]. In contrast, Pth null fetuses were initially reported to have shortened tibial metaphyseal lengths, shorter metacarpals and metatarsals, smaller vertebrae, reduced trabecular bone volumes, and fewer osteoclasts and osteoblasts [190]. In addition, there were modest changes in expression of genes involved in chondrocyte maturation and apoptosis, mineralization, and vascular invasion of the growth plate [190]. Overall, the structural changes were largely seen in bone parameters whereas the cartilaginous indices were no different from wild-type siblings. However, when the Pth null was back-crossed from the original C57BL/6 strain to Black Swiss in order that it could be compared to the Hoxa3 null mice, no shortening of the long bones or alteration in trabecular bone volumes or skeletal morphology was observed [49]. Similarly, skeletal morphology was normal in Gcm2 null fetuses which have little or no circulating PTH [49]. However, in these studies of Pth null and Gcm2 null fetuses within the Black Swiss background, gene expression was not examined in the skeletal growth plates. The discrepancy between the shortened skeletal phenotype of the Pth null in a C57BL/6 background and its normal skeletal lengths on the Black Swiss background may be explainable by several factors. First, calcium is a growth factor and nutrient for the skeleton and the ambient level is z0.25 mmol/L higher in fetal and adult mice of the Black Swiss background compared to C57BL/6 [11]. The authors of the original Pth null report later noted that manipulating the calcium content of the diet had a profound effect on the skeletal phenotype of young Pth null mice [283]. Second, the ionized calcium of Pth null fetuses is likely lower in the C57BL/6 background than in the Black Swiss background, although no measurements were done in the

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original background (the blood calcium of Pthrp nulls is 0.12 mmol/L lower in C57BL/6 than in Black Swiss) [11]. Third, the genetic background of the inbred C57BL/6 strain is different from the outbred Black Swiss, and genetic differences are known to affect the phenotype of single gene deletions (the Pthr1 null dies at day 10 of gestation in C57BL/6 but survives to term in Black Swiss) [45]. Thus, it is possible that alterations in serum calcium, or genetic differences in background strain, may explain the inconsistent findings in the Pth null mouse. As in the adult, the fetal skeleton participates in the regulation of calcium homeostasis through resorption which helps maintain the concentration of calcium in the blood. If the available supply of calcium from the maternal circulation is inadequate then calcium may be mobilized from the fetal skeleton, ultimately compromising its strength. For example, maternal hypocalcemia due to parathyroidectomy or calcitonin infusion induces secondary hyperparathyroidism, bone resorption, and reduced mineralization in rat fetuses [77e81]. Several observations from knockout mice also support a role for the fetal skeleton in calcium homeostasis. The ionized calcium is lower in Pthr1 null than Pthrp null fetuses, despite placental calcium transport being increased in Pthr1 nulls and decreased in Pthrp nulls [11]. Lack of bone responsiveness to the aminoterminal portion of PTH and PTHrP may well, therefore, contribute to the hypocalcemia in mice without PTH1R. Placement of a constitutively active PTH1R receptor into the growth plates of Pthrp null fetuses not only reverses the chondrodysplasia [281], but results in a higher fetal blood calcium level [unpublished data]. Car null fetuses have a higher ionized calcium than normal, and this is maintained at least in part through increased PTH-stimulated bone resorption [12]. The skeletal calcium and magnesium content of Car null skeletons is significantly reduced as compared to their siblings, and bone resorption marker deoxypyridinoline is increased [12, unpublished observations]. Gp130 null fetuses have modest hypocalcemia (intermediate between wild-type siblings and the maternal calcium concentration) associated with reduced trabecular bone mass, lower osteoblast activity, and increased osteoclasts, despite having no reduction in the rate of placental calcium transfer [264]. That the parathyroids play an important role in utero in directing skeletal mineralization was first determined by thyroparathyroidectomy in fetal lambs, which caused hypocalcemia, inconstant changes in serum phosphorus, decreased skeletal calcium content, and rachitic changes [284,285]. These skeletal effects were partly reversed or prevented by infusions of calcium and phosphorus sufficient to normalize the calcium
phosphorus product, confirming that it was the

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hypocalcemia that caused the reduced mineralization [284]. More recent study of mice engineered to lack PTH or parathyroids has confirmed the importance of the ambient level of blood calcium in directing skeletal mineralization. Hoxa3 null and Pthr1 null fetuses have the lowest blood calcium and for each the skeletal calcium content is reduced about 25% (adjusted for decreased size of Pthr1 nulls); the magnesium content is reduced about 15% [40]. Pth null and Gcm2 null fetuses have less severe hypocalcemia with the blood calcium reduced to the maternal level, and the skeletal content of calcium and magnesium were each reduced about 15% [49]. Lack of PTH may have reduced bone mineralization by reducing the concentrations of calcium and other minerals presented to the skeletal surface, rather than through direct effects on osteoblasts or other bone cells. PTHrP may not directly regulate skeletal mineralization. In Pthrp null fetuses, the blood calcium was reduced to the maternal level but, unlike Pth null and Gcm2 null fetuses that had the same ionized calcium, skeletal mineral content was normal [40]. However, the Pthrp null skeleton is abnormally mineralized: normally cartilaginous portions of the skeleton (such as ribs and sternum) become mineralized bone, other portions mineralize earlier in gestation than normal [44], and close examination of the skeletal growth plates have shown them to be hypermineralized [190]. It has not been possible to remove the abnormally calcified cartilaginous skeleton before the analysis of skeletal mineral content to determine whether the mineral content of normal bone is reduced, normal, or increased. Although PTHrP may not directly regulate skeletal mineralization, it serves an indirect role by regulating placental calcium transfer and controlling the terminal differentiation in chondrocytes before bone formation can be initiated [276]. In summary, normal mineralization of the fetal skeleton requires intact fetal parathyroid glands and higher levels of calcium in the fetal circulation as compared to the adult. While both PTH and PTHrP may be involved in regulating skeletal mineralization, PTH plays the more direct and critical role in maintaining the appropriate circulating mineral concentrations (Fig. 11.13).

FETAL RESPONSE TO MATERNAL HYPERPARATHYROIDISM In humans, maternal primary hyperparathyroidism has been associated with adverse fetal outcomes in up to 80% of cases [286], including a 30% rate of spontaneous abortion or stillbirth [287,288], a 50% rate of tetany, and a 25% rate of death in the neonatal period [287,289]. These adverse outcomes are thought to result from maternal

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has a higher rate of maternal and fetal complications [297,303,304]. However, whether fetal and neonatal complications are truly reduced by surgical intervention during the second trimester is uncertain because many of those early cases had a form of primary hyperparathyroidism that is uncommon today (symptomatic, severe hypercalcemia, nephrocalcinosis or renal insufficiency), and reporting bias may have led to cases with the most adverse fetal and neonatal outcomes being published. Mild maternal hypercalcemia has been followed without operative intervention or adverse fetal or neonatal outcomes [305,306], and third trimester surgery has been performed successfully [307,308]. Ultimately the management decisions should involve a multidisciplinary team that includes an experienced parathyroid surgeon. Symptomatic and severe disease should preferably be operated upon in the second trimester, whereas mild asymptomatic disease diagnosed in the third trimester may be observed until after delivery.

FETAL RESPONSE TO MATERNAL HYPOPARATHYROIDISM

FIGURE 11.13 Schematic model 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 that will later be broken down and replaced by bone. In the absence of PTHrP (Pthrp null) a severe chondrodysplasia results but the skeleton is normally mineralized. PTH directs the accretion of mineral by the developing bone matrix indirectly by regulating the blood calcium; PTH may also have direct effects on the fetal skeleton. In the absence of PTH (Hoxa3 null, Pth null, and Gcm2 null), the cartilaginous template is normal but the bone that subsequently forms is severely undermineralized.

hypercalcemia which, in turn, causes suppression of the fetal parathyroid glands that can last for several months after birth or be permanent [287,288,290,291]. Maternal hypercalcemia due to familial hypocalciuric hypercalcemia has also suppressed fetal and neonatal parathyroid function [292e295]. This is consistent with animal models in which acute [296] and chronic [12] elevations of the maternal serum calcium suppress the fetal PTH level (see Fig. 11.1). Surgical correction of primary hyperparathyroidism during the second trimester is generally recommended [288,297e299]. Elective surgery at this time point is well tolerated and appears to reduce the rate of adverse events when compared to historical cases [297,298,300,301] or to mothers whose hypercalcemia was medically managed [302]. Third trimester surgery

Maternal hypoparathyroidism in human pregnancy has been associated with the development of intrauterine, fetal hyperparathyroidism. This 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 [119,260,261,309,310]. Spontaneous abortion, stillbirth and neonatal death have also been reported [311e313]. Similar skeletal findings have been reported in fetuses and neonates of women with pseudohypoparathyroidism [314,315], renal tubular acidosis [316], and chronic renal failure [317]. Although these skeletal changes have been interpreted to indicate fetal secondary hyperparathyroidism, no serum measurements of intact PTH (or PTHrP) have been reported for this condition. The serum calcium level has been reported to be normal. These changes in human fetal skeletons differ from what has been found in animal models of maternal hypocalcemia (discussed above), 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 following summary models.

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REFERENCES

Blood Calcium Regulation The fetal blood calcium is set at a level higher than maternal through the actions of PTHrP and PTH acting in concert (among other potential factors). The parathyroid CaR suppresses PTH in response to the high fetal serum calcium, but this low level of PTH remains important for maintaining a normal blood calcium and facilitating normal mineral accretion by the skeleton. Calcitriol synthesis and secretion are suppressed due to low PTH and high blood calcium and phosphorus. PTH and PTHrP are both present in the fetal circulation and act to regulate independently but additively the fetal blood calcium. PTH has the greater effect but neither ligand can make up for 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.

Placental Calcium Transfer

[3] [4] [5]

[6]

[7]

[8]

[9]

[10]

Calcium is actively transferred across the placenta. Both PTHrP and PTH are synthesized within the placenta and may act locally to stimulate placental calcium transfer; whether PTHrP and PTH produced within the parathyroids influences placental function remains unclear. Since placental calcium transfer decreases in the absence of PTHrP, but not in the absence of PTH, it would appear that PTHrP plays a more significant role in regulating this process.

[11]

[12]

[13]

[14]

Skeletal Mineralization PTHrP acts locally within the growth plate to direct endochondral bone development, and outside of bone to affect mineralization by contributing to the regulation of the blood calcium and placental calcium transfer. In contrast, 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 or above, and possibly by direct actions on osteoblasts within the bone matrix. PTH has the more critical role in maintaining skeletal mineral accretion as compared to PTHrP, since mineralization is decreased significantly in the absence of PTH or parathyroids, but not in the absence of PTHrP. The fetal hypercalcemia is needed to achieve full mineralization of the fetal skeleton by term; when the blood calcium is reduced to the maternal level, skeletal mineralization is significantly reduced.

References

[15]

[16]

[17]

[18]

[19]

[20] [21]

[1] Givens MH, Macy IC. The chemical composition of the human fetus. J Biol Chem 1933;102:7e17. [2] David L, Anast CS. Calcium metabolism in newborn infants. The interrelationship of parathyroid function and calcium,

[22]

magnesium, and phosphorus metabolism in normal, sick, and hypocalcemic newborns. J Clin Invest 1974;54:287e96. Schauberger CW, Pitkin RM. Maternal-perinatal calcium relationships. Obstet Gynecol 1979;53:74e6. Schedewie HK, Odell WD, Fisher DA, et al. Parathormone and perinatal calcium homeostasis. Pediatr Res 1979;13:1e6. Pitkin RM, Cruikshank DP, Schauberger CW, Reynolds WA, Williams GA, Hargis GK. Fetal calcitropic hormones and neonatal calcium homeostasis. Pediatrics 1980;66:77e82. Delivoria-Papadopoulos M, Battaglia FC, Bruns PD, Meschia G. Total, protein-bound, and ultrafilterable calcium in maternal and fetal plasmas. Am J Physiol 1967;213:363e6. Fleischman AR, Lerman S, Oakes GK, Epstein MF, Chez RA, Mintz DH. Perinatal primate parathyroid hormone metabolism. Biol Neonate 1975;27:40e9. Pitkin RM, Reynolds WA, Williams GA, Kawahara W, Bauman AF, Hargis GK. Maternal and fetal parathyroid hormone responsiveness in pregnant primates. J Clin Endocrinol Metab 1980;51:1044e7. Garel JM, Barlet JP. Calcium metabolism in newborn animals: the interrelationship of calcium, magnesium, and inorganic phosphorus in newborn rats, foals, lambs, and calves. Pediatr Res 1976;10:749e54. Wadsworth JC, Kronfeld DS, Ramberg Jr CF. Parathyrin and calcium homeostasis in the fetus. Biol Neonate 1982;41:101e9. Kovacs CS, Lanske B, Hunzelman JL, Guo J, Karaplis AC, Kronenberg HM. Parathyroid hormone-related peptide (PTHrP) regulates fetal-placental calcium transport through a receptor distinct from the PTH/PTHrP receptor. Proc Natl Acad Sci USA 1996;93:15233e8. Kovacs CS, Ho-Pao CL, Hunzelman JL, et al. Regulation of murine fetal-placental calcium metabolism by the calciumsensing receptor. J Clin Invest 1998;101:2812e20. Kovacs CS, Kronenberg HM. Maternal-fetal calcium and bone metabolism during pregnancy, puerperium and lactation. Endocr Rev 1997;18:832e72. Math F, Davrainville JL. Postnatal variations of extracellular free calcium levels in the rat. Influence of undernutrition. Experientia 1979;35:1355e6. Moniz CF, Nicolaides KH, Tzannatos C, Rodeck CH. Calcium homeostasis in second trimester fetuses. J Clin Pathol 1986;39:838e41. Delvin EE, Glorieux FH, Salle BL, David L, Varenne JP. Control of vitamin D metabolism in preterm infants: feto-maternal relationships. Arch Dis Child 1982;57:754e7. Caple IW, Heath JA, Care AD, Heaton C, Farrugia W, Wark JD. The regulation of osteoblast activity in fetal lambs by placental calcium transport and the fetal parathyroid glands. In: Jones CT, editor. Fetal and Neonatal Development. Ithaca, NY: Perinatal Press; 1988. p. 90e3. MacIsaac RJ, Heath JA, Rodda CP, et al. Role of the fetal parathyroid glands and parathyroid hormone-related protein in the regulation of placental transport of calcium, magnesium and inorganic phosphate. Reprod Fertil Dev 1991;3:447e57. Thomas ML, Anast CS, Forte LR. Regulation of calcium homeostasis in the fetal and neonatal rat. Am J Physiol 1981;240: E367-E72. Krukowski M, Smith JJ. pH and the level of calcium in the blood of fetal and neonatal albino rats. Biol Neonate 1976;29:148e61. Krukowski M, Smith JJ. Acidosis, hypercalcemia, and hyperphosphatemia in rat fetuses near term and effects of maternal acid/base loading. Proc Soc Exp Biol Med 1979;162:359e64. Kovacs CS. Fetal mineral homeostasis. In: Glorieux FH, Pettifor JM, Ju¨ppner H, editors. Pediatric Bone: Biology and Diseases. San Diego: Academic Press; 2003. p. 271e302.

PEDIATRIC BONE

268

11. FETAL MINERAL HOMEOSTASIS

[23] Lima MS, Kallfelz F, Krook L, Nathanielsz PW. Humeral skeletal development and plasma constituent changes in fetuses of ewes maintained on a low calcium diet from 60 days of gestation. Calcif Tissue Int 1993;52:283e90. [24] Halloran BP, De Luca HF. Effect of vitamin D deficiency on skeletal development during early growth in the rat. Arch Biochem Biophys 1981;209:7e14. [25] Miller SC, Halloran BP, DeLuca HF, Jee WS. Studies on the role of vitamin D in early skeletal development, mineralization, and growth in rats. Calcif Tissue Int 1983;35:455e60. [26] Brommage R, DeLuca HF. Placental transport of calcium and phosphorus is not regulated by vitamin D. Am J Physiol 1984;246:F526eF9. [27] Bourdeau A, Manganella G, Thil-Trubert CL, Sachs C, Cournot G. Bioactive parathyroid hormone in pregnant rats and fetuses. Am J Physiol 1990;258:E549eE54. [28] Ibrahim MM, Thomas ML, Forte LR. Maternal-fetal relationships in the parathyroidectomized rat. Intestinal calcium transport, serum calcium, immunoreactive parathyroid hormone and calcitonin. Biol Neonate 1984;46:89e97. [29] Ho C, Conner DA, Pollak MR, et al. A mouse model of human familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Nat Genet 1995;11:389e94. [30] Li YC, Pirro AE, Amling M, et al. Targeted ablation of the vitamin D receptor: an animal model of vitamin D dependent rickets type II with alopecia. Proc Natl Acad Sci USA 1997;94:9831e5. [31] Kovacs CS, Woodland ML, Fudge NJ, Friel JK. The vitamin D receptor is not required for fetal mineral homeostasis or for the regulation of placental calcium transfer. Am J Physiol Endocrinol Metab 2005;289:E133e44. [32] Burnette JC, Simpson DM, Chandler Jr DC, Bawden JW. Fetal blood calcium response to maternal parathyroid and vitamin D administration in guinea pigs. J Dent Res 1968;47:444e6. [33] Krukowski M, Lehr D. Parathyroid hormone and the placental barrier. Arch Int Pharmacodyn 1963;146:245e65. [34] Garel JM, Pic P, Jost A. Action de la parathormone chez de foetus de rat. Ann Endocrinol 1971;32:253e62. [35] Reynolds WA, Pitkin RM, Wezeman FH. Calcitonin effects in primate pregnancy. Am J Obstet Gynecol 1975;122:212e8. [36] Northrop G, Misenhimer HR, Becker FO. Failure of parathyroid hormone to cross the nonhuman primate placenta. Am J Obstet Gynecol 1977;129:449e53. [37] Chalon S, Garel JM. Plasma calcium control in the rat fetus. III. Influence of alterations in maternal plasma calcium on fetal plasma calcium level. Biol Neonate 1985;48:329e35. [38] Garel JM, Gilbert M, Besnard P. Fetal growth and 1,25-dihydroxyvitamin D3 injections into thyroparathyroidectomized pregnant rats. Reprod Nutr Dev 1981;21:961e8. [39] Gilbert M, Besnard P, Garel JM. Effects of maternal parathyroid glands and vitamin D3 metabolites on fetal growth and fetal liver glycogen stores in thyro-parathyroidectomized pregnant rats. Biomedicine 1980;32:93e9. [40] Kovacs CS, Chafe LL, Fudge NJ, Friel JK, Manley NR. PTH regulates fetal blood calcium and skeletal mineralization independently of PTHrP. Endocrinology 2001;142:4983e93. [41] Kovacs CS, Manley NR, Moseley JM, Martin TJ, Kronenberg HM. Fetal parathyroids are not required to maintain placental calcium transport. J Clin Invest 2001;107:1007e15. [42] Suzuki Y, Kovacs CS, Takanaga H, Peng JB, Landowski CP, Hediger MA. Calcium TRPV6 is involved in murine maternalfetal calcium transport. J Bone Miner Res 2008;23:1249e56. [43] Loughead JL, Mimouni F, Tsang RC. Serum ionized calcium concentrations in normal neonates. Am J Dis Child 1988;142:516e8.

[44] Karaplis AC, Luz A, Glowacki J, et al. Lethal skeletal dysplasia from targeted disruption of the parathyroid hormone-related peptide gene. Genes Dev 1994;8:277e89. [45] Lanske B, Karaplis AC, Lee K, et al. PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth. Science 1996;273:663e6. [46] Reitz RE, Daane TA, Woods JR, Weinstein RL. Calcium, magnesium, phosphorus, and parathyroid hormone interrelationships in pregnancy and newborn infants. Obstet Gynecol 1977;50:701e5. [47] Garel JM, Milhaud G, Jost A. [Hypocalcemic and hypophosphatemic action of thyrocalcitonin in fetal rats]. C R Acad Sci Hebd Seances Acad Sci D 1968;267:344e7. [48] Garel JM, Pic P. Evolution of phosphatemia in the rat fetus during the late stages of gestation. Biol Neonate 1972;21:369e74. [49] Simmonds CS, Karsenty G, Karaplis AC, Kovacs CS. Parathyroid hormone regulates fetal-placental mineral homeostasis. J Bone Miner Res 2010;25:594e605. [50] Lachenmaier-Currle U, Harmeyer J. Placental transport of calcium and phosphorus in pigs. J Perinat Med 1989;17:127e36. [51] Care AD, Caple IW, Singh R, Peddie M. Studies on calcium homeostasis in the fetal Yucatan miniature pig. Lab Anim Sci 1986;36:389e92. [52] Bogden JD, Thind IS, Kemp FW, Caterini H. Plasma concentrations of calcium, chromium, copper, iron, magnesium, and zinc in maternal and cord blood and their relationship to low birth weight. J Lab Clin Med 1978;92:455e62. [53] Mellor DJ, Matheson IC. Variations in the distribution of calcium, magnesium and inorganic phosphorus within chronically catheterized sheep conceptuses during the last eight weeks of pregnancy. Q J Exp Physiol Cogn Med Sci 1977;62:55e63. [54] Barlet JP, Davicco MJ, Moncaup M, Lefaivre J. Foetal plasma magnesium levels during maternal hypo- or hypermagnesaemia in ewes. Br J Nutr 1979;42:559e66. [55] Garel JM, Barlet JP. The effects of calcitonin and parathormone on plasma magnesium levels before and after birth in the rat. J Endocrinol 1974;61:1e13. [56] McDonald KR, Fudge NJ, Woodrow JP, et al. Ablation of calcitonin/calcitonin gene related peptide-a impairs fetal magnesium but not calcium homeostasis. Am J Physiol Endocrinol Metab 2004;287:E218eE26. [57] Saggese G, Baroncelli GI, Bertelloni S, Cipolloni C. Intact parathyroid hormone levels during pregnancy, in healthy term neonates and in hypocalcemic preterm infants. Acta Paediatr Scand 1991;80:36e41. [58] Hillman LS, Slatopolsky E, Haddad JG. Perinatal vitamin D metabolism. IV. Maternal and cord serum 24,25-dihydroxyvitamin D concentrations. J Clin Endocrinol Metab 1978;47:1073e7. [59] Allgrove J, Adami S, Manning RM, O’Riordan JL. Cytochemical bioassay of parathyroid hormone in maternal and cord blood. Arch Dis Child 1985;60:110e5. [60] Thie´baud D, Janisch S, Koelbl H, et al. Direct evidence of a parathyroid related protein gradient between the mother and the newborn in humans. Bone Miner 1993;23:213e21. [61] Khosla S, Johansen KL, Ory SJ, O’Brien PC, Kao PC. Parathyroid hormone-related peptide in lactation and in umbilical cord blood. Mayo Clin Proc 1990;65:1408e14. [62] Dvir R, Golander A, Jaccard N, et al. Amniotic fluid and plasma levels of parathyroid hormone-related protein and hormonal modulation of its secretion by amniotic fluid cells. Eur J Endocrinol 1995;133:277e82. [63] Rubin LP, Posillico JT, Anast CS, Brown EM. Circulating levels of biologically active and immunoreactive intact parathyroid hormone in human newborns. Pediatr Res 1991;29:201e7.

PEDIATRIC BONE

REFERENCES

[64] Seki K, Furuya K, Makimura N, Mitsui C, Hirata J, Nagata I. Cord blood levels of calcium-regulating hormones and osteocalcin in premature infants. J Perinat Med 1994;22:189e94. [65] Fairney A, Jackson D, Clayton BE. Measurement of serum parathyroid hormone, with particular reference to some infants with hypocalcaemia. Arch Dis Child 1973;48:419e24. [66] Seino Y, Ishida M, Yamaoka K, et al. Serum calcium regulating hormones in the perinatal period. Calcif Tissue Int 1982;34:131e5. [67] Papantoniou NE, Papapetrou PD, Antsaklis AJ, Kontoleon PE, Mesogitis SA, Aravantinos D. Circulating levels of immunoreactive parathyroid hormone-related protein and intact parathyroid hormone in human fetuses and newborns. Eur J Endocrinol 1996;134:437e42. [68] Seki K, Wada S, Nagata N, Nagata I. Parathyroid hormonerelated protein during pregnancy and the perinatal period. Gynecol Obstet Invest 1994;37:83e6. [69] Wieland P, Fischer JA, Trechsel U, et al. Perinatal parathyroid hormone, vitamin D metabolites, and calcitonin in man. Am J Physiol 1980;239:E385eE90. [70] Tsang RC, Chen IW, Friedman MA, Chen I. Neonatal parathyroid function: role of gestational age and postnatal age. J Pediatr 1973;83:728e38. [71] Collignon H, Davicco MJ, Barlet JP. Calcitonin mRNA expression and plasma calciotropic hormones in fetal lambs. Domest Anim Endocrinol 1996;13:269e76. [72] Garel JM, Dumont C. Distribution and inactivation of labeled parathyroid hormone in rat fetus. Horm Metab Res 1972;4:217e21. [73] Garel JM. Distribution of labeled parathyroid hormone in rat fetus. Horm Metab Res 1972;4:131e2. [74] Leroyer-Alizon E, David L, Anast CS, Dubois PM. Immunocytological evidence for parathyroid hormone in human fetal parathyroid glands. J Clin Endocrinol Metab 1981;52:513e6. [75] Senior PV, Heath DA, Beck F. Expression of parathyroid hormone-related protein mRNA in the rat before birth: demonstration by hybridization histochemistry. J Mol Endocrinol 1991;6:281e90. [76] MacIsaac RJ, Caple IW, Danks JA, et al. Ontogeny of parathyroid hormone-related protein in the ovine parathyroid gland. Endocrinology 1991;129:757e64. [77] Rebut-Bonneton C, Garel JM, Delbarre F. Parathyroid hormone, calcitonin, 1,25-dihydroxycholecalciferol, and basal bone resorption in the rat fetus. Calcif Tissue Int 1983;35:183e9. [78] Rebut-Bonneton C, Demignon J, Amor B, Miravet L. Effect of calcitonin in pregnant rats on bone resorption in fetuses. J Endocrinol 1983;99:347e53. [79] Sinclair JG. Fetal rat parathyroids as affected by changes in maternal serum calcium and phosphorus through parathyroidectomy and dietary control. J Nutr 1942;23:141e52. [80] Garel JM, Geloso-Meyer A. [Fetal hyperparathyroidism in rats following maternal hypoparathyroidism]. Rev Eur Etud Clin Biol 1971;16:174e8. [81] Chalon S, Garel JM. Plasma calcium control in the rat fetus. I. Influence of maternal hormones. Biol Neonate 1985;48:313e22. [82] MacIsaac RJ, Horne RS, Caple IW, Martin TJ, Wintour EM. Effects of thyroparathyroidectomy, parathyroid hormone, and PTHrP on kidneys of ovine fetuses. Am J Physiol 1993;264:E37e44. [83] Barlet JP, Champredon C, Coxam V, Davicco MJ, Tressol JC. Parathyroid hormone-related peptide might stimulate calcium secretion into the milk of goats. J Endocrinol 1992;132:353e9. [84] Barlet JP, Davicco MJ, Rouffet J, Coxam V, Lefaivre J. Short communication: parathyroid hormone-related peptide does not stimulate phosphate placental transport. Placenta 1994;15:441e4.

269

[85] Fleischman AR, Rosen JF, Cole J, Smith CM, DeLuca HF. Maternal and fetal serum 1,25-dihydroxyvitamin D levels at term. J Pediatr 1980;97:640e2. [86] Hollis BW, Pittard WB. Evaluation of the total fetomaternal vitamin D relationships at term: evidence for racial differences. J Clin Endocrinol Metab 1984;59:652e7. [87] Care AD, Ross R, Pickard DW, et al. Calcium homeostasis in the fetal pig. J Dev Physiol 1982;4:85e106. [88] Noff D, Edelstein S. Vitamin D and its hydroxylated metabolites in the rat. Placental and lacteal transport, subsequent metabolic pathways and tissue distribution. Horm Res 1978;9:292e300. [89] Tanaka Y, Halloran B, Schnoes HK, DeLuca HF. In vitro production of 1,25-dihydroxyvitamin D3 by rat placental tissue. Proc Natl Acad Sci USA 1979;76:5033e5. [90] Weisman Y, Sapir R, Harell A, Edelstein S. Maternal-perinatal interrelationships of vitamin D metabolism in rats. Biochim Biophys Acta 1976;428:388e95. [91] Ross R, Care AD, Robinson JS, Pickard DW, Weatherley AJ. Perinatal 1,25-dihydroxycholecalciferol in the sheep and its role in the maintenance of the transplacental calcium gradient. J Endocrinol 1980;87:17Pe8P. [92] Chalon S, Garel JM. Plasma calcium control in the rat fetus. II. Influence of fetal hormones. Biol Neonate 1985;48:323e8. [93] Hillman LS, Haddad JG. Human perinatal vitamin D metabolism. I. 25-Hydroxyvitamin D in maternal and cord blood. J Pediatr 1974;84:742e9. [94] Haddad Jr JG, Boisseau V, Avioli LV. Placental transfer of vitamin D3 and 25-hydroxycholecalciferol in the rat. J Lab Clin Med 1971;77:908e15. [95] Viljakainen HT, Saarnio E, Hytinantti T, et al. Maternal vitamin D status determines bone variables in the newborn. J Clin Endocrinol Metab 2010;95:1749e57. [96] Horwitz MJ, Tedesco MB, Sereika SM, Hollis BW, GarciaOcana A, Stewart AF. Direct comparison of sustained infusion of human parathyroid hormone-related protein-(1e36). J Clin Endocrinol Metab 2003;88:1603e9. [97] Horwitz MJ, Tedesco MB, Sereika SM, et al. Continuous PTH and PTHrP infusion causes suppression of bone formation and discordant effects on 1,25(OH)2 vitamin D. J Bone Miner Res 2005;20:1792e803. [98] Gardella T, Ju¨ppner H, Bringhurst FR, Potts Jr JT. Receptors for parathyroid hormone (PTH) and PTH-related protein. In: Bilezikian JP, Raisz LG, Martin TJ, editors. Principles of Bone Biology. 3rd ed. New York: Academic Press; 2008. p. 555e76. [99] Wittelsberger A, Rosenblatt M. Parathyroid hormone-receptor interactions. In: Bilezikian JP, Raisz LG, Martin TJ, editors. Principles of Bone Biology. 3rd ed. New York: Academic Press; 2008. p. 595e637. [100] Fudge NJ, Kovacs CS. Pregnancy up-regulates intestinal calcium absorption and skeletal mineralization independently of the vitamin D receptor. Endocrinology 2010;151:886e95. [101] Halloran BP, DeLuca HF. Vitamin D deficiency and reproduction in rats. Science 1979;204:73e4. [102] Glazier JD, Mawer EB, Sibley CP. Calbindin-D9K gene expression in rat chorioallantoic placenta is not regulated by 1,25dihydroxyvitamin D3. Pediatr Res 1995;37:720e5. [103] Verhaeghe J, Thomasset M, Brehier A, Van Assche FA, Bouillon R. 1,25(OH)2D3 and Ca-binding protein in fetal rats: relationship to the maternal vitamin D status. Am J Physiol 1988;254:E505eE12. [104] Marche P, Delorme A, Cuisinier-Gleizes P. Intestinal and placental calcium-binding proteins in vitamin D-deprived or -supplemented rats. Life Sci 1978;23:2555e61.

PEDIATRIC BONE

270

11. FETAL MINERAL HOMEOSTASIS

[105] Kovacs CS. Fetus, Neonate and Infant. In: Feldman D, Pike WJ, Adams JS, editors. Vitamin D. 3rd ed. New York: Academic Press; 2011. [106] Campbell DE, Fleischman AR. Rickets of prematurity: controversies in causation and prevention. Clin Perinatol 1988;15:879e90. [107] Pereira GR, Zucker AH. Nutritional deficiencies in the neonate. Clin Perinatol 1986;13:175e89. [108] Specker BL. Do North American women need supplemental vitamin D during pregnancy or lactation? Am J Clin Nutr 1994;59:484Se90S. [109] Brooke OG, Brown IR, Bone CD, et al. Vitamin D supplements in pregnant Asian women: effects on calcium status and fetal growth. Br Med J 1980;280:751e4. [110] Maxwell JP, Miles LM. Osteomalacia in China. J Obstet Gynaecol Br Empire 1925;32:433e73. [111] Mohapatra A, Sankaranarayanan K, Kadam SS, Binoy S, Kanbur WA, Mondkar JA. Congenital rickets. J Trop Pediatr 2003;49:126e7. [112] Innes AM, Seshia MM, Prasad C, et al. Congenital rickets caused by maternal vitamin D deficiency. Paediatr Child Health 2002;7:455e8. [113] Maiyegun SO, Malek AH, Devarajan LV, Dahniya MH. Severe congenital rickets secondary to maternal hypovitaminosis D: a case report. Ann Trop Paediatr 2002;22:191e5. [114] Begum R, Coutinho ML, Dormandy TL, Yudkin S. Maternal malabsorption presenting as congenital rickets. Lancet 1968;1:1048e52. [115] Park W, Paust H, Kaufmann HJ, Offermann G. Osteomalacia of the mother e rickets of the newborn. Eur J Pediatr 1987;146:292e3. [116] Ford JA, Davidson DC, McIntosh WB, Fyfe WM, Dunnigan MG. Neonatal rickets in Asian immigrant population. Br Med J 1973;3:211e2. [117] Moncrieff M, Fadahunsi TO. Congenital rickets due to maternal vitamin D deficiency. Arch Dis Child 1974;49:810e1. [118] Teotia M, Teotia SP, Nath M. Metabolic studies in congenital vitamin D deficiency rickets. Indian J Pediatr 1995;62:55e61. [119] Sann L, David L, Thomas A, Frederich A, Chapuy MC, Francois R. Congenital hyperparathyroidism and vitamin D deficiency secondary to maternal hypoparathyroidism. Acta Paediatr Scand 1976;65:381e5. [120] Beck-Nielsen SS, Brock-Jacobsen B, Gram J, Brixen K, Jensen TK. Incidence and prevalence of nutritional and hereditary rickets in southern Denmark. Eur J Endocrinol 2009;160(3):491e7. [121] Rauch F. Etiology and treatment of hypocalcemic rickets in children. In: Rose BD, ed. UpToDate 173. Welesley, MA: UpToDate; 2009. [122] Bouillon R, Verstuyf A, Mathieu C, et al. Vitamin D resistance. Best Pract Res Clin Endocrinol Metab 2006;20:627e45. [123] Takeda E, Yamamoto H, Taketani Y, Miyamoto K. Vitamin Ddependent rickets type I and type II. Acta Paediatr Jpn 1997;39:508e13. [124] Kitanaka S, Takeyama K, Murayama A, et al. Inactivating mutations in the 25-hydroxyvitamin D3 1alpha-hydroxylase gene in patients with pseudovitamin D-deficiency rickets. N Engl J Med 1998;338:653e61. [125] Silver J, Landau H, Bab I, et al. Vitamin D-dependent rickets types I and II. Diagnosis and response to therapy. Isr J Med Sci 1985;21:53e6. [126] Teotia M, Teotia SP. Nutritional and metabolic rickets. Indian J Pediatr 1997;64:153e7. [127] Johnson JA, Grande JP, Roche PC, Kumar R. Ontogeny of the 1,25-dihydroxyvitamin D3 receptor in fetal rat bone. J Bone Miner Res 1996;11:56e61.

[128] Kovacs CS. Calcium and bone metabolism during pregnancy and lactation. J Mammary Gland Biol Neoplas 2005;10:105e18. [129] Durand D, Barlet JP, Braithwaite GD. The influence of 1,25dihydroxycholecalciferol on the mineral content of foetal guinea pigs. Reprod Nutr Dev 1983;23:235e44. [130] Moore ES, Langman CB, Favus MJ, Coe FL. Role of fetal 1,25dihydroxyvitamin D production in intrauterine phosphorus and calcium homeostasis. Pediatr Res 1985;19:566e9. [131] Bergwitz C, Juppner H. Regulation of phosphate homeostasis by PTH, vitamin D, and FGF23. Annu Rev Med 2010;61:91e104. [132] Shimada T, Kakitani M, Yamazaki Y, et al. Targeted ablation of Fgf23 demonstrates an essential physiological role of FGF23 in phosphate and vitamin D metabolism. J Clin Invest 2004;113:561e8. [133] Takaiwa M, Aya K, Miyai T, et al. Fibroblast growth factor 23 concentrations in healthy term infants during the early postpartum period. Bone 2010;47:256e62. [134] Leroyer-Alizon E, David L, Dubois PM. Evidence for calcitonin in the thyroid gland of normal and anencephalic human fetuses: immunocytological localization, radioimmunoassay, and gel filtration of thyroid extracts. J Clin Endocrinol Metab 1980;50:316e21. [135] Samaan NA, Anderson GD, Adam-Mayne ME. Immunoreactive calcitonin in the mother, neonate, child and adult. Am J Obstet Gynecol 1975;121:622e5. [136] Kovarik J, Woloszczuk W, Linkesch W, Pavelka R. Calcitonin in pregnancy [letter]. Lancet 1980;1:199e200. [137] Woloszczuk W, Kovarik J, Pavelka P. Calcitonin in pregnant women and in cord blood. Gynecol Obstet Invest 1981;12:272e6. [138] Garel JM, Care AD, Barlet JP. A radioimmunoassay for ovine calcitonin: an evaluation of calcitonin secretion during gestation, lactation and foetal life. J Endocrinol 1974;62:497e509. [139] Garel JM, Milhaud G, Sizonenko P. [Thyrocalcitonin and the placental barrier in rats]. C R Acad Sci Hebd Seances Acad Sci D 1969;269:1785e7. [140] Garel JM, Barlet JP. Calcitonin in the mother, fetus and newborn. Ann Biol Anim Biochim Biophys 1978;18:53e68. [141] Care AD, Caple IW, Abbas SK, Pickard DW. The effect of fetal thyroparathyroidectomy on the transport of calcium across the ovine placenta to the fetus. Placenta 1986;7:417e24. [142] Hoff AO, Catala-Lehnen P, Thomas PM, et al. Increased bone mass is an unexpected phenotype associated with deletion of the calcitonin gene. J Clin Invest 2002;110:1849e57. [143] Allgrove J, Manning RM, Adami S, Chayen J, O’Riordan JL. Biologically active parathyroid hormone in foetal and maternal plasma [abstract]. Clin Sci 1981;60:11P. [144] Abbas SK, Ratcliffe WA, Moniz C, et al. The role of parathyroid hormone-related protein in calcium homeostasis in the fetal pig. Exp Physiol 1994;79:527e36. [145] Care AD. Development of endocrine pathways in the regulation of calcium homeostasis. Baillieres Clin Endocrinol Metab 1989;3:671e88. [146] Orloff JJ, Reddy D, de Papp AE, Yang KH, Soifer NE, Stewart AF. Parathyroid hormone-related protein as a prohormone: posttranslational processing and receptor interactions. Endocr Rev 1994;15:40e60. [147] Philbrick WM, Wysolmerski JJ, Galbraith S, et al. Defining the roles of parathyroid hormone-related protein in normal physiology. Physiol Rev 1996;76:127e73. [148] Wu TL, Vasavada RC, Yang K, et al. Structural and physiologic characterization of the mid-region secretory species of parathyroid hormone-related protein. J Biol Chem 1996;271:24371e81. [149] Ratcliffe WA, Thompson GE, Abbas SK, Care AD. Studies on the metabolic clearance of parathyroid hormone-related protein in pregnant, nonpregnant and fetal animals. In: Cohn DV,

PEDIATRIC BONE

REFERENCES

[150]

[151]

[152]

[153]

[154]

[155]

[156]

[157]

[158]

[159]

[160]

[161]

[162]

[163]

[164]

[165]

Gennari C, Tashjian Jr AH, editors. Calcium Regulating Hormones and Bone Metabolism: Basic and Clinical Aspects. 11th ed. New York: Elsevier Science Publishers; 1992. p. 69e75. Lee K, Deeds JD, Segre GV. Expression of parathyroid hormonerelated peptide and its receptor messenger ribonucleic acids during fetal development of rats. Endocrinology 1995;136:453e63. Karmali R, Schiffmann SN, Vanderwinden JM, et al. Expression of mRNA of parathyroid hormone-related peptide in fetal bones of the rat. Cell Tissue Res 1992;270:597e600. Burton DW, Brandt DW, Deftos LJ. Parathyroid hormonerelated protein in the cardiovascular system. Endocrinology 1994;135:253e61. Abbas SK, Pickard DW, Illingworth D, et al. Measurement of parathyroid hormone-related protein in extracts of fetal parathyroid glands and placental membranes. J Endocrinol 1990;124:319e25. Ferguson JE, Gorman JV, Bruns DE, et al. Abundant expression of parathyroid hormone-related protein in human amnion and its association with labor. Proc Natl Acad Sci USA 1992;89:8384e8. Bowden SJ, Emly JF, Hughes SV, et al. Parathyroid hormonerelated protein in human term placenta and membranes. J Endocrinol 1994;142:217e24. Ferguson JE, Seaner R, Bruns DE, et al. Expression of parathyroid hormone-related protein and its receptor in human umbilical cord: evidence for a paracrine system involving umbilical vessels. Am J Obstet Gynecol 1994;170:1018e24. Wysolmerski JJ, Philbrick WM, Dunbar ME, Lanske B, Kronenberg H, Broadus AE. Rescue of the parathyroid hormone-related protein knockout mouse demonstrates that parathyroid hormone-related protein is essential for mammary gland development. Development 1998;125:1285e94. Rubin LP, Kovacs CS, De Paepe ME, Tsai SW, Torday JS, Kronenberg HM. Arrested pulmonary alveolar cytodifferentiation and defective surfactant synthesis in mice missing the gene for parathyroid hormone-related protein. Dev Dyn 2004;230:278e89. Weatherley AJ, Ross R, Pickard DW, Care AD. The transfer of calcium during perfusion of the placenta and intact and thyroparathyroidectomized sheep. Placenta 1983;4:271e7. Rodda CP, Kubota M, Heath JA, et al. Evidence for a novel parathyroid hormone-related protein in fetal lamb parathyroid glands and sheep placenta: comparisons with a similar protein implicated in humoral hypercalcaemia of malignancy. J Endocrinol 1988;117:261e71. Mueller SO, Korach KS. Estrogen receptors and endocrine diseases: lessons from estrogen receptor knockout mice. Curr Opin Pharmacol 2001;1:613e9. Vidal O, Lindberg MK, Hollberg K, et al. Estrogen receptor specificity in the regulation of skeletal growth and maturation in male mice. Proc Natl Acad Sci USA 2000;97:5474e9. Windahl SH, Andersson G, Gustafsson JA. Elucidation of estrogen receptor function in bone with the use of mouse models. Trends Endocrinol Metab 2002;13:195e200. Lubahn DB, Moyer JS, Golding TS, Couse JF, Korach KS, Smithies O. Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene. Proc Natl Acad Sci USA 1993;90:11162e6. Couse JF, Curtis SW, Washburn TF, et al. Analysis of transcription and estrogen insensitivity in the female mouse after targeted disruption of the estrogen receptor gene. Mol Endocrinol 1995;9:1441e54.

271

[166] Kong YY, Yoshida H, Sarosi I, et al. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature 1999;397:315e23. [167] Li J, Sarosi I, Yan XQ, et al. RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism. Proc Natl Acad Sci USA 2000;97:1566e71. [168] Mizuno A, Amizuka N, Irie K, et al. Severe osteoporosis in mice lacking osteoclastogenesis inhibitory factor/osteoprotegerin. Biochem Biophys Res Commun 1998;247:610e5. [169] Bucay N, Sarosi I, Dunstan CR, et al. Osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev 1998;12:1260e8. [170] Beamer WG, Donahue LR, Rosen CJ, Baylink DJ. Genetic variability in adult bone density among inbred strains of mice. Bone 1996;18:397e403. [171] Thakker RV. Genetic regulation of parathyroid gland development. In: Bilezikian JP, Raisz LG, Martin TJ, editors. Principles of Bone Biology. 3rd ed. New York: Academic Press; 2008. p. 1415e29. [172] Blackburn CC, Manley NR. Developing a new paradigm for thymus organogenesis. Nat Rev Immunol 2004;4:278e89. [173] Phitayakorn R, McHenry CR. Incidence and location of ectopic abnormal parathyroid glands. Am J Surg 2006;191:418e23. [174] Liu Z, Farley A, Chen L, et al. Thymus-associated parathyroid hormone has two cellular origins with distinct endocrine and immunological functions. Submitted 2010. [175] Gilmour JR. The embryology of the parathyroid glands, the thymus and certain associated remnants. J Pathol Bacteriol 1937;45:507e22. [176] Gordon J, Bennett AR, Blackburn CC, Manley NR. Gcm2 and Foxn1 mark early parathyroid- and thymus-specific domains in the developing third pharyngeal pouch. Mech Dev 2001;103:141e3. [177] Liu Z, Yu S, Manley NR. Gcm2 is required for the differentiation and survival of parathyroid precursor cells in the parathyroid/ thymus primordia. Dev Biol 2007;305:333e46. [178] Su D, Ellis S, Napier A, Lee K, Manley NR. Hoxa3 and Pax1 regulate epithelial cell death and proliferation during thymus and parathyroid organogenesis. Dev Biol 2001;236:316e29. [179] Van Dyke JH. Aberrant parathyroid tissue and the thymus: Postnatal development of accessory parathyroid glands in the rat. Anat Rec 1959;134:185e203. [180] Manley NR, Capecchi MR. The role of Hoxa-3 in mouse thymus and thyroid development. Development 1995;121:1989e2003. [181] Gu¨nther T, Chen ZF, Kim J, et al. Genetic ablation of parathyroid glands reveals another source of parathyroid hormone. Nature 2000;406:199e203. [182] Tucci J, Russell A, Senior PV, Fernley R, Ferraro T, Beck F. The expression of parathyroid hormone and parathyroid hormonerelated protein in developing rat parathyroid glands. J Mol Endocrinol 1996;17:149e57. [183] Selvanayagam P, Graves K, Cooper C, Rajaraman S. Expression of the parathyroid hormone-related peptide gene in rat tissues. Lab Invest 1991;64:713e7. [184] Huan J, Olgaard K, Nielsen LB, Lewin E. Parathyroid hormone 7-84 induces hypocalcemia and inhibits the parathyroid hormone 1-84 secretory response to hypocalcemia in rats with intact parathyroid glands. J Am Soc Nephrol 2006;17:1923e30. [185] Docherty HM, Ratcliffe WA, Heath DA, Docherty K. Expression of parathyroid hormone-related protein in abnormal human parathyroids. J Endocrinol 1991;129:431e8. [186] Asa SL, Henderson J, Goltzman D, Drucker DJ. Parathyroid hormone-like peptide in normal and neoplastic human endocrine tissues. J Clin Endocrinol Metab 1990;71:1112e8.

PEDIATRIC BONE

272

11. FETAL MINERAL HOMEOSTASIS

[187] Danks JA, Ebeling PR, Hayman JA, et al. Immunohistochemical localization of parathyroid hormone-related protein in parathyroid adenoma and hyperplasia. J Pathol 1990;161:27e33. [188] Ikeda K, Weir EC, Mangin M, et al. Expression of messenger ribonucleic acids encoding a parathyroid hormone-like peptide in normal human and animal tissues with abnormal expression in human parathyroid adenomas. Mol Endocrinol 1988;2:1230e6. [189] Matsushita H, Hara M, Honda K, et al. Inhibition of parathyroid hormone-related protein release by extracellular calcium in dispersed cells from human parathyroid hyperplasia secondary to chronic renal failure and adenoma. Am J Pathol 1995;146:1521e8. [190] Miao D, He B, Karaplis AC, Goltzman D. Parathyroid hormone is essential for normal fetal bone formation. J Clin Invest 2002;109:1173e82. [191] Barri M, Abbas SK, Pickard DW, et al. Fetal magnesium homeostasis in the sheep. Exp Physiol 1990;75:681e8. [192] Kovacs CS, Chafe LL, Woodland ML, McDonald KR, Fudge NJ, Wookey PJ. Calcitropic gene expression suggests a role for intraplacental yolk sac in maternal-fetal calcium exchange. Am J Physiol Endocrinol Metab 2002;282. E721-E32. [193] Chattopadhyay N, Baum N, Bai M, et al. Ontogeny of the extracellular calcium-sensing receptor in rat kidney. Am J Physiol 1996;271:F736eF43. [194] Widdowson EM, Dickerson JW. Chemical composition of the body. In: Comar CL, Bronner F, editors. Mineral Metabolism: An Advanced Treatise, V. II, The Elements, Part A. New York: Academic Press; 1964. p. 1e247. [195] Comar CL. Radiocalcium studies in pregnancy. Ann NY Acad Sci 1956;64:281e98. [196] Care AD. The placental transfer of calcium. J Dev Physiol 1991;15:253e7. [197] Garel JM. Hormonal control of calcium metabolism during the reproductive cycle in mammals. Physiol Rev 1987;67:1e66. [198] Fisher GJ, Kelley LK, Smith CH. ATP-dependent calcium transport across basal plasma membranes of human placental trophoblast. Am J Physiol 1987;252:C38e46. [199] Borke JL, Caride A, Verma AK, et al. Calcium pump epitopes in placental trophoblast basal plasma membranes. Am J Physiol 1989;257:C341eC6. [200] Stulc J, Stulcova B, Smid M, Sach I. Parallel mechanisms of Caþþ transfer across the perfused human placental cotyledon. Am J Obstet Gynecol 1994;170:162e7. [201] Symonds HW, Sansom BF, Twardock AR. The measurement of the transfer of calcium and phosphorus from foetus to dam in the sheep using a whole body counter. Res Vet Sci 1972;13:272e5. [202] Ramberg Jr CF, Delivoria-Papadopoulos M, Crandall ED, Kronfeld DS. Kinetic analysis of calcium transport across the placenta. J Appl Physiol 1973;35:682e8. [203] Johnson JA, Kumar R. Renal and intestinal calcium transport: roles of vitamin D and vitamin D-dependent calcium binding proteins. Semin Nephrol 1994;14:119e28. [204] Shamley DR, Opperman LA, Buffenstein R, Ross FP. Ontogeny of calbindin-D28K and calbindin-D9K in the mouse kidney, duodenum, cerebellum and placenta. Development 1992;116:491e6. [205] Glazier JD, Atkinson DE, Thornburg KL, et al. Gestational changes in Ca2þ transport across rat placenta and mRNA for calbindin9K and Ca2þ-ATPase. Am J Physiol 1992;263:R930eR5. [206] Bruns ME, Fausto A, Avioli LV. Placental calcium binding protein in rats. Apparent identity with vitamin D-dependent calcium binding protein from rat intestine. J Biol Chem 1978;253:3186e90.

[207] Delorme AC, Marche P, Garel JM. Vitamin D-dependent calcium-binding protein. Changes during gestation, prenatal and postnatal development in rats. J Dev Physiol 1979;1:181e94. [208] Delorme AC, Danan JL, Ripoche MA, Mathieu H. Biochemical characterization of mouse vitamin D-dependent calciumbinding protein. Evidence for its presence in embryonic life. Biochem J 1982;205:49e57. [209] Tuan RS, Bigioni N. Ca2þ-activated ATPase of the mouse chorioallantoic placenta: developmental expression, characterization and cytohistochemical localization. Development 1990;110:505e13. [210] Kutuzova GD, Akhter S, Christakos S, Vanhooke J, KimmelJehan C, Deluca HF. Calbindin D(9k) knockout mice are indistinguishable from wild-type mice in phenotype and serum calcium level. Proc Natl Acad Sci USA 2006;103:12377e81. [211] Benn BS, Ajibade D, Porta A, et al. Active intestinal calcium transport in the absence of transient receptor potential vanilloid type 6 and calbindin-D9k. Endocrinology 2008;149:3196e205. [212] Lee GS, Lee KY, Choi KC, et al. Phenotype of a calbindin-D9k gene knockout is compensated for by the induction of other calcium transporter genes in a mouse model. J Bone Miner Res 2007;22:1968e78. [213] Prasad V, Okunade G, Liu L, Paul RJ, Shull GE. Distinct phenotypes among plasma membrane Ca2þ-ATPase knockout mice. Ann NY Acad Sci 2007;1099:276e86. [214] Okunade GW, Miller ML, Pyne GJ, et al. Targeted ablation of plasma membrane Ca2þ-ATPase (PMCA) 1 and 4 indicates a major housekeeping function for PMCA1 and a critical role in hyperactivated sperm motility and male fertility for PMCA4. J Biol Chem 2004;279:33742e50. [215] Moreau R, Daoud G, Masse A, Simoneau L, Lafond J. Expression and role of calcium-ATPase pump and sodium-calcium exchanger in differentiated trophoblasts from human term placenta. Mol Reprod Dev 2003;65:283e8. [216] Strid H, Powell TL. ATP-dependent Ca2þ transport is upregulated during third trimester in human syncytiotrophoblast basal membranes. Pediatr Res 2000;48:58e63. [217] Rurak DW. Development and function of the placenta. In: Harding R, Bocking AD, editors. Fetal Growth and Development. Cambridge, UK: Cambridge University Press; 2001. p. 17e43. [218] Steven DH, editor. Anatomy of the placental barrier. In: Comparative Placentation: Essays in Structure and Function. London, UK: Academic Press; 1975. p. 25e57. [219] Kirby DR, Bardbury S. The hemo-chorial mouse placenta. Anat Rec 1965;152:279e82. [220] Enders AC. A comparative study of the fine structures of the trophoblast in several hemochorial placentas. Am J Anat 1965;116:29e68. [221] Ramsey EM. The Placenta: Human and Animal. New York: Praeger; 1982. [222] Robinson NR, Atkinson DE, Jones CJ, Sibley CP. Permeability of the near-term rat placenta to hydrophilic solutes. Placenta 1988;9:361e72. [223] Wooding FBP, Morgan G, Jones GV, Care AD. Calcium transport and the localisation of calbindin-D9k in the ruminant placenta during the second half of pregnancy. Cell Tissue Res 1996;285:477e89. [224] Morgan G, Wooding FB, Care AD, Jones GV. Genetic regulation of placental function: a quantitative in situ hybridization study of calcium binding protein (calbindin-D9k) and calcium ATPase mRNAs in sheep placenta. Placenta 1997;18:211e8. [225] Nikitenko L, Morgan G, Kolesnikov SI, Wooding FB. Immunocytochemical and In situ hybridization studies of the

PEDIATRIC BONE

REFERENCES

[226]

[227]

[228]

[229]

[230]

[231] [232]

[233]

[234]

[235]

[236]

[237]

[238]

[239]

[240]

[241]

[242]

distribution of calbindin D9k in the bovine placenta throughout pregnancy. J Histochem Cytochem 1998;46:679e88. Jones GV, Taylor KR, Morgan G, Wooding FB, Care AD. Aspects of calcium transport by the ovine placenta: studies based on the interplacentomal region of the chorion. Placenta 1997;18:357e64. Malassine A, Frendo JL, Evain-Brion D. A comparison of placental development and endocrine functions between the human and mouse model. Hum Reprod Update 2003;9:531e9. Cross JC, Rossant J. Development of the embryo. In: Harding R, Bocking AD, editors. Fetal Growth and Development. Cambridge, UK: Cambridge University Press; 2001. p. 1e16. Duval M. Le placenta des rongeurs. Trosie`me partie. Le placenta de la souris et du rat. J Anat Physiol Normales Patholog Homme Anim 1891;27:24e73:344e95, 515e612. Duval M. Le placenta des ronguers. Quatrie`me partie. Le placenta du cochon d’Inde. J Anat Physiol Normales Patholog Homme Anim 1892;28:58e98:333e453. Jollie WP. Development, morphology, and function of the yolksac placenta of laboratory rodents. Teratology 1990;41:361e81. Bruns ME, Kleeman E, Mills SE, Bruns DE, Herr JC. Immunochemical localization of vitamin D-dependent calcium-binding protein in mouse placenta and yolk sac. Anat Rec 1985;213:514e7:32e5. Mathieu CL, Burnett SH, Mills SE, Overpeck JG, Bruns DE, Bruns ME. Gestational changes in calbindin-D9k in rat uterus, yolk sac, and placenta: implications for maternal-fetal calcium transport and uterine muscle function. Proc Natl Acad Sci USA 1989;86:3433e7. Shamley DR, Veale G, Pettifor JM, Buffenstein R. Trophoblastic giant cells of the mouse placenta contain calbindin-D9K but not the vitamin D receptor. J Endocrinol 1996;150:25e32. Ogura Y, Takakura N, Yoshida H, Nishikawa SI. Essential role of platelet-derived growth factor receptor alpha in the development of the intraplacental yolk sac/sinus of Duval in mouse placenta. Biol Reprod 1998;58:65e72. Bruns ME, Wallshein V, Bruns DE. Regulation of calciumbinding protein in mouse placenta and intestine. Am J Physiol 1982;242:E47e52. Dunne FP, Ratcliffe WA, Mansour P, Heath DA. Parathyroid hormone related protein (PTHrP) gene expression in fetal and extra-embryonic tissues of early pregnancy. Hum Reprod 1994;9:149e56. Bruns ME, Ferguson II JE, Bruns DE, et al. Expression of parathyroid hormone-related peptide and its receptor messenger ribonucleic acid in human amnion and chorion-decidua: implications for secretion and function. Am J Obstet Gynecol 1995;173:739e46. Beck F, Tucci J, Senior PV. Expression of parathyroid hormonerelated protein mRNA by uterine tissues and extraembryonic membranes during gestation in rats. J Reprod Fertil 1993;99:343e52. Ferguson II JE, Seaner RM, Bruns DE, Iezzoni JC, Bruns ME. Expression and specific immunolocalization of the human parathyroid hormone/parathyroid hormone-related protein receptor in the uteroplacental unit. Am J Obstet Gynecol 1998;179:321e9. van de Stolpe A, Karperien M, Lowik CW, et al. Parathyroid hormone-related peptide as an endogenous inducer of parietal endoderm differentiation. J Cell Biol 1993;120:235e43. Karperien M, van Dijk TB, Hoeijmakers T, et al. Expression pattern of parathyroid hormone/parathyroid hormone related peptide receptor mRNA in mouse postimplantation embryos indicates involvement in multiple developmental processes. Mech Dev 1994;47:29e42.

273

[243] Bradbury RA, Sunn KL, Crossley M, et al. Expression of the parathyroid Ca(2þ)-sensing receptor in cytotrophoblasts from human term placenta. J Endocrinol 1998;156:425e30. [244] Tanamura A, Nomura S, Kurauchi O, Furui T, Mizutani S, Tomoda Y. Purification and characterization of 1,25(OH)2D3 receptor from human placenta. J Obstet Gynaecol 1995;21:631e9. [245] Stumpf WE, Sar M, Narbaitz R, Huang S, DeLuca HF. Autoradiographic localization of 1,25-dihydroxyvitamin D3 in rat placenta and yolk sac. Horm Res 1983;18:215e20. [246] Jousset V, Legendre B, Besnard P, Segond N, Jullienne A, Garel JM. Calcitonin-like immunoreactivity and calcitonin gene expression in the placenta and in the mammary gland of the rat. Acta Endocrinol (Copenh) 1988;119:443e51. [247] Balabanova S, Kruse B, Wolf AS. Calcitonin secretion by human placental tissue. Acta Obstet Gynecol Scand 1987;66:323e6. [248] Nicholson GC, D’Santos CS, Evans T, et al. Human placental calcitonin receptors. Biochem J 1988;250:877e82. [249] Lafond J, Simoneau L, Savard R, Lajeunesse D. Calcitonin receptor in human placental syncytiotrophoblast brush border and basal plasma membranes. Mol Cell Endocrinol 1994;99:285e92. [250] Weatherley AJ, Ross R, Pickard DW, Care AD. The transfer of calcium during perfusion of the placenta and intact and thyroparathyroidectomized sheep. Placenta 1983;4:271e7. [251] Robinson NR, Sibley CP, Mughal MZ, Boyd RD. Fetal control of calcium transport across the rat placenta. Pediatr Res 1989;26:109e15. [252] Stulc J, Stulcova B. Transport of calcium by the placenta of the rat. J Physiol (Lond) 1986;371:1e16. [253] Bond H, Baker B, Boyd RD, et al. Artificial perfusion of the fetal circulation of the in situ mouse placenta: methodology and validation. Placenta 2006;27(Suppl. A):S69e75. [254] Bond H, Dilworth MR, Baker B, et al. Increased maternofetal calcium flux in parathyroid hormone-related protein-null mice. J Physiol 2008;586:2015e25. [255] Barlet JP. Calcitonin may modulate placental transfer of calcium in ewes. J Endocrinol 1985;104:17e21. [256] Kovacs CS. Fetal mineral homeostasis and skeletal mineralization. In: Massaro EJ, Rogers JM, editors. The Skeleton: Biochemical, Genetic, and Molecular Interactions in Development and Homeostasis. Totowa, NJ: Humana Press; 2004. p. 293e306. [257] Care AD, Dutton A, Mott JC, Robinson JS, Ross R. Studies of the transplacental calcium gradient in sheep [abstract]. J Physiol (Lond) 1979;290:19Pe20P. [258] Durand D, Barlet JP. Influence de la calcitonine maternelle sur la calce´mie foetale chez la brebis. Ann Endocrinol 1981;42:8C. [259] Barlet JP. Prolactin and calcium metabolism in pregnant ewes. J Endocrinol 1985;107:171e5. [260] Stuart C, Aceto Jr T, Kuhn JP, Terplan K. Intrauterine hyperparathyroidism. Postmortem findings in two cases. Am J Dis Child 1979;133:67e70. [261] Loughead JL, Mughal Z, Mimouni F, Tsang RC, Oestreich AE. Spectrum and natural history of congenital hyperparathyroidism secondary to maternal hypocalcemia. Am J Perinatol 1990;7:350e5. [262] Abbas SK, Pickard DW, Rodda CP, et al. Stimulation of ovine placental calcium transport by purified natural and recombinant parathyroid hormone-related protein (PTHrP) preparations. Q J Exp Physiol 1989;74:549e52. [263] Care AD, Abbas SK, Pickard DW, et al. Stimulation of ovine placental transport of calcium and magnesium by mid-molecule fragments of human parathyroid hormone-related protein. Exp Physiol 1990;75:605e8.

PEDIATRIC BONE

274

11. FETAL MINERAL HOMEOSTASIS

[264] Shin HI, Divieti P, Sims NA, et al. Gp130-mediated signaling is necessary for normal osteoblastic function in vivo and in vitro. Endocrinology 2004;145:1376e85. [265] Riddle RC, Macica CM, Clemens TL. Vascular, cardiovascular, and neurologic actions of parathyroid-related protein. In: Bilezikian JP, Raisz LG, Martin TJ, editors. Principles of Bone Biology. 3rd ed. New York: Academic Press; 2008. p. 733e48. [266] Farrugia W, de Gooyer T, Rice GE, Moseley JM, Wlodek ME. Parathyroid hormone(1e34) and parathyroid hormone-related protein(1e34) stimulate calcium release from human syncytiotrophoblast basal membranes via a common receptor. J Endocrinol 2000;166:689e95. [267] Ross R, Florer J, Halbert K, McIntyre L. Characterization of 1,25dihydroxyvitamin D3 receptors and in vivo targeting of [3H]1,25(OH)2D3 in the sheep placenta. Placenta 1989;10:553e67. [268] Chalon S, Garel JM. 1,25-Dihydroxyvitamin D3 injections into rat fetuses: effects on fetal plasma calcium, plasma phosphate and mineral content. Reprod Nutr Dev 1983;23:567e73. [269] Durand D, Braithwaite GD, Barlet JP. The effect of 1ahydroxycholecalciferol on the placental transfer of calcium and phosphate in sheep. Br J Nutr 1983;49:475e80. [270] Barlet JP, Davicco MJ, Coxam V. Calcitonin modulates parathyroid hormone related peptide-stimulated calcium placental transfer. In: Cohn DV, Gennari C, Tashjian AHJ, editors. Calcium Regulating Hormones and Bone Metabolism. Amsterdam: Elsevier; 1992. p. 124e8. [271] Shaw AJ, Mughal MZ, Maresh MJ, Sibley CP. Effects of two synthetic parathyroid hormone-related protein fragments on maternofetal transfer of calcium and magnesium and release of cyclic AMP by the in-situ perfused rat placenta. J Endocrinol 1991;129:399e404. [272] Stulc J, Stulcova B. Placental transfer of phosphate in anaesthetized rats. Placenta 1996;17:487e93. [273] Davicco MJ, Coxam V, Lefaivre J, Barlet JP. Parathyroid hormone-related peptide increases urinary phosphate excretion in fetal lambs. Exp Physiol 1992;77:377e83. [274] Brace RA. Amniotic fluid dynamics. In: Creasy RK, Resnik R, editors. MaternaleFetal Medicine: Principles and Practice. Philadelphia: W.B. Saunders; 1994. p. 106e14. [275] Yang Y. Skeletal morphogenesis during embryonic development. Crit Rev Eukaryot Gene Expr 2009;19:197e218. [276] Karsenty G, Kronenberg HM, Settembre C. Genetic control of bone formation. Annu Rev Cell Dev Biol 2009;25:629e48. [277] Lee K, Deeds JD, Bond AT, Ju¨ppner H, Abou-Samra AB, Segre GV. In situ localization of PTH/PTHrP receptor mRNA in the bone of fetal and young rats. Bone 1993;14:341e5. [278] Vortkamp A, Lee K, Lanske B, Segre GV, Kronenberg HM, Tabin CJ. Indian hedgehog and parathyroid hormone-related protein regulate the rate of cartilage differentiation. Science 1996;273:613e22. [279] Karaplis AC, He B, Nguyen MT, et al. Inactivating mutation in the human parathyroid hormone receptor type 1 gene in Blomstrand chondrodysplasia. Endocrinology 1998;139:5255e8. [280] Oostra RJ, van der Harten JJ, Rijnders WP, Scott RJ, Young MP, Trump D. Blomstrand osteochondrodysplasia: three novel cases and histological evidence for heterogeneity. Virchows Arch 2000;436:28e35. [281] Schipani E, Lanske B, Hunzelman J, et al. Targeted expression of constitutively active PTH/PTHrP receptors delays endochondral bone formation and rescues PTHrP-less mice. Proc Natl Acad Sci USA 1997;94(25):13689e94. [282] Weir EC, Philbrick WM, Amling M, Neff LA, Baron R, Broadus AE. Targeted overexpression of parathyroid hormonerelated peptide in chondrocytes causes chondrodysplasia and

[283]

[284]

[285]

[286]

[287] [288]

[289] [290]

[291]

[292]

[293]

[294]

[295]

[296]

[297]

[298] [299]

[300] [301] [302] [303] [304]

delayed endochondral bone formation. Proc Natl Acad Sci USA 1996;93:10240e5. Miao D, He B, Lanske B, et al. Skeletal abnormalities in Pth-null mice are influenced by dietary calcium. Endocrinology 2004;145:2046e53. Aaron JE, Abbas SK, Colwell A, et al. Parathyroid gland hormones in the skeletal development of the ovine foetus: the effect of parathyroidectomy with calcium and phosphate infusion. Bone Miner 1992;16:121e9. Aaron JE, Makins NB, Caple IW, Abbas SK, Pickard DW, Care AD. The parathyroid glands in the skeletal development of the ovine foetus. Bone Miner 1989;7:13e22. Schnatz PF, Curry SL. Primary hyperparathyroidism in pregnancy: evidence-based management. Obstet Gynecol Surv 2002;57:365e76. Ludwig GD. Hyperparathyroidism in relation to pregnancy. N Engl J Med 1962;267:637e42. Shangold MM, Dor N, Welt SI, Fleischman AR, Crenshaw Jr MC. Hyperparathyroidism and pregnancy: a review. Obstet Gynecol Surv 1982;37:217e28. Wagner G, Transhol L, Melchior JC. Hyperparathyroidism and pregnancy. Acta Endocrinol (Copenh) 1964;47:549e64. Bruce J, Strong JA. Maternal hyperparathyroidism and parathyroid deficiency in the child, with account of effect of parathyroidectomy on renal function, and of attempt to transplant part of tumor. Q J Med 1955;24:307e19. Better OS, Levi J, Grief E, Tuma S, Gellei B, Erlik D. Prolonged neonatal parathyroid suppression. A sequel to asymptomatic maternal hyperparathyroidism. Arch Surg 1973;106:722e4. Thomas BR, Bennett JD. Symptomatic hypocalcemia and hypoparathyroidism in two infants of mothers with hyperparathyroidism and familial benign hypercalcemia. J Perinatol 1995;15:23e6. Marx SJ, Attie MF, Levine MA, Spiegel AM, Downs Jr RW, Lasker RD. The hypocalciuric or benign variant of familial hypercalcemia: clinical and biochemical features in fifteen kindreds. Medicine (Baltimore) 1981;60:397e412. Powell BR, Buist NR. Late presenting, prolonged hypocalcemia in an infant of a woman with hypocalciuric hypercalcemia. Clin Pediatr (Phila) 1990;29:241e3. Page LA, Haddow JE. Self-limited neonatal hyperparathyroidism in familial hypocalciuric hypercalcemia. J Pediatr 1987;111:261e4. Kaplan EL, Burrington JD, Klementschitsch P, Taylor J, Deftos L. Primary hyperparathyroidism, pregnancy, and neonatal hypocalcemia. Surgery 1984;96:717e22. Delmonico FL, Neer RM, Cosimi AB, Barnes AB, Russell PS. Hyperparathyroidism during pregnancy. Am J Surg 1976;131:328e37. Johnstone RE, Kreindler T. Hyperparathyroidism during pregnancy. Obstet Gynecol 1972;40:580e5. Wilson DT, Martin T, Christensen R, Yee AH, Reynolds C. Hyperparathyroidism in pregnancy: case report and review of the literature. Can Med Assoc J 1983;129:986e9. Rubin A, Chaykin L, Ludwig GD. Maternal hyperparathyroidism and pregnancy. J Am Med Assoc 1968;206:128e30. Higgins RV, Hisley JC. Primary hyperparathyroidism in pregnancy. A report of two cases. J Reprod Med 1988;33:726e30. Kelly TR. Primary hyperparathyroidism during pregnancy. Surgery 1991;110:1028e33. discussion 33e4. Kristoffersson A, Dahlgren S, Lithner F, Jarhult J. Primary hyperparathyroidism in pregnancy. Surgery 1985;97:326e30. Carella MJ, Gossain VV. Hyperparathyroidism and pregnancy: case report and review. J Gen Intern Med 1992;7:448e53.

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APPENDIX

[305] Lueg MC, Dawkins WE. Primary hyperparathyroidism and pregnancy. South Med J 1983;76:1389e92. [306] Lowe DK, Orwoll ES, McClung MR, Cawthon ML, Peterson CG. Hyperparathyroidism and pregnancy. Am J Surg 1983;145:611e4. [307] Schnatz PF. Surgical treatment of primary hyperparathyroidism during the third trimester. Obstet Gynecol 2002;99:961e3. [308] Haenel LC, Mayfield RK. Primary hyperparathyroidism in a twin pregnancy and review of fetal/maternal calcium homeostasis. Am J Med Sci 2000;319:191e4. [309] Bronsky D, Kiamko RT, Moncada R, Rosenthal IM. Intra-uterine hyperparathyroidism secondary to maternal hypoparathyroidism. Pediatrics 1968;42:606e13. [310] Aceto Jr T, Batt RE, Bruck E, Schultz RB, Perz YR. Intrauterine hyperparathyroidism: a complication of untreated maternal hypoparathyroidism. J Clin Endocrinol Metab 1966;26:487e92. [311] Eastell R, Edmonds CJ, de Chayal RC, McFadyen IR. Prolonged hypoparathyroidism presenting eventually as second trimester abortion. Br Med J (Clin Res Ed) 1985;291:955e6. [312] Anderson GW, Musselman L. The treatment of tetany in pregnancy. Am J Obstet Gynecol 1942;43:547e67. [313] Kehrer E. Die geburtschilflich-gyna¨kologische bedeutung der tetanie. Arch Gynaek 1913;99:372e447. [314] Vidailhet M, Monin P, Andre M, Suty Y, Marchal C, Vert P. [Neonatal hyperparathyroidism secondary to maternal hypoparathyroidism]. Arch Fr Pediatr 1980;37:305e12. [315] Glass EJ, Barr DG. Transient neonatal hyperparathyroidism secondary to maternal pseudohypoparathyroidism. Arch Dis Child 1981;56:565e8. [316] Savani RC, Mimouni F, Tsang RC. Maternal and neonatal hyperparathyroidism as a consequence of maternal renal tubular acidosis. Pediatrics 1993;91:661e3. [317] Levin TL, States L, Greig A, Goldman HS. Maternal renal insufficiency: a cause of congenital rickets and secondary hyperparathyroidism. Pediatr Radiol 1992;22:315e6. [318] Gu¨nther T, Chen ZF, Kim J, et al. Genetic ablation of parathyroid glands reveals another source of parathyroid hormone. Nature 2000;406:199e203.

APPENDIX Hoxa3 null [40,41,180] Deletion of Hoxa3 gene which results in abnormalities in tissues deriving from the third and fourth pharyngeal arches, including absence of parathyroids and thymus. Circulating PTH is absent while plasma PTHrP is normal. Lethal at birth.

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Gcm2 null [49,174,318] Deletion of gene Gcm2 which results in absence of parathyroids but detectable PTH in the circulation; plasma PTHrP is normal. The thymus has been deduced to contain hyperplastic parathyroid tissue which produces near-normal circulating PTH levels; however, the Gcm2 null thymus has not been examined to confirm this, and studies in Gcm2 null fetuses indicate that no parathyroid tissue is present in the thymus. Not lethal although many hypocalcemic deaths occur in the postnatal weeks. Pth null [190] Deletion of gene encoding PTH which results in hyperplastic parathyroids but undetectable circulating PTH and normal plasma PTHrP. Not lethal although many hypocalcemic deaths occur in the postnatal weeks. Pthrp null [11,44] Deletion of gene encoding PTHrP, resulting in severe chondrodysplasia, abnormally calcified ribs, surfactant deficiency, and other abnormalities. PTH upregulates threefold. Lethal at birth. Pthr1 null [11,45] Deletion of gene encoding PTH/ PTHrP receptor (also known as PTH1 receptor), resulting in absence of biological effects of amino-terminal PTH and PTHrP (and high levels of both peptides). Lethal between mid-gestation and after birth, depending upon the genetic background. Car null [12,29] Deletion of the gene encoding the calcium-sensing receptor which results in fetal hyperparathyroidism with reduced PTHrP. Lethal 2e3 weeks postnatal; heterozygotes are hypercalcemic but live normal life spans. Ctcgrp null [56,142] Deletion of the gene encoding calcitonin and calcitonin gene-related peptide-alpha. Non-lethal and normal at birth; adults are fertile and live normal life spans. Vdr null [30,31] Deletion of the gene encoding the vitamin D receptor results in a murine form of vitamin D dependent rickets type II. Non-lethal and normal at birth, but results in significant hypocalcemia, secondary hyperparathyroidism, osteomalacia and reduced fertility in adults.

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12

Radiology Judith E. Adams 1, Zulf Mughal 2, John Damilakis 3, Amaka C. Offiah 4 1

Consultant Radiologist, Department of Clinical Radiology, Central Manchester University Hospitals NHS Foundation Trust, The Royal Infirmary, Manchester, UK, 2 Consultant in Paediatric Bone Disorders & Honorary Senior Lecturer in Child Health, Royal Manchester Children’s Hospital, Central Manchester University Hospitals NHS Foundation Trust, Manchester, UK, 3 Associate Professor, University of Crete, Faculty of Medicine, Department of Medical Physics, Iraklion, Crete, Greece, 4 HEFCE Clinical Senior Lecturer, Academic Unit of Child Health, Sheffield Children’s NHS Foundation Trust, Sheffield, UK

IMAGING TECHNIQUES, INTERPRETATION, AND STRATEGIES

• differentiating between benign and malignant lesions where possible • monitoring the effect of treatment and disease.

Introduction Imaging plays an important role in the identification and management of bone and soft-tissue diseases in children. Of all the imaging techniques, radiography has been established for the longest period, since x-rays were discovered by Roentgen in December 1895. Definitive diagnosis of many bone and joint disorders in children, such as fractures, dysplasias and metabolic bone diseases, require no additional imaging [1]. However, the past 40 years have brought an expansion in imaging techniques, which now include radionuclide scanning (RNS), computed tomography (CT), ultrasonography (US) and magnetic resonance imaging (MRI). CT, US and subsequently MRI have greatly improved the identification and characterization of soft-tissue lesions, which might not be visible on radiographs [2]. Some of these techniques may be expensive and may not be widely available in some countries (e.g. MRI) or may involve a considerable dose of ionizing radiation to the patient (e.g. CT). All require skill and experience in their execution and interpretation, particularly in children, and for resources and techniques to be used effectively there must be close collaboration and discussion between clinician and radiologist [3]. The role of imaging includes: • confirming that a skeletal lesion is present • defining whether a lesion is single or multiple • defining the features and extent of a lesion so that a diagnosis may be made

Pediatric Bone, Second Edition DOI: 10.1016/B978-0-12-382040-2.10012-7

Imaging Methods Radiography Radiographs are probably still the most widely used imaging technique in musculoskeletal imaging in pediatric practice, despite the expansion in modern imaging methods over past decades [1]. In radiography, x-rays are passed through the body; some are absorbed, the amount depending on the atomic number and thickness of the tissue through which the x-rays pass. A differential pattern of x-rays reaches the fluorescent screens of the cassette, in which is held the radiographic film. The fluorescence emitted by the screens is dependent on the incident x-rays and this fluorescence alters the silver crystals in the radiographic film. The film is then processed to obtain the radiographic image. Most xrays are absorbed in the tissues containing high atomic number material (bone and calcium) and these structures appear white (radiodense) on the radiograph. In tissues where there is little absorption (air and fat) the radiograph appears black (radiolucent) (Fig. 12.1). In soft-tissue structures (muscle) where the absorption of x-rays varies between these two high and low extremes, the radiographic images will show intermediate intensities of gray. The use of cassettes was an important development because it reduced the radiation dose to the patient from that delivered originally in the past, when x-rays directly altered the silver crystals of radiographic plates. Radiographs demonstrate lesions of bones well,

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(A)

(B)

FIGURE 12.1

Radiographs: in an adolescent of the (A) normal pelvis AP showing growth plates at the proximal femora and greater trochanter, and in which bowel gas is black (radiolucent) and bones are white (radiodense), with soft tissues being various levels of gray between these two extremes; (B) lateral hip; as a radiograph is a 2D image of a 3D object, two views at right angles are often required to show abnormalities in two planes.

but often are inadequate for defining soft-tissue abnormalities, where US, CT and MRI hold significant advantages. Radiographs are two-dimensional images of threedimensional anatomical structures; therefore, two radiographic views at right angles (e.g. anteroposterior [AP] and lateral) are often required to define clearly the anatomical site and extent of a bone lesion (see Fig. 12.1). Destruction in cortical bone is better visualized when the x-ray beam is tangential, rather than vertical, to the cortical area of destruction. Loss of trabecular bone can be more difficult to define and up to 40% of the trabecular bone might be destroyed before it is evident on a radiograph. Radiography, therefore, can be insensitive to subtle bone destruction and a normal radiographic examination does not exclude the presence of a bone lesion. In particular, detection may be difficult in areas of complex anatomy (such as

the wrist or foot) or where bone is obscured by overlying structures (such as the sacrum on an AP view of the pelvis); cross-sectional imaging (CT or MRI) have higher diagnostic sensitivity. When radiographic hard copy images are scrutinized, it is essential that the viewing conditions are optimum, with proper x-ray viewing boxes in a room with subdued lighting. Over the past decade or more, technical developments have resulted in replacement of screen-film radiography by digital detectors in computed radiography (CR) and digital radiography (DR). In these, the image is captured electronically rather than on hard copy film, as part of a picture archiving and communications system (PACS). There has been some loss of spatial resolution with CR when compared to that of film/screen radiography and steps must be taken to minimize dose [4]. However, the advantages of such digital imaging are a wide dynamic range and image enhancement, manipulation, transmission and storage, which enable improved image presentation and reduced rates of repeat exposures [5]. Radiographic screening (fluoroscopy) allows imaging in real time, and motion (such as that of the bowel or joints) can be studied, but carries a higher radiation dose than does radiography. All techniques that use ionizing radiation should be performed only when appropriate clinical indications exist. In radiographic examinations, gonadal shielding should be used whenever possible in children and young adults. However, the shield must not cover anatomical sites of clinical diagnostic relevance, as this would then require additional exposure when the film has to be repeated. To ensure that the patient’s radiation dose is reduced to a minimum, the best radiographic technique should be used from the outset to define the suspected pathology, and examinations should not be repeated unnecessarily. Strengths: widely available, relatively inexpensive, low radiation dose when gonads and other radiosensitive organs (e.g. breast, thyroid) are not included in the field of the primary x-ray beam, generally good spatial resolution, mobile units available. Limitations: relatively poor contrast resolution, overlap of anatomical structures.

Other Imaging Techniques that use Ionizing Radiation Computed Tomography (CT) This technique was introduced by Hounsfield in 1972 for imaging of the brain, transforming the practice of neuroradiology [6]. The potential of CT for imaging elsewhere in the body was soon realized and generalpurpose CT scanners were introduced in 1975, with one of the important clinical applications being in

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musculoskeletal disorders. In CT, the x-ray beam is finely collimated (slice thickness originally being 0.5e10 mm) and those x-rays that pass through the body are recorded by sensitive detectors. The x-ray tube and detectors rotate around the patient and, with the aid of powerful computing, the transmitted radiation is presented in analog (gray-scale picture) form. High atomic number structures (bone) are white and have a high attenuation value (250e1000 HU [Hounsfield Units]), air has a low attenuation value ( 600 to 1000 HU), as does fat ( 100 HU), with muscle having an attenuation value of approximately 50 HU. The Hounsfield scale of CT is set around water measuring 0 HU. By altering the attenuation level (mid-HU value) and range (extent of gray scale) at which the image is viewed, the tissue to be displayed can be determined. To view bone, the window level is set at about 250 HU with a wide window width (1000 HU). For soft-tissue structures, a level of 50 HU and a width of 250 HU are selected. For air-filled structures (e.g. lung) a level 600 HU and a width 1000 HU are used. To avoid missing lesions, it is essential to use appropriate window settings and interrogate the CT images on screen. Contrast medium (CM) enhancement can aid diagnosis; weak (4%) barium solution or gastrografin, administered orally at an appropriate time before scanning, is required when scanning the abdomen or pelvis to opacify bowel loops so that they are not misinterpreted as soft-tissue masses. CM administered intravenously as a bolus or by continuous infusion (known as dynamic post-contrast scanning) can provide useful information as to the vascularity of a lesion and its relationship to adjacent vascular structures. Original CT scanners obtained adjacent individual 2D sections but technological developments (slip-ring technology and multiple rings of detectors) enable continuous spiral (helical) scanning to be performed (multidetector spiral CT; MDCT). These enable rapid scanning (e.g. approximately 20 seconds for contiguous sections through the thorax) which is advantageous in children to avoid movement artifact, improved spatial resolution, dynamic contrast enhanced scanning and three-dimensional volume acquisition [7,8] (Fig. 12.2). Despite methods being introduced to reduce ionizing radiation dose and make faster scanning possible through autotube current regulation and dual source scanning [9,10], CT for imaging involves a considerable ionizing radiation dose which is of particular consideration when the technique is applied in children (Table 12.1) [11e19]. For quantitative computed tomography (QCT) for measurement of bone mineral density (BMD), lower doses of radiation are used, both centrally and peripherally, than in CT used for imaging [20,21]. Strengths: rapid scanning; high contrast resolution makes CT superior to radiography for soft-tissue

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imaging; cross-sectional anatomical display overcomes the problem of overlapping structures in radiographs; allows quantitative assessments for composition and dimensions (e.g. QCT); data can be manipulated to give coronal, sagittal, or three-dimensional volumetric images (see Fig. 12.2). Limitations: relatively high radiation dose and cost, spatial resolution not as good as radiographs when applied to central, as opposed to peripheral, skeletal sites. Radionuclide Scanning (RNS) Radionuclide bone scanning was introduced in the early 1960s. Since that time, developments in radiopharmaceutical agents and scanning techniques have led to significant improvements in the spatial resolution of radionuclide images which has been particularly advantageous when applied to children [22]. 99Tcmlabeled phosphate compounds (such as methylene diphosphonate [MDP]) are administered intravenously and are taken up in the skeleton between 2 and 4 hours. Photon emission from the whole skeleton, or localized sites, is recorded by a scintillation camera. The uptake of radionuclide in bone is determined by the rate of bone turnover and the integrity of the blood supply. Any process that alters the balance between bone resorption and bone formation (or alters vascularity) can cause abnormalities of the bone scan, with regions of increased activity (“hot spots”) or decreased activity (photon-deficient “cold spots”). RNS is very sensitive to abnormalities in the skeleton and can be particularly helpful in detection of pathologic changes in a symptomatic site when radiographs have shown no abnormality. Even small areas of increased activity are easy to detect. Unfortunately, RNS is non-specific because a variety of conditions (including primary and secondary bone tumors, infections, fractures and adjacent synovitis) may cause increased uptake of radionuclide in the juvenile skeleton. The distribution of abnormality may suggest certain diseases; multiple and scattered “hot spots” throughout the skeleton in the presence of normal radiographs suggest metastases. Ideally, radionuclide scans should be interpreted and reported in conjunction with the companion radiographs. In the normal skeleton, there may be variable uptake of radionuclide because of regional differences in bone turnover, for example in the epiphyses and metaphyses of the growth plate in children (Fig. 12.3). Single photon emission CT (SPECT) applies tomographic technology to radionuclide scanning, enabling a cross-sectional image to be obtained and enhancing conspicuity of a lesion. The radionuclide bone scan is sensitive to detecting Looser’s zones in rickets/osteomalacia that may not be evident radiographically. If there is associated secondary

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(B)

(A)

(C)

FIGURE 12.2 Computed tomography (CT): of a child with a congenital deformity of the foot. MDCT enables adjacent (A) transverse axial thin volumetric sections (e.g. 1.2 mm, 0.6 mm) to be acquired rapidly and from which can be reconstructed (B) coronal or sagittal reformations or (C) 3D volumetric images with appropriate thresholding (bone in this image) to be obtained.

hyperparathyroidism, there will be generalized increase in uptake of the radionuclide by the skeleton (“super scan”), with elevation of the bone/soft tissue ratio. There will be poor renal uptake of the radionuclide if the cause of the rickets and osteomalacia and vitamin D deficiency (absence of 1,25(OH)2 being produced) is chronic kidney disease. As mesenchymal tumors have somatostatin receptors, and may be associated with tumor-induced osteomalacia, indium-111 labeled pentetreotide has become an important imaging technique to identify the presence and confirm the site of such tumors, which

may otherwise prove elusive to clinical localization (22e24), but there are few data of use of this radionuclide scanning in children. In the past, if there was an abnormal area of uptake on the RNS it was then helpful to perform CT or MRI targeted at the area of abnormality, since these imaging methods offer higher spatial resolution and superior anatomical detail of the site and size of the lesion. However, technical developments in scanners now combine the radionuclide positron emission tomography (PET) with CT through image co-registration [25].

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TABLE 12.1

281

Effective dose (mSV) from Diagnostic X-Ray Examinations Performed on Pediatric Patients Being Investigated for Musculoskeletal Diseases Patient age (years)

Examination type

1

5

10

15

Reference

Lateral

13.9

12.2

11.8

11.8

[11]

AP

18.0

15.0

14.1

13.9

PA

16.1

14.0

13.1

12.8

PA

6.0

7.0

11.0

N/A

[12]

Radiography Skull

Chest

a

Lateral

32.2

N/A

N/A

N/A

[13]

Abdomen

AP

80.0

90.0

150.0

150.0

[12]

Pelvis

AP

9.0

26.0

N/A

N/A

[14]

Thoracic spine

AP

N/A

N/A

262.0b

142.0

[15]

N/A

N/A

b

489.0

288.0

1915

6981

8110

8274

[16]

521

3874

5741

7175

[16]

799

5829

9369

11511

[16]

1114

8364

13234

16573

[16]

2840

3004

3136

3307

[17]

N/A

22.2

17.8

11.1

[18]

N/A

5.2

4.8

4.2

[18]

N/A

N/A

N/A

<3.0

[19]

Lateral Lumbar spine

AP Lateral

Computed tomography Head and neckc d

Chest

d

Abdomen and pelvis Trunk

d

Scintigraphy Bone scane Bone densitometry Dual x-ray absorptiometry Proximal femur (hip)f f

Whole body

High resolution pQCT Tibia a

Dose figures are the average for examinations performed in ten institutions. Dose figures are the average for examinations performed in two peditaric hospitals. Effective doses for helical CT examinations; adapted from Tzedakis et al. [16]. The applied tube potential values were 80 kV for 1-year and 120 kV for 5, 10 and 15-year-old children. The applied mAs values were 150 mAs, 220 mAs, 280 mAs and 280 mAs for 1, 5, 10 and 15-year-old children, respectively. Values include the effect of z overscanning on effective dose. Dose figures are the average for a male and female. d Effective doses for helical examinations; adapted from Tzedakis et al. [16]. The applied tube potential values were 120 kV for all scans. The applied mAs values were 20 mAs, 50 mAs, 85 mAs and 120 mAs for 1, 5, 10 and 15-year-old children, respectively. Values include the effect of z overscanning on effective dose. Dose figures are the average for a male and female. e Adapted from Smith et al. [17]. A dose of 95 MBq, 170 MBq, 280 MBq and 450 MBq was considered for a 1-year-old, 5-year-old, 10-year-old and 15-year-old child correspondingly. f Doses were estimated for Hologic DXA devices. Values are given for scans lengths adjusted to the size of the child’s body. b c

FIGURE 12.3 Radionuclide scan (RNS): in a normal child. 99Tcmlabeled phosphate compounds are given intravenously and are taken up in sites of the skeleton where there is increased blood flow or increased osteoblastic activity or both. Photon emission from the whole skeleton, or localized sites, is recorded by a scintillation camera. Scanning within minutes of administration of the radiopharmaceutical will give blood pool images (right images). For bone uptake scanning must be delayed for between 2 and 4 hours (left images) and images are captured in the anterior and posterior projections. In children, there is normally increased uptake in the metaphyses as illustrated, which is a cause of false positive and negative diagnoses, and the technique involves a high radiation dose to the bone marrow in children. The RNS is a sensitive technique which is good for surveying the entire skeleton. However, the increased uptake is not specific and can occur in a number of different etiologies (e.g. bone tumors, infections, fractures and adjacent synovitis).

Radionuclide scanning in children is of limited value, because there is high uptake in the normal metaphyses of the growing skeleton (see Fig. 12.3). In addition, the examination carries a significant ionizing radiation dose to the bone marrow (see Table 12.1). Strengths: sensitive to identify sites of abnormal skeletal uptake, so good general skeletal survey technique; gives functional information (e.g. tumor uptake). Limitations: delayed imaging required (2e4 hours), poor spatial resolution, high radiation dose to bone marrow, long scan time so prone to movement artifact in children.

Imaging Techniques that do not Use Ionizing Radiation Ultrasonography (US) This technique was first used clinically in the late 1960s, mainly in obstetric practice and prenatal

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diagnosis. Its potential for diagnosis elsewhere in the body was soon recognized, and ultrasound scanners are now widely available. They are relatively inexpensive to purchase compared with other imaging equipment. Unlike other cross-sectional imaging techniques, ultrasound scanners can be small and mobile. A US scanner can, therefore, be transported to examine patients in the emergency department, operating room and intensive care unit. In US scanning, sound waves are emitted from a transducer that is held against the skin surface. The transducer is “coupled” to the skin with lubricating jelly to avoid artifacts from intervening air. The sound waves penetrate the body, are reflected by surfaces, and are received by the transducer. The anatomy of a section of the body is displayed on a monitor and can be recorded (Fig. 12.4). As the technique does not use ionizing radiation, US is particularly well suited to application in children [26,27]. Depth of penetration of the ultrasound beam is determined by the frequency. Probes of 3e5 MHz are used to examine musculoskeletal disorders. Higher frequency probes of 7.5e10 MHz have excellent spatial resolution and are ideal for imaging

FIGURE 12.4 Ultrasound scan: (longitudinal; head to left) of the normal hip of a child. Reflections in the lower half of the image are US reflections from the femoral head and shaft, with an anterior gap which is the growth plate. The anterior reflections are from adjacent muscle groups. US does not use ionizing radiation, a particular advantage in children, and can be used for real-time imaging (e.g. of joint movements) and is particularly good for imaging fluid (water is usually transonic [no sound waves reflected] so can be used to identify joint effusions).

superficial structures. Modern scanners allow real-time imaging so that physiologic motion and arterial pulsation can be observed. Doppler ultrasound enables quantitative measurement of flow in blood vessels and tumors, which can enhance tissue-specific diagnosis. The use of US in the diagnosis of musculoskeletal disorder has increased over the years and continues to do so. US is also useful in examining large joints (such as the hip) (Fig. 12.4) for effusions, applicable particularly in children. US gives an indication of the composition of a mass and provides information on the relationship of the mass to adjacent structures, particularly vessels. Although US is widely available, inexpensive and harmless at the energies used in clinical practice, the quality of the images obtained and their interpretation are dependent on the skill and expertise of the operator. Strengths: portable, no ionizing radiation, relatively inexpensive, real time imaging (e.g. joint mobility). Limitations: operator dependency. Magnetic Resonance Imaging (MRI) This technique uses the principles of magnetism instead of ionizing radiation, an advantage in pediatric imaging [28e31]. MRI depends on intrinsic tissue parameters that reflect the chemical characteristics of that tissue. MRI is the phenomenon in which certain atoms can absorb and re-emit radiofrequency waves of a specific energy when in a magnetic field. It is principally the distribution of hydrogen ions (protons), abundant in biologic tissues, which contributes to MR images. MR imaging was introduced into clinical practice in the early 1980s. Clinical whole-body scanners operate with a field strength of 1.5 T (Tesla), although an increasing number of higher field strength (3 T) scanners are becoming available [32], which improve spatial resolution and scan speed, particularly relevant to pediatric imaging, but there are also limitations of such high magnetic field strengths (artifacts, noise, involuntary muscle twitching). Pulses of radiofrequency radiation are applied and the magnitude of the nuclear signal following this pulse is measured as relaxation times. Variations in pulse sequences can alter tissue contrast, which is determined by T1 and T2 relaxation times and proton density. A great number of pulse sequences have been developed to potentiate differences in tissue contrast and enhance the conspicuity of pathologic lesions. In bone and soft-tissue lesions, T1- and T2weighted (T1W and T2W) images are usually acquired, and tissues may have specific signal intensities: Fat: gives high intensities signals on T1W and T2W images Fluid: gives low intensities signals on T1W and high signals on T2W

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IMAGING TECHNIQUES, INTERPRETATION, AND STRATEGIES

Cartilage: gives intermediate signal intensity on T1W and high signal on T2W Cortical bone and fibrous tissue: gives low signal and muscle intermediate signal in both sequences. The signal intensity of hemorrhage depends on the age of the hematoma: Hyperacute: (<24 hours; oxyhemoglobin) intermediate signal T1W; high signal T2W Acute: (1e3 days; deoxyhemoglobin) low signal on both T1W and T2W Subacute: early (>3 days; intracellular methemoglobin) high on T1W; low on T2W Subacute: late (>7 days extracellular methemoglobin) high signal on both T1W and T2W Chronic hemorrhage: (>14 days; hemosiderin/ferritin) low signal on both T1W and T2W. The high contrast sensitivity of MRI makes it the imaging method of choice for defining soft-tissue margins and marrow changes within bones. Vessels that contain flowing blood appear as signal voids on MRI and are easily identified. MR angiography can be performed without the use of intravenous contrast media. Images can be obtained in multiple planes (Fig. 12.5), and paramagnetic contrast agents (gadolinium-labeled DTPA), which shorten T1 and T2 relaxation times of adjacent protons, are given intravenously to perform contrast-enhanced scans. To maximize the signal-to-noise ratio and spatial resolution in MR images, specialized surface transmitterreceiver coils have been designed for scanning specific anatomical sites, such as the extremities. The advent of faster MRI scanning techniques (gradient-echo) enables the acquisition of three-dimensional data; this has important applications in musculoskeletal disorders. Strengths: no ionizing radiation, high tissue contrast sensitivity, images obtained directly in any anatomical plane, indication of tissue composition. Limitations: high cost, limited availability in some countries, artifacts from motion (cardiac and bowel) and ferromagnetic objects, inferior to CT in demonstrating changes in cortical bone, and the technique cannot be used in patients who have ferromagnetic clips or cardiac pacemakers. Claustrophobia e about 2% of patients cannot tolerate the tunnel of the MRI scanner; technical challenges if anesthetic is required to maintain patient to be still during scanning to avoid motion artifact, which may be the case with young children. Bone Densitometry Methods of quantitative assessment of the skeleton are covered in detail in Chapter 13. Dual energy x-ray absorptiometry (DXA) was introduced in the late 1980s and is the method which is most widely utilized

283

in both adults and children [33]. DXA has the advantages of providing measurements in clinically relevant sites (proximal femur, lumbar spine L1e4, forearm) and, more recently, lateral femur in children with leg contractures [34]. Whole body DXA (excluding head) provides information of total and regional bone mineral content (BMC), BMD, fat mass and fat free (muscle) mass, and should be performed in children [35,36]. The ionizing radiation doses from DXA are extremely small and not much more than a few hours of natural background radiation [21] (see Table 12.1). Limitations of DXA are that integral (cortical and trabecular) bone is measured and the measurement is an “areal” density (g/cm2) and thus size dependent, underestimating BMD in small bones and overestimating BMD in large bones, a particular problem in growing children [36]. Various methods have been suggested to correct for this (e.g. calculating bone mineral apparent density [BMAD] and others) and for which there are reference data [33,37,38]. QCT has advantages over DXA in that it provides separate measures of cortical and trabecular bone in the central and peripheral skeleton and is a true volumetric density (mg/cm3) and so not size dependent [20]. Although the radiation dose for QCT of the lumbar spine is higher than for DXA, a lower dose technique than for imaging can be used to minimize exposure. High resolution (HR) scanning can be used at peripheral sites (in plane spatial resolution 82 mm) to examine trabecular and cortical structure [39] and this technique is increasingly being applied in pediatric research studies [40]. However, QCT remains currently predominantly a research technique. Digital X-Ray Radiogrammetry (DXR) Metacarpal morphometry and radiogrammetry are the oldest methods for quantitative assessment of the skeleton, and were usually applied to the second metacarpal of the non-dominant hand on a radiograph (metacarpal index [MI]). Although the method was inexpensive and widely available, and provided useful research and epidemiologic information, it was labor intensive and imprecise. MI was replaced with the current established methods of bone mineral densitometry (DXA, QCT, QUS) (see Chapter 13). With the application of modern computer vision techniques (active shape/appearance models [ASM/AAM]), metacarpal morphometric analysis has been rejuvenated, with improvement in precision and evidence that the method can be applied to studies in adults and children [41]. A radiograph of the hand involves a negligible ionizing radiation dose (see Table 12.1) and is often performed in children to assess bone age and DXR can be applied retrospectively or prospectively to these images [42].

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(B)

((A) (A (A) A)

(C)

FIGURE 12.5 Magnetic resonance imaging (MRI): of the cervical spine and brain in a child with type IV mucopolysaccharidosis (Morqio’s disease), with hydrocephalus, hypoplasia of the odontoid, which can lead to atlanto-axial instability, and abnormal shape and marrow signal of the vertebral bodies. Imaging in any plane is feasible and different sequences alter signal from various tissues; (A) sagittal T1W image showing cerebrospinal fluid (CSF) to be low signal (dark); (B) sagittal T2W image in which CSF is high signal (white); (C) T1W coronal image showing dilated cerebral ventricles (low signal CSF) related to the hydrocephalus.

Bone Aging Bone-age assessment from a radiograph of the nondominant hand is important in clinical practice and research studies of skeletal development in children, and is covered in detail in Chapter 14. There are three main techniques used for determining bone age from a PA radiograph of the hand: Tanner-Whitehouse (TW) II [43] and III [44], Greulich-Pyle (GP) [45] and

the Fels [46] methods. In the TW and GP methods, the patient’s hand radiograph is compared to an atlas of hand radiographs acquired in normal children of the same chronological age. The Fels method uses a more complex series of measurements that are compared to a reference set of measurements. The TW and GP methods assess maturity of distal radius and ulna, metacarpals, phalanges and the carpal bones excluding

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the pisiform; the Fels method uses all the same bones, but includes the pisiform and adductor sesamoid of the first metacarpal. The GP method is the more simple and quick method to perform and is the one most widely used in clinical practice [47], whereas the TW and Fels method are favored for research studies [48]. Assessment of bone age has the potential for automation applying computer vision techniques [49]. Ionizing Radiation Doses and Regulations It is recognized that exposure to ionizing radiation can have adverse effects which are either stochastic (those that occur by chance and consist primarily of cancer and genetic effects but for which there is no threshold dose of exposure to ionizing radiation) or deterministic (damage by ionizing radiation where a dose threshold exists, and for which the severity of damage increases with increasing dose above that threshold). In diagnostic imaging, it is the former effects which are generally relevant. To minimize the dose of ionizing radiation exposure to workers, the general public and patients, the International Commission on Radiation Protection (ICRP) in 1977 adopted the policy of as low as reasonably achievable (ALARA). This is particularly relevant in radiological imaging of children in which there must be close collaboration between pediatricians, radiologists and medical physicists to perform the optimum imaging technique using the minimum radiation dose to define the clinical problem [50,51]. There should be strong clinical justification for requesting an imaging examination which uses ionizing radiation; if there is an alternative technique which does not use ionizing radiation (e.g. US, MRI) and which could define the clinical problem equally as well, or better, then that technique should be used in preference, if feasible. When imaging techniques which use ionizing radiation are clinically justified and performed, then technical factors (kVp and mAs) and radiation dose limitation procedures (gonadal protective shielding if the area of diagnostic interest is not covered, shorter exposure time, well collimated x-ray beam to expose only the relevant anatomical region required, low dose rate, quality assurance [QA] programs) must be implemented. A large number of imaging procedures are carried out in children, but radiographs are still the most widely used [52]. Fluoroscopy and image guided interventional procedures carry higher doses of radiation than radiography [53]. There is increasing use of CT, in both adults and children, and this now contributes most to medical radiation exposure to the population [51]. Technical developments of MDCT have improved spatial resolution but this can be at the cost of increased radiation dose. However, efforts have been made to minimize exposure in CT (automatic

modulation to adjust x-ray tube current according to anatomical site being scanned; lower currents required in sites where there is air [lungs] rather than solid tissues [pelvis]). Lower tube currents can be used in children than in adults as children are smaller. The dose to which a patient is exposed is generally expressed as effective dose (ED) which takes into account the absorbed dose and a weighting factor, defined by the ICRP, and dependent on the radiosensitivity of the organ exposed [51]. The radiation doses involved in imaging of musculoskeletal diseases are given in Table 12.1 [11e19,21,54].

IMAGING STRATEGIES A Radiological Approach to Skeletal Dysplasias The Skeletal Survey When a skeletal dysplasia is suspected, a full radiographic skeletal survey (as opposed to images of selected sites) is recommended. This allows the radiologist to identify all the important positive and negative findings, and increases the likelihood of reaching a definitive diagnosis. The recommended baseline dysplasia skeletal survey consists of the following: anteroposterior (AP) and lateral skull; lateral thoracolumbar spine; AP pelvis (including lumbar spine); one upper limb; one lower limb; AP chest; dorso-plantar (DP) left hand. This series of radiographs should be acquired for any child suspected of having a constitutional disorder of bone, regardless of the clinician’s differential diagnosis, except in the following circumstances, when additional imaging may be obtained (before or after reviewing the initial survey): 1. “Babygram” (an AP and lateral head to foot radiograph): in preterm fetuses and stillbirths because of their small size 2. Both upper and lower limbs: when there is limb asymmetry or suspected epiphyseal stippling 3. Cervical spine: in suspected or diagnosed conditions associated with cervical instability 4. Coned views: for more detailed assessment: sites of suspected abnormality (e.g. epiphyseal stippling) 5. Other family members: ascertainment of radiological features at different ages by imaging other affected family members may help reach a diagnosis 6. Repeat survey: if the diagnosis is uncertain, a repeat survey may help. There is no benefit in repeating the survey within 12 months of the initial survey or imaging previously normal sites. Interpretation of the Skeletal Survey Although collectively constitutional bone disorders are relatively common, individual disorders are rare.

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(B)

(A)

(C)

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FIGURE 12.6 Radiographic skeletal survey: in a 13-year old boy with craniometaphyseal dysplasia. (A) Lateral skull; (B) AP forearm; (C) AP tibia and fibula; (D) DP hand. There is sclerosis of the skull base with absent pneumatization of sinuses, the metaphyses are wide and there is bowing of the long bones and undertubulation of the bones of the hand.

The radiologist needs to approach the images in a systematic way to identify the abnormalities and reach a diagnosis [55].

ANATOMICAL LOCALIZATION

Some conditions are named based entirely on sites affected, e.g. ischiopubicpatella syndrome,

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craniometaphyseal dysplasia (Fig. 12.6). However, while identification of the general sites affected gives a starting point, e.g. if a search of databases or textbooks is required to reach a diagnosis, it is usually necessary to classify the condition further, for example metaphyseal chondrodysplasia (types Schmid or McKusick). Therefore, rather than simply identifying affected sites, more precise interpretation of the radiographic abnormalities is required. BONES

Consider the five ‘Ss’: structure, shape, size, sum and soft tissues. Structure: determine the general appearance of the bones, e.g. density, exostoses and enchondromata. Consider whether all bones are affected or whether abnormality is limited to certain bones or sites of bones. Is increased bone density uniform, or does it appear as bone islands, striations or a combination of both (Fig. 12.7)? Shape: there is a myriad of terms used to describe the shape of bones in skeletal dysplasias. Examples include the “telephone receiver” femora of thanatophoric dysplasia (Fig. 12.8A,B), and the diamond-shaped vertebral bodies and “dumbbell” long bones of metatropic dysplasia (Fig. 12.8C,D). Size: abnormalities of size are self-explanatory and include overgrowth or retarded growth. The latter may be relative (e.g. fibula short in comparison to tibia in hypochondroplasia, but long in campomelic dysplasia) or absolute (e.g. the short metacarpals and phalanges of Albright’s hereditary osteodystrophy). Selective shortening of metacarpals and phalanges occurs in the various brachydactyly syndromes. The term “hypoplasia” only applies when a bone is relatively short compared to other bones in that individual. A child with constitutional short stature will have fingers that

are short compared with another child of similar age, but normal for his/her height, and so not hypoplastic. The ribs are short in the short rib polydactyly group of disorders. Sum: bones may be too many, too few, or fused. Polydactyly is associated with a considerable number of dysplasias and may be pre-axial (side of the thumb), post-axial (side of the little finger) or meso-axial (central). An absent patella is a feature of the nailepatella and ischiopubicpatella syndromes. An absent radius occurs in TAR (thrombocytopenia absent radius) syndrome, HolteOram syndrome and Fanconi’s anemia e a differentiating feature is that the thumb is always present in TAR, while it is abnormal or absent in HolteOram and Fanconi syndrome. Soft tissues: may be reduced (atrophic) or increased (hypertrophic) in size, there may be contractures or calcification. Soft tissue involvement may change both the diagnosis and the prognosis. For example, calcification within phleboliths changes the diagnosis of Ollier’s diseases to Maffucci’s syndrome. The former condition is benign, while the latter is associated with a significant risk of malignant change (30% or more). COMPLICATIONS AND CHARACTERISTIC FINDINGS

Complications may be of the condition (e.g. fractures, atlanto-axial instability, and malignancy) or secondary to treatment (e.g. needle tract infection). Characteristic findings: some conditions have radiographic findings that once identified are pathognomonic, e.g. “dripping candle wax” appearance of melorrheostosis, the lace-like iliac crests of Dygve Melchior Clausen syndrome, the “snail-like” appearance of the pelvis in Schnekenbecken dysplasia, the iliac horns of the nailepatella syndrome or the constellation of features (dysostosis multiplex) present in the storage disorders (Figs 12.5 and 12.9).

(A)

(B)

FIGURE 12.7 Density of bones: showing metaphyseal striations in a patient with osteopathia striata in proximal femora on (A) AP pelvis and cranial sclerosis on (B) AP skull.

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(A)

(B) (C)

(D)

FIGURE 12.8 Shape of bones: (A) AP and (B) lateral radiographs of a fetus with thanatophoric dysplasia (TD) with bowed “telephone receiver” femora, short ribs, micromelia, platyspondyly and horizontal trident acetabulae. The normal skull vault indicates TD type 1. The skull is cloverleaf shaped in TD type 2. A 2-year-old child with metatropic dysplasia (C) lateral spine showing platyspondyly with diamond shaped vertebral bodies. (D) Lateral femur showing micromelia with dumb-bell shaped limbs due to broad metaphyses.

DEAD OR ALIVE

Making the Diagnosis

The severity of a condition, how early it presents (e.g. in utero) and whether or not it is lethal will sometimes help in reaching a final diagnosis, e.g. heterozygous achondroplasia will present with short femora in utero only in the third trimester, homozygous achondroplasia will present before this time, and thanatophoric dysplasia is incompatible with life. Severe osteogenesis imperfecta (OI) (type II and severe type III) will present in utero (Fig. 12.10); milder types may not present until childhood.

Reaching a diagnosis is important to giving the correct genetic advice and to enable prognosis to be determined. Complications associated with the disorder can be pre-empted and perhaps prevented. The gamut sections of textbooks may be used, but databases (e.g. London Dysmorphology Database) [56] that allow searches to be made based on identified abnormalities are particularly useful. Expert opinion (e.g. European Skeletal Dysplasia Network [ESDN]) [57] may be sought, but it may not always be possible

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(A) (B)

FIGURE 12.9 Diagnostic features: in a 3-year-old boy with biochemically confirmed mucopolysaccharidosis type II. Radiographs show the features of (mild/moderate) dysostosis multiplex. (A) Lateral skull with macrocephaly; (B) PA hand with mild proximal pointing of the second to fourth metacarpals.

to reach a conclusive diagnosis. In such cases, it is better to give no diagnosis than to give the wrong diagnosis.

GENETIC DISORDERS Osteogenesis Imperfecta (see Chapter 19) Antenatal diagnosis (US and fetal MRI) of the severe OI types (types II and III) is possible, and requires identification of diminished skull vault ossification and/or bowed or fractured bones, particularly the long bones [58]. Postnatal radiographic features, common to all types, include generalized osteopenia, multiple Wormian bones in the skull vault, vertebral crush fractures, slender (gracile) long bones, and bowing and fractures of long bones and ribs. Metaphyseal fractures (as seen in child abuse) may occur, but are not common. The diagnosis of OI is mainly based on clinical criteria. Radiographs may help to confirm the diagnosis, and by identifying fractures will assist clinical management of the patient, including orthopedic intervention and the decision to commence bisphosphonate therapy. The key radiograph that should be obtained is the lateral spine (to identify vertebral fractures). Wormian bones are present in approximately 50% of patients, and while their presence may help to confirm the

diagnosis, it does not impact on management. If the clinical diagnosis is uncertain, then a full skeletal survey is recommended. Subtypes of OI that are readily distinguished through their radiographic features include types I, II (a, b and c), III, IV and V. Type VI requires histological confirmation. Classification of OI types VII and VIII is predominantly based on results of genetic mutation analysis. A move has been made away from this derivation of OI types based on a mixture of radiological and genetic phenoypes, towards one based only on radiological phenotype [59e61]. Types VII and VIII are now classified as autosomal recessive types with an indication of the identified mutation. Type I: affected patients have mild/moderate radiographic changes. They may present in infancy with fractures but the rate of fracture tends to decrease with age. Type II: is the perinatally lethal type of OI. The radiographic changes are severe. There are three subtypes (IIA, IIB and IIC). Angulation of tibiae and fibulae is common to all subtypes. Distinguishing radiographic features are: Type IIA: absent skull vault ossification, multiple confluent healing rib fractures leading to wide ribs, wide and crumpled “concertina” femora.

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Type III (Fig. 12.11): of all non-lethal forms of OI, this is the most severe, with radiographic features including slender “ribbon” ribs, “cod fish” vertebrae (biconcave end plates due to fracture), basilar invagination, platybasia, kyphoscoliosis and protrusio acetabuli. Type IV: the radiographic changes are mild to moderate. Basilar invagination is common. Like type I, the number of fractures decreases as the child gets older. The presence of white sclerae renders the differentiation between this diagnosis and child abuse more difficult. Type V: radiographic findings evolve with time. In infancy, the radiographs demonstrate dense metaphyses with metaphyseal spurs. With increasing age there is progressive ossification of the interosseous membranes (Fig. 12.12). Fractures heal with hyperplastic callus which may be so profuse as to mimic malignancy. Type VI: by demonstrating an abnormal pattern of lamellar bone under polarized light, qualitative histology allows a definitive diagnosis. However, there are no distinguishing radiographic features, which are moderate to severe. Other Recessive Types of OI [56]* Cartilage-associated protein (CRTAP):

FIGURE 12.10 Osteogenesis imperfecta type IIB: the diagnosis may be made prenatally. AP radiograph of a 21-week fetus with slender ribs, crumpled fractured femora. The severity of changes at this gestational age is in keeping with OI type IIB despite absence of the characteristic angulation of tibiae and fibulae.

Type IIB (see Fig. 12.10): in contrast to IIA, neonates will have (some) ossification of the skull vault. They also have slender “beaded” ribs, bowing of femora and fractures of long bone. It is often difficult to differentiate mild IIB from severe type III e the distinction occasionally being made based on patient’s survival. Type IIC: This subtype is not common. Like OI IIA, there is absent ossification of the skull vault. Like IIB there are multiple rib fractures e more discrete than in IIA. Distal pointing of the femora has been described and affected fetuses have a deformed pelvis. A distinctive feature is severe osteopenia with sclerotic stippling.

i. Previously classified as type VII: these patients have variable rhizomelic shortening, otherwise no distinguishing findings (moderate to severe radiographic findings). ii. OI types IIB and IIC. Genetic mutations of: LEPRE1: i. Previously classified as OI type VIII: severe and like type II; may be perinatally lethal. ii. OI types IIB and IIC. PPIB, SERPINH1 and FKBP10: These mutations cause severe or perinatally lethal forms of OI. There is increasing use of cyclical bisphosphonates therapy in children with conditions which cause osteoporosis, including osteogenesis imperfecta [61e64]. Such treatment can interfere with modeling of the metaphyses [65] and result in transverse dense lines (“zebra” lines) [66] (see Fig. 12.11C). These are thought to be horizontally arranged trabeculae which tend to regress with time following remodeling [65].

*

It is worth noting that while CRTAP, LEPRE1 and PPIB mutations have been identified in patients with OI types IIB and IIC, to date all patients with OI type IIA who have undergone mutation analysis have had COL1A1 or COL1A2 mutations.

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(A) (B)

(D)

(E)

(C)

FIGURE 12.11 Osteogenesis imperfecta Type III: (A) lateral skull of a 3-week old infant with a thin vault and a few Wormian bones. A 4year-old girl (B) chest radiograph, (C) lateral femur, (D) PA forearm all showing generalized osteopenia, multiple fractures and slender ribs. The multiple dense metaphyseal bands are related to intermittent bisphosphonate therapy (bisphosphonate lines) administered through the central line evident on the chest radiograph and (E) frontal skull radiograph with multiple Wormian bones.

Differential Diagnosis ANTENATAL

Causes of reduced skull vault ossification: hypophosphatasia (see Chapter 28); achondrogenesis. Causes of slender bones/fractures: in utero e hypomobility syndromes. Causes of femoral bowing: campomelic dysplasia; kyphomelic dysplasia; Sto¨veeWiedermann dysplasia.

FIGURE 12.12

Osteogenesis imperfecta type V: radiograph of forearm shows interosseous membrane ossification which results in limitation of pronation and supination.

POSTNATAL

Causes of reduced bone density (Table 12.2): idiopathic juvenile osteoporosis; glucocorticoid therapy;

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Causes of Osteoporosis in Children (Incomplete Listing)

Primary

Juvenile Idiopathic of young adults

Secondary Endocrine

Glucocorticoid excess Hyperthyroidism Hyperparathyroidism Growth hormone deficiency (childhood onset)

nutritional and endocrine disorders; osteoporosis pseudoglioma syndrome. Causes of multiple fractures: physical child abuse (non-accidental injury [NAI]); copper deficiency; Bruck syndrome. Causes of Wormian bones: HajdueCheney syndrome.

Hypophosphatemia (see Chapter 26)

Nutritional

Intestinal malabsorption Chronic liver disease Vitamin C deficiency (scurvy)

Hereditary

Osteogenesis imperfecta Homocystinuria Marfan syndrome EhlerseDanlos syndrome

Hematological

Thalassaemia Sickle cell disease Gaucher’s

Other

Rheumatoid arthritis Cancer and its therapies

This autosomal dominant, autosomal recessive or Xlinked disorder [67] leads to renal tubular dysfunction and urinary loss of phosphate. The radiographic changes are those of rickets (Fig. 12.13). In childhood, there is generalized mild reduction in bone density, coarse trabecular pattern, wide growth plates and cupped and frayed metaphyses. There is bowing of the tubular bones. Widening of the costochondral junctions gives the “rickety rosary”. In older children and adults, the bones may be of increased density with calcification/ossification of tendons and entheses (bone/ligament junctions). In children, the radiographic changes of rickets are most severe at the sites of rapid growth (knee, wrist); therefore, if clinical suspicion is low, radiographs of these sites may suffice for initial screening.

(B)

(A)

FIGURE 12.13 Hypophosphatemia: a 14 year old; radiographs of (A) lateral skull showing thickening of the skull vault with prominent convolutional markings as a result of premature sutural fusion; (B) forearm shows changes of rickets at the distal radius and ulna with metaphyseal irregularity and wide growth plates.

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Differential Diagnosis Other causes of rickets; metaphyseal chondrodysplasias (particularly type Schmid).

Albright’s Hereditary Osteodystrophy; Pseudohypoparathyroidism; PseudoPseudohypoparathyroidism (see Chapter 21) These patients have a characteristic round face, short stature and, in most cases, obesity with intellectual impairment [68]. Characteristic radiographic findings are in the hands and feet with premature fusion and cone-shaped epiphyses of metacarpals, metatarsals and phalanges which are short in length (Fig. 12.14). This typically affects the third and fourth digits, but may involve any of the digits to varying degrees and in any combination e including affecting all digits. The capital femoral epiphyses are small with a reduced height, and slipping of the capital femoral epiphysis, coxa vara and valga has been described. There is failure of widening of the inter-pedicular distances of the lumbar spine. In the skull, there may be thickening of the calvarium with widened diploe¨ and hyperostosis frontalis interna.

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Intracranial calcification (secondary to prolonged hypocalcemia) may be present. A full skeletal survey is required to exclude other causes of brachydactyly. Differential Diagnosis Brachydactyly E; acrodysostosis; acromesomelic dysplasia; trichorhinophalangeal syndrome; multiple epiphyseal dysplasia / pseudoachondroplasia.

Hypophosphatasia (see Chapter 28) Subtypes of this rare disorder include lethal perinatal, benign perinatal, infantile (Fig. 12.15), childhood, adult and odontohypophosphatasia [69]. Both perinatal subtypes have similar radiographic findings. However, these resolve spontaneously in the benign form. Perinatal: poor mineralization of the skeleton with some non-ossified bones and absent ossification of pedicles of the spine. There is poor ossification of the skull vault, irregular ossification of metaphyses and bowing of long bones with diaphyseal spurs and dimpling overlying the sites of the spurs. Infantile/adult: although infants have wide anterior fontanels, they subsequently develop premature closure of sutures with a “copper beaten” skull due to raised intracranial pressure (see Fig. 12.15C). The metaphyses show well-demarcated defects in mineralization and there may be bowing of long bones. Bone pain and fractures are characteristic. Poor dentition is a feature and there may be early loss of milk and permanent teeth. Odontohypophosphatasia: included here for completeness, is a dental diagnosis associated with premature loss of fully rooted milk teeth. The skeleton is usually not affected. For neonates and smaller infants, AP and lateral “babygrams” may be performed. However, a full skeletal survey is recommended in older infants, children and adults. Differential Diagnosis Fetal/neonatal: achondrogenesis (various types); osteogenesis imperfecta. Older age groups: osteogenesis imperfecta; rickets (including healing rickets); metaphyseal chondrodysplasias.

Hyperphosphatasia

FIGURE 12.14 Albright’s hereditary osteodystrophy (pseudohypoparathyroidism): hand radiograph shows short metacarpal and phalanges due to premature fusion of cone-shaped epiphyses.

Idiopathic hyperphosphatasia is a rare autosomal recessive bone disorder, characterized by excessive bone resorption and bone formation. The radiographic appearances include widening (undertubulation) and bowing of the diaphyses with disorganized trabeculae and thickened cortices, vertebral osteoporosis, acetabular protrusion and thickening of the skull vault, due

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(C)

(B) (A)

FIGURE 12.15 Hypophosphatasia: radiographs of 30-month-old male; (A) lateral tibia and fibula; (B) forearm showing generalized osteopenia, metaphyseal defects; and (C) lateral skull showing prominent convolutional markings in the vault as a result of premature fusion of the sutures.

to widening of the diploic space associated with absent pneumatization of the paranasal sinuses and sclerosis of facial bones. There is considerable variability in phenotype, with some cases diagnosed in infancy and others in later childhood. Most cases appear to arise from inactivating mutations in the gene encoding osteoprotegerin, a product of osteoblasts that is critically involved in osteoclastogenesis [70]. Treatment with inhibitors of bone resorption (calcitonin or bisphosphonates) is successful in ameliorating some aspects of the disorder. Differential Diagnosis Infantile cortical hyperostosis; endosteal hyperostosis; polyostotic fibrous dysplasia; osteogenesis imperfecta; Paget’s disease in affected adults.

Sclerosing Bone Dysplasias (see Chapter 20) Sclerosing bone disorders can be subdivided according to their clinical presentation, the primarily affected cell type, and the cellular pathways [71]. Osteoclastrich osteopetrosis in most cases is related to mutations in genes required for osteoclast function, whereas osteoclast-poor forms are related to abnormalities in osteoclast differentiation. Osteosclerosis can also be caused by increased bone formation either through abnormalities in transforming growth factor-beta signaling (Camurati-Engelman disease, osteopoikilosis) or caused by mutations in genes involved in the Wnt

pathway, which regulates osteoblast differentiation (endosteal hyperostosis, sclerosteosis, van Buchem disease, high bone-mass syndrome, osteopathia striata). Osteopetrosis (Fig. 12.16) Three major subtypes of this condition are recognized: severe (infantile), intermediate (juvenile) and mild (adult) [72]. The severe form presents in infancy (or at birth) with generalized increased bone density. With progressive narrowing of bone medullary spaces there is increasing bone marrow failure. Thickening of the skull base leads to compression of the cranial nerves with resultant visual and hearing disturbances. Radiographs may demonstrate relative metaphyseal lucency, “bone in bone” appearance (Fig. 12.16B) and/or a “harlequin” appearance of the orbits. Increased bone density, but with tendency to fracture, is a feature of the juvenile and adult forms of the disease (Fig. 12.16B). With the abnormality of osteoclastic function the ends of the long bones are abnormally modeled (Fig. 12.16D). When associated with carbonic anhydrase deficiency intracranial calcification occurs in juvenile osteopetrosis. The diagnosis of autosomal dominant adult osteopetrosis may be made incidentally. Generalized osteosclerosis is present in type I, whereas axial sclerosis with a “rugger jersey” spine is typical of type II (see Fig. 12.16C).

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(A)

(B)

(D)

(C)

FIGURE 12.16 Osteopetrosis: infantile (A) 6-month-old boy with generalized increase in bone density and irregular metaphyses. (B) AP pelvic radiograph of a previously healthy girl who sustained a transverse subtrochanteric fracture of the left femur following a trivial fall; note the “bone within a bone” appearance of the iliac wings and (C) lateral spine radiograph showing the classical “rugger jersey” spine; the diagnosis of autosomal dominant osteopetrosis was made in this patient; (D) AP forearm showing generalized increased bone density with abnormal modeling of the distal radius due to failure of normal osteoclast function.

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MAJOR DIFFERENTIAL DIAGNOSIS

Infants: dysosteosclerosis; sclerosteosis. Older age groups: pycnodysostosis; Paget’s disease in adults; osteoblastic metastases. Pycnodysostosis Radiographic features of this condition [73] include generalized increased bone density with over tabulation of long bones (slender diaphyses). Skull radiographs demonstrate micrognathia, multiple Wormian bones, large wide-open anterior fontanel and sutures and an obtuse mandibular angle. The distal ends of the clavicles are hypoplastic and in the hands and feet there is pointing or acro-osteolysis of distal phalanges. The iliac bones are narrow but, in contrast to cleidocranial dysplasia, there is normal ossification of the pubic bones. DIFFERENTIAL DIAGNOSIS

Osteopetrosis; cleidocranial dysplasia; mandibuloacral dysplasia; causes of acro-osteolysis.

RICKETS AND METABOLIC BONE DISORDERS Rickets Rickets is a disease of the growing child and remains a common problem in many parts of the world. Clinical and radiological changes of rickets arise from impaired mineralization of the metaphyseal growth plates of long bones due to inadequate concentrations of calcium and phosphate in the extracellular fluid. Mineralization of bone osteoid and the growth plate during enchondral ossification depends on the normal availability of vitamin D, calcium, phosphorus, alkaline phosphatase and a normal pH prevailing in the body environment. If there is a deficiency of these substances, for any reason, or if there is severe systemic acidosis, then mineralization of bone will be defective. There will be a qualitative abnormality of bone (in contrast to osteoporosis, which is predominantly a quantitative abnormality of bone), with reduction in the mineral/osteoid ratio, resulting in rickets in children and osteomalacia in adults. A large number of different diseases (vitamin D deficiency, hypophosphatemia, hypocalcemia, hypophosphatasia and severe acidosis) can cause the same radiological abnormalities of rickets and osteomalacia [23]. Privational or nutritional rickets arises from chronic dietary calcium deficiency, vitamin D deficiency or combination of both these factors [74] (see Chapters 8 and 23). Causes of rickets arising from vitamin D deficiency, and inherited defects in renal synthesis of 1,25dihydroxyvitamin D (1,25(OH)2D) or resistance to its

actions have been reviewed (see Chapters 8, 25, 27 and 29). Secondary hyperparathyroidism occurs in any cause of rickets which results in reduction in serum calcium, and plays a pivotal role in the pathogenesis of renal osteodystrophy (see Chapter 29). High serum parathyroid hormone (PTH) concentration in these conditions will result in demineralization of the skeleton and phosphaturia and hypophosphatemia, which further contributes to impaired mineralization of the growth plate. A number of genetic disorders result in chronic hypophosphatemia, secondary to impaired renal reabsorption of filtered phosphate [76]. Failure of apoptosis of hypertrophic chondrocytes in the growth plate due to low serum phosphorus concentration [77,78] appears to be the common pathophysiological pathway in rickets arising from renal phosphorus wastage due to conditions which result in elevated serum fibroblastic growth factor 23 (FGF-23) concentrations [76], inactivating mutations in the SLC34A3 gene encoding NaPi-IIc in renal tubules (67) or elevated serum PTH (deficiency of vitamin D and calcium, vitamin D dependent rickets type 1 and type 2) [74,75]. Radiological Features of Rickets These depend on a number of factors including: age of the child, severity of rickets, rate of growth of long bones and presence and degree of secondary hyperparathyroidism [23]. As rickets is a disease of a growing child, radiological manifestations are more likely to be present at rapidly growing ends of long bones, e.g. the distal, rather than proximal, end of the femur. Thus, a good quality AP radiograph of the knee or PA of the wrist is helpful in the diagnosis of rickets. The earliest radiological sign of rickets is loss of the crisp line, produced by the zone of provisional calcification, at the interface of the epiphyseal growth plates and the metaphyses of long bones. This zone becomes frayed or “brush like”, and in more advanced stages of rickets it becomes concave or “cup” shaped (Fig. 12.17). The metaphysis also becomes wider (flared) than normal (Fig. 12.17A). These metaphyseal changes tend to be more marked in toddlers (Fig 12.17) than in adolescents (Fig. 12.18) with rickets. Rachitic bone is soft and bends; radiographs of legs show limb deformities of weight-bearing bones of genu valgum (knock knees) (Fig. 12.18) or genu varum (bowlegs). These deformities tend to modify the radiological features of rickets. For example, in genu varum, the mechanical axis shifts medially and this causes greater stress to be placed on the medial aspects of the femur and tibia. This results in more florid radiological changes of rickets on medial, rather than the lateral, aspects of metaphyses of these bones (Fig. 12.19). In severe cases of rickets, radiographs may show pathological fractures, through Looser’s zones (see Fig. 12.17C,D), which may be confused with child abuse [79,80].

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(A)

(C)

(D)

(B)

FIGURE 12.17 Vitamin D deficiency rickets in toddlers: radiographs of (A) the wrist which demonstrates cupping and fraying of the metaphyses of the radius and ulna; (B) the chest radiograph showing rachitic changes (expansion and poor mineralization) at the anterior ends of the ribs (rachitic ‘rosary’) and rickets at the left proximal humeral metaphysis; (C) forearm and (D) lateral femur with severe rickets, pathological fractures (through Looser’s zones) and intense secondary hyperparathyroidism indicated by hazy, disorganized trabecular pattern and cortical tunneling.

In rickets arising from conditions associated with secondary hyperparathyroidism, bones show generalized osteopenia with coarsening and haziness of the trabecular pattern and cortical tunneling (see Figs 12.17C,D; 12.20). Other radiological features of secondary hyperparathyroidism include subperiosteal and endosteal bone resorption, periosteal reaction along diaphyses of long bones and occasionally cystic bone lesions due to “brown tumors” (Fig. 12.20). Radiographs of children with X-linked hypophosphatemic rickets (XLH) do not show changes of hyperparathyroidism unless they have been treated with excessive amounts of inorganic phosphate supplements and inadequate amounts of calcitriol or alphacalcidiol (see Chapter 26).

These patients tend to have thicker cortices and coarse trabecular pattern, in spite of absence of secondary hyperparathyroidism (see Fig. 12.19). The earliest sign of healing of rickets is calcification of the zone of provisional calcification, resulting in sclerosis of the metaphyses (Fig. 12.21A,C). With appropriate treatment, there is gradual reversal of all the radiological features of rickets over 4e6 months (Fig. 12.21).

Other Conditions Fibrous Dysplasia Fibrous dysplasia (FD) (see Chapter 22) is a skeletal disorder caused by replacement of normal bone by

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(B)

(A)

FIGURE 12.18

Vitamin D deficiency rickets in an adolescent: AP radiographs of (A) the knee of a 10-year-old girl. There is genu valgum deformity (knock knees) due to the bones being soft and bending, and changes of rickets at the metaphyses of the femora and tibiae; (B) lower leg in an adolescent boy with genu valgum and mild changes of rickets at the metaphyses. In older children and adolescents the cupping and fraying of the metaphyses is less florid than in toddlers.

benign fibrous connective tissue containing trabeculae of immature, non-lamellar bone. FD may affect a single skeletal site (monostotic FD) or multiple sites (polyostotic [FD], McCuneeAlbright syndrome [MAS]) [81], usually in variable combinations with characteristic irregular pigmented cafe´-au-lait skin lesions and endocrine abnormalities (thyrotoxicosis, precocious puberty, acromegaly and Cushing’s syndrome). Both FD and MAS are caused by postzygotic activating missense mutations of the gene encoding for heterotrimeric G protein a-subunit (GNAS1) (see Chapter 22). The skeletal radiographic manifestations of MAS include “ground-glass” appearance, radiolucent lesions, endosteal resorption, deformity and pathological fracture through affected bone. These patients may also have hypophosphatemic rickets/osteomalacia, caused by excessive synthesis of FGF-23.

Osteoporosis and Fragility Fractures Assessment of surrogate measures of bone strength parameters, such as bone mineral content (BMC) and bone mineral density (BMD) measured by densitometric techniques (see Chapter 13) aid the assessment of

children with conditions associated with increased propensity to low trauma fracture. However, good quality conventional radiographs remain important in the initial evaluation of children who have suffered fragility fractures. In fact, vertebral fractures can occur despite normal spine BMD [82]. Detailed assessment and management of children with primary and secondary causes of osteoporosis (see Table 12.2), who are prone to fragility fractures, are discussed in Chapters 18 and 19. Long bone and vertebral fractures are an important cause of morbidity in children with chronic disorders, such as cerebral palsy, rheumatic diseases and those treated with oral glucocorticoids. Children and adolescents with cerebral palsy and other disabling neuromuscular disorders are prone to fractures that occur with minimal trauma, or during normal activities such as dressing. The skeleton of a growing healthy child continuously adapts to increasing mechanical loading from larger and stronger muscles by increasing bone mass and altering bone geometry, which increase bone strength. By contrast, disabled children with cerebral palsy or neuromuscular disorders, who have reduced muscle forces and inability to participate in normal load-bearing activities, fail to

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(A)

(B)

FIGURE 12.19 X-linked hypophosphatemic rickets (XLH): (A) standing AP radiograph of the legs of a girl showing genu varum (bowlegs), relatively thick cortices, lack of osteopenia and no radiological changes of secondary hyperparathyroidism. At the knee, changes of rickets are more florid on the medial aspects of the distal femoral and proximal tibial metaphyses. (B) AP radiograph of the knee of a boy showing genu varum deformity, relatively thick cortices, slightly coarse trabecular pattern, lack of osteopenia and no radiological changes of secondary hyperparathyroidism. The changes of rickets are more florid on the medial aspects of distal femoral and proximal tibial metaphyses.

generate strain magnitudes as occur in bones of healthy children [83]. This leads to reduced periosteal bone expansion resulting in narrow long bones (Fig. 12.22). Further immobilization, for example after surgery, results in endosteal bone loss with thinning of cortices. These narrow and thin long bones have increased propensity to fracture from bending and torsional loads. Those with impaired mobility (especially if they have never stood or walked), stiff or contracted joints, quadriplegia or with poor nutrition are at greater risk of such fragility fractures. The mid-diaphyses and metaphyses of the distal femur, proximal tibia and proximal humerus are common fracture sites. Immobilization after fracture or surgery is also an important risk factor for fracture (Fig. 12.22).

Vertebral fractures occur in children with both primary and secondary osteoporosis (see Table 12.2). The earliest radiological sign is thinning and loss of non-weightbearing horizontal trabecular struts in the vertebral bodies, resulting in prominence of remaining vertical trabeculae giving a striated appearance. The cortical shell and endplates often appear accentuated. Lateral spine radiographs may show changes consistent with fractures of vertebral bodies (Fig. 12.23). The diagnosis of vertebral fractures in children has generally been made by observational assessment from lateral spinal radiographs. In adults, there is an extensive literature on how vertebral fractures should be defined [84,85], as consistency is required in therapeutic trials assessing the efficacy of osteoporotic therapies. Methods which have been

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(A)

(B)

(C)

FIGURE 12.20 Rickets and secondary hyperparathyroidism. In chronic renal failure: radiographs of (A) AP knee of a 4-year-old girl with severe secondary hyperparathyroidism (serum PTH 541 pg/mL; reference value 10e60 pg/mL) showing rachitic changes at the distal femoral metaphyses, genu valgum and coarse trabecular pattern; (B) middle finger of a boy on hemodialysis showing subperiosteal erosions, diagnostic of hyperparathyroidism, which give the bones an irregular outline. In prematurity: (C) periosteal reaction along the forearm of an infant with secondary hyperparathyroidism due to prematurity, loss of small bowel due to necrotizing enterocolitis. Small cystic lesions in the distal humeral shaft are “brown tumors” of hyperparathyroidism.

applied are six point morphometry [86], the semiquantitative (SQ) method [87] and the algorithm-based qualitative (ABQ) technique [88]. Six point morphometry will not differentiate between vertebral fractures and vertebral deformities (e.g. Scheuermann’s disease, normal variants such as short vertebral height and “cupid’s bow”, rickets/osteomalacia) which is a problem in adults, and may also be so in children [84,85,89,90]. Careful scrutiny of the vertebral endplate helps to differentiate vertebral fractures, in which the endplate is irregular and depressed due to microfractures, from vertebral deformities in which the endplate is clear and well defined [88]. One of these quantitative methods (SQ) has recently been applied to the study of the prevalence of vertebral fracture in children treated with glucocorticoid therapy for treatment of rheumatic disorders and acute lymphoblastic leukemia in Canada (the Steroid-Associated Osteoporosis in the Pediatric Population [STOPP]) [91,92]. There have been technical developments in DXA (fan beam x-ray source and bank of detectors; “C” arm which can be moved through 90 ) which have not only shortened scan time and improved spatial resolution but also have enabled single and dual energy images of the

spine (PA and lateral) to be obtained for vertebral fracture assessment (VFA) [93,94]. This is used increasingly in adults, but to date there are no reports of its application in children, although there would be a strong rationale for doing so, as the radiation dose is considerably lower at approximately 1/100 of the doses involved in conventional spinal radiographs [95,96]. Occasionally, radiographs of children presenting with fractures of long bones, occurring after trivial injury, reveal a sclerosing bone disorder, such as osteopetrosis (see Fig. 12.16).

Osteopathy of Prematurity Essentially, the changes are those of rickets in a premature infant [97,98], and are usually identified between 6 and 12 weeks after birth (Fig. 12.24). The radiographic findings may be categorized into three grades of increasing severity: Grade 1: loss of dense white metaphyseal line, submetaphyseal lucency, cortical thinning. Grade 2: as in grade 1 with additional changes of rickets (frayed, irregular, cupped and splayed metaphyses).

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(A)

(B)

(C)

FIGURE 12.21 Healing of rickets with treatment: radiographs of (A) the wrist of a boy with vitamin D deficiency rickets showing sclerosis of metaphyseal region, 2 months after treatment with oral cholecalciferol and of forearm of a girl with vitamin D dependent rickets type 1 (B) before and (C) 5 months after treatment with calcitriol with almost complete reversal of severe radiological features of rickets, including healing of pathological fractures.

(A)

FIGURE 12.22

Osteoporosis: radiographs of (A) an infant with congenital muscular dystrophy showing generalized osteopenia and slender femoral diaphysis with thin cortices. The fracture in the distal femoral shaft arose during normal handling of the child. (B) The knee of a 12-year-old girl with cerebral palsy and bilateral hip dislocation; the fracture in the distal femoral shaft came to light on the day her hip spica was removed.

(B)

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(B)

FIGURE 12.23

Osteoporotic vertebral fractures: in a boy with Duchenne muscular dystrophy treated with oral glucocorticoids; (A) at baseline a lateral scan projection radiograph for QCT which shows osteopenia but with no evidence of vertebral fractures and (B) lateral spinal radiograph 2.5 years later; he has developed anterior compression fractures of several vertebral bodies. Both images show profound osteopenia with “picture framing” of the vertebral bodies due to accentuation of the cortices and endplates.

Grade 3: Rickets and fractures. Differential Diagnosis Child abuse; inclusion cell (I cell) disease.

A RADIOLOGICAL APPROACH TO NONACCIDENTAL INJURY Imaging Modalities

FIGURE 12.24 Osteopathy of prematurity: AP radiograph of a preterm infant on the special care baby unit with generalized osteopenia and multiple healing rib fractures due to osteopathy of prematurity.

Skeletal RNS requires expertise to produce the high quality images that are required, and may lead to false negatives at the metaphyses (“hot” in normal children) and skull (no increased tracer uptake in skull fractures), or false positive metaphyseal fractures (because of the normal increased tracer uptake at the metaphyses in children) (see Fig. 12.3). Such scans involve a high radiation dose as the bone marrow is irradiated and it is not possible to date the age of fractures. Although RN scans are helpful in identifying unsuspected rib and diaphyseal fractures, they should be used (if at all) as an adjunct to, and should not replace, the radiographic skeletal survey. Skeletal Survey

Generally, any child in whom child abuse is suspected will have radiographs of the skeleton and cross-sectional imaging (CT and/or MRI) of the brain. Cranial and abdominal US may be performed, depending on the protocols of individual departments and presentation of affected children. Increasingly, CT and MRI are being utilized as alternatives to radiography for the identification of skeletal injury, particularly in post-mortem cases, and both have a role when intra-abdominal injury is suspected.

When child abuse is suspected, a full skeletal survey should be performed according to relevant guidelines, but would usually include [99,100]: • • • • • • •

AP and left and right oblique radiographs of the chest AP and lateral skull AP limbs (or individual long bones) PA hands DP feet Lateral spine, including cervical spine AP abdomen, to include the pelvis and lower ribs

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A RADIOLOGICAL APPROACH TO NON-ACCIDENTAL INJURY

• PA chest radiograph in 10e14 days for identification of previously unidentified rib fractures. It is important that the imaging system used is of high technical quality, that there is careful positioning and xray collimation of each anatomical site imaged, that additional views (in different planes) are obtained, either of known or clinically suspicious sites of injury or of abnormal sites identified on the initial radiographs. Whenever possible, a pediatric radiologist should review the skeletal survey for appropriate image quality. Double reporting is highly recommended. In addition to the views listed above, individual cases may require any of the following: • Lateral radiographs to evaluate displacement of an identified shaft fracture • Coned views of metaphyses (particularly if poor views were obtained initially) • Towne’s (AP 30 ) view of the skull (for suspected occipital injury) • Delayed views of identified abnormality; these can confirm a fracture which might become more obvious with time due to osteolysis along the fracture line or callus formation, exclude a normal variant as a cause of the abnormality and aid in dating the fracture by the callus formation present • Skeletal survey of siblings and other children under the age of two years with the same carers. Post-Mortem Imaging A full skeletal survey should be performed as part of the forensic investigation into the cause of death of either a sudden unexpected death in infancy (SUDI) or an obvious case of physical abuse. Abnormal bones are then resected, decalcified and subjected to further high radiation and resolution imaging in a cabinet x-ray system (Faxitron x-ray LLC, Lincolnshire, IL, USA) and histopathological examination.

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radiologist to date the fracture. Soft tissue injury first becomes apparent radiographically around 12 hours after the fracture has occurred, increasing over the next 3 or 4 days. Soft tissue injury will then gradually subside, and should have resolved by 10 days after the initial trauma. METAPHYSEAL FRACTURES

Although traditionally divided into two types based on radiological appearance; namely “corner” and “bucket-handle” fractures, they are in fact the same lesion with the radiological appearance depending on the radiographic projection (Fig. 12.25). They result from direct shearing and/or twisting forces. Although metaphyseal fractures classically present in child abuse, they may also occur in rickets, scurvy, in some skeletal dysplasias and rarely in osteogenesis imperfecta. An important normal variant to consider is the metaphyseal spur. DIAPHYSEAL FRACTURES

These occur four times more frequently in abuse than metaphyseal fractures, but are of lower specificity for abuse than metaphyseal fractures because they also occur more commonly following accidental trauma. Any bone may be fractured, but the likelihood of abuse increases in a non-ambulant child, particularly when there is no history of injury, or an inappropriate or inconsistent history is given. Spiral fractures result from twisting forces, while transverse and oblique fractures occur secondary to direct impact or indirect forces as the limb is pulled or the child is swung. The major differential diagnoses are of a toddler’s traumatic fracture of the tibia and osteogenesis imperfecta. The features of other underlying conditions such as rickets or scurvy, if of sufficient severity to lead to fractures, will be apparent on the radiographs. SKULL FRACTURES

Specific Sites Depending on the age of the study population, an estimated 10e70% of physically abused children manifest some form of skeletal trauma. Furthermore, fractures are second only to soft tissue injury as the commonest presentation of physical child abuse. SOFT TISSUE INJURY

Hemorrhage associated with a fracture seeps into the soft tissues immediately beginning to obliterate the fascial planes between subcutaneous fat and muscle. This is then followed by edema/inflammation, which further obliterates the soft tissue planes. There may not necessarily be an underlying fracture. However, when there is a fracture, loss of soft tissue planes allows the

Features of a skull fracture that increase the suspicion of abuse are those reflecting high impact trauma, and include non-parietal fractures, complex fractures (especially if both sides of the skull are affected), multiple fractures, fractures greater than 3 mm in width, growing fractures, depressed fractures (especially occipital) and fractures associated with intracranial injury. Skull fractures (see Fig. 12.25A) may occur in the absence of intracranial injury, and conversely intracranial injury may occur in the absence of a skull fracture. CT of the head is therefore mandatory whenever abuse is suspected. Skull radiographs may be omitted from the skeletal survey only if thin section MDCT with 3D reformatting on a bone algorithm is performed to obtain adequate spatial resolution.

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(A)

(B)

(C)

(D)

FIGURE 12.25 Non-accidental injury: fractures are often multiple and of varying ages: (A) lateral skull in a 3-week-old abused male with a right parietal skull fracture. Note the associated scalp swelling indicating that this fracture is less than 10 days old; (B) AP femora showing periosteal reaction along both femoral shafts. Metaphyseal fractures do not heal with periosteal reaction. The periosteum surrounding both femora has been damaged during the same gripping and twisting forces that caused the metaphyseal fractures. Subperiosteal new bone formation differs from physiological periosteal reaction by extending beyond the diaphysis and being more than 4 mm thick; (C) right and (D) left AP femora in an 11-week-old abused infant. There are healing metaphyseal fractures of both distal femora, both proximal tibiae and right distal tibia. Note also the angulated fracture of the left proximal fibula. Metaphyseal irregularity of the left proximal fibula may be related to the angulated metadiaphyseal fracture or represent a separate metaphyseal fractures. RIB FRACTURES

Because they are rarely seen in infants, rib fractures, whether single or multiple, have a high specificity for abuse. Fractures of the shafts result from compressive

forces in the anteroposterior or side-to-side direction. Costochondral fractures usually result from direct blows and have a worse prognosis, being associated with visceral or intra-abdominal solid organ damage.

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Cardiopulmonary resuscitation causes rib fractures in approximately 1% of infants, and then usually affects the anterior arcs of third to fifth ribs. Acute rib fractures are easily missed on initial chest radiography and, in surviving patients, a follow-up radiograph in 10e14 days significantly increases the identification of rib fractures due to the presence of healing callus. The differential diagnosis includes accidental trauma, cardiopulmonary resuscitation and osteogenesis imperfecta. SUBPERIOSTEAL NEW BONE FORMATION

In the context of abuse, subperiosteal new bone formation occurs either as a result of the normal healing process of a shaft fracture or of stripping of the periosteum as the affected limb is exposed to gripping and twisting forces (see Fig. 12.25B). Important differential diagnoses include physiological periosteal reaction (symmetrical, appears between the ages of 4 weeks and 4 months and is restricted to the shafts). OTHER FRACTURES

Fractures that are present less often, but which have a high specificity for abuse, include fractures of the acromion and body of the scapula, vertebral bodies, metacarpals, superior pubic rami and metatarsals and epiphyseal fracture dislocations. Radiological Dating of Fractures This is by no means an exact science, and varies depending on the age of the child (healing is faster in younger children) and other factors such as whether or not the fracture has been stabilized. When dating fractures a time range must always be cited and the following is a guide (see Fig 12.25): Soft tissue swelling: 12 hours to 10 days. Subperiosteal new bone: the earliest this is present is 7 days following the traumatic episode. Irregular fracture margin: the initially distinct margins of an acute fracture become irregular and widened as a result of macrophage activity. The earliest this is seen is 7 days. Soft callus: a gradual and ongoing process of osteoid calcification is present from about 1e6 weeks. Hard callus: increasing consolidation of the fracture with the fracture line disappearing about 6e8 weeks after the fracture occurred. Remodeling: the process by which the normal alignment and shape of the bone is restored. In undisplaced fractures of long bones and ribs, remodeling is more or less complete by 12 weeks after a fracture. Metaphyseal fractures: these do not heal with callus formation. The fractured bone is gradually incorporated into the adjacent bone, a process which is usually

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complete by 4 weeks and certainly by 6 weeks after the fracture. Skull fractures: cannot be dated. Overlying scalp swelling may infer a fracture that has occurred in the preceding 10 days (see Fig. 12.25A).

References [1] Swischuk LE. Emergency pediatric imaging: current status and update. Semin Ultrasound CT MR 2007;28:158e68. [2] Carty H, Brunelle F, Stringer DA, Kao S, editors. Imaging Children. 2nd ed. Edinburgh: Elsevier Churchill Livingstone; 2005. [3] Jackson S, Adams JE. Imaging techniques, interpretation and strategies. In: Bullough PG, editor. Orthopaedic Pathology. 5th ed. St Louis: Mosby Elsevier; 2009. p. 53e79. [4] Willis CE. Strategies for dose reduction in ordinary radiographic examinations using CR and DR. Pediatr Radiol 2004;34(Suppl. 3):S196e200. [5] Cowen AR, Davies AG, Kengyelics SM. Advances in computed radiography systems and their physical imaging characteristics. Clin Radiol 2007;62:1132e41. [6] Hounsfield GN. Computerized transverse axial scanning (tomography). 1. Description of system. Br J Radiol 1973;46:1016e22. [7] Salamipour H, Jimenez RM, Brec SL, Chapman VM, Kalra MK, Jaramillo D. Multidetector row CT in pediatric musculoskeletal imaging. Pediatr Radiol 2005;35:555e64. [8] Fayad LM, Johnson P, Fishman EK. Multidetector CT of musculoskeletal disease in the pediatric patient: principles, techniques, and clinical applications. Radiographics 2005;25:603e18. [9] Kalender WA, Buchenau S, Deak P, et al. Technical approaches to the optimisation of CT. Phys Med 2008;24:71e9. [10] Kalender WA, Quick HH. Recent advances in medical physics. Eur Radiol 2011;21:501e4. [11] Mazonakis M, Damilakis J, Raissaki M, Gourtsoyiannis N. Radiation dose and cancer risk to children undergoing skull radiography. Pediatr Radiol 2004;34:624e9. [12] Huda W. Assessment of the problem: pediatric doses in screen-film and digital radiography. Pediatr Radiol 2004;34(Suppl. 3):S173e82. [13] Hintenlang K, Williams J, Hintenlang D. A survey of radiation dose associated with pediatric plain-film chest x-ray examinations. Pediatr Radiol 2002;32:771e7. [14] Geleijns J, Broerse J, van Vliet M, Lopez M, Zonderland H. Assessment of effective dose in paediatric radiology. A survey at 14 Dutch hospitals. Radiat Prot Dosim 2000;90:135e40. [15] Yakoumakis EN, Tsalafoutas IA, Aliberti M, et al. Radiation doses in common x-ray examinations carried out in two dedicated paediatric hospitals. Radiat Prot Dosim 2007;124: 348e52. [16] Tzedakis A, Damilakis J, Perisinakis K, Karantanas A, Karabekios S, Gourtsoyiannis N. Influence of z overscanning on normalized effective doses calculated for pediatric patients undergoing multidetector CT examinations. Med Phys 2007;34:1163e75. [17] Smith T, Evans K, Lythgoe M, Anderson P, Gordon I. Dosimetry of pediatric radiopharmaceuticals: uniformity of effective dose and a simple aid for its estimation. J Nucl Med 1997;38:1982e7. [18] Blake G, Naeem M, Boutros M. Comparison of effective dose to children and adults from dual x-ray absorptiometry examinations. Bone 2006;38:935e42.

PEDIATRIC BONE

306

12. RADIOLOGY

[19] Burrows M, Liu D, McKay H. High resolution peripheral QCT imaging of bone microstructure in adolescents. Osteoporos Int 2010;21:515e20. [20] Adams JE. Quantitative computed tomography. Eur J Radiol 2009;71:415e24. [21] Damilakis J, Adams JE, Guglielmi G, Link TM. Radiation exposure in x-ray-based imaging techniques used in osteoporosis. Eur Radiol 2010;20:2707e14. [22] Nadel HR. Pediatric bone scintigraphy update. Semin Nucl Med 2010;40:31e40. [23] Adams JE. Diagnosis and management e radiology. In: Feldman D, Pike JW, Adams JS, editors. Vitamin D. 3rd ed. San Diego: Elsevier; 2011. In press. [24] Jan de Beur SM, Streeten EA, Civelek AC, et al. Localisation of mesenchymal tumours by somatostatin receptor imaging. Lancet 2002;359:761e3. [25] Hesse E, Moessinger E, Rosenthal H, et al. Oncogenic osteomalacia: exact tumor localization by co-registration of positron emission and computed tomography. J Bone Miner Res 2007;22:158e62. [26] Karmazyn B. Ultrasound of pediatric musculoskeletal disease: from head to toe. Semin Ultrasound CT MR 2011;32:142e50. [27] Keller MS. Musculoskeletal sonography in the neonate and infant. Pediatr Radiol 2005;35:1167e73. [28] Jaramillo D, Laor T. Pediatric musculoskeletal MRI: basic principles to optimize success. Pediatr Radiol 2008;38:379e91. [29] Balassy C, Ho¨rmann M. Role of MRI in paediatric musculoskeletal conditions. Eur J Radiol 2008;68:245e58. [30] Darge K, Jaramillo D, Siegel MJ. Whole-body MRI in children: current status and future applications. Eur J Radiol 2008;68:289e98. [31] MacKenzie JD, Vasanawala SS. State-of-the-art in pediatric body and musculoskeletal magnetic resonance imaging. Semin Ultrasound CT MR 2010;31:86e99. [32] Meyer JS, Jaramillo D. Musculoskeletal MR imaging at 3 T. Magn Reson Imaging Clin N Am 2008;16:533e45. [33] Adams JE, Bishop N. Dual energy x-ray absorptiometry (DXA) in adults and children. In: Rosen C, editor. Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. 7th ed. American Society of Bone and Mineral Research (ASBMR); 2009. p. 152e8. [34] Zemel BS, Stallings VA, Leonard MB, et al. Revised pediatric reference data for the lateral distal femur measured by Hologic Discovery/Delphi dual-energy x-ray absorptiometry. J Clin Densitom 2009;12:207e18. [35] Bishop N, Braillon P, Burnham J, et al. Dual-energy x-ray aborptiometry assessment in children and adolescents with diseases that may affect the skeleton: the 2007 ISCD Pediatric Official Positions. J Clin Densitom 2008;11:29e42. [36] Adams JE. Dual energy x-ray absorptiometry. In: Grampp S, editor. Radiology of Osteoporosis. 2nd ed. Berlin: Springer; 2008. p. 105e24. [37] Gordon CM, Bachrach LK, Carpenter TO, et al. Dual energy xray absorptiometry interpretation and reporting in children and adolescents: the 2007 ISCD Pediatric Official Positions. J Clin Densitom 2008;11:43e58. [38] Ward KA, Ashby RL, Roberts SA, Adams JE, Mughal MZ. UK reference data for the Hologic QDR Discovery dual energy xray absorptiometry scanner in healthy children aged 6e17 years. Arch Dis Child 2007;92:53e9. [39] Krug R, Burghardt AJ, Majumdar S, Link TM. High-resolution imaging techniques for the assessment of osteoporosis. Radiol Clin North Am 2010;48:601e21.

[40] McKay H, Liu D, Egeli D, Boyd S, Burrows M. Physical activity positively predicts bone architecture and bone strength in adolescent males and females. Acta Paediatr 2011;100:97e101. [41] Adams JE. Radiogrammetry and radiographic absorptiometry. Radiol Clin North Am 2010;48:531e40. [42] van Rijn RR, Grootfaam DS, Lequin MH, et al. Digital radiogrammetry of the hand in a pediatric and adolescent Dutch Caucasian population: normative data and measurements in children with inflammatory bowel disease and juvenile chronic arthritis. Calcif Tissue Int 2004;74:342e50. [43] Tanner J, Whitehouse R, Cameron N, Marshall W, Healy M, Goldstein H. Assessment of Skeletal Maturity and Prediction of Adult Height (TW2 Method). 2nd ed. London: Academic Press, Harcourt Brace Jovanovich; 1983. [44] Tanner J, Healy M, Goldstein H, Cameron N. Assessment of Skeletal Maturity and Prediction of Adult Height (TW3 Method). 3rd ed. London: Saunders; 2001. [45] Greulich W, Pyle S. Radiographic Atlas of Skeletal Development of the Hand and Wrist. 2nd ed. Stanford: Stanford University Press; 1959. [46] Roche A, Chumlea W, Thissen D. Assessing the Skeletal Maturity of the Hand-wrist: FELS method. Springfield, IL: Charles C. Thomas; 1988. [47] Bull RK, Edwards PD, Kemp PM, Fry S, Hughes IA. Bone age assessment: a large scale comparison of the Greulich and Pyle, and Tanner and Whitehouse (TW2) methods. Arch Dis Child 1999;81:172e3. [48] Vignolo M, Milani S, Cerbello G, Coroli P, Dibattista E, Aicardi G. Fels, Greulich-Pyle, and Tanner-Whitehouse boneage assessments in a group of Italian children and adolescents. Am J Hum Biol 1992;4:493e500. [49] Thodberg HH. Clinical review: an automated method for determination of bone age. J Clin Endocrinol Metab 2009;94:2239e44. [50] Lancini F, Madsen MT, Kao S. The effects of radiation on the fetus and child. In: Carty H, Brunelle F, Stringer DA, Kao S, editors. Imaging Children. 2nd ed. Edinburgh: Elsevier Science Ltd; 2005. p. 7e22. [51] Huda W. Radiation dose and image quality. In: Carty H, Brunelle F, Stringer DA, Kao S, editors. Imaging Children. 2nd ed. Edinburgh: Elsevier Science Ltd; 2005. p. 23e31. [52] Dorfman AL, Fazel R, Einstein AJ, et al. Use of medical imaging procedures with ionizing radiation in children: a populationbased study. Arch Pediatr Adolesc Med 2011;165:458e60. [53] Strauss KJ, Kaste SC. ALARA in pediatric interventional and fluoroscopic imaging: striving to keep radiation doses as low as possible during fluoroscopy of pediatric patients e a white paper executive summary. J Am Coll Radiol 2006;3:686e8. [54] Mazrani W, McHugh K, Marsden PJ. The radiation burden of radiological investigations. Arch Dis Child 2007;92:1127e31. [55] Offiah AC, Hall CM. Radiological diagnosis of constitutional disorders of bone. As easy as A, B, C? Pediatr Radiol 2003;33: 153e61. [56] http://openlibrary.org/books/OL115439M/London_dysmor phology_database. [57] http://www.esdn.org/eug/Home accessed 03/04/2011. [58] Morgan JA, Marcus PS. Prenatal diagnosis and management of intrauterine fracture. Obstet Gynecol Surv 2010;65:249e59. [59] Van Dijk FS, Pals G, Van Rijn RR, Nikkels PGJ, Cobben JM. Classification of osteogenesis imperfecta revisited. Eur J Med Genet 2010;53:1e5. [60] Michou L, Brown JP. Genetics of bone diseases: Paget’s disease, fibrous dysplasia, osteopetrosis, and osteogenesis imperfecta. Joint Bone Spine 2010;78:252e8.

PEDIATRIC BONE

307

REFERENCES

[61] Bishop N. Characterising and treating osteogenesis imperfecta. Early Hum Dev 2010;86:743e6. [62] Shaw NJ, Bishop NJ. Bisphosphonate treatment of bone disease. Arch Dis Child 2005;90:494e9. [63] Rauch F, Glorieux FH. Treatment of children with osteogenesis imperfecta. Curr Osteoporos Rep 2006;4:159e64. [64] Bishop N. Primary osteoporosis. Endocr Dev 2009;16:157e69. [65] Land C, Rauch F, Glorieux FH. Cyclical intravenous pamidronate treatment affects metaphyseal modeling in growing patients with osteogenesis imperfecta. J Bone Miner Res 2006;21:374e9. [66] Al Muderis M, Azzopardi T, Cundy P. Zebra lines of pamidronate therapy in children. J Bone Joint Surg Am 2007;89:1511e6. [67] Bastepe M, Juppner H. Inherited hypophosphatemic disorders in children and the evolving mechanisms of phosphate regulation. Rev Endoc Metab Disord 2008;9:171e80. [68] Wilson LC, Hall CM. Albright’s hereditary osteodystrophy and pseudohypoparathyroidism. Semin Musculoskelet Radiol 2002;6:447e57. [69] E. Mornet. Hypophosphatasia. Best Pract Res Clin Rheumatol 2008;1:113e27. [70] Cundy T. Idiopathic hyperphosphatasia. Semin Musculoskelet Radiol 2002;6:307e12. [71] de Vernejoul MC, Kornak U. Heritable sclerosing bone disorders: presentation and new molecular mechanisms. Ann NY Acad Sci 2010;1192:269e77. [72] Stoker DJ. Osteopetrosis. Semin Musculoskelet Radiol 2002;6:299e305. [73] Soliman AT, Ramadan MA, Sherif A, Aziz Bedair ES, Rizk MM. Pycnodysostosis: clinical, radiologic and endocrine evaluation and linear growth after growth hormone therapy. Metabolism 2001;50:905e11. [74] Pettifor JM. Vitamin D &/or calcium deficiency rickets in infants & children: a global perspective. Indian J Med Res 2008;127:245e9. [75] Malloy PJ, Feldman D. Genetic disorders and defects in vitamin D action. Endocrinol Metab Clin North Am 2010;39:333e46. [76] Prie´ D, Friedlander G. Genetic disorders of renal phosphate transport. N Engl J Med 2010;362:2399e409. [77] Sabbagh Y, Carpenter TO, Demay MB. Hypophosphatemia leads to rickets by impairing caspase-mediated apoptosis of hypertrophic chondrocytes. Proc Natl Acad Sci USA 2005;102:9637e42. [78] Tiosano D, Hochberg Z. Hypophosphatemia: the common denominator of all rickets. J Bone Miner Metab 2009;27:392e401. [79] Senniappan S, Elazabi A, Doughty I, Mughal MZ. Case 2: Fractures in under-6-month-old exclusively breast-fed infants born to immigrant parents: nonaccidental injury? (case presentation). Diagnosis: Pathological fractures secondary to vitamin D deficiency rickets in under-6-months-old, exclusively breast-fed infants, born to immigrant parents. Acta Paediatr 2008;97:836e7. 992e3. [80] Chapman T, Sugar N, Done S, Marasigan J, Wambold N, Feldman K. Fractures in infants and toddlers with rickets. Pediatr Radiol 2010;40:1184e9. [81] Chapurlat RD, Orcel P. Fibrous dysplasia of bone and McCuneAlbright syndrome. Best Pract Res Clin Rheumatol 2008;22:55e69. [82] Sbrocchi AM, Rauch F, Matzinger M, Feber J, Ward LM. Vertebral fractures despite normal spine bone mineral density

[83]

[84] [85]

[86]

[87]

[88]

[89]

[90]

[91]

[92]

[93]

[94]

[95]

[96] [97]

[98] [99] [100]

in a boy with nephrotic syndrome. Pediatr Nephrol 2011;26:139e42. Ward KA, Caulton JM, Adams JE, Mughal MZ. Perspective: cerebral palsy as a model of bone development in the absence of postnatal mechanical factors. J Musculoskelet Neuronal Interact 2006;6:154e9. Ferrar L, Jiang G, Adams J, Eastell R. Identification of vertebral fractures: an update. Osteoporos Int 2005;16:717e28. Link TM, Guglielmi G, van Kuijk C, Adams JE. Radiologic assessment of osteoporotic vertebral fractures: diagnostic and prognostic implications. Eur Radiol 2005;15:1521e32. Guglielmi G, Diacinti D, van Kuijk C, et al. Vertebral morphometry: current methods and recent advances. Eur Radiol 2008;18:1484e96. Genant HK, Wu CY, van Kuijk C, Nevitt MC. Vertebral fracture assessment using a semiquantitative technique. J Bone Miner Res 1993;8:1137e48. Jiang G, Eastell R, Barrington NA, Ferrar L. Comparison of methods for the visual identification of prevalent vertebral fracture in osteoporosis. Osteoporos Int 2004;15:887e96. Adams JE. Osteoporosis. In: Pope TL, Bloem HL, Beltran J, Morrison WB, Wilson D, editors. Imaging of the Musculoskeletal System. Philadelphia: Saunders Elsevier; 2008. p. 1489e508. Adams JE. Osteoporosis. In: Weissman B, editor. Imaging of Arthritis and Metabolic Bone Disease. Philadelphia: Saunders Elsevier; 2009. p. 601e21. Halton J, Gaboury I, Grant R, et al. Canadian STOPP Consortium. Advanced vertebral fracture among newly diagnosed children with acute lymphoblastic leukemia: results of the Canadian Steroid-Associated Osteoporosis in the Pediatric Population (STOPP) research program. J Bone Miner Res 2009;24:1326e34. Huber AM, Gaboury I, Cabral DA, et al. Canadian SteroidAssociated Osteoporosis in the Pediatric Population (STOPP) Consortium. Prevalent vertebral fractures among children initiating glucocorticoid therapy for the treatment of rheumatic disorders. Arthritis Care Res (Hoboken) 2010;62:516e26. Genant HK, Li J, Wu CY, Shepherd JA. Vertebral fractures in osteoporosis: a new method for clinical assessment. J Clin Densitom 2000;3:281e90. Rea JA, Li J, Blake GM, Steiger P, Genant HK, Fogelman I. Visual assessment of vertebral deformity by x-ray absorptiometry: a highly predictive method to exclude vertebral deformity. Osteoporos Int 2000;11:660e8. Schousboe JT, Vokes T, Broy SB, et al. Vertebral Fracture Assessment: the 2007 ISCD Official Positions. J Clin Densitom 2008;11:92e108. Lewiecki EM. Bone densitometry and vertebral fracture assessment. Curr Osteoporos Rep 2010;8:123e30. Koo WW, Gupta JM, Nayanar VV, Wilkinson M, Posen S. Skeletal changes in preterm infants. Arch Dis Child 1982;57:447e52. Sharp M. Bone disease of prematurity. Early Hum Dev 2007;83:653e8. Offiah AC, Hall CM, editors. Radiological Atlas of Child Abuse. Abingdon: Radcliffe Publishing Ltd; 2009. Offiah A, van Rijn RR, Perez-Rossello JM, Kleinman PK. Skeletal imaging of child abuse (non-accidental injury). Pediatr Radiol 2009;39:461e70.

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Non-invasive Techniques for Bone Mass Measurement Mary B. Leonard 1, Laura K. Bachrach 2 1

Associate Professor of Pediatrics and Epidemiology, Department of Pediatrics, The Children’s Hospital of Philadelphia, Department of Biostatistics and Epidemiology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA 2 Professor of Pediatrics, Department of Pediatrics, Stanford University School of Medicine, Palo Alto, CA, USA

INTRODUCTION Bone mass is a function of bone size and volumetric bone mineral density (BMD) and is a key determinant of bone strength. Peak bone mass is established in early adulthood and depends on the bone mass acquired throughout skeletal growth and development. Greater peak bone mass counteracts the inevitable bone loss due to aging, menopause, and varied chronic diseases in adulthood. Therefore, bone mass accrual during childhood and adolescence has important implications for lifelong bone health. In 2007, the International Society of Clinical Densitometry conducted a Pediatric Position Development Conference to address skeletal health assessment in children and adolescents [1e5]. Although heritability estimates for bone mass, structure and density range from 45 to 85%, currently identified bone genetic markers explain only a small portion of the variation in individual bone mass [6e9]. Many additional factors influence bone acquisition during infancy and childhood, such as sex, the timing of the onset of puberty, calcium and vitamin D nutrition, physical activity and obesity. Epidemiology studies in large cohorts have provided insights into determinants of bone mass during childhood. Recent studies have highlighted the importance of the intrauterine environment and maternal factors, such as maternal smoking, physical activity, and nutrition (especially calcium and vitamin D) on fetal bone acquisition and long-term bone health [10,11]. Finally, randomized clinical trials have demonstrated that physical activity and calcium intake impact bone acquisition in children. Chronic diseases during childhood and adolescence pose numerous threats to bone acquisition [12]. The

Pediatric Bone, Second Edition DOI: 10.1016/B978-0-12-382040-2.10013-9

impact may be immediate, resulting in fragility fractures, or delayed, caused by suboptimal peak bone mass accrual with subsequent fractures in adulthood. Risk factors for impaired bone accrual include malnutrition, cachexia, endocrine disturbances, and chronic inflammation. Mobility and biomechanical loading are important determinants of bone accrual and may be compromised in children with prolonged illness. Glucocorticoids, the mainstay of treatment in numerous pediatric illnesses, inhibit bone formation. Other therapies, such as anticonvulsants, immunosuppressive medications, chemotherapy, and radiation therapy, also adversely affect bone during growth.

INDICATIONS FOR BONE MASS MEASUREMENTS As recognition of the importance of bone accrual during growth and development has increased, so has the demand for non-invasive methods to assess bone mass in children and adolescents. Researchers have sought a means to define the tempo and magnitude of bone accrual and the factors modulating peak bone mass in healthy youth and in chronic disease. With this information, investigators hope to develop interventions that will optimize bone accrual in healthy children. For clinicians, densitometry is viewed as a means of screening the bone health of young patients with one of myriad chronic diseases associated with low bone mass and fractures early in life. The goal of bone assessment in this setting is to identify patients at risk for skeletal fragility and to monitor the response to therapy.

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In 2007, the International Society of Clinical Densitometry (ISCD) conducted a Pediatric Position Development Conference to address skeletal health assessment in children and adolescents, ages 5 to 19 years (www. iscd.org). The Conference culminated in a series of publications addressing dual energy x-ray absorptiometry (DXA) prediction of fracture and definition of osteoporosis; DXA assessment in pediatric diseases that may affect the skeleton; DXA interpretation and reporting; and peripheral quantitative computed tomography (pQCT) measurement [1e5]. The guidelines recommended DXA scans as part of a comprehensive skeletal health assessment in patients at increased risk of fracture. Specifically, the guidelines recommended DXA assessment at the time of clinical presentation in children with primary bone diseases, or in those with potential secondary bone disease, such as those due to chronic inflammatory disease, endocrine disturbances, or childhood cancer. DXA assessment was recommended at fracture presentation in children with chronic immobilization due to neuromuscular disease if contractures did not prevent the safe and appropriate positioning of the child. Finally, the recommended minimum time interval for repeating a bone density measurement to monitor diseases processes or treatment with a bone-active medication was 6 months. The sections below provide further details regarding other aspects of the official positions.

ASSESSMENT OF BONE QUALITY IN CHILDREN The 2000 National Institutes of Health Osteoporosis Prevention, Diagnosis, and Therapy Consensus Development Conference Statement defined osteoporosis as a skeletal disorder characterized by compromised bone strength predisposing to an increased risk of fracture [13]. Bone strength reflects the integration of two main features: BMD and bone quality. Bone quality refers to bone architecture, turnover, damage accumulation, and mineralization. A variety of non-invasive methods is available to assess the peripheral, central, or entire skeleton. The vast majority of studies that assessed bone acquisition in neonates, infants and children were based on DXA measures of bone mineral content (BMC), projected bone area, and areal bone mineral density (g/cm2). More recently, studies utilizing QCT in the spine and pQCT in the radius and tibia have characterized trabecular and cortical volumetric BMD (g/cm3) and cortical geometry in healthy children and in those with chronic disease. Finally, advanced imaging techniques, such as high-resolution pQCT (HR-pQCT) can be used to assess trabecular microarchitecture. While advances in the non-invasive assessment of bone mass have provided greater insight into components of bone

quality in children, these methods are not able to assess bone turnover, microdamage accumulation and mineralization. Rauch and Schoenau proposed the following approach to the assessment of BMD during childhood and adolescence [14]. Volumetric BMD is considered in three distinct levels. First, material BMD reflects the degree of mineralization of the organic bone matrix and is the amount of mineral divided by the volume of the bone matrix, excluding marrow spaces, osteonal canals, lacunae, and canaliculi. Assessment of material BMD requires a bone biopsy specimen and can be measured using quantitative backscattered electron imaging [15]. Second, compartment BMD is the amount of mineral divided by the volume of the trabecular or cortical compartments, including marrow spaces, osteonal canals, lacunae, and canaliculi, and is a function of the material BMD and bone volume fraction. The trabecular compartment is defined as the space within the endocortical surface; the cortical compartment is limited by the periosteal and endosteal surfaces. Trabecular and cortical compartment BMD can be measured by QCT techniques. Third, total BMD is the amount of mineral divided by the volume enclosed by the periosteal bone surface. These definitions are illustrated in Figure 13.1. Importantly, DXA does not capture any of these three levels of BMD because DXA is a two-dimensional technique that measures the BMC within the projected bone area, generating areal BMD. The implications of this distinction are discussed in further detail below. Whole bone structural strength is determined not only by BMD, but also by the geometry of the bone. Bone size and the distribution of bone mass influence the resistance of bone to fracture. Cortical bone mass is distributed about a central axis to provide maximum strength with a minimum of material [16]. For example, the section modulus provides an estimate of resistance to torsional and flexural stress. Section modulus of a bone relates the periosteal radius (Rp) to the endosteal radius (Re) and is proportional to [(Rp4
Re4)/Rp], highlighting the exponential effect of small changes in bone dimensions on strength. Currently available non-invasive techniques vary considerably in their ability to quantify cortical bone geometry. Whole body DXA bone area relative to height has been proposed as an estimate of periosteal dimensions [17,18]. Similarly, specialized software, termed hip structural analysis (HSA) [19], has been used to estimate geometric parameters such as the moment of inertia from conventional DXA data. In contrast, QCT techniques provide direct measures of periosteal and endosteal circumference and can be used to estimate section modulus and other measures of bone strength that correlate well with in vitro fracture studies [20].

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FIGURE 13.1 Figures AeE illustrate the three levels of volumetric BMD. The mineral mass, shown in gray, that determines the material BMD and compartment BMD in trabecular (A and B) and cortical (C and D) bone is identical (mass 1 ¼ mass 2), but the volumes differ. The compartment volumes include marrow space (B) and osteonal canals, lacunae and canaliculi (D); therefore, the material BMD is greater than the compartment BMD. The total BMD (E) can be applied to the entire bone, a portion of the bone (e.g. the distal end) or a section through the bone. (From Rauch F, Schoenau E. Changes in bone density during childhood and adolescence: an approach based on bone’s biological organization. J Bone Miner Res. 2001;16:597-604.)

The interpretation of bone measurements in children is complicated by linear growth and sex-, race- and maturation- specific increases in trabecular and cortical volumetric BMD and cortical geometry e with important implications for bone strength [21e23]. These processes evolve at varying rates in different regions of the skeleton, with appendicular growth preceding spinal mineral acquisition [24]. Furthermore, within a given region of interest, trabecular and cortical compartments respond variably to sex steroids, calcium intake, mechanical loading, diseases and medications. Therefore, the accurate interpretation of bone mass measurements in children requires consideration of these factors.

CLASSIFICATION OF BONE HEALTH IN CHILDREN DXA is widely accepted as a quantitative measure of skeletal status. In older adults, DXA estimates of areal BMD are sufficiently robust predictors of osteoporotic fractures that can be used to define the disease. The World Health Organization criteria for the diagnosis of osteoporosis in adults is based on a T-score, the comparison of

a DXA BMD result with the average BMD of young adults at the time of peak bone mass [25]. The risk of bone fractures is estimated to double with each standard deviation (SD) that DXA BMD falls below the mean. The current ISCD Adult Official Positions concluded that osteoporosis may be diagnosed in postmenopausal women and in men aged 50 and older if the T-score of the lumbar spine, total hip or femoral neck is
2.5 or less [26]. While the T-score is a standard component of DXA BMD results, it is clearly inappropriate to assess skeletal health in children through comparison with peak adult bone mass. Rather, children are assessed relative to age, bone size, or body size, expressed as a Z-score. In adults, a history of low impact fractures is part of the criteria for diagnosing severe osteoporosis. Low impact fractures are defined as fractures that occur after a fall from standing height or less. This definition is often difficult to apply to fractures in children that occur during play or sports activities, and there are no established definitions of low impact fractures in children. Despite the growing body of published normative DXA data in children, there are insufficient data to support a definition of osteoporosis in children based on DXA results. Fractures occur commonly in otherwise healthy children with a peak incidence during early

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adolescence around the time of the pubertal growth spurt [27]. Peak gains in bone size preceded peak gains in BMC in a longitudinal sample of boys and girls, supporting the theory that the dissociation between skeletal expansion and skeletal mineralization results in a period of relative bone weakness [28]. The data presented in the DXA Fracture Discrimination section below suggest that low DXA BMD and BMC are contributing factors for fractures in healthy children; however, bone geometry and non-skeletal factors such as sports participation, body size, and sedentary activities also contribute to fracture risk. Furthermore, the relations between DXA BMC, bone geometry and fracture risk in children with chronic illness may be different than those observed in healthy children and have not been adequately characterized. The dissociation between DXA BMD and fracture risk in chronic disease is illustrated in adults treated with glucocorticoids; at similar levels of DXA BMD, postmenopausal women treated with glucocorticoids, as compared with non-users of glucocorticoids, had significantly greater risks of fracture [29]. In the absence of data to define a fracture threshold in children, the ISCD Pediatric Position Development Conference advised the following [4]. First, the diagnosis of osteoporosis in children and adolescents should not be made on the basis of densitometric criteria alone. Rather, the diagnosis requires the presence of both a clinically significant fracture history, and low bone mass. A clinically significant fracture history is one or more of the following: a long bone fracture of the lower extremities; a vertebral compression fracture; and two or more long bone fractures of the upper extremities. And, low bone mass is defined as a DXA BMC or areal BMD Z-score that is less than or equal to
2.0, adjusted for age, sex, and body size, as appropriate. While these guidelines represent an important first step in the classification of bone health in children, the utility of this approach is not yet proven and the appropriate adjustments for body size have not been established.

RADIATION DOSE IN BONE DENSITOMETRY Radiation exposure is an important consideration in pediatric diagnostic imaging. The 11th report on carcinogens published in 2005 by the US Department of Health and Human Services concluded that “x-radiation and gamma radiation are known to be human carcinogens based on sufficient evidence in humans” [30]. In children, this is especially important as children are more susceptible to radiation-induced cancer and have a longer life expectancy, resulting in a greater risk of developing radiation-induced fatal cancer [31]. Table 13.1 summarizes the effective dose for the different imaging modalities

TABLE 13.1

Values of Effective Doses for Irradiation from Different Sources

Type of irradiation

Effective dose (mSv)

Hand radiograph for radiogrammetry [33]

<0.20

DXA [34] Posteroanterior lumbar spinea 5 year old

9.1

10 year old

7.1

15 year old

5.0 a

Proximal femur 5 year old

7.4

10 year old

5.9

15 year old

3.9

b

Total body

5 year old

5.2e10.5

10 year old

4.8e9.6

15 year old

4.2e8.4

QCT bone mineral measurement [35,36] Single slice L1eL3 with (10 mm slice thickness)

90

3D QCT spine L1eL2 (10 cm scan length)

1500

pQCT forearm/tibia on single slice scanner

<3 per slice

High resolution pQCT forearm

<5 per slice

Natural background radiation per day

6.6

a

The DXA results for the hip and spine were obtained using Hologic Discovery and QDR scanners and represent the Express mode with scaled scan lengths. Significantly higher doses were observed with the Fast or Array modes. b The DXA results for the whole body were obtained using Hologic Discovery and QDR scanners and represent the range of doses observed between the different models. The QCT data are based on studies in adults and adapted from references [35,36]

described below. The effective dose depends on the absorbed radiation, the size of the irradiated volumes, the relative sensitivity of the irradiated organs, and the radiation energies [32]. Background radiation effective dose averages approximately 2400 mSv per year, or 6.6 mSv per day [33].

NON-INVASIVE MEASUREMENT TECHNIQUES Radiogrammetry and Radiographic Absorptiometry Radiogrammetry was first described in 1960 and is a quantitative method for assessing cortical bone

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geometry in tubular bones [37]. Measurements were initially made with a ruler or fine vernier needle calipers and scores have been published for diverse skeletal sites, such as the radius, humerus, clavicle, tibia and femur [38e41]. This method is most commonly applied to the second metacarpal of the non-dominant hand on a posteroanterior radiograph. An attraction of this method in children is that it makes use of standard radiographs of the non-dominant hand that are often obtained in children with abnormal growth or bone metabolism to estimate bone age. Measurements include the periosteal width (D); the medullary endosteal width (d); the cortical index, which is the ratio between these measurements (D
d/D); and the combined cortical thickness (D
d). 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 [42]. Manual measurements had poor precision, with coefficients of variation (CV) of 10% or worse [31,41]. Current applications of radiogrammetry have taken advantage of advancing technologies in computerized image processing to provide automated measurements of bone dimensions (Figure 13.2) [31,43]. The introduction of automated computed techniques has improved precision to 0.5% [44]. In addition, digital

FIGURE 13.2 Radiograph of the left hand: regions of interest have been automatically positioned in the mid-shafts of the second to fourth metacarpals. (From van Rijn RR, Van Kuijk C. Of small bones and big mistakes; bone densitometry in children revisited. Eur J Radiol. 2009;71:432e9.)

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x-ray radiogrammetry (DXR) provides an estimate of BMD from basic geometric measurements. Radiogrammetry has been used to study the effect of calcium intake on bone mass in adolescent girls [45] and to assess determinants of bone mass in preadolescent girls [46]. In the latter study, skeletal age was found to be the major determinant of bone mass measured at the second metacarpal. More recently, normative DXR data have been published in Dutch [47] and German cohorts [48,49], demonstrating expected sex, age and maturation differences. In addition, DXR was used to demonstrate bone deficits in children with inflammatory bowel disease and juvenile idiopathic arthritis, and in children treated for leukemia or growth hormone deficiency [47,50,51]. Of note, DXR fails in approximately 4% of children’s hand radiographs, especially in younger children less than 6 years of age [47]. A second method for obtaining bone measurements using plain radiographs is radiographic absorptiometry. The photographic density of a bone on a radiograph is approximately proportional to the mass of bone located in the x-ray beam. This technique involves obtaining a radiograph of a peripheral skeleton site simultaneously with an aluminum or hydroxyapatite reference wedge placed near the bone [52e55]. The image is captured electronically, and bone mineral “density” is calculated in arbitrary units using the 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 is approximately 1% and this technique demonstrates an excellent correlation between ash weight and the BMC values expressed in arbitrary units [56,57]. In a study of 1190 children, ages 6.8e10.7 years, Trouerbach et al. showed that BMC measured by radiographic absorptiometry increased with skeletal age and that girls had higher BMD at the diaphyseal and metaphyseal sites than boys [58]. In contrast, another study showed no sex differences in children and small increases in BMD until the age of 11.5 years [59]. Thereafter, bone density increased markedly in both boys and girls. Recently, normative data for radiographic absorptiometry have been published using skeletal age groups rather than chronological ages [60]. In general, the data indicate that BMD remained fairly constant until skeletal age 12 years in boys and 10 years in girls, when it markedly increased. Radiogrammetry and radiographic absorptiometry are derived from hand radiographs, which result in minimal radiation exposure (see Table 13.1).

Dual-Energy X-Ray Absorptiometry DXA is the most widely available and commonly employed technique worldwide for the assessment of

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bone mass [61]. The advantages of DXA include rapid scan times, a low ionizing radiation dose, and the increasing availability of reference data, as detailed below. 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 [62]. Two energy settings are used to optimize the separation of mineralized and soft tissue components in the area analyzed. The low-energy photons are attenuated by the soft tissue surrounding the bone, whereas the highenergy photons are attenuated by bone and soft tissue. 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 high-energy measurements. Pixel by pixel attenuation values are converted to areal BMD by comparison with a bone mineral phantom. Bone area is calculated by summing the pixels within the bone edges, as defined by software algorithms. BMC is calculated by multiplying mean areal BMD by the projected bone area. DXA may be applied to the whole body, or to skeletal regions of interest, such as the posteroanterior or lateral spine, the proximal femur and the ulna and radius. Of note, children with physical disabilities such as cerebral palsy and muscular dystrophy have low bone mass and increased fracture risk [63,64]. The performance of spine and whole body DXA in these children is difficult due to scoliosis and limb contractures. In order to address the difficulties in obtaining clinically meaningful assessments of BMD in these children, a new technique was developed utilizing DXA measurements of the distal femur projected in the lateral plane [65,66]. Advantages of this technique are that the femur is the most common site of fracture in disabled children, children with severe contractures can be comfortably positioned, and metallic fixation is rarely utilized in this region. Further, subregional analyses allow separate assessment of regions rich in cortical versus cancellous bone (Figure 13.3). The different DXA manufacturers have different approaches to calibration that prohibit the interchangeability of results from different instruments [67]. Hologic instruments derive the two photonenergies by rapid switching of the x-ray tube potential and employ a synchronized rotating filter to provide internal calibration on a pixel-by-pixel basis. In contrast, GE-Lunar and Norland use a constant potential x-ray source and absorption edge 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

FIGURE 13.3 The DXA lateral distal femur scan is analyzed for three regions of interest. Region 1 (R1) is the anterior distal metaphysis that is predominantly trabecular bone. Region 2 (R2) is the metadiaphysis and is composed of both trabecular and cortical bone. Region 3 is the diaphysis and is composed of cortical bone. (From Zemel BS, Stallings VA, Leonard MB et al. Revised pediatric reference data for the lateral distal femur measured by Hologic Discovery/Delphi dualenergy X-ray absorptiometry. J Clin Densitom. 2009;12:207e18.)

results from one scanner to another. In 1994, Genant et al. proposed a universal standardization based on patient and phantom cross-calibration results [68]. In a follow-up study conducted in 2010, the investigators demonstrated that the standardized BMD values derived using the universal standardized equations were equivalent within 1.0% for hip but significantly different for spine for state-of-art fan-beam DXA Hologic and GE-Lunar systems. Precision of DXA BMD measurements has been extensively studied both in vitro and in vivo, demonstrating a high degree of reproducibility in adults [67,69,70]. Results vary depending on the site measured: lumbar spine CV have been reported to be as low as 0.4 to 1.7%, the forearm varies between 0.9 and 1.9%, total body values are 0.6 to 0.7%, and the femur has the highest CV% at 0.5 to 2.6%. Fewer precision data are available in children. Leonard et al. recently conducted a study of the reproducibility of BMD measurements

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in children at the spine, hip and whole body, as well as of whole body measurements of BMC, lean body mass and fat mass [71]. The data are summarized in Table 13.2. The authors also used the measured precision to estimate the time interval that needs to elapse before a statistically significant change in a DXA variable can be detected. For example, for spine BMD, a significant increase should be observable after 6 months for boys over the age of 11 years. For younger boys, more than 12 months has to elapse before anticipated changes can be detected with confidence. Therefore, the time intervals required to elapse before a conclusion can be made concerning the significance of observed differences between successive measurements of BMD or body composition in children depend upon the age of the child. 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 [72e75]. DXA scanning systematically underestimated ashing data; however, the DXA measurements and ashing values were highly correlated (r ¼ 0.98e0.99). Additional in vivo studies demonstrated that accuracy at each skeletal site depends greatly on the soft tissue surrounding the bone [76]. Radiation exposure from DXA exams is low, as indicated by dosimetry studies in adults [77] and in pediatric subjects [34,78]. While doses are low, reported values vary widely as a function of scan manufacturer, scan mode, and hardware (pencil beam vs. fan beam). The radiation effective dose in an adult for a spine and hip examination with current systems is between 1 and 20 mSv, depending on the make, model and scan mode used [79]. Because the exposure factors (tube voltage, filtration, tube current, scan width and scan length) are optimized for adults, the effective dose received by children is higher than adults [34]. This is because children are thinner and the doses to internal TABLE 13.2

organs are higher when there is less attenuation of radiation by overlying tissues. In addition, current fan beam systems result in exposure of a greater proportion of the body to the x-ray beam in children. In 2006, Blake et al. conducted a comparison of the effective dose to children and adults from DXA examinations using four different Hologic DXA scanners (Hologic Discovery and QDR models) [34]. The effective dose for spine (hip) examinations performed with the Express mode using the default adult scan lengths were 16.1 (9.8), 11.1 (6.7), 5.6 (3.9) and 4.4 (3.1) mSv for a 5-, 10- and 15-year-old child and adult, respectively. However, when the scan lengths were adjusted appropriately, the child doses were reduced to 9.1 (7.4), 7.1 (5.9) and 5.0 (3.7) mSv. The effective dose for the Fast and Array modes were 1.5- and 3-fold greater, respectively. Effective doses for whole body scans for a 5-, 10- and 15-year-old child and adult varied from 5.2 to 10.5, 4.8 to 9.6, 4.2 to 8.4, and 4.2 to 8.4 mSv depending on the Discovery model. Using the infant whole body mode (only available on the A-model), they were 7.5 mSv for a 1-year-old and 8.9 mSv for a neonate. The data in Table 13.1 represent the results using the express mode with scaled scan lengths. Limitations of DXA in Infants and Children DXA has several limitations that are pronounced in the assessment of infants and children (Table 13.3). These include the impact of pencil beam vs. fan beam methods, difficulties in scan acquisition due to limitations in the bone edge detection software in infants and children, and difficulties in the interpretation of DXA results in children with variable body size, body composition and skeletal maturation. A study of children referred for enrollment in a pediatric osteoporosis protocol based on low DXA spine BMD highlights the importance of these limitations [109]: overall 80% had at least one error in interpretation of the DXA scan. Ultimately, only 26% retained the diagnosis of low BMD.

DXA Precision According to Scan Site and Subject Age <10 years of age

10e18 years of age

DXA site

Technique precision

CV (%)

Technique precision

L1eL4 spine BMD

0.0083 g/cm2

1.2

0.0052 g/cm2

0.7

Total femur BMD

0.0126 g/cm2

1.6

0.0094 g/cm2

1.0

Whole body BMD

0.0087 g/cm2

1.0

0.0086 g/cm2

0.9

Whole body BMC

12.9 g

1.3

17.6

1.2

Whole body lean mass

201 g

0.9

251

0.7

Whole body fat mass

172 g

2.2

189

1.9

Data generated in 32 children using a Hologic Discovery A densitometer (software version 12.3.3) with three scans per subject. Adapted from [71]

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CV (%)

316 TABLE 13.3

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Limitations of DXA Techniques in Infants and Children

Scan acquisition

Fan beam results in magnification error with apparent differences in bone area and BMC as body size varies with growth [80,81]. The further the bone is from the x-ray source (e.g. due to increases in soft tissue), the lower the estimate of the bone area and BMC

Scan analysis

Software developed to improve bone detection in the infant and child resulted in significantly different results for bone size, BMC and body composition [82e85] Difficult to define landmarks and region of interest in the immature hip [86]

Reference data [18,87e103]

Variable hardware and software across published reference data sets Limited data in infants and young children Analysis methods not standardized Not all reference data are sex specific or race specific, with important implications for results [104] Inconsistent mathematical approaches to non-linear and heteroscedastic data

Interpretation

Unable to distinguish between cortical and trabecular bone Underestimates volumetric density in children with short stature [105,106] with consequent over diagnosis of bone deficits [107] Difficult to interpret in children with delayed growth and maturation [108]

completed more rapidly in a single sweep of the x-ray arm down the region of interest. The advantage of fanbeam technology is the generation of a higher resolution image with a substantially lower scan time. For example, the lumbar spine can be scanned in 30 seconds with the fan beam, in contrast to greater than 3 minutes for the pencil-beam. This has important practical implications in younger children where movement artifacts are more difficult to prevent. Importantly, the fan beam method leads to magnification as function of the distance between the bone and the x-ray source (Figure 13.4) [80]. The closer the body part is to the x-ray source, the greater the magnification. For serial densitometric measurements, increases in soft tissue during normal growth could increase a bone’s distance from the fan-beam source and result in apparent reductions in area and BMC. Cole et al. recently reported a correction method based on waist girth, a common anthropometric measure [81]. This correction was applied to DXA data obtained in a cohort of premenarcheal gymnasts and non-gymnasts, increasing the observed areal BMD differences between these two groups. Other studies have demonstrated that these errors may result in biased estimates of BMC and bone area in subjects across the wide range of body sizes present in the pediatric spectrum [80,113]. The most recent advance is the introduction of the narrow fan beam bone densitometer. This machine uses a narrow fan beam x-ray source that scans in

More recently, software bone detection algorithms have improved, robust sex- and race-specific reference data have been published [110], and strategies to adjust for the confounding effects of body size have been developed [111]. Importantly, the ISCD Pediatric Position Development Conference statement includes recommendations for standardized reporting of DXA results [3]. DXA HARDWARE AND SCAN ACQUISITION

Changes in DXA x-ray beam technology improved image resolution and decreased scan time; however, these newer technologies result in greater radiation exposure and introduce magnification errors that impact measures of bone size [112]. Originally, DXA scanners used a collimated beam of x-rays combined with sequential detectors or a single detector that moved back and forth across the patient in a raster pattern as the beam traveled down the region of interest. This pencil beam method produced the most accurate measure of bone geometry. The new generation fanbeam systems use a slit collimator to generate a beam that fans across the patient in conjunction with a linear array of detectors so that the scan acquisition could be

FIGURE 13.4

A bone that is positioned closer to the surface of the scanning table (height H1), and hence to the x-ray source, will experience a wider fan-beam angle and thus will be seen by the detector array as having a larger diameter D1 (and, incidentally, larger area and BMC) than a bone that is farther from the source (D2 at height H2). (From: Cole JH, Dowthwaite JN, Scerpella TA, van der Meulen MC. Correcting fan-beam magnification in clinical densitometry scans of growing subjects. J Clin Densitom. 2009;12:322e9.)

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a rectilinear fashion, similar to the pencil beam machines. However, the beam is wider and can therefore cover the body more rapidly. Recent cross-calibration studies demonstrated no detectable magnification error between the old-generation pencil beam scanners and the new narrow fan beam machines [114]. BONE EDGE DETECTION

The difference between the x-ray attenuation characteristics of incompletely mineralized bone and the surrounding soft tissues in preterm infants, term infants, and young children are not as distinct as in adults. A study conducted with a software program (XRVT, Norland Corp., Fort Atkinson, WI) that allows adjustment of the bone detection thresholds, evaluated DXA results in the forearm of preterm and term infants across a range of bone detection thresholds [82]. All scans could be analyzed using the lowest threshold; however, only 12 of 45 scans could be analyzed using the standard higher threshold due to incomplete bone maps. The threshold choice significantly affected the DXA results; a higher threshold resulted in lower BMC and greater BMD results compared with lower thresholds. A gold standard measure of BMC is needed to assess the accuracy of DXA. Chemical analyses of the whole piglet carcass have been used as a gold standard measure of whole body BMC because young piglets are similar to infants in body size and composition. Brunton et al. used an early generation DXA machine (QDR 1000, Hologic, Inc. Bedford, MA) to compare piglet whole body BMC and body composition estimated by the Pediatric software (Hologic, Inc., PedWB, version 5.35) with chemical analyses of the whole carcass in small (1.6 kg) and large (6 kg) piglets [115]. The authors concluded that the accuracy and precision of DXA estimates of BMC were acceptable when the body weight of the piglet (and presumably an infant) was approximately 6 kg. A subsequent study by the same investigators evaluated an upgraded infant software version (Hologic, Inc., InfWB, version 5.56) that included a revised algorithm which assessed BMC in small local regions of tissue [116]. This is a potentially superior method because soft-tissue variations in one part of the body do not affect the measures of bone or body composition in another part of the body [117]. Re-analysis of the scans with the infant software resulted in improved accuracy of the estimation of total BMC and fat mass in the small piglets; however, BMC detection was still incomplete and variable compared with ash BMC. Other investigators confirmed that the infant software was significantly more accurate than the pediatric software [118]. Extensions of comparisons of the pediatric and infant software to preterm infants showed that the

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BMC results were threefold lower when acquired with the pediatric software compared to the infant software when used in preterm infants at the time of hospital discharge (mean weight 2.1 kg) [117]. However, when scanned again 6 weeks later (at a mean weight of 4.1 kg), BMC estimates did not differ. These findings are consistent with the piglet studies described above: the pediatric software resulted in incomplete detection of BMC in smaller infants but performed well in larger infants. Koo and colleagues have conducted a series of experiments to assess the validity of DXA techniques in infants and small children [83,119e123]. In a comparison of the commercial infant whole body software (v5.71p) and multiple versions of the adult whole body software; the adult software resulted in significantly lower estimates of BMC and greater BMD in infants less than 10 kg [123]. A cross-validation study using software vKH6 (the software from Hologic, Inc. was modified by Koo et al. [119] and is not commercially available) demonstrated that measured and predicted BMC and body composition measures were highly correlated and there were no significant differences in the residuals from predicted versus measured DXA values between the larger piglets (1.94e21.1 kg) and the smaller piglets (0.60e1.58 kg) [120]. Similar issues complicate DXA measures of bone density in young children. Pediatric DXA images frequently could not be analyzed with earlygeneration software due to failure of the bone edge detection algorithm to identify and measure all bones completely. In one series, the DXA spine scan could not be analyzed using standard software (QDR 2000, Hologic, Inc., Bedford, MA.) in 40% of chronically ill children less than 12 years of age and in younger healthy children, particularly those less than 6 years of age [84]. Subsequent software modifications improved detection of low density bone in children and severely osteopenic adults; however, this modification increased the detection of lower density bone and resulted in a systematically greater increase in measured bone area than BMC; hence, the BMD measurements obtained with the new software were consistently lower than those obtained with the standard software [84]. The magnitude of this effect was clinically significant, averaging 0.7 SD. More recent modifications in the whole body bone detection software also resulted in significantly lower BMD results and the magnitude of the difference was progressively greater with decreasing weight below 40 kg [85]. Figure 13.5 illustrates the large magnitude of the effect in smaller children; among children weighing 10 kg, the new software resulted in a 25% lower whole body BMD compared with the prior version.

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In conclusion, it cannot be overemphasized that these differences in hardware and software will impact interpretation of longitudinal pediatric DXA studies, as well as existing pediatric whole body bone reference databases. Investigators must know which DXA model, hardware and software version they are using, and which techniques produced any reference database they may use for comparison. CONFOUNDING EFFECT OF BONE SIZE

A significant limitation of DXA is the reliance on projected two-dimensional measures of BMD. DXA expresses BMD as the amount of mineral divided by the area scanned (g/cm2). This is not a true volumetric density (g/cm3) since the bone thickness in the direction of the beam is not measured. Bones of larger width and height also tend to be thicker. Because this third dimension is not factored into DXA estimates of areal BMD, DXA systematically underestimates the bone density of shorter individuals. This is especially important in the assessment of children with threats to bone acquisition that are also associated with poor growth. Poor growth may result in the appearance of low areal BMD for age when the volumetric BMD is normal [107]. For this reason, some investigators advocate that areal BMD should not be used in growing children [14,120,124].

FIGURE 13.5

The newer whole body software (Hologic, Inc., version 12.1) resulted in significantly lower estimates of whole body BMD compared with version 11.2 among subjects less than 40 kg body weight. The BMD results from the two software versions fall on the line that identifies subjects greater than 40 kg, and the BMD results from version 12.1 are progressively lower than the results from version 11.1 in subjects weighing less than 40 kg. (From Shypailo RJ, Ellis KJ. Bone assessment in children: comparison of fan-beam DXA analysis. J Clin Densitom. 2005;8:445-53.

The confounding effect of skeletal size on DXA measures is well recognized. The ISCD Official Position for Skeletal Health Assessment in Children and Adolescents states: “In children with linear growth or maturational delay, spine and total body less head BMC and areal BMD results should be adjusted for absolute height or height age, or compared to pediatric reference data that provide age-, sex-, and heightspecific Z-scores” [3]. Currently, pediatric reference data for determining height-specific Z-scores for spine or total body BMC or BMD are not widely available. A commonly used approach is to substitute skeletal age or “height age” (the age at which a child’s height is the median height-for-age on the growth chart) for chronological age to adjust for short stature. An important concern with the use of BMC or BMD relative to height, or height-age is that short-for-age children will be compared to children of similar height who are younger and less physically mature. Similarly, the use of bone age may not adequately address this issue of bone size. Varied analytic strategies have been proposed to estimate vertebral volumetric BMD from projected bone dimensions and BMC. The technique developed by Carter et al. is based on the observation that vertebral BMC scaled proportionate to the projected bone area to the 1.5 power [125]. Therefore, vertebral volume is estimated as (area)1.5 and bone mineral apparent density (BMAD) is defined as [BMC/ (area)1.5]. Kroger et al. proposed an alternative estimate of vertebral volume: the lumbar body is assumed to have a cylindrical shape and volume of the cylinder is calculated as (p)(radius2)(height), which is equivalent to (p)((width/2)2)(area/width)) [126,127]. This approach was validated by comparison with magnetic resonance (MR) measures of vertebral dimensions in 32 adults [128]; DXA-derived volumetric BMD correlated moderately well with BMD based on MR-derived estimates of vertebral volume (r ¼ 0.68). Although these methods provide estimates of vertebral volume, the BMC includes the bone content of cortical shell of the vertebral body, as well as the superimposed cortical spinous processes. These two approaches have been used in numerous pediatric studies to assess the effects of preterm birth [129,130], puberty [105,131], ethnicity [87,132,133], gene polymorphisms [134], and physical activity [135e137] on spine volumetric BMD in healthy children, to assess the effects of calcium deficiency and milk avoidance [138,139] and hypovitaminosis D [140], to assess the effects of varied chronic diseases associated with poor growth [141e152] and to assess therapies [146,148,153]. A recent study by Wren et al. sought to evaluate the usefulness of DXA spine correction factors based on

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NON-INVASIVE MEASUREMENT TECHNIQUES

published geometric formulas and anthropometric parameters, compared with three-dimensional QCT [154]. Subject height, weight, body mass index (BMI), skeletal age, and Tanner stage were assessed in 84 healthy children. Two geometric calculations based on DXA spine results were used to estimate volumetric BMD: (1) BMAD [125] and (2) areal BMD/bone height. DXA and QCT BMC were highly correlated (r2 ¼ 0.94). However, DXA areal BMD correlated significantly more strongly with QCT volume (r2 ¼ 0.68) than with QCT density (r2 ¼ 0.39), illustrating the confounding effect of bone size on DXA areal BMD results. The use of DXA correction factors only slightly improved the density correlations (r2 ¼ 0.49 for BMAD; r2 ¼ 0.55 for areal BMD/bone height). The correlations between QCT volumetric BMD and DXA estimates were particularly poor for subjects in Tanner stages 1e3 (r2 ¼ 0.02 for areal BMD; r2 ¼ 0.13 for BMAD; r2 ¼ 0.27 for areal BMD/bone height). In contrast, multiple regression accounting for the anthropometric and developmental parameters greatly improved the agreement between the DXA and CT densities (r2 ¼ 0.91). These results suggest that DXA BMC is a more accurate and reliable measure than DXA BMD for assessing bone acquisition, particularly for prepubertal children and those in the early stages of sexual development. Use of DXA BMD would be reasonable if adjustments for body size, pubertal status, and skeletal maturity are made, but these additional assessments add significant complexity to research studies, and to clinical interpretation. It is not known if these volumetric techniques provide better estimates of fracture risk compared with areal BMD in healthy children or children with chronic disease. These geometric approaches are not readily applied to the complex shape of the whole skeleton. Alternative approaches for the assessment of whole body BMC and BMD include sex-specific centile curves for age, heightspecific means and standard deviations, and z-score prediction models [18,88e93,155,156]. In addition, the observed strong correlation between muscle mass and whole body BMC has prompted numerous investigators to advocate a multistage algorithm for the assessment of DXA whole body bone data relative to muscle mass in children [157e159]. Proposed strategies include assessing bone area relative to height and BMC relative to bone area [18], assessing BMC relative to height and age [116], assessing BMC relative to body weight or lean mass [157,158,160e162], and multistaged prediction models for BMC incorporating age, ethnicity, height, weight, bone area and pubertal stage [155,163]. Despite the recent widespread availability of lumbar spine and whole body reference data (summarized below), there is lack of consensus regarding the most appropriate strategy for the interpretation of two dimensional DXA BMC and bone area results across

319

children of differing body size and body composition. Zemel et al. recently published a critical appraisal of these methods using data obtained with Hologic, Inc. (Bedford, MA) bone densitometers (QDR4500A, QDR4500W and Delphi A models) in over 1500 children from the multicenter Bone Mineral Density in Childhood Study, with validation in a separate sample of healthy children [111,164]. Spine and whole body BMC and BMD-for-age Z-scores were compared with Z-scores generated using height age, height-specific reference data (i.e. BMC or BMD relative to height), and BMAD-for-age. In addition, adjustment for height Z-score was proposed as an alternative approach that would simultaneously consider both height and age. These analyses confirmed that use of height-age or the assessment of BMC or BMD relative to height resulted in a systematic bias: older healthy children with height Z-scores less than
1.0 had BMC of BMD Z-scores greater than expected relative to height or height-age. BMAD-for-age Z-scores also did not fully remove the effects of height status on areal BMD. In contrast, adjustment for height Z-score was the only approach for which the effects of short (or tall) stature were not significant (Figure 13.6). The publication provides the equations necessary to adjust sex- and age-specific BMC and BMD Z-scores for height Z-score. Therefore, this technique can be readily applied in clinical practice to assess the degree to which a low BMC or BMD Zscore can be attributed to short stature. Further validation of this approach using other bone outcomes such as fracture is needed. As discussed below, quantitative studies using receiver operating characteristics (ROC) curves to assess the ability of these varied strategies to discriminate between fracture and non-fracture cases are imperative in order to identify the best analytic approach to the interpretation of whole body DXA data for research and clinical applications. DXA Reference Data Comparisons to appropriate pediatric bone reference data are essential to determine the clinical impact of nutritional deficiencies and childhood disease on bone development, to monitor changes in bone mass, and to identify patients for treatment protocols. Misclassification of a bone health results may lead to unnecessary testing, parental anxiety, alteration in treatment of the underlying disease, and initiation of needless treatment or the absence of an effective intervention. The 2007 ISDC Pediatric Position Development Conference advised that an appropriate reference data set must include a sample of the general healthy population sufficiently large to characterize the normal variability in bone measures that takes into consideration sex, age and race/ethnicity [3]. An additional important

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13. NON-INVASIVE TECHNIQUES FOR BONE MASS MEASUREMENT

FIGURE 13.6

Impact of methods to adjust for difference in stature. The distributions of whole body BMC Z-scores are shown in short (height Z-score <
1), average (
1height Z-score1) and tall (height Z-score >1) children and adolescents. Whole body BMC for age Z-scores in the first panel demonstrate the significant effect of growth status with lower Z-scores observed in the short participants and higher Z-scores in the tall participants, relative to average participants. BMC Z-scores calculated using height age, BMC-for-height (BMCfor-Ht), and BMAD resulted in biased estimates. In contrast, adjustment for height Z-score (HAZ-adj) resulted in a similar Z-score distribution among short, average, and tall healthy children, suggesting an unbiased adjustment. (From Zemel BS, Leonard MB, Kelly A et al. Height adjustment in assessing dual energy x-ray absorptiometry measurements of bone mass and density in children. J Clin Endocrinol Metab. 2010;95:1265-73.)

attribute is use of the most current measurement technology with standardized data acquisition. Although there are numerous publications describing DXA measures of BMC and BMD relative to age in healthy children, few have the attributes needed to serve as a reference [87,89,92,94-98]. For example, earlier studies of bone mineralization in healthy children were conducted using single or dual-photon absorptiometry [165,166], or DXA in a pencil beam mode [87,99]. While these studies were instrumental in describing determinants of bone acquisition, they cannot be used as reference data for current research studies or clinical care due to the changes in bone density assessment technology. A systematic comparison of published pediatric DXA BMD normative data generated with Hologic, Inc. (Bedford, MA) scanners in 1999 revealed differences in the age-specific means and standard deviations for BMD across five studies [89,97,99e101]. These differences had a significant impact on the diagnosis of osteopenia in children with chronic diseases [104]. Importantly, use of reference data that were not sex specific resulted in significantly greater misclassification of males as having osteopenia [104]. Reference data for Hologic, Inc. scanners have improved markedly in recent years. Sex- and race (black vs. non-black)-specific reference data for BMC of the whole body and lumbar spine and BMD of the whole body, lumbar spine, total hip, femoral neck, and forearm relative to age were recently reported in 1544 children, age 6 to 16 years enrolled in the multicenter study “Bone Mineral Density in Childhood Study” (BMDCS) [110]. Figure 13.7 illustrates the sex differences in lumbar spine BMC for age percentiles within non-black participants. DXA scans were performed using Hologic, Inc. bone densitometers (QDR4500A, QDR4500W, and

Delphi A models). Scans were performed on a single densitometer at each center. The software versions used for acquisition varied from version 11.1 to 12.3 depending on the anatomic scan site. Of note, the Hologic, Inc. pediatric reference data on the scanners, as of 2010, include the BMDCS data as well as supplemental data obtained at other centers to provide reference data for ages 3 to 21 years. Reference data for whole body bone and body composition for children ages 8 through 20 years are also available for Hologic systems based on data collected in the National Health and Nutrition Examination Survey between 1999 and 2004 [167]. Adolescents between 12 and 19 years of age were over sampled to provide more reliable estimates for this group. The whole body DXA scans were acquired with a QDR 4500A fan beam densitometer and analyses of all exams were performed using Hologic Discovery software version 12.1. Sex-specific reference curves were developed for Non-Hispanic Whites, Blacks, and MexicanAmericans. Reference charts detail (1) total and subtotal (excludes head) whole body BMC and BMD results relative to age, (2) total and subtotal (excludes head) whole body BMC and BMD results relative to height, (3) total lean mass relative to height, (4) subtotal body BMC relative to lean mass, and (5) subtotal BMC relative to lean mass. Reference data for percent body fat and lean mass index (lean mass/height2) relative to age are also included. Lastly, Zemel et al. recently published sex- and racereference curves for lateral distal femur BMD based on Hologic Discovery/Delphi scans in 821 healthy children, aged 5 to 18 years [161]. The new lateral distal femur Z-scores were strongly and significantly associated with weight, body mass index, and spine and

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321

FIGURE 13.7 Lumbar spine BMC for age in non-black girls and non-black boys. Smoothed curves are shown for the 3rd, 25th, 50th, 75th and 97th percentiles. The plotted points represent the corresponding empirical percentile values for a given age group. (From Kalkwarf HJ, Zemel BS, Gilsanz V et al. The bone mineral density in childhood study: bone mineral content and density according to age, sex, and race. J Clin Endocrinol Metab. 2007;92:2087e99.)

whole body BMD Z-scores. The authors concluded that the comparability of lateral density femur measurements to other BMD assessment modes supports this alternative site in children with disabilities, who are particularly prone to low trauma fractures of long bones, and for whom traditional DXA measurement sites are not feasible. An additional consideration is the mathematical approach used to capture the non-linearity of gains in bone mass and body composition with age during growth and development, and the greater variability with increasing age (heteroscedasticity), as evident in Figure 13.7. The most common approach to characterize reference data is to define the mean and a standard deviation. However, when the distribution of values for a given age is skewed, it is inappropriate to use the standard deviation to characterize the variability. Furthermore, variability is not fixed and increases with age and puberty. Some statisticians recommend transforming bone mineral data using parametric regression modeling [88]. This technique becomes particularly important when a large data set is not available for more data intensive techniques. The LMS method is more appropriate when data are both skewed and heteroscedastic [169], as is the case with bone mass and body composition data. The LMS method uses a power transformation to normalize data. The optimal power to obtain normality is calculated, and the trend is summarized by a smooth curve (L). Smoothed curves for the mean (M) and coefficient of variation (S) by age are acquired, and these three measures are used to describe the data distribution. The disadvantages of the LMS approach are that it requires large amounts of data,

and that multiple adjustments (e.g. age and height) cannot be made simultaneously. The BMDCS [110], lateral distal femur [168], and body composition [167] reference curves described above were generated using the LMS technique. Reference data are also available for GE Lunar Corp (Madison, WI) Densitometers. GE-Lunar DXA systems provide pediatric reference data for the spine, femur and total body for children aged 5 to 19 years. Whole body reference data are available with and without the head [170]. In the USA, reference data have incorporated data from the Bone Mineral Density in Childhood Study described above [110]. Although these reference curves were based on data collected using Hologic systems, the reference curves for both black and non-black children were converted to Lunar values and are available in GE-Lunar software version 13. Data from several pediatric and adult sources were used to convert QDR pediatric values to GE-Lunar-equivalent values [171]. GE-Lunar software uses the approach proposed by Molgaard et al. [18] and expresses total body BMC relative to age, height (narrow bones), and bone area (light bones), and height as function of age (short bones). In addition, this software provides body composition Z-scores for lean body mass for height and BMC for lean body mass following Crabtree et al. [158]. Lastly, reference data in infants are limited. However, Kalkwarf et al. recently reported lumbar spine data in 269 healthy infants and toddlers, aged 1 to 36 months using the software developed by Hologic, Inc. to analyze lumbar spine DXA scans in children less than 36 months of age [172]. BMC and BMD increased with age and similar increases were observed relative to weight and

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length. No sex differences in BMC or BMD were evident when statistically adjusting for age, length and weight. The authors concluded that age specific reference values for lumbar spine BMC, but not BMD, need to be sex specific, and there was no apparent need to develop race specific reference data for infants and toddlers. Given ongoing accumulation of pediatric reference data, DXA users may need to contact their dealer to determine the reference data source installed on a given machine, and to identify additional relevant databases. For example, the BMDCS will publish cross-sectional and longitudinal reference data across a wider age range in the coming years.

Fracture Discrimination Quantitative data using ROC curves to assess the ability of varied DXA sites and analytic strategies to discriminate between children with and without a fracture are very limited. These studies are imperative in order to identify the best analytic approach to the interpretation of DXA data for research and clinical applications. In 2007, the ISCD convened a Task Force to review the evidence that DXA measures are predictive of fractures in apparently healthy children [4]. Studies in infants were not included. At the time of their review, 11 studies were identified. In three of these, the fractures were assessed prospectively after a baseline DXA scan [173e175]. In the remainder, the DXA scans were obtained after the fracture occurred [176e183]. The largest prospective study was conducted in over 6000 children at 10 years of age [173], demonstrating a weak inverse relationship between baseline total body less head (TBLH) BMD and subsequent fracture risk with an odds ratio (OR) per standard deviation lower BMD of 1.12 (95% CI 1.02e1.25). However, the relationship was stronger using TBLH BMC adjusted for bone area, height, and weight (OR 1.89; 95% CI 1.18e3.04). Fracture risk was unrelated to TBLH bone area; however, an inverse association was observed between fracture risk and TBLH area adjusted for height and weight (OR 1.51; 95% CI 1.17e1.95). Based on these 11 studies, the expert panel concluded that (1) low ultradistal and one-third radius areal BMD and low spine areal BMD and BMAD adjusted for age and sex were associated with distal forearm fracture; and (2) the following were associated with fractures at all sites combined: low BMC of TBLH adjusted for height and gender; low BMC of TBLH adjusted for age, height, weight, and bone area; and low bone area of TBLH adjusted for age, height, and weight. There was little evidence that BMAD measures were more closely associated with fractures than areal BMD [173,174,181]. More recently, Flynn et al. conducted a prospective study of DXA scans at age 8 in 183 children; fracture

rates were ascertained over the subsequent 8 year interval [184]. DXA scans were performed at hip, spine and total body, and reported as BMC, BMD, and BMAD. There was a total of 63 fractures, with 68% of fractures in the upper limb. In analyses adjusted for height, weight, age and sex, total body BMC, spine BMC, total body BMD, total body BMAD, and spine BMD (hazard ratios per SD ranged from 2.47 to 1.53) were all significantly associated with upper limb fracture risk. Similar, but weaker associations were present for total fractures. Hip results were not associated with fracture. In a related study, DXA scans were performed at age 19 years in 991 otherwise healthy men and compared in the 304 (31%) with a history of fracture during childhood and adolescence, to those without a fracture history [185]. Men with prevalent fracture had significantly lower BMD in the spine, total femur, total body and radius with unadjusted odds ratios per SD of 1.22 to 1.26. The odds ratios were greater (1.35 to 1.50) when adjusted for age, adult physical activity, smoking, calcium intake height and weight. As detailed below, the unadjusted and adjusted odds ratios were higher for pQCT measures of radius and trabecular volumetric BMD. Lastly, Kalkwarf et al. conducted a DXA and pQCT study in children aged 5 to 16 years with a forearm fracture (n ¼ 224) and injured controls with no fracture on x-ray (n ¼ 200) within a mean of 28 days of the injury [186]. ROC curves were generated for each outcome and the area under the curve (AUC) was calculated to reflect the potential of that bone measure to identify individuals at risk of fracture. ROC curves are commonly used to evaluate the performance of a diagnostic test: an AUC of 1 indicates a perfect test while an ACU of 0.5 indicates a test that is equivalent to chance. While the adjusted means were significantly lower in the fracture cases compared with controls for DXA (spine BMD and BMAD, total hip and femoral BMD and BMC, one-third radius BMD, and TBLH BMC and bone area) and pQCT radius (distal total vBMD, midshaft total BMC, cortical vBMD, and cortical area) scans, the ROC results were disappointing. The AUC varied from 0.56 to 0.59 when just considering the DXA or pQCT bone measures, and from 0.63 to 0.65 when considering bone measures and injury severity. Further details are provided below in the section of fracture discrimination by QCT, and in Figure 13.12. DXA data on fracture discrimination in healthy children may not be applicable to children with chronic disease; however, data are very limited. In a small series of 32 children with chronic disease, vertebral compression fractures were poorly predicted by single spine BMD measurements [187]. The largest study to date was conducted in 619 children, aged 6 to 18 years,

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with muscular dystrophy or moderate to severe cerebral palsy [188]. There was a significant correlation between fracture history and BMD Z-scores in the distal lateral femur; 35% to 42% of those with BMD Z-scores less than
5 had fractured compared with 13% to 15% of those with BMD Z-scores greater than
1. Risk ratios were 1.06 to 1.15 (95% C.I. 1.04e1.22) per standard deviation decrement in BMD Z-score. Prospective studies are currently underway to assess further the utility of DXA scans for fracture prediction in children with chronic diseases, such as glucocorticoid-induced osteoporosis.

Quantitative Computed Tomography In recent years, the recognition of the advantages or QCT over DXA (i.e. distinct measures of trabecular and cortical volumetric BMD and geometry) has resulted in significantly increased use of QCT in bone research studies in children and adults. CT utilizes x-rays and generates an image that is based on the linear x-ray absorption coefficients of the tissues in the x-ray path. The transverse anatomic sections provide a three-dimensional image unobscured by overlying structures. All CT scanners are calibrated to the x-ray attenuation of water. 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 corresponds to the attenuation coefficient of air. Conversion of BMD to g/cm3 is performed using linear regression to relate the HU number of the bone to that of the compartments of the calibration standard that are included in the scan field (Figure 13.8).

FIGURE 13.8 Vertebral tomogram for QCT bone density assessment. The reference calibration phantom is placed under the patient and scanned simultaneously. (From Brunader R, Shelton DK. Radiologic bone assessment in the evaluation of osteoporosis. Am Fam Physician. 2002;65:1357e64.)

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QCT provides measures of compartment BMD (see Figure 13.1). For example, the QCT values for trabecular volumetric BMD reflect not only the material BMD of the trabecular bone, but also the number and thickness of the trabeculae and the amount of marrow per pixel. Spine QCT is an established technique for measuring BMD in the axial skeleton [189]. A lateral scout view is used to identify the scan sites. The original CT scanners use a rotateetranslate scan mode to obtain single 10 mm thick slices through the middle of each vertebral body (usually three to four vertebrae between T12 and L4) with the gantry angled parallel to the vertebral end plates. A region of interest (ROI) is positioned in the interior portion of trabecular bone of the vertebral body for analysis and the average density across the vertebrae is calculated. The time required for this procedure is approximately 10 minutes. QCT methods using these single slices (two-dimensional) through each vertebra do not require the same image quality as scans performed for diagnostic imaging and therefore, require a lower radiation dose compared with higher resolution techniques (see Table 13.1). Gilsanz and colleagues have conducted a series of spine QCT studies in children and adolescents, characterizing important sex and race differences in trabecular volumetric BMD with growth and development [190e194]. In addition to measurements of volumetric BMD, QCT provides measures of the height and the cross-sectional area of each vertebral body. The heights of the anterior, middle, and posterior portions of the vertebral bodies are measured on lateral scout view, whereas the area of the vertebral body is calculated on CT scans corresponding to an ROI in each vertebral body and excluding structures behind the most anterior margin of the spinal canal. This technique has been used to characterize sex differences in vertebral body size in children and adolescents [195]. More recently, technical advances, such as complete and multiple rings of detectors and spiral rotation of the x-ray tube, resulted in rapid acquisition of images of volumes of tissue [35,196]. The thorax and abdomen can be scanned in 20 seconds. This technique allows the acquisition of data from which single slice twodimensional (ROI) or three-dimensional volumetric (volume of interest [VOI]) measures can be made. Such volumetric methods enable analysis of the hip, which was not feasible with the single slice ROI technique due to poor precision [35]. With increasing use of multidetector CT, there is a trend to scan two complete vertebrae between T12 and L3 to minimize radiation exposure, and the acquisition slice width is narrower (1e3 mm) which improves spatial resolution. In spiral multidetector CT, automodulation techniques are used to reduce radiation exposure by adapting the x-ray tube current to the individual subject and anatomic

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site [197]; however, these volumetric methods results in substantially greater radiation exposure compared with other imaging techniques [36] (see Table 13.1). Clinical QCT scans have also been used to assess the appendicular skeleton in health and disease. For, example, Gilsanz and colleagues conducted a series of studies in the midshaft of the femur, demonstrating race and sex differences in cortical dimensions in healthy children [191,194]. The precision of QCT bone measurement is excellent in children. CV for measures of spine trabecular BMD, vertebral body height, and vertebral cross-sectional area were reported as 1.5, 1.3, and 0.8%, respectively [191]. The CV for repeated QCT measurements of cortical volumetric BMD, cortical bone area, and the cross-sectional area of the femur ranged from 0.6 and 1.5% [191]. Dedicated peripheral QCT (pQCT) scanners were developed to measure trabecular, cortical and total volumetric BMD, cortical dimensions, and fat and muscle cross-sectional area in the radius and tibia (StratecMedizintechnik, Pforzheim, Germany). These table-top scanners are smaller, portable, less expensive and result in substantially less radiation exposure compared with conventional QCT (see Table 13.1). Calibration of the system is not achieved simultaneously at each scan, but it is done on a routine basis with a specifically designed phantom. A scout view is performed to localize the endplate and growth plate (if not yet fused) of the tibia or radius, a reference line is placed relative to the endplate or growth plate, and scan sites are selected as a percentage of bone length. Individual 2 mm thick tomographic slices are taken at these selected sites. The metaphyseal regions are rich in trabecular bone and the shafts are essentially entirely cortical bone. Since the majority of childhood fractures occur in the long bones, there is good rationale for obtaining measures of bone structure and volumetric BMD at these sites. The pixel size for the newer pQCT devices is as small as 0.4 mm. Scanning time ranges from 2 to 3 minutes in smaller children to 4 to 5 minutes in adults. Figure 13.9 illustrates images acquired at the 3% tibia metaphysis in (A) a healthy female with normal trabecular volumetric BMD and (C) an age and race-matched female with Crohn disease and a low trabecular volumetric BMD. The figure also illustrates images acquired at the 38% tibia diaphysis in (B) a healthy female with a normal cortical section modulus and (D) an age and race-matched female with Crohn disease and a low cortical section modulus. Trabecular and cortical bone respond differently to stimuli such as sex hormones, nutrition, mechanical loading, and disease effects (e.g. inflammatory cytokines or glucocorticoid therapy). Therefore, pQCT allows more specific classification of normal and abnormal

bone mass acquisition in childhood, compared with DXA. For example, studies in healthy children have been used to characterize sex, pubertal and race effects on bone density, bone structure and the relations between bone, muscle and fat [21,198e206], the impact of physical activity interventions [207,208] and calcium supplementation [209], and genetic associations [9,204,210,211]. Studies in children with chronic diseases have demonstrated discrete effects of disease processes and treatments on cortical and trabecular bone. For example, pQCT studies conducted in children with chronic inflammatory conditions, such as Crohn disease and juvenile idiopathic arthritis, demonstrated significant deficits in trabecular volumetric BMD and cortical dimensions with preserved cortical volumetric BMD, compared with healthy controls [212,213]. In contrast high-dose chronic glucocorticoid therapy in childhood nephrotic syndrome was associated with lower trabecular and higher cortical volumetric BMD, compared with healthy controls [214]. This study highlights an important strength of pQCT: pQCT captured the opposing effects of glucocorticoids to increase and decrease cortical and trabecular volumetric BMD respectively in children treated with glucocorticoids. However, the DXA PA spine areal BMD was not different in children with nephrotic syndrome compared with controls, suggesting that the superimposition of the cortical and trabecular structures concealed the glucocorticoid effects. pQCT has also been used to characterize bone deficits in non-inflammatory conditions, such as scoliosis [215] and cerebral palsy [216] and to document the effects of therapies, such as growth hormone [217] or pamidronate [218]. pQCT bone measurements show good precision. The CV values for trabecular volumetric BMD and cortical volumetric BMD measurements were 0.8 and 1.1%, respectively in one study [219]. Another group of investigators reported that the CV for short-term precision ranged from 0.5% to 1.6% for pQCT volumetric BMD and cortical geometry outcomes in children and adolescents [214]. Although conventional QCT and pQCT measurements of bone are able separately to assess the density of cortical and trabecular bones, these scans are subject to variable pixel sizes and partial volume effects, as detailed below. The accurate segmentation and analysis of trabecular architecture and cortical bone requires adequate spatial resolution. The CT image is formed by 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. The spatial resolution of CT also depends on slice width, noise, and the modulation transfer function (which characterizes

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FIGURE 13.9 Tibia pQCT images at the 3% metaphysis site (A and C) and 38% diaphysis site (B and D). Image A was obtained in a healthy 16-year-old Caucasian female with a trabecular vBMD Z-score relative to age, sex and race of 0.0, and image C was obtained in a 16-year-old Caucasian female with Crohn disease and a trabecular vBMD Z-score of
3.4. Image B was obtained in a healthy 14-year-old Caucasian female with a section modulus Z-score relative to sex, race and tibia length of 0.0 and image D was obtained in a 14-year-old Caucasian female with Crohn disease and a section modulus Z-score of
2.9.

the spatial frequency response [220]. Therefore, the pixel size is usually smaller than the true spatial resolution [35]. Trabecular thickness ranges from 60 to 150 mm and the separation between trabeculae ranges from 300 to 1000 mm [221]. In spine QCT, radiation doses limit the spatial resolution to approximately 160e300 mm. Although individual trabeculae cannot be defined, finite element analyses of spine QCT VOI data in adults have been used to identify sites within the vertebral body at highest risk for fracture [222], and to document the structural changes and reductions in “at-risk” bone tissue following treatment with bone-active medications [223,224]. To our knowledge, finite element analysis has not been applied to spine QCT data in children. The standard pQCT scanners described above have a resolution of z400 mm, and therefore lack the ability accurately to assess

bone microarchitecture. However, the newest generation of high-resolution pQCT (HR-pQCT) scanners was developed to image trabecular and cortical structure in peripheral skeletal sites in humans in vivo (Xtreme CT, Scanco Medical, Zurich, Switzerland). Figure 13.10 illustrates HR-pQCT images in the radius and tibia. The scan is performed on a segment spanning 9.02 mm. Data are obtained using a 3D stack of 110 high-resolution CT slices. Although the current resolution of 82 mm does not permit direct measurement of the microstructural parameters, methods have been developed to estimate trabecular parameters from the 3D grayscale image [225]. HR-pQCT demonstrates excellent correlation with ex vivo mCT imaging (resolution 20 mm or better) [226], and HRpQCT can be used to construct microfinite element models of bone strength [227].

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FIGURE 13.10 High-resolution pQCT provides detailed images of bone geometry and microarchitecture at the radius (left) and tibia (right). The scout view illustrates the reference line position (solid line) and the measurement site (dotted line). (Adapted from Figure 2 in Nickolas TL, Stein E, Cohen A et al. Bone mass and microarchitecture in CKD patients with fracture. J Am Soc Nephrol. 2010;21:1371-80.)

HR-pQCT studies conducted in children and adolescents to characterize trabecular and cortical architecture according to pubertal stage [228,229] demonstrate an association between impact loading physical activity and greater trabecular area, density and number in healthy adolescents [230], demonstrate significant differences in bone architecture between the radius and tibia [231], and demonstrate that late menarche is associated with lower cortical volumetric BMD and cortical thickness [232,233]. The study conducted by Kirmani et al. [228] used HR-pQCT to investigate the structural basis for the greater incidence of forearm fractures observed during the adolescent growth spurt in epidemiology studies [27]. Total bone strength, estimated using microfinite element models of bone strength, increased linearly with greater bone age, with males showing greater bone strength than females after midpuberty [228]. However, the proportion of load borne by cortical bone, and the ratio of cortical to trabecular bone volume, decreased transiently during mid- to late puberty in both sexes, with apparent cortical porosity peaking during this time. The authors noted that this mirrors the incidence of distal forearm fractures in prior studies. While HR-pQCT scans may provide additional important insights, studies demonstrating superior fracture discrimination compared with DXA and pQCT have not been performed in children. Limitations of QCT in Children The cost and inaccessibility of whole body CT scanners has markedly limited the use for bone measurements in

the spine. In addition, although with low settings, spine QCT exams can be performed with a low level of radiation exposure, the dose continues to be higher than that associated with DXA. Whole body CT scanners are expensive, large, non-portable 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, creating a lack of accessibility. These disadvantages have partially been overcome by smaller, less expensive pQCT scanners. pQCT measurements in children present unique challenges due to the small bone size, the presence of the growth plate, the changing shape of the metaphysis during growth, and the consequent difficulties obtaining repeated measures in the same anatomic location in longitudinal studies (Table 13.4) [5]. These challenges are discussed at length in the 2007 ISCD report [5]. NON-STANDARDIZED SCAN PROTOCOLS AND MULTIPLE OUTCOME MEASURES

The majority of pQCT studies have used Stratec XCT devices (models XCT900, SCT2000, and XCT3000). Presently, there are no studies comparing manufacturers, devices and software for their impact on pediatric bone assessment. The Stratec software requires the selection of analysis modes and thresholds, and these parameters affect analysis results [236]. Furthermore, the software derives numerous measures of volumetric BMD (e.g. total BMD, cortical BMD and trabecular

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TABLE 13.4

Limitations of QCT Techniques in Infants and Children

Scan acquisition

Placement of the reference line, scan speed, voxel size and selected scan sites impact pQCT results Cortical volumetric BMD may be underestimated due to partial volume effects [234] Growth in the metaphysis prohibits measures at the same site over time at appendicular sites [235] There is a lack of data comparing different devices

Scan analysis

pQCT scan protocols (thresholds, analysis modes) vary across studies with significant differences in bone results [236] pQCT software generates numerous outcome measures and derived estimates of bone strength that vary across studies

Reference data

Existing spine QCT reference data may not be applicable to scans acquired using spiral techniques [192] There are no pQCT reference data with adequate representation of pediatric age and sex groups to characterize normal variation Age ranges, measurement sites, outcome measures and analysis protocols differ markedly across pQCT studies Published pQCT studies do not always provide sufficient information describing the analysis modes and reference line placement Limited data in young children and no data in infants

Interpretation

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Geometry measures are confounded by bone length [21,213] Large variations in trabecular bone morphology associated with skeletal development limit the interpretation of pQCT studies based on a single slice [235]

BMD), bone geometry (e.g. total area, trabecular area, cortical area, cortical thickness, periosteal circumference, endosteal circumference, marrow cross-sectional area), and surrogate measures of bone strength (e.g. polar moment of inertia, section modulus, stress-strain index, and bone strength index) in the radius and tibia [5]. Because pQCT is a research tool at individual centers, scan acquisition procedures (e.g. reference line placement, scan speed, voxel size and measurement sites), analysis protocols, and measurement outcomes have not been standardized. PARTIAL VOLUME EFFECTS

The cortical bones of children are smaller and thinner than those of adults, and are more subject to partial volume effects. Partial volume effects are a function of the resolution of the image (voxel size) and the size of the bone being measured. Figure 13.11 illustrates the etiology of partial volume effects in the cortical diaphysis: partial volume effects are due to the fact that voxels

FIGURE 13.11

Illustration of partial volume effects in cortical bone. Partial volume effects are due to the fact that voxels located close to the bone edge are more likely to be comprised of both bone and soft tissue. Voxels that overlap bone and soft tissue will have a lower density value than the voxels that are attenuated by bone only within the bone envelope. Smaller bones will have a higher proportion of voxels close to the bone edge and may thereby appear to have a lower density due to this artifact. (From Zemel B, Bass S, Binkley T et al. Peripheral quantitative computed tomography in children and adolescents: the 2007 ISCD Pediatric Official Positions. J Clin Densitom. 2008;11:59e74.)

located close to the bone edge are more likely to be comprised of both bone and soft tissue. Voxels that overlap bone and soft tissue will have a lower density value than the voxels that are attenuated by bone only within the bone envelope. Smaller bones will have a higher proportion of voxels close to the bone edge and may thereby appear to have a lower cortical compartment volumetric BMD due to this artifact. The tibia is larger than the radius and pediatric studies are frequently done in the tibia to minimize partial volume effects. Similarly, measures of cortical volumetric BMD are best performed in the bone diaphysis where the cortical bone is substantially thicker compared with the metaphysis. A pQCT study conducted using a bone phantom concluded that the minimum thickness necessary for an accurate density evaluation of cortical bone by QCT is 2.0 to 2.5 mm; below this threshold, QCT values decline in a linear way relative to width [234]. CHANGES IN REFERENCE LINES AND MEASUREMENT SITES WITH GROWTH

The performance of pQCT measures in the metaphysis is complicated by variable metaphyseal length

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FIGURE 13.12

Risk of forearm fracture associated with a 1 standard deviation lower Z-score for bone outcome measures derived from pQCT and DXA scans. The vertical dash is the odds ratio and the horizontal line represents the 95% confidence interval. (From Kalkwarf HJ, Laor T, Bean JA. Fracture risk in children with a forearm injury is associated with volumetric bone density and cortical area (by peripheral QCT) and areal bone density (by DXA). Osteoporos Int. 2010.)

relative to tibia length, and changes in metaphyseal dimensions with growth. In addition, trabecular volumetric BMD declines rapidly as one moves proximally from the growth plate [235,237] and this pattern of decay varies between individuals and within individuals over time. Therefore, large variations in bone morphology associated with skeletal development may limit the interpretation of pediatric pQCT studies based on a single slice. Lee et al. characterized the variability in pQCT measures of trabecular volumetric BMD along the metaphysis in 35 children with cerebral palsy, aged 6 to 12 years [235]. The patterns of decay in metaphyseal trabecular bone density were different in all subjects, and the density changed from the physis to the shaft at an average rate of 16.8% per 1 mm. Importantly, the slopes of the density curve changed in some children over a 6-month interval. While there was a high correlation (r2 ¼ 0.88) between the BMD of a single slice located a fixed distance from the growth plate and the overall mean metaphysis density, the BMD values over a 6-month interval were only moderately correlated (r2 ¼ 0.58). Of note, the investigators did not have measures of tibia length at each time point and it is not known if the use of measures based on tibia length

(e.g. 3% of tibia length) would have improved within subject correlations over time. CONFOUNDING EFFECTS OF BONE LENGTH

An important advantage of QCT techniques is the generation of three-dimensional measures that are not confounded by body size. While this is true for measures of volumetric BMD, the interpretation of measures of cortical geometry and muscle and fat area should consider bone length. Prior studies have shown that cortical BMC, cortical cross-sectional area, periosteal circumference, endosteal circumference, stressestrain index, section modulus, cross-sectional moment of inertia, and muscle area and fat area are all highly correlated with tibia length (all P<0.0001) in children and adolescents [214]. Because measures of bone mass and geometry are proportional to the length of the bone, children with poor growth (i.e. shorter tibia and radius) will have lower values for these measures. Most reference data are presented relative to age rather than limb length. Therefore, the interpretation of size-dependent pQCT measures in children with advanced or delayed growth is problematic. One approach is to use LMS methods to generate sex- and race-specific Z-scores for

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cortical geometry and muscle and fat area relative to tibia length [212e214]. It is especially important to consider limb length when assessing the functional muscleebone unit. This was illustrated in a study of children with juvenile idiopathic arthritis (JIA) [213]. Tibia pQCT scans were obtained in 101 patients with JIA and 830 healthy control subjects; all were aged 5 to 22 years. Multivariate models were used to assess cortical section modulus relative to muscle cross-sectional area. The comparison of JIA patients with low muscle area to healthy controls without adjustment for group differences in age and tibia length resulted in the appearance that section modulus was paradoxically greater than expected relative to muscle area. This is because a JIA patient with muscle deficits was older and had a longer tibia (and consequently, greater section modulus) than a healthy control with a similar muscle area. In models adjusted for age and tibia length, JIA was associated with lower section modulus relative to muscle area. Reference Data Gilsanz et al. recently compiled spine QCT data in 1222 healthy white participants, aged 5 to 21 years, scanned between 1992 and 2006 using the same mineral reference phantom [192]. The study demonstrated that the percentage increase in trabecular volumetric BMD with age was comparable in males and females, but occurred significantly earlier in females. The authors provided the mean and standard deviation results for males and females according to age. Although the authors anticipate that these data would be highly correlated with values using the spiral three-dimensional techniques, the values from the latter may be slightly higher [238,239]. Access to adequate pQCT reference data is severely hampered by lack of standardization of measurement hardware and software, reference line placement, measurement site (e.g. percentage or fixed distance from the reference line), scan acquisition protocols (e.g. voxel size, scan speed), and analysis protocols (e.g. thresholds and algorithms to define the bone edge). In fact, the majority of publications provide insufficient details regarding these procedures. As reviewed by the ISCD, in the radius, investigators have used regions of interest at 4%, 6%, 10%, 15%, 20%, 33%, 50% or 65% of ulnar length, and at 7 mm from the endplate. Similarly, tibia measurements have been obtained at 8%, 10%, 20%, 38%, 50%, 65%, or 66% of tibia length, or at 10 mm from the distal endplate. The ISCD report includes a detailed summary of normative data [5] e concluding that the comparative data have several shortcomings. The measurement sites and protocols were not always well described or standardized, outcome measures vary across studies, the majority of studies

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either had a large representation of children in a narrow age range, or a large age range with a very small number of subjects within each age/sex category, and samples were composed of predominantly Caucasian children. The ISCD report highlights the need to quantify differences in pQCT models and software versions, and the need for standardization and documentation of all measurement procedures. Fracture Discrimination As detailed above, Kalkwarf et al. conducted a DXA and pQCT study in children aged 5 to 16 years with a forearm fracture and injured controls with no fracture [186]. The odds ratio and 95% confidence intervals for each bone measure by pQCT or DXA that significantly differed between cases and controls are summarized in Figure 13.12. While numerous measures were significant, fracture discrimination was poor; the AUC varied from 0.56 to 0.59. Darelid et al. conducted a study in 991 young men, aged 18.9  0.6 years, enrolled in the population based Gothenburg Osteoporosis and Obesity Determinants Study [185]. On review of medical records, 304 had an x-ray verified fracture during childhood. Areal BMD of the whole body, femoral neck, spine and radius was measured by DXA and cortical and trabecular volumetric BMD and bone size were measured by tibia and radius pQCT. Men with prevalent fractures had significantly lower DXA areal BMD at all measured sites than men without fracture. Men with a prevalent fracture had markedly lower trabecular volumetric BMD (radius 6.6%, P<0.0001; tibia 4.5%, P<0.0001) and a marginally lower cortical volumetric BMD (radius 0.4%, P ¼ 0.0012; tibia 0.3%, P ¼ 0.015), compared with men without fracture. However, cortical cross-sectional area and periosteal circumference did not differ between fracture and non-fracture subjects. In a logistic regression analysis adjusted for age, height, weight, calcium intake, smoking, and physical activity, pQCT trabecular volumetric BMD was associated with fracture but DXA areal BMD was not. Every SD decrease in trabecular volumetric BMD of the radius and tibia was associated with a 1.46-fold greater fracture prevalence. Further studies are needed to determine if assessment of trabecular volumetric BMD enhances prediction of fractures during growth in healthy subjects, and those with chronic disease.

Quantitative Ultrasound Quantitative ultrasound (QUS) methods have been developed to assess bone health in peripheral skeletal sites such as the calcaneus, phalanges of the hand, and tibia. QUS devices have several advantages compared to x-ray-based techniques in terms of cost and health

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risks. QUS equipment is smaller in size and usually mobile. On average, the cost of the device is substantially less than a DXA scanner and maintenance is less expensive. The absence of ionizing radiation and the short scan time makes the method suitable for use even in small subjects. The use of QUS equipment does not require a controlled environment or specific authorization by health or radiation safety authorities. In addition, large reference datasets are available at selected skeletal sites. Ultrasound is a mechanical wave with frequencies extending from 20 kHz to 100 MHz. Its energy is transmitted through a medium (i.e. 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 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 [240]. 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 ¼ (E/r)1/2, where E is the modulus of elasticity (a measure of resistance to deformation), and r 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 e 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. 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, the investigators concluded that the bone architecture was responsible for the scattering mechanism [241]. 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 was attributed mainly to absorption [241,242]. Conversely, in other

studies, energy losses by scattering in the internal structure of cancellous bone or friction at the bone marrow interfaces were considered the main cause of attenuation [243e245]. Attenuation strongly depends on the frequency of the ultrasound wave employed. With the use of a lowfrequency range (200e600 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 (attenuation vs. frequency) is the BUA value, given in decibels per megahertz. 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 an increase in bone porosity led to the identification of an amplitude-related measurement of SoS. This amplitude-dependent parameter (AD-SoS) is able to magnify the differences in SoS as measured in diverse bone samples (i.e. normal or osteoporotic bone) [246,247]. AD-SoS is expressed in meters per second. In other QUS devices, ultrasound velocity is measured as apparent velocity of ultrasound (AVU), which differs from true velocity by approximately 100 m/s. 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 [248,249]. 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/s, expressed in microseconds [250]. Some QUS devices also provide combined measurements called stiffness index and quantitative ultrasound index. These parameters are mathematically calculated from both SoS and BUA values. Prior pediatric studies have reported these indices, including normative data in varied populations [251,252]. More recently, Baroncelli et al. reported pediatric applications of two additional phalangeal QUS variables based on the morphology of the ultrasound graphic trace: (1) energy was extrapolated from the area under the ultrasound signal received, and (2) weighted-slope was derived from the angular coefficient of the regression line fitting the top point of the peaks of the ultrasound signal [253]. The first QUS devices measured velocity and attenuation in the heel, and for years ultrasound investigations

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concentrated on this site. The number 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 ultrasound equipment consists of two transducers, a transmitter and a receiver (Figure 13.13) [254]. At the

phalangeal and calcaneus sites, the transducers are placed on opposites sides of the bone. The ultrasound wave produced by the transmitter crosses the bone and is received by the second transducer. All of the devices that study the crossing of bone by ultrasound waves 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, a coupling medium is used to enhance the transmission of the ultrasound wave between the transducers and the skin. In general, QUS systems use water or ultrasound gel as coupling substances. Because the ultrasound wave crosses the bone being examined, the information pertains to both cortical and cancellous portions at these sites. Alternatively, QUS multisite devices combine the transmitter and the receiver in one probe and can be used to measure the ultrasound velocity longitudinally along the cortical bone, as illustrated in the tibia in Figure 13.13. These types of measures provide information about cortical bone only [255,256]. The values of reproducibility for the various skeletal sites are shown in Table 13.5. 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 [257]. Improved precision is obtained with the newer devices (using coupled or in-line transducers) by rotating the probe

TABLE 13.5 Site

Reproducibility of Ultrasound Measurements at Different Skeletal Sites CV%

CV% in children

BUA

0.8e2.5

1.4e5.4

SoS

0.2e0.6%

0.14e0.64

Patella

1.5

0.49

Tibia

0.2e0.7

0.43

Radius

0.4e0.8

Phalanges

0.5e1.1

Calcaneus

FIGURE 13.13 Schematic representation of QUS devices used to assess bone status in children. The images on the left illustrate the probes and transducers with the hashed arrows representing the principle pathways of the ultrasound waves from the emitter transducer to the receiving transducer. The images to the right illustrate the region of interest. (From Baroncelli GI. Quantitative ultrasound methods to assess bone mineral status in children: technical characteristics, performance, and clinical application. Pediatr Res. 2008;63:220e8.)

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0.55

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around the long bone and by averaging the readings of several scans. Bone size and soft tissue thickness may affect the measurement of QUS variables. Cheng et al. reported that calcaneal length correlated significantly with SoS using a scanner from one manufacturer, while heel width correlated significantly with SoS using a scanner by a different manufacturer [258]. Heel width also correlated significantly with BUA with one scanner but not the other. The authors concluded that variation in the region of interest and bone size might affect the accuracy of QUS measurements since the calcaneus is heterogeneous both in terms of its external geometry and its internal structure and density. In growing children, it is estimated that only 6% of AD-SoS measures in the proximal phalanges of the hand are related to finger width [259]. In a study of SoS measured in the tibia midshaft in 1689 healthy children, SoS correlated positively with body height and weight, but negatively with the length of the tibia [260]. The thickness of the surrounding soft tissues in the heel [261], proximal phalange of the hand [262], and tibia or radius [263] may also influence QUS variables. Measures of phalangeal SoS can be corrected for the overlying soft tissue by including measures of the soft tissue area of the first interdigital space [259]. BTT is largely independent of soft tissue bias in the phalanges of the hand [264]. There are numerous studies assessing the relations between QUS parameters, age, pubertal status and anthropometry. The comparison of results is complicated by the heterogeneous skeletal sites, by the use of different QUS devices within the same site, and by differences in SoS and BUA results in the same study. For example, one study compared the results obtained with two different QUS devices that measured SoS at the calcaneus and the tibia [265]. The correlation between calcaneal and tibial ultrasound was poor (r ¼ 0.29; P<0.01), demonstrating that QUS measurements are site specific. Studies comparing tibia and radius SoS demonstrated a low (r ¼ 0.39, P<0.02) [263] or good correlation (r ¼ 0.77, P<0.05) [266]. In another example, SoS in the thumb and patella increased with age and peaked at 20 to 25 years of age, while SoS in the calcaneus showed no increase after puberty [265]. Anthropometric measures, age and pubertal stage influenced QUS measures in most studies of the calcaneus, patella, proximal phalanges of the hand, tibia and radius. These data indicate that skeletal growth and sex-dependent differences in skeletal maturation are important determinants of QUS measurements. The majority of early studies were conducted in the calcaneus. Most studies of the calcaneus showed an increase in BUA [267e270] and an increase in SoS [265,268,270,271] with age. However, a study conducted in 3299 healthy Caucasian children, aged 6 to 18 years,

reported no correlation between the SoS and age, height, and weight [269]. Measurements of SoS at the cortical shaft of the tibia in large samples of healthy children and adolescents showed a strong age dependence [260,272e274]. A longitudinal study of QUS parameters at the patella site demonstrated that the patella AVU rate of change was greater at ages 10 to 13 years in girls and between 15 and 18 years in boys [275,276]. In 2006, Baroncelli et al. reported phalangeal QUS results in 3044 (1513 males and 1531 females) healthy subjects, aged 2 to 21 years [253]. The report provided reference data for phalangeal AD-SoS and BTT, both expressed as centiles and Z score, according to sex, age, height, weight, BMI, and pubertal stage. The ADSoS data are shown in Figure 13.14. In both sexes, ADSoS and BTT were significantly correlated (r ¼ 0.92, P<0.0001), and both parameters were correlated with all of the anthropometric variables (r ¼ 0.53e0.85, P<0.0001). Few studies have compared QUS measures with other techniques for bone mass measurement in children and adolescents. In a sample of 58 healthy youth, DXA total body BMD values were positively correlated with heel BUA measurements (r ¼ 0.74, P<0.001) [277]. In another study of 125 participants, DXA measurements obtained from the spine, the femoral neck, and the whole skeleton showed a moderate correlation with SoS and BUA values obtained at the calcaneus (r ¼ 0.23e0.58, P<0.008) [268]. Lequin et al. compared tibial QUS SoS values with BMD measured at the midphalanges by radiogrammetry in 563 children [278]. Multiple regression analysis showed that SoS correlated significantly with BMD for both boys (r ¼ 0.65, P<0.001) and girls (r ¼ 0.59, P<0.001). Most recently, Fricke et al. examined the associations between SoS as measured by QUS at the thumb, patella, and calcaneus, and trabecular volumetric BMD as measured by pQCT in 216 participants [279]. Linear regression analysis revealed that the prediction of SoS by volumetric BMD was weak (r2<0.1). Moreover, body height and measures of bone size had a stronger influence on SoS than volumetric BMD. The authors concluded that QUS is not a suitable method to assess volumetric BMD. Limitations 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 [280,281]. Additionally, QUS measurements seem to be related to bone

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FIGURE 13.14

QUS reference data for ADSoS measured at the distal end of the proximal phalangeal diaphysis of the hand (DBM Sonic, IGWEA, Carpi, Italy) in (A) 1513 healthy males and (B) 1531 healthy females, aged 2 to 21 years. The results are expressed as percentiles. (From Baroncelli GI, Federico G, Vignolo M et al. Crosssectional reference data for phalangeal quantitative ultrasound from early childhood to young-adulthood according to gender, age, skeletal growth, and pubertal development. Bone. 2006;39:159e73.)

size [282,283] 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. 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 phalanges. 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. Nonetheless, bone measurements by x-ray-based devices known to predict fracture risk have correlated poorly with those obtained with QUS [284]. Reference Data The low cost, lack of radiation exposure, and ease of measurement has facilitated the generation of

numerous large reference datasets. As shown in Figure 13.14, sex-specific reference curves are available for AD-SoS and BTT measured at the distal end of the proximal phalangeal diaphysis of the hand based on measures in 3044 healthy children and adolescents in Italy (DBM Sonic, IGEA, Carpi, Italy) [285]. Sex-specific reference values, expressed as means and SD for each year of age, are also available for BUA and SoS of the calcaneus based on measures in 3299 children, aged 6 to 18 years in Germany (Sahara, Hologic Inc., Waltham, MA) [269]. And, sex-specific reference values for SoS, expressed as means and SD for each year of age, measured by multisite quantitative ultrasound at the midshaft of the tibia and distal third of the radius are available based on data in 1085 healthy subjects, aged 0 to 18 years, in Israel (Sunlight Omnisense 7000P) [273]. Additional large datasets include tibia SoS data in 1689 healthy Japanese children and adolescents, aged 7 to 19 years [260], phalangeal AD-SoS and BTT in 1328 German children, aged 3 to 17 years [264], and

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calcaneal SoS, BUA and stiffness index in 726 healthy Chinese children and adolescents [252]. While these databases are large and provide sexspecific reference data, they are not applicable to measures obtained using scanners by different manufacturers and may not be applicable to children from other countries. Fracture Discrimination QUS has been used in large population based studies of fracture risk in the elderly. Two recent studies with ROC analyses reported that baseline QUS results predicted subsequent fracture. A study of 7000 elderly women reported that calcaneal QUS results were predictive of hip fracture risk, while phalanges QUS was not [286]. A study of 1455 men and women, aged 65 to 76 years, demonstrated that a 1 SD decrease in calcaneal BUA was associated with a twofold increase in fracture risk [287]. To our knowledge, prospective data relating QUS results to subsequent fractures are not available in children. However, a recent study in a population sample of over 4000 female army recruits suggests that QUS is informative in young adults [288]. Calcaneal QUS was measured at the beginning of basic training, and incident stress fractures were documented over the subsequent 8 weeks. Heel SoS was significantly related to the risk of stress fracture (P<0.0001); the area under the ROC curve was 0.70. The relative risk (RR) of fracture of those in the lowest quintile (Q1), compared with the highest quintile of SoS was 6.7. Jones et al. conducted a study in 415 adolescents (160 with a prevalent fracture) to describe the association between different measures of bone mass and fracture [289]. DXA measurement sites included the hip, spine, radius and total body, and QUS measures included calcaneal BUA, SoS, and stiffness, and radius SoS. The metacarpal index was calculated from a hand x-ray. Significantly lower DXA measures, heel BUA, and heel stiffness were observed in those with a history of upper limb fracture (all P<0.05), compared to those without a fracture. The odds ratios for fracture were 1.38 (95% C.I. 1.08, 1.77) for heel BUA and 1.34 (95% C.I. 1.06, 1.71) for heel stiffness for each 1 SD decrease in the QUS results. Despite significant correlations between all the bone mass measures, radius SoS and metacarpal index did not discriminate those with fracture from those without. These data require confirmation in larger, prospective studies.

SUMMARY The ultimate test of any measurement of bone mass is its ability to predict clinical bone fragility. In adults,

DXA areal BMD has been shown to be a very significant correlate of fracture. However, the risk of fracture for children whose bone densities fall 1 or more SDs below the mean for age, bone age, or body size has not been established. Furthermore, the ability of each of these modalities to predict fracture in children with chronic diseases has not been assessed. 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.

References [1] Baim S, Leonard MB, Bianchi ML, et al. Official positions of the International Society for Clinical Densitometry and executive summary of the 2007 ISCD Pediatric Position Development Conference. J Clin Densitom 2008;11:6e21. [2] Bishop N, Braillon P, Burnham J, et al. Dual-energy X-ray aborptiometry assessment in children and adolescents with diseases that may affect the skeleton: the 2007 ISCD Pediatric Official Positions. J Clin Densitom 2008;11:29e42. [3] Gordon CM, Bachrach LK, Carpenter TO, et al. Dual energy Xray absorptiometry interpretation and reporting in children and adolescents: the 2007 ISCD Pediatric Official Positions. J Clin Densitom 2008;11:43e58. [4] Rauch F, Plotkin H, DiMeglio L, et al. Fracture prediction and the definition of osteoporosis in children and adolescents: the ISCD 2007 Pediatric Official Positions. J Clin Densitom 2008;11:22e8. [5] Zemel B, Bass S, Binkley T, et al. Peripheral quantitative computed tomography in children and adolescents: the 2007 ISCD Pediatric Official Positions. J Clin Densitom 2008;11:59e74. [6] Arden NK, Baker J, Hogg C, Baan K, Spector TD. The heritability of bone mineral density, ultrasound of the calcaneus and hip axis length: a study of postmenopausal twins. J Bone Miner Res 1996;11:530e4. [7] Arden NK, Spector TD. Genetic influences on muscle strength, lean body mass, and bone mineral density: a twin study. J Bone Miner Res 1997;12:2076e81. [8] Koay MA, Tobias JH, Leary SD, Steer CD, Vilarino-Guell C, Brown MA. The effect of LRP5 polymorphisms on bone mineral density is apparent in childhood. Calcif Tissue Int 2007;81:1e9. [9] Paternoster L, Ohlsson C, Sayers A, et al. OPG and RANK polymorphisms are both associated with cortical bone mineral density: findings from a metaanalysis of the Avon longitudinal study of parents and children and Gothenburg osteoporosis and obesity determinants cohorts. J Clin Endocrinol Metab 2010;95:3940e8.. [10] Macdonald-Wallis C, Tobias JH, Davey Smith G, Lawlor DA. Parental smoking during pregnancy and offspring bone mass at age 10 years: findings from a prospective birth cohort. Osteoporos Int 2011;22:1809e19. [11] Cooper C, Westlake S, Harvey N, Javaid K, Dennison E, Hanson M. Review: developmental origins of osteoporotic fracture. Osteoporos Int 2006;17:337e47. [12] Klein GL, Fitzpatrick LA, Langman CB, et al. The state of pediatric bone: summary of the ASBMR pediatric bone initiative. J Bone Miner Res 2005;20:2075e81. [13] NIH. Osteoporosis Prevention, Diagnosis, and Therapy. NIH Consensus Statement 2000 March 27-29;17:1e36.

PEDIATRIC BONE

REFERENCES

[14] Rauch F, Schoenau E. Changes in bone density during childhood and adolescence: an approach based on bone’s biological organization. J Bone Miner Res 2001;16:597e604. [15] Roschger P, Paschalis EP, Fratzl P, Klaushofer K. Bone mineralization density distribution in health and disease. Bone 2008;42:456e66. [16] Turner CH. Basic biomechanical measurements of bone: a tutorial. Bone 1993;14:595e608. [17] Leonard MB, Shults J, Elliott DM, Stallings VA, Zemel BS. Interpretation of whole body dual energy X-ray absorptiometry measures in children: comparison with peripheral quantitative computed tomography. Bone 2004;34:1044e52. [18] Molgaard C, Thomsen BL, Prentice A, Cole TJ, Michaelsen KF. Whole body bone mineral content in healthy children and adolescents. Arch Dis Child 1997;76:9e15. [19] Beck TJ, Ruff CB, Warden KE, Scott Jr WW, Rao GU. Predicting femoral neck strength from bone mineral data. A structural approach. Invest Radiol 1990;25:6e18. [20] Liu D, Manske SL, Kontulainen SA, et al. Tibial geometry is associated with failure load ex vivo: a MRI, pQCT and DXA study. Osteoporos Int 2007;18:991e7. [21] Leonard MB, Elmi A, Mostoufi-Moab S, et al. Effects of sex, race, and puberty on cortical bone and the functional muscle bone unit in children, adolescents, and young adults. J Clin Endocrinol Metab 2010;95:1681e9. [22] Seeman E. From density to structure: growing up and growing old on the surfaces of bone. J Bone Miner Res 1997;12:509e21. [23] Seeman E, Delmas PD. Bone quality e the material and structural basis of bone strength and fragility. N Engl J Med 2006;25(354):2250e61. [24] Bass S, Delmas PD, Pearce G, Hendrich E, Tabensky A, Seeman E. The differing tempo of growth in bone size, mass, and density in girls is region-specific. J Clin Invest 1999;104:795e804. [25] WHO. The WHO Study Group: Assessment of fracture risk and its application to screening for postmenopausal osteoporosis. 1994; Geneva, Switzerland; 1994. [26] Lewiecki EM, Gordon CM, Baim S, et al. International Society for Clinical Densitometry 2007 Adult and Pediatric Official Positions. Bone 2008;43:1115e21. [27] Khosla S, Melton 3rd LJ, Dekutoski MB, Achenbach SJ, Oberg AL, Riggs BL. Incidence of childhood distal forearm fractures over 30 years: a population-based study. J Am Med Assoc 2003;290:1479e85. [28] Faulkner RA, Davison KS, Bailey DA, Mirwald RL, BaxterJones AD. Size-corrected BMD decreases during peak linear growth: implications for fracture incidence during adolescence. J Bone Miner Res 2006;21:1864e70. [29] Van Staa TP, Laan RF, Barton IP, Cohen S, Reid DM, Cooper C. Bone density threshold and other predictors of vertebral fracture in patients receiving oral glucocorticoid therapy. Arthritis Rheum 2003;48:3224e9. [30] NTP 11th Report on Carcinogens. Rep Carcinog 2005;11:1eA32. [31] van Rijn RR, Van Kuijk C. Of small bones and big mistakes; bone densitometry in children revisited. Eur J Radiol 2009;71:432e9. [32] Huda W, Bissessur K. Effective dose equivalents, HE, in diagnostic radiology. Med Phys 1990;17:998e1003. [33] Okkalides D, Fotakis M. Patient effective dose resulting from radiographic examinations. Br J Radiol 1994;67:564e72. [34] Blake GM, Naeem M, Boutros M. Comparison of effective dose to children and adults from dual X-ray absorptiometry examinations. Bone 2006;38:935e42. [35] Adams JE. Quantitative computed tomography. Eur J Radiol 2009;71:415e24.

335

[36] Engelke K, Adams JE, Armbrecht G, et al. Clinical use of quantitative computed tomography and peripheral quantitative computed tomography in the management of osteoporosis in adults: the 2007 ISCD Official Positions. J Clin Densitom 2008;11:123e62. [37] Barnett E, Nordin BE. The radiological diagnosis of osteoporosis: a new approach. Clin Radiol 1960;11:166e74. [38] Horsman A, Simpson M. The measurement of sequential changes in cortical bone geometry. Br J Radiol 1975;48:471e6. [39] Bloom RA, Pogrund H, Libson E. Radiogrammetry of the metacarpal: a critical reappraisal. Skeletal Radiol 1983;10:5e9. [40] Kalla AA, Meyers OL, Parkyn ND, Kotze TJ. Osteoporosis screening e radiogrammetry revisited. Br J Rheumatol 1989;28:511e7. [41] Adams JE. Radiogrammetry and radiographic absorptiometry. Radiol Clin North Am 2010;48:531e40. [42] Garn SM, Poznanski AK, Nagy JM. Bone measurement in the differential diagnosis of osteopenia and osteoporosis. Radiology 1971;100:509e18. [43] Jorgensen JT, Andersen PB, Rosholm A, Bjarnason NH. Digital X-ray radiogrammetry: a new appendicular bone densitometric method with high precision. Clin Physiol 2000;20:330e5. [44] Rosholm A, Hyldstrup L, Backsgaard L, Grunkin M, Thodberg HH. Estimation of bone mineral density by digital X-ray radiogrammetry: theoretical background and clinical testing. Osteoporos Int 2001;12:961e9. [45] Matkovic V, Fontana D, Tominac C, Goel P, Chesnut 3rd CH. 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 1990;52:878e88. [46] Ilich JZ, Hangartner TN, Skugor M, Roche AF, Goel PK, Matkovic V. Skeletal age as a determinant of bone mass in preadolescent females. Skeletal Radiol 1996;25:431e9. [47] van Rijn RR, Grootfaam DS, Lequin MH, et al. Digital radiogrammetry of the hand in a pediatric and adolescent Dutch Caucasian population: normative data and measurements in children with inflammatory bowel disease and juvenile chronic arthritis. Calcif Tissue Int 2004;74:342e50. [48] Bottcher J, Pfeil A, Schafer ML, et al. Normative data for digital X-ray radiogrammetry from a female and male German cohort. J Clin Densitom 2006;9:341e50. [49] Malich A, Freesmeyer MG, Mentzel HJ, et al. Normative values of bone parameters of children and adolescents using digital computer-assisted radiogrammetry (DXR). J Clin Densitom 2003;6:103e11. [50] Mentzel HJ, Blume J, Boettcher J, et al. The potential of digital X-ray radiogrammetry (DXR) in the assessment of osteopenia in children with chronic inflammatory bowel disease. Pediatr Radiol 2006;36:415e20. [51] van Rijn RR, Boot A, Wittenberg R, et al. Direct X-ray radiogrammetry versus dual-energy X-ray absorptiometry: assessment of bone density in children treated for acute lymphoblastic leukaemia and growth hormone deficiency. Pediatr Radiol 2006;36:227e32. [52] Hayashi Y, Yamamoto K, Fukunaga M, Ishibashi T, Takahashi K, Nishii Y. Assessment of bone mass by image analysis of metacarpal bone roentgenograms: a quantitative digital image processing (DIP) method. Radiat Med 1990;8:173e8. [53] Cosman F, Herrington B, Himmelstein S, Lindsay R. Radiographic absorptiometry: a simple method for determination of bone mass. Osteoporos Int 1991;2:34e8. [54] Seo GS, Shiraki M, Aoki C, et al. Assessment of bone density in the distal radius with computer assisted X-ray densitometry (CXD). Bone Miner 1994;27:173e82.

PEDIATRIC BONE

336

13. NON-INVASIVE TECHNIQUES FOR BONE MASS MEASUREMENT

[55] Adami S, Zamberlan N, Gatti D, et al. Computed radiographic absorptiometry and morphometry in the assessment of postmenopausal bone loss. Osteoporos Int 1996;6:8e13. [56] Yang SO, Hagiwara S, Engelke K, et al. Radiographic absorptiometry for bone mineral measurement of the phalanges: precision and accuracy study. Radiology 1994;192:857e9. [57] Gulam M, Thornton MM, Hodsman AB, Holdsworth DW. Bone mineral measurement of phalanges: comparison of radiographic absorptiometry and area dual X-ray absorptiometry. Radiology 2000;216:586e91. [58] Trouerbach WT, de Man SA, Gommers D, Zwamborn AW, Grobbee DE. Determinants of bone mineral content in childhood. Bone Miner 1991;13:55e67. [59] van Teunenbroek A, Mulder P, de Muinck Keizer-Schrama S, et al. Radiographic absorptiometry of the phalanges in healthy children and in girls with Turner syndrome. Bone 1995;17:71e8. [60] van Rijn RR, Lequin MH, van Leeuwen WJ, Hop WC, van Kuijk C. Radiographic absorptiometry of the middle phalanx (digit II) in a Caucasian pediatric population: normative data. Osteoporos Int 2000;11:240e7. [61] Blake GM, Fogelman I. Bone densitometry and the diagnosis of osteoporosis. Semin Nucl Med 2001;31:69e81. [62] Cullum ID, Ell PJ, Ryder JP. X-ray dual-photon absorptiometry: a new method for the measurement of bone density. Br J Radiol 1989;62:587e92. [63] Larson CM, Henderson RC. Bone mineral density and fractures in boys with Duchenne muscular dystrophy. J Pediatr Orthop 2000;20:71e4. [64] Henderson RC, Berglund LM, May R, et al. The relationship between fractures and DXA measures of BMD in the distal femur of children and adolescents with cerebral palsy or muscular dystrophy. J Bone Miner Res 2010;25:520e6. [65] Harcke HT, Taylor A, Bachrach S, Miller F, Henderson RC. Lateral femoral scan: an alternative method for assessing bone mineral density in children with cerebral palsy. Pediatr Radiol 1998;28:241e6. [66] Henderson RC, Lark RK, Newman JE, et al. Pediatric reference data for dual X-ray absorptiometric measures of normal bone density in the distal femur. Am J Roentgenol 2002;178:439e43. [67] Tothill P, Avenell A, Reid DM. Precision and accuracy of measurements of whole-body bone mineral: comparisons between Hologic, Lunar and Norland dual-energy X-ray absorptiometers. Br J Radiol 1994;67:1210e7. [68] Genant HK, Grampp S, Gluer CC, et al. Universal standardization for dual x-ray absorptiometry: patient and phantom cross-calibration results. J Bone Miner Res 1994;9:1503e14. [69] Hind K, Oldroyd B, Truscott JG. In vivo precision of the GE Lunar iDXA densitometer for the measurement of total body composition and fat distribution in adults. Eur J Clin Nutr 2010. Sep 15. [70] Johnson J, Dawson-Hughes B. Precision and stability of dualenergy X-ray absorptiometry measurements. Calcif Tissue Int 1991;49:174e8. [71] Leonard CM, Roza MA, Barr RD, Webber CE. Reproducibility of DXA measurements of bone mineral density and body composition in children. Pediatr Radiol 2009;39:148e54. [72] Ho CP, Kim RW, Schaffler MB, Sartoris DJ. Accuracy of dualenergy radiographic absorptiometry of the lumbar spine: cadaver study. Radiology 1990;176:171e3. [73] Hagiwara S, Engelke K, Yang SO, et al. Dual x-ray absorptiometry forearm software: accuracy and intermachine relationship. J Bone Miner Res 1994;9:1425e7. [74] Sabin MA, Blake GM, MacLaughlin-Black SM, Fogelman I. The accuracy of volumetric bone density measurements in dual x-ray absorptiometry. Calcif Tissue Int 1995;56:210e4. r.

[75] Sran MM, Khan KM, Keiver K, Chew JB, McKay HA, Oxland TR. Accuracy of DXA scanning of the thoracic spine: cadaveric studies comparing BMC, areal BMD and geometric estimates of volumetric BMD against ash weight and CT measures of bone volume. Eur Spine J 2005;14:971e6. [76] Svendsen OL, Hassager C, Skodt V, Christiansen C. Impact of soft tissue on in vivo accuracy of bone mineral measurements in the spine, hip, and forearm: a human cadaver study. J Bone Miner Res 1995;10:868e73. [77] Lewis MK, Blake GM, Fogelman I. Patient dose in dual x-ray absorptiometry. Osteoporos Int 1994;4:11e5. [78] Njeh CF, Samat SB, Nightingale A, McNeil EA, Boivin CM. Radiation dose and in vitro precision in paediatric bone mineral density measurement using dual X-ray absorptiometry. Br J Radiol 1997;70:719e27. [79] Njeh CF, Fuerst T, Hans D, Blake GM, Genant HK. Radiation exposure in bone mineral density assessment. Appl Radiat Isot 1999;50:215e36. [80] Cole JH, Scerpella TA, van der Meulen MC. Fan-beam densitometry of the growing skeleton: are we measuring what we think we are? J Clin Densitom 2005;8:57e64. [81] Cole JH, Dowthwaite JN, Scerpella TA, van der Meulen MC. Correcting fan-beam magnification in clinical densitometry scans of growing subjects. J Clin Densitom 2009;12:322e9. [82] Sievanen H, Backstrom MC, Kuusela AL, Ikonen RS, Maki M. Dual energy x-ray absorptiometry of the forearm in preterm and term infants: evaluation of the methodology. Pediatr Res 1999;45:100e5. [83] Koo WW, Hammami M, Shypailo RJ, Ellis KJ. Bone and body composition measurements of small subjects: discrepancies from software for fan-beam dual energy X-ray absorptiometry. J Am Coll Nutr 2004;23:647e50. [84] Leonard MB, Feldman HI, Zemel BS, Berlin JA, Barden EM, Stallings VA. Evaluation of low density spine software for the assessment of bone mineral density in children. J Bone Miner Res 1998;13:1687e90. [85] Shypailo RJ, Ellis KJ. Bone assessment in children: comparison of fan-beam DXA analysis. J Clin Densitom 2005;8:445e53. [86] McKay HA, Petit MA, Bailey DA, Wallace WM, Schutz RW, Khan KM. Analysis of proximal femur DXA scans in growing children: comparisons of different protocols for cross-sectional 8-month and 7-year longitudinal data. J Bone Miner Res 2000;15:1181e8. [87] Bachrach LK, Hastie T, Wang MC, Narasimhan B, Marcus R. Bone mineral acquisition in healthy Asian, Hispanic, black, and Caucasian youth: a longitudinal study. J Clin Endocrinol Metab 1999;84:4702e12. [88] Ellis KJ, Shypailo RJ, Hardin DS, et al. Z score prediction model for assessment of bone mineral content in pediatric diseases. J Bone Miner Res 2001;16:1658e64. [89] Faulkner RA, Bailey DA, Drinkwater DT, McKay HA, Arnold C, Wilkinson AA. Bone densitometry in Canadian children 8e17 years of age. Calcif Tissue Int 1996;59:344e51. [90] Binkley TL, Specker BL, Wittig TA. Centile curves for bone densitometry measurements in healthy males and females ages 5e22 yr. J Clin Densitom 2002;5:343e53. [91] Hannan WJ, Tothill P, Cowen SJ, Wrate RM. Whole body bone mineral content in healthy children and adolescents. Arch Dis Child 1998;78:396e7. [92] Maynard LM, Guo SS, Chumlea WC, et al. Total-body and regional bone mineral content and areal bone mineral density in children aged 8e18 y: the Fels Longitudinal Study. Am J Clin Nutr 1998;68:1111e7. [93] van der Sluis IM, de Ridder MA, Boot AM, Krenning EP, de Muinck Keizer-Schrama SM. Reference data for bone density

PEDIATRIC BONE

REFERENCES

[94]

[95]

[96]

[97]

[98]

[99]

[100] [101]

[102]

[103]

[104]

[105]

[106]

[107]

[108]

[109]

[110]

and body composition measured with dual energy x-ray absorptiometry in white children and young adults. Arch Dis Child 2002;87:341e7. discussion 7. Zanchetta JR, Plotkin H, Alvarez Filgueira ML. Bone mass in children: normative values for the 2e20-year-old population. Bone 1995;16(Suppl. 4):393Se9S. Sabatier JP, Guaydier-Souquieres G, Laroche D, et al. Bone mineral acquisition during adolescence and early adulthood: a study in 574 healthy females 10e24 years of age. Osteoporos Int 1996;6:141e8. McCormick DP, Ponder SW, Fawcett HD, Palmer JL. Spinal bone mineral density in 335 normal and obese children and adolescents: evidence for ethnic and sex differences. J Bone Miner Res 1991;6:507e13. Bonjour JP, Theintz G, Buchs B, Slosman D, Rizzoli R. Critical years and stages of puberty for spinal and femoral bone mass accumulation during adolescence. J Clin Endocrinol Metab 1991;73:555e63. Plotkin H, Nunez M, Alvarez Filgueira ML, Zanchetta JR. Lumbar spine bone density in Argentine children. Calcif Tissue Int 1996;58:144e9. Southard RN, Morris JD, Mahan JD, et al. Bone mass in healthy children: measurement with quantitative DXA. Radiology 1991;179:735e8. Henderson RC, Madsen CD. Bone density in children and adolescents with cystic fibrosis. J Pediatr 1996;128:28e34. Glastre C, Braillon P, David L, Cochat P, Meunier PJ, Delmas PD. Measurement of bone mineral content of the lumbar spine by dual energy x-ray absorptiometry in normal children: correlations with growth parameters. J Clin Endocrinol Metab 1990;70:1330e3. del Rio L, Carrascosa A, Pons F, Gusinye M, Yeste D, Domenech FM. Bone mineral density of the lumbar spine in white Mediterranean Spanish children and adolescents: changes related to age, sex, and puberty. Pediatr Res 1994;35:362e6. Braillon PM, Cochat P. Analysis of dual energy X-ray absorptiometry whole body results in children, adolescents and young adults. Appl Radiat Isot 1998;49:623e4. Leonard MB, Propert KJ, Zemel BS, Stallings VA, Feldman HI. Discrepancies in pediatric bone mineral density reference data: potential for misdiagnosis of osteopenia. J Pediatr 1999;135:182e8. Katzman DK, Bachrach LK, Carter DR, Marcus R. Clinical and anthropometric correlates of bone mineral acquisition in healthy adolescent girls. J Clin Endocrinol Metab 1991;73:1332e9. Prentice A, Parsons TJ, Cole TJ. Uncritical use of bone mineral density in absorptiometry may lead to size-related artifacts in the identification of bone mineral determinants. Am J Clin Nutr 1994;60:837e42. Wren TA, Liu X, Pitukcheewanont P, Gilsanz V. Bone densitometry in pediatric populations: discrepancies in the diagnosis of osteoporosis by DXA and CT. J Pediatr 2005;146:776e9. Leonard MB, Bachrach LK. Assessment of bone mineralization following renal transplantation in children: limitations of DXA and the confounding effects of delayed growth and development. Am J Transplant 2001;1:193e6. Gafni RI, Baron J. Overdiagnosis of osteoporosis in children due to misinterpretation of dual-energy x-ray absorptiometry (DEXA). J Pediatr 2004;144:253e7. Kalkwarf HJ, Zemel BS, Gilsanz V, et al. The bone mineral density in childhood study: bone mineral content and density according to age, sex, and race. J Clin Endocrinol Metab 2007;92:2087e99.

337

[111] Zemel BS, Leonard MB, Kelly A, et al. Height adjustment in assessing dual energy x-ray absorptiometry measurements of bone mass and density in children. J Clin Endocrinol Metab 2010;95:1265e73. [112] Crabtree N, Leonard MB, Zemel BS. Dual energy X-ray absorptiometry. In: Saywyer AJ, Bachrach LK, Fung EL, editors. Bone Densitometry in Growing Patients: Guidelines for Clinical Practice. Totowa, New Jersey: Humana Press; 2007. p. 41e57. [113] Leonard MB, Shults J, Zemel BS. DXA estimates of vertebral volumetric bone mineral density in children: potential advantages of paired posteroanterior and lateral scans. J Clin Densitom 2006;9:265e73. [114] Oldroyd B, Smith AH, Truscott JG. Cross-calibration of GE/ Lunar pencil and fan-beam dual energy densitometers e bone mineral density and body composition studies. Eur J Clin Nutr 2003;57:977e87. [115] Brunton JA, Bayley HS, Atkinson SA. Validation and application of dual-energy x-ray absorptiometry to measure bone mass and body composition in small infants. Am J Clin Nutr 1993;58:839e45. [116] Brunton JA, Weiler HA, Atkinson SA. Improvement in the accuracy of dual energy x-ray absorptiometry for whole body and regional analysis of body composition: validation using piglets and methodologic considerations in infants. Pediatr Res 1997;41:590e6. [117] Picaud JC, Lapillonne A, Pieltain C, et al. Software and scan acquisition technique-related discrepancies in bone mineral assessment using dual-energy X-ray absorptiometry in neonates. Acta Paediatr 2002;91:1189e93. [118] Picaud JC, Nyamugabo K, Braillon P, et al. Dual-energy X-ray absorptiometry in small subjects: influence of dual-energy X-ray equipment on assessment of mineralization and body composition in newborn piglets. Pediatr Res 1999;46:772e7. [119] Koo WW, Hammami M, Hockman EM. Use of fan beam dual energy x-ray absorptiometry to measure body composition of piglets. J Nutr 2002;132:1380e3. [120] Chauhan S, Koo WW, Hammami M, Hockman EM. Fan beam dual energy X-ray absorptiometry body composition measurements in piglets. J Am Coll Nutr 2003;22:408e14. [121] Koo WW, Hammami M, Hockman EM. Interchangeability of pencil-beam and fan-beam dual-energy X-ray absorptiometry measurements in piglets and infants. Am J Clin Nutr 2003;78:236e40. [122] Koo WW, Hammami M, Hockman EM. Validation of bone mass and body composition measurements in small subjects with pencil beam dual energy X-ray absorptiometry. J Am Coll Nutr 2004;23:79e84. [123] Koo WW, Hockman EM, Hammami M. Dual energy X-ray absorptiometry measurements in small subjects: conditions affecting clinical measurements. J Am Coll Nutr 2004;23:212e9. [124] Nelson DA, Koo WW. Interpretation of absorptiometric bone mass measurements in the growing skeleton: issues and limitations. Calcif Tissue Int 1999;65:1e3. [125] Carter DR, Bouxsein ML, Marcus R. New approaches for interpreting projected bone densitometry data. J Bone Miner Res 1992;7:137e45. [126] Kroger H, Kotaniemi A, Kroger L, Alhava E. Development of bone mass and bone density of the spine and femoral neckea prospective study of 65 children and adolescents. Bone Miner 1993;23:171e82. [127] Kroger H, Kotaniemi A, Vainio P, Alhava E. Bone densitometry of the spine and femur in children by dual-energy x-ray absorptiometry. Bone Miner 1992;17:75e85. [128] Kroger H, Vainio P, Nieminen J, Kotaniemi A. Comparison of different models for interpreting bone mineral density

PEDIATRIC BONE

338

[129]

[130]

[131]

[132]

[133]

[134]

[135]

[136]

[137]

[138]

[139]

[140]

[141]

[142]

[143]

[144]

[145]

[146]

13. NON-INVASIVE TECHNIQUES FOR BONE MASS MEASUREMENT

measurements using DXA and MRI technology. Bone 1995;17:157e9. Smith CM, Coombs RC, Gibson AT, Eastell R. Adaptation of the Carter method to adjust lumbar spine bone mineral content for age and body size: application to children who were born preterm. J Clin Densitom 2006;9:114e9. Backstrom MC, Kouri T, Kuusela AL, et al. Bone isoenzyme of serum alkaline phosphatase and serum inorganic phosphate in metabolic bone disease of prematurity. Acta Paediatr 2000;89:867e73. Haapasalo H, Kannus P, Sievanen H, et al. Development of mass, density, and estimated mechanical characteristics of bones in Caucasian females. J Bone Miner Res 1996;11:1751e60. Bhudhikanok GS, Wang MC, Eckert K, Matkin C, Marcus R, Bachrach LK. Differences in bone mineral in young Asian and Caucasian Americans may reflect differences in bone size. J Bone Miner Res 1996;11:1545e56. Wang MC, Aguirre M, Bhudhikanok GS, et al. Bone mass and hip axis length in healthy Asian, black, Hispanic, and white American youths. J Bone Miner Res 1997;12:1922e35. Boot AM, van der Sluis IM, de Muinck Keizer-Schrama SM, et al. Estrogen receptor alpha gene polymorphisms and bone mineral density in healthy children and young adults. Calcif Tissue Int 2004;74:495e500. Ward KA, Roberts SA, Adams JE, Mughal MZ. Bone geometry and density in the skeleton of pre-pubertal gymnasts and school children. Bone 2005;36:1012e8. Dyson K, Blimkie CJ, Davison KS, Webber CE, Adachi JD. Gymnastic training and bone density in pre-adolescent females. Med Sci Sports Exerc 1997;29:443e50. Morris FL, Naughton GA, Gibbs JL, Carlson JS, Wark JD. Prospective ten-month exercise intervention in premenarcheal girls: positive effects on bone and lean mass. J Bone Miner Res 1997;12:1453e62. Pettifor JM, Moodley GP. Appendicular bone mass in children with a high prevalence of low dietary calcium intakes. J Bone Miner Res 1997;12:1824e32. Rockell JE, Williams SM, Taylor RW, Grant AM, Jones IE, Goulding A. Two-year changes in bone and body composition in young children with a history of prolonged milk avoidance. Osteoporos Int 2005;16:1016e23 Lehtonen-Veromaa MK, Mottonen TT, Nuotio IO, Irjala KM, Leino AE, Viikari JS. Vitamin D and attainment of peak bone mass among peripubertal Finnish girls: a 3-y prospective study. Am J Clin Nutr 2002;76:1446e53. Neely EK, Marcus R, Rosenfeld RG, Bachrach LK. Turner syndrome adolescents receiving growth hormone are not osteopenic. J Clin Endocrinol Metab 1993;76:861e6. Bhudhikanok GS, Wang MC, Marcus R, Harkins A, Moss RB, Bachrach LK. Bone acquisition and loss in children and adults with cystic fibrosis: a longitudinal study. J Pediatr 1998;133:18e27. Bhudhikanok GS, Lim J, Marcus R, Harkins A, Moss RB, Bachrach LK. Correlates of osteopenia in patients with cystic fibrosis. Pediatrics 1996;97:103e11. Sood M, Hambleton G, Super M, Fraser WD, Adams JE, Mughal MZ. Bone status in cystic fibrosis. Arch Dis Child 2001;84:516e20. Takahashi Y, Minamitani K, Kobayashi Y, Minagawa M, Yasuda T, Niimi H. Spinal and femoral bone mass accumulation during normal adolescence: comparison with female patients with sexual precocity and with hypogonadism. J Clin Endocrinol Metab 1996;81:1248e53. Boot AM, Engels MA, Boerma GJ, Krenning EP, De Muinck Keizer-Schrama SM. Changes in bone mineral density, body

[147]

[148]

[149]

[150]

[151]

[152]

[153]

[154]

[155]

[156]

[157]

[158]

[159]

[160]

[161]

[162]

[163]

composition, and lipid metabolism during growth hormone (GH) treatment in children with GH deficiency. J Clin Endocrinol Metab 1997;82:2423e8. Bachrach LK, Marcus R, Ott SM, et al. Bone mineral, histomorphometry, and body composition in adults with growth hormone receptor deficiency. J Bone Miner Res 1998;13:415e21. van der Sluis IM, Boot AM, Hop WC, De Rijke YB, Krenning EP, de Muinck Keizer-Schrama SM. Long-term effects of growth hormone therapy on bone mineral density, body composition, and serum lipid levels in growth hormone deficient children: a 6-year follow-up study. Horm Res 2002;58:207e14. Abad V, Chrousos GP, Reynolds JC, et al. Glucocorticoid excess during adolescence leads to a major persistent deficit in bone mass and an increase in central body fat. J Bone Miner Res 2001;16:1879e85. Bielinski BK, Darbyshire P, Mathers L, Boivin CM, Shaw NJ. Bone density in the Asian thalassaemic population: a crosssectional review. Acta Paediatr 2001;90:1262e6. Salvatoni A, Mancassola G, Biasoli R, et al. Bone mineral density in diabetic children and adolescents: a follow-up study. Bone 2004;34:900e4. Daniels MW, Wilson DM, Paguntalan HG, Hoffman AR, Bachrach LK. Bone mineral density in pediatric transplant recipients. Transplantation 2003;76:673e8. Gandrud LM, Cheung JC, Daniels MW, Bachrach LK. Low-dose intravenous pamidronate reduces fractures in childhood osteoporosis. J Pediatr Endocrinol Metab 2003;16:887e92. Wren TA, Liu X, Pitukcheewanont P, Gilsanz V. Bone acquisition in healthy children and adolescents: comparisons of dualenergy x-ray absorptiometry and computed tomography measures. J Clin Endocrinol Metab 2005;90:1925e8. Horlick M, Wang J, Pierson Jr RN, Thornton JC. Prediction models for evaluation of total-body bone mass with dualenergy X-ray absorptiometry among children and adolescents. Pediatrics 2004;114:e337e45. Willing MC, Torner JC, Burns TL, et al. Percentile distributions of bone measurements in Iowa children: the Iowa Bone Development Study. J Clin Densitom 2005;8:39e47. Hogler W, Briody J, Woodhead HJ, Chan A, Cowell CT. Importance of lean mass in the interpretation of total body densitometry in children and adolescents. J Pediatr 2003;143:81e8. Crabtree NJ, Kibirige MS, Fordham JN, et al. The relationship between lean body mass and bone mineral content in paediatric health and disease. Bone 2004;35:965e72. Schoenau E, Neu CM, Beck B, Manz F, Rauch F. Bone mineral content per muscle cross-sectional area as an index of the functional muscle-bone unit. J Bone Miner Res 2002;17:1095e101. Tothill P, Hannan WJ. Bone mineral and soft tissue measurements by dual-energy X-ray absorptiometry during growth. Bone 2002;31:492e6. Goulding A, Taylor RW, Jones IE, McAuley KA, Manning PJ, Williams SM. Overweight and obese children have low bone mass and area for their weight. Int J Obes Relat Metab Disord 2000;24:627e32. Crabtree NJ, Boivin CM, Shaw NJ. Can body size related normative data improve the diagnosis of osteoporosis in children? (Abstract). J Bone Min Res 2001;16(Suppl. 1):S560. Burnham JM, Shults J, Semeao E, et al. Whole body BMC in pediatric Crohn disease: independent effects of altered growth, maturation, and body composition. J Bone Miner Res 2004;19:1961e8.

PEDIATRIC BONE

REFERENCES

[164] Zemel BS, Leonard MB, Kelly A, et al. Adjustment for height in the clinical assessment of DXA measures of bone mass and density in children. J Clin Endocrinol Metab 2010;95:1265e73. [165] Hui SL, Johnston Jr CC, Mazess RB. Bone mass in normal children and young adults. Growth 1985;49:34e43. [166] Bishop NJ, dePriester JA, Cole TJ, Lucas A. Reference values for radial bone width and mineral content using single photon absorptiometry in healthy children aged 4 to 10 years. Acta Paediatr 1992;81:463e8. [167] Kelly TL, Wilson KE, Heymsfield SB. Dual energy X-ray absorptiometry body composition reference values from NHANES. PLoS One 2009;4:e7038. [168] Zemel BS, Stallings VA, Leonard MB, et al. Revised pediatric reference data for the lateral distal femur measured by Hologic Discovery/Delphi dual-energy X-ray absorptiometry. J Clin Densitom 2009;12:207e18. [169] Cole TJ. The LMS method for constructing normalized growth standards. Eur J Clin Nutr 1990;44:45e60. [170] Wacker W, Barden H. Pediatric reference data for male and female total body and spine BMD and BMC. GE Lunar Corp (Madison, WI). Dallas: International Society of Clinical Densitometry; 2002. [171] Fan B, Wu X, Levine M, et al. Cross calibration of whole body scans between GE-Lunar and Hologic systems. J Clin Densitom 2009;12:385. [172] Kalkwarf HJ, Yolton K, Uetrecht U, Fletcher A, Heubi J. Babies have bones too: Development of reference values for bone mass and density of the lumbar spine of infants and toddlers considering age, size, sex, and race (Abstract). J Bone Miner Res 2010;25(suppl. 1). URL: www.asbmr.org/meetings/ Annualmeeting/AbstractDetails.aspx?aid=a74a6aaf-beøc-4883bdaf-9979bcdd4ccd. [173] Clark EM, Ness AR, Bishop NJ, Tobias JH. Association between bone mass and fractures in children: a prospective cohort study. J Bone Miner Res 2006;21:1489e95. [174] Ferrari SL, Chevalley T, Bonjour JP, Rizzoli R. Childhood fractures are associated with decreased bone mass gain during puberty: an early marker of persistent bone fragility? J Bone Miner Res 2006;21:501e7. [175] Goulding A, Jones IE, Taylor RW, Manning PJ, Williams SM. More broken bones: a 4-year double cohort study of young girls with and without distal forearm fractures. J Bone Miner Res 2000;15:2011e8. [176] Goulding A, Cannan R, Williams SM, Gold EJ, Taylor RW, Lewis-Barned NJ. Bone mineral density in girls with forearm fractures. J Bone Miner Res 1998;13:143e8. [177] Goulding A, Jones IE, Taylor RW, Williams SM, Manning PJ. Bone mineral density and body composition in boys with distal forearm fractures: a dual-energy x-ray absorptiometry study. J Pediatr 2001;139:509e15. [178] Ma DQ, Jones G. Clinical risk factors but not bone density are associated with prevalent fractures in prepubertal children. J Paediatr Child Health 2002;38:497e500. [179] Jones IE, Taylor RW, Williams SM, Manning PJ, Goulding A. Four-year gain in bone mineral in girls with and without past forearm fractures: a DXA study. Dual energy X-ray absorptiometry. J Bone Miner Res 2002;17:1065e72. [180] Goulding A, Grant AM, Williams SM. Bone and body composition of children and adolescents with repeated forearm fractures. J Bone Miner Res 2005;20:2090e6. [181] Jones G, Ma D, Cameron F. Bone density interpretation and relevance in caucasian children aged 9-17 years of age: insights from a population-based fracture study. J Clin Densitom 2006;9:202e9.

339

[182] Manias K, McCabe D, Bishop N. Fractures and recurrent fractures in children; varying effects of environmental factors as well as bone size and mass. Bone 2006;39:652e7. [183] Ma D, Jones G. The association between bone mineral density, metacarpal morphometry, and upper limb fractures in children: a population-based case-control study. J Clin Endocrinol Metab 2003;88:1486e91. [184] Flynn J, Foley S, Jones G. Can BMD assessed by DXA at age 8 predict fracture risk in boys and girls during puberty? an eightyear prospective study. J Bone Miner Res 2007;22:1463e7. [185] Darelid A, Ohlsson C, Rudang R, Kindblom JM, Mellstrom D, Lorentzon M. Trabecular volumetric bone mineral density is associated with previous fracture during childhood and adolescence in males: the GOOD study. J Bone Miner Res 2010;25:537e44. [186] Kalkwarf HJ, Laor T, Bean JA. Fracture risk in children with a forearm injury is associated with volumetric bone density and cortical area (by peripheral QCT) and areal bone density (by DXA). Osteoporos Int 2010. Jun 23. [187] Makitie O, Doria AS, Henriques F, et al. Radiographic vertebral morphology: a diagnostic tool in pediatric osteoporosis. J Pediatr 2005;146:395e401. [188] Henderson RC, Berglund LM, May R, et al. The relationship between fractures and DXA measures of BMD in the distal femur of children and adolescents with cerebral palsy or muscular dystrophy. J Bone Miner Res 2010;25:520e6. [189] Cann CE, Genant HK. Precise measurement of vertebral mineral content using computed tomography. J Comput Assist Tomogr 1980;4:493e500. [190] Gilsanz V, Gibbens DT, Roe TF, et al. Vertebral bone density in children: effect of puberty. Radiology 1988;166:847e50. [191] Gilsanz V, Kovanlikaya A, Costin G, Roe TF, Sayre J, Kaufman F. Differential effect of gender on the sizes of the bones in the axial and appendicular skeletons. J Clin Endocrinol Metab 1997;82:1603e7. [192] Gilsanz V, Perez FJ, Campbell PP, Dorey FJ, Lee DC, Wren TA. Quantitative CT reference values for vertebral trabecular bone density in children and young adults. Radiology 2009;250:222e7. [193] Gilsanz V, Roe TF, Mora S, Costin G, Goodman WG. Changes in vertebral bone density in black girls and white girls during childhood and puberty. N Engl J Med 1991;325:1597e600. [194] Gilsanz V, Skaggs DL, Kovanlikaya A, et al. Differential effect of race on the axial and appendicular skeletons of children. J Clin Endocrinol Metab 1998;83:1420e7. [195] Gilsanz V, Boechat MI, Roe TF, Loro ML, Sayre JW, Goodman WG. Gender differences in vertebral body sizes in children and adolescents. Radiology 1994;190:673e7. [196] Kalender WA. CT: the unexpected evolution of an imaging modality. Eur Radiol 2005;(Suppl. 4):D21e4. [197] Kalender WA, Wolf H, Suess C, Gies M, Greess H, Bautz WA. Dose reduction in CT by on-line tube current control: principles and validation on phantoms and cadavers. Eur Radiol 1999;9:323e8. [198] Wetzsteon RJ, Hughes JM, Kaufman BC, et al. Ethnic differences in bone geometry and strength are apparent in childhood. Bone 2009;44:970e5. [199] Xu L, Nicholson P, Wang Q, Alen M, Cheng S. Bone and muscle development during puberty in girls e a 7-year longitudinal study. J Bone Miner Res 2009;24:1693e8. [200] Binkley TL, Specker BL. Muscleebone relationships in the lower leg of healthy pre-pubertal females and males. J Musculoskelet Neuronal Interact 2008;8:239e43. [201] Cooper DM, Ahamed Y, Macdonald HM, McKay HA. Characterising cortical density in the mid-tibia: intra-individual

PEDIATRIC BONE

340

[202]

[203]

[204]

[205]

[206]

[207]

[208]

[209]

[210]

[211]

[212]

[213]

[214]

[215]

[216]

[217]

[218]

13. NON-INVASIVE TECHNIQUES FOR BONE MASS MEASUREMENT

variation in adolescent girls and boys. Br J Sports Med 2008;42:690e5. Farr JN, Chen Z, Lisse JR, Lohman TG, Going SB. Relationship of total body fat mass to weight-bearing bone volumetric density, geometry, and strength in young girls. Bone 2010;46:977e84. Wetzsteon RJ, Petit MA, Macdonald HM, Hughes JM, Beck TJ, McKay HA. Bone structure and volumetric BMD in overweight children: a longitudinal study. J Bone Miner Res 2008;23:1946e53. Havill LM, Mahaney MC, T LB, Specker BL. Effects of genes, sex, age, and activity on BMC, bone size, and areal and volumetric BMD. J Bone Miner Res 2007;22:737e46. Macdonald H, Kontulainen S, Petit M, Janssen P, McKay H. Bone strength and its determinants in pre- and early pubertal boys and girls. Bone 2006;39:598e608. Wang Q, Alen M, Nicholson P, et al. Growth patterns at distal radius and tibial shaft in pubertal girls: a 2-year longitudinal study. J Bone Miner Res 2005;20:954e61. Macdonald HM, Kontulainen SA, Khan KM, McKay HA. Is a school-based physical activity intervention effective for increasing tibial bone strength in boys and girls? J Bone Miner Res 2007;22:434e46. Specker B, Binkley T, Fahrenwald N. Increased periosteal circumference remains present 12 months after an exercise intervention in preschool children. Bone 2004;35:1383e8. Ward KA, Roberts SA, Adams JE, Lanham-New S, Mughal MZ. Calcium supplementation and weight bearing physical activity e do they have a combined effect on the bone density of pre-pubertal children? Bone 2007;41:496e504. Suuriniemi M, Kovanen V, Mahonen A, et al. COL1A1 Sp1 polymorphism associates with bone density in early puberty. Bone 2006;39:591e7. Suuriniemi M, Mahonen A, Kovanen V, et al. Association between exercise and pubertal BMD is modulated by estrogen receptor alpha genotype. J Bone Miner Res 2004;19:1758e65. Dubner SE, Shults J, Baldassano RN, et al. Longitudinal assessment of bone density and structure in an incident cohort of children with Crohn’s disease. Gastroenterology 2009;136:123e30. Burnham JM, Shults J, Dubner SE, Sembhi H, Zemel BS, Leonard MB. Bone density, structure, and strength in juvenile idiopathic arthritis: Importance of disease severity and muscle deficits. Arthritis Rheum 2008;58:2518e27. Wetzsteon RJ, Shults J, Zemel BS, et al. Divergent effects of glucocorticoids on cortical and trabecular compartment BMD in childhood nephrotic syndrome. J Bone Miner Res 2009;24:503e13. Cheung CS, Lee WT, Tse YK, et al. Generalized osteopenia in adolescent idiopathic scoliosiseassociation with abnormal pubertal growth, bone turnover, and calcium intake? Spine 2006;31:330e8. Binkley T, Johnson J, Vogel L, Kecskemethy H, Henderson R, Specker B. Bone measurements by peripheral quantitative computed tomography (pQCT) in children with cerebral palsy. J Pediatr 2005;147:791e6. Schweizer R, Martin DD, Haase M, et al. Similar effects of longterm exogenous growth hormone (GH) on bone and muscle parameters: a pQCT study of GH-deficient and small-forgestational-age (SGA) children. Bone 2007;41:875e81. Simm PJ, Briody J, McQuade M, Munns CF. The successful use of pamidronate in an 11-year-old girl with complex regional pain syndrome: response to treatment demonstrated by serial peripheral quantitative computerised tomographic scans. Bone 46:885e8.

[219] Neu CM, Manz F, Rauch F, Merkel A, Schoenau E. Bone densities and bone size at the distal radius in healthy children and adolescents: a study using peripheral quantitative computed tomography. Bone 2001;28:227e32. [220] Mori I, Machida Y. Deriving the modulation transfer function of CT from extremely noisy edge profiles. Radiol Phys Technol 2009;2:22e32. [221] Schnitzler CM, Biddulph SL, Mesquita JM, Gear KA. Bone structure and turnover in the distal radius and iliac crest: a histomorphometric study. J Bone Miner Res 1996;11:1761e8. [222] Eswaran SK, Gupta A, Keaveny TM. Locations of bone tissue at high risk of initial failure during compressive loading of the human vertebral body. Bone 2007;41:733e9. [223] Mawatari T, Miura H, Hamai S, et al. Vertebral strength changes in rheumatoid arthritis patients treated with alendronate, as assessed by finite element analysis of clinical computed tomography scans: a prospective randomized clinical trial. Arthritis Rheum 2008;58:3340e9. [224] Keaveny TM, Donley DW, Hoffmann PF, Mitlak BH, Glass EV, San Martin JA. Effects of teriparatide and alendronate on vertebral strength as assessed by finite element modeling of QCT scans in women with osteoporosis. J Bone Miner Res 2007;22:149e57. [225] Boutroy S, Bouxsein ML, Munoz F, Delmas PD. In vivo assessment of trabecular bone microarchitecture by highresolution peripheral quantitative computed tomography. J Clin Endocrinol Metab 2005;90:6508e15. [226] MacNeil JA, Boyd SK. Accuracy of high-resolution peripheral quantitative computed tomography for measurement of bone quality. Med Eng Phys 2007;29:1096e105. [227] Pistoia W, van Rietbergen B, Lochmuller EM, Lill CA, Eckstein F, Ruegsegger P. Estimation of distal radius failure load with micro-finite element analysis models based on threedimensional peripheral quantitative computed tomography images. Bone 2002;30:842e8. [228] Kirmani S, Christen D, van Lenthe GH, et al. Bone structure at the distal radius during adolescent growth. J Bone Miner Res 2009;24:1033e42. [229] Burrows M, Liu D, Moore S, McKay H. Bone microstructure at the distal tibia provides a strength advantage to males in late puberty: an HR-pQCT study. J Bone Miner Res 2010;25:1423e32. [230] McKay H, Liu D, Egeli D, Boyd S, Burrows M. Physical activity positively predicts bone architecture and bone strength in adolescent males and females. Acta Paediatr 2011;100(1):97e101. [231] Liu D, Burrows M, Egeli D, McKay H. Site specificity of bone architecture between the distal radius and distal tibia in children and adolescents: An HR-pQCT study. Calcif Tissue Int 2010;87:314e23. [232] Chevalley T, Bonjour JP, Ferrari S, Rizzoli R. Influence of age at menarche on forearm bone microstructure in healthy young women. J Clin Endocrinol Metab 2008;93:2594e601. [233] Chevalley T, Bonjour JP, Ferrari S, Rizzoli R. Deleterious effect of late menarche on distal tibia microstructure in healthy 20-year-old and premenopausal middle-aged women. J Bone Miner Res 2009;24:144e52. [234] Binkley TL, Specker BL. pQCT measurement of bone parameters in young children: validation of technique. J Clin Densitom 2000;3:9e14. [235] Lee DC, Gilsanz V, Wren TA. Limitations of peripheral quantitative computed tomography metaphyseal bone density measurements. J Clin Endocrinol Metab 2007;92:4248e53. [236] Kontulainen S, Liu D, Manske S, Jamieson M, Sievanen H, McKay H. Analyzing cortical bone cross-sectional geometry by peripheral QCT: comparison with bone histomorphometry. J Clin Densitom 2007;10:86e92.

PEDIATRIC BONE

REFERENCES

[237] Rauch F, Tutlewski B, Fricke O, et al. Analysis of cancellous bone turnover by multiple slice analysis at distal radius: a study using peripheral quantitative computed tomography. J Clin Densitom 2001;4:257e62. [238] Link TM, Koppers BB, Licht T, Bauer J, Lu Y, Rummeny EJ. In vitro and in vivo spiral CT to determine bone mineral density: initial experience in patients at risk for osteoporosis. Radiology 2004;231:805e11. [239] Theodorou DJ, Theodorou SJ, Andre MP, Kubota D, Weigert JM, Sartoris DJ. Quantitative computed tomography of spine: comparison of three-dimensional and two-dimensional imaging approaches in clinical practice. J Clin Densitom 2001;4:57e62. [240] Kaczmarek M, Pakula M, Kubik J. Multiphase nature and structure of biomaterials studied by ultrasounds. Ultrasonics 2000;38:703e7. [241] Wear KA. Anisotropy of ultrasonic backscatter and attenuation from human calcaneus: implications for relative roles of absorption and scattering in determining attenuation. J Acoust Soc Am 2000;107:3474e9. [242] Wear KA. Frequency dependence of ultrasonic backscatter from human trabecular bone: theory and experiment. J Acoust Soc Am 1999;106:3659e64. [243] McKelvie ML, Palmer SB. The interaction of ultrasound with cancellous bone. Phys Med Biol 1991;36:1331e40. [244] Nicholson PH, Strelitzki R, Cleveland RO, Bouxsein ML. Scattering of ultrasound in cancellous bone: predictions from a theoretical model. J Biomech 2000;33:503e6. [245] Strelitzki R, Nicholson PH, Paech V. A model for ultrasonic scattering in cancellous bone based on velocity fluctuations in a binary mixture. Physiol Meas 1998;19:189e96. [246] Benitez CL, Schneider DL, Barrett-Connor E, Sartoris DJ. Hand ultrasound for osteoporosis screening in postmenopausal women. Osteoporos Int 2000;11:203e10. [247] Njeh CF, Richards A, Boivin CM, Hans D, Fuerst T, Genant HV. Factors influencing the speed of sound through the proximal phalanges. J Clin Densitom 1999;2:241e9. [248] Heaney RP, Avioli LV, Chesnut III CH, Lappe J, Recker RR, Brandenburger GH. Osteoporotic bone fragility. Detection by ultrasound transmission velocity. J Am Med Assoc 1989;261:2986e90. [249] Heaney RP, Avioli LV, Chesnut III CH, Lappe J, Recker RR, Brandenburger GH. Ultrasound velocity, through bone predicts incident vertebral deformity. J Bone Miner Res 1995;10:341e5. [250] Wuster C, Albanese C, De Aloysio D, et al. Phalangeal osteosonogrammetry study: age-related changes, diagnostic sensitivity, and discrimination power. The Phalangeal Osteosonogrammetry Study Group. J Bone Miner Res 2000;15:1603e14. [251] Alwis G, Rosengren B, Nilsson JA , et al. Normative calcaneal quantitative ultrasound data as an estimation of skeletal development in Swedish children and adolescents. Calcif Tissue Int 2010;87:493e506. [252] Zhu ZQ, Liu W, Xu CL, Han SM, Zu SY, Zhu GJ. Ultrasound bone densitometry of the calcaneus in healthy Chinese children and adolescents. Osteoporos Int 2007;18:533e41. 253 Baroncelli GI, Battini R, Bertelloni S, et al. Analysis of quantitative ultrasound graphic trace and derived variables assessed at proximal phalanges of the hand in healthy subjects and in patients with cerebral palsy or juvenile idiopathic arthritis. A pilot study. Bone 2010;46:182e9. [254] Baroncelli GI. Quantitative ultrasound methods to assess bone mineral status in children: technical characteristics, performance, and clinical application. Pediatr Res 2008;63:220e8.

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[255] Foldes AJ, Rimon A, Keinan DD, Popovtzer MM. Quantitative ultrasound of the tibia: a novel approach for assessment of bone status. Bone 1995;17:363e7. [256] Orgee JM, Foster H, McCloskey EV, Khan S, Coombes G, Kanis JA. A precise method for the assessment of tibial ultrasound velocity. Osteoporos Int 1996;6:1e7. [257] Evans WD, Jones EA, Owen GM. Factors affecting the in vivo precision of broad-band ultrasonic attenuation. Phys Med Biol 1995;40:137e51. [258] Cheng S, Njeh CF, Fan B, et al. Influence of region of interest and bone size on calcaneal BMD: implications for the accuracy of quantitative ultrasound assessments at the calcaneus. Br J Radiol 2002;75:59e68. [259] Baroncelli GI, Federico G, Bertelloni S, de Terlizzi F, Cadossi R, Saggese G. Bone quality assessment by quantitative ultrasound of proximal phalanxes of the hand in healthy subjects aged 3e21 years. Pediatr Res 2001;49:713e8. [260] Kaga M, Takahashi K, Suzuki H, et al. Ultrasound assessment of tibial cortical bone acquisition in Japanese children and adolescents. J Bone Miner Metab 2002;20:111e5. [261] Chappard C, Camus E, Lefebvre F, et al. Evaluation of error bounds on calcaneal speed of sound caused by surrounding soft tissue. J Clin Densitom 2000;3:121e31. [262] Guglielmi G, Njeh CF, de Terlizzi F, et al. Palangeal quantitative ultrasound, phalangeal morphometric variables, and vertebral fracture discrimination. Calcif Tissue Int 2003;72:469e77. [263] Hartman C, Brik R, Tamir A, Merrick J, Shamir R. Bone quantitative ultrasound and nutritional status in severely handicapped institutionalized children and adolescents. Clin Nutr 2004;23:89e98. [264] Barkmann R, Rohrschneider W, Vierling M, et al. German pediatric reference data for quantitative transverse transmission ultrasound of finger phalanges. Osteoporos Int 2002;13:55e61. [265] Schonau E, Radermacher A, Wentzlik U, Klein K, Michalk D. The determination of ultrasound velocity in the os calcis, thumb and patella during childhood. Eur J Pediatr 1994;153:252e6. [266] Levine A, Mishna L, Ballin A, et al. Use of quantitative ultrasound to assess osteopenia in children with Crohn disease. J Pediatr Gastroenterol Nutr 2002;35:169e72. [267] Mughal MZ, Ward K, Qayyum N, Langton CM. Assessment of bone status using the contact ultrasound bone analyser. Arch Dis Child 1997;76:535e6. [268] Lum CK, Wang MC, Moore E, Wilson DM, Marcus R, Bachrach LK. A comparison of calcaneus ultrasound and dual X-ray absorptiometry in healthy North American youths and young adults. J Clin Densitom 1999;2:403e11. [269] Wunsche K, Wunsche B, Fahnrich H, et al. Ultrasound bone densitometry of the os calcis in children and adolescents. Calcif Tissue Int 2000;67:349e55. [270] Sawyer A, Moore S, Fielding KT, Nix DA, Kiratli J, Bachrach LK. Calcaneus ultrasound measurements in a convenience sample of healthy youth. J Clin Densitom 2001;4: 111e20. [271] Jaworski M, Lebiedowski M, Lorenc RS, Trempe J. Ultrasound bone measurement in pediatric subjects. Calcif Tissue Int 1995;56:368e71. [272] Lequin MH, van Rijn RR, Robben SG, Hop WC, van Kuijk C. Normal values for tibial quantitative ultrasonometry in caucasian children and adolescents (aged 6 to 19 years). Calcif Tissue Int 2000;67:101e5. [273] Zadik Z, Price D, Diamond G. Pediatric reference curves for multisite quantitative ultrasound and its modulators. Osteoporos Int 2003;14:857e62.

PEDIATRIC BONE

342

13. NON-INVASIVE TECHNIQUES FOR BONE MASS MEASUREMENT

[274] Pettinato AA, Loud KJ, Bristol SK, Feldman HA, Gordon CM. Effects of nutrition, puberty, and gender on bone ultrasound measurements in adolescents and young adults. J Adolesc Health 2006;39:828e34. [275] Lappe JM, Recker RR, Malleck MK, Stegman MR, Packard PP, Heaney RP. Patellar ultrasound transmission velocity in healthy children and adolescents. Bone 1995;16(Suppl): S251e256. [276] Lappe JM, Stegman M, Davies KM, Barber S, Recker RR. A prospective study of quantitative ultrasound in children and adolescents. J Clin Densitom 2000;3:167e75. [277] Mughal MZ, Langton CM, Utretch G, Morrison J, Specker BL. Comparison between broad-band ultrasound attenuation of the calcaneum and total body bone mineral density in children. Acta Paediatr 1996;85:663e5. [278] Lequin MH, van Rijn RR, Robben SG, van Leeuwen WJ, Hop WC, van Kuijk C. Quantitative tibial ultrasonometry versus radiographic phalangeal absorptiometry in a Caucasian pediatric population. Calcif Tissue Int 2001;68:323e9. [279] Fricke O, Tutlewski B, Schwahn B, Schoenau E. Speed of sound: relation to geometric characteristics of bone in children, adolescents, and adults. J Pediatr 2005;146:764e8. [280] Cadossi R, de Terlizzi F, Cane V, Fini M, Wuster C. Assessment of bone architecture with ultrasonometry: experimental and clinical experience. Horm Res 2000;54(Suppl. 1):9e18. [281] Njeh CF, Fuerst T, Diessel E, Genant HK. Is quantitative ultrasound dependent on bone structure? A reflection. Osteoporos Int 2001;12:1e15.

[282] Serpe LJ, Rho JY. Broadband ultrasound attenuation value dependence on bone width in vitro. Phys Med Biol 1996;41: 197e202. [283] Wu CY, Gluer CC, Jergas M, Bendavid E, Genant HK. The impact of bone size on broadband ultrasound attenuation. Bone 1995;16:137e41. [284] Krestan CR, Grampp S, Resch-Holeczke A, Henk CB, Imhof H, Resch H. Diagnostic disagreement of imaging quantitative sonography of the calcaneus with dual X-ray absorptiometry of the spine and femur. Am J Roentgenol 2001;177:213e6. [285] Baroncelli GI, Federico G, Vignolo M, et al. Cross-sectional reference data for phalangeal quantitative ultrasound from early childhood to young-adulthood according to gender, age, skeletal growth, and pubertal development. Bone 2006;39:159e73. [286] Krieg MA, Cornuz J, Ruffieux C, et al. Prediction of hip fracture risk by quantitative ultrasound in more than 7000 Swiss women > or ¼ 70 years of age: comparison of three technologically different bone ultrasound devices in the SEMOF study. J Bone Miner Res 2006;21:1457e63. [287] Moayyeri A, Kaptoge S, Dalzell N, et al. Is QUS or DXA better for predicting the 10-year absolute risk of fracture? J Bone Miner Res 2009;24:1319e25. [288] Lappe J, Davies K, Recker R, Heaney R. Quantitative ultrasound: use in screening for susceptibility to stress fractures in female army recruits. J Bone Miner Res 2005;20:571e8. [289] Jones G, Boon P. Which bone mass measures discriminate adolescents who have fractured from those who have not? Osteoporos Int 2008;19:251e5.

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C H A P T E R

14

Assessment of Maturation: Bone Age and Pubertal Assessment Noe¨l Cameron 1, David D. Martin 2 1

Centre for Global Health and Human Development, School of Sport, Exercise and Health Sciences, Loughborough University, Loughborough, Leicestershire, UK and 2 Paediatric Endocrinology and Diabetology, University Children’s Hospital, Tu¨bingen University, Tu¨bingen, Germany

BACKGROUND

Initial Considerations

The process of maturation is continuous throughout life e it begins at conception and ends at death. This chapter will concentrate on the assessment of the process of maturation from birth to childhood, i.e. that part of the total process that is intimately linked to physical growth. It is important, therefore, 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 organised, specialised, and mature state”. The end-point of maturation, within the context of the growth, is the attainment of adulthood, which we define as a “functionally mature individual”. Functional maturation, in a biological context, implies the ability to procreate successfully and raise offspring who themselves will successfully procreate. We know that, 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, 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 themselves dependent on the norms in a particular society. Thus, in their broadest context, maturation and growth are intimately related and both must reach functional and structural endpoints that provide the opportunity for successful procreation.

Pediatric Bone, Second Edition DOI: 10.1016/B978-0-12-382040-2.10014-0

In order to understand how maturation can be assessed, it is important first to appreciate that maturation is not linked to time in a chronological sense. In other words, one year of chronological time is not equivalent to one year of maturational “time”. This is perhaps best illustrated in Figure 14.1 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, the proportion and distribution of subcutaneous fat, and the development of the skeleton and musculature, that result in sexually dimorphic body shapes in adulthood. While each individual has passed through the same chronological time span, they have done so at very different rates of maturation. Secondly, 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. The same applies to skeletal maturity, although recent developments in the assessment of skeletal maturation based on active appearance models now reflect continuous bone age changes [4]. Thirdly, there is variability of maturation within the individual. For instance, while skeletal and secondary sexual maturation are associated they are not correlated so significantly that one can categorically associate

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FIGURE 14.1 Three normal boys aged 14.75 years and three normal girls aged 12.75 years demonstrating dramatically different stage of maturation. (From Tanner JM. Growth and endocrinology of the adolescent. In: Gardner L, ed. Endocrine and Genetic Diseases of Childhood, 2nd edn. Philadelphia: WB Saunders; 1975.)

a particular stage of sexual maturation with a particular skeletal “age” [5,6]. Fourthly, 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, we have a process of “uneven maturation”.

PEDIATRIC BONE

BACKGROUND

Fifthly, 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 Figure 14.1 for instance, the females are aged exactly 12.75 years and the males 14.75 years yet their levels of secondary sexual development are similar. Sixthly, 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 considerations, the 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 relationship between maturity and size, have governed the development of techniques for the assessment of maturation. The Concept of Time Roy M. Acheson [7] elegantly described the problem of “time” within the development of skeletal maturity assessment methods. 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 to 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 [7].

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 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 [8] one is faced with the final “standards” for males and females which correspond to an “age” of 18 years but which in fact represent full maturity or the maturity to be found in any individual who has achieved total epiphyseal fusion regardless of his or her actual chronological age. Acheson [9,10], and Tanner and colleagues [11e14] overcame this problem in the assessment of skeletal maturity by moving away from an “age”-based method to develop the “bone-specific scoring” techniques in which numerical scores were assigned to each bone rather than a bone “age”. Acheson’s earlier attempt, (the “Oxford Method”) gave scores of 1, 2, 3, etc. to each stage, but did not account for the fact that the differences between scales were not equivalent; the “difference” between stage 1 and stage 2 did not represent the same advancement in

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maturity, as 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 which can be rated on a maturity scale from zero percent maturity to 100% maturity. Without dwelling on the mathematics, which are given in detail by Tanner and colleagues [13,14], the principle is an important one and should be applied to any new system of assessing maturity. In addition, the bonespecific scoring approach can be applied to an appropriate sample of radiographs from any population to derive maturity norms. Maturity Indicators The development of the concept of maturity indicators by Wingate Todd [15], based on the pioneering work of Milo Hellman in 1928 [16], was fundamental in developing methods to assess accurately skeletal maturity. Prior to 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 [17,18] or from a skeletal survey of each child [19], 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 [20e22]. 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 [23] and the latter method because only the carpus was used which, as we now appreciate, is not representative of overall maturity. Wingate Todd identified “determinators of maturity” within the changing radiological appearance of long and short bones [15]. Greulich and Pyle [8] 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”. While 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 14.2 illustrates the maturity indicators for the developing radius identified by Greulich and Pyle (GP) [8] and Tanner et al. (TW) [11]. 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 while Tanner et al. [11] described eight. It is apparent, however, that visually at least the indicators of Tanner and his colleagues are not dramatically different from those of Greulich and Pyle. Maturity indicators must conform to certain prerequisites if they are to be useful. They must possess the quality of universality in that they must be present in all normal children and they must appear sequentially, and in the

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FIGURE 14.2

Maturity indicators for the radius identified by Greulich and Pyle (GP) and Tanner and Whitehouse (TW).

same sequence, in all children. Roche and his colleagues added to these criteria while developing the Fels handewrist method [24,25]. 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 ought to reflect a continuous process of maturation rather than a discontinuous process) and completeness (its prevalence in the population). Maturational Variation Maturational variation covers two aspects: (a) the variation of maturation within a process; and (b) the variation of maturation between processes. The former aspect may be observed within sexual maturation from the data published by Marshall and Tanner [5,6] on British children. They illustrated variation by investigating the percentage of girls or boys within 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 assessment method to allow for intra-individual variation. Within clinical situations, for instance, the difficulties in accurately rating the various stages of breast, genitalia, or pubic hair development within the Tanner five-point scale, have led to the combination of the stages into a three or four-point “pubertal” staging technique (see below). Thus, variations within individuals between the different aspects of secondary sexual development are impossible to quantify and, in terms of research to investigate variability in maturation, the pubertal staging technique loses significant sensitivity. The variation of maturation between different aspects of maturity presents difficulties in implying a general maturational level to the individual. Entry into the early stages of puberty is not, for instance, associated with any particular level of skeletal maturity except in the broadest sense. 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. An 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

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BACKGROUND

menarche tends to occur at a skeletal age of 12.5e14.0 “years” regardless of chronological age. There is also a very good correlation between bone age retardation/advancement and age at peak height velocity, as calculated by Gasser et al. [26], or age at menarche (Figures 14.3 and 14.4, unpublished data from our analysis of the First Zurich Longitudinal Study) and between bone age and age at peak height velocity. All boys have their maximum height velocity at a bone age of about 13.5 years but those with delayed bone age at the age of 9 will have a lower peak height velocity at 13.5 years of bone age and those with an advanced bone age at 9 years will have a higher peak height velocity at 13.5 years of bone age (Figure 14.5). Sexual Dimorphism Ideally, any method that assesses maturity should be able to assess the same process of maturation in both males and females. While that criterion is true of skeletal maturity assessment methods and also for dental maturation and perhaps for 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 having 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 yet their position within the pattern of growth is quite different and thus their association with other aspects of maturation also differs. Menarche occurs following peak height velocity and

a bone age of 13 years (see Figure 14.4) and towards the latter part of secondary sexual development, i.e. in breast stage 3, 4, or 5 [5]. Relatively sparse data on spermarche identify its occurrence at approximately 14 years in boys, which would be in the early or middle part of the adolescent growth spurt and thus indicative of an earlier stage of pubertal 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. Size does, however, enter assessment as a maturity indicator as a ratio measure. For example, the maturity indicator for stage D in the radius of the TW2/3 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 mid-puberty. 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. Clinicians, however, use the above measures as indicators of normal testicular growth and of the initial and middle stages of pubertal development. Similarly, the uterus and ovary volumes can be measured by ultrasound and there are cut-offs marking the initiation of pubertal development [27]. Although size does not play a role in the assessment of maturity, it is important to note that there is of course a correlation between body height and maturity: a small Girls 17

16

16

15

15

14

14

Spurt age

Spurt age

Boys 17

13

13

12

12

11

11

10

10

9 –4

–2

0

2

4

9 –4

Bone age retardation

–2

0

2

4

Bone age retardation

FIGURE 14.3 Correlation between age at peak height velocity (growth spurt) and BoneXpert GP bone age retardation. The retardation is defined as CA-BA at the visit with BA closest to 15.5 years for boys and 13.5 years for girls. In other words, what is effectively plotted is the relationship between age of growth spurt and age of epiphyseal fusion. (Thodberg and Martin, unpublished data from the First Zurich Longitudinal Study.)

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14. ASSESSMENT OF MATURATION: BONE AGE AND PUBERTAL ASSESSMENT

Girls 17

16

16

15

15

14

14 Menarche

Start of testicle growth

Boys 17

13 12

13 12

11

11

10

10

9 –4

–2 0 2 Bone age retardation

9 –4

4

–2 0 2 Bone age retardation

4

FIGURE 14.4 Correlation between age start of testicular growth (left diagram) or age at menarche (right diagram) and BoneXpert GP bone age retardation (defined as in the prevoius figure), i.e. the plot shows the relationship between age of testicular growth/menarche and age of epiphyseal fusion. Menarche is strongly related to epiphyseal fusion, with a noticeable outlier. (Thodberg and Martin, unpublished data from the First Zurich Longitudinal Study.) 1ZLS − boys 12 Advanced Average Delayed

Advanced Average Delayed 10

Height Velocity wrt BA (cm/yr)

Height Velocity wrt BA (cm/yr)

10

8

6

4

2

0

FIGURE 14.5 The height increase per BoneXpert BA year in children of the First Zurich Longitudinal Study. The children are divided into tertiles of advanced, normal and delayed bone age according to the BA at age 9 for boys and age 8 for girls. (Martin and Thodberg, unpublished data from the First Zurich Longitudinal Study.)

1ZLS − girls

12

8

6

4

2

8

10

12

14

16

0

8

10

Bone Age (yr)

12

14

16

Bone Age (yr)

child will tend to have a lower skeletal maturity than a tall child of the same age (Figure 14.6).

THE METHODS OF ASSESSMENT

technique of Greulich and Pyle [8]; the TannereWhitehouse bone-specific scoring technique [11e14]; and the Fels handewrist method [24,25]. All use the left hand and wrist to estimate a skeletal age or bone age, yet the latter two are different both in concept and in method from the former.

Skeletal Development

Atlas Techniques

Three techniques are most commonly used in clinical situations to estimate skeletal maturity: the atlas

The Atlas technique has its origins in the pioneer work of Dr T. Wingate Todd, who published an Atlas

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THE METHODS OF ASSESSMENT

12 11

Bona age (y)

10 9 8 7 BoneXpert BA (y)

6

Tanner-Whitehouse BA (y)

Greulich-Pyle BA (y)

2

2

2

r = 0.34

r = 0.30

r = 0.25

5 125

130

135 140 Height (cm)

145

150

125

130

135 140 Height (cm)

145

150

125

130

135 140 Height (cm)

145

150

FIGURE 14.6 Automatic BoneXpert GP bone age (BA) (left), manual Greulicheyle BA (middle) and manual TW3 BA (right) by height in the 110 normal boys from the First Zurich Longitudinal Study who all had an x-ray taken within 2 weeks of their ninth birthday: All show a weak correlation between BA and stature. (Martin and Thodberg, unpublished data from the First Zurich Longitudinal Study.)

of Skeletal Maturity in 1937 [15]. A “Skiagraphic Atlas” showing the development of the bones of the hand and wrist had, in fact, been published in London in 1898 by a surgeon called 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 handewrist radiographs of 1000 children from the Brush Foundation Study of Human Growth and Development, which started in 1929 in Cleveland, Ohio. The children were only admitted to the study on the application of a pediatrician and were thus, in the Midwest 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 described. The appearance of these indicators was taken as typical for a healthy child of that age and sex. Having described these indicators for each bone and each age, the series was re-examined to identify radiographs showing, for every bone, the modal maturity for that 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 those standards that appeared in the Atlas. Continuing Todd’s work, Drs William Walter 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 [8,29e31).

That for the hand and wrist is the best known and is referred to universally as the “Greulich-Pyle Atlas”. Bone-specific Scoring Techniques Bone-specific techniques were developed in an attempt to overcome the two main disadvantages of the Atlas techniques. These were 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 handewrist. Similarly, the acceptance of “age” from the standards implied the acceptance of a chronological time series. THE OXFORD METHOD

Roy M. Acheson, working on radiographs from about 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” [7]. In essence, he identified maturity indicators and assigned scores ranging from 0, when no center was present, through 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 bonespecific systems, depended upon assigning a number or score to each maturity indicator or combination of maturity indicators. However, Acheson’s scores were

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arbitrary e 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 some way 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 his colleagues to develop their techniques a few years later. THE TANNEReWHITEHOUSE METHOD

In 1959 and 1962, J.M. Tanner and R.H. 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 [11e13] and most recently as TW3 [14]. 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 ought to be the same. This common score, with suitable standardization, would be the individual’s maturity. To arrive at a practical technique, a variety of modifications to this rationale had to be made. In addition, Tanner and his colleagues were highly critical of the method and how it operated in practice e in how well it served the pediatric and research communities for whom it was intended. Their monitoring of the system promoted the various modifications that resulted in TW2 and most recently TW3. The underlying rationale of the TannereWhitehouse 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 his 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; they therefore omitted rays 2 and 4 from the final calculations. Further, 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 arrive at population “standards” that related bone maturity scores to chronological ages. The resulting curve of bone maturity score against age was sigmoid demonstrating a non-linear relationship between skeletal maturity and chronological age. There were three objections to TW1. First, some of the maturity indicators involved the assessment of size relations between bones which may be altered by pathological conditions and thus violates the requirement of universality in the selection of maturity indicators. Secondly, by constraining the number of maturity indicators to eight, Tanner et al. were weakening their system by of necessity ignoring the fact that some bones may exhibit greater or fewer maturity indicators than the eight required by the TW1 system. Thirdly, 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 known neither to play a major role in growth in height nor in epiphyseal fusion. Tanner and his colleagues took cognisance of these criticisms in their development of the TW2 system which was in use in Europe for the next 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)). Also, the last two stages of R and U were merged, so that there are eight stages for R (BeI) and seven for U (BeH). 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 his colleagues felt that: (1) the development of each bone reflects primarily a single process defined as maturation; (2) the scores from each of the bones in a particular individual should, with suitable standardization, be the same and this common score would be the individual’s maturity. In practice, the scores of the various bones are not identical, one of the most important reasons being the large gaps between successive events in a single bone. Tanner et al. therefore defined the

PEDIATRIC BONE

THE METHODS OF ASSESSMENT

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 bone scores about their mean value and secondly, the scores are constrained to avoid the solution in which perfect agreement is reached by giving the same score to every stage. Table 14.1 illustrates this procedure. Two rival systems of scores are illustrated, labeled L and M, and 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 is, however, 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. [11] generalized this by using all the bones and all the stages and by adding up the total disagreement sum of squares over all members of a large standardizing 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 TannereWhitehouse technique [13,14] or in the Biometrika paper of Healy and Goldstein [32].) The TannereWhitehouse 2 skeletal maturity system was thus thought to address the disadvantages of both the GreulichePyle Atlas method and the Oxford method. It allowed an assessment of skeletal maturity that was age independent and, because of the three TABLE 14.1

Two Alternative Systems for Scoring Maturity in the Bones of the Hand and Wrist

Scores for system L

Scores for system M

Stage

Stage

Bone

A

B

C

A

B

C

R

0

10

20

0

8

16

U

0

10

25

5

12

26

M

0

12

17

0

9

14 Scores

Observed stages

System L

System M

R

B

10

8

U

A

0

5

M

C

17

14

9

9

146

42

Mean score Sum of squares of deviations

Data from Tanner et al. [13]. Two rival systems of scores are illustrated, labeled L and M, and 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 is, however, 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.

351

systems available from a single rating (TW2 (20), RUS, CARPAL), allowed considerable flexibility both in the assessment and the monitoring of skeletal maturity. Tanner and his colleagues have since published an updated method now known as TW3 [14]. It is almost 25 years since the second edition of TW2 and, like all systems within growth research that rely on source samples from a particular historical time, Tanner and his colleagues were acutely aware of the secular trend. That trend has been a recognized aspect of generational differences in human growth for many years. The secular trend affects both growth in overall size and in maturity such that size gets larger and maturational events occur earlier with each succeeding generation. Thus, rate of skeletal maturation will also have advanced and bone-specific scoring techniques ought to reflect or allow for that advancement. In addition, some important conceptual advances have occurred in the last 20 years. One of these is that it is now widely recognized that “standards” and “references” are not the same beast. 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. it is 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 differences between TW2 and TW3. The most important thing, however, is that the descriptions and manual ratings of the stages of the bones were not altered. They remained 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. This is because it was felt that the mixture of the carpal maturity scores with the RUS maturity scores was not of major value. Skeletal maturity of the carpal bones in isolation appears to give different information about the process of maturity. The radius, ulna and short bones (RUS) are certainly more useful both in terms of reflecting general skeletal maturity and in the prediction of adult height. Secondly, the source samples for the reference charts were updated so as to reflect the norms for more recent samples of children from Europe and North America. Thus, the conversion to bone age also changed particularly from about 10 years onwards. The third and fourth changes related to the height prediction technique rather than the assessment of

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skeletal maturity per se. RUS bone score was now used rather than bone age in the prediction equations and the source sample was improved by using more appropriate data from the First Zurich Longitudinal Growth Study. However, using the same children of the First Zurich Longitudinal Growth study, it has recently been shown that the GP system as well as the BoneXpert GP system are better correlated with growth potential [33], and thus slightly better predictors of adult height [34]. Interestingly, omitting radius and ulna (which are weighted at 20% each in the TW3 system) actually further improved adult height prediction [33]. The new bone age, EA90 (to reflect the European and American sources) or TW3, was based on data from samples of children in Europe and North America and Japan assessed in the 1970s, 1980s and 1990s. These included Belgian data (N¼21 174 boys, 10 000 girls) from the Leuven growth study, Spanish children (N¼2000 with over 5000 radiographs) from the Bilbao study, Japanese children (N¼1000) from Tokyo, Italian children (950 boys and 880 girls) from Genoa, Argentinian data from the early 1970s and data from Project Heartbeat of about 1000 normal EuropeaneAmerican children in Texas (for details of these samples see Tanner et al. [14]). The new EA90 bone age values were chosen to match these populations 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 Figure 14.7. At preadolescent ages, the scores for boys vary very little between the systems. After 9 years of age in boys and

from about 5 years in girls the differences increase quite dramatically so that, for instance, a boy scoring 405 would have had a TW2 bone age of 13 years and a TW3 bone age of 11.7 years. This difference of a year to 18 months is pretty consistent during adolescence reflecting the relative advancement of the EA90 sample compared to the TW2 sample. The RocheeWainereThissen Technique In 1975, Roche, Wainer and Thissen published a technique to estimate the skeletal maturity of the knee [35]. Roche, in particular, was critical of the handewrist 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 [36]. In addition, the usefulness of the handewrist techniques was limited at early ages when few centers were visible and in later ages when some areas (e.g. the carpus) reach 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 ought to give a more appropriate estimation of skeletal maturity, but this may not actually be the case. However, Roche was to change his opinion in the next decade and, with his colleagues Cameron Chumlea and David Thissen, he produced

60 40 20 0

TW2 – TW3

–20

Boys

– 40 – 60

Girls

– 80 –100 –120 –140 –160 –180

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

Bone Age, yrs

FIGURE 14.7

Differences between TW2 and TW3 bone maturity scores for whole year chronological age from 2 to 16 years. (The TW3 score has been subtracted from the TW2 score, thus negative values indicate advancement of TW3 over TW2. For example, an average 12-year-old boy would have bone maturity score of 361 on TW2 and 427 on TW3. The difference is therefore 66 (361427¼66). Thus, in the TW3 sample this level of maturity has been reached at an earlier age than in the TW2 sample and TW3 is advanced in relation to TW2.)

PEDIATRIC BONE

353

RELIABILITY

a handewrist scoring technique in 1988 known as the Fels handewrist method [24,25]. THE FELS HANDeWRIST TECHNIQUE

The theoretical basis for the Fels handewrist method is little different from that of the earlier TannereWhitehouse methods. Roche and his colleagues went through the laborious process of identifying suitable 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 rather dated and susceptible to the problems of secular change. From a possible 130 maturity indicators taken from the literature, 98 were finally selected that conformed to the criteria of universality, discriminative ability, reliability, validity and completeness. In addition to graded indicators, Roche and his colleagues also used metric ratios of lengths of radius, ulna, metacarpals and phalanges. Roche et al. [24,25] maintain that the Fels method differs from previous methods in terms of the observations made, the chronological ages when 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 [24]. 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 idea of the confidence of the estimated age. The GreulichePyle atlas technique and the TW2 and Fels scoring systems are the result of attempts to quantify skeletal maturation from different theoretical standpoints. The younger TW2 and Fels systems gained much from studying the disadvantages of the GreulichePyle system and attempted to overcome them. There are advantages to all three 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. BONEXPERT

Tanner recognized that skeletal maturation assessment was something that a computer should be able to do better than a human operator [36]. By using advanced mathematical methods and taking advantage of the significant increase in computer power over the last 20 years, the physicist Hans Henrik Thodberg was able to develop the BoneXpert (BX) method for automatic determination of skeletal maturation. This method applies the holistic aspects of the GP method (where the examiner is encouraged to take all information into account and even to develop a personal rating

approach) in a bone-specific way. Using a training set of over 3000 bones, principle component analysis of shape, intensities and Gabor texture energies were used for a statistical distillation of the parameter combinations that were relevant to each bone age. BoneXpert assesses bone age via a triple-layer process [38,39]: Layer A: using a generative model able to generate artificial but realistic images of all allowed bone shapes and densities, the borders of 15 bones are reconstructed: the five metacarpals, the phalanges of fingers 1, 3 and 5, and the radius and ulna. Abnormal bones are automatically rejected. Layer B: the intrinsic bone age is determined for 13 of these 15 bones. Metacarpals 2 and 4 are only used to determine bone mass by radiogrammetry on the three middle metacarpals e hence the same RUS bones are used as in the TW3 system. Bones with an intrinsic bone age deviating by more than 2.4 years from the average of all the bones are rejected. If fewer than eight bones are accepted, the entire image is rejected and no skeletal maturity values are reported. Layer C: the intrinsic bone age is transformed into a GreulichePyle bone age based on the calibration to a training set of 1097 radiographs with manual ratings [39]. The intrinsic skeletal maturities can also be transformed to continuous TW bone stages, from which SMS and then TW2 and TW3 bone ages can be computed using the standard TW method. The computer extracts visual information in a manner different from the human eye, so it was important to conduct extensive validation studies on both healthy children, and children with the common diagnoses of short stature (Turner syndrome, GHD, etc.). TW transformation still awaits full validation, whereas the GP transformation has been validated in several ways, showing that the system is robust and consistent and that BoneXpert bone age has a better correlation with growth potential than does TW3 [39e42]. Finally, the BoneXpert adult height prediction model, based on BoneXpert GP bone age, was presented in 2009 and shown to give a more accurate adult height prediction than the manual TW3 adult height prediction model on the same children [43]. The BoneXpert adult height prediction model was further validated on the children of the Third Zurich Longitudinal Study, all offspring of a parent from the First Zurich Longitudinal Study [43], on the Bjo¨rk study [34,44] and on the French Longitudinal Study [45] with equally good results.

RELIABILITY Tanner et al. [14] provide a detailed description of the comparative reliability of the different manual

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14. ASSESSMENT OF MATURATION: BONE AGE AND PUBERTAL ASSESSMENT

systems. Experienced raters tend to obtain 95% confidence limits of  0.5 to  0.6 “years” based on two standard errors of measurement [14]. Thissen [46] provides examples that suggest a somewhat greater error when using the Fels handewrist technique but, like the reliability of TW methods, that may be because of the chronological age of the specific example chosen; reliability tends to be greater or smaller depending on chronological age because, at certain ages, small changes in maturity indicators can result in large changes in skeletal age. Thus a miss-rating of one of these indicators will result in an inflated standard error of measurement from a testeretest reliability study. The testeretest reliability of BoneXpert is 0 years, i.e. the program rates the same image identically each time. Using the comparison of left and right hands of the same children taken at the same time and assuming all variability between the hands was due to BoneXpert alone, the reliability (SD on a single determination, multiply by 2 to get 95% confidence interval) of BoneXpert was 0.17 years [40]. Using the triplet method (assuming that the second of three bone age ratings in a series should lie on a straight line between the other two when plotted against age and assuming that all digression from the straight line was due exclusively to BoneXpert), the reliability of BoneXpert was measured at 0.18 years [4].

COMPARABILITY OF THE ATLAS, BONE-SPECIFIC AND AUTOMATIC METHODS In the most recent comprehensive comparison of the Atlas and Bone-specific techniques, Aicardi and his colleagues [47] compared the GreulichePyle, Tannere Whitehouse and Fels methods using a sample of 589 children (250 girls, 339 boys) aged from 2 to 15 years of chronological age. These Italian children from Genoa were admitted to a clinical pediatrics department for investigations of growth, obesity, and acute diseases. While 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 handewrist methods with an average deviation of 0.11 years. This was followed by the Fels handewrist (0.32 years), TannereWhitehouse RUS (0.35 years) and finally GreulichePyle (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 (TWeRUS), and 0.04 (GP). Naturally, such studies are rare because of the generally uncommon situation in which children have both handewrist and knee radiographs taken in the course of hospital entry. Aicardi et al. [47] concluded that further comparisons are required to decide on whether

handewrist or knee methods are most useful in clinical settings and particularly when there is a concern for growth potential. Tanner and his colleagues include a discussion on population differences in skeletal maturity in the most recent TW3 method [14] and good historical data are provided by Eveleth and Tanner’s review of growth globally [48,49]. 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. While 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 handewrist and/or knee and, in the case of the Fels handewrist method, the availability of the appropriate software. Of the more recent methods, the TannereWhitehouse technique is most often used within European settings and the Fels handewrist technique within North American settings but that situation probably reflects marketing and familiarity rather than scientific considerations. 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 would seem sensible to use the most recent methods and those that expose the child to the least radiation dosage. Being more recent, it would appear that the TannereWhitehouse and Fels handewrist methods would be preferred over the GreulichePyle and the RWT knee techniques. Yet the quick and intuitive GreulichePyle method has remained very popular around the world, and recent work has shown that the automated BoneXpert GreulichePyle as well as the manual GreulichePyle methods actually correlated slightly better with growth potential than did the TW RUS bone age [32] and were thus better predictors of final height [38].

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 described as the Tanner Scale or the Tanner Staging Technique. It was developed by Tanner [50] and was based on the work of Reynolds and Wines [51] and Nicolson and Hanley [52]. Tanner [50] divided 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

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SECONDARY SEXUAL DEVELOPMENT

stages B1eB5, genitalia development in stages G1eG5, pubic hair development in stages PH1ePH5 and axillary hair development in stages AleA3. The following descriptions of the stages of development of the breasts, genitalia and pubic hair are to be found in Tanner [50] and Marshall and Tanner [5,6] and are produced here to accompany the illustrations of secondary sexual development (Figures 14.8, 14.9, and 14.10).

Breast Development Stage 1: Pre-adolescent: 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.

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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: Pre-adolescent: testes, scrotum and penis are of about 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 penis: this 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. Stage 5: Genitalia adult in size and shape.

Pubic Hair development Stage 1: Pre-adolescent: 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 chiefly 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 in type, but the area covered by it is still considerably smaller than in the adult. 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.

Clinical Evaluations

FIGURE 14.8 Breast development in girls according to the Tanner staging technique. (From Tanner JM. Growth at Adolescence, 2nd edn. Oxford: Blackwell Scientific Publications; 1962.)

The assessment of secondary sexual development is a standard clinical procedure and, at such times, 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 crosssectional. This practical difficulty has led to the amalgamation of some of the stages to create pubertal stages. These pubertal stages are either on a three- or four-point scale and combine breast/genitalia development with pubic hair development [53,54]. In the three-point

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FIGURE 14.9 Genitalia development in boys according to the Tanner staging technique. (From Tanner JM. Growth at Adolescence, 2nd edn. Oxford: Blackwell Scientific Publications; 1962.)

technique for instance, stage P1 represents the prepubertal state (B1/G1; PH1), and stages P2 (B2e4/G2e4; PH2e4) and P3 (B5/G5; PH5) the mid-pubertal and postpubertal states. 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 Tanner [5,6], suggests that it may be misleading to expect stage synchronization in as many as 50% of normal children.

Self-assessment of Pubertal Status 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 on normal children who participate in growth studies, as opposed to those being clinically assessed, are problematical from both ethical and subject compliance viewpoints. In order to overcome this problem, the procedure of “self-assessment” has been developed and validated in a number of studies. 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 in photographic or line drawing styles as long as the contents are clear. To each picture of each stage is appended an explanation, in the language of the participant, of what the stage represents. The participant is instructed to remove whatever clothing is necessary in order for them to be able to observe properly their pubic hair/genitalia or pubic hair/breast development in the mirrors. The participant then marks on a separate sheet their stage of development and seals that sheet within an envelope on which

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the more controlled environment of a physician’s surgery as opposed to a school. The main reason for low correlations and thus poor validity in any setting with any group of participants is likely to be centered around 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/she has to do, then validity is high. Less effort in explaining procedures leads to lower validity. The procedure that should be adopted is that the observer should explain the procedure thoroughly to the participant using appropriate (non-scientific) 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 assessments by the observer is, of course, an ideal but requires a full and carefully considered argument to obtain the appropriate ethical permission.

Age at Menarche

FIGURE 14.10 Pubic hair development in boys and girls according to the Tanner staging technique. (From Tanner JM. Growth at Adolescence, 2nd edn. Oxford: Blackwell Scientific Publications; 1962.)

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 upon the age of the participants (e.g. early or late adolescence) [55], gender [55,56], the setting in which assessments are performed (e.g. school or clinic) [57,58], ethnicity [59], and whether they are a distinct diagnostic group such as cystic fibrosis [60] or anorexia nervosa [61] or socially-disadvantaged [62]. 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 while girls have been more consistent with experts [56]. The amount of attention given to explaining the required procedure appears to be of major importance. Thus, excellent rating agreement between physicians and adolescents have been found in clinical settings, with kappa coefficients between 0.66 and 0.91, [58,63,64] but rather less agreement in school settings (kappa¼0.35e0.42; correlations¼0.25e0.52) [57,58]. Improved agreement in clinical settings probably reflects

Age at menarche is usually obtained 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 on a sample of girls will produce a classical dose response sigmoid curve that may be used graphically to 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 a 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 the girl was at school or on holiday, 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 asked and the age at which they report menarche e the older the women the younger they believe they were. Such results have been found in both developed and developing countries and cast a seed of doubt about the reliability of retrospective methods beyond the teenage and early adult years. Prospective methods are 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 periods.

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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. That is seldom possible except in clinical situations and it is thus more likely that status quo and retrospective methods are the technique 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 the accuracy of recalled ages at this particular event. From a clinical point of view, it may be interesting to know that a girl has on average 6.62.2(SD) cm left to grow from the time of menarche [43].

Secondary Sexual Events in Boys While 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 time taken for the voice to be consistently in a lower register, and the logistical complications involved in the assessment of spermarche. Testicular volume, using the Prader orchidometer [65], is commonly the only measure of male secondary sexual development outside the rating scales previously mentioned, though other measurement techniques have been described to estimate testicular volume [66]. The detection of spermatozoa in the urine has been proposed as a quick, non-invasive method to assess the functional state of the maturing gonad and may be useful as a screening technique in population studies [67e72]. Its use, however, may be limited because longitudinal [71,73] and cross-sectional [70] studies have shown that spermaturia is a discontinuous phenomenon.

References [1] Bogin B. Patterns of Human Growth. Cambridge: Cambridge University Press; 1999. [2] Tove´e MJ, Maisey DS, Emery JL, Cornelissen PL. Visual cues to female physical attractiveness. Proc R Soc B: Biol Sci 1999;266:211. [3] Zaadstra BM, Seidell JC, Van Noord PA, et al. Fat and female fecundity: prospective study of effect of body fat distribution on conception rates. Br Med J 1993;306:484.

[4] Martin DD, Sato K, Sato M, Thodberg HH, Tanaka T. Validation of the BoneXpert method for automated determination of bone age on Japanese children. Horm Res Paediatr 2010;73:398e404. [5] Marshall WA, Tanner JM. Variations in pattern of pubertal changes in girls. Arch Dis Child 1969;44:291. [6] Marshall WA, Tanner JM. Variations in the pattern of pubertal changes in boys. Br Med J 1970;45:13. [7] Acheson RM. Maturation of the skeleton. In: Falkner F, editor. Human Development. Philadelphia: Saunders; 1966. p. 465e502. [8] Greulich W, Pyle S. Radiographic Atlas of the Skeletal Development of the Hand and Wrist. 2nd ed. Stanford: Stanford University Press; 1959. [9] Acheson RM. A method of assessing skeletal maturity from radiographs: A report from the Oxford Child Health Survey. J Anat 1954;88:498. [10] Acheson RM. The Oxford method of assessing skeletal maturity. Clin Orthop 1957;10:19e39. [11] Tanner JM, Whitehouse RH, Healy MJR. A New System for Estimating Skeletal Maturity from the Hand and Wrist, with Standards Derived from a Study of 2,600 Healthy British Children. Part II: The Scoring System. Paris: International Children’s Centre; 1962. [12] Tanner JM. Assessment of Skeletal Maturity and Prediction of Adult Height (TW2 method). London: Academic Press; 1975. [13] Tanner JM, Whitehouse RH, Cameron N, Marshall WA, Healy MJR, Goldstein H. Assessment of Skeletal Maturity and Prediction of Adult Height. London: Academic Press; 1983. [14] Tanner JM, Healy M, Goldstein H, Cameron N. Assessment of Skeletal Maturity and Prediction of Adult Height (TW3 Method). 3rd ed. London: WB Saunders Harcourt Publishers Ltd; 2001. [15] Todd TW. Atlas of Skeletal Maturation. St Louis: Mosby; 1937. [16] Hellman M. Ossification of epiphyseal cartilages in the hand. Am J Phys Anthrop 1928;11:223e57. [17] Rotch TM. 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 Assoc Am Phys 1909;24:603e30. [18] Bardeen CR. The relation of ossification to physiological development. J Radiol 1921;2:1e8. [19] Sontag LW, Lipford J. The effect of illness and other factors on the appearance pattern of skeletal epiphyses. J Pediatr 1943;23:391e409. [20] Lowell F, Woodrow H. Some data on anatomical age and its relation to intelligence. Pedagog Semin 1922;29:1e15. [21] Carter TM. Technique and devices used in radiographic study op the wrist bones of children. J Educ Psychol 1926;17:237e47. [22] Flory CD. Osseous development in the hand as an index of skeletal development. Monogr Soc Res Child Devel 1936:13. [23] Pryor JW. The hereditary nature of variation in the ossification of bones. Anat Rec 1907;1:84e8. [24] Roche AF, Chumlea C, Thiessen D. Assessing the Skeletal Maturity of the HandeWrist:Fels Method. Springfield, Illinois: CC Thomas; 1988. [25] Chumlea WC, Roche AF, Thissen D. The FELS method of assessing the skeletal maturity of the handewrist. Am J Hum Biol 1989;1:175e83. [26] Gasser T, Sheehy A, Largo RH. Statistical characterization of the pubertal growth spurt. Ann Hum Biol 2001;28:395e402. [27] Haber HP, Ranke MB. Pelvic ultrasonography in Turner syndrome: standards for uterine and ovarian volume. J Utra Med 1999;18:271.

PEDIATRIC BONE

REFERENCES

[28] Poland J. Skiagraphic Atlas Showing the Development of the Bones of the Wrist and Hand: For the Use of Students and Others. London: Smith, Elder, & Co; 1898. [29] Greulich W, Pyle S. Radiographic Atlas of the Skeletal Development of the Hand and Wrist. 2nd ed. Stanford: Stanford University Press; 1950. [30] Pyle SI, Hoerr NL. A Radiographic Standard of Reference for the Growing Knee. Springfield: CC Thomas; 1955. [31] Pyle SI, Hoerr NL. A Radiographic Standard of Reference for the Growing Knee. 2nd ed. Springfield: CC Thomas; 1959. [32] Healy MJR, Goldstein H. An approach to the scaling of categorized attributes. Biometrika 1976;63:219e29. [33] Thodberg HH, Jenni OG, Ranke MB, Martin DD. Validation of bone age methods through prediction of final adult height. Horm Res Paediatr; 2010;74:15e22. [34] Thodberg HH, Juul A, Lomholt J, et al. Adult Height Prediction Models. The Handbook of Growth and Growth Monitoring in Health and Disease. Springer; 2011. [35] Roche AF, Wainer H, Thissen D. Skeletal Maturity: the Knee Joint as a Biological Indicator. London: Plenum Medical Book Company; 1976. [36] Roche AF. Associations between the rates of maturation of the bones of the hand-wrist. Am J Phys Anthropol 1970;33:3. [37] Tanner JM, Gibbons R. Automatic bone age measurement using computerized image analysis. J Pediatr Endocrinol 1994;7:141e5. [38] Thodberg HH, Kreiborg S, Juul A, Pedersen KD. The BoneXpert method for automated determination of skeletal maturity. IEEE Trans Med Imag 2009;28:52e66. [39] Martin DD, Deusch D, Schweizer R, Binder G, Thodberg HH, Ranke MB. Clinical application of automated GreulichePyle bone age determination in children with short stature. Pediatr Radiol 2009;39:598e607. [40] Martin DD, Neuhof J, Jenni OG, Ranke MB, Thodberg HH. Automatic determination of left and right hand bone age. Horm Res Paediatr 2010;74:50e5. [41] Thodberg HH. An automated method for determination of bone age. J Clin Endocrinol Metab 2009;94:2239e44. [42] van Rijn RR, Lequin MH, Thodberg HH. Automatic determination of Greulich and Pyle bone age in healthy Dutch children. Pediatr Radiol 2009:39591e7. [43] Thodberg HH, Jenni OG, Caflisch J, Ranke MB, Martin DD. Prediction of adult height based on automated determination of bone age. J Clin Endocrinol Metab 2009;94:4868e74. [44] Bjo¨rk A. The use of metallic implants in the study of facial growth in children: method and application. Am J Phys Anthropol 1968;29:243e54. [45] Martin DD, Schittenhelm J, Ranke MB, Binder G, Thodberg HH. Validation of a new adult height prediction method based on automated determination of bone age in a French population. Hormone Research in Pediatrics 2010;69(suppl.3):135. [46] Thissen D. Statistical estimation of skeletal maturity. Am J Hum Biol 1989;1:2. [47] Aicardi G, Vignolo M, Milani S, Naselli A, Magliano P, Garzia P. Assessment of skeletal maturity of the handewrist and knee: a comparison among methods. Am J Hum Biol 2000;12:610e5. [48] Eveleth PB, Tanner JM. Worldwide Variation in Human Growth. Cambridge: Cambridge University Press; 1976. [49] Eveleth PB, Tanner JM. Worldwide Variation in Human Growth. 2nd ed. Cambridge: Cambridge University Press; 1990. [50] Tanner JM. Growth at Adolescence: with a General Consideration of the Effects of Hereditary and Environmental Factors upon Growth and Maturation from Birth to Maturity. Oxford: Wiley-Blackwell; 1962.

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[51] Reynolds EL, Wines JV. Physical changes associated with adolescence in boys. Am J Dis Child 1948;75:329e50. [52] Nicolson AB, Hanley C. Indices of physiological maturity: derivation and interrelationships. Child Devel 1953;24:3e38. [53] Kulin HE, Bwibo N, Mutie D, Santner SJ. The effect of chronic childhood malnutrition on pubertal growth and development. Am J Clin Nutr 1982;36:527. [54] Chaning-Pearce SM, Solomon L. A longitudinal study of height and weight in black and white Johannesburg children. S Afr Med J 1986;70:743. [55] Varona-Lopez W, Guillemot M, Spyckerelle Y, Mulot B, Deschamps JP. Self assessment of the stages of sex maturation in male adolescents. Pediatrie 1988;43:245. [56] Sarni P, De Toni T, Gastaldi R. Validity of self-assessment of pubertal maturation in early adolescents. Minerva Pediatr 1993;45:397. [57] Wu WH, Lee CH, Wu CL. Self-assessment and physicians’ assessment of sexual maturation in adolescents’ in Taipei. Zhonghua Minguo xiao er ke yi xue hui za zhi 1993;34:125e31. [58] Schlossberger NM, Turner RA, Irwin CE. Validity of self-report of pubertal maturation in early adolescents. J Adolesc Health 1992;13:109e13. [59] Hergenroeder AC, Hill RB, Wong WW, Sangi-Haghpeykar H, Taylor W. Validity of self-assessment of pubertal maturation in African American and European American adolescents. J Adolesc Health 1999;24:201e5. [60] Boas SR, Falsetti D, Murphy TD, Orenstein DM. Validity of selfassessment of sexual maturation in adolescent male patients with cystic fibrosis. J Adolesc Health 1995;17:42e5. [61] Hick KM, Katzman DK. Self-assessment of sexual maturation in adolescent females with anorexia nervosa. J Adolesc Health 1999;24:206e11. [62] Hardoff D, Tamir A. Self-assessment of pubertal maturation in socially disadvantaged learning-disabled adolescents. J Adolesc Health 1993;14:398. [63] Duke PM, Litt IF, Gross RT. Adolescents’ self-assessment of sexual maturation. Pediatrics 1980;66:918. [64] Brooks-Gunn J, Warren MP, Rosso J, Gargiulo J. Validity of selfreport measures of girls’ pubertal status. Child Devel 1987:829e41. [65] Prader A. Testicular size: assessment and clinical importance. Triangle. Sandoz J Med Sci 1966;7:240. [66] Daniel WA. Testicular volumes of adolescents. J Pediatr 1982;101:1010e2. [67] Schaefer F, Marr J, Seidel C, Tilgen W, Scharer K. Assessment of gonadal maturation by evaluation of spermaturia. Arch Dis Child 1990;65:1205. [68] Baldwin BT. The determination of sex maturation in boys by a laboratory method. J Comp Psychol 1928;8:29e38. [69] Richardson DW, Short RV. Time of onset of sperm production in boys. J Biosoc Sci Suppl 1978;5:15. [70] Hirsch M, Shemesh J, Modan M, Lunenfeld B. Emission of spermatozoa. Age of onset. Int J Androl 1979;2:289e98. [71] Nielsen CT, Skakkebaek NE, Richardson DW, et al. Onset of the release of spermatozia (spermarche) in boys in relation to age, testicular growth, pubic hair, and height. Clin Endocrinol Metab 1986;62:532. [72] Kuhn HE, Frontera MA, Demers LM, Bartholomew MJ, Lloyd TA. The onset of sperm production in pubertal boys: relationship to gonadotropin excretion. Arch Pediat Adolesc Med 1989;143:190. [73] Hirsch M, Lunenfeld B, Modan M, Ovadia J, Shemesh J. Spermarche e The age of onset of sperm emission. J AdolescHealth Care 1985;6:35e9.

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Biochemical Markers of Bone Metabolism Nick Shaw, Wolfgang Ho¨gler Birmingham Children’s Hospital and University of Birmingham, Birmingham, UK

INTRODUCTION When assessing a child for a potential bone disorder, even detailed clinical examination, biochemical tests of calciumephosphate metabolism and bone imaging sometimes do not provide a sufficient answer towards the underlying pathology. There and then the question may arise whether the patient has a particular defect in bone formation or bone resorption. The ultimate and most accurate technique is to take a bone biopsy and perform histomorphometry. Transiliac bone biopsy needs preparation, general anesthesia, a skilled clinician or surgeon to perform it, and samples need to be sent off to one of the few specialized units worldwide who can analyze and interpret them. Naturally, clinicians and researchers therefore have been looking for a way to measure bone metabolism through measurement in blood and urine. 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. 15.1, Table 15.1). A large number of commercially available bone metabolism marker assays, primarily designed for diagnosis and follow up of metabolic bone diseases in adults, has been employed in children as well. In this chapter, we review the clinical areas of interest, the possible applications and limitations of using these markers in the pediatric setting.

osteoporosis and fractures [1,2], to monitor antiresorptive therapy [3,4] and also have a promising role in metastatic bone disease [5]. In children, these markers are released into the circulation during three different physiological processes: growth in bone length (bone elongation); growth in bone width (bone modeling by periosteal expansion); and bone remodeling (turnover). All bone markers measured in children reflect the sum of these three processes. Growth in bone length 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. 15.2). 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

WHAT IS BONE METABOLISM IN CHILDREN? Before dealing with individual indicators of bone metabolism, it is important to consider what an ideal bone marker reflects, and the differences between children and adults. In adults, bone turnover markers mainly represent bone remodeling and are commonly used as independent predictors of the risk of

Pediatric Bone, Second Edition DOI: 10.1016/B978-0-12-382040-2.10015-2

FIGURE 15.1 Diagram of bone formation and resorption markers on the diaphysis of a growing tubular bone. The diagram demonstrates periosteal expansion on the outside of cortical bone (left) and endocortical resorption on the inside (right). Reproduced with permission from Seibel MJ et al. The use of molecular markers of bone turnover in the management of patients with metastatic bone disease. Clin Endocrinol 2008;68:839-49.

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Copyright Ó 2012 Elsevier Inc. All rights reserved.

362 TABLE 15.1

15. BIOCHEMICAL MARKERS OF BONE METABOLISM

Commonly Measured Bone Turnover Markers

Marker

Acronyms

Clinical source

Procollagen type 1 N-terminal propeptide

P1NP

Serum

Procollagen type 1 C-terminal propeptide

P1CP

Serum

Osteocalcin

OC

Serum

Bone alkaline phosphatase

BALP

Serum

Pyridinoline

PYD

Urine or serum

Deoxypyridinoline

DPD

Urine or serum

N-terminal cross-linked telopeptide

NTX

Urine or serum

C-terminal cross-linked telopeptide

CTX

Urine or serum

Tartrate-resistant acid phosphatase

TRAP

Serum

Bone formation

Bone resorption

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 [6]. 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. Growth in bone width occurs by a process that Frost [7] termed bone modeling. Bone modeling involves the

FIGURE 15.3 Bone growth in width. Reproduced with permission from S. Karger AG, Basel CH and Caudex Medical, Oxford, UK; for Schoenau E et al. From bone biology to bone analysis. Horm Res 2004;61:257e69.

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. 15.3). Thereby, the outer circumference of a long bone or a vertebral body is gradually increased. At the same time, osteoclasts 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 FIGURE 15.2 Bone growth in length. Reproduced with permission from S. Karger AG, Basel CH and Caudex Medical, Oxford, UK; for Schoenau E et al. From bone biology to bone analysis. Horm Res. 2004;61:257e69.

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WHAT IS BONE METABOLISM IN CHILDREN?

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 [8] called remodeling. Remodeling consists of successive cycles of bone resorption and formation on the same bone surface (Fig. 15.4). The basic features of this process are identical for trabecular and cortical bone [9]. A group of osteoclasts removes a small quantity (“packet”) of bone tissue which, after a reversal phase, is replaced

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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” [9]. 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

FIGURE 15.4

Bone remodeling units continuously turn over cortical bone (A) and trabecular bone (B). The principles are the same for both intracortical and trabecular remodeling. Osteoclasts dig tunnels (cortical bone) or trenches (trabecular bone) which are subsequently filled by osteoclastic bone formation. A remodeling unit in trabecular bone moves parallel to the surface and corresponds exactly to half of a remodeling unit in cortical bone (hemiosteonal remodeling). The remodeling balance is near zero, meaning a similar amount of bone is resorbed and refilled. Reproduced with permission from Caudex Medical, Oxford, UK. Schoenau E, et al. Prediction of growth response to GH treatment: A reference guide, 2001.

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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 always to postulate such a drastic derangement in the 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. From birth to late adolescence, during any given time interval, the relative contributions of endochondral bone formation, modeling, and remodeling to the total amount of bone turned over 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 the essential difference to the situation in adults, in whom bone metabolic activity is basically limited to remodeling. Thus, all biochemical measurements in adults reflect the activity of different bone cells during coupled remodeling, and markers are regarded as remodeling, or turnover, markers. In the assessment of changes in the three main processes of bone metabolism, pediatric bone specialists are bound to the two-dimensional measurement of bone mass using dual energy x-ray absorptiometry or the three-dimensional measurement of bone geometry, mass and density using quantitative computed tomography. The use of biochemical bone markers complements these physical measures by providing a dynamic picture of whole body bone turnover that can be repeated at much shorter intervals. This dynamic assessment allows early detection of effects of disease or treatment long before changes in bone mass or progression in bone disease can be accurately ascertained. Normative curves are thus a prerequisite tool for evaluating children with metabolic bone diseases.

BIOCHEMICAL CONSIDERATIONS 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, creatinine itself is subject to considerable biological variation and change with age as muscle mass increases. Thus, if creatinine excretion is reduced, for example, due to decreased muscle mass [10], 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 osteoblasts. The most commonly used formation markers in pediatric research and clinical practice are alkaline phosphatase and its isoforms, osteocalcin, and the procollagenbreakdown products PICP and PINP (see Table 15.1).

Alkaline Phosphatase 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-non-specific ALP gene; both isoforms differ only in their patterns of post-translational glycosylation [11]. Many methods have been developed for the determination of bonespecific ALP (BALP), but specificity remains a problem for all these assays [12]. Total BALP can be further separated into isoforms that are specific for cortical and trabecular bone, respectively [13,14]. However, more research is needed, using isoelectric focusing or highperformance liquid chromatography (HPLC) [15] to identify whether certain isoforms are more prevalent in certain clinical conditions and provide information on 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, the lack of tissue-nonspecific ALP causes hypophosphatasia, a severe and often lethal mineralization defect that will hopefully soon be treatable [16]. The clearance of ALP from serum may be transiently impaired after viral infections in young children (transient hyperphosphatasemia), leading to excessively high total ALP serum activity without any detectable consequences [17]. Despite ALP’s role in bone formation and mineralization,

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elevated ALP is also the cheapest diagnostic marker in all forms of rickets, a demineralization disorder, which normalizes with treatment, another indicator that its physiologic role is not fully understood.

Osteocalcin Osteocalcin, or bone Gla (g-carboxyglutamic acidcontaining) protein, is a 5.8-kDa non-collagenous protein that is exclusively synthesized by osteoblasts [11]. The molecule is gamma-carboxylated intracellularly under the influence of vitamin K. It is secreted and incorporated into the organic bone matrix during the matrix mineralization phase. 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 significant instability and by difficulties in distinguishing between the various molecular forms that are found in the circulation [18]. Intact osteocalcin is metabolized primarily by the kidneys and it is excreted into the urine, where several different osteocalcin fragments have been identified [19]. Until relatively recently, the function of osteocalcin was uncertain. However, recent studies suggest that osteocalcin is involved in the regulation of energy metabolism [20]. An initial observation demonstrated that transgenic mice with inactivation of osteocalcin had a phenotype with increased bone mass due to increased bone formation [21]. This finding suggests that osteocalcin normally functions to limit bone formation without impairing bone resorption or mineralization. In addition, transgenic mice lacking osteocalcin had evidence of increased visceral fat associated with insulin resistance and glucose intolerance in early life. Such mice also have evidence of decreased beta cell proliferation, insulin production and energy expenditure. These features are associated with decreased levels of adiponectin, an adipokine known to enhance insulin sensitivity (Fig. 15.5). The phenotype of osteocalcin null mice is the opposite of that seen in Esp-null mice. Esp, which is also known as the protein tyrosine phosphatase (PTP) receptor type gene, is expressed in osteoblasts and encodes the protein tyrosine phosphatase termed osteotesticular PTP (OST-PTP). Esp null mice have a high rate of neonatal death due to hypoglycemia as a result of insulin overproduction by hyperplastic beta cells and increased insulin sensitivity. OST-PTP seems to be necessary for post-translation metabolic inactivation of osteocalcin, involving gamma-carboxylation of glutamic acid residues. As a consequence Esp null mice have more uncarboxylated osteocalcin and may therefore be better protected against obesity and diabetes [22]. These new data

FIGURE 15.5 Regulation of energy metabolism by the skeleton. Reproduced with permission from S. Karger AG, Basel; for Lieben L et al. Bone and metabolism: a complex crosstalk. Horm Res 2009;71(Suppl. 1): 134-8.

suggest that bone acts as an endocrine tissue that regulates metabolic homeostasis. Studies in humans have demonstrated that osteocalcin is inversely associated with the homeostasis model assessment for insulin resistance (HOMA-IR) [23]. Studies have suggested that osteocalcin may be a predictor of cardiovascular health, due to its role in insulin secretion, and presumably vascular calcification. Reduced serum total osteocalcin has been shown to be inversely related to the presence of diabetes and the metabolic syndrome in humans [20,24]. Further studies are required to elucidate the role of osteocalcin on energy metabolism in humans.

Procollagen Type I Propeptides Collagen type I is by far the most abundant protein synthesized by osteoblasts [11]. After secretion into the extracellular space, the procollagen type I C- and Nterminal propeptides (PICP and PINP) are cleaved off and released into the circulation. The remaining collagen molecule will undergo several processing 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 [25]. This uptake can be disturbed, resulting in very high serum levels of PICP [26]. PINP is also taken up by liver endothelial cells, but this occurs via the scavenger receptor [27]. Radioimmunoassays have been developed to measure both PICP and PINP in serum samples [28e30]. Although collagen type I is found in many

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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 [11]. The appeal of PICP and PINP as markers of bone formation is that they reflect the activity of a crucial and well-characterized 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 post-translational modification of bone type I collagen and are released upon bone matrix degradation during osteoclastic bone resorption. The positions of these compounds within the intact collagen fibril are schematically represented in Figure 15.6. The most commonly used markers of bone resorption are collagen breakdown products and osteoclastic enzymes (see Table 15.1).

Type I Collagen Degradation Compounds Pyridinoline (PYD) and deoxypyridinoline (DPD) are generated from hydroxylysine and lysine during the extracellular maturation of the collagen fibril [31]. Pyridinium cross-links are interchain bonds that stabilize the collagen fibril (see Fig. 15.6). PYD is the prevalent cross-link in bone, but it is also found in articular cartilage and other connective tissues [31]. DPD is more specific for bone because its extraosseous concentrations are comparatively lower [11]. In the serum of children, the fraction of free cross-links is approximately 16e18% and the remainder is peptide bound [32]. In

urine, the free fraction is approximately 40% and the peptide-bound forms represent approximately 60%. Excretion of urinary cross-links is thought to be a better marker of bone resorption than other collagen breakdown products like hydroxyproline, because cross-links are released only during collagen degradation, are not metabolized, and are independent of nutritional collagen intake [11]. Pediatric reference data are available for urinary PYD and DPD [30,33]. Various immunoassays have been developed to quantify cross-links [11], including assays using antibodies against the pyridinium structures [34,35], or against the cross-link containing C-terminal telopeptide of type I collagen (ICTP) [36]. More commonly used nowadays are assays using 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 [37,38]. Serum and urine assays and pediatric reference data for NTX and CTX are available [30,39e42]. Hydroxylysine glycosides, residues of collagen breakdown, like galactosyl-hydroxylysine (Gal-Hyl) have received less attention than cross-links as markers of bone resorption in children and adolescents probably because they can only be determined by HPLC. Despite development of an immunoassay to quantify urinary Gal-Hyl [43], this marker has not received widespread attention.

Osteoclastic Enzymes Tartrate-resistant acid phosphatase 5b (TRAP5b) is special among markers of bone resorption in that it is an enzyme derived from the osteoclast that can be quantified in serum samples [11]. TRAP5b is involved in bone FIGURE 15.6 Bone resorption markers derived from type 1 collagen. Reproduced with permission from Caudex Medical, Oxford, UK; for Schoenau E et al (2001). Prediction of growth response to GH treatment: A reference guide.

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matrix degradation and released into the circulation during the resorption process itself or after detachment of the osteoclast from the bone surface, and later degraded to fragments. A variety of immunoassays have been developed [44,45] but pediatric reference data are sparse [42,46]. Cathepsin K is a lysosomal cysteine protease found in osteoclasts. This enzyme is needed to create the typical acidic environment for osteoclastic bone resorption, including the ability to breakdown cartilage (collagen and elastin). Lack of this enzyme causes pycnodysostosis, an osteosclerotic condition associated with short stature. Enzyme-linked immunosorbent assays (ELISA) for serum cathepsin K have been developed but no pediatric studies are available to date. Drug development for osteoporosis currently focuses on antibody treatment against this enzyme.

Osteocytic Proteins Sclerostin, the gene product of the sclerostin gene (SOST), and Dickkopf 1 (DKK-1) are proteins, mainly expressed by osteocytes. These proteins act as inhibitors to the Wnt signaling pathway and thus inhibit bone formation. Mechanical loading in rats and mice reduces sclerostin levels leading to greater bone formation [47], whereas immobilization in humans increases sclerostin levels, leading to bone loss [48]. Sclerostin is thought to be the crucial link between osteocytic mechanosensing and osteoblastic bone formation, and can be measured in serum by ELISA. Sclerostin antibodies are currently in development as a new anabolic treatment for osteoporosis. If successful, serum sclerostin may become one of the most important bone markers in the future, though its clinical application in children and adolescents will need to be established.

The OPG/RANKL/RANK System The control of osteoclastogenesis is complex. Osteoprotegerin (OPG) is a soluble receptor secreted by many cell types including osteoblasts. OPG acts as a decoy receptor, capturing RANKL (receptor activator for nuclear factor kB ligand). RANKL activates osteoclastogenesis through the RANK receptor. RANKL is mostly cell bound, but also circulates in a soluble form (sRANKL). As a consequence, OPG knockout or RANKL overexpression lead to osteoporosis, whereas OPG overexpression or RANKL knockout lead to osteopetrosis with impaired dental eruption [49]. RANKL antibodies are in development for therapy of osteoporosis. RANKL mRNA expression is stimulated by the inflammatory cytokines, parathyroid hormone (PTH),

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1,25 (OH)2 vitamin D3 and steroids, and has helped to explain how chronic inflammatory conditions induce osteoporosis. Serum assays for OPG and sRANKL are available but results currently lack the consistency for clinical utility even in adults. It is questionable whether the serum markers really reflect the activity in the bone microenvironment, to what extent they originate from non-skeletal sources in different conditions, and there is a number of technical limitations [50]. Current research focuses on OPG and sRANKL more as cardiovascular risk factors, in particular arterial calcification [51].

PEDIATRIC REFERENCE DATA Biological Variation Several markers of bone and collagen metabolism show circadian variations, usually with higher values at night than during the day [52e55]. For example, PICP in children varies by 10e20%, osteocalcin by 30e60%, and Gal-Hyl by 30% over a 24-hour period, although bone ALP does not show significant circadian variation [56]. 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 [54,57e59] but limited information is available 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. Overnight fasting has been recommended for adults before bone marker sampling [2,60], but this is often impracticable for infants, younger children or the chronically ill. In addition, the clinical impact of feeding vs fasting in adults was reportedly small, apart from serum CTX [2,60,61], and detailed information is missing for most bone marker assays, in particular for children. However, as monitoring is the main purpose of using bone markers, the individual one-off measurement is much less important than the course over time. Using reference curves, clinicians can chose which regimen (fasting or not) is best for the individual patient and then should stick to the chosen regimen for all subsequent measurements.

Clinical Considerations Normal pediatric reference ranges for serum markers of bone formation and resorption are a prerequisite for the assessment of metabolic bone disorders and for the monitoring of antiresorptive therapy or

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disease progression. Skeletal growth and puberty lead to considerable changes in raw levels of bone formation and resorption markers with age, demonstrated by their correlation with growth velocity [62e66]. Thus, any longitudinal measurement in a patient necessitates comparison relative to the physiologically changing reference curves. The known high intraindividual variation in bone marker concentrations and their release during different anabolic and catabolic processes preclude their use for one-off diagnostic purposes [42,62,63]. A considerable number of markers of bone and collagen turnover have been designed but no single test fulfils all the criteria for an ideal marker. In addition, no marker in children is specific for any of the three different biological processes of remodeling, modeling and epiphyseal growth [64]. Bone marker concentrations can be similar in a child with high bone remodeling and low growth rate and in a normally growing child. Therefore, knowledge of growth velocity and pubertal development is necessary in the correct interpretation of markers. We would recommend using a set of different formation and resorption markers as the preferred approach in the longitudinal assessment of bone diseases and in the monitoring of antiresorptive or growth modulating therapies.

Growth Spurts and Sex Differences Infancy and puberty, as periods of rapid growth, generally cause greater serum concentrations of bone markers [42,46,56,66e69]. These findings indicate that both bone formation and resorption are accelerated during periods of growth spurt. Not surprisingly, later peaks were observed for BALP, ICTP and TRAP5b in boys, reflecting their later pubertal development and thus bone mass accrual [42]. In both boys and girls, concentrations of most bone markers declined during late puberty with lowest values in the transition to adulthood. Since growth and puberty usually are completed by late adolescence, markers of bone formation and resorption converge into adult values. One has to extrapolate our data for ages >17 years because subject numbers were low in this age group.

Creating Reference Curves Establishing pediatric reference ranges for bone markers and assessing their relation to sex, age and anthropometric data requires a large population of healthy children. A number of previous normative studies partly suffered from low subject numbers. In addition, the routine use of these data has also been hindered by the lack of applying appropriate curve fitting procedures. Curve fitting is essential, because

of the frequently skewed distribution of bone marker data, the age-related changes that occur within individual age groups and the different variation between age groups. These changes complicate the interpretation of longitudinal results and the monitoring of children in intervention studies. Of a large number of available pediatric reference studies, probably the most comprehensive is that of Rauchenzauner et al. [42], who provide age- and sex-specific reference curves for a total of five different bone markers that were derived from a large cohort of healthy children aged 2 months to 18 years, enabling the calculation of sex- and age-specific SD scores (Fig. 15.7).

BONE MARKERS DURING NORMAL DEVELOPMENT Pre- and Perinatal Bone Metabolism A number of studies gained 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 [70e76], showing greater osteocalcin levels in fetal than in maternal blood. Cord blood osteocalcin increases after 22e27 weeks of gestation and decreases thereafter [75]. Similarly, concentrations of PICP, PINP, and ICTP in cord blood decrease with gestational age [77e81]. There is no relationship between fetal and maternal bone marker levels at birth [70,76,82e84]. In term newborns small for gestational age, osteocalcin cord blood concentrations were lower than those in newborns appropriate for gestational age [73]. Maternal smoking negatively influences birth weight, but also alkaline phosphatase levels [85e87]. 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. Bone markers have also been quantified in surplus amniotic fluid obtained during amniocentesis. Again, levels of collagen turnover markers decreased with gestational age [88,89]. Interpretation of such data is difficult because it is unknown how these collagen markers enter amniotic fluid and how they are eliminated. However, confirmation comes from the probably most reliable studies using fetal blood sampling by intrauterine cordocentesis. These results confirm that bone and collagen metabolism decreases from 18 weeks of gestation to birth. This pattern was found for total ALP activity [90] as well as for PICP and ICTP [91], reflecting similar changes in the rate of intrauterine weight gain [92]. Limited data are available on the clinical utility of biochemical bone markers prenatally or at birth. Early

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FIGURE 15.7 (A) Backtransformed reference curves for bone formation markers OC and BALP in boys (filled circles) and girls (open circles). Curves represent the 50th centile (straight lines) and 3rd/97th centile (dotted lines). (B) Backtransformed reference curves for bone resorption markers ICTP, CTX, and TRAP5b in boys (filled circles) and girls (open circles). Curves represent the 50th centile (straight lines) and 3rd/97th centile (dotted lines). Reproduced, with permission from Rauchenzauner M et al. Sex- and age-specific reference curves for serum markers of bone turnover in healthy children from 2 months to 18 years. J Clin Endocrinol Metabol 2007;94:443-9. PEDIATRIC BONE

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case reports suggested that bone markers may help in the prenatal diagnosis of congenital bone disorders, such as hypophosphatasia and osteogenesis imperfecta [89,93]. Markers of collagen metabolism have also been used in studies assessing the effect of obstetric interventions, such as antenatal dexamethasone therapy, on fetal bone development [78,94,95]. In contrast, attempts to gain insight into the pathophysiology of bone metabolism in infants of diabetic mothers have been largely unsuccessful [79,88].

Postnatal Bone Metabolism In the first 24 hours after delivery, 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 [96]. Probably the best studied aspect of early skeletal changes is the decline in neonatal serum calcium levels after the placental supply of calcium is cut off, leading to a secondary increase in parathyroid hormone levels [97,98]. This increase occurs within hours after birth and during this time declining formation markers OC and PICP have been reported [72], whereas bone resorption remains similar or even increases [99]. This scenario appears plausible when skeletal adaptation is considered from a nutritional standpoint (calcium mobilization after interruption of placental supply and low initial intake) or from a biomechanical standpoint (bone loss in a disuse situation). The following 6e12 days represent a transitional growth period. This is characterized by large individual variations [100] in body weight and hydration state that complicate the assessment of the postnatal growth pattern. Due to the large measurement errors during this period, reliable growth data are sparse and inconsistent for the first days of life. Along with these metabolic changes and the postnatal resumption in growth velocity, the available studies show a parallel increase in bone marker concentrations after birth [71,74,80,101e106). Again, most studies on this topic were performed in preterm infants, who typically miss the phase of peak bone mineral accretion during the last trimester and postnatal life in these infants is often complicated by serious illness. However, practically all studies in term babies also show increasing concentrations of serum and urine formation and resorption markers during the first days of life [71,85,99,101,107,108] although the urinary levels may partly reflect increasing collagen type I degradation or a decrease in creatinine excretion during the immediate postnatal period.

Infancy and Childhood 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 [37,42,56,109e114]. After 4 years of age, serum bone marker levels usually remain stable until puberty [42,56,67,82,112]. Urinary resorption markers related to creatinine tend to decrease after 4 years of age until puberty [33,37,115], but this may reflect increasing creatinine excretion rather than decreasing bone resorption [116].

Puberty All three mechanisms contributing to bone turnover in growing individuals (longitudinal growth, modeling, and remodeling) accelerate during puberty [117,118]. 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 [118], the peak in bone marker levels is earlier and less marked in girls [42,42,56,82,114,119,120]. However, most studies on urinary resorption markers normalized to creatinine failed to detect a pubertal peak [54,57,115,119] because the increase in the excretion of collagen breakdown products is offset by the concomitant increase in creatinine excretion. After pubertal stage 5, bone marker levels decrease to adult levels [42,116].

BONE AND COLLAGEN MARKERS IN METABOLIC BONE DISEASES Interpretation and 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 skeleton e osteoblasts 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. 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. 15.8). In the literature, bone formation markers are often interpreted as if they reflected only the activity level of osteoblasts, but the number of

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Determinants of whole body bone formation activity. Formation markers mirror the sum activity of all osteoblasts at all skeletal regions. Reproduced, with personal permission from Rauch F.

osteoblasts is probably a much more important determinant of whole body bone formation activity [9]. 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 (see Fig. 15.8): (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 e total body bone mass and the average bone surface per bone mass ratio (see Fig. 15.8). 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. The advantage of breaking down whole body osteoblast activity into these factors is that each of 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-to-bone 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. A fundamental difficulty in the use and interpretation of bone turnover markers in pediatric practice is the fact that children grow with appropriate increases in length and width of bones to accommodate this process. Thus, bone turnover markers reflect the three processes of longitudinal bone growth, bone modeling and remodeling without being able to separate these individual components. This is different to the situation in adults where bone turnover markers essentially reflect bone remodeling. As a consequence of these differences, the levels of bone turnover markers in children are higher than those in adults. Thus, any process that affects growth in a child will lead to a change in the level of bone turnover markers. This has to be considered when interpreting results of studies that have examined bone turnover markers following a therapeutic intervention. It is sometimes mistakenly interpreted that the intervention has demonstrated a beneficial effect on bone formation. In a one year study of the use of growth hormone in children with juvenile idiopathic arthritis, increases in plasma osteocalcin were seen and interpreted as reflecting an increase in bone formation [121]. However, their correlation with height velocity was also noted and thus they were reflecting longitudinal bone growth. In another longitudinal study of children with juvenile idiopathic arthritis, the bone turnover markers, bone alkaline phosphatase and ICTP, were found to be predictors of the change in total body bone mineral content over 2 years with lower values in the patients compared to controls concluding that there is evidence of reduced bone turnover in the children with arthritis

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[122]. However, it is likely that the children with arthritis would have been growing more slowly than the controls thus accounting for the reduced levels of bone turnover markers as well as total body bone mineral content another growth dependant variable. 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. 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 directly to reflect changes in bone metabolism because bone structure and mass can be assumed to remain similar during the study interval.

CLINICAL AREAS OF INTEREST Use in Adult Practice The use of bone turnover markers in adult practice is well established and they have been in use for at least 15 years. Here they are used for several different clinical indications including the monitoring of treatment response in metastatic bone disease and Paget’s disease. However, their principal use is within the field of osteoporosis where they have been utilized to assess fracture risk often in conjunction with measurements of bone density and to monitor the use of bisphosphonate treatment [4]. It is recognized that accelerated bone turnover is associated with a higher fracture risk. Their principal use at present is the monitoring of osteoporosis therapy. As the reduction in fracture risk is often related to reductions in bone resorption, it is more appropriate to monitor such markers than bone density. They are also usefully monitored to document compliance with bisphosphonate medication. However, they are not currently recommended as part of routine clinical practice in most osteoporosis guidelines. Their main use has been in the context of clinical trials with large numbers of subjects rather than in an individual patient. There remain concerns about their variability, the lack of quality control programs for laboratories performing the analyses and the lack of valid reference ranges for many countries [123]. In the context of research, studies in normal volunteers placed on bed rest have demonstrated rapid increases in bone resorption markers seen within 24 hours which is sustained over several weeks of

prolonged bed rest [124]. Bone formation markers are reduced at the same time indicating the mechanism of bone loss induced by immobility is primarily due to increased bone resorption. Similar changes have been seen in spinal cord injury patients [125]. Here bisphosphonate treatment can reduce the excretion of urinary NTX and prevent bone loss [126]. Another study undertaken in healthy volunteers has shown that resistive vibration exercise can attenuate the increase in bone resorption markers and stimulate an increase in bone formation markers thus reducing the extent of bone loss [127].

Growth Prediction Since all three mechanisms that characterize skeletal growth (endochondral bone formation, modeling, and remodeling) involve the formation and degradation of bone matrix, it is not surprising that various biochemical parameters derived from bone and collagen metabolism correlate with longitudinal growth of children. Unlike insulin-like growth factor 1 (IGF-1), none of the available bone markers can be used for diagnostic purposes because of their great variability. However, the early change in bone marker concentrations following growth hormone (GH) treatment in children with GH deficiency or idiopathic short stature gives a useful prediction of growth velocity response to treatment after one year [62]. The individual therapeutic effect on growth is quite variable and it would be helpful to have a way to evaluate responsiveness after a short period of therapy. Compared with OC, ICTP and CTX, the change in BALP values after 3 months of GH therapy gave the best prediction of growth velocity response [63,128]. Nonetheless, the prediction of one individual marker is generally too imprecise to serve as a basis for clinical decisions [116]. In fact, a statistically significant association between a marker and growth velocity is not equivalent to a clinically relevant prediction. More important is the confidence interval of the prediction. Scho¨nau et al. [129] described a growth prediction model that, as well as bone age retardation, pretreatment IGF-1 level and height velocity after 3 months, included urinary DPD concentrations after 1 month. The model was later validated [130], and competes with other prediction models that focus on titrating GH against IGF-1 levels [131] or growth velocity response [132,133]. Using the change of a set of bone markers during GH therapy, including IGF-1 [134,135], may better help differentiate a true response to GH treatment from non-responders, a group which itself needs to be defined first in terms of growth velocity [136]. Such an approach could allow early GH dose adjustments or even GH withdrawal in non-responders. Future research needs to focus not only on responsiveness, dose adjustment and efficacy of GH, but also

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whether such prediction models could help reduce unnecessary treatment with its social and economic burdens.

Bone Marker Studies in Osteogenesis Imperfecta Osteogenesis imperfecta is characterized by bone fragility and decreased bone mass. Detailed densitometric and bone histomorphometric analyses have been published [137,138]. 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 [139e141]. 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 [142]. 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 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 [138] 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 TABLE 15.2

373

half the normal amount of bone. As discussed earlier, bone marker levels also depend on bone mass, bone surface-to-mass ratio, and cell number-to-bone surface ratio. These parameters can only be determined by histomorphometry. As expected, osteoblasts of osteogenesis imperfecta patients form bone at only half the normal rate [138]. However, the number of osteoblasts relative to bone surface area is very much increased, resulting in increased surface-based bone formation rates (Table 15.2). 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 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 [10], 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. Bone turnover markers have been much more effectively used in the longitudinal monitoring of the effect of bisphosphonates in children with osteogenesis imperfecta. In one of the earliest studies of the use of intravenous pamidronate in this condition, the bone turnover markers alkaline phosphatase and the N-telopeptide of type 1 collagen were shown to decrease within 3e4 months of treatment initiation and were reduced by 13 and 26% of baseline values respectively per year [143]. Another study, which examined the longitudinal change in bone turnover markers in 69 children and adolescents receiving monthly pamidronate treatment, found no correlations with improvements in bone density, mobility or pain [144].

Bone Formation Rates in Osteogenesis Imperfecta Type III on Increasing Levels of Biological Organization

Bone formation rate relative to

Rate of bone formation* (in % of age-specific mean value)

Commentary

Osteoblast number

49

Genetic defect in osteoblast function

Bone surface area

140

Very high osteoblast number more than compensates low activity of single cell

Bone mass

225

A high bone-surface-to-mass ratio leads to even higher bone mass related bone formation rate

External bone size

66

Extremely low bone mass leads to low bone size related bone formation rate

* Calculated from Rauch et al. [138]

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A study which examined the effect of discontinuation of pamidronate treatment showed that, after 2 years, the levels of the bone resorption marker NTX increased by 28% in serum and 27% in urine but were still 41% lower than the average value seen in healthy children [145]. Thus, there was still evidence of a continuing biological effect of pamidronate within the skeleton on bone turnover after 2 years (Fig. 15.9). Monitoring of bone turnover markers are therefore useful in children receiving long-term bisphosphonate treatment to help ensure there is no evidence of oversuppression of bone turnover.

Impact of Chronic Disease and Treatment The interpretation of biochemical markers of bone turnover in children with chronic disease is compounded by the fact that such conditions are often associated with reductions in growth velocity and delayed pubertal maturation. As has been demonstrated earlier, these markers are closely related to growth velocity and increase during the early stages of puberty. It is difficult to determine the impact of the condition on bone remodeling independent of its effects on longitudinal bone growth and bone modeling. Thus, for example, there are several studies that have examined the impact of glucocorticoids used in the treatment of a chronic condition on bone turnover. A study in children with leukemia treated with dexamethasone or prednisolone demonstrated marked reductions in bone alkaline phosphatase and urine deoxypyridinoline excretion [146]. However such changes were also accompanied by slowing of growth velocity. Similar results have been seen in children with asthma receiving inhaled corticosteroids [147].

FIGURE 15.9 Effect of discontinuation of pamidronate on bone mass and urinary NTX concentrations. Reproduced with permission from Rauch F et al. Pamidronate in children and adolescents with osteogenesis imperfecta: effect of treatment discontinuation. J Clin Endocrinol Metabol 2006;91:2168-74.

One study undertaken in children with Crohn’s disease has attempted to adjust for the confounding effects of growth and pubertal status to determine the independent effects on bone formation and resorption [148]. In a group of healthy controls, pubertal stage, height velocity and whole body bone mineral content at baseline and accrual over the subsequent 6 months were the significant variables that determined levels of bone alkaline phosphatase and urine deoxypyridinoline/creatinine ratios accounting for 77e80% of the variability. Sequential multivariate analysis was then used to examine differences in bone markers in subjects with Crohn’s disease and controls by adjusting for differences in growth, pubertal maturation and bone mass accrual. Adjustment for these factors demonstrated that subjects with Crohn’s disease still had significantly lower levels of bone alkaline phosphatase and higher levels of urine deoxypyridinoline excretion indicating that they had reduced bone formation and increased bone resorption reflecting the effects of glucocorticoid therapy or inflammatory cytokines on osteoblast suppression and promoting osteoclastogenesis. Thus, it is possible to adjust for key variables that determine the level of bone markers in growing children but, clearly, this requires a more sophisticated analysis than has been employed in many pediatric studies. Therefore, to interpret future studies of bone markers in children and adolescents, it is important to know about pubertal status, height velocity, whole body bone mineral content and its speed of accrual. The effect of inhaled steroids on bone development is less obvious than that of systemic steroids. A cross-sectional study of asthmatic children did not detect an effect of inhaled beclomethasone on the bone formation markers bone ALP and osteocalcin [149]. In a more sensitive longitudinal study, PICP and osteocalcin, but not bone ALP, decreased within 1 month of starting treatment with inhaled beclomethasone [150]. One month of treatment with 800 mcg of inhaled budesonide had a similar effect and resulted in significantly decreased levels of PICP, PINP, ICTP, PYD, DPD, and NTX [151]. Even lower dose budesonide (200 mg per day) led to detectable suppression of these collagen markers [147]. 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.

Aromatase Inhibitors The use of drugs that block the synthesis of estrogen by inhibiting the activity of the aromatase enzyme has attracted interest because of the recognized effects of

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estrogen on longitudinal bone growth. The potential inhibition of the effect of estrogen at the growth plate has been considered as a therapeutic maneuver to prolong longitudinal bone growth and improve adult height. Examples of studies undertaken using the aromatase inhibitor letrozole include boys with constitutional delay in growth and puberty [152] and adolescent boys with idiopathic short stature. In this latter group, a 2-year randomized placebo controlled study [153] showed increases in both bone formation and bone resorption markers in the placebo group but, in the letrozole treated group, although there was an initial increase in the bone resorption marker (U-INTP), it subsequently declined with no consistent increases seen in the bone formation markers serum PINP and alkaline phosphatase. Thus, it was felt that prolonged aromatase inhibition had a suppressive effect on both bone formation and resorption. It is currently

375

not clear how such an observation relates to the finding of mild vertebral body abnormalities in the letrozoletreated group [154].

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 [155,156] (Fig. 15.10). The value of other markers is less certain. Because the contribution of the liver isoform to total ALP activity is negligible in children with increased bone turnover, who do not simultaneously suffer from cholestatic liver disease [12], the determination of bone-specific ALP does not offer any advantage in rickets. Osteocalcin levels do not show a consistent pattern in children with vitamin

FIGURE 15.10 Changes in bone marker concentrations 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, n¼14; 10 weeks, n¼10; 12 weeks, n¼14; 14 weeks, n¼1. The rectangular area represents mean 2 SD of controls for each biochemical bone marker: a, P¼not 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 Baroncelli GI et al. Bone turnover in children with vitamin D deficiency rickets before and during treatment. Acta Paediatr. 2000;89:513e8.

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D or calcium deficiency rickets [157e160]. Levels of PICP and ICTP are mildly elevated before treatment [114,155,160].

EhlerseDanlos Syndrome Type VI EhlerseDanlos syndrome type VI is a rare disorder affecting connective tissue in many organs [161] and is caused by a deficiency in lysylehydroxylase, an enzyme involved in the post-translational 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 [31], lack of lysylehydroxylation 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 EhlerseDanlos syndrome type VI [162].

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 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 simply to 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 [116]. 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 non-invasive manner and thus can be determined repeatedly in short time intervals. 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.

Acknowledgments The authors wish to acknowledge Eckhard Scho¨nau and Frank Rauch who gave us permission to use sections of Chapter 14 in the first edition of Pediatric Bone.

References [1] Miller PD. Bone density and markers of bone turnover in predicting fracture risk and how changes in these measures predict fracture risk reduction. Curr Osteoporos Rep 2005;3:103e10. [2] Nishizawa Y, Nakamura T, Ohta H, et al. Guidelines for the use of biochemical markers of bone turnover in osteoporosis. J Bone Miner Metab 2004;2005(23):97e104. [3] Bonnick SL, Shulman L. Monitoring osteoporosis therapy: bone mineral density, bone turnover markers, or both? Am J Med 2006;119(Suppl. 1):S25e31. [4] Hochberg MC, Greenspan S, Wasnich RD, Miller P, Thompson DE, Ross PD. Changes in bone density and turnover explain the reductions in incidence of nonvertebral fractures that occur during treatment with antiresorptive agents. J Clin Endocrinol Metab 2002;87:1586e92. [5] Hannon RA, Eastell R. Bone markers and current laboratory assays. Cancer Treat Rev 2006;32(Suppl. 1):7e14. [6] Baron R. Anatomy and ultrastructure of bone. In: Favus M, editor. Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. Philadelphia: Lippincott-Raven; 1996. p. 3e10.

PEDIATRIC BONE

377

REFERENCES

[7] Frost HM. Skeletal structural adaptations to mechanical usage (SATMU): 1. Redefining Wolff’s law: the bone modeling problem. Anat Rec 1990;226:403e13. [8] Frost HM. Skeletal structural adaptations to mechanical usage (SATMU): 2. Redefining Wolff’s law: the remodeling problem. Anat Rec 1990;226:414e22. [9] Parfitt AM. Bone-forming cells in clinical conditions. In: Hall B, editor. The Osteoblast and Osteocyte. Caldwell, NJ: Telford; 1990. p. 351e429. [10] Heymsfield SB, Arteaga C, McManus C, Smith J, Moffitt S. Measurement of muscle mass in humans: validity of the 24-hour urinary creatinine method. Am J Clin Nutr 1983;7:478e94. [11] Calvo MS, Eyre DR, Gundberg CM. Molecular basis and clinical application of biological markers of bone turnover. Endocr Rev 1996;17:333e68. [12] Rauch F, Middelmann B, Cagnoli M, Keller KM, Schonau E. Comparison of total alkaline phosphatase and three assays for bone-specific alkaline phosphatase in childhood and adolescence. Acta Paediatr 1997;86:583e7. [13] Magnusson P, Larsson L, Magnusson M, Davie MW, Sharp CA. Isoforms of bone alkaline phosphatase: characterization and origin in human trabecular and cortical bone. J Bone Miner Res 1999;14:1926e33. [14] Magnusson P, Sharp CA, Farley JR. Different distributions of human bone alkaline phosphatase isoforms in serum and bone tissue extracts. Clin Chim Acta 2002;325:59e70. [15] Sharp CA, Linder C, Magnusson P. Analysis of human bone alkaline phosphatase isoforms: comparison of isoelectric focusing and ion-exchange high-performance liquid chromatography. Clin Chim Acta 2007;379:105e12. [16] Whyte MP. Physiological role of alkaline phosphatase explored in hypophosphatasia. Ann NY Acad Sci 2010;1192:190e200. [17] Schonau E, Herzog KH, Bohles HJ. Transient hyperphosphatasaemia of infancy. Eur J Pediatr 1988;148:264e6. [18] Masters PW, Jones RG, Purves DA, Cooper EH, Cooney JM. Commercial assays for serum osteocalcin give clinically discordant results. Clin Chem 1994;40:358e63. [19] Matikainen T, Kakonen SM, Pettersson K, et al. Demonstration of the predominant urine osteocalcin fragments detectable by twosite immunoassays. J Bone Miner Res 1999;14:431e8. [20] Saleem U, Mosley Jr TH, Kullo IJ. Serum osteocalcin is associated with measures of insulin resistance, adipokine levels, and the presence of metabolic syndrome. Arterioscler Thromb Vasc Biol 2010;30:1474e8. [21] Bennett CN, Ouyang H, Ma YL, et al. Wnt10b increases postnatal bone formation by enhancing osteoblast differentiation. J Bone Miner Res 2007;22:1924e32. [22] Lieben L, Callewaert F, Bouillon R. Bone and metabolism: a complex crosstalk. Horm Res 2009;71(Suppl. 1):134e8. [23] Pittas AG, Harris SS, Eliades M, Stark P, Dawson-Hughes B. Association between serum osteocalcin and markers of metabolic phenotype. J Clin Endocrinol Metab 2009;94:827e32. [24] Yeap BB, Chubb SA, Flicker L, et al. Reduced serum total osteocalcin is associated with metabolic syndrome in older men via waist circumference, hyperglycemia, and triglyceride levels. Eur J Endocrinol 2010;163:265e72. [25] Smedsrod B, Melkko J, Risteli L, Risteli J. Circulating C-terminal propeptide of type I procollagen is cleared mainly via the mannose receptor in liver endothelial cells. Biochem J 1990;271:345e50. [26] Sorva A, Tahtela R, Risteli J, et al. Familial high serum concentrations of the carboxyl-terminal propeptide of type I procollagen. Clin Chem 1994;40:1591e3. [27] Melkko J, Hellevik T, Risteli L, Risteli J, Smedsrod B. Clearance of NH2-terminal propeptides of types I and III procollagen is

[28]

[29]

[30] [31] [32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

a physiological function of the scavenger receptor in liver endothelial cells. J Exp Med 1994;179:405e12. Melkko J, Niemi S, Risteli L, Risteli J. Radioimmunoassay of the carboxyterminal propeptide of human type I procollagen. Clin Chem 1990;36:1328e32. Melkko J, Kauppila S, Niemi S, et al. Immunoassay for intact amino-terminal propeptide of human type I procollagen. Clin Chem 1996;42:947e54. Yang L, Grey V. Pediatric reference intervals for bone markers. Clin Biochem 2006;39:561e8. Eyre DR, Paz MA, Gallop PM. Cross-linking in collagen and elastin. Annu Rev Biochem 1984;53:717e48. Colwell A, Eastell R. The renal clearance of free and conjugated pyridinium cross-links of collagen. J Bone Miner Res 1996;11:1976e80. Husain SM, Mughal Z, Williams G, et al. Urinary excretion of pyridinium crosslinks in healthy 4e10 year olds. Arch Dis Child 1999;80:370e3. Black D, Duncan A, Robins SP. Quantitative analysis of the pyridinium crosslinks of collagen in urine using ion-paired reversedphase high-performance liquid chromatography. Anal Biochem 1988;169:197e203. Seyedin SM, Kung VT, Daniloff YN, et al. Immunoassay for urinary pyridinoline: the new marker of bone resorption. J Bone Miner Res 1993;8:635e41. Risteli J, Elomaa I, Niemi S, Novamo A, Risteli L. Radioimmunoassay for the pyridinoline cross-linked carboxy-terminal telopeptide of type I collagen: a new serum marker of bone collagen degradation. Clin Chem 1993;39:635e40. Bollen AM, Eyre DR. Bone resorption rates in children monitored by the urinary assay of collagen type I cross-linked peptides. Bone 1994;15:31e4. Bonde M, Qvist P, Fledelius C, Riis BJ, Christiansen C. Immunoassay for quantifying type I collagen degradation products in urine evaluated. Clin Chem 1994;40:2022e5. Clemens JD, Herrick MV, Singer FR, Eyre DR. Evidence that serum NTx (collagen-type I N-telopeptides) can act as an immunochemical marker of bone resorption. Clin Chem 1997;43:2058e63. Rosenquist C, Fledelius C, Christgau S, et al. Serum crosslaps one step ELISA. First application of monoclonal antibodies for measurement in serum of bone-related degradation products from C-terminal telopeptides of type I collagen. Clin Chem 1998;44:2281e9. Mora S, Prinster C, Proverbio MC, et al. Urinary markers of bone turnover in healthy children and adolescents: age-related changes and effect of puberty. Calcif Tissue Int 1998;63:369e74. Rauchenzauner M, Schmid A, Heinz-Erian P, et al. Sex- and agespecific reference curves for serum markers of bone turnover in healthy children from 2 months to 18 years. J Clin Endocrinol Metab 2007;92:443e9. Leigh SD, Ju HS, Lundgard R, Daniloff GY, Liu V. Development of an immunoassay for urinary galactosylhydroxylysine. J Immunol Methods 1998;220:169e78. Halleen J, Hentunen TA, Hellman J, Vaananen HK. Tartrateresistant acid phosphatase from human bone: purification and development of an immunoassay. J Bone Miner Res 1996;11:1444e52. Halleen JM, Alatalo SL, Suominen H, Cheng S, Janckila AJ, Vaananen HK. Tartrate-resistant acid phosphatase 5b: a novel serum marker of bone resorption. J Bone Miner Res 2000;15:1337e45. Chen CJ, Chao TY, Janckila AJ, Cheng SN, Ku CH, Chu DM. Evaluation of the activity of tartrate-resistant acid phosphatase

PEDIATRIC BONE

378

[47]

[48]

[49] [50]

[51]

[52] [53]

[54]

[55]

[56]

[57]

[58]

[59]

[60]

[61]

[62] [63]

15. BIOCHEMICAL MARKERS OF BONE METABOLISM

isoform 5b in normal Chinese children e a novel marker for bone growth. J Pediatr Endocrinol Metab 2005;18:55e62. Robling AG, Niziolek PJ, Baldridge LA, et al. Mechanical stimulation of bone in vivo reduces osteocyte expression of Sost/ sclerostin. J Biol Chem 2008;283:5866e75. Gaudio A, Pennisi P, Bratengeier C, et al. Increased sclerostin serum levels associated with bone formation and resorption markers in patients with immobilization-induced bone loss. J Clin Endocrinol Metab 2010;95:2248e53. Khosla S. Minireview: the OPG/RANKL/RANK system. Endocrinology 2001;142:5050e5. Rogers A, Eastell R. Circulating osteoprotegerin and receptor activator for nuclear factor kappaB ligand: clinical utility in metabolic bone disease assessment. J Clin Endocrinol Metab 2005;90:6323e31. Quercioli A, Montecucco F, Bertolotto M, et al. Coronary artery calcification and cardiovascular risk: the role of RANKL/OPG signalling. Eur J Clin Invest 2010;40:645e54. Heuck C, Skjaerbaek C, Wolthers OD. Diurnal rhythm of serum osteocalcin in normal children. Acta Paediatr 1998;87:930e2. Fujimoto S, Kubo T, Tanaka H, Miura M, Seino Y. Urinary pyridinoline and deoxypyridinoline in healthy children and in children with growth hormone deficiency. J Clin Endocrinol Metab 1995;80:1922e8. Rauch F, Schnabel D, Seibel MJ, et al. Urinary excretion of galactosyl-hydroxylysine is a marker of growth in children. J Clin Endocrinol Metab 1995;80:1295e300. Saggese G, Baroncelli GI, Bertelloni S, Cinquanta L, DiNero G. Twenty-four-hour osteocalcin, carboxyterminal propeptide of type I procollagen, and aminoterminal propeptide of type III procollagen rhythms in normal and growth-retarded children. Pediatr Res 1994;35:409e15. Tobiume H, Kanzaki S, Hida S, et al. Serum bone alkaline phosphatase isoenzyme levels in normal children and children with growth hormone (GH) deficiency: a potential marker for bone formation and response to GH therapy. J Clin Endocrinol Metab 1997;82:2056e61. Marowska J, Kobylinska M, Lukaszkiewicz J, Talajko A, Rymkiewicz-Kluczynska B, Lorenc RS. Pyridinium crosslinks of collagen as a marker of bone resorption rates in children and adolescents: normal values and clinical application. Bone 1996;19:669e77. Shaw NJ, Dutton J, Fraser WD, Smith CS. Urinary pyridinoline and deoxypyridinoline excretion in children. Clin Endocrinol (Oxf) 1995;42:607e12. Tillmann V, Gill MS, Thalange NK, et al. Short-term changes in growth and urinary growth hormone, insulin-like growth factor-I and markers of bone turnover excretion in healthy prepubertal children. Growth Horm IGF Res 2000;10:28e36. Clowes JA, Hannon RA, Yap TS, Hoyle NR, Blumsohn A, Eastell R. Effect of feeding on bone turnover markers and its impact on biological variability of measurements. Bone 2002;30:886e90. Qvist P, Christgau S, Pedersen BJ, Schlemmer A, Christiansen C. Circadian variation in the serum concentration of C-terminal telopeptide of type I collagen (serum CTx): effects of gender, age, menopausal status, posture, daylight, serum cortisol, and fasting. Bone 2002;31:57e61. Crofton PM, Kelnar CJ. Bone and collagen markers in paediatric practice. Int J Clin Pract 1998;52:557e65. de Ridder CM, Delemarre-van de Waal HA. Clinical utility of markers of bone turnover in children and adolescents. Curr Opin Pediatr 1998;10:441e8.

[64] Schonau E, Rauch F. Markers of bone and collagen metabolismproblems and perspectives in paediatrics. Horm Res 1997;48(Suppl. 5):50e9. [65] Szulc P, Seeman E, Delmas PD. Biochemical measurements of bone turnover in children and adolescents. Osteoporos Int 2000;11:281e94. [66] van Coeverden SC, Netelenbos JC, de Ridder CM, Roos JC, Popp-Snijders C, Delemarre-Van de Waal HA. Bone metabolism markers and bone mass in healthy pubertal boys and girls. Clin Endocrinol (Oxf) 2002;57:107e16. [67] Crofton PM, Wade JC, Taylor MR, Holland CV. Serum concentrations of carboxyl-terminal propeptide of type I procollagen, amino-terminal propeptide of type III procollagen, cross-linked carboxyl-terminal telopeptide of type I collagen, and their interrelationships in schoolchildren. Clin Chem 1997;43:1577e81. [68] Crofton PM, Evans N, Taylor MR, Holland CV. Serum crosslaps: pediatric reference intervals from birth to 19 years of age. Clin Chem 2002;48:671e3. [69] Kubo T, Tanaka H, Inoue M, Kanzaki S, Seino Y. Serum levels of carboxyterminal propeptide of type I procollagen and pyridinoline crosslinked telopeptide of type I collagen in normal children and children with growth hormone (GH) deficiency during GH therapy. Bone 1995;17:397e401. [70] Yamaga A, Taga M, Hashimoto S, Ota C. Comparison of bone metabolic markers between maternal and cord blood. Horm Res 1999;51:277e9. [71] Delmas PD, Glorieux FH, Delvin EE, Salle BL, Melki I. Perinatal serum bone Gla-protein and vitamin D metabolites in preterm and fullterm neonates. J Clin Endocrinol Metab 1987;65:588e91. [72] Loughead JL, Mimouni F, Ross R, Tsang RC. Postnatal changes in serum osteocalcin and parathyroid hormone concentrations. J Am Coll Nutr 1990;9:358e62. [73] Namgung R, Tsang RC. Factors affecting newborn bone mineral content: in utero effects on newborn bone mineralization. Proc Nutr Soc 2000;59:55e63. [74] Pittard III WB, Geddes KM, Hulsey TC, Hollis BW. Osteocalcin, skeletal alkaline phosphatase, and bone mineral content in very low birth weight infants: a longitudinal assessment. Pediatr Res 1992;31:181e5. [75] Seki K, Furuya K, Makimura N, Mitsui C, Hirata J, Nagata I. Cord blood levels of calcium-regulating hormones and osteocalcin in premature infants. J Perinat Med 1994;22:189e94. [76] Shima M, Seino Y, Tanaka Y, Yabuuchi H, Tsutsumi C, Moriuchi S. Bone gamma-carboxyglutamic acid containing protein in the perinatal period. Acta Paediatr Scand 1985;74:674e7. [77] Hytinantti T, Rutanen EM, Turpeinen M, Sorva R, Andersson S. Markers of collagen metabolism and insulin-like growth factor binding protein-1 in term infants. Arch Dis Child Fetal Neonatal Ed 2000;83:F17e20. [78] Kajantie E, Hytinantti T, Koistinen R, et al. Markers of type I and type III collagen turnover, insulin-like growth factors, and their binding proteins in cord plasma of small premature infants: relationships with fetal growth, gestational age, preeclampsia, and antenatal glucocorticoid treatment. Pediatr Res 2001;49:481e9. [79] Ogueh O, Khastgir G, Studd J, Jones J, Alaghband-Zadeh J, Johnson MR. Maternal and fetal plasma levels of markers of bone metabolism in gestational diabetic pregnancies. Early Hum Dev 1998;53:155e61. [80] Seibold-Weiger K, Wollmann HA, Ranke MB, Speer CP. Plasma concentrations of the carboxyterminal propeptide of type I procollagen (PICP) in preterm neonates from birth to term. Pediatr Res 2000;48:104e8. [81] Saarela T, Risteli J, Kauppila A, Koivisto M. Type I collagen markers in cord serum of appropriate vs. small for gestational

PEDIATRIC BONE

REFERENCES

[82]

[83]

[84]

[85]

[86]

[87]

[88]

[89]

[90]

[91]

[92] [93]

[94]

[95]

[96] [97] [98]

[99]

[100] [101]

age infants born during the second half of pregnancy. Eur J Clin Invest 2001;31:438e43. Cole DE, Carpenter TO, Gundberg CM. Serum osteocalcin concentrations in children with metabolic bone disease. J Pediatr 1985;106:770e6. Ogueh O, Khastgir G, Studd J, Jones J, Alaghband-Zadeh J, Johnson MR. The relationship of fetal serum markers of bone metabolism to gestational age. Early Hum Dev 1998;51:109e12. Yasumizu T, Kato J. Concentrations of serum markers of type I collagen synthesis and degradation and serum osteocalcin in maternal and umbilical circulation. Endocr J 1996;43:191e5. Ho¨gler W, Schmid A, Raber G, et al. Perinatal bone turnover in term human neonates and the influence of maternal smoking. Pediatr Res 2003;53:817e22. Kuhnert BR, Kuhnert PM, Lazebnik N, Erhard P. The effect of maternal smoking on the relationship between maternal and fetal zinc status and infant birth weight. J Am Coll Nutr 1988;7:309e16. Jauniaux E, Biernaux V, Gerlo E, Gulbis B. Chronic maternal smoking and cord blood amino acid and enzyme levels at term. Obstet Gynecol 2001;97:57e61. Harrast SD, Kalkwarf HJ. Effects of gestational age, maternal diabetes, and intrauterine growth retardation on markers of fetal bone turnover in amniotic fluid. Calcif Tissue Int 1998;62:205e8. Kauppila S, Tekay A, Risteli L, Koivisto M, Risteli J. Type I and type III procollagen propeptides in amniotic fluid of normal pregnancies and in a case of mild osteogenesis imperfecta. Eur J Clin Invest 1998;28:831e7. Nava S, Bocconi L, Zuliani G, Kustermann A, Nicolini U. Aspects of fetal physiology from 18 to 37 weeks’ gestation as assessed by blood sampling. Obstet Gynecol 1996;87:975e80. Ogueh O, Wright EM, Jones J, Alaghband-Zadeh J, Nicolaides KH, Johnson MR. Fetal bone metabolism in normal and rhesus isoimmunised pregnancies. Br J Obstet Gynaecol 2001;108:986e92. Dunn PM. A perinatal growth chart for international reference. Acta Paediatr Scand Suppl 1985;319:180e7. Tongsong T, Pongsatha S. Early prenatal sonographic diagnosis of congenital hypophosphatasia. Ultrasound Obstet Gynecol 2000;15:252e5. Saarela T, Risteli J, Kauppila A, Koivisto M. Effect of short-term antenatal dexamethasone administration on type I collagen synthesis and degradation in preterm infants at birth. Acta Paediatr 2001;90:921e5. Saarela T, Risteli J, Koivisto M. Effects of short-term dexamethasone treatment on collagen synthesis and degradation markers in preterm infants with developing lung disease. Acta Paediatr 2003;92:588e94. Rauch F, Schoenau E. The developing bone: slave or master of its cells and molecules? Pediatr Res 2001;50:309e14. Kruse K. [Perinatal calcium metabolism. Physiology and pathophysiology]. Monatsschr Kinderheilkd 1992;140(Suppl. 1):S1e7. Kovacs CS, Kronenberg HM. Maternal-fetal calcium and bone metabolism during pregnancy, puerperium, and lactation. Endocr Rev 1997;18:832e72. Pratico G, Caltabiano L, Palano GM, Zingale A. [Normal levels of collagen-type-I telopeptide in the first 90 days of life]. Pediatr Med Chir 1998;20:193e5. Largo RH, Wa¨lli R, Duc G, Fanconi A, Prader A. Evaluation of perinatal growth. Helv paediat Acta 1980;35:419e36. Mora S, Prinster C, Bellini A, et al. Bone turnover in neonates: changes of urinary excretion rate of collagen type I cross-linked peptides during the first days of life and influence of gestational age. Bone 1997;20:563e6.

379

[102] Shiff Y, Eliakim A, Shainkin-Kestenbaum R, Arnon S, Lis M, Dolfin T. Measurements of bone turnover markers in premature infants. J Pediatr Endocrinol Metab 2001;14:389e95. [103] Naylor KE, Eastell R, Shattuck KE, Alfrey AC, Klein GL. Bone turnover in preterm infants. Pediatr Res 1999;45:363e6. [104] Younoszai MK, Haworth JC. Excretion of hydroxyproline in urine by premature and normal full-term infants and those with intrauterine growth retardation during the first three days of life. Pediatr Res 1968;2:17e21. [105] Crofton PM, Shrivastava A, Wade JC, et al. Bone and collagen markers in preterm infants: relationship with growth and bone mineral content over the first 10 weeks of life. Pediatr Res 1999;46:581e7. [106] Bhandari V, Fall P, Raisz L, Rowe J. Potential biochemical growth markers in premature infants. Am J Perinatol 1999;16:339e49. [107] Tsukahara H, Watanabe Y, Hirano S, Tsubokura H, Kimura K, Mayumi M. Assessment of bone turnover in term and preterm newborns at birth: measurement of urinary collagen crosslink excretion. Early Hum Dev 1999;53:185e91. [108] Crofton PM, Hume R. Alkaline phosphatase isoenzymes in the plasma of preterm and term infants: serial measurements and clinical correlations. Clin Chem 1987;33:1783e7. [109] Crofton PM. Wheat-germ lectin affinity electrophoresis for alkaline phosphatase isoforms in children: age-dependent reference ranges and changes in liver and bone disease. Clin Chem 1992;38:663e70. [110] Lieuw AF, Sierra RI, Specker BL. Carboxy-terminal propeptide of human type I collagen and pyridinium cross-links as markers of bone growth in infants 1 to 18 months of age. J Bone Miner Res 1995;10:849e53. [111] Lutchman EC, Hardwick TA, Biener R, Chowdhury HA, Trout JR, Shapses SA. Longitudinal study of urinary hydroxy-pyridinium cross-links and growth in healthy infants: higher values with breastfeeding and after daytime sleep. Exp Clin Endocrinol Diabetes 1998;106:51e6. [112] Trivedi P, Risteli J, Risteli L, Hindmarsh PC, Brook CG, Mowat AP. Serum concentrations of the type I and III procollagen propeptides as biochemical markers of growth velocity in healthy infants and children and in children with growth disorders. Pediatr Res 1991;30:276e80. [113] Michaelsen KF, Johansen JS, Samuelson G, Price PA, Christiansen C. Serum bone gamma-carboxyglutamic acid protein in a longitudinal study of infants: lower values in formula-fed infants. Pediatr Res 1992;31:401e5. [114] Saggese G, Bertelloni S, Baroncelli GI, Di Nero G. Serum levels of carboxyterminal propeptide of type I procollagen in healthy children from 1st year of life to adulthood and in metabolic bone diseases. Eur J Pediatr 1992;151:764e8. [115] Rauch F, Schonau E, Woitge H, Remer T, Seibel M. Urinary excretion of hydroxy-pyridinium cross-links of collagen reflects skeletal growth velocity in normal children. Exp Clin Endocrinol 1994;102:94e7. [116] Rauch F, Georg M, Stabrey A, et al. Collagen markers deoxypyridinoline and hydroxylysine glycosides: pediatric reference data and use for growth prediction in growth hormone-deficient children. Clin Chem 2002;48:315e22. [117] Parfitt AM, Travers R, Rauch F, Glorieux FH. Structural and cellular changes during bone growth in healthy children. Bone 2000;27:487e94. [118] Tanner JM, Whitehouse RH. Clinical longitudinal standards for height, weight, height velocity, weight velocity, and stages of puberty. Arch Dis Child 1976;51:170e9. [119] Blumsohn A, Hannon RA, Wrate R, et al. Biochemical markers of bone turnover in girls during puberty. Clin Endocrinol (Oxf) 1994;40:663e70.

PEDIATRIC BONE

380

15. BIOCHEMICAL MARKERS OF BONE METABOLISM

[120] Hertel NT, Stoltenberg M, Juul A, et al. Serum concentrations of type I and III procollagen propeptides in healthy children and girls with central precocious puberty during treatment with gonadotropin-releasing hormone analog and cyproterone acetate. J Clin Endocrinol Metab 1993;76:924e7. [121] Rooney M, Davies UM, Reeve J, Preece M, Ansell BM, Woo PM. Bone mineral content and bone mineral metabolism: changes after growth hormone treatment in juvenile chronic arthritis. J Rheumatol 2000;27:1073e81. [122] Lien G, Selvaag AM, Flato B, et al. A two-year prospective controlled study of bone mass and bone turnover in children with early juvenile idiopathic arthritis. Arthritis Rheum 2005;52:833e40. [123] Meier C, Seibel MJ, Kraenzlin ME. Use of bone turnover markers in the real world: are we there yet? J Bone Miner Res 2009;24:386e8. [124] Heer M, Baecker N, Mika C, Boese A, Gerzer R. Immobilization induces a very rapid increase in osteoclast activity. Acta Astronaut 2005;57:31e6. [125] Zehnder Y, Luthi M, Michel D, et al. Long-term changes in bone metabolism, bone mineral density, quantitative ultrasound parameters, and fracture incidence after spinal cord injury: a cross-sectional observational study in 100 paraplegic men. Osteoporos Int 2004;15:180e9. [126] Zehnder Y, Risi S, Michel D, et al. Prevention of bone loss in paraplegics over 2 years with alendronate. J Bone Miner Res 2004;19:1067e74. [127] Armbrecht G, Belavy DL, Gast U, et al. Resistive vibration exercise attenuates bone and muscle atrophy in 56 days of bed rest: biochemical markers of bone metabolism. Osteoporos Int 2010;21:597e607. [128] Crofton PM, Stirling HF, Schonau E, Kelnar CJ. Bone alkaline phosphatase and collagen markers as early predictors of height velocity response to growth-promoting treatments in short normal children. Clin Endocrinol (Oxf) 1996;44:385e94. [129] Scho¨nau E, Westermann F, Rauch F, et al. A new and accurate prediction model for growth response to growth hormone treatment in children with growth hormone deficiency. Eur J Endocrinol 2001;144:13e20. [130] Land C, Blum WF, Shavrikova E, Kloeckner K, Stabrey A, Schoenau E. Predicting the growth response to growth hormone (GH) treatment in prepubertal and pubertal children with isolated GH deficiency e model validation in an observational setting (GeNeSIS). J Pediatr Endocrinol Metab 2007;20:685e93. [131] Cohen P, Rogol AD, Howard CP, Bright GM, Kappelgaard AM, Rosenfeld RG. Insulin growth factor-based dosing of growth hormone therapy in children: a randomized, controlled study. J Clin Endocrinol Metab 2007;92:2480e6. [132] Bakker B, Frane J, Anhalt H, Lippe B, Rosenfeld RG. Height velocity targets from the national cooperative growth study for first-year growth hormone responses in short children. J Clin Endocrinol Metab 2008;93:352e7. [133] Ranke MB, Lindberg A. Observed and predicted growth responses in prepubertal children with growth disorders: guidance of growth hormone treatment by empirical variables. J Clin Endocrinol Metab 2010;95:1229e37. [134] Buckway CK, Guevara-Aguirre J, Pratt KL, Burren CP, Rosenfeld RG. The IGF-I generation test revisited: a marker of GH sensitivity. J Clin Endocrinol Metab 2001;86:5176e83. [135] Schwarze CP, Wollmann HA, Binder G, Ranke MB. Short-term increments of insulin-like growth factor I (IGF-I) and IGF-binding protein-3 predict the growth response to growth hormone (GH) therapy in GH-sensitive children. Acta Paediatr Suppl 1999;88:200e8.

[136] Scho¨nau E. Growth prediction with biochemical markers and its consequences. Eur J Endocrinol 1997;137:603e4. [137] Lund AM, Molgaard C, Muller J, Skovby F. Bone mineral content and collagen defects in osteogenesis imperfecta. Acta Paediatr 1999;88:1083e8. [138] Rauch F, Travers R, Parfitt AM, Glorieux FH. Static and dynamic bone histomorphometry in children with osteogenesis imperfecta. Bone 2000;26:581e9. [139] Brenner RE, Schiller B, Vetter U, Ittner J, Teller WM. Serum concentrations of procollagen I C-terminal propeptide, osteocalcin and insulin-like growth factor-I in patients with non-lethal osteogenesis imperfecta. Acta Paediatr 1993;82:764e7. [140] Proszynska K, Wieczorek E, Olszaniecka M, Lorenc RS. Collagen peptides in osteogenesis imperfecta, idiopathic juvenile osteoporosis and EhlerseDanlos syndrome. Acta Paediatr 1996;85:688e91. [141] Lund AM, Hansen M, Kollerup G, Juul A, Teisner B, Skovby F. Collagen-derived markers of bone metabolism in osteogenesis imperfecta. Acta Paediatr 1998;87:1131e7. [142] Brenner RE, Vetter U, Bollen AM, Morike M, Eyre DR. Bone resorption assessed by immunoassay of urinary cross-linked collagen peptides in patients with osteogenesis imperfecta. J Bone Miner Res 1994;9:993e7. [143] Glorieux FH, Bishop NJ, Plotkin H, Chabot G, Lanoue G, Travers R. Cyclic administration of pamidronate in children with severe osteogenesis imperfecta. N Engl J Med 1998;339:947e52. [144] Astrom E, Magnusson P, Eksborg S, Soderhall S. Biochemical bone markers in the assessment and pamidronate treatment of children and adolescents with osteogenesis imperfecta. Acta Paediatr 2010. [145] Rauch F, Munns C, Land C, Glorieux FH. Pamidronate in children and adolescents with osteogenesis imperfecta: effect of treatment discontinuation. J Clin Endocrinol Metab 2006;91: 1268e74. [146] Ahmed SF, Tucker P, Mushtaq T, Wallace AM, Williams DM, Hughes IA. Short-term effects on linear growth and bone turnover in children randomized to receive prednisolone or dexamethasone. Clin Endocrinol (Oxf) 2002;57:185e91. [147] Heuck C, Heickendorff L, Wolthers OD. A randomised controlled trial of short term growth and collagen turnover in asthmatics treated with inhaled formoterol and budesonide. Arch Dis Child 2000;83:334e9. [148] Tuchman S, Thayu M, Shults J, Zemel BS, Burnham JM, Leonard MB. Interpretation of biomarkers of bone metabolism in children: impact of growth velocity and body size in healthy children and chronic disease. J Pediatr 2008;153:484e90. [149] Konig P, Hillman L, Cervantes C, et al. Bone metabolism in children with asthma treated with inhaled beclomethasone dipropionate. J Pediatr 1993;122:219e26. [150] Sorva R, Turpeinen M, Juntunen-Backman K, Karonen SL, Sorva A. Effects of inhaled budesonide on serum markers of bone metabolism in children with asthma. J Allergy Clin Immunol 1992;90:808e15. [151] Sorva R, Tahtela R, Turpeinen M, et al. Changes in bone markers in children with asthma during inhaled budesonide and nedocromil treatments. Acta Paediatr 1996;85:1176e80. [152] Wickman S, Kajantie E, Dunkel L. Effects of suppression of estrogen action by the p450 aromatase inhibitor letrozole on bone mineral density and bone turnover in pubertal boys. J Clin Endocrinol Metab 2003;88:3785e93. [153] Hero M, Makitie O, Kroger H, Nousiainen E, Toiviainen-Salo S, Dunkel L. Impact of aromatase inhibitor therapy on bone turnover, cortical bone growth and vertebral morphology in pre- and peripubertal boys with idiopathic short stature. Horm Res 2009;71:290e7.

PEDIATRIC BONE

381

REFERENCES

[154] Hero M, Toiviainen-Salo S, Wickman S, Makitie O, Dunkel L. Vertebral morphology in aromatase inhibitor-treated males with idiopathic short stature or constitutional delay of puberty. J Bone Miner Res 2010;25:1536e43. [155] Baroncelli GI, Bertelloni S, Ceccarelli C, Amato V, Saggese G. Bone turnover in children with vitamin D deficiency rickets before and during treatment. Acta Paediatr 2000;89:513e8. [156] Klein GL. Nutritional rickets and osteomalacia. In: Favus M, editor. Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. New York: Raven Press; 1996. p. 301e5. [157] Greig F, Casas J, Castells S. Changes in plasma osteocalcin concentrations during treatment of rickets. J Pediatr 1989;114:820e3. [158] Oginni LM, Worsfold M, Sharp CA, Oyelami OA, Powell DE, Davie MW. Plasma osteocalcin in healthy Nigerian children and

[159] [160]

[161]

[162]

in children with calcium-deficiency rickets. Calcif Tissue Int 1996;59:424e7. Kruse K, Kracht U. Evaluation of serum osteocalcin as an index of altered bone metabolism. Eur J Pediatr 1986;145:27e33. Scariano JK, Walter EA, Glew RH, et al. Serum levels of the pyridinoline crosslinked carboxyterminal telopeptide of type I collagen (ICTP) and osteocalcin in rachitic children in Nigeria. Clin Biochem 1995;28:541e5. Steinmann B, Royce P, Superti-Furga A. The Ehlers-Danlos syndrome. In: Royce P, Steinmann B, editors. Connective Tissue and its Heritable Disorders: Molecular, Genetic and Medical Aspects. New York: Wiley-Liss; 1993. p. 351e407. Steinmann B, Eyre DR, Shao P. Urinary pyridinoline cross-links in Ehlers-Danlos syndrome type VI. Am J Hum Genet 1995;57:1505e8.

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Pediatric Bone Histomorphometry Frank Rauch Shriners Hospital for Children, Montreal, Quebec, Canada

INTRODUCTION Bone biopsies can be useful for establishing a diagnosis in an individual patient with a bone disorder. They can also be used for investigating disease characteristics or treatment effects. Biopsy samples can be assessed qualitatively or quantitatively. The quantitative analysis of bone specimens is called bone histomorphometry. Bone histomorphometry is a key tool for studying bone tissue. Both the activity of bone metabolism and the amount and distribution of bone tissue can be analyzed with unsurpassed resolution. When tetracycline labeling is performed prior to biopsy, bone histomorphometry offers the unique possibility to study bone cell function in vivo. Importantly for pediatric use, the growth process does not directly interfere with the measurements. Bone histomorphometry is also an excellent educational tool. The insight derived from studying bone tissue can be used better to understand results of indirect methods, such as bone densitometry or biochemical markers of bone metabolism. Knowledge of bone tissue is crucial to put the disparate findings of molecular and cellular studies into perspective. Despite these advantages, bone histomorphometry is underused in pediatrics. This may be partly due to the fact that histomorphometry requires an invasive procedure to obtain a bone sample, is labor intensive, and needs special equipment and expertise. Other reasons may include overestimation of the utility of non-invasive bone diagnostics and lack of information about what bone histomorphometry does. Bone tissue is very hard and for that reason is more difficult to process than soft tissues. In routine pathology, bone tissue is therefore usually decalcified and thus converted into a soft tissue. However, this leads to the loss of important information about bone mineralization and bone cell activity. To assess metabolic

Pediatric Bone, Second Edition DOI: 10.1016/B978-0-12-382040-2.10016-4

bone disorders, it is therefore generally more informative to analyse samples undecalcified. This chapter summarizes the methodology of bone histomorphometry and highlights the tissue-level characteristics of normal and abnormal bone development. The aim is to open this field to the non-specialized reader with an interest in pediatric bone disorders. More detailed accounts of methodology can be found elsewhere [1,2].

BASIC CONCEPTS Between birth and adulthood, bones undergo considerable increases in size. The most frequently assessed aspect of this process is longitudinal bone growth, as reflected by body height. The increase in bone length is mostly due a mechanism called endochondral ossification [3]. The primary effector cells of this process are growth plate chondrocytes. These cells continuously divide and synthesize a cartilaginous matrix which, in a stepwise process, is subsequently converted into osseous tissue. Longitudinal bone growth and endochondral ossification are a traditional focus of interest in pediatric research. However, histomorphometry does not usually deal with endochondral ossification and therefore this process is not described in more detail here. Bone histomorphometry mostly provides information on two other aspects of bone development, bone remodeling and bone modeling. These tissue-based mechanisms of bone development and maintenance have received far less attention in pediatrics than longitudinal bone growth. Bone created by endochondral ossification is continuously renewed by a process named remodeling [4]. Remodeling consists of successive cycles of bone resorption and formation on the same bone surface. The basic features of this process are identical for trabecular and cortical bone [4]. A group of osteoclasts removes a small quantity (“packet”) of bone tissue

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which, after a reversal phase, is replaced by a team of osteoblasts. The entire group of cells involved in this process is named remodeling unit or basic multicellular unit. The fact that osteoblast activity is linked to previous osteoclast action has been named “coupling” [4]. The difference in the amounts of bone which are removed and added in one remodeling cycle is called “remodeling balance”. The remodeling balance is typically close to zero so that there is no or little net effect on the amount of bone. However, the remodeling process renews the bone tissue and thereby prevents tissue damage from accumulating [5]. Bone growth in width occurs through a different mechanism, called modeling [4]. Bone modeling involves the same set of effector cells as bone remodeling, osteoclasts and osteoblasts. However, while in remodeling both cells types are sequentially active on the same bone surface, osteoclasts and osteoblasts act on different surfaces during modeling. During bone growth in width, osteoblasts are typically located on the outer (periosteal) surface of a bone cortex, where they deposit bone matrix and later mineralize it. Thereby, the outer circumference of a long bone or a vertebral body is increased. At the same time, osteoclasts located on the inner (endocortical) surface of the cortex resorb bone, thus increasing the size of the marrow cavity. Since osteoblasts are active without interruption in bone modeling, much more rapid increases in the amount of bone tissue can occur than in bone remodeling. Osteoclasts usually remove less bone tissue than is deposited by osteoblasts during modeling [4]. Therefore, modeling leads to a net increase in the amount of bone tissue. For example, the difference between osteoblastic matrix deposition and osteoclastic bone resorption leads to cortical thickening. Modeling and remodeling are not just abstract concepts of bone metabolism, but are reflected in the histoanatomy of cortical bone.

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. 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. 16.1). This bone is easily accessible, does 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 [6]. It is important to note that horizontal transiliac samples are required for histomorphometric evaluation. Vertical samples (from the iliac crest downwards) cannot be used because of the presence of the growth plate. 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. 16.2). The inner diameter of the trochar should be at least 5 mm. We are using 5 mm needles in children below 12 years of age and 6 mm needles in children 12 years or older, unless their height is below the third percentile. In children and adolescents, the biopsy procedure is usually performed under general anesthesia. This procedure does not have side effects other than transient local discomfort [7]. Patients are allowed to get out of bed after 3 hours and can usually be discharged on the same day. The operator’s experience is an important factor in obtaining an adequate sample and in keeping intervention-related morbidity to a minimum.

METHODOLOGY Bone 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 sites other than the ilium is limited because reference data are only available for the ilium. Quantitative bone histomorphometry requires an intact biopsy specimen of good quality. This implies

FIGURE 16.1 procedure.

PEDIATRIC BONE

Anatomic location for the transiliac bone biopsy

385

METHODOLOGY

FIGURE 16.2

View of a 5-mm trephine for transiliac bone biopsy.

Full histomorphometric analysis requires prior in vivo 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. Figure 16.3 shows the labeling schedule. The tetracycline compound used is tetracycline-HCl (brand names differ from country to country, e.g. TetracyclineÒ; SumycinÒ, AchromycinÒ, and several other brand names) at a dose of 20 mg/kg body weight per day,

FIGURE 16.3 Schedule for prebiopsy tetracycline labeling.

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16. PEDIATRIC BONE HISTOMORPHOMETRY

with a maximum dosage of 1500 mg per day. The daily amount is given orally in two doses. The drug is given for two days in both label courses. The two courses are separated by an interlabel time of 10 days. Bone biopsy is performed 4e6 days after the last administration of tetracycline. 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 Montreal Shriners Hospital, 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 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 hours but should not exceed 10 days because the tetracycline labels are washed out when fixation is too long. Following fixation, the specimen is dehydrated in absolute ethanol and embedded in methylmethacrylate. Cutting mineralized bone requires a special microtome. For each specimen, two to five series of undecalcified, 6e10-mm-thick consecutive sections should be cut at least 150 mm apart. The sections are then deplastified to allow for optimal staining. The most widely used staining methods for histomorphometric analysis are toluidine blue and Masson Goldner trichrome. The sections that will be used for fluorescence microscopy are 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 40e50 mm2. Measurement Procedure The actual histomorphometric analysis requires a high-quality microscope that is suitable for fluorescence

microscopy. In the early years of the technique, 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 identify correctly the individual histoanatomical components.

HISTOMORPHOMETRIC MEASURES Definitions Histomorphometric measures follow a standardized and well-defined terminology that was introduced in 1987 [8]. Articles published before that time 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 [8]. 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. Flat cells of the osteoblast lineage that cover quiescent nonperiosteal bone surfaces are referred to as lining cells. The term osteoclast is restricted to multinucleated 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. 16.4). The terms cancellous and trabecular are usually used interchangeably. The outer delimitation of the cortex is called the periosteal surface, and the inner border is the endocortical surface (Fig. 16.4). 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

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387

HISTOMORPHOMETRIC MEASURES

FIGURE 16.4

Schematic representation of a bone biopsy section. The different types of bone surfaces are indicated.

histologic section in fact reflects a volume. In order to highlight the three-dimensional nature of bone, threedimensional terminology is favored when reporting histomorphometric 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 scheme: source e 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 16.1): 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 of histomorphometry. Structural Parameters The overall size of an intact biopsy specimen is expressed as core width (C.Wi) (Fig. 16.5), which is the

mean distance between the two periosteal surfaces of the sample. C.Wi thus reflects the thickness of the ilium, but also depends on the angle between biopsy needle and ileal surface. Ideally, the needle should be perpendicular to the ilium, but this is not always easy to achieve. 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 two observers may disagree where the border between the two compartments should be 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 (see Fig. 16.5). 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 ¼ 100%  BV/TV). The two surface-to-volume ratios (BS/BV and BS/TV) are important for establishing the link between bone surfacebased 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 surfacerelated activity of bone cells is quite similar [9]. 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) or trabecular

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388 TABLE 16.1

16. PEDIATRIC BONE HISTOMORPHOMETRY

The Most Commonly used Histomorphometric Parameters

Parameter

Abbreviation

Structural parameters Core width

C.Wi

Cortical width

Ct.Wi

Cortical porosity

Ct.Po

Bone volume/tissue volume

BV/TV

Trabecular thickness

Tb.Th

Trabecular number

Tb.N

Trabecular separation

Tb.Sp

Static formation parameters

FIGURE 16.5 Basic histomorphometric parameters of bone

Osteoid thickness

O.Th

Osteoid surface/bone surface

OS/BS

Osteoid volume/bone volume

OV/BV

Osteoblast surface/bone surface

Ob.S/BS

Osteoblast surface/osteoid surface

Ob.S/OS

Wall thickness

W.Th

structure.

from a single two-dimensional section. In any case, analysis of trabecular bone architecture has not been applied to pediatric histomorphometry. Static Formation Parameters

Dynamic formation parameters Mineralizing surface/bone surface

MS/BS

Mineralizing surface/osteoid surface

MS/OS

Mineral apposition rate

MAR

Adjusted apposition rate

Aj.AR

Mineralization lag time

Mlt

Osteoid maturation time

Omt

Bone formation rate/bone surface

BFR/BS

Bone formation rate/bone volume

BFR/BV

Activation frequency

Ac.f

Formation period

FP

Static resorption parameters Eroded surface/bone surface

ES/BS

Osteoclast surface/bone surface

Oc.S/BS

Number of osteoclasts/bone perimeter

N.Oc/B.Pm

separation, can be mathematically derived from Tb.Th and Tb.N. This parameter is often included in histomorphometric reports, although it does not provide any additional information once Tb.Th and Tb.N are known. Apart from these classic structural parameters, a set of indices has been developed to describe quantitatively the architecture of trabecular bone [10]. These approaches are limited by the fact that the three-dimensional architecture of a bone cannot be reconstructed

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. 16.6). Osteoid thickness (O.Th) corresponds to the mean distance between the surface of the osteoid seam facing the bone marrow on the one hand and the interface between unmineralized and mineralized bone on the other (Fig. 16.6). This interface is called the mineralization front, because mineral is rapidly incorporated into the organic bone matrix at that location. Osteoid seams do not end abruptly, so some minimum width must be specified. According to the procedures established at the Montreal Shriners Hospital, osteoid seams above 1.5 mm are included in the analysis [6]. 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 continuously flatter and those that remain on the bone surface turn into lining cells. Thus, there is a continuum between flat osteoblasts and lining cells, and it is necessary to indicate the criteria used to distinguish between the two cell types. We use a definition of osteoblasts as cells that are directly apposed to osteoid and exhibiting a definite Golgi apparatus. Osteoblast surface per osteoid surface (Ob.S/OS) is the percentage of osteoid surface that is covered by

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HISTOMORPHOMETRIC MEASURES

389

FIGURE 16.6 Schematic representation of a remodeling site in trabecular bone. Osteoclasts in the front dig a trench across the bone surface, which is then refilled by a team of osteoblasts.

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) (see Fig. 16.6). W.Th should not be confused with cortical thickness, with which it 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 (see Fig. 16.6), 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 [11]. 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 [8]. This is usually easily visible under polarized light. 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 at the tissue level. This underscores the importance of performing tetracycline labeling prior to biopsy. 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 [8]. 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/OS. 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 [12] 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 [13]. 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 [9]. It is often mistakenly assumed

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16. PEDIATRIC BONE HISTOMORPHOMETRY

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 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 at the boneebone 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” [14]. In addition, many of the locations that are classified as presenting an eroded aspect are the result of aborted resorption attempts rather than of remodeling-linked osteoclast action [14]. 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 osteoclastebone 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 [8]. 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. Osteoclast surface and number are more useful indicators of bone resorption than eroded surface, because their interpretation is less ambiguous. However, these are still imperfect indicators of bone resorptive activity, as they reflect only the amount of osteoclasts, but not their function. Thus, bone histomorphometry provides far less information on bone resorption than it does on bone formation. Reproducibility of Histomorphometric Measures Only one study has evaluated the reproducibility of bone histomorphometric measures in children and adolescents [6]. Structural parameters showed a variability

of 5e10%, whereas variations were highest for cellular parameters (20e30%). Reproducibility was best for two primary parameters of osteoblast team function e mineral apposition rate and wall thickness (4 and 5%, respectively). The variability of repeated measurements was smaller in children than in adults [15,16], which may be explained by the higher bone turnover in children. This effect reduces the sampling error for parameters of bone formation and resorption.

PEDIATRIC BONE HISTOMORPHOMETRY IN HEALTH AND DISEASE Reference Data for Pediatric Iliac Bone Histomorphometry Reference data have been published based on results from 58 individuals between 1.5 and 22.9 years of age who underwent surgery for reasons independent of abnormalities in bone development and metabolism [6]. The results are shown in Table 16.2. 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. Some parameters are more likely to vary with methodology than others. 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. According to the protocol used in the reference data study, all osteoid seams with a width above 1.5 mm are measured. If a higher cutoff is used, then higher results for osteoid thickness and lower values for osteoid surface extent will be obtained. Wall thickness depends on the type of staining. Procedures are available to stain specifically the cement line [17]. Wall thickness can then be simply measured as the distance between the cement line and the nearest bone surface. In the reference data study, wall thickness was quantified on Goldner stained sections under polarized light as the distance from quiescent bone surfaces to the abrupt change in collagen fiber orientation [18]. This method tends to yield higher values than when staining of the cement line is performed.

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TABLE 16.2

Reference Data (Mean  SD) for Iliac Bone Histomorphometry from 1.5 to 23 Years

Age (years)

1.5e6.9

7.0e10.9

11.0e13.9

14.0e16.9

17.0e22.9

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 (mm)

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 (mm)

481  112

453  62

464  78

461  70

404  77

O.Th (mm)

5.8  1.4

5.9  1.1

6.7  1.7

6.3  1.0

6.9  1.2

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 (mm)

33.9  3.8

40.6  3.0

45.1  6.9

44.4  3.2

41.1  2.5

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 (mm/d)

1.04  0.17

0.95  0.07

0.87  0.09

0.81  0.09

0.75  0.09

Aj.AR (mm/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

5.7  1.3

6.5  1.0

7.6  1.8

7.6  1.2

9.4  2.3

BFR/BS (mm /mm /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

Structural

Static formation

Dynamic formation

Omt (d) 3

2

Static resorption

Cellular parameters depend on the degree of cellular preservation, which is influenced by sample fixation and staining technique. In the reference data study, biopsy specimens were fixed in 10% phosphate-buffered formalin (pH 7.1) and kept at room temperature for 48e72 hours. They were then dehydrated in increasing concentrations of ethanol, cleared with xylene and embedded in methylmethacrylate. After sectioning, samples were deplastified with ethylene glycol monoethyl acetate to allow for optimal staining. Measurements of cells were performed using toluidine stained sections.

Mineralizing surface and mineral apposition rate vary with the labeling substance, labeling schedule, and dosage used [19]. The labeling schedule in use at the Shriners Hospital for Children in Montreal is shown in Figure 16.3. As mentioned earlier, eroded surface is a subjective measure [14]. In the reference data study, eroded surface was measured in toluidine-stained sections. Bone surfaces with scalloped or ragged appearance of the boneebone marrow interface are included, whether or not osteoclasts are present. This includes also shallow

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16. PEDIATRIC BONE HISTOMORPHOMETRY

excavations, which are identified by the presence of eroded lamellae at the bone surface.

Development of Human Bone Tissue A histomorphometric study on the proximal femoral metaphysis of 35 fetuses and newborns showed that trabecular bone volume increased between the second and third trimester of pregnancy [20]. It appears that bone mineralizes far more quickly during fetal development than in postnatal life. In addition, trabeculae of fetal bone thicken at a speed that is far more rapid than would be compatible with remodeling. It thus appears that modeling, not remodeling, is the predominant mechanism responsible for the development of femoral metaphyseal cancellous bone in utero. Between 2 and 20 years of age, the trabecular bone volume of iliac bone increases markedly [6]. 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 5% of the total amount of bone turned over [18]. 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 are very high in 2e5-year-old children, decrease until approximately age 8 or 9 years, and then increase again to a pubertal peak [18]. After the age of puberty, the values decline to the low adult ranges. Interestingly, wall thickness increases with age, whereas mineral apposition rate decreases 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, iliac bone development is characterized by an outward modeling drift [18,21]. Both cortices move through tissue space in parallel by reciprocal activities of their surfaces [21]. 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 [18,21]. 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 thickening during the growth period. This mode of development also implies that a large proportion of iliac trabeculae are 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. Cortical bone undergoes intracortical remodeling. When intracortical remodeling activity is elevated, cortical porosity is high, because a higher proportion of osteons are “under construction” at any given time point and therefore still have a larger osteonal canal than mature osteons. Intracortical remodeling activity decreases after 14 years of age. Consequently, cortical porosity decreases after that age [21]. Similar to what is observed on the endocortical and periosteal surfaces of the internal and the external iliac cortex, metabolic activity also differs on the intracortical surfaces of the two cortices [21]. Intracortical remodeling activity is higher on the osteonal surfaces of the internal cortex than of the external cortex [21]. Even within the same osteon, bone formation activity favors the side of the osteonal canal that faces towards the periosteum [22]. The general features of iliac bone development are similar between black and white children [23]. However, it appears that during pubertal development cortical thickness increases more in black than in white individuals [23]. This difference in cortical thickness persists into adulthood and may explain why blacks have fewer fractures than whites [24]. The mode of iliac 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. Similarly, the cells residing within the same osteonal canal are exposed to the same environment of soluble factors, but bone formation still is predominantly observed on the surface facing the periosteum. These observations are more in line with the idea that bone modeling and remodeling are governed by signals from osteocytes that are transmitted to the surfaces via the canalicular network [25].

Disorders of Bone Mineralization In the growing skeleton, mineralization occurs in two different types of tissue e 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 by evaluating signs and symptoms of the typical growth plate defects. Osteomalacia is also associated with clinical, radiological and biochemical abnormalities, but is more precisely evaluated in iliac bone specimens. The physiological process of mineralization represents the incorporation of mineral (calcium, phosphorus,

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and others) into organic bone matrix after it has been synthesized and deposited by osteoblasts [12,26]. 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) [26]. This leads to an accumulation of unmineralized bone matrix because osteoblasts continue to secrete osteoid for some time. The aspect of tetracycline labels reflects the severity of the mineralization defect. In a mild mineralization disorder, the proportion of tetracycline-labeled osteoid is decreased and the distance between 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 mm 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 [6], the cutoff values for osteoid thickness and mineralization lag time would be calculated as approximately 9 mm and 25 days, respectively [27]. 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 Mineralization In calcipenic forms of rickets and osteomalacia, hyperparathyroidism occurs in addition to the mineralization

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defect. This secondary hyperparathyroidism leads to increased bone turnover and is characterized by 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. Paratrabecular fibrosis can be seen in severe forms of hyperparathyroid bone disease. 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 improvement. 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 low-calcium, 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 Mineralization 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 [33e36]. 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.

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Cancellous bone volume is high in most patients, but this includes a large amount of unmineralized matrix. The mineralized fraction of cancellous bone is typically at the mean for age [35]. X-linked hypophosphatemic rickets can be distinguished histologically from most other causes of osteomalacia because osteocytes are surrounded by a halo of unmineralized bone [37]. 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 in general, but hypomineralized periosteocytic lesions do not occur [40]. In tumor-induced osteomalacia, trabecular bone surfaces are almost completely covered with thick layers of osteoid, and osteoblast activity is low. In one case, normalized osteoid thickness and vigorous bone formation activity were found 5 months after removal of the underlying tumor [41]. In a case of autosomal recessive hypophosphatemic rickets caused by a DMP1 mutation, excessive osteoid was present not only on trabecular surfaces and osteons but also surrounding osteocyte lacunae [42]. The histological picture is indistinguishable from that created by X-linked hypophosphatemic rickets.

Osteopenic Disorders Osteogenesis Imperfecta Types I, III, and IV Osteogenesis imperfecta (OI) is a heritable bone fragility disorder which, in the majority of cases, is caused by mutations in COL1A1 or COL1A2, the genes that encode the two collagen type I alpha chains, alpha 1 and alpha 2 (see Chapter 18). Osteogenesis imperfecta has been classically divided into four clinical types [43]. 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. In a study on 70 children with OI types I, III, and IV between 1.5 and 13.5 years of age, the external size of the biopsy core did not increase with age, and cortical width was generally markedly below normal. Because external bone size and cortical width during growth

are determined by modeling processes [18], 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. 16.7). Low trabecular number can result from either increased loss or decreased production of trabeculae. There was no evidence that children with OI lose secondary trabeculae because trabecular number remained constant with age (Fig. 16.7). 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. Inadequate trabecular thickening in OI is caused by a defect in bone remodeling. In the control group, each remodeling cycle added 2.8 mm more bone than it resorbed. In OI type I, the positive balance was only 1.1 mm 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. 16.8). 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. 16.8). The cause of increased recruitment 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. There are two general classes of mutations in type I collagen that result in OI. The first are mutations that cause a failure to synthesize the products of one COL1A1 allele and thus lead to haploinsufficiency [44]. The second class of mutations are those that result in the synthesis of collagen molecules with structural abnormalities. This is most frequently caused by the substitution of glycine by another amino acid in the triple helical domain of either the alpha 1 or the alpha 2 chain [45]. The relationship between genotype and histomorphometric phenotype in OI types I, III and IV was examined in a study on 96 patients [46].

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Core width (outer bone size) and trabecular bone volume were similar between patients with haploinsufficiency mutations and those with helical glycine mutations, but cortical width was about 50% higher in the haploinsufficiency group. Osteoid surface, osteoblast surface, bone formation rate, eroded surface and osteoclast number varied significantly with genotype and for each parameter were lowest in the haploinsufficiency group. This suggests that bone mass differences between these genotypic groups are mainly caused by differences of bone modeling on periosteal and endocortical bone surfaces and less by differences in trabecular bone metabolism. For helical mutations, there was no obvious relationship between the type of substituting amino acid or the position of the mutation and histomorphometric parameters. The data also suggest that the classification of OI according to phenotypic types provides a better reflection of histological disease severity than the grouping of patients according to mutation type. OI Types Identified by Bone Histology and Histomorphometry: OI Types V and VI

FIGURE 16.7 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.

Many OI patients present unusual clinical features. One of these is hyperplastic callus formation, which can appear spontaneously, following fracture, or with intramedullary rodding. 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. 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 [47]. 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

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FIGURE 16.8

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Bone formation abnormalities in OI on three levels of biological organization.

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 [48]. 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. Histological Phenocopy of OI Type I without Collagen Mutation: OI type VII OI type VII is an autosomal recessive form of OI that is caused by mutations in the CRTAP gene [49]. 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 [50]. 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 surfacebased 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

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report shows that there are characteristic differences between the two disorders at the tissue level [51]. 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. 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 [52]. Both structural 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 Syndromes 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 tissuelevel characteristics of a disease would be helpful. However, only a few reports have included histomorphometric data on skeletal dysplasias. In Camurati Engelman disease, cortical width of long-bone diaphyses is increased. Iliac bone histomorphometry in a 9-year-old boy with this disorder showed a low amount of trabecular bone [53]. In

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osteopathia striata with cranial sclerosis, an increased trabecular bone volume per tissue volume but normal bone metabolism was found in a 14-year-old girl [54]. In idiopathic hyperphosphatasia, bone turnover is markedly increased and can be slowed down with intravenous pamidronate [55]. A novel type of bone fragility disorder was identified in three children based on bone histological appearance [56]. Apart from recurrent lower limb fractures these patients had a paucity of the birefringent pattern of normal lamellar bone in their iliac bone specimens. Quantitative histomorphometric analysis demonstrated osteomalacia with a prolonged mineralization lag time in the presence of a decreased mineral apposition rate. The pathogenetic basis for this disorder remains to be elucidated. Fibrous Dysplasia An analysis of 27 iliac bone samples from children with fibrous dysplasia showed that, in dysplastic lesions, trabeculae were clearly thinner and increased in number [27]. Osteoid indices, osteoblast surface per bone surface, and mineralization lag time were elevated in dysplastic areas. Patients with fibrous dysplasia often have slightly low serum phosphorus concentrations, but there was no detectable effect of serum phosphorus levels and histomorphometric indices in dysplastic bone areas. In non-dysplastic bone tissue, low serum phosphorus levels were associated with mildly increased osteoid thickness and prolonged mineralization lag time. It was concluded that the mild systemic mineralization defect of fibrous dysplasia warrants treatment with oral phosphorus supplementation if signs of rickets are absent. Many patients with fibrous dysplasia are treated with intravenous bisphosphonates [57]. However, a histomorphometric study in seven patients who had received pamidronate for a mean of 2.2 years did not find any discernible effect of the treatment on the dysplastic bone tissue [58]. Osteopetrosis Bone histology in autosomal recessive infantile osteopetrosis is characterized by a large amount of calcified cartilage and usually an abnormally high number of osteoclasts [59e61]. However, in a few patients, osteoclasts are absent, presumably depending on the underlying genetic defect [61]. The histological abnormalities of autosomal dominant osteopetrosis are relatively mild [62]. Cortical width and cancellous bone volume are only slightly increased, and bone formation and resorption indices show only subtle deviations from normal.

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Secondary 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 (see Chapter 29). Secondary Osteopenic Disorders in Children and Adolescents ACUTE LYMPHOBLASTIC LEUKEMIA

Children with newly diagnosed untreated acute lymphoblastic leukemia can have low bone density and vertebral compression fractures [63]. One study assessed iliac bone biopsy samples in 23 such patients [64]. In patients below 10 years of age, the amount of trabecular bone was low, whereas trabecular bone volume was normal in older children. There was evidence for low bone remodeling activity but no mineralization defect was found. INFLAMMATORY BOWEL DISEASE

Transiliac biopsies were taken at the time of diagnostic endoscopy in 20 patients (age: 14.2  2.7 years) with newly diagnosed inflammatory bowel disease [65]. Bone histomorphometry revealed low cortical width and low metabolic bone activity, with preservation of trabecular bone volume and absence of osteomalacia. BETA THALASSEMIA

In a histomorphometric study on 17 children and adolescents with suboptimally treated beta thalassemia disease, a normal amount of trabecular bone was found [66]. However, osteoid thickness was markedly increased and iron deposits appeared along mineralization fronts and osteoid surfaces. It was concluded that iron overload led to focal osteomalacia, and thus contributed to the bone disease in these patients. BURN INJURIES

Bone mass can be low in children following severe burns. Histomorphometric studies have shown severely suppressed bone formation activity in the weeks following a burn injury [67e69]. There was no indication of increased bone resorption. The overall histomorphometric picture did not change when children with

burn injuries were treated with pamidronate [69,70], but this treatment nevertheless was associated with a preservation of bone mass.

Use of Histomorphometry in Treatment Studies Effects of Bisphosphonate Treatment in OI In children with OI, the effect of bisphosphonates on bone tissue has been studied in some detail. In a study on 45 children and adolescents with OI type I, III and IV (age 1.4e17.5 years), iliac bone biopsies were obtained at the start of intravenous pamidronate treatment as well as after 2 to 4 years (mean 2.4 years) of therapy [71]. The main bone mass relevant change during the treatment period was an increase in cortical thickness of 88%. The amount of trabecular bone increased by an average of 46%. This was due to a higher trabecular number, whereas trabecular thickness remained stable. Bone surface-based indicators of cancellous bone remodeling decreased by 26e75%. There was no evidence for a mineralization defect in any of the patients. These results suggested that, in growing children with OI, pamidronate had a twofold effect. With regard to remodeling, bone resorption and formation are coupled and consequently both processes were inhibited. However, during modeling of cortical bone, osteoclasts and osteoblasts are active on different surfaces and are thus uncoupled. Therefore, resorption was selectively targeted by pamidronate, and continuing bone formation could increase cortical width. Similar results were found in a study on 24 young children with OI who had all received pamidronate treatment since the first or second year of life [72]. Their histomorphometric results after 3 years of pamidronate treatment were compared to those of a historical control group of untreated children with severe OI who were matched for OI type and age. Iliac bone histomorphometry showed 61% higher cortical width and 89% higher cancellous bone volume in pamidronate-treated patients. Bone formation rate per bone surface in the pamidronate group was only 17% that of untreated patients. Thus, pamidronate treatment started in infancy leads to a marked increase in the amounts of both cortical and trabecular bone, but also suppressed bone turnover markedly. The bone-tissue effects of pamidronate in the newer types of OI were largely similar to those found in “classical” OI types. In seven children with OI type V, 2 years of pamidronate treatment were associated with an average increase of 86% in cortical thickness [73]. Cortical thickness also increased (þ53%) after 3 years of intravenous pamidronate in children and adolescents with OI type VI [74]. However, the mineralization defect which is the characteristic feature of OI type VI, did not change during pamidronate treatment. In OI type VII, cortical

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REFERENCES

width increased with pamidronate treatment, but trabecular bone volume did not change significantly [75]. The longer-term effects of pamidronate treatment on the bone tissue were evaluated in a longitudinal study of 25 children and adolescents with OI who had received intravenous pamidronate for >4 years [76]. Iliac bone biopsies were performed at treatment start, after 2.70.5 years (meanSD), and after 5.50.7 years of therapy. Mean cortical width and cancellous bone volume increased by 87% and 38%, respectively, between baseline and the first time point during treatment. Thereafter, cortical width did not change significantly, but there was a trend towards higher cancellous bone volume. Average bone formation rate on trabecular surfaces decreased by 70% after pamidronate treatment was initiated, and showed a trend towards a further decline in the second part of the study interval. These results indicate that the gains that can be achieved with pamidronate treatment appear to be largely realized in the first 2e4 years. Little is known about the bone tissue-level effects of bisphosphonate compounds other than pamidronate. One study examined the efficacy and safety of oral risedronate in the treatment of pediatric patients with mild OI [77]. Iliac bone biopsies were performed at the end of the 2-year study period. Histomorphometric analysis of these transiliac bone biopsies did not show a significant treatment difference in cortical width, trabecular bone volume, or parameters of bone turnover. These results suggest that the skeletal effects of oral risedronate are weaker than those that are commonly observed with intravenous pamidronate treatment. Side effects of Bisphosphonate Treatment One of the radiological features of intravenous bisphosphonate treatment in growing children is the appearance of transverse lines in the metaphyses of long bones. These lines were examined in the case of a child with OI type VII, where the iliac bone biopsy had inadvertently included part of the iliac growth plate and the adjacent metaphysis [78]. It was seen that these metaphyseal lines corresponded to horizontal trabeculae that were undergoing active remodeling, rather than “frozen growth plate cartilage” as had been hypothesized before. Changes in the appearance of osteoclasts have previously been noted in children receiving pamidronate and have been interpreted as signs of toxicity [71,79]. A study analyzed osteoclast parameters in paired iliac bone specimens before and after 2e4 years of cyclical intravenous pamidronate therapy in 44 pediatric OI patients and found that intravenous pamidronate of young OI patients leads to an increase in osteoclast size [80]. However, the presence of large osteoclasts was not associated with detectable untoward clinical effects. The presence of particularly large osteoclasts

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may indicate increased responsiveness to the action of pamidronate.

INDICATIONS FOR BONE BIOPSY AND HISTOMORPHOMETRY IN PEDIATRIC BONE DISORDERS 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 diagnose and treat correctly 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 presence of hypomineralized periosteocytic lesions. Bone histology is also 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 non-invasive methods for studying the amount, distribution, and metabolism of bone are fraught with technical limitations and uncertainties regarding the interpretation of results. The availability of histomorphometric 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.

References [1] Eriksen EF, Axelrod DW, Melsen F. Bone Histomorphometry. New York: Raven Press; 1994. [2] Recker R. Bone Histomorphometry: Techniques and Interpretation. Boca Raton: CRC Press; 1983.

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[3] Rauch F. Bone growth in length and width: the yin and yang of bone stability. J Musculoskelet Neuronal Interact 2005;5:194e201. [4] Rauch F. Watching bone cells at work: what we can see from bone biopsies. Pediatr Nephrol 2006;21:457e62. [5] Allen MR, Burr DB. Bisphosphonate effects on bone turnover, microdamage, and mechanical properties: What we think we know and what we know that we don’t know. Bone 2010. [6] Glorieux FH, Travers R, Taylor A, et al. Normative data for iliac bone histomorphometry in growing children. Bone 2000;26:103e9. [7] Hernandez JD, Wesseling K, Pereira R, Gales B, Harrison R, Salusky IB. Technical approach to iliac crest biopsy. Clin J Am Soc Nephrol 2008;3(Suppl. 3):S164e9. [8] Parfitt AM, Drezner MK, Glorieux FH, et al. Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res 1987;2:595e610. [9] Parfitt AM. Misconceptions (2): turnover is always higher in cancellous than in cortical bone. Bone 2002;30:807e9. [10] Compston JE. Connectivity of cancellous bone: assessment and mechanical implications [editorial]. Bone 1994;15:463e6. [11] Parfitt AM. Osteonal and hemi-osteonal remodeling: the spatial and temporal framework for signal traffic in adult human bone. J Cell Biochem 1994;55:273e86. [12] Parfitt AM. Renal bone disease: a new conceptual framework for the interpretation of bone histomorphometry. Curr Opin Nephrol Hypertens 2003;12:387e403. [13] Parfitt AM, Simon LS, Villanueva AR, Krane SM. Procollagen type I carboxy-terminal extension peptide in serum as a marker of collagen biosynthesis in bone. Correlation with Iliac bone formation rates and comparison with total alkaline phosphatase. J Bone Miner Res 1987;2:427e36. [14] Parfitt AM. Morphometry of bone resorption: introduction and overview. Bone 1993;14:435e41. [15] Chavassieux PM, Arlot ME, Meunier PJ. Intersample variation in bone histomorphometry: comparison between parameter values measured on two contiguous transiliac bone biopsies. Calcif Tissue Int 1985;37:345e50. [16] de Vernejoul MC, Kuntz D, Miravet L, Goutallier D, Ryckewaert A. Bone histomorphometric reproducibility in normal patients. Calcif Tissue Int 1981;33:369e74. [17] Bain SD, Impeduglia TM, Rubin CT. Cement line staining in undecalcified thin sections of cortical bone. Stain Technol 1990;65:159e63. [18] Parfitt AM, Travers R, Rauch F, Glorieux FH. Structural and cellular changes during bone growth in healthy children. Bone 2000;27:487e94. [19] Parfitt AM, Foldes J, Villanueva AR, Shih MS. Difference in label length between demethylchlortetracycline and oxytetracycline: implications for the interpretation of bone histomorphometric data. Calcif Tissue Int 1991;48:74e7. [20] Salle BL, Rauch F, Travers R, Bouvier R, Glorieux FH. Human fetal bone development: histomorphometric evaluation of the proximal femoral metaphysis. Bone 2002;30:823e8. [21] Rauch F, Travers R, Glorieux FH. Cellular activity on the seven surfaces of iliac bone: a histomorphometric study in children and adolescents. J Bone Miner Res 2006;21:513e9. [22] Rauch F, Travers R, Glorieux FH. Intracortical remodeling during human bone development e A histomorphometric study. Bone 2007;40:274e80. [23] Schnitzler CM, Mesquita JM, Pettifor JM. Cortical bone development in black and white South African children: iliac crest histomorphometry. Bone 2009;44:603e11.

[24] Schnitzler CM, Mesquita JM. Cortical bone histomorphometry of the iliac crest in normal black and white South African adults. Calcif Tissue Int 2006;79:373e82. [25] Hughes JM, Petit MA. Biological underpinnings of Frost’s mechanostat thresholds: the important role of osteocytes. J Musculoskelet Neuronal Interact 2010;10:128e35. [26] Rauch F. The rachitic bone. Endocr Dev 2003;6:69e79. [27] Terpstra L, Rauch F, Plotkin H, Travers R, Glorieux FH. Bone mineralization in polyostotic fibrous dysplasia: histomorphometric analysis. J Bone Miner Res 2002;17:1949e53. [28] Marie PJ, Pettifor JM, Ross FP, Glorieux FH. Histological osteomalacia due to dietary calcium deficiency in children. N Engl J Med 1982;307:584e8. [29] Schnitzler CM, Pettifor JM, Patel D, Mesquita JM, Moodley GP, Zachen D. Metabolic bone disease in black teenagers with genu valgum or varum without radiologic rickets: a bone histomorphometric study. J Bone Miner Res 1994;9:479e86. [30] Pettifor JM, Marie PJ, Sly MR, et al. The effect of differing dietary calcium and phosphorus contents on mineral metabolism and bone histomorphometry in young vitamin D-replete baboons. Calcif Tissue Int 1984;36:668e76. [31] Pettifor JM, Schnitzler CM, Ross FP, Moodley GP. Endemic skeletal fluorosis in children: hypocalcemia and the presence of renal resistance to parathyroid hormone. Bone Miner 1989;7:275e88. [32] Teotia M, Teotia SP, Singh KP. Endemic chronic fluoride toxicity and dietary calcium deficiency interaction syndromes of metabolic bone disease and deformities in India: year 2000. Indian J Pediatr 1998;65:371e81. [33] Glorieux FH, Marie PJ, Pettifor JM, Delvin EE. Bone response to phosphate salts, ergocalciferol, and calcitriol in hypophosphatemic vitamin D-resistant rickets. N Engl J Med 1980;303:1023e31. [34] Marie PJ, Glorieux FH. Histomorphometric study of bone remodeling in hypophosphatemic vitamin D-resistant rickets. Metab Bone Dis Relat Res 1981;3:31e8. [35] Marie PJ, Glorieux FH. Stimulation of cortical bone mineralization and remodeling by phosphate and 1,25dihydroxyvitamin D in vitamin D-resistant rickets. Metab Bone Dis Relat Res 1981;3:159e64. [36] Marie PJ, Glorieux FH. Bone histomorphometry in asymptomatic adults with hereditary hypophosphatemic vitamin Dresistant osteomalacia. Metab Bone Dis Relat Res 1982;4:249e53. [37] Frost HM. Some observations on bone mineral in a case of vitamin D-resistant rickets. Henry Ford Hosp Med Bull 1958;6:300e10. [38] Harrell RM, Lyles KW, Harrelson JM, Friedman NE, Drezner MK. Healing of bone disease in X-linked hypophosphatemic rickets/osteomalacia. Induction and maintenance with phosphorus and calcitriol. J Clin Invest 1985;75:1858e68. [39] Marie PJ, Glorieux FH. Relation between hypomineralized periosteocytic lesions and bone mineralization in vitamin Dresistant rickets. Calcif Tissue Int 1983;35:443e8. [40] Gazit D, Tieder M, Liberman UA, Passi-Even L, Bab IA. Osteomalacia in hereditary hypophosphatemic rickets with hypercalciuria: a correlative clinical-histomorphometric study. J Clin Endocrinol Metab 1991;72:229e35. [41] Ward LM, Rauch F, White KE, et al. Resolution of severe, adolescent-onset hypophosphatemic rickets following resection of an FGF-23-producing tumour of the distal ulna. Bone 2004;34:905e11. [42] Feng JQ, Ward LM, Liu S, et al. Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism. Nat Genet 2006;38:1310e5.

PEDIATRIC BONE

REFERENCES

[43] Rauch F, Glorieux FH. Osteogenesis imperfecta. Lancet 2004;363:1377e85. [44] Byers PH. Collagens: building blocks at the end of the development line. Clin Genet 2000;58:270e9. [45] Marini JC, Forlino A, Cabral WA, et al. Consortium for osteogenesis imperfecta mutations in the helical domain of type I collagen: regions rich in lethal mutations align with collagen binding sites for integrins and proteoglycans. Hum Mutat 2007;28:209e21. [46] Rauch F, Lalic L, Roughley P, Glorieux FH. Relationship between genotype and skeletal phenotype in children and adolescents with osteogenesis imperfecta. J Bone Miner Res 2010;25:1367e74. [47] Glorieux FH, Rauch F, Plotkin H, et al. Type V osteogenesis imperfecta: a new form of brittle bone disease. J Bone Miner Res 2000;15:1650e8. [48] Glorieux FH, Ward LM, Rauch F, Lalic L, Roughley PJ, Travers R. Osteogenesis imperfecta type VI: a form of brittle bone disease with a mineralization defect. J Bone Miner Res 2002;17:30e8. [49] Morello R, Bertin TK, Chen Y, et al. CRTAP is required for prolyl 3-hydroxylation and mutations cause recessive osteogenesis imperfecta. Cell 2006;127:291e304. [50] Ward LM, Rauch F, Travers R, et al. Osteogenesis imperfecta type VII: an autosomal recessive form of brittle bone disease. Bone 2002;31:12e8. [51] Rauch F, Travers R, Norman ME, Taylor A, Parfitt AM, Glorieux FH. Deficient bone formation in idiopathic juvenile osteoporosis: a histomorphometric study of cancellous iliac bone. J Bone Miner Res 2000;15:957e63. [52] Rauch F, Travers R, Norman ME, Taylor A, Parfitt AM, Glorieux FH. The bone formation defect in idiopathic juvenile osteoporosis is surface specific. Bone 2002;31:85e9. [53] Bondestam J, Mayranpaa MK, Ikegawa S, Marttinen E, Kroger H, Makitie O. Bone biopsy and densitometry findings in a child with Camurati-Engelmann disease. Clin Rheumatol 2007;26:1773e7. [54] Ward LM, Rauch F, Travers R, et al. Osteopathia striata with cranial sclerosis: Clinical, radiological, and bone histological findings in an adolescent girl. Am J Med Genet 2004;129A:8e12. [55] Cundy T, Wheadon L, King A. Treatment of idiopathic hyperphosphatasia with intensive bisphosphonate therapy. J Bone Miner Res 2004;19:703e11. [56] Munns CF, Rauch F, Travers R, Glorieux FH. Three children with lower limb fractures and a mineralization defect: a novel bone fragility disorder? Bone 2004;35:1023e8. [57] Chapurlat RD, Orcel P. Fibrous dysplasia of bone and McCuneAlbright syndrome. Best Pract Res Clin Rheumatol 2008;22:55e69. [58] Plotkin H, Rauch F, Zeitlin L, Munns C, Travers R, Glorieux FH. Effect of pamidronate treatment in children with polyostotic fibrous dysplasia of bone. J Clin Endocrinol Metab 2003;88:4569e75. [59] Shapiro F, Key LL, Anast C. Variable osteoclast appearance in human infantile osteopetrosis. Calcif Tissue Int 1988;43:67e76. [60] Cournot G, Trubert-Thil CL, Petrovic M, et al. Mineral metabolism in infants with malignant osteopetrosis: heterogeneity in plasma 1,25-dihydroxyvitamin D levels and bone histology. J Bone Miner Res 1992;7:1e10. [61] Del Fattore A, Peruzzi B, Rucci N, et al. Clinical, genetic, and cellular analysis of 49 osteopetrotic patients: implications for diagnosis and treatment. J Med Genet 2006;43:315e25. [62] Bollerslev J, Steiniche T, Melsen F, Mosekilde L. Structural and histomorphometric studies of iliac crest trabecular and cortical bone in autosomal dominant osteopetrosis: a study of two radiological types. Bone 1989;10:19e24.

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[63] Halton J, Gaboury I, Grant R, et al. Advanced vertebral fracture among newly diagnosed children with acute lymphoblastic leukemia: results of the Canadian Steroid-Associated Osteoporosis in the Pediatric Population (STOPP) research program. J Bone Miner Res 2009;24:1326e34. [64] Leeuw JA, Koudstaal J, Wiersema-Buist J, Kamps WA, Timens W. Bone histomorphometry in children with newly diagnosed acute lymphoblastic leukemia. Pediatr Res 2003;54:814e8. [65] Ward LM, Rauch F, Matzinger MA, Benchimol EI, Boland M, Mack DR. Iliac bone histomorphometry in children with newly diagnosed inflammatory bowel disease. Osteoporos Int 2010;21:331e7. [66] Mahachoklertwattana P, Sirikulchayanonta V, Chuansumrit A, et al. Bone histomorphometry in children and adolescents with beta-thalassemia disease: iron-associated focal osteomalacia. J Clin Endocrinol Metab 2003;88:3966e72. [67] Klein GL, Herndon DN, Goodman WG, et al. Histomorphometric and biochemical characterization of bone following acute severe burns in children. Bone 1995;17:455e60. [68] Klein GL, Bi LX, Sherrard DJ, et al. Evidence supporting a role of glucocorticoids in short-term bone loss in burned children. Osteoporos Int 2004;15:468e74. [69] Klein GL, Wimalawansa SJ, Kulkarni G, Sherrard DJ, Sanford AP, Herndon DN. The efficacy of acute administration of pamidronate on the conservation of bone mass following severe burn injury in children: a double-blind, randomized, controlled study. Osteoporos Int 2005;16:631e5. [70] Przkora R, Herndon DN, Sherrard DJ, Chinkes DL, Klein GL. Pamidronate preserves bone mass for at least 2 years following acute administration for pediatric burn injury. Bone 2007;41:297e302. [71] Rauch F, Travers R, Plotkin H, Glorieux FH. The effects of intravenous pamidronate on the bone tissue of children and adolescents with osteogenesis imperfecta. J Clin Invest 2002;110:1293e9. [72] Munns CF, Rauch F, Travers R, Glorieux FH. Effects of intravenous pamidronate treatment in infants with osteogenesis imperfecta: clinical and histomorphometric outcome. J Bone Miner Res 2005;20:1235e43. [73] Zeitlin L, Rauch F, Travers R, Munns C, Glorieux FH. The effect of cyclical intravenous pamidronate in children and adolescents with osteogenesis imperfecta Type V. Bone 2006;38:13e20. [74] Land C, Rauch F, Travers R, Glorieux FH. Osteogenesis imperfecta type VI in childhood and adolescence: Effects of cyclical intravenous pamidronate treatment. Bone 2007;40:638e44. [75] Cheung MS, Glorieux FH, Rauch F. Intravenous pamidronate in osteogenesis imperfecta type VII. Calcif Tissue Int 2009;84:203e9. [76] Rauch F, Travers R, Glorieux FH. Pamidronate in children with osteogenesis imperfecta: histomorphometric effects of longterm therapy. J Clin Endocrinol Metab 2006;91:511e6. [77] Rauch F, Munns CF, Land C, Cheung M, Glorieux FH. Risedronate in the treatment of mild pediatric osteogenesis imperfecta: a randomized placebo-controlled study. J Bone Miner Res 2009;24:1282e9. [78] Rauch F, Travers R, Munns C, Glorieux FH. Sclerotic metaphyseal lines in a child treated with pamidronate: histomorphometric analysis. J Bone Miner Res 2004;19:1191e3. [79] Whyte MP, Wenkert D, Clements KL, McAlister WH, Mumm S. Bisphosphonate-induced osteopetrosis. N Engl J Med 2003;349:457e63. [80] Cheung MS, Glorieux FH, Rauch F. Large osteoclasts in pediatric osteogenesis imperfecta patients receiving intravenous pamidronate. J Bone Miner Res 2009;24:669e74.

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C H A P T E R

17

A Diagnostic Approach to Skeletal Dysplasias Sheila Unger 1, Andrea Superti-Furga 2, David L. Rimoin 3 1

University of Lausanne, Service of Medical Genetics, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland 2 University of Lausanne, Division of Pediatrics, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland 3 Medical Genetics Birth Defects Center, Cedars-Sinai Health System and Department of Pediatrics and Medicine, UCLA School of Medicine, Los Angeles, California, USA

INTRODUCTION 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 400 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]. 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. Currently, only after a limited differential diagnosis has been established should confirmatory molecular investigations be considered. However, with the development of new sequencing technologies and their rapid expansion into clinical practice, it is likely that this

Pediatric Bone, Second Edition DOI: 10.1016/B978-0-12-382040-2.10017-6

algorithm will change over the course of the next years. It is increasingly probable that patients will arrive for their consultation with their genome already sequenced and as clinicians we will be asked which of the identified “variants” could explain the phenotype. This chapter reviews this sequence of diagnostic steps and outlines some of the more important radiographic findings.

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. 17.1). As awareness of these conditions grew, their numbers expanded to more than 400 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 remains 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

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17. A DIAGNOSTIC APPROACH TO SKELETAL DYSPLASIAS

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

FIGURE 17.1

Differences in 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 severely affected.

dysplasias rather than a succinct grouping of the varied and numerous disorders (Table 17.1). A framework in which to classify the skeletal dysplasias on the basis of their molecular defects has 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]. This type of grouping is more helpful to developmental biologists than to physicians. 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 non-lethal 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

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 non-lethal 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 fairly constant and can be used in developing a differential diagnosis. Increasingly, both lethal and non-lethal 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 or CHST3 deficiency) [16,17]. 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 [18]. 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) [19]. 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) [20]. 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 [21]. Specific learning disabilities have been

PEDIATRIC BONE

TABLE 17.1

Classification of Genetic Skeletal Disorders

Group/name of disorder

Inheritance

MIM No.

Locus

Gene

Protein

Thanatophoric dysplasia type 1 (TD1)

AD

187600

4p16.3

FGFR3

FGFR3

Thanatophoric dysplasia type 2 (TD2)

AD

187601

4p16.3

FGFR3

FGFR3

Severe achondroplasia with developmental delay and acanthosis nigricans (SADDAN)

AD

see 187600

4p16.3

FGFR3

FGFR3

Achondroplasia

AD

100800

4p16.3

FGFR3

FGFR3

Hypochondroplasia

AD

146000

4p16.3

FGFR3

FGFR3

Camptodactyly, tall stature and hearing loss syndrome (CATSHL)

AD

187600

4p16.3

FGFR3

FGFR3

Hypochondroplasia-like dysplasia(s)

AD, SP

1. FGFR3 CHONDRODYSPLASIA GROUP

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Achondrogenesis type 2 (ACG2; Langer-Saldino)

AD

200610

12q13.1

COL2A1

Type 2 collagen

Platyspondylic dysplasia, Torrance type

AD

151210

12q13.1

COL2A1

Type 2 collagen

Hypochondrogenesis

AD

200610

12q13.1

COL2A1

Type 2 collagen

Spondyloepiphyseal dysplasia congenital (SEDC)

AD

183900

12q13.1

COL2A1

Type 2 collagen

Spondyloepimetaphyseal dysplasia (SEMD) Strudwick type

AD

184250

12q13.1

COL2A1

Type 2 collagen

Kniest dysplasia

AD

156550

12q13.1

COL2A1

Type 2 collagen

Spondyloperipheral dysplasia

AD

271700

12q13.1

COL2A1

Type 2 collagen

Mild SED with premature onset arthrosis

AD

12q13.1

COL2A1

Type 2 collagen

SED with metatarsal shortening (formerly Czech dysplasia)

AD

609162

12q13.1

COL2A1

Type 2 collagen

Stickler syndrome type 1

AD

108300

12q13.1

COL2A1

Type 2 collagen

Stickler syndrome type 2

AD

604841

1p21

COL11A1

Type 11 collagen alpha-1 chain

Marshall syndrome

AD

154780

1p21

COL11A1

Type 11 collagen alpha-1 chain

Fibrochondrogenesis

AR

228520

1p21

COL11A1

Type 11 collagen alpha-1 chain

Otospondylomegaepiphyseal dysplasia (OSMED), recessive type

AR

215150

6p21.3

COL11A2

Type 11 collagen alpha-2 chain

HISTORY AND PHYSICAL EXAMINATION

2. TYPE 2 COLLAGEN GROUP AND SIMILAR DISORDERS

Stickler-like syndrome(s) 3. TYPE II COLLAGEN GROUP

405

(Continued)

Classification of Genetic Skeletal DisordersdCont’d

Group/name of disorder

Inheritance

MIM No.

Locus

Gene

Protein

Otospondylomegaepiphyseal dysplasia (OSMED), dominant type (Weissenbacher-Zweymu¨ller syndrome, Stickler syndrome type 3)

AD

215150

6p21.3

COL11A2

Type 11 collagen alpha-2 chain

Achondrogenesis type 1B (ACG1B)

AR

600972

5q32-33

DTDST

SLC26A2 sulfate transporter

Atelosteogenesis type 2 (AO2)

AR

256050

5q32-33

DTDST

SLC26A2 sulfate transporter

Diastrophic dysplasia (DTD)

AR

222600

5q32-33

DTDST

SLC26A2 sulfate transporter

MED, autosomal recessive type (rMED; EDM4)

AR

226900

5q32-33

DTDST

SLC26A2 sulfate transporter

SEMD, PAPSS2 type

AR

603005

10q23-q24

PAPSS2

PAPS-Synthetase 2

Chondrodysplasia with congenital joint dislocations, CHST3 type (recessive Larsen syndrome)

AR

608637

10q22.1

CHST3

Carbohydrate sulfotransferase 3; chondroitin 6sulfotransferase

EhlerseDanlos syndrome, CHST14 type (“musculo-skeletal variant”)

AR

601776

15q14

CHST14

Carbohydrate sulfotransferase 14; dermatan 4sulfotransferase

Dyssegmental dysplasia, SilvermaneHandmaker type

AR

224410

1q36-34

PLC (HSPG2)

Perlecan

Dyssegmental dysplasia, RollandeDesbuquois type

AR

224400

1q36-34

PLC (HSPG2)

Perlecan

SchwartzeJampel syndrome (myotonic chondrodystrophy)

AR

255800

1q36-34

PLC (HSPG2)

Perlecan

SED, Kimberley type

AD

608361

15q26

AGC1

Aggrecan

SEMD, Aggrecan type

AR

612813

15q26

AGC1

Aggrecan

Familial osteochondritis dissecans

AD

165800

15q26

AGC1

Aggrecan

Frontometaphyseal dysplasia

XLD

305620

Xq28

FLNA

Filamin A

Osteodysplasty MelnickeNeedles

XLD

309350

Xq28

FLNA

Filamin A

Otopalatodigital syndrome type 1 (OPD1)

XLD

311300

Xq28

FLNA

Filamin A

Otopalatodigital syndrome type 2 (OPD2)

XLD

304120

Xq28

FLNA

Filamin A

Terminal osseous dysplasia with pigmentary defects (TODPD)

XLD

300244

Xq28

FLNA

Filamin A

Atelosteogenesis type 1 (AO1)

AD

108720

3p14.3

FLNB

Filamin B

Atelosteogenesis type 3 (AO3)

AD

108721

3p14.3

FLNB

Filamin B

Larsen syndrome (dominant)

AD

150250

3p14.3

FLNB

Filamin B

406

TABLE 17.1

4. SULFATION DISORDERS GROUP

6. AGGRECAN GROUP

7. FILAMIN GROUP AND RELATED DISORDERS

17. A DIAGNOSTIC APPROACH TO SKELETAL DYSPLASIAS

PEDIATRIC BONE

5. PERLECAN GROUP

Spondylo-carpal-tarsal dysplasia

AR

272460

3p14.3

FLNB

Filamin B

Spondylo-carpal-tarsal dysplasia

AR

272460

Franck-terHaar syndrome

AR

249420

5q35.1

SH3PXD2B

TKS4

Serpentine fibula e polycystic kidney syndrome

AD?

600330

Metatropic dysplasia

AD

156530

12q24.1

TRPV4

Transient receptor potential cation channel, subfamily V, member 4

Spondyloepimetaphyseal dysplasia, Maroteaux type (Pseudo-Morquio syndrome type 2)

AD

184095

12q24.1

TRPV4

Transient receptor potential cation channel, subfamily V, member 4

Spondylometaphyseal dysplasia, Kozlowski type

AD

184252

12q24.1

TRPV4

Transient receptor potential cation channel, subfamily V, member 4

Brachyolmia, autosomal dominant type

AD

113500

12q24.1

TRPV4

Transient receptor potential cation channel, subfamily V, member 4

Familial digital arthropathy with brachydactyly

AD

606835

12q24.1

TRPV4

Transient receptor potential cation channel, subfamily V, member 4

4p16

EVC1

EvC gene 1

4p16

EVC2

EvC gene 2

8. TRPV4 GROUP

Chondroectodermal dysplasia (Ellisevan Creveld)

AR

225500

Short ribs e polydactyly syndrome (SRPS) type 1/3 (SaldinoeNoonan/VermaeNaumoff)

AR

263510

11q22.3

DYNC2H1

Dynein, cytoplasmic 2, heavy chain 1

SRPS type 1/3 (SaldinoeNoonan/VermaeNaumoff)

AR

263510

3q25.33

IFT80

Intraflagellar transport 80 (homolog of)

SRPS type 1/3 (SaldinoeNoonan/VermaeNaumoff)

AR

263510

SRPS type 2 (Majewski)

AR

263520

SRPS type 4 (Beemer)

AR

269860

Oral-facial-digital syndrome type 4 (MohreMajewski)

AR

258860

Asphyxiating thoracic dysplasia (ATD; Jeune)

AR

208500

3q25.33

IFT80

Intraflagellar transport 80 (homolog of)

Asphyxiating thoracic dysplasia (ATD; Jeune)

AR

208500

11q22.3

DYNC2H1

Dynein, cytoplasmic 2, heavy chain 1

Asphyxiating thoracic dysplasia (ATD; Jeune)

AR

208500

Thoracolaryngopelvic dysplasia (Barnes)

AD

187760

HISTORY AND PHYSICAL EXAMINATION

PEDIATRIC BONE

9. SHORT-RIBS DYSPLASIAS (WITH OR WITHOUT POLYDACTYLY) GROUP

10. MULTIPLE EPIPHYSEAL DYSPLASIA AND PSEUDOACHONDROPLASIA GROUP Pseudoachondroplasia (PSACH)

AD

177170

19p13.1

COMP

COMP

Multiple epiphyseal dysplasia (MED) type 1 (EDM1)

AD

132400

19p13.1

COMP

COMP (Continued)

407

408

TABLE 17.1 Classification of Genetic Skeletal DisordersdCont’d Group/name of disorder

Inheritance

MIM No.

Locus

Gene

Protein

Multiple epiphyseal dysplasia (MED) type 2 (EDM2)

AD

600204

1p32.2-33

COL9A2

Collagen 9 alpha-2 chain

Multiple epiphyseal dysplasia (MED) type 3 (EDM3)

AD

600969

20q13.3

COL9A3

Collagen 9 alpha-3 chain

Multiple epiphyseal dysplasia (MED) type 5 (EDM5)

AD

607078

2p23-24

MATN3

Matrilin 3

Multiple epiphyseal dysplasia (MED) type 6 (EDM6)

AD

120210

6q13

COL9A1

Collagen 9 alpha-1 chain

Stickler syndrome, recessive type

AR

120210

6q13

COL9A1

Collagen 9 alpha-1 chain

Familial hip dysplasia (Beukes)

AD

142669

4q35

Multiple epiphyseal dysplasia with microcephaly and nystagmus (Lowry-Wood)

AR

226960

Metaphyseal dysplasia, Schmid type (MCS)

AD

156500

6q21-22.3

COL10A1

Collagen 10 alpha-1 chain

Cartilage-hair hypoplasia (CHH; metaphyseal dysplasia, McKusick type)

AR

250250

9p13

RMRP

RNA component of RNAse H

Metaphyseal dysplasia, Jansen type

AD

156400

3p22-21.1

PTHR1

PTH/PTHrP receptor 1

Eiken dysplasia

AR

600002

3p22-21.1

PTHR1

PTH/PTHrP receptor 1

Metaphyseal dysplasia with pancreatic insufficiency and cyclic neutropenia (ShwachmaneBodianeDiamond syndrome, SBDS)

AR

260400

7q11

SBDS

SBDS protein

Metaphyseal anadysplasia type 1

AD, AR

309645

11q22.2

MMP13

Matrix metalloproteinase 13

Metaphyseal anadysplasia type 2

AR

20q13.12

MMP9

Matrix metalloproteinase 9

Metaphyseal dysplasia, Spahr type

AR

250400

Metaphyseal acroscyphodysplasia (various types)

AR

250215

Genochondromatosis (type 1/type 2)

AD/SP

137360

Metaphyseal chondromatosis with D-2-hydroxyglutaric aciduria

AR/SP

see 271550

Spondyloenchondrodysplasia (SPENCD)

AR

271550

19p13.2

ACP5

Tartrate-resistant acid phosphatase (TRAP)

Odontochondrodysplasia (ODCD)

AR

184260

Spondylometaphyseal dysplasia, Sutcliffe type or corner fractures type

AD

184255

Multiple epiphyseal dysplasia (MED), other types

PEDIATRIC BONE

12. SPONDYLOMETAPHYSEAL DYSPLASIAS (SMD)

17. A DIAGNOSTIC APPROACH TO SKELETAL DYSPLASIAS

11. METAPHYSEAL DYSPLASIAS

SMD with severe genu valgum

AD

184253

SMD with cone-rod dystrophy

AR

608940

SMD with retinal degeneration, axial type

AR

602271

Dysspondyloenchondromatosis

SP

Cheiro-spondyloenchondromatosis

SP

13. SPONDYLO-EPI-(META)-PHYSEAL DYSPLASIAS (SE(M)D) AR

223800

18q12-21.1

DYM

Dymeclin

Immuno-osseous dysplasia (Schimke)

AR

242900

2q34-36

SMARCAL1

SWI/SNF-related regulator of chromatin subfamily A-like protein 1

SED, WolcotteRallison type

AR

226980

2p12

EIF2AK3

Translation initiation factor 2-alpha kinase-3

SEMD, Matrilin type

AR

608728

2p23-p24

MATN3

Matrilin 3

SEMD, short limb e abnormal calcification type

AR

271665

1q23

DDR2

Discoidin domain receptor family, member 2

SED tarda, X-linked (SED-XL)

XLR

313400

Xp22

SEDL

Sedlin

Spondylo-megaepiphyseal-metaphyseal dysplasia (SMMD)

AR

613330

4p16.1

NKX3-2

NK3 Homeobox 2

Spondylodysplastic EhlerseDanlos syndrome

AR

612350

11p11.2

SLC39A13

Zinc transporter ZIP13

SPONASTRIME dysplasia

AR

271510

SEMD with joint laxity (SEMD-JL) leptodactylic or Hall type

AD

603546

SEMD with joint laxity (SEMD-JL) Beighton type

AR

271640

Platyspondyly (brachyolmia) with amelogenesis imperfecta

AR

601216

Late onset SED, autosomal recessive type

AR

609223

Brachyolmia, Hobaek and Toledo types

AR

271530, 271630

Achondrogenesis type 1A (ACG1A)

AR

200600

14q32.12

TRIP11

Golgi-microtubule-associated protein, 210-KD; GMAP210

Schneckenbecken dysplasia

AR

269250

1p31.3

SLC35D1

Solute carrier family 35 member D1; UDPglucuronic acid/UDP-N-acetylgalactosamine dual transporter

Spondylometaphyseal dysplasia, Sedaghatian type

AR

250220

Severe spondylometaphyseal dysplasia (SMD Sedaghatian-like)

AR

7q11

SBDS

SBDS gene, function still unclear

Opsismodysplasia

AR

HISTORY AND PHYSICAL EXAMINATION

PEDIATRIC BONE

DyggveeMelchioreClausen dysplasia (DMC)

14. SEVERE SPONDYLODYSPLASTIC DYSPLASIAS

(Continued)

409

258480

410

TABLE 17.1

Classification of Genetic Skeletal DisordersdCont’d

Group/name of disorder

Inheritance

MIM No.

Locus

Gene

Protein

Trichorhinophalangeal dysplasia types 1/3

AD

190350

8q24

TRPS1

Zinc finger transcription factor

Trichorhinophalangeal dysplasia type 2 (LangereGiedion)

AD

150230

8q24

TRPS1 and EXT1

Zinc finger transcription factor and Exostosin 1

Acrocapitofemoral dysplasia

AR

607778

2q33-q35

IHH

Indian hedgehog

Cranioectodermal dysplasia (LevineSensenbrenner) type 1

AR

218330

3q21

IFT122

Intraflagellar transport 122 (Chlamydomonas, homolog of)

Cranioectodermal dysplasia (LevineSensenbrenner) type 2

AR

613610

2p24.1

WDR35

WD repeat-containing protein 35

Geleophysic dysplasia

AR

231050

9q34.2

ADAMTSL2

ADAMTS-like protein 2

Geleophysic dysplasia, other types

AR

Acromicric dysplasia

AD

102370

Acrodysostosis

AD

101800

Angel-shaped phalango-epiphyseal dysplasia (ASPED)

AD

105835

SaldinoeMainzer dysplasia

AR

266920

Acromesomelic dysplasia type Maroteaux (AMDM)

AR

602875

9p13-12

NPR2

Natriuretic peptide receptor 2

Grebe dysplasia

AR

200700

20q11.2

GDF5

Growth and Differentiation Factor 5

Fibular hypoplasia and complex brachydactyly (Du Pan)

AR

228900

20q11.2

GDF5

Growth and Differentiation Factor 5

Acromesomelic dysplasia with genital anomalies

AR

609441

4q23-24

BMPR1B

Bone morphogenetic protein receptor 1B

Acromesomelic dysplasia, OseboldeRemondini type

AD

112910

15. ACROMELIC DYSPLASIAS

17. MESOMELIC AND RHIZO-MESOMELIC DYSPLASIAS Dyschondrosteosis (LerieWeill)

Pseudo-AD

127300

Xpter-p22.32

SHOX

Short stature e homeobox gene

Langer type (homozygous dyschondrosteosis)

Pseudo-AR

249700

Xpter-p22.32

SHOX

Short stature e homeobox gene

Omodysplasia

AR

258315

13q31-q32

GPC6

Glypican 6

Robinow syndrome, recessive type

AR

268310

9q22

ROR2

Receptor tyrosine kinase-like orphan receptor 2

Robinow syndrome, dominant type

AD

180700

Mesomelic dysplasia, Korean type

AD

2q24-32

Duplication in HOXD gene cluster

17. A DIAGNOSTIC APPROACH TO SKELETAL DYSPLASIAS

PEDIATRIC BONE

16. ACROMESOMELIC DYSPLASIAS

Mesomelic dysplasia, Kantaputra type

AD

156232

Mesomelic dysplasia, Nievergelt type

AD

163400

Mesomelic dysplasia, KozlowskieReardon type

AR

249710

Mesomelic dysplasia with acralsynostoses (VerloeseDavidePfeiffer type)

AD

600383

Mesomelic dysplasia, Savarirayan type (triangular tibia-fibular aplasia)

SP

605274

Campomelic dysplasia (CD)

AD

Stu¨veeWiedemann dysplasia

AR

2q24-32

Duplications in HOXD gene cluster

8q13

SULF1 and SLCO5A1

Heparan sulfate 6-O-endosulfatase 1 and solute carrier organic anion transporter family member 5A1

114290

17q24.3-25.1

SOX9

SRY-box 9

601559

5p13.1

LIFR

Leukemia Inhibitory Factor Receptor

18. BENT BONES DYSPLASIAS

Kyphomelic dysplasia, several forms

211350

PEDIATRIC BONE

3-M syndrome (3M1)

AR

273750

6p21.1

CUL7

Cullin 7

3-M syndrome (3M2)

AR

612921

2q35

OBSL1

Obscurin-like 1

KennyeCaffey dysplasia type 1

AR

244460

1q42-q43

TBCE

Tubulin-specific chaperone E

KennyeCaffey dysplasia type 2

AD

127000

Microcephalicosteodysplastic primordial dwarfism type 1/3 (MOPD1)

AR

210710

2q

Microcephalicosteodysplastic primordial dwarfism type 2 (MOPD2; Majewski type)

AR

210720

21q

PCNT2

Pericentrin 2

IMAGE syndrome (intrauterine growth retardation, metaphyseal dysplasia, adrenal hypoplasia, and genital anomalies)

XL/AD

300290

Osteocraniostenosis

SP

602361

HallermanneStreiff syndrome

AR

234100

HISTORY AND PHYSICAL EXAMINATION

19. SLENDER BONE DYSPLASIA GROUP

20. DYSPLASIAS WITH MULTIPLE JOINT DISLOCATIONS Desbuquois dysplasia (with accessory ossification center in digit 2)

AR

251450

17q25.3

CANT1

Desbuquois dysplasia with short metacarpals and elongated phalanges (Kim type)

AR

251450

17q25.3

CANT1

Desbuquois dysplasia (other variants with or without accessory ossification center)

AR

Pseudodiastrophic dysplasia

AR

264180

411

(Continued)

Classification of Genetic Skeletal DisordersdCont’d

Group/name of disorder

Inheritance

MIM No.

Locus

Gene

Protein

CDP, X-linked dominant, ConradieHu¨nermann type (CDPX2)

XLD

302960

Xp11

EBP

Emopamil-binding protein

CDP, X-linked recessive, brachytelephalangic type (CDPX1)

XLR

302950

Xp22.3

ARSE

Arylsulfatase E

CHILD (congenital hemidysplasia, ichthyosis, limb defects)

XLD

308050

Xp11

NSDHL

NAD(P)H steroid dehydrogenase-like protein

CHILD (congenital hemidysplasia, ichthyosis, limb defects)

XLD

308050

Xq28

EBP

Emopamil-binding protein

Greenberg dysplasia

AR

215140

1q42.1

LBR

Lamin B receptor, 3-beta-hydroxysterol delta (14)-reductase

Rhizomelic CDP type 1

AR

215100

6q22-24

PEX7

Peroxisomal PTS2 receptor

Rhizomelic CDP type 2

AR

222765

1q42

DHPAT

Dihydroxyacetonephosphateacyltransferase (DHAPAT)

Rhizomelic CDP type 3

AR

600121

2q31

AGPS

Alkylglycerone-phosphate synthase (AGPS)

CDP tibial-metacarpal type

AD/AR

118651

AstleyeKendall dysplasia

AR?

412

TABLE 17.1

21. CHONDRODYSPLASIA PUNCTATA (CDP) GROUP

Blomstrand dysplasia

AR

215045

3p22-21.1

PTHR1

PTH/PTHrP receptor 1

Desmosterolosis

AR

602398

1p33-31.1

DHCR24

3-beta-hydroxysterol delta-24-reductase

Caffey disease (including infantile and attenuated forms)

AD

114000

17q21-22

COL1A1

Collagen 1, alpha-1 chain

Caffey disease (severe variants with prenatal onset)

AR

114000

Raine dysplasia (lethal and non-lethal forms)

AR

259775

7p22

FAM20C

23. INCREASED BONE DENSITY GROUP (WITHOUT MODIFICATION OF BONE SHAPE) Osteopetrosis, severe neonatal or infantile forms (OPTB1)

AR

259700

11q13

TCIRG1

Subunit of ATPase proton pump

Osteopetrosis, severe neonatal or infantile forms (OPTB4)

AR

611490

16p13

CLCN7

Chloride channel 7

Osteopetrosis, infantile form, with nervous system involvement (OPTB5)

AR

259720

6q21

OSTM1

Grey lethal / Osteopetrosis associated transmembrane protein

Osteopetrosis, intermediate form, osteoclast-poor (OPTB2)

AR

259710

13q14.11

RANKL (TNFSF11)

Receptor activator of NF-kappa-B ligand (Tumor necrosis factor ligand superfamily, member 11)

17. A DIAGNOSTIC APPROACH TO SKELETAL DYSPLASIAS

PEDIATRIC BONE

22. NEONATAL OSTEOSCLEROTIC DYSPLASIAS

AR

612302

18q21.33

RANK (TNFRSF11A)

Receptor activator of NF-kappa-B

Osteopetrosis, intermediate form (OPTB6)

AR

611497

17q21.3

PLEKHM1

Pleckstrin homology domain-containing protein, family M, member 1

Osteopetrosis, intermediate form (OPTA2)

AR

259710

16p13

CLCN7

Chloride channel pump

Osteopetrosis with renal tubular acidosis (OPTB3)

AR

259730

8q22

CA2

Carbonic anhydrase 2

Osteopetrosis, late-onset form type 1 (OPTA1)

AD

607634

11q13.4

LRP5

Low density lipoprotein receptor-related protein 5

Osteopetrosis, late-onset form type 2 (OPTA2)

AD

166600

16p13

CLCN7

Chloride channel 7

Osteopetrosis with ectodermal dysplasia and immune defect (OLEDAID)

XL

300301

Xq28

IKBKG (NEMO)

Inhibitor of kappa light polypeptide gene enhancer, kinase of

Osteopetrosis, moderate form with defective leukocyte adhesion (LAD3)

AR

612840

11q12

FERMT3 (KIND3)

Fermitin 3 (Kindlin 3)

Osteopetrosis, moderate form with defective leukocyte adhesion

AR

612840

11q13

RASGRP2 (CalDAGGEF1)

Rasguanyl nucleotide-releasing protein 2

Pyknodysostosis

AR

265800

1q21

CTSK

Cathepsin K

Osteopoikilosis

AD

155950

12q14

LEMD3

LEM domain-containing 3

Melorheostosis with osteopoikilosis

AD

155950

12q14

LEMD3

LEM domain-containing 3

Osteopathiastriata with cranial sclerosis (OSCS)

XLD

300373

Xq11.1

WTX

FAM123B

Melorheostosis

SP

Dysosteosclerosis

AR

224300

Osteomesopyknosis

AD

166450

Osteopetrosis with infantile neuroaxonal dysplasia

AR?

600329

HISTORY AND PHYSICAL EXAMINATION

PEDIATRIC BONE

Osteopetrosis, infantile form, osteoclast-poor with immunoglobulin deficiency (OPTB7)

24. INCREASED BONE DENSITY GROUP WITH METAPHYSEAL AND/OR DIAPHYSEAL INVOLVEMENT AD

123000

5p15.2-14.2

ANKH

Homolog of mouse ANK (ankylosis) gene

Diaphyseal dysplasia CamuratieEngelmann

AD

131300

19q13

TGFbeta1

Transforming growth factor beta 1

Hematodiaphyseal dysplasia Ghosal

AR

231095

7q34

TBXAS1

Thromboxane A synthase 1

Hypertrophic osteoarthropathy

AR

259100

4q34-35

HPGD

15-alpha-hydroxyprostaglandin dehydrogenase

Pachydermoperiostosis (hypertrophic osteoarthropathy, primary, autosomal dominant)

AD

167100

Oculodentoosseous dysplasia (ODOD) mild type

AD

164200

6q22-23

GJA1

Gap junction protein alpha-1

Oculodentoosseous dysplasia (ODOD) severe type

AR

257850

Osteoectasia with hyperphosphatasia (juvenile Paget’s disease)

AR

239000

8q24

OPG

Osteoprotegerin (Continued)

413

Craniometaphyseal dysplasia, autosomal dominant type

Classification of Genetic Skeletal DisordersdCont’d Inheritance

MIM No.

Locus

Gene

Protein

Sclerosteosis

AR

269500

17q12-21

SOST

Sclerostin

Endosteal hyperostosis, van Buchem type

AR

239100

17q12-21

SOST

Sclerostin

Trichodentoosseous dysplasia

AD

190320

17q21

DLX3

Distal-less homeobox 3

Craniometaphyseal dysplasia, autosomal recessive type

AR

218400

6q21-22

Diaphyseal medullary stenosis with bone malignancy

AD

112250

9p21-p22

Craniodiaphyseal dysplasia

AD

122860

Craniometadiaphyseal dysplasia, Wormian bone type

AR

–

Endosteal sclerosis with cerebellar hypoplasia

AR

213002

LenzeMajewski hyperostotic dysplasia

SP

151050

Metaphyseal dysplasia, BrauneTinschert type

XL

605946

Pyle disease

AR

265900

COL1A1: Collagen 1 alpha-1 chain, COL1A2: Collagen 1 alpha-2 chain, CRTAP: CartilageAssociated Protein, LEPRE1: leucineprolineenriched proteoglycan (leprecan) 1, PPIB: peptidylprolylisomerase B (cyclophilin B), FKBP10: FK506 binding protein 10, SERPINH: serpin peptidase inhibitor clade H 1, SP7: SP7 transcription factor (Osterix)

25. OSTEOGENESIS IMPERFECTA AND DECREASED BONE DENSITY GROUP PEDIATRIC BONE

Osteogenesis imperfecta, non-deforming form (OI type 1)

AD

COL1A1, COL1A2

Osteogenesis imperfecta, perinatal lethal form (OI type 2)

AD, AR

COL1A1, COL1A2, CRTAP, LEPRE1, PPIB

Osteogenesis imperfecta, progressively deforming type (OI type 3)

AD, AR

COL1A1, COL1A2, CRTAP, LEPRE1, PPIB, FKBP10, SERPINH1

Osteogenesis imperfecta, moderate form (OI type 4)

AD, AR

COL1A1, COL1A2, CRTAP, FKBP10, SP7

Osteogenesis imperfecta with calcification of the interosseous membranes and/or hypertrophic callus (OI type 5)

AD

610967

Bruck syndrome type 1 (BS1)

AR

259450

17p12

FKBP10

FK506 binding protein 10

Bruck syndrome type 2 (BS2)

AR

609220

3q23-24

PLOD2

Procollagenlysyl hydroxylase 2

Osteoporosis-pseudoglioma syndrome

AR

259770

11q12-13

LRP5

LDL-receptor related protein 5

Calvarial doughnut lesions with bone fragility

AD

126550

Idiopathic juvenile osteoporosis

SP

259750

ColeeCarpenter dysplasia (bone fragility with craniosynostosis)

SP

112240

Spondylo-ocular dysplasia

AR

605822

Osteogenesis imperfecta, other types

17. A DIAGNOSTIC APPROACH TO SKELETAL DYSPLASIAS

Group/name of disorder

414

TABLE 17.1

Osteopenia with radiolucent lesions of the mandible

AD

166260

EhlerseDanlos syndrome, progeroid form

AR

130070

5q35

B4GALT7

Xylosylprotein 4-beta-galactosyltransferase deficiency

Gerodermaosteodysplasticum

AR

231070

1q24.2

GORAB

SCYL1-binding protein 1

Cutis laxa, autosomal recessive form, type 2B (ARCL2B)

AR

612940

17q25.3

PYCR1

Pyrroline-5-carboxylate reductase 1

Cutis laxa, autosomal recessive form, type 2A (ARCL2A) (Wrinkly skin syndrome)

AR

278250, 219200

12q24.3

ATP6VOA2

ATPase, Hþ transporting, lysosomal, V0 subunit A2

SingletoneMerten dysplasia

AD

182250

Hypophosphatasia, perinatal lethal and infantile forms

AR

241500

1p36.1-p34

ALPL

Alkaline phosphatase, tissue non-specific (TNSALP)

Hypophosphatasia, adult form

AD

146300

1p36.1-p34

ALPL

Alkaline phosphatase, tissue non-specific (TNSALP)

Hypophosphatemic rickets, X-linked dominant

XLD

307800

Xp22

PHEX

X-linked hypophosphatemia membrane protease

Hypophosphatemic rickets, autosomal dominant

AD

193100

12p13.3

FGF23

Fibroblast growth factor 23

Hypophosphatemic rickets, autosomal recessive, type 1 (ARHR1)

AR

241520

4q21

DMP1

Dentin matrix acidic phosphoprotein 1

Hypophosphatemic rickets, autosomal recessive, type 2 (ARHR2)

AR

613312

6q23

ENPP1

Ectonucleotidepyrophosphatase/ phosphodiesterase 1

Hypophosphatemic rickets with hypercalciuria, X-linked recessive

XLR

300554

Xp11.22

CLCN5

Chloride channel 5

Hypophosphatemic rickets with hypercalciuria, autosomal recessive (HHRH)

AR

241539

9q34

SLC34A3

Sodium-phosphate cotransporter

Neonatal hyperparathyroidism, severe form

AR

239200

3q13.3-21

CASR

Calcium-sensing receptor

Familial hypocalciurichypercalcemia with transient neonatal hyperparathyroidism

AD

145980

3q13.3-21

CASR

Calcium-sensing receptor

Calcium pyrophosphate deposition disease (familial chondrocalcisnosis) type 2

AD

118600

5p15.2-14.2

ANKH

Homolog of mouse ANK (ankylosis) gene

26. ABNORMAL MINERALIZATION GROUP

HISTORY AND PHYSICAL EXAMINATION

PEDIATRIC BONE

27. LYSOSOMAL STORAGE DISEASES WITH SKELETAL INVOLVEMENT (DYSOSTOSIS MULTIPLEX GROUP) Mucopolysaccharidosis type 1H / 1S

AR

607014

4p16.3

IDA

Alpha-1-Iduronidase

Mucopolysaccharidosis type 2

XLR

309900

Xq27.3-28

IDS

Iduronate-2-sulfatase

Mucopolysaccharidosis type 3A

AR

252900

17q25.3

HSS

Heparan sulfate sulfatase

Mucopolysaccharidosis type 3B

AR

252920

17q21

NAGLU

N-Ac-beta-D-glucosaminidase

415

(Continued)

416

TABLE 17.1

Classification of Genetic Skeletal DisordersdCont’d Inheritance

MIM No.

Locus

Gene

Protein

Mucopolysaccharidosis type 3C

AR

252930

8p11-q13

HSGNAT

Ac-CoA: alpha-glucosaminide N-acetyltransferase

Mucopolysaccharidosis type 3D

AR

252940

12q14

GNS

N-Acetylglucosamine 6-sulfatase

Mucopolysaccharidosis type 4A

AR

253000

16q24.3

GALNS

Galactosamine-6-sulfate sulfatase

Mucopolysaccharidosis type 4B

AR

253010

3p21.33

GLBI

beta-Galactosidase

Mucopolysaccharidosis type 6

AR

253200

5q13.3

ARSB

Arylsulfatase B

Mucopolysaccharidosis type 7

AR

253220

7q21.11

GUSB

beta-Glucuronidase

Fucosidosis

AR

230000

1p34

FUCA

alpha-Fucosidase

alpha-Mannosidosis

AR

248500

19p13.2-12

MANA

alpha-Mannosidase

beta-Mannosidosis

AR

248510

4q22-25

MANB

beta-Mannosidase

Aspartylglucosaminuria

AR

208400

4q23-27

AGA

Aspartyl-glucosaminidase

GMI gangliosidosis, several forms

AR

230500

3p21-14.2

GLB1

beta-Galactosidase

Sialidosis, several forms

AR

256550

6p21.3

NEU1

Neuraminidase (sialidase)

Sialic acid storage disease (SIASD)

AR

269920

6q14-q15

SLC17A5

Sialin (sialic acid transporter)

Galactosialidosis, several forms

AR

256540

20q13.1

PPGB

beta-Galactosidase protective protein

Multiple sulfatase deficiency

AR

272200

3p26

SUMF1

Sulfatase-modifying factor-1

Mucolipidosis II (I-cell disease), alpha/beta type

AR

252500

12q23

GNPTAB

N-Acetylglucosamine 1-phosphotransferase, alpha/beta subunits

Mucolipidosis III (Pseudo-Hurler polydystrophy), alpha/beta type

AR

252600

12q23

GNPTAB

N-Acetylglucosamine 1-phosphotransferase, alpha/beta subunits

Mucolipidosis III (Pseudo-Hurler polydystrophy), gamma type

AR

252605

16p13

GNPTG

N-Acetylglucosamine 1-phosphotransferase, gamma subunit

Familial expansileosteolysis

AD

174810

18q22.1

RANK (TNFRSF11A)

Mandibuloacral dysplasia type A

AD

248370

1q21.2

LMNA

Lamin A/C

Mandibuloacral dysplasia type B

AR

608612

1p34

ZMPSTE24

Zinc metalloproteinase

Progeria, HutchinsoneGilford type

AD

176670

1q21.2

LMNA

Lamin A/C

TorgeWinchester syndrome

AR

259600

16q13

MMP2

Matrix metalloproteinase 2

HajdueCheney syndrome

AD

102500

1p13

NOTCH2

Notch homolog protein 2

Multicentric carpal-tarsal osteolysis with and without nephropathy

AD

166300

28. OSTEOLYSIS GROUP

17. A DIAGNOSTIC APPROACH TO SKELETAL DYSPLASIAS

PEDIATRIC BONE

Group/name of disorder

Lipomembraneous osteodystrophy with leukoencephalopathy (presenile dementia with bone cysts; Nasu-Hakola)

AR

221770

6p21.2

TREM2

Triggering receptor expressed on myeloid cells 2

Lipomembraneous osteodystrophy with leukoencephalopathy (presenile dementia with bone cysts; NasueHakola)

AR

221770

19q13.1

TYROBP

Tyro protein tyrosine kinase-binding protein

29. DISORGANIZED DEVELOPMENT OF SKELETAL COMPONENTS GROUP AD

133700

8q23-24.1

EXT1

Exostosin-1

Multiple cartilaginous exostoses 2

AD

133701

11p12-11

EXT2

Exostosin-2

Multiple cartilaginous exostoses 3

AD

600209

19p

Cherubism

AD

118400

4p16

SH3BP2

SH3 domain-binding protein 2

Fibrous dysplasia, polyostotic form

SP

174800

20q13

GNAS1

Guanine nucleotide-binding protein, alphastimulating activity subunit 1

Progressive osseous heteroplasia

AD

166350

20q13

GNAS1

Guanine nucleotide-binding protein, alphastimulating activity subunit 1

Gnathodiaphyseal dysplasia

AD

166260

11p15.1-14.3

TMEM16E

Transmembrane protein 16E

Metachondromatosis

AD

156250

12q24

PTPN11

Protein-tyrosine phosphatase nonreceptortype 11

Osteoglophonic dysplasia

AD

166250

8p11

FGFR1

Fibroblast growth factor receptor 1

Fibrodysplasiaossificansprogressiva (FOP)

AD, SP

135100

2q23-24

ACVR1

Activin A (BMP type 1) receptor

Neurofibromatosis type 1 (NF1)

AD

162200

17q11.2

NF1

Neurofibromin

Carpotarsalosteochondromatosis

AD

127820

Cherubism with gingival fibromatosis (Ramon syndrome)

AR

266270

Dysplasia epiphysealishemimelica (Trevor)

SP

127800

Enchondromatosis (Ollier)

SP

166000

Enchondromatosis with hemangiomata (Maffucci)

SP

166000

HISTORY AND PHYSICAL EXAMINATION

PEDIATRIC BONE

Multiple cartilaginous exostoses 1

30. OVERGROWTH SYNDROMES WITH SKELETAL INVOLVEMENT Weaver syndrome

SP/AD

277590

Sotos syndrome

AD

117550

5q35

NSD1

Nuclear receptor-binding su-var, enhancer of zeste, and trithorax domain protein 1

MarshalleSmith syndrome

SP

602535

19p13.3

NFIX

nuclear factor I/X

Proteus syndrome

SP

176920

Marfan syndrome

AD

154700

15q21.1

FBN1

Fibrillin 1

417

(Continued)

418

TABLE 17.1

Classification of Genetic Skeletal DisordersdCont’d Inheritance

MIM No.

Locus

Gene

Protein

Congenital contractural arachnodactyly

AD

121050

5q23.3

FBN2

Fibrillin 2

LoeyseDietz syndrome types 1A and 2A

AD

609192, 610168,

9q22

TGFBR1,

TGFbeta receptor subunit 1

LoeyseDietz syndrome types 1B and 2B

AD

608967, 610380

3p22

TGFBR2

TGFbeta receptor subunit 2

Overgrowth syndrome with 2q37 translocations

SP

–

2q37

NPPC

Natriuretic peptide precursor C

Overgrowth syndrome with skeletal dysplasia (NishimuraeSchmidt, endochondral gigantism)

SP?

31. GENETIC INFLAMMATORY/RHEUMATOID-LIKE OSTEOARTHROPATHIES

PEDIATRIC BONE

Progressive pseudorheumatoid dysplasia (PPRD; SED with progressive arthropathy)

AR

208230

6q22-23

WISP3

WNT1-inducible signaling pathway protein 3

Chronic infantile neurologic cutaneous articular syndrome (CINCA) / neonatal onset multisystem inflammatory disease (NOMID)

AD

607115

1q44

CIAS1

Cryopyrin

Sterile multifocal osteomyelitis, periostitis, and pustulosis (CINCA/NOMID-like)

AR

147679

2q14.2

IL1RN

Interleukin 1 receptor antagonist

Chronic recurrent multifocal osteomyelitis with congenital dyserythropoietic anemia (CRMO with CDA; Majeed syndrome)

AR

609628

18p11.3

LPIN2

Lipin 2

Hyperostosis/hyperphosphatemia syndrome

AR

610233

2q24-q31;

GALNT3

UDP-N-acetyl-alpha-Dgalactosamine:polypeptide Nacetylgalactosaminyltransferase 3

Infantile systemic hyalinosis/Juvenile hyaline fibromatosis (ISH/JHF)

AR

236490

4q21

ANTXR2

Anthrax toxin receptor 2

RUNX2

Runt related transcription factor 2

32. CLEIDOCRANIAL DYSPLASIA AND ISOLATED CRANIAL OSSIFICATION DEFECTS GROUP Cleidocranial dysplasia

AD

119600

6p21

CDAGS syndrome (craniosynostosis, delayed fontanel closure, parietal foramina, imperforate anus, genital anomalies, skin eruption)

AR

603116

22q12-q13

YuniseVaron dysplasia

AR

216340

Parietal foramina (isolated)

AD

168500

11q11.2

ALX4

Aristaless-like 4

Parietal foramina (isolated)

AD

168500

5q34-35

MSX2

Muscle segment homeobox 2

See also: pycnodysostosis, wrinkly skin syndrome, and several others

17. A DIAGNOSTIC APPROACH TO SKELETAL DYSPLASIAS

Group/name of disorder

33. CRANIOSYNOSTOSIS SYNDROMES AD

101600

8p12

FGFR1

Fibroblast growth factor receptor 1

Pfeiffer syndrome (FGFR2-related)

AD

101600

10q26.12

FGFR2

Fibroblast growth factor receptor 2

Apert syndrome

AD

101200

10q26.12

FGFR2

Fibroblast growth factor receptor 2

Craniosynostosis with cutis gyrata (BeareeStevenson)

AD

123790

10q26.12

FGFR2

Fibroblast growth factor receptor 2

Crouzon syndrome

AD

123500

10q26.12

FGFR2

Fibroblast growth factor receptor 2

Crouzon-like craniosynostosis with acanthosisnigricans (Crouzonodermoskeletal syndrome)

AD

612247

4p16.3

FGFR3

Fibroblast growth factor receptor 3

Craniosynostosis, Muenke type

AD

602849

4p16.3

FGFR3

Fibroblast growth factor receptor 3

AntleyeBixler syndrome

AR

201750

7q11.23

POR

Cytochrome P450 oxidoreductase

Craniosynostosis Boston type

AD

604757

5q35.2

MSX2

MSX2

SaethreeChotzen syndrome

AD

101400

7p21.1

TWIST1

TWIST

ShprintzeneGoldberg syndrome

AD

182212

BallereGerold syndrome

AR

218600

8q24.3

RECQL4

RECQ Protein-like 4

Carpenter syndrome

AR

201000

RAB23

34. DYSOSTOSES WITH PREDOMINANT CRANIOFACIAL INVOLVEMENT AD

154500

5q32

TCOF1

Treacher Collins-Franceschetti syndrome 1

Mandibulo-facial dysostosis (Treacher Collins, FranceschettieKlein)

AD

154500

13q12.2

POLR1D

Polymerase (RNA) I polypeptide D

Mandibulo-facial dysostosis (Treacher Collins, FranceschettieKlein)

AR

154500

6p21.1

POLR1C

Polymerase (RNA) I polypeptide C

Oral-facial-digital syndrome type I (OFD1)

XLR

311200

Xp22.3

CXORF5

chr. X open reading frame 5

Weyeracrofacial (acrodental) dysostosis

AD

193530

4p16

EVC1

Ellis-van Creveld 1 protein

Endocrine-cerebro-osteodysplasia (ECO)

AR

612651

6p12.3

ICK

Intestinal cell kinase

Craniofrontonasal syndrome

XLD

304110

Xq13.1

EFNB1

Ephrin B1

Frontonasal dysplasia, type 1

AR

136760

1p13.3

ALX3

Aristaless-like-3

Frontonasal dysplasia, type 2

AR

613451

11p11.2

ALX4

Aristaless-like-4

Frontonasal dysplasia, type 3

AR

613456

12q21.3

ALX1

Aristaless-like 1

Hemifacial microsomia

SP/AD

164210

Miller syndrome (postaxial acrofacial dysostosis)

AR

263750

16q22

DHODH

Dihydroorotate dehydrogenase

Acrofacial dysostosis, Nager type

AD/AR

154400

Acrofacial dysostosis, Rodriguez type

AR

201170

419

Mandibulo-facial dysostosis (Treacher Collins, FranceschettieKlein)

HISTORY AND PHYSICAL EXAMINATION

PEDIATRIC BONE

Pfeiffer syndrome (FGFR1-related)

(Continued)

Classification of Genetic Skeletal DisordersdCont’d

Group/name of disorder

Inheritance

MIM No.

Locus

Gene

Protein

420

TABLE 17.1

35. DYSOSTOSES WITH PREDOMINANT VERTEBRAL WITH AND WITHOUT COSTAL INVOLVEMENT AD

176450

7q36

HLXB9

Homeobox gene HB9

Spondylocostal dysostosis type 1 (SCD1)

AR

277300

19q13

DLL3

Delta-like 3

Spondylocostal dysostosis type 2 (SCD2)

AR

608681

15q26

MESP2

Mesoderm posterior (expressed in) 2

Spondylocostal dysostosis type 3 (SCD3)

AR?

609813

7p22

LFNG

Lunatic fringe

Spondylocostal dysostosis type 4 (SCD4)

AR

17p13.1

HES7

Hairy-and-enhancer-of-split-7

15q26

MESP2

Mesoderm posterior (expressed in) 2

8q22.1

GDF6

Growth and differentiation factor 6

PEDIATRIC BONE

Spondylothoracic dysostosis

AR

KlippeleFeil anomaly with laryngeal malformation

AD

Spondylocostal/thoracic dysostosis, other forms

AD/AR

Cerebro-costo-mandibular syndrome (rib gap syndrome)

AD/AR

117650

Cerebro-costo-mandibular-like syndrome with vertebral defects

AR

611209

17q25

COG1

Component of oligomeric Golgi complex 1

Diaphanospondylodysostosis

AR

608022

7p14

BMPER

Bone morphogenetic protein-binding endothelial cell precursor-derived regulator

Ischiopatellar dysplasia (small patella syndrome)

AD

147891

17q21-q22

TBX4

T-box gene 4

Small patella-like syndrome with clubfoot

AD

5q31

PITX1

Paired-like homeodomain transcription factor 1 (pituitary homeobox 1)

Nail-patella syndrome

AD

161200

9q34.1

LMX1B

LIM homeobox transcription factor 1

Genitopatellar syndrome

AR?

606170

Ear-patella-short stature syndrome (MeiereGorlin)

AR

224690

2q35-36

IHH

Indian Hedgehog

BMPR1B

Bone Morphogenetic Protein Receptor, 1B

BMP2

Bone Morphogenetic Protein Type 2

20q11.2

GDF5

Growth and Differentiation Factor 5

9q22

ROR2

Receptor Tyrosine Kinase-like Orphan Receptor 2

148900

36. PATELLAR DYSOSTOSES

37. BRACHYDACTYLIES (WITH OR WITHOUT EXTRASKELETAL MANIFESTATIONS) Brachydactyly type A1

AD

112500

Brachydactyly type A1

AD

Brachydactyly type A2

AD

112600

Brachydactyly type A2

AD

112600

Brachydactyly type A2

AD

112600

Brachydactyly type A3

AD

112700

Brachydactyly type B

AD

113000

5p31 4q23

17. A DIAGNOSTIC APPROACH TO SKELETAL DYSPLASIAS

Currarino triad

AD

611377

17q

NOG

Noggin

Brachydactyly type C

AD, AR

113100

20q11.2

GDF5

Growth and Differentiation Factor 5

Brachydactyly type D

AD

113200

2q31

HOXD13

Homeobox D13

Brachydactyly type E

AD

113300

12p11.22

PTHLH

Parathyroid hormone-like hormone (Parathyroid hormone related peptide, PTHRP)

Brachydactyly type E

AD

113300

2q31

HOXD13

Homeobox D13

Brachydactyly e mental retardation syndrome

AD

600430

2q37.3

HDAC4

Histone deacetylase 4

Hyperphosphatasia with mental retardation, brachytelephalangy, and distinct face

AR

1p36.11

PIGV

Phosphatidylinositol-glycan biosynthesis class V protein (GPI mannosyltransferase 2)

Brachydactyly e hypertension syndrome (Bilginturan)

AD

112410

12p12.2-11.2

Brachydactyly with anonychia (Cooks syndrome)

AD

106995

17q24.3

SOX9

SRY e box 9

Microcephaly-oculo-digito-esophageal-duodenal syndrome (Feingold syndrome)

AD

164280

2p24.1

MYCN

nMYC oncogene

Hand-foot-genital syndrome

AD

140000

7p14.2

HOXA13

Homeobox A13

Brachydactyly with elbow dysplasia (Liebenberg syndrome)

AD

186550

Keutel syndrome

AR

245150

12p13.1-12.3

MGP

Matrix Gla protein

Albright hereditary osteodystrophy (AHO)

AD

103580

20q13

GNAS1

Guanine nucleotide binding protein of adenylatecyclase e subunit

RubinsteineTaybi syndrome

AD

180849

16p13.3

CREBBP

CREB-Binding Protein

RubinsteineTaybi syndrome

AD

180849

22q13

EP300

E1A-Binding Protein, 300-KD

CateleManzke syndrome

XLR?

302380

Brachydactyly, Temtamy type

AR

605282

Christian type brachydactyly

AD

112450

CoffineSiris syndrome

AR

135900

Mononen type brachydactyly

XLD?

301940

Poland anomaly

SP

173800

TBX3

T-box gene 3 Nipped-B-like

HISTORY AND PHYSICAL EXAMINATION

PEDIATRIC BONE

Brachydactyly type B2

38. LIMB HYPOPLASIA e REDUCTION DEFECTS GROUP Ulnar-mammary syndrome

AD

181450

de Lange syndrome

AD

122470

5p13.1

NIPBL

Fanconi anemia (see note below)

AR

227650

(several)

(several) (Continued)

421

Classification of Genetic Skeletal DisordersdCont’d Inheritance

MIM No.

Locus

Gene

Protein

Thrombocytopenia-absent radius (TAR)

AR?/AD?

274000

1q21.1

(several)

Thrombocythemia with distal limb defects

AD

3q27

THPO

Thrombopoietin

HolteOram syndrome

AD

142900

12q24.1

TBX5

T-box gene 5

Okihiro syndrome (Duane e radial ray anomaly)

AD

607323

20q13

SALL4

SAL-like 4

Cousin syndrome

AR

260660

1p13

TBX15

T-box gene 15

Roberts syndrome

AR

268300

8p21.1

ESCO2

Homolog of Establishment of Cohesion e 2

Split-hand-foot malformation with long bone deficiency (SHFLD1)

AD

119100

1q42.2-q43

Split-hand-foot malformation with long bone deficiency (SHFLD2)

AD

610685

6q14.1

Split-hand-foot malformation with long bone deficiency (SHFLD3)

AD

612576

17p13.1

Tibial hemimelia

AR

275220

Tibial hemimelia-polysyndactyly-triphalangeal thumb

AD

188770

Acheiropodia

AR

200500

7q36

LMBR1

Putative receptor protein

Tetra-amelia

XL

301090

Tetra-amelia

AR

273395

17q21

WNT3

Wingless-type MMTV integration site family, member 3

Ankyloblepharon-ectodermal dysplasia-cleft lip/palate (AEC)

AD

106260

3q27

P63 (TP63)

Tumor Protein p63

Ectrodactyly-ectodermal dysplasia cleft-palate syndrome Type 3 (EEC3)

AD

604292

3q27

P63 (TP63)

Tumor Protein p63

Ectrodactyly-ectodermal dysplasia cleft-palate syndrome type 1 (EEC1)

AD

129900

7q11.2-12.3

Ectrodactyly-ectodermal dysplasia-macular dystrophy syndrome (EEM)

AR

225280

16q22

CDH3

Cadherin 3

Limb-mammary syndrome (including ADULT syndrome)

AD

603273

3q27

P63 (TP63)

Tumor Protein p63

Split hand-foot malformation, isolated form, type 4 (SHFM4)

AD

605289

3q27

P63 (TP63)

Tumor Protein p63

Split hand-foot malformation, isolated form, type 1 (SHFM1)

AD

183600

7q21.3-22.1

Split hand-foot malformation, isolated form, type 2 (SHFM2)

XL

313350

Xq26

17. A DIAGNOSTIC APPROACH TO SKELETAL DYSPLASIAS

PEDIATRIC BONE

Group/name of disorder

422

TABLE 17.1

Split hand-foot malformation, isolated form, type 3 (SHFM3)

AD

600095

10q24

Dactylin

Split hand-foot malformation, isolated form, type 5 (SHFM5)

AD

606708

2q31

Al-AwadiRaaseRothschild limbepelvis hypoplasiaaplasia

AR

276820

3p25

WNT7A

Wingless-type MMTV integration site family, member 7A

Fuhrmann syndrome

AR

228930

3p25

WNT7A

Wingless-type MMTV integration site family, member 7A

RAPADILINO syndrome

AR

266280

8q24.3

RECQL4

RECQ Protein-like 4

AdamseOliver syndrome

AD/AR

100300

Femoral hypoplasia-unusual face syndrome (FHUFS)

SP/AD?

134780

Femur-fibula-ulna syndrome (FFU)

SP?

228200

Hanhart syndrome (Hypoglossia-hypodactylia)

AD

103300

Scapulo-iliac dysplasia (Kosenow)

AD

169550

7q36

SHH

Sonic Hedgehog

7q36

SHH

Sonic Hedgehog

HISTORY AND PHYSICAL EXAMINATION

FBXW4

39. POLYDACTYLY-SYNDACTYLY-TRIPHALANGISM GROUP PEDIATRIC BONE

AD

174400

Preaxialpolydactyly type 1 (PPD1)

AD

174400

Preaxialpolydactyly type 2 (PPD2)/ Triphalangeal thumb (TPT)

AD

174500

Preaxialpolydactyly type 3 (PPD3)

AD

174600

Preaxialpolydactyly type 4 (PPD4)

AD

174700

7p13

GLI3

Gli-Kruppel Family Member 3

Greigcephalopolysyndactyly syndrome

AD

175700

7p13

GLI3

Gli-Kruppel Family Member 3

PallistereHall syndrome

AD

146510

7p13

GLI3

Gli-Kruppel Family Member 3

Synpolydactyly (complex, fibulin1-associated)

AD

608180

22q13.3

FBLN1

Fibulin 1

Synpolydactyly

AD

186000

2q31

HOXD13

Homeobox D13

TowneseBrocks syndrome (renal-ear-anal-radial syndrome)

AD

107480

16q12.1

SALL1

SAL-like 1

Lacrimo-auriculo-dento-digital syndrome (LADD)

AD

149730

10q26.12

FGFR2

Fibroblast growth factor receptor 2

Lacrimo-auriculo-dento-digital syndrome (LADD)

AD

149730

4p16.3

FGFR3

Fibroblast growth factor receptor 3

Lacrimo-auriculo-dento-digital syndrome (LADD)

AD

149730

5p13-p12

FGF10

Fibroblast growth factor 10

Acrocallosal syndrome

AR

200990

7p13

Acro-pectoral syndrome

AD

605967

7q36

Acro-pectoro-vertebral dysplasia (F-syndrome)

AD

102510

2q36

Mirror-image polydactyly of hands and feet (LaurineSandrow syndrome)

AD

135750

7q36

SHH

Sonic Hedgehog

423

Preaxialpolydactyly type 1 (PPD1)

(Continued)

Group/name of disorder

Inheritance

MIM No.

Locus

Gene

Protein

CenanieLenz syndactyly

AR

212780

11p11.2

LRP4

low density lipoprotein receptor-related protein 4

CenanieLenz like syndactyly

SP (AD?)

15q13-q14

GREM1, FMN1

Gremlin 1, Formin 1

Oligosyndactyly, radio-ulnar synostosis, hearing loss and renal defects syndrome

SP (AR?)

15q13-q14

FMN1

Formin 1

Syndactyly, MalikePercin type

AD

609432

17p13.3

STAR syndrome (syndactyly of toes, telecanthus, anoand renal malformations)

XL

300707

Xq28

Syndactyly type 1 (IIIeIV)

AD

185900

2q34-36

Syndactyly type 3 (IVe-V)

AD

185900

6q21-23

GJA1

Syndactyly type 4 (IeV) Haas type

AD

186200

7q36

SHH

Syndactyly type 5 (syndactyly with metacarpal and metatarsal fusion)

AD

186300

2q31

HOXD13

Syndactyly with craniosynostosis (Philadelphia type)

AD

601222

2q35-36.3

Syndactyly with microcephaly and mental retardation (Filippi syndrome)

AR

272440

Meckel syndrome type 1

AR

249000

17q23

Meckel syndrome type 2

AR

603194

11q

Meckel syndrome type 3

AR

607361

8q21

TMEM67

Meckel syndrome type 4

AR

611134

12q

CEP290

Meckel syndrome type 5

AR

611561

16q12.1

RPGRIP1L

Meckel syndrome type 6

AR

612284

4p15

CC2D2A

424

TABLE 17.1 Classification of Genetic Skeletal DisordersdCont’d Mirror-image polydactyly of hands and feet (LaurineSandrow syndrome)

FAM58A

PEDIATRIC BONE

MKS1

40. DEFECTS IN JOINT FORMATION AND SYNOSTOSES Multiple synostoses syndrome type 1

AD

186500

17q22

NOG

Noggin

Multiple synostoses syndrome type 2

AD

186500

20q11.2

GDF5

Growth and Differentiation Factor 5

Multiple synostoses syndrome type 3

AD

612961

13q11-q12

FGF9

Proximal symphalangism type 1

AD

185800

17q22

NOG

Noggin

Proximal symphalangism type 2

AD

185800

20q11.2

GDF5

Growth and Differentiation Factor 5

Radio-ulnar synostosis with amegakaryocytic thrombocytopenia

AD

605432

7p15-14.2

HOXA11

Homeobox A11

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Sonic Hedgehog

HISTORY AND PHYSICAL EXAMINATION

reported in hypochondroplasia and achondroplasia, but their significance remains controversial [22]. 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 [23,24]. DyggveeMelchioreClausen and dysosteosclerosis are both rare dysplasias associated with severe to profound mental retardation [25,26]. 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 [27]. Certain dysplasias are more common among certain ethnic groups, such as cartilage-hair hypoplasia in the Amish [28] and spondyloepimetaphyseal dysplasia with joint laxity in the South African Afrikaner population [29]. 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

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determine the upper segment and then calculating the upper segment-to-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 disorderspecific growth curves. Specialized growth curves have been developed for achondroplasia, pseudoachondroplasia, spondyloepiphyseal dysplasia congenita, and diastrophic dysplasia [13,30]. 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 ConradieHu¨nermann) [31] (Fig. 17.2). 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 (Ellisevan Creveld syndrome) [32]. 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 [33] (Fig. 17.3). In general, children with skeletal dysplasias do not show dysmorphic features of the head and neck, but one important feature is the PierreeRobin sequence seen in the type II collagenopathies and campomelic dysplasia [9] (Fig. 17.4).

FIGURE 17.2 A 21-year-old woman with chondrodysplasia punctata ConradieHu¨nermann 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 clubfeet, 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).

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(A)

(B)

FIGURE 17.3 (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.

(A)

(B)

FIGURE 17.4 (A) A newborn with campomelic dysplasia and typical craniofacial features. He has midface hypoplasia, protuberant eyes, and PierreeRobin 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 PierreeRobin sequence. She has a proven type II collagenopathy with a 9 base pair deletion in exon 41.

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

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, lateral thoracic and lumbar spine,

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B

FIGURE 17.5 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.

thorax, pelvis with hips, long bones, and hands [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. 17.5). This pattern of features is specific to PSACH and sufficient for making the diagnosis [34]. 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 [35]. This pattern of affected regions helps to differentiate CHH from the other metaphyseal dysplasias and nutritional rickets [36]. The pattern is key to the diagnosis because few radiographic features are specific. One notable exception is the finding of iliac horns (Fig. 17.6) in nail-patella syndrome, which are essentially pathognomonic for the disorder [37]. The distribution of affected areas can also be an important indicator, for example, in brachyolmia (Fig. 17.7) the radiographic abnormalities are limited to the spine [38]. 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 [39,40] (Fig. 17.8). Osteopenia is seen in another group of disorders, including osteogenesis imperfecta and hypophosphatasia [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

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FIGURE 17.6 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.

(A)

chondrodysplasia punctata, have multiple coronal clefts [42]. One of the more specific findings in the spine is the “double hump” seen in DyggvveeMelchioreClausen syndrome [25] (Fig. 17.9). Again, it is important to keep in mind the “fourth dimension” or the evolution of findings over time [43]. 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 [44,45]. 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

(B)

FIGURE 17.7 (A) Lateral radiograph of the lumbar spine of an adult individual with brachyolmia showing generalized mild platyspondyly and irregularity (proven TRPV4 mutation). (B) Lateral radiograph of the spine in a child with metatropic dysplasia (a molecularly related condition but with much more severe changes). There is significant platyspondyly and kyphosis and at this age increased intervertebral distance.

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FIGURE 17.9

Lateral radiograph of the lumbar spine of a teenage girl with DyggveeMelchioreClausen 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 of the few skeletal dysplasias associated with developmental delay.

FIGURE 17.8

AP radiograph of the left hand of a 4.5-year-old boy with pyenodyostosis. 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.

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 diastrophic dysplasia and Desbuquois dysplasia [33,46]. 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 [47]. The classic example is type 12 cone epiphyses in the trichorhinophalangeal disorders [48] (Fig. 17.10).

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 syndrome) [49]. 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 [50]. Several children with acampomelic campomelic dysplasia due to point mutations in SOX9 or chromosomal rearrangement have been reported [51,52] (Fig. 17.11). Campomelia is also seen in other dysplasias, such as StuveeWiedemann syndrome [53], and more commonly as a reflection of fractures/bone fragility in osteogenesis imperfecta [54]. Campomelia can also be seen in nonskeletal dysplastic conditions, such as MeckeleGruber syndrome, presumably as a consequence of fetal hypokinesia [55]. Like campomelia, chondrodysplasia punctata is a radiographic sign and not a specific diagnostic entity [56]. 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,

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(A)

(B)

FIGURE 17.10

(A) Radiograph of the left hand of a 3-year-old boy with LangereGiedion 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).

intrauterine infections, chromosomal abnormalities, and some metabolic diseases [57]. Punctate epiphyses disappear with age as the multiple calcified centers coalesce, reinforcing the need for an early and complete skeletal survey if a dysplasia is suggested. Radiographic views of the pelvis can also be important in the differential diagnosis. In a child with ATD,

FIGURE 17.11 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.

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 [58]. Although the pelvic abnormalities are clinically silent, they are diagnostically important (Fig. 17.12). 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 [59,60].

FIGURE 17.12 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).

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BIOCHEMICAL INVESTIGATIONS Biochemical investigations are not often useful but, in certain instances, can be invaluable. Classic examples of dysplasias diagnosed in this manner are the mucopolysaccharidoses 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 [61]. 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

FIGURE 17.14 Radiograph of the left hand of a 10-month-old boy with Hurler disease (a-iduronidase deficiency). Of note is the marked proximal pointing of the metacarpals, resembling a sharpened pencil.

in Morquio syndrome and, along with the platyspondyly, result in the gibbus deformity, which is frequently the presenting sign of the disorder [62] (Fig. 17.13). There are also characteristic changes in the hands, including short proximally pointed metacarpals and bullet-shaped phalanges (Fig. 17.14). Wide ribs that narrow posteriorly are a frequent sign of dysostosis multiplex [61] (Fig. 17.15). Abnormalities in sterol metabolism have been recognized as causing several forms of chondrodysplasia punctata, including chondrodysplasia punctata ConradieHu¨nermann and congenital hemidysplasia with ichthyosis and limb defects [31,63]. Sterol analysis was useful to show that these were metabolically related disorders and is now used for confirmation of diagnosis [63]. Another example of biochemical analysis is the measurement of GNAS1 function in the diagnosis of Albright hereditary osteodystrophy [64]. Quantitative analysis of this protein’s activity in the erythrocyte membrane has been used for diagnosis prior to gene discovery [65].

CARTILAGE HISTOLOGY

FIGURE 17.13

Lateral radiograph of a 4.5-year-old boy 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 dysostosis multiplex, with flattening and midanterior beaking. There is a kyphosis of approximately 15 .

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

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useful criteria for the distinction and diagnosis of bone dysplasias: 1. Where is the primary abnormality e in bone tissue (e.g. osteogenesis imperfecta), in cartilage tissue (e.g. achondrogenesis 1b and 2), or at the growth plate (thanatophoric dysplasia)? 2. Is the extracellular matrix affected or is it microscopically normal? 3. Are the chondrocytes morphologically normal or do they show changes in shape (e.g. spindle shaped as in fibrochondrogenesis or ballooned as in collagen 2 dysplasias)? 4. Within the growth plate, are the relative widths of the columnar zone, the hypertrophic chondrocyte zone, and the provisional calcification zone correct?

FIGURE 17.15 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.

Routine hematoxylin and eosin staining is of limited value because of the poor affinity of cartilage matrix for these dyes. Whenever possible, AzaneMallory 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.

FIGURE 17.16 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 fibro-osseous 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 20 . (See color plate section.)

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MOLECULAR BASIS

FIGURE 17.17 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 section.)

Fibrochondrogenesis is a lethal disorder named for its unusual histological pattern [66]. 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 [66,67]. The molecular basis of the disorder was recently elucidated using whole genome single nucleotide polymorphism (SNP) genotyping and surprisingly mutations were found in COL11A1 [68]. The columnar zone is reduced in width in the thanatophoric dysplasia/achondroplasia group, whereas it can be markedly wider than normal in hypophosphatasia [69] (Fig. 17.16). The importance of cartilage 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) [70,71] (Fig. 17.17). 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 dyssegmental dysplasia to the perlecan gene by virtue of histologic analogies to a perlecan mouse knockout) but misleading in others [72]. 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 II defect or a metabolic defect leading to cellular demise in diastrophic dysplasia. The intermediate DTDST defect, atelosteogenesis 2, was separated from severe diastrophic dysplasia despite radiographic and histologic evidence of a close relationship between the two.

MOLECULAR BASIS As the molecular basis has become known for increasingly more skeletal dysplasias, mutation analysis has

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become an invaluable 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 CHH and that hair anomalies and immunodeficiency are not obligate features of CHH [73]. Similarly, molecular analysis has revealed that Ehlerse Danlos syndrome type 7 is caused by splicing mutations in the type 1 collagen genes, thus explaining the phenotypic overlap between this disorder and osteogenesis imperfecta [74].

PRENATAL DETECTION OF SUSPECTED SKELETAL DYSPLASIA Ultrasonographic examination during pregnancy is a 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 non-lethal or variably lethal skeletal dysplasias are increasingly detected [15,75]. The most common findings prompting suspicion of a skeletal dysplasia are short limbs for gestational age or polyhydramnios [76]. 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 [15]. Increasingly, CT scans are being employed as part of the diagnostic strategy despite lack of evidence that this improves diagnostic accuracy [77] (Fig. 17.18). Prenatally, it is most important to determine whether or not the fetus actually has a skeletal dysplasia and, if 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 [78] (Fig. 17.19). 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 [78]. However, not all short limbs are due to dysplasia, and

FIGURE 17.18

Fetal CT done at 30 weeks’ gestation after defective spinal ossification was noted on ultrasound. This image is a particularly nice demonstration of findings associated with type II collagen disorders: defective vertebral body ossification, short limbs, rounded upper border of the iliac wings, and delayed pubic bone ossification. Image generously provided by Drs K. Maeda and T. Kaji, University of Tokushima, Japan.

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 [79]. 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, 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 [80]. 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

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CONCLUSION

(A)

(B)

FIGURE 17.19 (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. TDI causes bent/angulated femurs. TD2 is associated with cloverleaf skull caused by multiple craniosynostoses and relatively straight femurs.

(A)

(B)

FIGURE 17.20 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.

type II collagenopathies [81] (Fig. 17.20). 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. clubfeet and contractures), and congenital heart defects [79]. Accurate prenatal diagnosis of skeletal dysplasias remains problematic. In order to ensure appropriate counseling, post-termination 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 pattern-recognition 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. Pattern-recognition skills will

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perhaps become even more important in the years to come when we will be presented with a barrage of genomic information and be asked to pinpoint the cause of the patient’s disorder. Even before the advent of targeted therapies, establishment of a precise and correct diagnosis is important for appropriate counseling regarding potential complications, expected adult height, and recurrence risk.

References [1] Warman M, Cormier-Daire V, Hall C, et al. Nosology of genetic skeletal disorders: 2010 Revision. Am J Med Genet 2011 (in press). [2] Ala-Kokko L, Baldwin CT, Moskowitz RW, Prockop DJ. 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 1990;87:6565e8. [3] Lachman RS. Radiologic and imaging assessment of the skeletal dysplasias. In: Kelnar CJH, Savage MO, Stirling HF, Saenger P, editors. Growth Disorders. London: Chapman & Hall; 1998. p. 251e64. [4] Andersen PE, Hauge M. Congenital generalised bone dysplasias: A clinical, radiological, and epidemiological survey. J Med Genet 1989;26:37e44. [5] Orioli IM, Castilla EE, Barbosa-Neto JG. The birth prevalence rates for the skeletal dysplasias. J Med Genet 1986;23:328e32. [6] Spranger J. Changes in clinical practice with the unravelling of diseases: Connective tissue disorders. J Inherit Metab Dis 2001;24:117e26. [7] International Working Group on Constitutional Diseases of Bone. International classification of osteochondrodysplasias. Eur J Pediatr 1992;151:407e15. [8] Butler NR. The classification and registration of bone dysplasias. Postgrad Med J 1977;53:427e8. [9] Spranger J, Winterpacht A, Zabel B. The type II collagenopathies: a spectrum of chondrodysplasias. Eur J Pediatr 1994;153:56e65. [10] Superti-Furga A, Bonafe L, Rimoin DL. Molecular-pathogenetic classification of genetic disorders of the skeleton. Am J Med Genet 2002;106:282e93. [11] Rimoin DL. Molecular defects in the chondrodysplasias. Am J Med Genet 1996;63:106e10. [12] Jones KL. Smith’s Recognizable Patterns of Human Malformation. 6th ed. Philadelphia: Elsevier Saunders; 2006. [13] Horton WA, Rotter JI, Rimoin DL, Scott CI, Hall JG. Standard growth curves for achondroplasia. J Pediatr 1978;93:435e8. [14] McKeand J, Rotta J, Hecht JT. Natural history study of pseudoachondroplasia. Am J Med Genet 1996;63:406e10. [15] Krakow D, Alanay Y, Rimoin LP, et al. Evaluation of prenatal-onset osteochondrodysplasias by ultrasonography: a retrospective and prospective analysis. Am J Med Genet 2008;149A:1917e24. [16] Schultz C, Langer LO, Laxova R, Pauli RM. Atelosteogenesis type III: Long term survival prenatal diagnosis, and evidence for dominant transmission. Am J Med Genet 1999;83:28e42. [17] Unger S, Lausch E, Rossi A, et al. Phenotypic features of carbohydrate sulfotransferase 3 (CHST3) deficiency in 24 patients: congenital dislocations and vertebral changes as principal diagnostic features. Am J Med Genet 2010;152A:2543e9. [18] Hall JG. Arthrogryposis multiplex congenita: Etiology, genetics, classification, diagnostic approach, and general aspects. J Pediatr Orthop 1997;B6:159e66. [19] Makitie O, Pukkala E, Kaitila I. Increased mortality in cartilagehair hypoplasia. Arch Dis Child 2001;84:65e7.

[20] Ozcay F, Derbent M, Demirhan B, Tokel K, Saatci U. A family with Jeune syndrome. Pediatr Nephrol 2001;16:623e6. [21] Todorov AB, Scott CI, Warren AE, Leeper JD. Developmental screening tests in achondroplastic children. Am J Med Genet 1981;9:19e23. [22] Thompson NM, Hecht JT, Bohan TP, et al. Neuroanatomic and neuropsychological outcome in school-age children with achondroplasia. Am J Med Genet 1999;88:145e53. [23] Bellus GA, Bamshad MJ, Przylepa KA, et al. Severe achondroplasia with developmental delay and acanthosis nigricans (SADDAN): Phenotypic analysis of a new skeletal dysplasia caused by a Lys650Met mutation in fibroblast growth factor receptor 3. Am J Med Genet 1999;85:53e65. [24] Tavormina PL, Bellus GA, Webster MK, et al. A novel skeletal dysplasia with developmental delay and acanthosis nigricans is caused by a Lys650met mutation in the fibroblast growth factor receptor 3 gene. Am J Hum Genet 1999;64:722e31. [25] Toledo SP, Saldanha PH, Lamego C, Mourao PA, Dietrich CP, Mattar E. Dyggve-Melchior-Clausen syndrome: Genetic studies and report of affected sibs. Am J Med Genet 1979;4:255e61. [26] Pascual-Castroviejo I, Casas-Fernandez C, Lopez-Martin V. X-linked dysosteosclerosis: Four familial cases. Eur J Pediatr 1977;126:127e38. [27] Vajo Z, Francomano CA, Wilkin DG. The molecular and genetic basis of fibroblast growth factor receptor 3 disorders: The achondroplasia family of skeletal dysplasias, Muenke craniosynostosis and Crouzon syndrome with acanthosis nigricans. Endocr Rev 2000;21:23e39. [28] McKusick VA, Eldridge R, Hostetler J, Egeland JA, Ruangwit U. Dwarfism in the Amish. II. Cartilage-hair hypoplasia. Bull Johns Hopkins Hosp 1965;116:285e326. [29] Beighton P, Kozlowski K, Gericke G, Wallis G, Grobler L. Spondyloepimetaphyseal dysplasia with joint laxity and severe, progressive kyphoscoliosis. S Afr Med J 1983;64:772e5. [30] Horton WA, Hall JG, Scott CI, Pyeritz RE, Rimoin DL. Growth curves for height for diastrophic dysplasia, spondyloepiphyseal dysplasia congenita, and pseudoachondroplasia. Am J Dis Child 1982;136:316e9. [31] Kelley RI, Wilcox WG, Smith M, Kratz LE, Moser A, Rimoin DL. Abnormal sterol metabolism in patients with ConradieHunermanneHapple syndrome and sporadic lethal chondrodysplasia punctata. Am J Med Genet 1999;83:213e9. [32] Digilio MC, Marino B, Giannotti A, Dallapiccola B. Atrioventricular canal defect and postaxial polydactyly indicating phenotypic overlap of Ellisvan Creveld and Kaufman-McKusick syndromes. Pediatr Cardiol 1997;18:74e5. [33] Rossi A, Superti-Furga A. Mutations in the diastrophic dysplasia sulfate transporter (DTDST) gene (SLC26A2); 22 novel mutations, mutation review, associated skeletal phenotypes, and diagnostic relevance. Hum Mutat 2001;17:159e71. [34] Wynne-Davies R, Hall CM, Young ID. Pseudoachondroplasia: Clinical diagnosis at different ages and comparison of autosomal dominant and recessive types. A review of 32 patients (26 kindreds). J Med Genet 1986;23:425e34. [35] Glass RBJ, Tifft CJ. Radiologic changes in infancy in McKusick cartilage hair hypoplasia. Am J Med Genet 1999;86:312e5. [36] Thacher TD, Fischer PR, Pettifor JM, Lawson JO, Manaster BJ, Reading JC. Radiographic scoring method for the assessment of the severity of nutritional rickets. J Trop Pediatr 2000;46:132e9. [37] Karabulut N, Ariyurek M, Erol C, Tacal T, Balkanci F. Imaging of “iliac horns” in nail-patella syndrome. J Cornput Assist Tomogr 1996;20:530e1. [38] Shohat M, Lachman R, Gruber H, Rimoin DL. Brachyolmia: radiographic and genetic evidence of heterogeneity. Am J Med Genet 1989;33:209e19.

PEDIATRIC BONE

REFERENCES

[39] Wilson CJ, Vellodi A. Autosomal recessive osteopetrosis: Diagnosis, management, and outcome. Arch Dis Child 2000;83:449e52. [40] Sedano HD, Gorlin RJ, Anderson VE. Pycnodysostosis: Clinical and genetic considerations. Am J Dis Child 1968;116:70e7. [41] Byers PH. Osteogenesis imperfecta: Perspectives and opportunities. Curr Opin Pediatr 2000;12:603e9. [42] Westvik J, Lachman RS. Coronal and sagittal clefts in skeletal dysplasias. Pediatr Radiol 1998;28:764e70. [43] Giedion A. The weight of the fourth dimension for the diagnosis of genetic bone disease. Pediatr Radiol 1994;24:387e91. [44] Whyte MP, Gottesman GS, Eddy MC, McAlister WH. X-linked recessive spondyloepiphyseal dysplasia tarda. Clinical and radiographic evolution in a 6-generation kindred and review of the literature. Medicine (Baltimore) 1999;78:9e25. [45] Langer LO, Beals RK, Scott CI. Sponastrime dysplasia: Diagnostic criteria based on five new and six previously published cases. Pediatr Radiol 1997;27:409e14. [46] Jequier S, Perreault G, Maroteaux P. Desbuquois syndrome presenting with severe neonatal dwarfism, spondyloepiphyseal dysplasia and advanced carpal bone age. Pediatr Radiol 1992;22:440e2. [47] Giedion A. Acrodysplasias. Clin Orthop Rei Res 1976;114:107e15. [48] Giedion A. Phalangeal cone-shaped epiphyses of the hand: Their natural history, diagnostic sensitivity, and specificity in cartilage hair hypoplasia and the trichorhinophalangeal syndromes I and II. Pediatr Radiol 1998;28:751e8. [49] Patton MA, Afzal AR. Robinow syndrome. J Med Genet 2002;39:305e10. [50] Mansour S, Hall CM, Pembrey ME, Young ID. A clinical and genetic study of campomelic dysplasia. J Med Genet 1995;32:415e20. [51] Thong M-K, Scherer G, Kozlowski K, Haan E, Morris L. Acampomelic campomelic dysplasia with SOX9 mutation. Am J Med Genet 2000;93:421e5. [52] Ninomiya S, Narahara K, Tsuji K, Yokoyama Y, Ito S, Seino Y. Acampomelic campomelic syndrome and sex reversal associated with de novo t(12;17) translocation. Am J Med Genet 1995;56:31e4. [53] Cormier-Daire V, Munnich A, Lyonnet S, et al. Presentation of six cases of Stuve-Wiedemann syndrome. Pediatr Radiol 1998;28:776e80. [54] Thompson EM. Non-invasive prenatal diagnosis of osteogenesis imperfecta. Am J Med Genet 1993;45:201e6. [55] Majewski F, Stob H, Goecke T, Kemperdick H. Are bowing of long tubular bones and preaxial polydactyly signs of the Meckel syndrome? Hum Genet 1983;65:125e33. [56] Poznanski AK. Punctate epiphyses: A radiological sign, not a disease. Pediatr Radiol 1994;24:418e24. [57] Patel MS, Callahan JW, Zhang S, et al. Early-infantile galactosialidosis: Prenatal presentation and postnatal follow-up. Am J Med Genet 1999;85:38e47. [58] Burn J, Hall C, Marsden D, Matthew DJ. Autosomal dominant thoracolaryngopelvic dysplasia: Barnes syndrome. J Med Genet 1986;23:345e9. [59] Borochowitz Z, Jones KL, Silbey R, Adomian G, Lachman R, Rimoin DL. A distinct lethal neonatal chondrodysplasia with snail-like pelvis: Schneckenbecken dysplasia. Am J Med Genet 1986;25:47e59. [60] Cormire-Daire V, Savarirayan R, Lachman RS, et al. “Baby rattle” pelvis dysplasia. Am J Med Genet 2001;100:37e42. [61] Spranger JW. Catabolic disorders of complex carbohydrates. Postgrad Med J 1977;53:441e8. [62] Northover H, Cowie RA, Wraith JE. Mucopoly-saccharidosis type IVA (Morquio syndrome): A clinical review. J Inherit Metab Dis 1996;19:357e65.

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[63] Grange DK, Kratz LE, Braverman NE, Kelley RI. CHILD syndrome caused by deficiency of 3b-hydroxysteroid-D8, D7isomerase. Am J Med Genet 2000;90:328e35. [64] Patten JL, Johns DR, Valle D, et al. Mutation in the gene encoding the stimulatory G protein of adenylate cyclase in Albright’s hereditary osteodystrophy. N Engl J Med 1990;322:1412e9. [65] Levine MA, Downs RW, Singer M, Marx SJ, Aurbach GD, Spiegel AM. Deficient activity of guanine nucleotide regulatory protein in erythrocytes from patients with pseudohypoparathyroidism. Biochem Biophys Res Commun 1980;94:1319e24. [66] Lazzaroni-Fossati F, Stanescu V, Stanescu R, Serra G, Magliano P, Maroteaux P. Fibrochondrogenesis. Arch Fr Pediatr 1978;35:1096e104. [67] Whitley CB, Langer LO, Ophoven J, et al. Fibrochondrogenesis: Lethal, autosomal recessive chondrodysplasia with distinctive cartilage histology. Am J Med Genet 1984;19:256e7. [68] Tompson SW, Bacino CA, Safina NP, et al. Fibrochondrogenesis results from mutations in the COL11A1 type XI collagen gene. Am J Hum Genet 2010;87:708e12. [69] Wilcox WR, Tavormina PL, Kkrakow D, et al. Molecular, radiologie, and histopathologic correlations in thanatophoric dysplasia. Am J Med Genet 1998;78:274e81. [70] Superti-Furga A. A defect in the metabolic activation of sulfate in a patient with achondrogenesis IB. Am J Hum Genet 1994;55:1137e45. [71] Korkko J, Cohn DH, Ala-Kokko L, Krakow D, Prockop DJ. Widely distributed mutations in the COL2A1 gene produce achondrogenesis type II/hypochondrogenesis. Am J Med Genet 2000;92:95e100. [72] Arikawa-Hirasawa E, Wilcox WR, Le AH, et al. Dyssegmental dysplasia, Silverman-Handmaker type, is caused by functional null mutations of the perlecan gene. Nat Genet 2001;27:431e4. [73] Bonafe L, Schmitt K, Eich G, Giedion A, Superti-Furga A. RMRP gene sequence analysis confirms a cartilage-hair hypoplasia variant with only skeletal manifestations and reveals a high density of single nucleotide polymorphisms. Clin Genet 2002;61:146e51. [74] Weil D, D’Alessio M, deWet W, Cole WG, Chan D, Bateman JF. A base substitution in the exon of a collagen gene causes alternative splicing and generates a structurally abnormal polypeptide in a patient with Ehlers-Danlos syndrome type VIL. EMBO J 1989;8:1705e10. [75] Superti-Furga A, Unger S. Prenatal diagnosis of disorders of bone and connective tissue. In: Milunsky A, Milunsky J, editors. Genetic Disorders and the Fetus: Diagnosis, Prevention, and Treatment. New Jersey, USA: Wiley-Blackwell; 2010. p. 680e704. [76] Tretter AE, Saunders RC, Meyers CM, et al. Antenatal diagnosis of lethal skeletal dysplasias. Am J Med Genet 1998;75:518e22. [77] Cassart M, Massez A, Cos T, et al. Contribution of threedimensional computed tomography in the assessment of fetal skeletal dysplasia. Ultrasound Obstet Gynecol 2007;29:537e43. [78] Ramus RM, Martin LB, Twickler DM. Ultrasonographic prediction of fetal outcome in suspected skeletal dysplasias with use of the femur length-to-abdominal circumference ratio. Am J Obstet Gynecol 1998;179:1348e52. [79] Gaffney G, Manning N, Boyd PA, Ra V, Gould S, Chamberlain P. Prenatal sonographic diagnosis of skeletal dysplasias e A report of the diagnostic and prognostic accuracy in 35 cases. Prenatal Diagn 1998;18:357e62. [80] Hassan J, Sepulveda W, Teixeira J, Garrett C, Fisk NM. Prenatal sonographic diagnosis of cleidocranial dysostosis. Prenatal Diagn 1997;17:770e2. [81] Turner GM, Twining P. The facial profile in the diagnosis of fetal abnormalities. Clin Radiol 1993;47:389e95.

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The Spectrum of Pediatric Osteoporosis Maria Luisa Bianchi 1, Francis H. Glorieux 2 1

Bone Metabolism Unit, Istituto Auxologico Italiano IRCCS, Milan, Italy 2 Professor of Surgery, Pediatrics and Human Genetics, McGill University; Adjunct Professor of Pediatrics, University of Montreal; Director of Research, Shriners Hospital for Children, Genetics Unit, Montreal, Quebec, Canada

INTRODUCTION Osteoporosis is the most common metabolic bone disorder in adults, and remains a major health problem worldwide [1,2]. There is increasing awareness that osteoporosis may also affect children and adolescents, either because of intrinsic skeletal defects (primary osteoporosis) or as a complication of other diseases or their treatment (secondary osteoporosis) [3]. The purpose of this chapter is to provide an approach to the diagnosis and treatment of pediatric osteoporosis, on the basis of the current literature.

DEFINITION AND DIAGNOSIS OF OSTEOPOROSIS IN PEDIATRIC PATIENTS Osteoporosis is defined in adults as low bone mineral mass and deterioration of bone microarchitecture, with an increased risk of fragility (atraumatic) fractures. It is preceded by a less severe condition named osteopenia (scarcity of bone), the simple reduction of bone mass for gender and age. In children, speaking of “deterioration” of bone tissue may be inappropriate, as the bone fragility of pediatric osteoporosis is more likely to result from failure to develop an adequate bone microarchitecture during growth. Moreover, unlike in adults, low bone mass alone is currently not considered a strong predictor of fracture risk in children and adolescents and, as discussed below, a diagnosis of osteoporosis in children is only made in the presence of low bone mass and a history of fragility fractures. Osteoporosis should not be confused with osteomalacia (usually called rickets in children), which is a mineralization defect of bone tissue. Both osteomalacia

Pediatric Bone, Second Edition DOI: 10.1016/B978-0-12-382040-2.10018-8

and osteoporosis are associated with low bone mass (“bone mineral content”, BMC) and bone mineral density (BMD), but due to completely different mechanisms. Osteoporosis is caused either by insufficient formation or increased resorption of bone tissue. Osteomalacia is an impaired mineralization of bone matrix, due to different causes, mostly related to a deficiency of vitamin D. Various methods have been employed to assess bone health in children and adolescents. Evaluation of bone pain and mobility, and x-ray confirmation of fractures are often the first diagnostic step. Low bone mass or osteoporosis are detected by bone densitometry, quantitative computed tomography (QCT), peripheral quantitative computed tomography (pQCT), quantitative ultrasound (QUS) and, in special cases, bone histology/histomorphometry. The assessment of the basic bone metabolism parameters, such as serum/ urinary levels of calcium, phosphate, vitamin D, parathyroid hormone (PTH), is a useful diagnostic complement, while that of bone turnover markers can be useful in special conditions [4]. A detailed discussion of non-invasive techniques for bone mass measurement is presented in Chapter 13, of bone turnover markers in Chapter 15, and of bone histomorphometry in Chapter 16. Currently, the most widely used tool for the assessment of bone mineralization and the diagnosis of osteoporosis at any age is dual-energy x-ray absorptiometry (DXA). DXA is the preferred method for quantifying BMC and BMD because of its precision, reproducibility, speed, minimal exposure to radiation, and worldwide availability. The existence of pediatric normative data for different populations makes it highly suitable for children [5e7]. Its limitation is that it cannot measure bone volume, so that only an “areal” BMD (aBMD) can

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be calculated (see below). Only DXA will be discussed in some detail in this chapter. QCT is used only in special cases, because of the high radiation dose. pQCT allows independent measurement of bone size and volumetric BMD (vBMD), but has limited use in pediatrics because of scarce availability of pQCT instruments, few reference data for children, lack of uniformity in measurement site, scan acquisition and analysis, and outcome measures. The requirement that the patient must remain still for several minutes to complete the test is a further drawback. QUS is appealing because of the absence of ionizing radiations, lower cost, and portability. However, the available devices evaluate different bone parameters at different skeletal sites and the results are not easily comparable, normative data for children are scarce, and the method’s validity has not yet been established.

Basic Aspects of DXA Densitometry DXA measures the BMC (in grams) of the scanned bone, but can only measure the projection area of the scanned bone in the coronal plane, not the bone volume. Thus, DXA can only calculate aBMD (BMC in grams per cm2 of bone projection area) and not vBMD (BMC in grams per cm3 of bone volume). For mathematical reasons, aBMD overestimates the true (volumetric) BMD as bone size increases or, in other words, if two bones of equal vBMD are analyzed with DXA, the smaller bone will have a lower aBMD than the larger bone [5e7]. In adults, aBMD is a reasonable surrogate for vBMD, since BMC may change but bone size remains relatively stable after the end of growth, which means that any observed changes in aBMD will reflect changes in vBMD. This is not valid for children and adolescents, in which both BMC and bone size are progressively changing and, for this reason, the interpretation of aBMD values in children and adolescents is much more complex than in adults. This regards both the comparison of a child with a control population and the follow up of a subject during growth. Ideally, only gender- and age-matched children of equal body (bone) size should be compared, and since this is usually impossible, appropriate corrections for size must be applied. If aBMD (or aBMD change) is interpreted without considering bone size, the true density (or change) may be overestimated and a diagnosis of low bone density (or of a decrease in bone density) may be missed. To overcome this limitation, some methods to estimate vBMD with DXA have been proposed. For example, a vertebral body can be likened to a cube or a cylinder [8,9], so that an approximate volume can be calculated from the projection height and width, and a bone mineral “apparent” density

(BMAD) can be obtained by dividing the BMC by the estimated volume of bone in the region of interest. Another simple correction is to correct BMC for the subject’s height. This correction has been accepted at a recent consensus conference on pediatric densitometry [7], but no correction method is universally accepted.

The Diagnosis of Osteoporosis For adults, specific diagnostic criteria for osteopenia and osteoporosis based on DXA densitometry have been developed. According to the World Health Organization, an aBMD measurement of 2.5 SD or more below the mean of gender-matched healthy young adults (T-score
2.5) indicates osteoporosis. A T-score comprised between 1 and 2.5 indicates osteopenia [2]. To date, there are no established criteria to define osteoporosis in pediatric patients and there is no consensus on a diagnosis of osteoporosis based solely on the BMD value. Moreover, since the T-score is the result of the comparison of a subject’s BMD with the mean BMD of healthy young adults, it can only be used for adults who have already reached their peak bone mass (PBM). It is obviously incorrect to use the T-score in young patients who have not yet achieved their PBM. The Z-score (i.e. the number of SD deviations that a subject’s BMD is below the mean value of ageand gender-matched healthy controls) must be used instead. In adults, BMD is considered a strong predictor of fracture risk, and for postmenopausal women a 1 SD reduction in BMD from the healthy young adult mean corresponds to a two- to threefold increased risk for fractures [2]. In growing subjects, there is only preliminary evidence that BMD is a predictor of fracture risk [10,11]. Several reports, both old and new, suggest that children with low BMD have a higher fracture rate, and that forearm and wrist fractures are associated with low BMD [12e16]. There are also some prospective studies showing an association between BMD and fracture risk in children. For example, Goulding et al. showed that for each 1 SD reduction in total body (TB) BMD, there was a doubling of risk for new fractures in young girls [10]. In a British prospective study on 6213 children aged 9.9 years, studied with DXA and followed for 2 years, the fracture risk was found to be increased by 89% for each 1 SD decrease of BMC adjusted for height and weight. A particularly interesting result of this study was the predictive meaning of TB BMD, which was inversely related to the risk of fractures [17]. In a Swiss prospective study (125 girls followed for 8.5 years), the cumulative incidence of fractures was 46.4%, forearm and wrist being the most common sites. Decreased bone mass gain in the axial and appendicular

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skeleton and reduced vertebral size were observed in girls who sustained fractures at the time of pubertal maturity. The authors concluded that fractures during childhood may be predictive of future low PBM and increased bone fragility [18]. A recent review and meta-analysis of studies on children up to 16 years of age concluded that there is some evidence for an association between low BMD and fractures: however, considering the limitations of the available studies and their possible biases, large prospective cohort studies are still needed for definitive conclusions [19]. Until more data are available, the clinical risk of reduced BMD in pediatric patients cannot be as precisely assessed as in adults. Moreover, the fracture threshold in children is likely to vary at different skeletal sites, as bone mineral accrual and bone growth proceed at different rates [20,21]. Finally, it is possible that different diseases produce different fracture thresholds, also depending on age and duration of disease, but there are no data on these aspects. Currently, most experts agree that the diagnosis of osteoporosis in growing subjects cannot be solely based on the BMD Z-score. While Z-score values below 2 are generally considered a serious warning, a child with low BMD should not be labeled with osteoporosis unless there is a history of atraumatic (fragility) fractures [7], which indicates that the skeleton has not been able to withstand the normal mechanical challenges (growth and muscle force), because of inadequate bone mass and/or architecture. The clinical relevance of uncomplicated low BMD in the young and its long-term consequences remain difficult to evaluate, and the diagnosis of “osteopenia” should be avoided. In conclusion, DXA densitometry should serve as a guide to monitor the patient’s BMD gain (or lack thereof) over time, in response to therapy or in the context of evolving disease. It should be interpreted cautiously, considering the patient’s age, gender, body size, pubertal stage and ethnicity. Correlation with overt clinical indices of skeletal health such as fractures, bone pain, limb deformity and impaired mobility should always be sought. Bone turnover markers cannot be used for the diagnosis of osteoporosis, but may aid in the monitoring of patients receiving antiresorptive drugs. Bone markers are difficult to interpret in children, due to the wide range of normal values and the changes related to age and Tanner stage [4]. Iliac crest bone biopsies in children are useful for the histological characterization of different forms of osteogenesis imperfecta (OI) (see Chapter 19), and may also be used for the study of bisphosphonate effect at the tissue level [22]. Pediatric normative data for iliac crest bone histology and histomorphometry have been published [23]. However, bone biopsies are invasive

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procedures that should be limited to selected cases and performed only at specialized centers.

THE MECHANOSTAT MODEL: MUSCLEeBONE INTERACTION IN BONE GROWTH AND DEVELOPMENT Understanding the process of bone growth and development is preliminary to understanding the pathogenesis of osteoporosis. Skeletal growth and development has two key aspects: the accumulation of bone minerals and the organization of bone macro- and microarchitecture. The last two decades of biomedical research have produced a huge mass of new information on the cellular and molecular aspects of bone biology, and many biochemical and environmental factors have been recognized as determinants or regulators of bone development. However, this approach, focused on the role of individual factors and on specific aspects of bone biology, has not provided any meaningful insight into what really happens at the “macro” organ level. What can be derived from it is a conceptually simple but unsatisfactory “cumulative model”. Bone development is presented as the cumulative effect of a large number of interacting factors (genetic, hormonal, nutritional, environmental, behavioral) that activate bone cells first to build (the bone “modeling” of growth) and then to maintain (the bone “remodeling” of maturity) bone tissue [24]. The integration of bone mass and bone architecture results in bone “strength”, that is, the resistance of bone to the external insults that may cause fractures. According to the cumulative model, during growth and development, as much mineral as possible should be accumulated in the skeleton e bone minerals becoming both a factor of bone’s physical strength and the body’s reserves of calcium. And the observation that, somewhere between 20 and 30 years of age, an individual will reach his/her “peak” value of bone mass, has led to the conclusion that maximizing this value is the best strategy to prevent osteoporosis in later age [25]. However, if bone is considered at the organ level e that is, in the light of its physiological properties and functions e an integrated model is needed to explain how the accumulation of bone minerals (i.e. the accretion of bone mass) is controlled, and how a healthy bone achieves and regulates its final shape and its most important property, its structural “strength”. The cumulative model of bone development is unsatisfactory as it does not explain “how” bone architecture is determined and “when” the accumulation process will cease because bone mass is considered adequate. According to this model, bone development is

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essentially a blind process, and the hypothesis that all the needed information is present in the genetic pool has insurmountable problems [24]. From an evolutionary point of view, there is a high selection pressure to develop strong bones, able to resist all the ordinary mechanical stresses without breaking, since the fracture of a major bone in a wild-living animal is most likely to cause death. Bone strength, however, cannot be simply achieved by maximizing the BMC, because excessive body weight would be an evolutionary hindrance, decreasing running speed and increasing energy expenditure. In fact, another solution is possible: bone strength can be achieved by optimizing bone architecture at both the macroscopic and the microscopic level, thus limiting the increase in weight to what is strictly necessary. Like most if not all biological processes, the “control” of bone growth and development is likely to be achieved through a feedback system and, in the past 15 years, a functional model, based on Frost’s “mechanostat” hypothesis, has been proposed [24e27]. This model is based on the concept that the greater the bone strength, the lesser the bone deformations (strain) caused by external mechanical forces. The proposed homeostatic negative feedback loop is that bone deformations beyond certain thresholds (“mechanostat set points”) are sensed by the osteocytes, the primary mechanosensory cells of bone, that in turn send signals to activate osteoblasts and osteoclasts. If mechanical strain descends below a certain threshold, the excess mineral mass can be disposed of, and bone is resorbed by osteoclasts. Vice versa, if bone becomes exposed to higherthan-normal mechanical strain and another threshold is exceeded, bone formation by osteoblasts is stimulated to increase bone strength. This process will go on until bone strain is brought close to the set point and an acceptable level of bone strength is achieved (Fig. 18.1). The model is in accordance with many

observations and correctly predicts many aspects of bone physiology and physiopathology [27]. At the cellular level, mechanical strains provide the information about exactly where mineralized bone should be added to bring bone deformation below the set point, or where, conversely, unnecessary bone mineral could be removed. In the words of Rauch and Schoenau, “the required mechanical strength of bone determines its mass and architecture, not vice versa.” [24]. The functional model of bone development distinguishes two different phases. The first is the genesis of the basic shape of bones, according to a genetically determined plan, during embryonic development. In the soft tissue environment in which bone templates are built, diffusible factors called “morphogens” are thought to send signals, along diffusion gradients, to the involved cells, determining their developmental fate and role in bone building. Such diffusible signals, however, can have only a very limited role in the second phase, the growth and development of mineralized bone tissue. In this phase, the mechanostat model is able to explain how activated bone cells, by locally adding or removing mineralized bone to keep the mechanical strain close to the set point, will build and maintain bone mass and internal architecture (bone modeling and remodeling). The mechanostat model can also explain how bone strength can be maintained or increased during growth, when both bone length and muscular force increase, generating greater bone strain. Body weight (gravitational force) was once thought to be the main mechanical force acting on bone to stimulate bone modeling and/or remodeling. The current evidence is that, throughout life, muscle contraction is quantitatively more important than body weight, and that it already acts during embryonic and fetal development, when bone mineralization starts [28,29]. The importance of hormonal, nutritional, and environmental/ behavioral FIGURE 18.1

The feedback regulated model of bone growth and development based on Frost’s “mechanostat” hypothesis. (Redrawn from Fricke O, Schoenau E. The “Functional MuscleeBone Unit”: probing the relevance of mechanical signals for bone development in children and adolescents. Growth Horm IGF Res 2007;17:1e9.)

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factors, however, must not be overlooked. They act in concert with genetic factors to determine longitudinal bone growth and muscle mass, and they might also alter the mechanostat set points, or the width of the “tolerance zone” around the set points, and consequently affect osteoblast and osteoclast activity. The study of children and adolescents has made us aware of the limitations of bone densitometry. BMC by itself has no meaning, as it is clearly dependent on bone (body) size: it will be different in perfectly healthy bones of different size. Areal BMD is difficult to interpret, especially in growing patients: as explained above, it cannot be used to compare bones of different size (either in different subjects or in the follow up of the same subject), because it will overestimate the actual (volumetric) BMD in larger bones. But also vBMD cannot be considered a good indicator of bone strength, at least in long bones: as noted by Schoenau, a thick rod is more resistant to mechanical stress than a thin rod of the same material (thus, with equal density), so that also vBMD will correlate with bone strength only for a given bone size [30]. If the main force behind bone development is muscle force, a special attention to the changes in muscular mass, as well as the study of bone and muscle as a functional unit, is clearly justified. Thus, the concept of the “functional muscleebone unit” has been developed [30]. Under this assumption, the study of bone and muscle should go strictly together in the evaluation of bone health in children and adolescents. A simple twostep diagnostic algorithm, based on two “yes/no” questions (Is muscle mass adequate for body height? Is BMC adequate for muscle mass?) has been proposed to distinguish between normality, primary bone defect, secondary bone defects, and mixed bone defects [31]. The muscleebone model is able to explain the increase in bone mass and bone strength during childhood, and also why certain physical exercises can prevent bone loss during immobilization. To explain tentatively why exercise does not continue to increase bone strength after puberty, a role of the joint cartilage as a “third agent” cooperating with the muscleebone unit has recently been hypothesized [32]. This short presentation was only meant to offer a general picture of a very complex, and still rapidly evolving, model of bone growth and development. It can be added that very recently, Hughes and Petit further elucidated the role of the osteocytes in determining the mechanostat set points. When mechanical stimuli fall below a certain threshold, as happens in immobilization and disuse, osteocytes undergo apoptosis, and bone resorption follows. In the presence of customary mechanical loading, osteocyte apoptosis is prevented, survival is promoted, and bone mass is maintained. If the strain stimuli are higher than another

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threshold, osteocytes will release sufficient anabolic factors to stimulate net bone formation. Osteocytes appear also to have a critical role, not yet fully understood, in bone remodeling and microdamage repair. Osteocyte apoptosis is observed before bone resorption in response to microdamage, but it is not clear how bone formation is stimulated in the damaged area. One hypothesis is that the dying osteocytes send “turn-over” signals that activate their nearby living companions to start bone formation [33].

THE SPECTRUM OF PEDIATRIC OSTEOPOROSIS Osteoporosis in childhood is usually suspected when a patient presents with frequent and/or low trauma fractures, chronic bone pain, or an incidental finding of “possible osteopenia” on plain x-rays. Sometimes the skeletal health of children with known risk factors e such as neuromuscular disorders, chronic glucocorticoid (GC) use, endocrinopathies or poor nutrition e may be evaluated even before clinical symptoms are present, unveiling the presence of low bone mass or subtle vertebral compression. It is convenient to distinguish “primary osteoporosis”, resulting from an intrinsic skeletal defect, and “secondary osteoporosis”, due to underlying illness or treatment [3]. A list of the most common forms of pediatric osteoporosis is presented in Table 18.1.

Primary Osteoporosis Primary osteoporosis can be further divided into two main groups: the heritable disorders of connective tissue, (e.g. 01) 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, but the disease is classified as a primary osteoporosis because of the lack of extraskeletal manifestations. The list of disorders characterized by primary osteoporosis is continuously growing and the more common are presented in Table 18.2. 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 bone literature is OI, because of its higher frequency and the new treatment options for children with severe forms. OI is separately presented in Chapter 19.

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Most Common Forms of Pediatric Osteoporosis

Primary osteoporosis 1. Heritable disorders of connective tissue a. Osteogenesis imperfecta b. Bruck syndrome c. Osteoporosis-pseudoglioma syndrome d. EhlerseDanlos syndrome e. Marfan syndrome f. Homocystinuria g. Cutis laxa h. Menkes disease/occipital horn syndrome i. Congenital contractural arachnodactyly 2. Idiopathic juvenile osteoporosis Secondary osteoporosis 1. Neuromuscular diseases a. Cerebral palsy b. Duchenne muscular dystrophy c. Prolonged immobilization, including spinal cord injury and limb disuse d. Rett syndrome 2. Chronic illnesses a. Leukemia and other childhood cancers b. Rheumatic diseases c. Eating disorders d. Inflammatory bowel diseases, celiac disease and other malabsorption syndromes e. Cystic fibrosis f. Nephropathies g. Thalassemia h. Primary biliary cirrhosis i. AIDS j. Organ transplantation 3. Endocrine and reproductive disorders a. Diabetes mellitus b. Thyroid disorders c. Growth hormone deficiency d. Disorders of puberty e. Hypogonadism f. Turner syndrome g. Hyperprolactinemia h. Hyperparathyroidism i. Cushing syndrome 4. Iatrogens a. Glucocorticoids b. Antiepileptic drugs (phenytoin, phenobarbital, carbamazepine, sodium valproate) c. Anticoagulants (heparin, warfarin, low molecular weight heparin) d. Methotrexate e. Calcineurin inhibitors (cyclosporine and tracolimus) f. Antiretrovirals g. Medroxyprogesterone acetate h. GnRH agonists i. L-thyroxine suppressive therapy j. Radiotherapy 5. Inborn errors of metabolism a. Lysinuric protein intolerance b. Glycogen storage diseases c. Galactosemia d. Gaucher disease 6. Other a. Severe burns (based on current literature)

BRUCK SYNDROME

Bruck syndrome (BS) is a rare, autosomal recessive disorder, with few cases reported worldwide. The BS phenotype shares features with OI, including bone fragility, deformity of the spine and extremities, low bone mass, wormian bones, and blue or white sclerae [34e36]. Despite the shared features, BS is clearly distinguishable from OI by the prominent finding of congenital joint contractures and the absence of the typical alterations in type I collagen that are found in OI [37]. BS is due to a deficiency of a bone-specific telopeptidyl lysyl hydroxylase, probably encoded in the chromosome 17pl2 region, that is responsible for the formation of type I collagen cross-links in bone, but not in ligaments or cartilage. The lysine residues within the telopeptide region of type I collagen are underhydroxylated, leading to aberrant cross-linking [34]. Recently, the molecular consequences of three point mutations in lysyl hydroxylase 2 (LH2) were studied and the significant reduction of collagen telopeptide hydroxylation was explained by the low activity of mutant LH2 [38]. Moreover, mutations in FKBP10 (a gene that encodes FKBP65, an extracellular matrix binding protein whose mutations affect type I procollagen secretion) have recently been described [36]. Regarding treatment, Shaheen et al. reported that two brothers with BS sustained fewer fractures after being treated with a parenteral bisphosphonate [36]. Andiran et al. treated a boy with multiple fractures with cyclic pamidronate and reported an increase in BMD, a significant decrease of pain and a decreased number of new fractures (only two during 2 years of treatment) [39]. OSTEOPOROSIS-PSEUDOGLIOMA SYNDROME

The osteoporosis-pseudoglioma syndrome (OPPG) is an autosomal recessive condition, with an estimated population incidence of 1 per 2 000 000 [40]. The phenotypic features resemble those of 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 or infancy-onset blindness. The ocular defect, which resembles a pseudoglioma, arises from persistent hyperplasia of the vitreous, possibly because of failure of the primary vitreal vasculature to undergo involution during embryogenesis [41]. The Osteoporosis-Pseudoglioma Collaborative Group demonstrated that homozygous (likely loss-of-function) mutations in the LRP5 gene, encoding low-density lipoprotein receptor-related protein 5 (LRP5), are responsible for the OPPG phenotype [41] and, more recently, it has been demonstrated that these mutations impair the Wnt and Norrin signal transduction [40,42].

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TABLE 18.2

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Major Causes of Primary Osteoporosis in Childhood and Adolescence

Disease

Inheritance

Mutations

Type IeIV

AD/AR

Mutations involving type I collagen (COL1A1/COL1A2 genes)

Type V

AD

Unknown

Type VI

Unknown

Unknown

Type VII

AR

Hypomorphic expression of cartilage-associated protein (CRTAP) gene

Type VIII

AR

LEPRE1 gene mutation

Type IX

AR

PPIB gene mutation

Classical type (EDS I and II)

AD

The 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 V collagen (COL5A1 gene) Mutations in TNX-B gene, encoding tenascin-X Other mutations unknown

Vascular type (EDS IV)

AD

Mutations in type III collagen (COL3A1 gene)

Kyphoscoliosis type (EDS VI)

AR

Mutations in lysyl hydroxylase (PLOD1 gene)

Arthrochalasia type (EDS VIIa and VIIb)

AD

Mutations in COL1A1/COL1A2 genes altering the procollagen N-peptidase cleavage site of the alpha1 and alpha2 chains of type I collagen

Dermatosparaxis type (EDS VIIc)

AR

Mutations in ADAMTS-2 gene, encoding procollagen N-proteinase

Osteogenesis imperfecta

EhlerseDanlos syndrome

Progeroid EDS, Periodontitis type, Fibronectin-deficient type, X-linked Type V)

Unknown

Marfan syndrome

AD

Mutations in FBN1 gene, one of two genes encoding fibrillin-1 (the main structural component of elastin-associated cross-links) Rare mutations in COL1A2 gene Rare mutations in TGFBR2 gene, encoding TGF-beta receptor 2

Homocystinuria

AR

Mutations in CBS gene, encoding cystathionine beta-synthase, resulting in elevated plasma homocysteine levels Hyperhomocysteinemia may damage fibrillin-1

Bruck syndrome

AR

Mutations in PLOD2 gene encoding bone-specific telopeptidyl lysyl hydroxylase, which interferes with collagen cross-link formation in bone (but not ligaments or cartilage) Mutations FKBP10 gene

Cutis laxa

AR

Lysyl oxidase deficiency (mutations in LOX gene), with abnormal synthesis of collagen and elastin cross-links Mutations in the fibulin gene (FBLN5, FBLN4, EFEMP2)

AD

Mutations in the elastin (ELN) gene

Menkes disease/Occipital horn syndrome

XLR

Mutations in the gene (ATP7A) encoding copper-transporting ATPase, with lysyl oxidase deficiency and aberrant synthesis of collagen cross-links

Osteoporosis-pseudoglioma syndrome

AR

Mutations in the LRP5 gene, expressed in osteoblasts and associated with transduction of Wnt signaling

Congenital contractural arachnodactyly

AD/AR

Mutations in FBN2 gene encoding fibrillin-2, a structural protein in bone matrix

Idiopathic juvenile osteoporosis

-

Unknown

AD: autosomal dominant; AR: autosomal recessive; XLR: X-linked recessive

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At least 12 different homozygous [40,41] and 15 heterozygous [40,43,44] LRP5 mutations have been described in OPPG. No phenotypic distinctions have been reported in heterozygotes compared to homozygotes [40]. Obligate heterozygotes have been found to have modestly reduced bone mass but no eye pathology [41,45]. Inactivating mutations in LRP5 can also cause familial exudative vitreoretinopathy (FEVR), a disease characterized by premature arrest of the development of retinal vasculature. Retinal avascularity leads to complications such as folding and neovascularization of the retina, which in turn leads to retinal detachment and blindness in most cases. FEVR has been classically described without an osteoporotic phenotype [46]. However, on routine BMD scans, most patients with FEVR (those with LRP5 mutations) have reduced bone mass or osteoporosis, suggesting that OPPG and FEVR are part of a single phenotypic spectrum [46]. LRP5 is expressed by developing and mature osteoblasts, and affects bone mineral accrual during Wntmediated osteoblastic proliferation and differentiation. The mutant LRP5 reduces bone mass accrual in murine models, and children with OPPG demonstrate very thin cortices and few trabeculae on iliac crest samples [44]. The importance of LRP5 as a modulator of bone mass accrual is further substantiated by the observation that an activating mutation in the LRP5 gene results in an autosomal dominant “high bone mass trait” [47]. Children with OPPG may have severe osteoporosis with significant pain from vertebral and extremity fractures. Antiresorptive therapies have been explored, and recently described [48e51]. Zacharin et al. treated three children (age 9e11 years) with vertebral fractures with pamidronate or clodronate, and obtained improvement in pain, mobility and size of vertebral bodies after 2 years of therapy. There were no new fractures, growth and puberty proceeded normally, and the drugs were well tolerated. The authors concluded that bisphosphonate therapy is justified in patients with OPPG with symptomatic vertebral fractures [48]. Streeten et al. treated four children (age 2e8 years) with different bisphosphonates (BPs) for 1.5 to 6.5 years. After trying oral risedronate, intravenous (i.v.) pamidronate and oral alendronate, they found that the best improvement in BMD Z-scores was obtained with alendronate (1 mg/ kg/day). A boy who sustained a new fracture during pamidronate treatment, was given teriparatide (20 mg subcutaneously every other day for 15 months) but did not have any BMD increase [49]. Barros et al. observed increased BMD and decreased fracture rate in two brothers treated with pamidronate for 3 years [50]. Bayram et al. treated a 21-year-old woman with pamidronate and observed decreased bone pain, improved mobility, and increased BMD at lumbar spine (LS) and femoral neck (FN) [51].

EHLERSeDANLOS SYNDROME

EhlerseDanlos syndrome (EDS) is a group of phenotypically and genetically heterogeneous inherited connective tissue disorders, characterized by defects in various extracellular matrix proteins including collagens and small leucine-rich proteoglycans (e.g. decorin), leading to tissue fragility of the skin, ligaments, blood vessels and internal organs. EDS affects about 1 in 5000 individuals [52]. Patients with EDS have in common joint and skin hyperlaxity and easy bruising. Other variable characteristics of the disease include recurrent joint dislocations, fragile (cigarette paperlike) scars, mitral and triscuspid valve prolapse, kyphoscoliosis, fragility of the ocular, uterine, cardiovascular and gastrointestinal systems. Severe pain, related to hypermobility, dislocations, and previous surgery, is very common and is associated with moderate to severe functional impairment [52,53]. In 1998, the classification of EDS was revised by Beighton et al. and six subtypes were defined [54]. The major forms and their underlying genetic defects are presented in Table 18.2. A deletion of the TNX gene (encoding tenascin-X, a connective tissue protein developmentally associated with collagen fibrils) has been associated with EDS [55], and Zweers et al. confirmed that tenascin-X deficiency can result in connective tissue abnormalities, manifesting as classical EDS with autosomal recessive inheritance [56]. These findings demonstrate that EDS is more than a simple alteration of collagen. 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. Only a few studies have addressed the bone health of EDS patients. Once, osteoporosis was not considered a cardinal feature of EDS, but subsequent studies have found reductions in bone mass at LS [57], FN [58], or both [59]. Dolan et al. compared 23 patients (mean age 38.515.5 years) with healthy controls. Prior fractures were 10 times more common in EDS, with 86.9% of patients reporting a total of 47 low-impact fractures, compared to 8.7% of controls. A significant reduction in BMD was found at FN and LS [59]. Carbone et al. found no difference in LS BMD nor in biochemical markers of bone and mineral metabolism between 23 adult patients with EDS III and controls. EDS patients had a significantly reduced FN BMD with respect to controls, but this difference disappeared after adjustment for body height, weight and physical activity levels [60]. The reasons for these discrepant findings may be partly due to the disease classification. In the study by Carbone et al. [60], all patients had EDS III and thus were a more homogeneous group than those in prior

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studies [57e59], and patients with co-morbidities affecting bone and mineral metabolism were not included. A recent study reported osteoporosis in all of 11 patients (children, adolescents and young adults) with EDS who underwent BMD measurement [61]. This observation and the increased fracture rate reported by Dolan et al. [59] suggest reduced bone strength, but comprehensive studies of bone health in sufficient numbers of patients with the various forms of EDS are needed. MARFAN SYNDROME

Marfan syndrome (MFS) is a common autosomal dominant condition with complete penetrance, characterized by variable skeletal, ocular and cardiovascular manifestations. Arachnodactyly (long, thin, hyperextensible fingers) is one of the most prominent clinical features. Pectus carinatum or excavatum, arm span/ height ratio >1.05, wrist and thumb signs, are the other major diagnostic criteria. MFS occurs in 2e3/ 10 000 live births, without gender predilection. About 25% of cases originate from new mutations, without familiarity [52,62]. The basic defect in MFS is linked to mutations in the FBN1 gene (chromosome 15), one of two genes encoding the glycoprotein fibrillin-1. Fibrillin is the main structural component of the elastin-associated microfibrils in the extracellular matrix, and is also present in bone. It has been hypothesized that a molecular defect may alter the distribution of mechanically induced strain or interfere with calcium binding [63,64]. Rarely, mutations in COL1A2 (one of the genes encoding type I collagen) have been described [65]. Recent genetic and mousemodel studies have focused on the interaction between fibrillin and transforming growth factor beta (TGFbeta), a ubiquitous mediator of inflammation, and there is evidence that a defect in fibrillin may lead to reduced binding and increased activity of TGF-beta. Also, missense mutations in TGFBR2, which encodes TGFbeta receptor 2, have been found in patients with type 2 MFS [66]. All these findings show that the critical features of MFS, including the cardiovascular involvement that determines the life expectancy of these patients, result from abnormal TGF-beta signaling. Lowering the TGF-beta levels with pharmacologic agents that block angiotensin II type 1 receptors is currently being investigated as a promising treatment [67]. Notwithstanding these advances, we are still far from a complete understanding of the pathophysiology of MFS [66]. The diagnosis of MFS has always been problematic. The old Berlin nosology of 1988 was superseded by the Ghent nosology of 1996 [68], that in its latest revision [69] puts more weight on the cardiovascular

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manifestations and defines aortic root aneurysm and ectopia lentis as the cardinal clinical features. Osteoporosis has been reported in both adults [70,71] and children with MFS [64]. Kohlmeier et al. described nine boys and girls (age 9.9e17.5 years) with MFS who were tall for age but of normal weight. All were moderately active and had no history of atraumatic fractures. BMD correlated with age, height and pubertal development as expected. FN aBMD was reduced, and LS aBMD showed a downward trend [64]. These authors also found that 32 women with MFS had both axial and appendicular osteopenia [71], whereas Carter et al. noted only axial osteoporosis in both women and men [70]. Tobias et al. found normal hip and LS BMD in postmenopausal women with MFS [72]. LeParc et al. observed a BMD reduction at hip and wrist in 60 patients with MFS (mean age 32.9  9.3 years). LS was not studied because 30% of the patients had had spine surgery. The reduction was similar at both sites and in both genders. However, the authors observed that the clinical relevance of the low BMD remains to be clarified, considering that the fracture rate in MFS patients was similar to that of a healthy young adult population [73]. Giampietro et al., evaluating 51 patients (30 adults) with MFS, found that FN BMD was significantly reduced only in adult male patients [74]. On the contrary, in the largest published study on bone and MFS, involving 130 subjects (mean age 34.7  10.7) who met the Ghent diagnostic criteria [68], both males and females had reduced BMD at hip and wrist with respect to healthy controls matched for gender, age, height and body mass index (BMI) [75]. Considering that these studies have been conducted before and after the publication of the Ghent nosology, these discrepancies may be due to differences in the diagnostic criteria for the syndrome. The fracture risk associated with a reduced BMD in MFS has not been determined yet. Fractures have been reported in 16/48 (33%) of adults with the syndrome, but site and degree of trauma were not indicated, and the fractures were attributed to the characteristic joint hypermobility of MFS [76]. No patient in the study by Kohlmeier et al. reported a history of non-traumatic fractures, although 50% of women and 12.5% of children sustained peripheral traumatic fractures [64]. In two other studies, the fracture incidence (including both trauma-related or low-impact fractures) ranged from 10% to 24.6%, and the fracture history was not correlated with BMD values [73,75]. Further studies are required to determine the clinical significance of reduced bone mass in children and adults with MFS. Whether poor exercise is related to the low bone mass of children with MFS has not been determined. Musculoskeletal pain and the recommendation to avoid contact sports because of risk of aortic rupture are likely to

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448

18. THE SPECTRUM OF PEDIATRIC OSTEOPOROSIS

interfere with physical activity and reduce mechanical loading. These aspects have not been addressed formally, though Kohlmeier et al. reported that none of their MFS subjects participated in vigorous or competitive sport [64]. HOMOCYSTINURIA

Homocystinuria is an autosomal recessive connective tissue disease, due to defects in the methionine metabolism leading to an elevated plasma concentration of homocysteine. Clinically, mental delay, ectopia lentis, marfanoid habitus, osteoporosis and early onset thrombotic vascular disease are observed. The reported prevalence is 1:344 000 worldwide. The occurrence of homocystine in urine may result from a number of different genetic defects, the most common of which is a deficiency in cystathionine beta-synthetase. This enzyme is important in the trans-sulfuration pathway, responsible for a key step in the conversion of methionine to cysteine. Homocysteine is an intermediate in this conversion, and defective cystathionine betasynthetase activity leads to an accumulation of methionine and homocysteine in plasma [77]. The multisystem toxicity of hyperhomocysteinemia is attributed to its spontaneous chemical reaction with many biologically important molecules, primarily proteins. Irreversible homocysteinylation of proteins leads to cumulative damage and progressive clinical manifestations. The glycoprotein fibrillin-1 is particularly susceptible to homocysteine attack through homocysteinylation of the epidermal growth factor (EGF)-like domains of the molecule [78]. Fibrillin-1, the main structural component of elastin microfibrils, is found in the medial layer of all elastic arteries, as well as in bone, cartilage, skin, and the suspensory ligament of the lens e all structures that are compromised in both MFS and homocystinuria. The fibrillin-1 gene mutations that give rise to the MFS phenotype support the hypothesis that fibrillin1 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. Recent data strongly suggest that structural and functional modifications as well as degradation processes of fibrillin-1 in the connective tissues play a major role in the pathogenesis of homocystinuria [79]. The detrimental effect of homocysteine on bone was observed in various cellular and animal studies [80,81]. Herrmann et al. recently demonstrated a tissue-specific accumulation of homocysteine, prevalently bound to extracellular collagen, in the bones of hyperhomocystinemic animals. This accumulation was accompanied by significant bone loss and reduced bone strength [82]. In addition, hyperhomocysteinemia seems to reduce the methylation capacity in bone, an additional

pathogenetic mechanism of potential relevance. Moreover, a very recent study not only confirmed the negative effects of homocysteine on bone, but also demonstrated for the first time that moderate hyperhomocysteinemia may induce structural changes in the growth plate during endochondral ossification in rat embryos. These changes may lead to lower bone density in bones that use cartilage as a template [83]. 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. Yap and Naughten reported their 25 years of experience with 25 Irish homocystinuric patients (age 2.5e23 years), diagnosed through a national screening program. By radiological examination, they found that osteoporosis was present in 33% of patients who were non-compliant with medical therapy, while it was absent in all of the 18 compliant patients [84]. A report of osteoporosis among six adult patients (age 27e48 years) with a late diagnosis of homocystinuria showed significant reduction in BMD at the FN, and an even greater reduction at LS [85]. Surprisingly, the osteoporosis was not more severe in the oldest patients compared to the younger ones. The treatment of patients with homocystinuria due to a defect in cystathionine beta-synthetase includes vitamin B6, low protein and low methionine diet, and adjuvant therapy with betaine and/or folate and vitamin B12 supplementation. Betaine lowers plasma homocysteine by augmenting homocysteine methylation to methionine [86], and plasma methionine concentration should be monitored in all persons receiving betaine. Until now, there are no systematic studies on bone density and fractures in patients affected by homocystinuria, and the clinical significance of the low bone mass and its relation to fracture risk in this disease are unknown. There are only a few case reports on the treatment of low bone density in homocystinuria. Gahl et al. reported that in five homocystinuric patients (age 5e32 years) treatment with oral betaine (3 g b.i.d.), in a double-blind, placebo-controlled, 2-year crossover study, significantly reduced the mean plasma homocystine levels, but did not increase the low LS BMD, measured by QCT [87]. Recently, multiple vertebral fractures and reduced BMD (measured by DXA) have been observed in a 22-year-old woman. A once-a-year i.v. infusion of zoledronic acid was started, without adverse effects, but the results are not yet available [88]. Idiopathic Juvenile Osteoporosis IJO is a rare, self-limiting disorder first described in detail by Dent and Friedman in 1965 [89]. Since then,

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only slightly over 100 cases have been reported, mostly from Poland [90,91]. IJO typically presents in previously healthy children during the 2e3 years preceding puberty [92], although it has been reported at 3 years of age [93]. There is no gender selection, and the disease does not seem heritable. Children report a gradual onset of pain, that may become severe, in the back, hips, knees and feet. Sometimes they have difficulty walking [94]. Vertebral compression fractures are frequent and may significantly compromise the length of the upper body segment. Long bone fractures, usually metaphyseal, may also be present. While physical examination is often normal, kyphosis, scoliosis, pectus carinatum, long bone deformity and difficult ambulation may be present. Radiological studies may show evidence of abnormal, newly formed bone in the metaphyseal areas (neoosseous osteoporosis), appearing as a radiolucent, submetaphyseal band. Long bones usually have normal length and cortical width, while wedge-shaped or biconcave vertebrae may be evident. Proszynska et al. showed that carboxyterminal propeptide of type I pro-collagen levels were higher in patients with IJO than in those with OI [95]. Low insulin-like growth factor 1 (IGF-1) and normal IGFBP3 (third fraction of IGF binding proteins) were reported in 12 subjects (age 7e18 years) affected by IJO. IGF-1 levels correlated with TB and LS BMD [96]. Other changes in biochemical markers of bone and mineral metabolism have been inconsistently reported in the literature [92,97] and, to date, no specific laboratory hallmarks of the disorder have been identified. As such, IJO remains a diagnosis of exclusion, once other primary and secondary causes of osteoporosis have been considered. Differentiating IJO from a mild form of OI may be difficult. The discriminating features (which may or may not be present) are presented in Table 18.3. The most remarkable feature of IJO is the spontaneous remission which occurs over 2e5 years, usually around the time of puberty, but disability may persist in adulthood [92]. The underlying pathogenesis remains unclear. The histomorphometric study of iliac crest bone biopsies in patients with IJO showed decreased cancellous bone volume and very low bone formation rates on cancellous surfaces, suggesting a disorder of osteoblast performance. The altered bone remodeling in children with IJO primarily affected the bone surfaces in contact with bone marrow [98]. 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 very young patients with the disorder have also been reported [93].

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Pludowski et al., using DXA for the first systematic study of a large number of patients with IJO (N¼61; 34 girls, age 13.6  3.1 years, age range 7e18.9; 27 boys, age 14.3  3.3, range 5e18.9), found that BMD was significantly reduced in both boys and girls with respect to age- and gender-matched healthy controls. LS BMD was more reduced than TB BMD. However, while BMD was very low, the indicators of muscular status (lean body mass [LBM], and the ratios LS BMC/LBM, TB BMC/LBM, height/LBM and LBM/weight) were normal, confirming that IJO is a primary bone disorder, characterized by “a transitory interruption of the normal response of bone tissue to load-generated muscle strains” during the acute phase (“active IJO”) characterized by pain and fractures [91]. 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. reported clinical improvement with calcitriol therapy [97], and Glorieux et al. found an increase in trabecular bone volume and bone formation rate in patients treated with long-term sodium fluoride [99]. Calcitonin therapy was attempted in a patient with an elevated 1,25-dihydroxyvitamin D (1,25OH2D) level, but there was no clinical improvement [100]. It should be noted that some patients have low levels of 1,25OH2D [97]. Impressive results were observed in a 13-year-old boy following 2 months of treatment with i.v. pamidronate. A positive calcium balance quickly ensued after initiation of therapy; and iliac crest bone biopsy, taken at the beginning of treatment and 2 months after its end, showed thin trabeculae with poor marrow cellularity before treatment, followed by a marked increase in new osteoblasts after treatment [101]. The role of spontaneous recovery in this patient’s clinical improvement is of course unknown. I.v. pamidronate led to similar results in five patients with pediatric osteoporosis, one of whom had IJO. This patient (an 11-year-old girl) presented with painful vertebral fractures, for which she received three pamidronate treatments over one week. There was a rapid onset of pain relief within 2 weeks of the initial treatment [102]. A boy (aged 8 years 3 months) with IJO and multiple fractures, especially vertebral, had a dramatic clinical improvement within 2 weeks after starting therapy with clodronate (i.v. every 3 months for 2 years). After 6 months of treatment, radiological improvement with healing of fractures and rebuilding of the vertebral plates was evident. BMD increased to normal within one year and growth velocity was accelerated. After 24 months, treatment was stopped. One year later, there was a recurrence of back pain and knee pain, and a tibial fracture was discovered. BMD had returned below normal.

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Different Characteristics of Idiopathic Juvenile Osteoporosis (IJO) and Osteogenesis Imperfecta (OI)

Feature

IJO

OI

Age at onset

In most cases, 2e3 years before puberty

(Depending on the type) in fetal life, at birth, during childhood

Family history

Negative

Often positive

Duration

Spontaneous remission after 2e5 years, generally during puberty

Lifelong; in some forms improvement during puberty

Skeletal manifestations

Reduced upper/lower segments ratio

Frequent fractures of long bones

Metaphyseal fractures common

Metaphyseal fractures rare

Normal stature

Short or normal stature

Neo-osseous osteoporosis*

Gracile bones Wormian bones Thin ribs Hyperplastic callus formation (OI type V)

Extraskeletal manifestations

Long bones straight, normal cortices

Long bone deformity, thin cortices

None

Blue sclerae Dentinogenesis imperfecta Joint hyperlaxity Deafness Cardiac lesions

Bone histology

Genetic/molecular studies

Decreased bone turnover

Increased bone turnover

Lamellation normal Surface-specific remodeling and modeling abnormalities

Lamellation normal in OI types IeIV Abnormal lamellar pattern in OI types V, VI

Unknown molecular defect

Type I collagen abnormalities often detected in OI types IeIV, due to mutations in COL1A1/COL1A2 genes Mutations in CRTAP gene (OI type VII) Mutations in LEPRE1 gene (OI type VIII) Mutations in PPIB gene (OI type IX)

* Considered pathognomonic of IJO

Reinstating clodronate treatment led again to rapid improvement [103]. 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, by avoiding 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 BPs may be justified in selected cases of IJO, when patients present with significant pain secondary to vertebral or limb fractures. As a final note, two cases of pregnancy in teenage girls with a previous history of IJO have been reported. In the first, the girl had a normal pregnancy and the newborn was normal [104]. In the second, a 19-year-old

girl, notwithstanding calcium and vitamin D supplementation, sustained three vertebral fractures in the third trimester, before delivering a healthy baby. She had a marked BMD decrease during pregnancy, and was left with height loss, an abnormal posture due to vertebral fractures, and severe back pain. High bone turnover was present both before and after pregnancy. The authors concluded that in this young woman bone turnover showed an exaggerated response to pregnancy, with bone resorption predominating over formation [105].

Secondary Osteoporosis The term “secondary osteoporosis” is used to define the forms of osteoporosis that are consequent or related to other disorders or their treatment (see Table 18.1).

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In recent years, bone metabolism alterations, reduced BMD, and even fragility fractures have been reported in a continuously growing list of chronic conditions, particularly those in which new and aggressive treatments have led to longer survival and improved long-term outcome e e.g. transplants, leukemia, cystic fibrosis (CF), Duchenne muscular dystrophy (DMD). The very success of the new treatment protocols has focused the attention not only on the short-term “emergency” aspects, but also on the prevention, evaluation and management of the medium- and long-term complications, both those due to the primary disease itself and those related to treatments. In consideration of the growing list of diseases/disorders brought to the attention of pediatric bone specialists, it is not possible to cover all of them exhaustively, and only an overview of the more important and frequent forms will be presented. Moreover, it must be underlined that reports on the treatment of most forms of secondary osteoporosis are often encouraging, but most studies are uncontrolled and on small patient samples. Neuromuscular Diseases Muscular strength has a strong influence on the developing bone. Mechanical loads and muscular activity induce important changes in bone mass and architecture during growth, when the skeleton has maximum plasticity and is continuously adapted to the changing needs of a growing body [106]. Recently, many randomized trials demonstrated the effects of physical activity on bone during childhood and adolescence [107e110]. Conversely, skeletal unloading produced by prolonged bed rest or cast immobilization leads to bone loss in adults and failure to accrue bone mass in children [111]. In this section, the major causes of abnormal muscle force and its consequences will be presented. CEREBRAL PALSY

Cerebral palsy (CP) is the most common physical disability of childhood. CP is a non-progressive neurologic condition caused by brain injury suffered before complete cerebral development (i.e. during the first 2 years of life) [112]. It is characterized by permanent motor impairment and, depending on severity, by different degrees of physical and mental dysfunction (e.g. epilepsy, cognitive delay, speech and sensory impairment). The estimated prevalence is 2/1000 live births [113]. In 70e80% of cases, CP originates prenatally and its precise cause remains unknown. Only about 6% of CP cases are considered secondary to complications during delivery, such as asphyxia. In 10e30% of patients, CP is

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acquired postnatally, the most common causes being bacterial meningitis, viral encephalitis, hyperbilirubinemia, and brain and spinal cord injuries. The disorder has been classified according to the predominant motor abnormality into a number of categories, including spastic, dyskinetic, ataxic, hypotonic and mixed forms [114]. Spastic CP is the most common subtype, with hemi- and diplegia being frequent presentations. Besides CP, there are other diseases that may cause motor disability in childhood: neural tube defects, neuromuscular disorders, and various chromosomal anomalies/genetic diseases like, for example, Rett syndrome, a severe form of motor disability caused by mutations in the X-linked MECP2 gene. CP may determine a number of painful skeletal complications, including scoliosis, joint subluxation and dislocation, torsional bone deformities, and fractures. A recent systematic review, reported a 12e23% prevalence of fractures, mainly of the long bones, and an incidence of 2.7e4.5% per year in children with CP [115]. A similar incidence of fractures (4.3% per year) was reported for Rett syndrome [116]. The management, in particular that of femur fractures, is very complex, since the frequent presence of a spastic muscle tone can cause an increased risk of shortening or malunion at the fracture site [117]. In a retrospective study on 37 patients with CP who had sustained 54 fractures from minimal trauma (74% in the femoral shaft and supracondylar region), the main causes for fractures were long, fragile lever arms and stiffness due to contractures in the major joints, especially knees and hips [118]. Bischof et al., studying 20 subjects with fractures out of a group of 88 patients with CP (age 6e29 years, median 17.5), observed that severity of the disease, low vitamin D levels, use of anticonvulsant drugs were related to the presence and number of fractures [119]. More recently, Leet et al., studying 418 children with CP, found that 50 had sustained fractures, and that older age at the time of the first fracture and valproic acid use were predictive of future fractures [120]. In another study, out of 364 CP children, 46 had sustained 62 fractures at baseline, and 20 sustained 24 new fractures during the follow up (median duration 1.6 years). History of previous fractures, gastrostomy, and high body fat were the identified risk factors [121]. According to another study, anticonvulsant therapy is the most relevant risk factor for fractures in non-ambulant children with CP [122]. Finally, Maruyama et al., evaluating 525 physically disabled children (age 6e15 years), of which 66.3% had CP, found no difference in fracture prevalence between genders, and reported that older age and joint contractures in the lower limbs were independently associated with fractures [123].

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Taken together, all these observations allow a better definition of patients with a high fracture risk, and the implementation of specific interventions to prevent fractures. A very recent study reported that the BMD Z-score is helpful to identify high-risk subjects. In 619 children (age 6e18 years) affected by CP (N ¼ 507) or muscular dystrophy (N ¼ 112), the distal femur BMD Z-score, measured with DXA, was strongly correlated with fracture history, and the fracture risk increased by 6e15% with each unit decrease in BMD Z-score [124]. Low bone mass is a characteristic feature of CP, well documented by several studies [125e129], and when it is associated with a history of fragility fractures, a diagnosis of osteoporosis is justified. The pathogenesis of osteoporosis in CP is complex and multifactorial. Muscle disuse and diminished muscle load upon the developing skeleton are obvious major factors [130]. In addition, several complications of CP (feeding difficulties, gastrointestinal reflux, aspiration syndromes, respiratory infections, seizures, contractures) lead to conditions potentially negative for bone health, such as inflammation, malnutrition, increased immobilization, and use of anticonvulsant drugs. The degree of preserved ambulation (muscle use) has been shown to correlate positively with bone mass in CP [125,126]. Lin et al. showed that BMC was significantly reduced in the affected limb of 19 children with spastic hemiplegic CP, compared to the healthy limb. Lean mass was reduced by 15% and BMD by 6% in both upper and lower paretic limbs [131]. Similar results were observed in nutritionally adequate patients with CP: BMC, BMD and lean mass were consistently lower in non-independent ambulators compared to independent ambulators [132]. The relevance of motor impairment was also demonstrated by a large study on 171 subjects (age 2e21 years) affected by moderate to severe spastic CP. BMD correlated with disease severity (expressed as neurologic impairment based on Gross Motor Functional Classification level). Weight Z-score, increasing age, prior fractures, anticonvulsant use, and feeding difficulties were identified as determinants of the BMD Z-score [127]. The importance of muscle loss in CP has also been highlighted by Ward et al. Weak muscles and immobility reduce loading and prevent normal bone development, leading to a “physiological osteopenia” with an increased fracture risk. The skeleton becomes incapable of withstanding daily activities or muscle spasms during an epileptic fit, and if fractures occur, the problems are compounded by forced immobilization, that leads to further bone loss [133]. In a study on 139 patients (age 3e15 years), nutritional status (as determined by caloric intake, skin fold thickness and BMI) was the second most important variable after ambulatory status. Pattern of involvement,

duration of immobilization in a cast, and reduced calcium intake (less than 500 mg/day) were additional, though less significant, adverse factors [134]. Vitamin D levels were studied in 125 non-institutionalized children with various forms of CP: 25-hydroxyvitamin D (25OHD) levels were significantly reduced, indicating a possible vitamin D deficiency [135]. In another study, children with CP and long-bone fractures had more severe biochemical and radiographic evidence of rickets compared to children with CP but without fractures. The fractures were attributed to vitamin D deficiency, possibly secondary to anticonvulsant use, which compounded the lack of sunlight exposure [119]. The influence of anticonvulsant therapy on bone development has been the source of much debate in a variety of clinical conditions, including CP. Some investigators reported an association between anticonvulsants and abnormal vitamin D metabolism [119,136], while others did not [135,137]. Rieger-Wettengl et al. evaluated 39 children with isolated epilepsy, receiving carbamazepine or valproic acid, and found that calcium and 25OHD levels were similar to those of controls. Trabecular vBMD at distal radius, evaluated with pQCT, was decreased, while BMC and grip strength were normal for age. The normal BMC, despite reduced trabecular vBMD, was attributed to a compensatory increase in cortical BMC. Bone turnover, as assessed by deoxypyridinoline, was elevated [138]. 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 in carbamazepine-treated patients receiving vitamin D supplementation [139], and it has since been suggested that the increased bone turnover is due to a direct effect of anticonvulsants on bone cells [140]. No studies on these aspects have been performed in children with CP. Epilepsy is associated with an increased fracture rate in adults, due to direct effects of antiepileptic drugs on bone, falls due to seizures, and reduced physical activity [141e143]. A reduced BMD has also been reported in children and adolescents with epilepsy [144], and some data indicate that longer duration of disease and male gender are associated with a greater BMD reduction [145]. The diagnosis of osteoporosis in CP is frequently made 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 BMD 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 patients with CP often prefer lying on their side, Harcke et al. tested the feasibility and accuracy of measuring BMD at four distal

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femur subregions in the lateral projection, with favorable results [146], and the method’s validity has been subsequently confirmed [124]. Recurrent fractures from minimal trauma strongly suggest the diagnosis of osteoporosis, even in the absence of BMD measurements. Ancillary data should include biochemical measurements of bone turnover 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 management of patients with CP is complex and, in recent years, different treatments have been proposed. Several studies were focused on the more obvious treatments, centered on physical activity. Chad et al. proposed a program of weight bearing for children with CP, and noted that after 8 months the FN BMC and vBMD and the total proximal femur BMC had increased significantly compared to controls [130]. Caulton et al., in a randomized controlled trial on 26 pre-pubertal non-ambulant children with CP (14 boys, 12 girls; age 4.3e10.8 years), found that longer periods of standing increased vertebral trabecular BMD, measured by QCT, but not proximal tibial trabecular BMD [147]. Two systematic reviews evaluated 10 studies on the effects of progressive resistance exercise on body structure and function: there was strong evidence about its efficacy in increasing the ability to generate muscle force in people with CP [148,149]. Unfortunately, these studies did not evaluate the effects on bone, but considering the relevance of muscular work in CP-related osteoporosis the results justify further specific trials. Recently, a short-term randomized trial evaluated the effects of whole-body vibration: 20 children with CP (age 6.2e12.3 years) were randomized to continue their school physiotherapy program or to add sidealternating whole-body vibration, nine minutes per school day for 6 months. Children treated with vibration therapy improved the walking speed in the 10-minute walk test, but there was no effect on aBMD at LS and distal femoral diaphysis [150]. Considering the small sample and the short duration of the study, no definitive conclusions can be drawn. Another recent trial used a global approach, including TB vibration, physiotherapy, resistance training and treadmill training. Retrospectively evaluating 78 children after 6 months on this program, highly significant changes in BMD, BMC, and muscle mass were observed, as well as significant changes for angle of verticalization, muscle force, and modified Gross Motor Function Measure [151]. Regarding vitamin D therapy, 13 children with severe spastic quadriplegic CP, receiving anticonvulsant therapy, were treated with calcitriol (0.25 mg/day) and calcium (500 mg/day) for 9 months. BMD measured

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by DXA showed significant increases from baseline values compared to untreated controls with similar degrees of CP [152]. Marked clinical improvement was also observed after 3 months of vitamin D supplementation (calciferol, 5000 IU/day) [119]. Considering that children with CP, like those with other physical disabilities, have profound osteopenia, and that the annual fracture rate is more than twice that encountered in healthy children, different studies considered treatment with BPs. However, most of these studies were not randomized, placebo-controlled trials, and were performed in small samples. A 20e40% increase in BMD was reported in three children with CP and fractures, treated with etidronate (one case) or pamidronate (two cases) for 12e18 months [153]. I.v. pamidronate was also used in a double-blind, placebocontrolled study on six pairs of non-ambulant children with CP. One member of each pair randomly received i.v. saline placebo while the other received pamidronate. After 18 months, the aBMD of distal femur (metaphyseal region) increased 89  21% in the pamidronate group, compared to 9  6% in the control group. Agenormalized aBMD Z-scores increased from 4.0  .6 to 1.8  1 in the pamidronate group while there was no significant change in the untreated group [154]. Plotkin et al. treated 23 non-ambulant children and adolescents with severe spastic quadriplegic CP and low BMD (mean age 10  5 years) with i.v. pamidronate for 2 days every 4 months for one year. LS and FN BMD increased significantly, and the BMD Z-scores increased by 1.6 at FN and 1.9 at LS [155]. In a recent study, 25 children (age 11 years) with severe quadriplegic CP and a history of at least one non-traumatic fracture were treated with i.v. pamidronate (for three consecutive days every 3e4 months) plus supplements of vitamin D. The fracture rate decreased from 30.6% per year before treatment, to 13.0% per year after treatment. In most participants, this effect lasted 4 years or longer. However, some of the children suffered a fracture soon after the drug was discontinued, suggesting that a longer treatment may be needed [156]. Other treatments have been studied, but only very preliminary data are available. A small pilot study on 10 patients indicated that 18 months of growth hormone (GH) therapy led to significant improvement in spinal BMD and linear growth [157]. Since vitamin K increases periosteal bone formation and suppresses endocortical bone resorption, it was given for 8 months (15 mg/ day) to an 8-year-old boy with right hemiplegia caused by fetal porencephaly. The results on tibial cortical bone were promising: in fact, the polar moment of inertia (an indicator of the resistance to torsion forces) showed an increase of 13.0% in the non-hemiplegic tibia and of 63.7% in the hemiplegic tibia [158].

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In conclusion, the skeletal health of children with CP represents an important area of future research given the burden of the frequent fractures to the patients and their families. The current literature suggests that a multidisciplinary approach to prevention and treatment (based on optimization of nutrition, weightbearing physical activity and medical intervention with antiosteoporotic agents) may lead to optimization of nutrition, weight-bearing physical activity and medical intervention with anti-osteoporotic agents may lead to significant improvements in the quality of life of these patients. DUCHENNE MUSCULAR DYSTROPHY

DMD, an X-linked recessive disorder due to a mutation of the dystrophin gene (at locus Xp21 in the short arm of the X chromosome), is the most frequent muscular disease in childhood. The severe reduction or absence of dystrophin, depending on the type of mutation, leads to the progressive degeneration of striated muscle, which is slowly substituted by fat and connective tissue. The process is characterized by a massive elevation of creatine kinase levels in the blood. The prevalence of DMD is estimated in 1:3500 births, with a tendency to a slight decrease recently, in response to genetic counseling. Proximal muscle weakness becomes evident in early childhood (usually before 3 years of age) and after a few years the child becomes progressively unable to walk. Generally, ambulation is completely lost at around 15 years of age. Most patients with DMD still die in early adulthood, even if the probability to survive to 30 years of age has considerably increased and in some studies is estimated at around 85% [159,160]. There is no definitive cure and, among the different drugs used to treat DMD, treatment with glucocorticoids (GCs) has clearly changed the course of the disease. GCs improve muscle strength, maintain cardiopulmonary function, prolong ambulation by 2 to 5 years, reduce the severity of scoliosis, and significantly extend life [161,162]. In recent years, different types, doses, and schemes of GC administration have been evaluated in clinical trials with the aim of reducing side effects (such as weight gain, cataracts, impaired growth, and behavioral changes), but no consensus has ever been reached. In clinical practice, GCs are currently started at a much earlier age than in the past, since the sooner they are given, the greater their effectiveness in slowing the disease progression. Fractures have been recognized as a complication of DMD many years ago, and since the mid-1970s, fractures of the long bones have been considered a serious obstacle in maintaining independent ambulation in these patients [163]. In recent years, the presence of low bone mass has been frequently reported, leading to a greater awareness of bone problems.

Currently, the risk of long-bone fractures in boys with DMD is estimated at approximately 20% and may be as high as 44%. However, prospective epidemiological studies on fractures in large cohorts of patients (on GC treatment or not) are still lacking. So¨derpalm et al. did not find a higher long-bone fracture rate in DMD patients versus healthy controls [164]. In a 15-year long study, Biggar et al. evaluated the effects of long-term treatment with deflazacort in 74 DMD boys (age 10e18 years), either on GC treatment or not, and observed a similar percentage (25%) of long-bone fractures among both treated and untreated boys [165]. However, vertebral fractures were not evaluated in these two studies. In another study on 41 boys with DMD, 66% of fractures involved the lower extremities, and aBMD correlated with the site of pathologic fractures, suggesting that the BMD reduction was associated with relevant clinical consequences [166]. The largest published study specifically designed to evaluate peripheral fractures in muscular diseases through a self-administered questionnaire, that involved 229 patients with DMD and other muscular diseases, confirmed that the fracture risk was increased in these patients (RR ¼ 1.9) with respect to age-matched healthy controls [167]. This study showed two other relevant aspects: low-energy fractures were present only in the patients, not in the controls, and almost 40% of the lower limb fractures led to permanent loss of function. Another study on a large DMD population (378 patients, age 1e25 years, median age 12 years) found that 20.9% of these patients had sustained fractures: 41% of fractures occurred between 8 and 11 years of age, and 48% in independently ambulant patients. Falling was the most common cause. It is very important to note that 20% of the ambulant patients and 27% of those using orthoses permanently lost mobility after the fracture [168]. Data on vertebral fractures are even less. In a retrospective study on 143 DMD boys, the GC-treated subjects had an increased fracture rate, long-bone fractures being 2.6 times more frequent than in untreated patients. Vertebral compression fractures occurred in 32% of the GC-treated group, whereas no vertebral fractures were found in the GC-naive group [169]. Vertebral fractures were also observed in a study on 33 boys with DMD: after 100 months of GC treatment, approximately 75% had sustained a vertebral fracture [170]. Chabot et al. studied 46 boys with DMD who received deflazacort over a 4-year period, and found that 26 (52%) of them suffered 37 fractures, of which 39% were vertebral compression fractures and the remainder long-bone fractures. Significant decrements in bone mass occurred over the study period [171]. On the contrary, a recent study, that used DXA to evaluate 25 boys (mean age 7.4 years) at baseline and after 30 months of GC treatment, concluded that GCs

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apparently had no detrimental effects on lumbar vertebrae [172]. Regarding bone density in DMD, there are only a few published studies. Almost all used DXA and concluded that BMC and BMD were lower than normal at different skeletal sites in DMD patients [164,166,167,170,172e174]. Some studies found a greater reduction of BMC and BMD at the lower limbs [166,172e174]. Finally, the relationship between bone density, mobility, and fractures has been poorly studied. In 41 boys with DMD, there was a significant BMD decrease in the lower limbs, where most fractures occurred [166]. Regarding the pathogenesis of osteoporosis in DMD, many factors have been considered but it is not clear whether the muscle dystrophy itself or other factors contribute to the weakening of bone. Muscle impairment significantly reduces weight-bearing activities during the crucial period of growth, and this can profoundly influence bone development and bone density. Animal studies have demonstrated that lean mass (muscle) is a significant determinant of BMD and bone strength, even when accompanied by a dystrophic phenotype [175]. In a study on 32 DMD boys, there was a significant BMD reduction at lower limbs and LS with respect to healthy controls, interpreted as the consequence of reduced weightbearing and muscular activity on bone [174]. GCs can certainly affect bone health in DMD, given their well-known negative effects on bone metabolism (for detailed information on GCs and bone, refer to “Iatrogenic causes of osteoporosis” later in this chapter). An increased frequency of fractures has been observed with GC treatment [168], even if short-term studies on deflazacort suggested that deflazacort may have a bone sparing effect in children [176]. On the other hand, GCs may also have an indirectly positive influence, by preserving muscle function and its stimuli on bone for a longer time. Recently, some preliminary studies discovered an involvement of cytokines, in particular TGF-beta and connective tissue growth factor (CTGF), in the replacement of the dystrophic muscle with fibrotic tissue [177,178]. Knowing the relevant role of cytokines in bone metabolism, it will not be a surprise if, within a few years, a cytokine involvement in the pathogenesis of bone loss in DMD is discovered. Considering the relatively recent interest in the bone complications in DMD and the lack of randomized placebo-controlled trials, the treatment of osteoporosis and fractures in DMD is currently based only on observations from clinical practice, general rules applied in other forms of secondary osteoporosis, and the few available studies, all on small patient samples [179]. A recent review of nutrition in DMD highlighted the importance of calcium and vitamin D supplements [180]. For the moment, only one study demonstrated

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that 25OHD (calcifediol) plus adequate dietary calcium intake seems to be effective in controlling bone turnover, correcting vitamin D deficiency, and increasing BMC and BMD in patients with DMD. In this prospective study on 33 patients on GC treatment (age 5e13 years), after 2 years of calcium-rich diet and calcifediol, BMC and BMD significantly increased in over 65% of the patients, and bone metabolism parameters and bone turnover markers normalized in 78.8% [181]. The only published study on BP use in DMD was conducted on 23 deflazacort-treated boys (age 6.9e15.6 years) who had only a slight decrease of the Z-score and no fractures, and received alendronate plus calcium and vitamin D supplements. After 2 years of treatment, they showed an improvement in TB and LS BMD Zscores, associated with younger age at baseline [182]. Currently, BP treatment with different drugs (pamidronate, risedronate), dosages, and administration routes (i.v., oral), is being used in DMD boys on long-term GCs in case of fractures, particularly vertebral fractures. Considering the relevance of bone problems in DMD, and the fact that fractures are a cause of premature loss of ambulation, fracture prevention in these patients is now recognized as a primary health issue. The development of novel therapies (such as mesenchymal stem cell therapy), and the continuous progress in understanding the pathogenetic mechanisms of bone loss underlines the necessity of specially designed controlled trials. IMMOBILIZATION AND LIMB DISUSE

Osteoporosis secondary to immobilization and disuse gives researchers a unique opportunity to study the skeleton’s adaptation to reduced muscle force. Several clinical conditions characterized by immobilization (from complete motor paralysis to temporary therapeutic recumbency), are associated with loss of muscle and bone mass. The results from studies on immobilization and limb disuse are remarkably concordant despite different methodologies, and clearly demonstrate the importance of weight bearing and muscular activity for skeletal health. In disuse osteoporosis, the reduction of mechanical stress on bone inhibits bone formation and accelerates bone resorption. Imaging diagnosis shows coarse trabecular pattern and thinning of cortical bones. The recent re-evaluation of the role of osteocytes allowed a new interpretation of the genesis of disuse osteoporosis. The osteocytes sense mechanical strain and translate it into signals to osteoblasts and osteoclasts, stimulating bone formation and/or resorption. Both intracellular (cAMP, cGMP) and extracellular (PGE2, IGF-1, IGF-2, TGF-beta) signal transmitters are involved [33,183]. A controlled study on healthy subjects evaluated the effect of voluntary bed rest for 3 months. There was

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a rapid and sustained increase in bone resorption and a subtler decrease in bone formation, demonstrated by histological and biochemical studies [184]. Dramatic changes in muscle mass and force, and significantly decreased BMD have been observed following 4e6 weeks of bed rest [185]. Szalay et al. studied postoperative bone loss in children undergoing lower extremity surgery requiring at least 4 weeks of non-weight-bearing or cast immobilization. Comparing preoperative and postoperative BMDs (measured by DXA), they showed that up to 34% of BMD can be lost in 4e6 weeks [186]. These findings demonstrated that prolonged bed rest is a very serious problem for children with a marginal BMD, like those affected by OI or other diseases leading to osteoporosis. Any period of forced immobilization further aggravates their frail bone health and increases the fracture risk for the future. Complete paralysis due to spinal cord injury (SCI) represents the most extreme case of immobilization, since neither muscle activity nor weight bearing are present below the level of the spinal lesion. An increased risk of osteoporosis is well documented in SCI and in older adolescents and adults the greatest decrements in bone mass occur within the first year following paralysis [187e189]. Bone loss begins immediately, at a rate of over 2% per month in the first 6 months, then 1% per month for the remainder of the year [190]. In a study aimed at evaluating body composition in SCI, a large group of adolescents affected by traumatic motor paralysis showed significantly decreased bone and lean tissue mass, measured by DXA. The greatest reduction of lean mass was in the lower extremities, followed by the trunk [191]. In another study that evaluated hip, distal femur and proximal tibia BMD for one year or more in 28 children with SCI, the BMD Z-scores for the FN, greater trochanter, and Ward’s triangle were about 40% lower than those of children without disability [192]. The measurement of LS BMD in SCI has given conflicting results, but LS BMD may be better preserved than that of lower limbs. In a recent study on young adults with chronic complete SCI, the BMD of the sublesional vertebrae was significantly decreased if the level of injury was at T6 or above [193]. As predicted, lesser degrees of bone loss occur with incomplete paralysis, due to residual muscle function. In children with lower extremity paralysis, and thus able to maintain erect posture, LS BMC may not be different from that of ambulatory controls [194]. Also, in principle, spasticity might decrease the risk of osteoporosis, but a recent study on 18 wheelchair-dependent adults, studied 14 years or more after the injury, found that the BMD reduction at hip was similar in those with severe spasticity and in those with mild or no spasticity, notwithstanding the greater muscle mass in the first group [195].

Low-impact fractures are more common in patients affected by SCI than in healthy subjects [190,196], and most often occur at distal femur. There are inconsistent data about whether bone loss in SCI could be prevented through therapeutic exercise (weight-bearing, passive standing, passive bicycling, etc.), electrical stimulation and bed positioning. Early interventions with passive weight bearing could decrease the early phase of bone loss immediately after SCI, but long-term results are uncertain [197]. Other studies found that passive weight bearing, like using long leg braces, or sitting/standing in standing frames, did not influence BMD at any site [198,199]. It has also been reported that intensive exercise could prevent bone loss in the upper, but not in the lower limbs [189]. Electrical muscle stimulation to elicit active contractions improved muscle function, but positive effects on bone are still uncertain. A very recent case report on three women with SCI reported that functional electrical stimulation for 6 months was able to maintain both aBMD (measured by DXA) and vBMD (measured by pQCT) at lower limbs: it maintained lean mass in both legs in one woman, and increased it in the other two [200]. Considering the increased bone resorption in prolonged immobilization, antiresorptive agents are indicated to reduce bone loss. Different BPs (etidronate, alendronate, pamidronate, clodronate, tiludronate) have been used in immobilized adults (also young adults), most often in conjunction with calcium and vitamin D supplements and have been able to prevent or attenuate BMD loss [201,202]. Two different regimens of i.v. pamidronate led to significant reductions in urinary N-terminal cross-linked telopeptide of type I collagen (NTx) excretion and in 24-hour urinary calcium excretion [203,204]. One of the few prospective, doubleblind, randomized, placebo-controlled studies compared weekly oral alendronate with placebo in 31 adult patients with acute SCI. At randomization, no significant differences in mobility were noted between the groups. After one year, TB, LS, and hip BMD (measured by DXA) and calcaneus BMD (measured by QUS) decreased in both groups, but less in the alendronate-treated group. Alendronate also induced significant reductions in urinary calcium excretion and in serum C-terminal cross-linked telopeptide of type I collagen (CTx) [205]. In another double-blind, randomized, placebo-controlled trial, 17 young adults with SCI were followed for one year after a single administration of either zoledronate or placebo. Using DXA for BMD evaluation and structural analysis of the proximal femur, the authors concluded that a single zoledronate infusion was able to reduce bone loss and maintain bone strength at the femur intertrochanteric and shaft sites for 12 months [206].

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Chronic Diseases A number of serious chronic disorders have been associated with bone morbidity in children, and with the improved long-term outcomes due to diagnostic and therapeutic advances, there has been increasing attention to the skeletal complications of chronic pediatric disease. The chronic illnesses most frequently associated with adverse effects on skeletal health in youth are discussed in this section. LEUKEMIA

Acute lymphoblastic leukemia (ALL) is the most common form of cancer in pediatric age. The incidence is 44 cases per million children in the age range 0e14 years. It is estimated that 25% of the new diagnosis of cancer in children are ALL. In the 1990s, in developed countries, the 5-year event-free survival rates for childhood with ALL were 70e83%, and the overall cure rate was about 80% [207]. For these reasons, most studies on the long-term outcome of childhood cancer are on ALL survivors. Bone morbidity associated with ALL and other childhood cancers has been a focus of attention in recent years [208], and has been observed in many ALL survivors, who have an almost doubled fracture rate [209]. Among the long-term complications associated with the treatment of ALL, osteopenia (bone mass loss) and osteonecrosis (avascular necrosis) are relatively common, in particular with prolonged GC treatment. Only osteopenia will be discussed here, since osteonecrosis is beyond the scope of this chapter. Musculoskeletal pain, gait abnormalities, osteopenia and fractures have been reported in children with ALL at diagnosis and during treatment [210]. Radiographs of painful regions show metaphyseal lucencies, sclerotic lesions and sites of periosteal reaction in many patients with bone pain at presentation. Different mechanisms have been proposed for the skeletal morbidity observed in ALL: bone infiltration by leukemic cells, paraneoplastic factors, and disordered mineral metabolism [210]. To date, only a few studies have evaluated the early skeletal alterations [211,212], but several groups have reported loss of bone mass during chemotherapy [210,213,214]. The greatest reduction in bone mass occurs during the first 6 months of therapy, consistent with the effect of GCs on bone [212,215]. A longitudinal study with quantitative ultrasonography (QUS) on 44 newly diagnosed children during treatment, demonstrated a rapid decrease of BMD during the first 6 months, and a significantly reduced BMD until the end of treatment [215] (for more information on drugs and bone, refer to “Iatrogenic causes of osteoporosis” later in this chapter).

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GCs are certainly involved in the reduction of BMD through their direct effects on bone cells and their indirect effects on the GH/IGF-1 axis, muscle strength, and calcium balance [216]. Methotrexate (MTX) is cytotoxic for osteoblasts and may act synergistically with GCs. Inactivity, nutritional deficiencies, renal electrolyte wasting, radiotherapy, and treatment-induced endocrine dysfunctions are additional factors for bone loss in ALL [217]. Other possible factors include vitamin D deficiency and hypomagnesemia. In a study by Halton et al., 65% of children with ALL had a decrease in BMD during treatment, and 39% sustained fractures, mainly at lower limbs. High levels of urinary NTx indicated increased bone resorption [210]. Van der Sluis et al. found a reduction of bone formation markers at diagnosis, while markers of bone resorption were normal at diagnosis but increased during treatment. ALL patients had a sixfold increase in fracture rate compared to healthy controls [212]. Pubertal patients may be more susceptible to skeletal insult during treatment than younger children, and BMC changes are strongly predictive of subsequent fractures [210]. Vertebral compression fractures are a serious complication of ALL in children. The prevalence and pattern of vertebral fractures, as well as their relationship to BMD and other clinical indices, have not been systematically studied. However, Halton et al., in a recent prospective study focused on vertebral fractures, observed that 29 (16%) of 186 children with ALL (age 1 month to 17 years; median age 5.3 years), evaluated within 30 days of diagnosis, had a total of 75 prevalent vertebral compression fractures, mostly in the mid-thoracic and thoracolumbar regions. LS BMD Z-score and back pain were associated with increased odds for fracture, and for every 1 SD reduction in LS BMD, the odds for fracture increased by 80%. The authors conclude that vertebral fractures are an under-recognized complication of ALL at diagnosis. Whether the fractures will resolve through bone growth during or after chemotherapy has not yet been determined [217]. Many studies have investigated bone alterations in survivors of childhood ALL [218e221], also to evaluate whether bone loss and abnormal mineral metabolism persist in the long term. Kadan-Lottick et al. studied 75 ALL survivors 11 to 82 months after diagnosis. Overall, the mean TB aBMD Z-score was normal, and a significant positive correlation was found between TB aBMD and years elapsed since start of maintenance therapy, after adjusting for risk status/age category, history of cranial irradiation, and total days of hospitalization. Patients receiving maintenance therapy within the last 6 months had a reduced bone mass and a doubled incidence of fractures [222]. Similar results are reported by van der Sluis et al. who showed that ALL survivors

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treated with high-dose MTX and dexamethasone, but not with cranial irradiation, had normal TB and LS BMD values 7.9 to 11.4 years after treatment [223]. Cranial irradiation is a special risk factor for bone morbidity in ALL [220], but fortunately, is less frequently used today. Other studies have shown that ALL survivors have a relatively lower BMD than the general population [222,224,225]. A recent study on 324 ALL survivors (under 21 years of age at initial diagnosis and in continuous remission for 2 years while off therapy) found that 23 (7.1%) had osteopenia or osteoporosis and 19 (5.9%) sustained 27 fractures [226]. The different bone status in ALL survivors may be explained by the different age at evaluation, different treatment protocols (drugs and doses), and different techniques for BMD measurement. According to the available studies, most survivors recovered bone mass as time off therapy grew longer, but severe BMD reduction may persist some years after completion of therapy [226]. In a study by Mandel et al. on 106 ALL survivors (age 7.8e30.6 years), evaluated 2.5 to 12.4 years after completion of therapy, only 23 had a reduced BMD compared to healthy controls. The authors concluded that most survivors of childhood ALL do recover normal BMD, while low BMD may persist in those who received the higher doses of MTX or GCs [227]. In conclusion, significant BMD reduction is frequently observed during ALL treatment, and may constitute an additional morbidity, carrying an increased fracture risk even for younger patients. ALL survivors are also at risk for other complications, such as hypothyroidism, hypogonadism and GH insufficiency, that may interfere with their bone health and their quality of life. Surveillance for endocrinopathies is thus required during and following ALL treatment to ensure appropriate interventions if any deficiencies appear. The current guidelines for patients treated with GCs, MTX or hematopoietic cell transplant recommend a baseline BMD evaluation at entry into long-term follow up, typically 2 years after completion of therapy [228,229]. Treatment of low BMD in ALL children follows the general rules: weight-bearing exercise, adequate dietary calcium intake, and calcium and vitamin D supplementation if necessary. Follow up of ALL must include endocrinologic evaluation and treatment of complications such as hypogonadism and GH deficiency [228]. More aggressive medical therapy with antiresorptive agents (BPs) to prevent bone mass decrease or fractures during ALL treatment has not been systematically studied, although pamidronate has been successfully used in ALL-associated hypercalcemia [230,231]. Finally,

educating ALL survivors to avoid smoking, alcohol, and excess caffeine intake is important as all these factors can favor BMD decrease. RHEUMATIC DISEASES

The epidemiology of rheumatologic diseases is quite variable in the different geographic areas. The worldwide prevalence of juvenile idiopathic arthritis (JIA) has been estimated at between 0.07 and 4.01 per 1000 children, with an annual incidence of 0.008e0.226 per 1000 children. Undiagnosed cases may partly account for this variability. The reported incidence of childhood-onset systemic lupus erythematosus (SLE) in the USA, Japan and Canada is 0.36e0.90 per 100 000 per year [232]. The association of inflammatory diseases of childhood e e.g. JIA, juvenile SLE, and juvenile dermatomyositis (JDMS) e with compromised skeletal health (osteopenia and osteoporosis) is well known. Chronic rheumatic diseases affect the skeleton through direct and indirect actions. The most important direct action on bone is due to inflammatory cytokines. Among the indirectly-acting factors, there are iatrogenic factors such as treatment with GCs, MTX or cyclosporine A (for detailed information on drugs and bone, refer to “Iatrogenic causes of osteoporosis” later in this chapter), reduced physical activity, poor nutrition, impaired growth, and delayed puberty. While chronic inflammation is present in all connective tissue diseases, rheumatoid arthritis (RA) is the condition best studied under this aspect. Both synovial and soluble cytokines are involved in RA, while only soluble cytokines are involved in other rheumatologic diseases. Cytokines promote osteoclast recruitment, activation and osteolytic action, and osteoclasts are probably the key players in bone loss in these diseases. Tumor necrosis factor alpha (TNF-alpha) is one of the most potent osteoclastogenic cytokines released in inflammation, and it stands foremost in the pathogenesis of RA [233]. Regarding the iatrogenic factors, GCs will be discussed separately. MTX is known to induce osteopenia when used at high dosages in children with malignancy, such as leukemia. The lower dosages used in JIA seem not associated with low bone mass [234,235]. A study showed a moderate bone mass increase in 32 children followed for 18 months, notwithstanding MTX treatment. Both absolute value and the increase in bone mass were not correlated with MTX dose or duration of therapy [235]. It is possible that the beneficial actions of MTX (reduced inflammation, increased mobility) can overcome its inhibitory effect on osteoblasts. An observational study did not find adverse effects of low doses of MTX on bone formation in adults (evaluated with bone turnover markers and bone formation indexes in

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bone biopsies) [236]. Cyclosporine A can cause a high turnover osteopenia [237,238]. Mobility can be significantly reduced in many rheumatologic diseases, depending upon disease severity. Madsen et al. examined the relationship between quadriceps muscle strength and bone status (assessed by QUS and DXA) in adults with RA. In multiple regression analysis, the quadriceps strength predicted BMD at hip and SOS at calcaneus independently of age, height, weight, disease duration, physical disability, pain, functional class and cumulative GC dose. They concluded that physical activity and muscle strengthening exercises can prevent bone loss in RA, at least partially [239]. Similar observations and conclusions may also be valid for children. Physical activity seems to be a positive determinant of BMD also in JIA, even if a large proportion of JIA patients have a reduced physical activity, especially weight-bearing activity [240,241]. There are some data on two groups of non-GC-treated children with JIA showing that decreased physical fitness (measured as either a timed walk or the number of performed sit-ups) correlated with, or was predictive of, low BMD [242]. JUVENILE IDIOPATHIC ARTHRITIS JIA, formerly called juvenile rheumatoid arthritis or juvenile chronic arthritis1, is a systemic inflammatory disorder characterized by chronic synovitis of the diarthrodial joints, with characteristic radiological features. Three major subsets are described: pauciarticular, polyarticular and systemic. JIA can appear at any age during childhood, more frequently in girls than in boys, although the gender ratio differs for the different subsets of the disease. The primary cause of the chronic inflammatory processes involving the synovial lining of diarthrodial joints e the hallmark of the disease e is not known. There is proliferation and infiltration of lymphocytes, plasma cells, and activated macrophages, indicating severe local immune reaction. Pro-inflammatory factors that stimulate the differentiation of osteoclast from hematopoietic precursors are also activated. Active arthritis reduces bone mineral accrual around affected joints (periarticular osteopenia), but may do so also at skeletal sites far from the diseased joints. Reed et al. observed that 24 (89%) of 27 children with active JIA had radius BMD 2 SD below the expected value for age. On a 3-year follow up, improvement in disease activity was associated with significant BMD increase, but patients with persistently active disease showed BMD decrease or no increase [243]. Supporting the

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hypothesis that disease activity impairs normal bone metabolism, many studies demonstrated that the reduction in bone mass increases with the duration, severity, activity of JIA, use of GCs, and loss of physical activity [241,244e246]. In a step-wise regression model, Pereira et al. observed a decrease in BMD in 50e60% of children with JIA and found that BMC was only correlated to lean body mass [246]. Atraumatic fractures may occur at an early age [247,248]. Vertebral fractures are more common in children who received a cumulative dose of 5 g of prednisone equivalents or more [248]. Multiple stress fractures (distal femur, calcaneus and sacrum) were described in a case report, and were attributed to the combined action of GCs, disease activity, and immobilization due to a previous traumatic fracture [249]. Murray et al. reported that 24 (23%) of 103 patients with systemic onset of JIA had at least one fracture, and that vertebral fractures accounted for 56% of the total. The risk of low BMD and fractures was higher in the presence of growth failure, erosive articular disease, and high cumulative GC dose [250]. In an earlier longitudinal study by Elsasser et al. on 63 children with JIA followed for 18 months, nine children had sustained a vertebral fracture at baseline and four sustained one during follow up. Spine crush fractures were associated with prolonged periods of bed rest, GC therapy, radial trabecular BMD more than 2 SD below normal and low serum levels of 25OHD [251]. In a recent population-based study on 1939 patients with childhood-onset arthritis and 207 072 controls, the patients with arthritis had a higher risk of both vertebral and non-vertebral fractures than controls, especially during adolescence and after 45 years of age [252]. Several studies found reduced bone mass at different skeletal sites in JIA, studied with different techniques (DXA, QUS, and pQCT) [243,246,247,253e256]. Most of them found reduced BMD in JIA children compared with healthy controls, but the values were not corrected for small size or delayed puberty. Kotaniemi et al., however, observed a significantly low BMD after adjustment for delayed skeletal and pubertal maturation or small bone size [247]. A later study on 105 adolescents with early-onset JIA (age 13e19 years, mean age at onset 2.8 years), assessed after a mean disease duration of 14.2 years, observed a persistent BMC reduction: 41% had low TB BMC and 34% had low TB BMD more than 11 years after disease onset. The reduction in TB BMC was related to duration of active disease, disease severity, bone resorption markers, weight, and height [256].

1

“Juvenile idiopathic arthritis” is currently the preferred nosological definition. However, the terms “juvenile rheumatoid arthritis” and “juvenile chronic arthritis” have been previously used and are still used by some authors. Notwithstanding the existence of subtle differences in the classification criteria, we chose to use JIA throughout the text, independently of the term used by the cited authors.

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Reduced levels of bone formation and bone resorption markers have been observed in children with active JIA both during GC and NSAID therapy [243,255]. Hillman et al. observed reduced bone formation markers (osteocalcin and bone specific alkaline phosphatase [BSAP]) in 44 children with active pauciarticular and polyarticular diseases [257]. Pepmueller et al. confirmed these results, and also found that disease activity markers were significantly correlated with decreases of bone formation markers, but not with bone resorption markers. The alterations of bone markers were accompanied by low bone mass, particularly low in the polyarticular subset [244]. Reed et al., in a study on 113 children with chronic rheumatic diseases (JIA, SLE, JDMS), confirmed the presence of reduced osteocalcin levels and concluded that this could be a sensitive index of reduced osteoblast activity and bone formation [258]. Marked linear growth retardation is frequently observed in JIA, and seems to be associated with disease-related factors, GC therapy, nutritional status, BMD, and earlier disease onset. Growth retardation, together with the adverse effects on bone remodeling, prevents the achievement of an optimal PBM, predisposing these subjects to increased fracture risk in adulthood. In a small group of 20 girls with active JIA, different values of bone mass were observed, depending on pubertal status: only postpubertal JIA girls had a lower bone mass than healthy girls matched for age and pubertal status [259], and similar results have been reported in another study on 38 girls [260]. On the contrary, Henderson et al. found low BMC in almost 30% of both prepubertal and postpubertal girls with mild to moderate JIA, never treated with GCs [234,261]. It has also been observed that adult subjects with a past history of JIA had a lower bone mass than matched controls [262,263]. There are only a few studies on the treatment of low bone mass and bone metabolism alterations in children with JIA and other rheumatic diseases, in particular controlled studies on preventive and treatment strategies. Calcium, vitamin D and 25OHD have been studied in pediatric patients with various rheumatic diseases, either treated with GCs or not. The available data on the efficacy of these treatments in reducing or preventing bone loss are inconclusive [264,265]. All these studies treated small numbers of patients with different characteristics (age, type, duration and severity of the disease) and the results are not comparable. However, three randomized studies recently evaluated the effects of calcium supplementation in JIA. In the first, a randomized double-blind placebo-controlled trial, 198 children and adolescents with JIA received either 1000 mg of Ca and 400 IU of vitamin D daily for 24 months, or placebo

calcium tablets and 400 IU of vitamin D. Those who received calcium showed a small but statistically significant increase in TB BMD compared with those on placebo [266]. According to another publication from the same study, a significant decrease in bone turnover markers was observed, after 12 months, only in the children who received calcium, and the authors concluded that calcium supplementation meets the physiological needs of JIA patients [267]. The third study, while studying calcium absorption, also evaluated the effects of calcium and vitamin D alone or in combination, compared with placebo, and reached different conclusions: only vitamin D3, with or without calcium, but not calcium alone, increased serum 25OHD and calcium levels. There was no effect on BMC [268]. In some specific conditions, the presence of osteoporosis (low bone mass plus fractures) requires a more aggressive treatment in children and adolescents as it does in adults, but only preliminary data on the efficacy of BPs are available. Shaw et al. treated one JIA patient, among four others with different diseases, with cyclical i.v. pamidronate because of severe back pain due to vertebral fractures, and observed pain relief and a high BMD increase (þ26%) after one year [102]. A few other studies evaluated the effects of different BPs on small numbers of patients with positive results [269e271]. Bianchi et al. used oral alendronate in 38 young patients (33 on long-term GC therapy), affected by diffuse connective tissue diseases and with low BMD or previous fragility fractures. After 12 months, the treated group showed a high BMD increase (þ14.9  9.8%), while 38 untreated controls (same age and same diseases, but not requiring GCs) had only a limited increase (þ2.6  5%). Fragility fractures were documented in 20% of patients prior to alendronate therapy, while no new fractures were sustained during the 12month treatment. Alendronate was well tolerated, with only minimal side effects, and growth was not influenced [272]. According to a recent review of the available evidence on the efficacy and safety of BPs in JIA children with low BMD and fragility fractures, all the selected studies (16 out of 94), reported that BP treatment for up to 3 years increased LS BMD from 4.5% to 19.1% with respect to baseline. The most common side effect was a flu-like reaction with i.v. BPs, after the first infusion, managed with paracetamol. The authors concluded that BPs are a promising treatment for osteoporosis in children with JIA, but the quality of evidence is variable and well-designed studies are needed [273]. Touati et al. observed a significant increase in bone turnover in 14 children with systemic JIA on longterm GC therapy, treated with GH for one year. However, bone turnover returned to the baseline velocity after GH discontinuation, and the BMC

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increase was not significant [274]. On the contrary, in a study on 20 children affected by JIA (17 of them treated with GCs), a 1-year course of GH therapy led to a significant increase in both BMC and height [275]. A prospective, randomized, placebo-controlled 3-year study on 30 children with JIA (15 on GH treatment, of whom three with vertebral fractures at baseline; 15 on placebo, two with vertebral fractures) reported inconclusive effects on BMD. Although the GH-treated group showed an improvement in BMD Z-score, it was not significant, and two additional patients had vertebral collapse [276]. A recent review concluded that GH treatment positively influences bone density, bone metabolism, bone geometry, and body composition [277]. However, given the limited experience, long-term controlled studies are needed to determine the risks and benefits of GH therapy in JIA and its real impact on bone. Siamopoulou et al. used salmon calcitonin and calcium supplements for 3 years in 10 children with severe JIA, observing an increase in LS BMD and a decrease in bone resorption markers [278]. Finally, Simonini et al. evaluated the effects of etanercept treatment for 1 year in 20 children with polyarticular JIA: there was a significant BMD increase (measured with QUS) with respect to baseline [279]. These positive results are waiting for confirmation by prospective, placebo-controlled trials on larger cohorts. There is no doubt that prevention and treatment of low bone mass and osteoporosis are mandatory for all JIA children who do not achieve rapid remission. Currently, there are no general guidelines, but the diagnostic and therapeutic options have been discussed in a recent review, with suggestions for introducing them into clinical practice [280]. JUVENILE SYSTEMIC LUPUS ERYTHEMATOSUS SLE is a multisystem autoimmune disorder characterized by the production of autoantibodies directed against various nuclear and cytoplasmic components of target cells. Vasculitis, partly due to the deposition of immune complexes, is typical, but inflammatory processes can involve any organ and system (skin, liver, kidney, serous membranes, heart, CNS and joints). SLE can affect children at any age. An increased incidence of bone loss and vertebral fractures is reported in adult women with SLE, but the available data on children and adolescents are less consistent and obtained from small samples. In a study by Castro et al., LS BMD was somewhat lower, but not significantly, in 16 girls (aged 6e17 years) with juvenile SLE with respect to controls [281], whereas, on the contrary, in a longitudinal study on 20 young patients with juvenile SLE (aged 5e25 years), Trapani et al. found that BMD was significantly lower than in controls, both at baseline and after 1 year, only

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in patients aged 19e25 years. There was a significant negative correlation between BMD and cumulative GC dose, but no correlation between BMD and disease activity or duration [282]. Finally, in a case report, Ozaki et al. observed that during GC therapy for SLE, an 11year-old girl presented multiple fish vertebra deformities, accompanied by a 5-cm height loss and severe low back pain [283]. JUVENILE DERMATOMYOSITIS JDMS is an autoimmune multisystem inflammatory disease that primarily affects skin and muscle. All muscular tissues can be involved, including the smooth muscle of gastrointestinal tract and the myocardium. Vasculitis of small vessels is the main feature of the disease in childhood. Calcinosis of the skin and subcutaneous tissues, muscles, tendons and ligaments is a late manifestation: it can appear while the inflammatory process is still active, or even years after the resolution of the active disease. JDMS used to be fatal in more than one third of affected children, but treatment with GCs and other drugs (MTX, cyclosporine A, azathioprine) has greatly improved outcome and survival. There are only a few studies on bone involvement in JDMS. Ellis et al., in a study of TB BMC in children with various diseases, evaluated 29 children affected by JDMS, and observed a reduced BMC Z-score in 27.6% of them [284]. In a study on 15 patients, of whom 10 had active disease, and five had inactive disease and had not taken GCs for 3e8 years, osteopenia or frank osteoporosis were observed in six patients with active disease and four on remission. Osteopenia persisted or worsened in seven of the 10 patients with active disease, while the other three had a significant BMD increase after being treated with BPs for vertebral compression fractures. The authors underlined that osteopenia usually worsens with active disease and may persist for several years after remission [285]. INFLAMMATORY BOWEL DISEASES AND CELIAC DISEASE

Malabsorption and inflammation are major causal factors of altered bone metabolism in inflammatory bowel diseases (IBD) and celiac disease. Impaired intestinal absorption is due to reduction of functional mucosa, especially in acute phases, and in Crohn’s disease also to surgical resection of ileum. Calcium malabsorption is caused by steatorrhea, alterations in calcium-transport mechanisms, and vitamin D deficiency (Fig. 18.2) [286]. The increased production of proinflammatory cytokines, including specific osteotropic cytokines involved in both normal and abnormal bone modeling/remodeling, may cause bone loss. For example, IL-1 (interleukin), IL-17 and TNF-alpha stimulate bone resorption by osteoclasts [287]. In addition, the

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FIGURE 18.2

Steps in vitamin D and calcium absorption and handling that may be altered in inflammatory bowel diseases and celiac disease. IBD, inflammatory bowel diseases; Ca, calcium; 25OH D, 25-hydroxyvitamin D; 1,25(OH)2 D, 1,25-dihydroxyvitamin D. (From Bianchi ML. Inflammatory bowel diseases, celiac disease, and bone. Arch Biochem Biophys 2010;503:54e65.)

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frequent use of GCs constitutes an additional risk, because of their well-known detrimental effects on bone [288,289] (refer also to “Iatrogenic causes of osteoporosis” later in this chapter). Both osteoporosis and osteomalacia are encountered in IBD and celiac disease. Osteoporosis is now the most frequent bone complication, while osteomalacia is progressively disappearing. INFLAMMATORY BOWEL DISEASES In most cases, IBD (Crohn’s disease and ulcerative colitis) occur in young adults, and one fourth of new diagnoses are made in subjects below 20 years of age. The peak incidence is between 10 and 40 years of age. The incidence of ulcerative colitis in industrialized countries is stable at around 0.7e3.6 per 100 000 children. That of Crohn’s disease is 1e4.9 per 100 000 children, but is increasing especially in north-western Europe and North America. The total incidence of IBD is 2.6e7.4 per 100 000 children. In general, the incidence of Crohn’s disease is about twice that of ulcerative colitis [290,291]. IBD are characterized by an intermittent course, with periodic exacerbations and remissions [291], and probably affect individuals with a genetic predisposition that alters some components of the innate and adaptive immune systems, normally active against intestinal microbes, misdirecting them to attack the bowel mucosa [292]. The prevalence of bone complications is difficult to estimate because clinical conditions, diagnostic facilities and treatment regimens are often different in the different studies and countries. The etiology of bone loss in IBD is still incompletely understood. Some in vitro studies have found that the serum of children with IBD contains osteoblast-inhibiting factors [293,294]. Moreover, young patients affected by IBD often suffer from inadequate nutritional intake, malabsorption, delayed puberty, growth retardation, and vitamin D deficiency, all of which have negative effects on bone development [295,296]. There is growing evidence that in IBD, inflammation per se contributes to an early onset of osteoporosis. Paganelli et al. studied 56 children with IBD and found that inflammation was an important determinant of bone loss, as shown by the correlation of BMAD with serum IL-6 and with disease activity indexes: 10 patients with Crohn’s disease were treated with infliximab and had a higher BMAD than those never treated, suggesting that anti-inflammatory treatment may have beneficial effects on bone density [297]. Several studies reported low BMD in children with IBD, often at time of diagnosis [296e303]. BMD seems to be lower in patients with Crohn’s disease compared to those with ulcerative colitis [304]. Ward et al. studied 20 children at diagnosis of IBD. Bone histomorphometry on transiliac bone biopsies

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showed a slightly reduced cortical width, and a low percentage of trabecular bone surface covered by osteoid or osteoclasts, suggesting reduced bone turnover, which was confirmed by biochemical bone markers. Height was normal, but LS BMD and TB BMC were moderately low for age and gender [298]. In a recent cross-sectional study on 143 IBD children and adolescents (98 with Crohn’s disease, 45 with ulcerative colitis; 29% newly diagnosed), Bechtold et al. observed lower height, weight, muscle mass, and total bone cross-sectional area (CSA, measured with pQCT), compared to age- and gender-matched healthy controls. In newly diagnosed patients, the ratio BMC/muscle CSA was higher than in those with longer disease duration. The trabecular BMD Z-score was at the lower normal level. The authors concluded that bone disease in children with IBD seems to be secondary to muscle wasting, which is already present at diagnosis. With longer disease duration, bone adapts to the lower muscle CSA [299]. In a study on 90 children and adolescents with IBD and 52 controls, Walther et al. found that 8% of girls and 20% of boys had a LS BMAD Z-score <2. According to this study, the prevalence of reduced BMD in pediatric patients with IBD is approximately the same as in adult patients, and can already be present before GC treatment [300]. Lopes et al. studied 40 children and adolescents with IBD and found low BMD in 10 (25%). They also identified GC cumulative dose, height-for-age Z-score, and BMI Z-score as independent risk factors associated with BMD [302]. In a retrospective study on 1649 patients from the PediIBD Consortium Registry, diagnosed with IBD before 18 years, osteopenia/osteoporosis (15%) followed arthritis among the most frequent extraintestinal manifestations of IBD after diagnosis [305]. There are also studies that did not find low BMD in patients with IBD. For example, in a study on children with Crohn’s disease, Burnham et al. showed that an apparently low TB BMC, measured by DXA, appeared normal after adjustment for gender, height, and lean body mass [306]. Studies on vitamin D deficiency, usually defined as serum 25OHD <15 ng/ml (37.5 nmol/L), are relatively few in children with IBD [295,307], and larger prospective controlled studies are needed. The causes of hypovitaminosis D in patients with IBD is unclear, and probably multifactorial (see Fig. 18.2). Serum 25OHD concentration in adult patients with IBD was not found consistently correlated with BMD, bone resorption, bone formation, or indicators of calcium homeostasis (such as PTH). Data for children are very scarce if not missing at all. The long-term significance of vitamin D deficiency is still unknown, and there are no sufficient studies or guidelines on treatment of hypovitaminosis D, maintenance of optimal vitamin D reserves, and effects of

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vitamin D supplementation on bone health or disease evolution in both adults and children with IBD. Pappa et al. found hypovitaminosis D in 45 of 130 patients with IBD (age 8e22 years) [308], and Sentongo et al. in 18 of 112 patients with Crohn’s disease (age 5e22 years) [307]. Recently, in 60 newly diagnosed children (39 Crohn, 21 ulcerative colitis), El-Matary et al. found serum levels of 25OHD significantly lower than in healthy controls [304]. Fractures are a well-know complication of IBDrelated osteoporosis. A large Canadian study on 6027 IBD patients, matched by year, age, gender, and postal area of residence to 6270 controls, found that the incidence of fractures (spine, hip, wrist/forearm, ribs) among IBD patients was 40% greater than in the general population [309]. In IBD children, actual fracture risk is unknown, and while there is evidence that reduced mineral accrual and low PBM are common in IBD, it is also unknown whether the onset of disease in childhood increases the risk of fractures in adult age [310]. A study on 733 young patients with Crohn’s disease and 488 with ulcerative colitis, and 3287 matched controls, did not observe a higher risk of fracture at any site. GC exposure was not associated with the occurrence of fractures [311]. Persad et al., in a survey of long bone fractures in children with IBD, compared to healthy sibling controls, found no statistically significant difference between the groups [312]. In a few cases of Crohn’s disease, severe osteoporosis with vertebral compression fractures was already present at diagnosis [313] and Semeao et al., reporting on the presence of vertebral fractures in five children with Crohn’s disease, suggested that BMD evaluation should be part of the standard medical care of these patients [314]. To prevent osteoporosis in IBD, it is necessary to keep the primary disease under control, to use systemic GCs at the minimum effective dose and for as short a time as possible, and to avoid calcium and vitamin D deficiency. All patients with IBD should receive osteoporosis prevention therapy at the start of GC therapy. Early osteoporosis prevention is justified by the fact that GCinduced bone loss is greatest in the first months of treatment and that the first months require the higher GC dose [315]. All patients with IBD should maintain an adequate calcium intake, about 1200e1500 mg/day. Calcium-rich foods should be preferred; calcium salts are only a second choice. Adequate calcium intake is recommended, commencing at the start of GC therapy [315], even if the effects of calcium and vitamin D intake on bone density are still uncertain. Although minimizing bowel inflammation is the main therapeutic goal in children and adolescents with IBD, it is essential to reduce the risk of complications such as growth failure, pubertal delay, and impaired bone mass accrual.

The effects of GH treatment for 1 year on growth velocity was studied in 10 children and adolescents with Crohn’s disease and poor height growth. GH increased height velocity and seemed to enhance bone mineralization [316], but the real impact of GH treatment can be assessed only with a prospective, randomized, controlled trial on a large cohort of children. Inflammation control may also be a treatment, or at least a co-treatment, for preventing or lessening bone loss in IBD. A recent study evaluated changes in bone formation and resorption markers, linear growth, and disease activity after infliximab therapy for 54 weeks in 112 children and adolescents with moderate to severe Crohn’s disease: significant increases in serum BSAP and NTx were observed, suggesting inhibition of TNFalpha action on osteoblasts. According to the authors, the increase in bone resorption markers (urinary CTx and serum deoxypyridinoline) reflects a coupling of bone formation and resorption and an increase in linear growth [317]. Another study that reported a higher BMAD with infliximab therapy in Crohn’s disease has been cited above [297]. CELIAC DISEASE Celiac disease is a chronic intestinal disorder characterized by an immune reaction to the gliadin fraction of gluten, a protein found in wheat, rye and barley. Gluten ingestion causes villous atrophy and inflammatory alterations of the small bowel mucosa, from the duodenum to the distal ileum. The disease occurs in genetically predisposed subjects and is often familial. In recent years, large screening studies have demonstrated that celiac disease has a much higher prevalence than estimated in previous surveys: up to 1% of the general population in Europe and the USA seems to be affected [318]. In the past, steatorrhea and malabsorption used to be the main presenting symptoms of celiac disease, but today the presentation is often atypical in both adults and children, with few if any intestinal symptoms at all [319]. Asymptomatic or atypical celiac disease may go unrecognized for many years, and several complications may develop, not only in adults but also in young subjects. Reduced BMD is unfortunately a common presentation from early adulthood and is often discovered by chance. There are only a few bone studies, mostly cross-sectional and on small samples, on pediatric patients. Low BMC has often been found at the time of diagnosis in celiac children, and even more in adolescents [320e323]. In patients with symptomatic celiac disease, osteomalacia or osteoporosis are secondary to reduced calcium absorption, due to villous atrophy, and/or to vitamin D deficiency and secondary hyperparathyroidism. General malnutrition may also have a role. Corazza

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et al. [324] found that malnutrition was present at the time of diagnosis in 67% of patients with overt celiac disease and in 31% of those with subclinical disease. This suggests that children and adolescents with undiagnosed celiac disease may suffer from an inadequate intake of calcium, protein and total calories in the years of maximal skeletal development and BMD accrual, which may lead to a low PBM and an increased risk of osteopenia/osteoporosis in older age. Additional risk factors for a less-than-optimal PBM value in young celiac patients have been recently highlighted. Celiac children have often a retarded growth. A decrease in growth stimulating factors, like IGF-1, is sometimes observed in celiac patients not on a glutenfree diet (GFD). A decrease in BSAP, IGF-1, IGF-binding protein, and serum CTx have been observed after a 4week gluten challenge in 54 children with celiac disease, who had previously been on GFD for at least 12 months [325]. The decrease in growth factors and bone markers was correlated with reduced body weight and increased intestinal mucosa inflammation. In particular, the decrease in IGF-1 and its binding protein was related to the degree of mucosal atrophy. This could be an explanation for the stunted growth observed in celiac children without clinical signs of malabsorption. A study discovered celiac disease in 12 (1.12%) out of 1066 children evaluated for short stature: after 1-year on GFD, nine of the celiac children showed an increased growth, while the remaining three showed an associated GH deficiency [326]. Thus, in children with celiac disease, a careful attention to growth is necessary even after starting GFD, and GH secretion should be evaluated in those with a good adherence to GFD but without catch-up growth. This aspect is relevant when evaluating BMD in celiac children, not only to avoid a misinterpretation of DXA data (apparently reduced BMD due to the short stature), but also because of the strong influence of GH on bone mass. An important aspect in growing patients with celiac disease is the frequent association, ranging from 2.4% to 10.4%, with type 1 diabetes (T1D) [327]. A recent study in children with T1D found that the presence of celiac autoimmunity is associated with a lower BMD [328]. Another risk factor is related to leptin, a hormone essential for body weight regulation, and also important in bone metabolism. Several studies observed that serum leptin level is correlated with BMD. The action of leptin is very complex and not fully known. This cytokine-like hormone, secreted by adipocytes, has both a direct anabolic effect on bone, via osteoblast stimulation, and an indirect catabolic effect, via central hypothalamic mechanisms and activation of the sympathetic nervous system. The complexity of its action is further demonstrated by its different effects on cortical and trabecular bone remodeling [329]. It has been recently

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discovered that celiac children have reduced serum leptin levels, and that GFD seems to be able to increase them: a significant correlation between leptin levels and BMI was found in these children [330]. Since cross-sectional and longitudinal studies in both adults and children have shown that BMD is positively correlated with body weight and BMI, it may be possible that increased leptin levels have positive effects on bone, due to increased body weight and leptin’s action on osteoblasts. Some epidemiological studies have brought to light the increased risk of fractures in celiac patients, although mainly in adults. Olmos et al. in a meta-analysis of eight studies, considered 20 955 celiac patients and 97 777 controls: 1819 fractures (8.7% prevalence) occurred in celiacs, versus 5955 (6.1%) in controls. The pooled odds ratio (OR) confirmed a significant association between fractures and celiac disease (OR ¼ 1.43) [331]. Very few data on fractures in celiac children are available. A Swedish general population-based study on 13 724 celiac patients and 65 627 controls concluded that celiacs, including children, had an increased risk of any-type fracture, and that the increased risk for hip fracture persisted 20 years after diagnosis of celiac disease (general hip fracture hazard ratio 2.1, for children 2.6; any-type fracture hazard ratio 1.4, for children 1.1) [332]. In children with a diagnosis of celiac disease, GFD is currently considered the most effective therapy also for bone. If strictly followed for the rest of life, it can resolve the intestinal inflammatory processes and improve bone development and BMD gain, even if the extent of recovery is variable, depending on many factors. Several studies have shown that GFD, started at an early age, can restore BMD to normal in children [320e322,333e336] and correct altered vitamin D metabolism [335]. According to these studies, only an early diagnosis of celiac disease, immediately followed by GFD, may allow the attainment of a normal bone mass. Mora et al. followed 14 celiac children for 1.28 years after starting GFD, and found that the annual increase in peripheral BMC was greater than in normal children, concluding that GFD was able to improve bone mineralization and restore BMC to normal [320]. These results were confirmed by a later study on a slightly larger group (22 children): both LS and TB BMD were normalized with long-term GFD [333]. Scotta et al. studied 66 celiac children and adolescents, and found a reduced LS and TB BMC and BMD only in those who had been on GFD for less than 12 months. They also observed that, when the diagnosis had been made after 2 years of age, the patients had lower values of BMI, fat mass and LS BMD [321]. Barera et al. found that TB BMD, fat mass and limb lean mass were lower in 29 celiac children at the time

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of diagnosis than in a matched control group, but that GFD for 1.2 years normalized the body composition in 20 patients (69%) [322]. The importance of GFD for bone density was confirmed by a further study of the same group [333]. Molteni et al. reported similar data in a group of young patients (13e28 years): normalized forearm BMD was only present in those with an early diagnosis and who had constantly followed a GFD since childhood [334]. Tau et al. following LS BMC and BMD in 24 children (16 aged less than 4 years) before and after GFD (average duration 14 months; range 3 monthse3.9 years) observed that 93% of children starting GFD before 4 years of age reached a normal LS BMD, while only 50% of children older at diagnosis and at start of GFD did so [336]. On the contrary, a recent study reported a persistently low BMD in some children notwithstanding long-term GFD [337]. It should be noted that long-term prospective studies are few, and there is still no evidence that an optimal PBM level can be achieved with GFD, or that it can be maintained for many years, as happens in normal subjects. Cellier et al. underlined the very important fact that many patients with celiac disease diagnosed in childhood, who resumed a normal diet during adolescence, may develop bone complications (severe osteopenia) in adult life even if remaining free of intestinal symptoms [338]. Calcium and vitamin D have a relevant role for bone health in celiac disease. Regarding calcium, it has been suggested that the daily intake in celiac disease should be higher than the recommended daily allowance because of latent malabsorption [339]. Few data are available on vitamin D metabolism in celiac disease. Challa et al. studied a very small sample of celiac children and measured serum calcium and vitamin D metabolites both at the time of diagnosis, and after 2e12 months on GFD. After GFD, serum calcium was significantly increased, while remaining within normal range; 1,25OH2D, initially high, was significantly decreased, reverting to normal; and 25OHD was also increased, although not significantly [335]. Another study observed that after 1 year on GFD, only the patients receiving supplements of calcium and 25OHD had a BMD increase [340]. In a study by Muzzo et al., 19 children with celiac disease, all with good compliance to GFD for at least 2 years, received a daily supplementation of 1000 mg of calcium and 400 IU of vitamin D for 24 months, and showed a significant increase of TB and FN BMD Z-score [341]. For supplementation, 25OHD may be preferable over the native vitamin D to correct deficiency, since celiac patients may have increased plasma turnover and fecal excretion of this key metabolite [342]. According to the current guidelines for osteoporosis in celiac disease [343,344], there is a general consensus

on the value of GFD, while the efficacy of calcium and vitamin D supplementation is still undemonstrated and requires further research. There are no studies on the calcium requirements and the type and dose of vitamin D supplements on sufficiently large groups of celiac patients to verify whether these supplements have a greater effect on BMD than GFD alone, or whether 25OHD should be preferred over native vitamin D. Also, the wisest use of bone densitometry in celiac patients, both at diagnosis and during follow up, is yet to be determined, and longitudinal, long-term studies on large cohorts of both adult and pediatric patients are needed. CYSTIC FIBROSIS

Cystic fibrosis is one of the most frequent autosomal recessive genetic diseases and is caused by mutations in a gene (CFTR) encoding the cystic fibrosis transmembrane conductance regulator protein (CFTR), a chloride channel that modulates salt and water transport across the epithelial cell membranes. The incidence is about 1:2000 newborns in Europe and 1:3000 white newborns in the USA. The estimated prevalence is 0.737/10 000 in Europe and 0.797/10 000 in the USA [345]. CFTR mutations alter the quantity, viscosity and/or salt concentration of all exocrine gland secretions, leading to severe pulmonary, pancreatic, hepatic, and gastrointestinal complications and premature death. In particular, lung involvement is characterized by chronic inflammation and infections, and respiratory insufficiency is the main cause of death. Survival depends on continuous supportive treatment and, in the last 30 years, thanks to new aggressive therapeutic strategies, the lifespan of CF patients has dramatically increased. More than 50% of patients are now expected to live up to 40 years of age [345], and this has brought into light long-term complications, such as diabetes and osteoporosis. CF is characterized by different genotypes and different disease severity. Delta-F508 mutations are associated with the highest mortality and the most severe clinical manifestations, including osteoporosis [346,347]. Several recent studies investigated the direct impact of CFTR mutations on bone (Fig. 18.3). Three studies demonstrated that CFTR-null mice have severe trabecular and cortical osteopenia due to decreased bone formation and increased bone resorption. In the absence of other causes of bone loss, such as malnutrition or pulmonary insufficiency, this suggested that loss of CFTR function has a direct effect on bone [348e350]. Shead et al. found CFTR expression in human osteoblasts, osteocytes and osteoclasts [351], and studying

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FIGURE 18.3 Architecture of the distal femur of control mice and mice with cystic fibrosis: (left) Cftr þ/þ control mice; (right) DF508-Cftr mice (C57BL/6 Cftrtm1Kth). The two-dimensional sectional scans (b,c,d) demonstrated thinner trabeculae and cortical thinning of bones in cystic fibrosis. (From Paradis J, Wilke M, Haston CK. Osteopenia in Cftr-deltaF508 mice. J Cyst Fibros. 2010;9:239e45.)

24 adult patients before, during, and after treatment of pulmonary infection, observed that systemic infectionrelated inflammation induced an increase in osteoclast number and activity, reversible with antibiotic therapy [352]. Finally, Le Heron et al. demonstrated that inhibition of CFTR channel function determined a significant decrease in osteoprotegerin (OPG) secretion and an increase in prostaglandin (PG) E2 secretion in cultures of human osteoblasts, leading to increased bone resorption [353]. In 88 adult patients, King et al. observed that, among other factors, delta-F508 mutation is associated with a reduced BMD, even if the mechanism remains unknown [346]. In a study on 136 young patients, all those who were homozygous for the CFTR gene had a marked decrease in BMD, whereas the heterozygotes showed milder bone involvement, even when carrying one delta-F508 mutation [354]. Larger CF populations must be studied for a conclusive evaluation of the genotype impact on bone. Beyond the genetic aspects, the pathogenesis of bone loss in CF is very complex and is due to different mechanisms, depending on disease severity and involvement of different organs. Among the major factors are malnutrition, intestinal calcium malabsorption, vitamin D and K insufficiency, pulmonary complications (chronic infections, systemic inflammation, respiratory failure), reduced physical activity, reduced IGF-1 levels, hypogonadism and delayed puberty, and GC use [355]. In adults, osteopenia and osteoporosis have been reported by many authors and fractures are also well documented [356e361]. Ribs and vertebrae (particularly thoracic) are the most common fracture sites. According

to Aris et al., the fracture rates (any site) are doubled in women (age 16e34 years) and men (age 25e45 years) with CF compared with the general population [358]. Spine and rib fracture rates seem to be much greater, 100-fold and 10-fold higher respectively [361], and these are often the cause of further decline in pulmonary function and increase in pulmonary infection exacerbations [358,361]. A recent meta-analysis on bone disease in adult CF patients, analyzing 12 studies with a total of 1055 patients, reported a pooled prevalence of 38% for osteopenia and 23.5% for osteoporosis. The pooled prevalence of radiological vertebral fractures was 14% and that of non-vertebral fractures 19.7% [362]. On the contrary, reports on fractures in children and adolescents with CF are few and inconclusive and, in most studies no fractures were observed, with the exception of adolescent females [363e367]. Also regarding the bone mineral status in children and young patients, much fewer and less consistent data are available than for adults [363,364,367e375]. Some authors have also observed a reduced BMD in prepubertal children [354,368,374], while others reported normal or almost normal values in childhood, with a progressive BMD reduction during adolescence [367,371,375]. In the cited study by Bianchi et al., only 46 of the 136 (34%) patients had a normal BMD (measured by DXA and corrected for body size), 44 (32%) had a moderate decrease, and 46 (34%) a marked decrease (Z-score <2) [354]. On the contrary, Conway et al., studying 107 children with well-preserved respiratory function (age 5e16 years), observed that BMD (measured with DXA and corrected for body size) was normal in over 90% of cases. The best predictors of

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BMD were BMI and FEV1, an index of lung function. The fracture rate was apparently not increased, as 20 out of 22 fractures (in 15 children) were associated with trauma [363]. At least in part, these conflicting results may be explained by the different characteristics of the patients (age range, pubertal status), the different degree of preserved respiratory function, and the different disease severity. The pathogenetic mechanisms leading to reduced bone mass in growing subjects with CF have been analyzed in several studies. In a 2-year study on 54 patients (aged 6e33 years), about one third had low BMD at baseline, but BMD normally increased during the 2 years and was correlated to weight and lung function. The study suggested that low BMD in CF depends on infections and nutritional parameters [376]. Both inadequate bone calcium deposition and excessive resorption seem to contribute to the reduced bone mass, although this has not been directly assessed in children. Calcium kinetics (bone calcium deposition and resorption rates, calcium balance, and markers of bone turnover) has been studied by Schulze et al. in 22 clinically stable girls with CF (age 7e18 years). There was a significant association of increased calcium absorption and serum leptin concentrations with the rates of calcium deposition, demonstrating the importance of nutritional status. Calcium deposition rates and serum osteocalcin levels were lower than those typically observed in healthy children. The authors concluded that altered bone turnover contributes to the reduction of bone mass in CF [377]. In a longitudinal study on 85 young patients with CF (age 5e18 years), Buntain et al. observed that gains in LS aBMD in children (5e10 years) and TB and total FN aBMD in adolescents (11e18 years) were significantly reduced with respect to controls, after adjusting for age, sex, and height Z-score. Lean tissue mass was significantly associated with TB and LS aBMD gains in all patients, and accounted for a significant proportion of the aBMD reduction. Lung function parameters were significantly associated with aBMD gains in adolescents [372]. A relationship between disease severity and bone mass was confirmed also by Lucidi et al., who found that in 82 CF patients (age 5e30 years), the spine BMAD Z-score was positively related to BMI, to the Shwachman-Kulczycki score of global health in CF, and to the Crispin-Norman radiological score of lung damage [364]. According to several studies on adults and on small samples of children, FEV1 seems to be the main determinant of bone density [354,365,375,378]. The impact of pulmonary function on bone mass can be explained by several reasons. In CF, with the worsening of pulmonary disease, physical activity is reduced, appetite is lost, resulting in weight reduction and muscle wasting,

chronic inflammation affects the bone remodeling processes, and GC use is increased e all factors directly contributing to bone mass loss. Regarding body composition, a positive association between pulmonary function tests and BMI or lean (muscle) mass was observed in 50 children with CF [379]. In accordance with the mechanostat model, muscular weakness may lead to BMD loss [380]. Suboptimal levels of vitamin D and vitamin K have been found in children with CF [374,381]. Grey et al. observed that 95% of 81 patients with CF (age 12.6  2.9 years), clinically stable for at least 3 months, had low levels of vitamin D and 82% had low levels of vitamin K [374]. Finally, there is growing evidence that pulmonary infection and chronic inflammation are associated with an increased production of inflammatory cytokines like TNF-alpha or IL-6, already known to have a direct role in bone loss in other chronic inflammatory diseases, such as asthma and RA [352,355,375]. Shead et al. observed significant changes in serum cytokines during the course of pulmonary infections and, in particular, a positive correlation between osteoclast number and serum TNF-alpha, and between resorption area and IL-6; and a negative correlation between osteoclast number and OPG [352]. In conclusion, as clearly demonstrated for adults with CF, a careful evaluation of bone status seems essential also in younger patients, to allow prevention, early recognition, and adequate treatment of bone metabolic derangements. However, while there is an increasing awareness of the problem and a general agreement on the principles [361,382], randomized controlled studies on the preventive and therapeutic strategies are still lacking. The national working group of the French Federation of CF Centers issued a consensus statement on bone health in patients with CF, with recommendations for screening and for supplementation with calcium and vitamins D and K. Further efforts to define the indications for treatment with BPs and anabolic agents are required [382]. In all CF patients, reduction of fracture risk is vitally important, since fractures lead to reduced mobility and significantly increase the risk of pulmonary infections. Moreover, rib and vertebral fractures severely limit the possibility of respiratory physical treatment, and osteoporotic bone fragility is a serious obstacle to major therapeutic interventions such as lung transplantation. Currently, however, even high-risk patients, such as candidates to lung transplantation, seem to be rarely investigated for bone fragility [383]. Regarding prevention and treatment of bone disease in CF, careful attention to nutrition is necessary, particularly to maintain muscle mass and to ensure a correct calcium and vitamin D intake. Physical activity is also

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important: a review of the effectiveness of physical exercise to improve bone mineral accrual in CF concluded that, even if controlled trials are lacking, possible benefits should be expected [384]. Calcium and vitamin D supplements have been recommended and used, but only a few short-term studies have been made [381,385], and a Cochrane review on vitamin D supplementation in CF concluded that there is “no evidence of benefit or harm” [386]. A recent study suggested that high doses of vitamin D (50 000 IU daily) are required to increase serum 25OHD levels in CF subjects, but the duration of treatment (28 days) and the follow up (6 months) were too short to draw any conclusions on the efficacy and safety of such treatment [387]. Only preliminary data exist about the efficacy of vitamin K in improving bone metabolism in young CF patients [388]. Precise recommendations regarding form, dose, schedule, and duration of treatment for both vitamin D and vitamin K are still lacking, and longitudinal studies in large cohorts of young CF patients are required. Hormonal replacement treatment (HRT) may be indicated in selected cases, with careful individualization. GH has been used with good results on height, weight, lean mass, BMC and quality of life, with persisting effects after withdrawal [389]. Sex hormone treatment in CF children is controversial, but may prove beneficial in cases of delayed puberty. Oral and i.v. BPs have been successfully used in adults in the presence of fragility fractures or significant BMD reduction, while waiting for solid transplants, or when long-term treatment with systemic GCs is being started [355,361,382]. In children and adolescents they have been rarely used, essentially only in the presence of vertebral fractures, and no controlled trials have been performed. It may also be added that liver transplantation improved nutritional status, lean mass, and TB and LS BMD in some adolescent patients [390]. Last but not least, special attention must be paid to GC therapy, as GCs affect bone metabolism and calcium homeostasis at many levels. Long-term GC treatment increases the risk of fractures even in young patients, and for this reason the minimum effective dose must be sought. For more information on GCs and bone, refer to “Iatrogenic causes of osteoporosis” later in this chapter. Eating Disorders Three major eating disorders are currently recognized: anorexia nervosa (AN), bulimia nervosa (BN), and eating disorders not otherwise specified (EDNOS). Females are much more affected than males. The onset

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is generally in mid- to late adolescence, although occurrence in younger children has also been reported [391]. AN is a chronic and severe disorder, characterized by refusal to maintain a minimal normal weight, intense fear of gaining weight or becoming fat (even if underweight), disturbed perception of body shape/size, and extreme dieting (typically 300e700 kcal/day) with significant weight loss. AN is often accompanied by compulsive over exercising and by other behaviors aimed at losing weight (e.g. self-induced vomiting, misuse of laxatives and diuretics). In girls, menstrual irregularity is often an early symptom and amenorrhea may precede weight loss in up to 20% of cases. AN carries a significant risk of premature death because of fatal cardiac arrhythmias, starvation, and suicide [391]. Recent studies suggested that AN is highly heritable and has a strong genetic component [392]. Two recent reviews reported a widely variable remission rate (29e84%), partly depending on the duration of follow up [393], and a recovery of normal weight and menstrual function in 35e86% of cases [394]. According to a recent review, the incidence of AN is from 4.2 to 8.3/ 100 000 person-years, with peak incidence rates for females aged 15e19 years. The lifetime prevalence of AN is estimated at 1.2e2.2%, with a male:female ratio of 1:10 to 1:15 [391]. BN is characterized by recurrent and/or uncontrollable binge eating (usually done in secrecy because of disgust or shame), followed by compensatory behavior to prevent weight gain. Unlike in AN, weight is often normal. Electrolyte imbalances and gastrointestinal problems may develop, but the risk of premature death is lower than in AN [391]. There are few epidemiological studies on BN: the annual incidence rate has been estimated at 11.5 and 13.5/100 000 person-years in the Netherlands and USA, respectively. The lifetime prevalence of BN among women aged 18 years or more was estimated at 1.5% in the USA, but may be as high as 2.3% according to a recent Finnish study. The male:female ratio for BN is estimated between 1:15 and 1:20 [391]. In AN, all the main hormonal axes (the hypothalamicpituitary-ovarian axis, the hypothalamic-pituitaryadrenal axis, the GH/IGF-1 axis, and the hypothalamic-pituitary-thyroid axis) are altered with obvious consequences for bone [395]. AN starting at an early age may lead to pubertal delay and primary amenorrhea, pubertal arrest with potential failure in attaining target height, or secondary amenorrhea [395]. All eating disorders are associated with significant medical and psychological morbidity, and reduced bone density and alterations of bone turnover are frequent complications [396,397]. This is not surprising, given the age at disease onset, most often mid-adolescence, prior to the attainment of PBM, and

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the number of risk factors for osteoporosis in these conditions. Caloric intake is severely restricted, and malnutrition/starvation leads to an inadequate intake of calcium, vitamin D and protein. Hypogonadotrophic hypogonadism, with estrogen and testosterone deficiency, results from starvation-induced hypothalamic dysfunction [398]. Hypercortisolemia [399] and reductions in circulating IGF-1 [398] have also been documented. Low levels of dehydroepiandrosterone (DHEA) or its sulfate have been reported in some [400] but not all [398] studies. Alterations of the neuroendocrine appetite modulators (leptin, ghrelin, peptide YY), known to affect bone metabolism, have also been demonstrated [401,402]. Excessive exercise or bed rest (e.g. during inpatient treatment) further contribute to the derangement of bone turnover. The association of AN with osteopenia and osteoporosis has been demonstrated in both adult women [396,399] and adolescent girls [398,403e407]. Osteopenia may develop within 6 months of disease onset [590] and a significant reduction in bone mass has been observed in adolescent girls less than 1 year after diagnosis [403]. An early diagnosis may help to prevent bone loss in girls with AN: in a study on 57 girls aged less than 18 years, 35 (62%) had normal BMD at diagnosis; weight at diagnosis and lean mass were the main predictors of bone loss [406]. Bredella et al. compared 10 adolescent girls with mild AN (age 13e18 years) with 10 normalweight girls, and found an abnormal bone structure of the distal radius of those with AN: trabecular number, volume and thickness were reduced, while trabecular separation was increased [407]. The same group observed that the altered bone structure at radius was associated with reduced BMD at LS (anteroposterior and lateral) and FN, but not at total hip. Endogenous IGF-1, leptin, and androgen levels predicted bone microarchitecture independently of BMI [408]. Confirming that adolescence is the critical period for bone mass accrual, Biller et al. found that adult women with an onset of amenorrhea before 18 years of age had a lower BMD than those who became amenorrheic after 18 years, independently of other variables like duration of amenorrhea [399]. In a study on 130 young anorexic women (mean age 24.4  0.5 years), a BMD T-score <1 was found at one or more skeletal sites in 92% of patients, and a BMD T-score <2.5 in 38% [409]. Osteopenia may persist after weight gain and resumption of regular menstrual cycles in anorexic women [410,411]. Miller et al., studying 75 anorexic women (age 18e40 years), observed a mean annual BMD increase of 3.1% at LS and 1.8% at hip in those who gained weight and resumed regular menses, while women who recovered menses without gaining weight showed LS BMD increase only, and those who gained weight without recovering menses showed hip BMD increase only.

Women who did not resume menstrual cycle nor gained weight had a BMD decrease of 2.7% at LS and 2.6% at hip [412]. Very few data are available for women with BN: BMI and a previous history of AN seem the major determinants of BMD [413]. There are relatively few studies on men and adolescent boys with eating disorders [414,415], and none on children. Mehler et al., in a retrospective study on 53 male patients with different eating disorders who had a BMD record, found that 19 (36%) had osteopenia and 14 (26%) osteoporosis at LS. BMD reduction was best predicted by BMI reduction and illness duration [414]. A study reported that boys with AN have low BMD and decreased bone turnover markers [415]. Long-term follow-up studies in AN demonstrated that BMD, and consequently the risk of osteoporosis, are significantly influenced by the duration of disease before recovery [410,416]. Soyka et al. found that, in female adolescents, AN duration was the most significant predictor of spinal BMD and suggested that reductions in circulating IGF-1 as well as sex steroid deficiencies were critical factors in the development of bone morbidity [398]. Over a mean follow up of 7.6 years, do Carmo et al. found a positive correlation between BMD and time since the first menstrual cycle post-amenorrhea in 15 adolescent girls [410]. The complex physiopathologic picture of bone disease in AN has not been fully clarified yet, but a recent article by Mehler and MacKenzie summarized the current knowledge. AN is characterized by an imbalance in bone turnover, with increased bone resorption without increased bone formation, which explains the significant bone loss (usually more severe at LS). Markers of bone resorption (e.g. NTx and deoxypyridinoline) are increased, while markers of bone formation, such as osteocalcin, are not. Serum levels of calcium, vitamin D, and PTH are normal. The amenorrhea of AN is associated with low circulating levels of estradiol and DHEA. Hepatic synthesis of IGF-1 is reduced, and its low levels impair osteoblast function (normal adolescents typically have high IGF-1 levels). Elevated levels of total and free serum cortisol and high 24-h urinary free cortisol excretion are observed. The levels of cortisol are inversely related to the levels of osteocalcin and, in general, hypercortisolism is associated with osteoporosis. On the contrary, serum pro-resorptive cytokines, such as TNF-alpha and IL-6, known to be associated with bone resorption in postmenopausal women, are not elevated in AN [417]. Many studies found an imbalance of bone turnover in AN, characterized by decreased bone formation and increased bone resorption, attributed to estrogen deficiency [398,404]. Studying bone status with DXA and QUS, and analyzing bone turnover markers (osteocalcin,

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BSAP, CTx) in 18 adolescent girls with AN, Oswie˛cimska et al. confirmed that both bone formation and resorption processes are altered [418]. According to Galusca et al., who evaluated bone turnover and hormones in 115 anorexic patients (60 adolescent girls aged 15.5e20 years and 55 young women aged 20e37 years), the uncoupling of bone turnover could be explained not only by the altered production of cortisol, IGF-1, GH and 17-beta-estradiol, but also by the action of free T3 and catecholamines, as well as by a direct, hormone-independent effect of undernutrition. For this reason, the treatment of AN-related osteoporosis should not be based only on weight gain, but should include multiple hormonal interventions with continuous adaptation during the follow up [419]. More recently, a decrease in the OPG/RANKL ratio has been observed in anorexic girls, and this could partly explain the increased bone loss [420]. Peptide YY has also been identified as an independent predictor of bone mass changes in AN [402]. And the role of leptin has been confirmed in a study on 103 young anorexic women (age 24.9  7.4 years), in which duration of amenorrhea and leptin level accounted for 27% of the variance of BMD [401]. In a preliminary study, Dosta´lova´ et al. hypothesized a role of macrophage inhibitory cytokine-1 (MIC-1, a member of the TGF-beta superfamily, strongly expressed in activated macrophages and adipocytes) in AN and the related bone loss [421]. Another very recent study by Fazeli et al. observed that in women with AN, body fat depots are low, but marrow fat mass is high, and is inversely associated with BMD. Since adipocytes and osteoblasts derive from the same mesenchymal stem cell, understanding how this differentiation process is regulated may also help to clarify the mechanisms of bone loss in AN. In this study, preadipocyte factor-1 (Pref-1), a member of the epidermal growth factor-like proteins involved in the regulation of adipocyte and osteoblast differentiation, was significantly higher in 20 women with AN (age 19e41 years) than in healthy controls, and Pref-1, IGF-1, IGF-BP2 and leptin levels were associated with marrow adiposity and LS BMD [422]. Bone disease in eating disorders may be severe enough to result in fragility fractures and an increased fracture risk throughout life. Anorexic women are reported to have a 2.6 to sevenfold increase in fracture risk with respect to healthy women of the same age [408,423e425]. According to a large study on all Danes diagnosed with eating disorders between 1977 and 1998 (2149 patients with AN, 1294 with BN, 942 with EDNOS), AN patients did not have an increased fracture risk before diagnosis with respect to controls, but the risk was increased by 98% after diagnosis, and the increase

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continued to persist more than 10 years after diagnosis. In patients with BN, there was a 31% increase in fracture risk before diagnosis, and a further small increase after diagnosis, but the risk returned to normal more than 1 year after diagnosis. These data suggest that BN may remain undiagnosed for a much longer period of time than AN, and that the skeletal damage is much more severe and persistent in AN [423]. In a population-based study from the USA, a follow up of anorexic subjects (2689 person-years of observation) demonstrated an increased risk for fractures (hip, LS, and forearm) later in life, with a 57% cumulative incidence of any fracture 40 years after diagnosis [424]. Another study reported that 30% of 214 young women with AN (age 17e45 years) had sustained fractures [425]. The therapeutic strategies for the recovery of bone mass in young patients with eating disorders, and in particular with AN, are still a matter of research and debate. In anorexic patients, osteoporosis treatment should begin with the restoration of a healthy weight. Several studies demonstrated increases in BMD associated with weight gain, using both DXA [426] and QCT [427]. Weight gain seems to be particularly important for the recovery of BMD at hip [428]. However, not all studies observed BMD improvements [404]. Adolescent girls who fail to gain weight show a continuous decrease in bone mass and density, and weight restoration with resumption of menses is considered essential for a recovery [411]. Menses usually reappear soon after an increase in weight: for example, Golden et al. reported that 86% of girls resumed normal menses within 6 months of achieving 90% of their ideal body weight [429]. A longitudinal Australian study demonstrated that normalization of LS vBMD in adolescent-onset disease is possible in about 3 years after the successful recovery of normal weight and menses [430]. On the contrary, in a prospective 1-year study on 26 adolescent and young women with AN (age 13e20 years), notwithstanding a mean weight gain of 10 kg and a significant height gain, there were no significant changes in LS and proximal femur BMD [431]. These contradictory results may be partly explained by the different follow-up duration, and more longterm prospective studies are required. For the moment, we should assume that the recovery of a normal nutritional and hormonal milieu is a necessary, but probably not sufficient, condition to overcome the deleterious effects of prolonged anorexia on bone, and that the complete reversibility of bone loss in adolescents with AN is uncertain [411,427]. In the general population, moderate physical exercise is a cornerstone of the prevention of osteoporosis. It is not clear what kind of physical activity can be safely recommended to subjects with AN, considering

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their tendency to over-exercise. Besides the effects of physical exercise on metabolic rate, caloric expenditure, and the menstrual cycle, high levels of exercise may continue to suppress appetite by increasing peptide YY [432]. Schulze et al. investigated 52 adolescent and young women (age 13e27 years), with a history of early onset of AN, 3e9 years after hospital discharge, and found that 50% had had a good recovery from AN, which correlated with BMC and BMD improvement. Regular physical activity had a positive effect on bone recovery, while the effect of estrogen replacement therapy was not significant. Duration of amenorrhea was negatively correlated with BMD (but not with BMC) [433]. An additional problem is related to the hospitalization of anorexic patients. Bed rest and graded nutritional therapy are often prescribed, but physical inactivity may lead to accelerated bone loss. A study on 28 adolescents and young women with AN (aged 13e21 years), reported that even short-term bed rest may have negative effects on bone, with suppressed bone formation and resorption and imbalance in bone turnover. The authors underlined the necessity of further studies to determine whether the protocols prescribing strict bed rest are appropriate [434]. Other therapeutic strategies have been explored, with variable results. Patients with AN may have profound estrogen deficiency, yet HRT has been disappointing. A double-blinded, randomized controlled trial reported that spinal bone mass (measured by QCT) was not 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% of their ideal weight [427]. In a study on 50 girls with AN, all were treated with the same standard treatment (including calcium and multivitamin supplementation), but 22 also received a daily oral contraceptive for almost 2 years. There was no significant increase in BMD (LS and hip) in the estrogen-treated group [435]. A multicenter randomized placebo-controlled trial compared the use of a triphasic oral contraceptive with placebo in 112 adolescent girls aged 11e17 years, and found no significant difference in LS or hip BMD between the groups after 1 year of treatment [436]. Another recent prospective study concluded that HRT for 2 years was not effective for the prevention of bone loss, and that the most predictive factor for the improvement of LS and hip BMD was weight increase [437]. Calcium and vitamin D intake does not appear to correlate with BMD nor to prevent reduction in bone mass [403,404,438], but Soyka et al. found vitamin D and calcium deficiency in about 50% of both adolescents

with AN and controls [404], and the correction of calcium and vitamin D deficiency is generally considered a basic intervention in eating disorders. Recent studies suggest that more aggressive medical therapy may be needed to treat severely malnourished patients with significant reductions in bone mass. A prospective, randomized, placebo-controlled trial on 60 osteopenic women with AN showed that recombinant human IGF-1 (rhIGF-1) produced a significant, though modest, increase of LS aBMD and lean mass, and that the combination with an antiresorptive agent (contraceptive pill) had synergistic effects [438]. Another recent study on 10 anorexic girls confirmed the efficacy of a rhIGF-1 (for 7e9 days) on bone formation [439]. In summary, on the basis of some recent reviews of the available literature [394,440,441], it can be concluded that in adolescent girls and women with AN and hypothalamic amenorrhea, HRT alone has only limited efficacy in improving bone mass, and should not be prescribed solely for this purpose. The best therapeutic strategy is to stimulate weight gain and resumption of the menstrual cycle through increased calorie intake. If necessary, calcium and vitamin D supplementation may be considered. It is also important to remember that in young girls, induction of puberty with estrogens and HRT can only be done with the aim of mirroring the normal evolution of puberty in healthy girls, and the form, dose and duration of treatment must be strictly individualized. Estrogens should be used with extreme caution in patients who have not yet reached their final height, because they will hasten epiphyseal closure [442]. It is still undetermined whether potent antiresorptive agents such as BPs, either alone or in combination with anabolic medications, should have a role in the treatment of AN-associated osteoporosis. In a shortterm randomized placebo-controlled study on 41 young anorexic women, both etidronate and calcium plus vitamin D were effective in increasing tibial BMD (measured with QUS) [443]. In a doubleblinded, randomized trial on 32 adolescents with AN (mean age 16.9  1.9 yr), alendronate (10 mg/day) was compared with placebo. All the participants also received 1200 mg of calcium and 400 IU vitamin D/ day plus the standard multidisciplinary treatment for AN. Only 21 subjects completed the 1-year study. FN and LS BMD showed a greater increase (respectively þ4.4% and þ3.5%) in the alendronate group than in the placebo group (þ2.3% and þ2.2%), but the results were not statistically significant. Body weight and weight gain were the most important determinants of BMD at the end of the study [444]. In an uncontrolled study, risedronate (5 mg/day) improved LS BMD (þ4.9% at 9 months) in 10 young women with AN [445].

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Endocrine Disorders DIABETES MELLITUS

T1D is the commonest form of diabetes in the pediatric age (over 90% of cases) [446]. The incidence of T1D in the 0e14 years age group is highly variable, from the highest rates in Sardinia (Italy) (37.8/100 000) and Finland (>40/100 000) to the lowest rates in Venezuela and China (0.1e4.5/100 000), and has been increasing in the last 50 years. The incidence is different in the different age subgroups, with a peak during puberty (10e14 years), and an earlier onset in girls than in boys [447]. Type 2 diabetes is also increasing in children in developed countries, because of the growing number of overweight or obese subjects [446], but to date, all studies on diabetes and bone during childhood and adolescence have considered only T1D. The pathogenesis of diabetic bone alterations is complex. Osteoblasts have receptors for insulin and IGF-1, and insulin, like IGF-1, promotes osteoblast replication and function [448]. Thus, the main effect of insulin deficiency on bone is decreased osteoblast recruitment, either by direct action or indirectly through other bone growth factors, and a decreased bone turnover is characteristic of diabetic osteopathy. Both the few studies on human bone biopsies [449] and those on diabetic rats [450] demonstrated a decrease in osteoblast number, osteoid formation, and bone mineral apposition rate, and a lesser decrease in the number of osteoclasts. Several studies reported reduced levels of osteocalcin (a bone formation marker) and IGF1 in T1D patients [451,452]. Persistently decreased bone formation by osteoblasts may determine low bone mass and osteoporosis. Decreased collagen synthesis in bone, cartilage and other tissues, and decreased collagen strength, with increased and abnormal collagen glycosylation and cross-linking, have also been observed in diabetic patients, with consequent bone fragility [453]. Moreover, vitamin D metabolism is altered in diabetes due to the effect of insulin on the synthesis of vitamin D binding protein, the renal 1-hydroxylation of 25OHD, and the concentration of vitamin D receptors in the duodenal mucosa, all contributing to a decreased synthesis of duodenal calbindin-D9K and to intestinal malabsorption of calcium. In summary, reduction in bone formation, changes in bone matrix structure, and impaired mineralization all contribute to a quantitative and qualitative deterioration of the diabetic bone. Osteopenia was first observed in diabetic children with x-rays over 80 years ago. In 1982, Wiske et al., using single photon absorptiometry and radiogrammetry, observed reduced bone mass in 78 young diabetics (mean duration of disease 6.7 years), without impaired growth or delayed bone maturation [454]. Subsequently, more precise methods such as DXA and pQCT have

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been used, and some authors reported a reduced BMD [452,455e457], while others observed a normal or slightly (but not significantly) reduced BMD [458e460]. A study found normal LS BMD in 23 children and adolescents 1e5 years after T1D onset [459], while another found reduced BMD in 23 prepubertal children a few months after diagnosis [457]. Still another, not only observed a lower BMD at total hip, FN, and TB in adolescent girls and young women with T1D (age 13e35 years), compared with age-matched healthy controls, but also found that these differences persisted 2 years later [452]. Brandao et al., in a recent cross-sectional study on 44 children with T1D (age 8.8  4.4 years, disease duration 6.6  3.9 years), compared with healthy matched controls, found normal LS BMD in most patients. Only two out of 44 subjects had a BMD Z-score <2.0 [460]. C ¸ amurdan et al. did not find a significant reduction of LS BMD in a group of 58 young patients (age 3.6e20.7 years). However, 14 subjects (24.1%) had BMD Z-score values between 1 and 2.5, and three had a Z-score <2.5. A deeper analysis revealed that glycosylated hemoglobin (HbA1c) and diabetes duration were the most important determinants of BMD: HbA1c value over 9.8% and diabetes duration over 3.6 years were cut-off values for osteopenia. Pubertal stage was another determinant of BMD, especially in newly diagnosed patients [461]. Several studies, even if conducted on small samples, demonstrated that poor control of hyperglycemia was associated with a lower LS BMD or BMAD [454,455,462,463]. On the contrary, Mastrandrea et al. found no correlation between BMD and diabetes control, growth factors, or metabolic bone markers [452]. Heilman et al. observed that an increased inflammatory status (high CRP levels) was associated with a lower BMD [462], and Moyer-Mileur et al. linked the low BMD to the alterations of the GH/IGF-1 axis induced by poor metabolic control: in this study on 11 adolescent girls, an earlier age at diagnosis was predictive of lower IGF-1, higher urine magnesium excretion, and lighter, thinner cortical bone (evaluated at tibia) [463]. These inconsistent observations may be due, at least in part, to three limitations. First, most studies were cross-sectional and on small samples. Second, many studies included very wide age ranges (adolescents and adults together). Third, data obtained from patients in different metabolic conditions are difficult to compare. Regarding the possible gender differences in bone involvement, two large cross-sectional studies gave different results. Le´ger et al. used DXA to study 127 patients with T1D (73 boys, 54 girls; age 6e20 years; diabetic for 3 years or more) and 319 matched controls, and observed a gender-related BMD difference. Diabetic girls, but not boys, had lower TB and LS BMC than

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controls. After adjustment for age, gender, pubertal stage and BMI, the ratio TB BMC/lean body mass was significantly lower in the diabetic patients than in controls. In both genders, large amounts of insulin and low serum levels of IGF-1 were independent determinants of low bone mass [464]. On the contrary, Va´zquez Ga´meza et al., comparing 66 patients (26 boys, 40 girls; age 3e17 years), and 327 age-matched controls, found no difference in BMD, measured with DXA, between genders. The BMD Z-score was between 1 and 2 in 10 patients (15.1%), and <2 in four (6.1%). Only the subgroup of older diabetic boys (15e17 years) had a significantly lower LS BMD than age-matched controls. The authors concluded that uncomplicated T1D may cause a smaller BMD increase during adolescence, potentially leading to a low PBM [465]. A very recent study by Saha et al. on 48 adolescents with T1D (26 girls, 22 boys; age 12e17.8 years), compared with healthy controls matched for age, gender, height, weight, and pubertal maturity, reported not only a difference between girls and boys, but also a smaller bone size in diabetic adolescents. BMD was evaluated at radius and tibia with pQCT, and at LS and proximal femur with DXA. Diabetic patients had lower BMC at all skeletal sites and smaller bone CSA at the radial and tibial diaphyses and at distal tibia, and boys had lower values than girls [466]. On the contrary, a smaller bone size in diabetic children but not in diabetic adolescents has been reported by Bechtold et al., who studied 42 girls and 46 boys (mean age 11.7  3.0 years; mean disease duration 5.6  3.7 years) and found that total and cortical bone CSA and muscle CSA (measured at radius with pQCT) were low in prepubertal but normal in adolescent patients. In 18 patients with early manifestation of T1D, there was greater bone involvement, with significantly reduced cortical BMD, total and cortical bone CSA, and muscle CSA [467]. The same authors, re-evaluating 41 of the 88 patients (19 girls, 22 boys; mean age 15.44  2.3 years) after about 5.5 years with pQCT, observed a normalization of total and cortical bone CSA and, considering the ratio BMC/muscle mass, concluded that bone size was well adapted to muscle mass [468]. However, it should be underlined that these two studies measured only an appendicular site (radius) and included only patients with good metabolic control, no evidence of diabetic retinopathy, neuropathy or nephropathy, no use of medications except insulin, no hospitalization or ketoacidosis in the preceding 6 months, and no restriction on physical activity. On the basis of these data, it can be concluded that gender, age at diagnosis, age at evaluation, and metabolic control, may all influence bone status and should be considered when comparing the results of different

studies, in addition to the site of BMC or BMD measurement. There are no data on the fracture risk of diabetic children/adolescents, and very few data on the longterm effects of bone alterations, in particular on the prevalence of osteoporosis and fractures later in life. The only study on a large diabetic population (24 605 patients: 12 551 men and 12 054 women, hospitalized for diabetes before 31 years of age) was conducted in Sweden and registered a total of 121 first hip fractures (70 in men and 51 in women), with a global cumulative probability of 65.8/1000. T1D patients had a greater than fourfold relative risk of hip fractures after 40 years of follow up [469]. Of course, from these data it is impossible to establish whether the attainment of a low PBM is responsible for the increased risk of hip fractures, also considering that in adult T1D patients many complications (ophthalmic, neurological, and cardiovascular events) may increase the risk of falls. Another relevant aspect to consider in young patients with T1D is the high frequency of low vitamin D levels reported by many studies, also from very sunny countries. In Brisbane, Australia, in a group of 47 diabetic children (age 12.6e14.6 years), 43% had
20 ng/ ml (50 nmol/L) of serum 25OHD [470]. A study from Qatar reported that in 170 T1D patients aged less than 16 years, serum 25OHD was 15.8  9.2 ng/ml (39.4  23 nmol/L) on average. Only 16 (9.4%) subjects were in the range 30e80 ng/ml (75e200 nmol/L), while 154 (90.6%) were below 30 ng/ml (75 nmol/L). In the same study also a high percentage of the 170 healthy controls were below 30 ng/ml [471]. Finally, some studies evaluated the influence of leptin on bone and body composition in children with T1D. Karagu¨zel et al. did not observe significant differences in serum leptin levels and body composition in children and adolescents with T1D on intensive insulin therapy, compared with healthy controls [472]. Kassem et al., studying 42 young adults (mean age 20.1  0.6 years) with childhood-onset T1D, observed a negative correlation between BMD Z-score or BMC with leptin, that persisted after adjustment for fat mass [473]. In summary, there are too few data to make conclusive considerations on the bone status of children and adolescents with T1D, especially regarding long-term consequences and fracture risk. BMD is not yet a routine evaluation in these patients, and larger prospective studies are needed to evaluate its usefulness. Currently, it can be recommended for selected patients, such as those with poor metabolic control. Counseling about adequate calcium intake, vitamin D levels, and regular physical activity is always advisable, also because diabetic children and adolescents, especially girls, seem to spend less time in physical activity than their healthy peers [474].

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Finally, it should be remembered that celiac disease is often associated with T1D [327]. Several studies have reported that patients with both diseases, particularly those who do not follow a strict GFD, have a higher reduction in BMD compared with patients with only T1D [328,475]. Patients with T1D and celiac disease must be considered at high risk for osteoporosis and need special attention to their bone health. THYROID DISORDERS

Physiological concentrations of thyroid hormones are essential for normal growth and skeletal development. Thyroid hormones influence the formation of both long bones (endochondral ossification) and flat bones (intramembranous ossification). In children, hypothyroidism causes growth retardation and impaired bone maturation, whereas hyperthyroidism causes accelerated growth, accelerated bone maturation, and premature fusion of growth plates. The bone effects of thyroid hormones now appear more complex than previously thought. Generally, bone loss and, in the case of growing subjects, reduced bone acquisition, was thought to be associated with the presence of overt hyperthyroidism (increased levels of thyroid hormones). However, recent data indicate that, at least in adults, bone alterations are present even in the presence of subclinical hyperthyroidism (normal levels of thyroid hormones with decreased levels of thyroid-stimulating hormone, TSH), and that hypothyroidism is also associated with reduced bone turnover and increased fracture risk [476e478]. HYPERTHYROIDISM Studies on bone cell cultures have demonstrated that T3 stimulates osteoblasts by binding to nuclear receptors, and that activated osteoblasts stimulate osteoclast activity [476]. Through this mechanism, hyperthyroidism leads to increased bone resorption and bone mass loss [479,480]. Biochemical markers of bone metabolism reflect the increased bone turnover, with elevated indices of bone formation (serum BSAP, osteocalcin, propeptide of type I collagen) and resorption (collagen cross-links pyridinoline and deoxypyridinoline) [481]. In addition, in hyperthyroidism, the levels of TNF-alpha and IL-6 are elevated, further stimulating osteoclast activity [482]. Thyrotoxicosis is less common in children than in adults, but the incidence has been increasing in the last 20 years. In different European countries, the incidence varies between 0.7/100 000 and 1.83/100 000 per year in subjects under 16 years of age; girls are more affected than boys, especially in the 10e14 years age group [483]. During childhood, Graves’ disease is the most frequent cause of thyrotoxicosis, followed by the transient toxic phase of chronic lymphocytic thyroiditis (Hashimoto’s thyroiditis). Activating TSH-receptor

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mutations and McCuneeAlbright syndrome are rare non-autoimmune causes. As expected, considering the action of thyroid hormones on bone, high bone resorption rates and low bone density have been demonstrated at diagnosis of hyperthyroidism in children and adolescents, as well as in adults [484e486]. Histomorphometric analyses of iliac crest in 40 untreated hyperthyroid patients demonstrated a marked increase in osteoclast activity at the cortical level, followed by a significant increase in cortical porosity [487]. In untreated adult patients with hyperthyroidism, many studies observed not only a decreased BMD but also an increased incidence of fractures, especially at the hip [484,485,488]. The available studies in children only evaluated BMD, and only in small samples. Both endogenous hyperthyroidism and excessive thyroid hormone replacement therapy have been associated with accelerated bone resorption and decrements in bone mass [486,489]. A recent study that used QCT to evaluate bone status in 18 children and adolescents with untreated hyperthyroidism revealed cortical bone loss at LS and hip with respect to a matched control group [490]. No longitudinal studies have systematically evaluated fracture risk. Mora et al., studying 13 girls at diagnosis of hyperthyroidism, found an increased bone turnover and a decreased LS and TB BMD with respect to healthy controls. There was an inverse correlation between the serum levels of free T4 and LS and TB BMD, and a direct correlation between free T4 and free T3 and urinary NTx levels. With methimazole treatment, NTx levels were normalized after 6 months, and remained stable thereafter. After 12 and 24 months of treatment, LS and TB BMD were also increased, and no longer different from those of controls, demonstrating that antithyroid treatment may be able to restore the physiologic conditions for achieving the best possible PBM [486]. Very similar data have been reported by Lucidarme et al. who, in a prospective study on 26 children with Graves’ disease, observed a significant reduction of BMD at LS and proximal femur and high bone turnover (increased osteocalcin) at diagnosis with respect to healthy controls. Eleven subjects (42%) had severe osteopenia (T-score <2). After 12 and 24 months of treatment with carbimazole, osteocalcin was normalized and there was a significant increase in BMD at both sites. No patients had continuing osteopenia [489]. These observations have been confirmed by another study on 50 adolescents and young adults (age 14e38 years) with thyrotoxicosis. Forty-six patients (92%) had a reduced LS BMD at baseline, but it increased significantly (þ4% on average) after treatment [491]. While recovery was rapidly achieved in these studies, following induction of euthyroidism, concern has been raised regarding the long-term efficacy of

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antithyroid medication for pediatric patients. Moreover, hyperthyroidism may not be adequately controlled in pediatric patients 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 [488]. When a disease with possible consequences on bone health develops during childhood, it is extremely important to know its long-term effects. Unfortunately, very few data are available for pediatric hyperthyroidism. Radetti et al. studied 28 patients (five male, 23 female; age 20.93.3 years) treated for many years with antithyroid drugs because of Graves’ disease, with onset at 11.82.9 years. The patients had normal BMD at LS and FN and the authors concluded that the attainment of a normal PBM is possible if Graves’ disease, beginning in childhood and adolescence, is appropriately treated [492]. Some authors have also underlined the fact that the therapeutic suppression of TSH without an elevation of thyroid hormones, as in subclinical hyperthyroidism, may be associated with severe osteoporosis, at least in adult patients [477,478]. This suggested that the bone loss occurring in hyperthyroidism might be due to TSH suppression rather than to high levels of thyroid hormones, and the hypothesis was confirmed by a study on TSH-receptor null mice [477,493]. However, the only published case report on pediatric patients (two male siblings aged 9.8 and 6.8 years with isolated TSH deficiency, due to mutation of the TSH beta-subunit gene) did not confirm these findings: in both children, treated with substitution doses of levothyroxine since birth, LS BMD, serum calcium, urinary calcium/creatinine ratio, and bone turnover markers (serum BSAP, osteocalcin, urinary NTx, CTx and deoxypyridinoline) were normal [494]. HYPOTHYROIDISM Untreated congenital hypothyroidism is characterized by growth arrest, epiphyseal dysgenesis, delayed bone age and short stature. Early replacement treatment with thyroxine increases growth velocity, allowing children to reach their predicted adult height. Two independent follow-up studies of children with congenital hypothyroidism, treated with thyroxine since birth, and evaluated after several years, showed that almost all of them had normal BMD with respect to healthy controls. Low body weight and low calcium intake could explain the few cases of reduced BMD in the second study [495,496]. On the contrary, seven adult women with congenital hypothyroidism, treated with substitution therapy since birth, had a low calcitonin concentration and a 10% reduction in radial BMD. The authors suggest that both calcitonin deficiency and

thyroid hormone treatment could have a role in bone loss [497]. In a study on 37 adolescents affected by congenital hypothyroidism and followed from the time of diagnosis (age 264 days) to age 17.81 years, long-term L-thyroxine therapy did not affect BMD, either evaluated by DXA at LS or by QUS at the proximal phalanges of hand. Careful monitoring of serum TSH and appropriate dose adjustments are required [498]. Pitukcheewanont et al. retrospectively reviewed the medical records of 32 girls and 13 boys with congenital hypothyroidism, treated with L-thyroxine, who had at least one BMD measurement with QCT. They found no difference in the femur trabecular BMD, cortical BMD, CSA or CBA between patients and age- and gendermatched healthy controls, and no significant differences between initial and subsequent BMD measurements, and concluded that in children with congenital hypothyroidism, thyroxine treatment seems to have little effect on bone [499]. On the contrary, in a recent cross-sectional study on 60 children and adolescents affected by congenital hypothyroidism (age 7e14 years; 41 prepubertal), the TB BMD, measured by DXA, was lower than in healthy controls, while height, weight and BMI did not show any significant difference [500]. To date, there are no data on the lifetime fracture risk for individuals with congenital hypothyroidism treated with long-term thyroxine replacement. GROWTH HORMONE DEFICIENCY

GH and its anabolic effector IGF-1 are essential for skeletal growth and for bone and muscle mass accrual. GH receptors are expressed by chondrocytes and osteoblasts, and throughout life, GH and IGF-1 regulate bone homeostasis, through a complex interaction with PTH and vitamin D, resulting in increased intestinal cell sensitivity to PTH, increased renal hydroxylation of 25OHD to 1,25OH2D, and enhanced intestinal calcium absorption [501e503]. Their action is maximal during puberty, when circulating levels increase markedly under the influence of sex hormones, and they strongly stimulate skeletal growth (linear growth, bone maturation, bone modeling and turnover, acquisition of bone mass by increasing osteoblast activity and collagen synthesis). In adulthood, they continue to regulate the rate of bone remodeling and are thus involved in the maintenance of bone mass. On this basis, GH-deficiency (GHD), a relatively uncommon cause of growth retardation, has been recognized as a possible cause of reduced bone mass and density. Two main forms of GHD are described, according to the age at onset: childhood-onset GHD (CO-GHD) and adult-onset GHD (AO-GHD). The cause of CO-GHD is often unknown (idiopathic GHD), and only this form will be discussed here.

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Cranial irradiation, neurosurgery, radiotherapy, injury are other causes [502]. A true GHD or an alteration of the GH/IGF-1 axis may also develop in many chronic diseases (leukemia, CF, renal failure, malabsorption, AN, etc.). The reported prevalence of CO-GHD is widely variable, from 1/3480 children to 1/5600 or even 1/30 000 [504]. Studies in adults with CO-GHD observed a reduced BMD, more in cortical (forearm) than in trabecular bone (spine). These data also support the hypothesis that the osteopenia observed in adults with CO-GHD may be caused by insufficient bone mass accrual during childhood and adolescence rather than by premature bone loss [505,506]. The effects of GHD on BMD in children have been challenging to discern. Since children with GHD are shorter and smaller (decreased muscle mass) than ageand gender-matched healthy children, their reduced body (bone) size carries a potential bias when DXA is used to evaluate BMD, as the value of aBMD may be misleading (see “Basic aspects of DXA densitometry” earlier in this chapter): correction for bone size (e.g. using BMAD as an estimate of vBMD) showed persistent BMD deficits [507,508]. On the contrary, in a study on 2209 short prepubertal children with idiopathic GHD, treated with GH, bone age and its progression seemed to be normal at least during the first year of treatment [509]. The reduction in bone mass seen in GHD has been attributed to impaired bone formation rather than to increased bone resorption. In children with GHD, treatment with recombinant human GH leads to an improvement in bone formation and an increase of BMD [507,508,510]. Boot et al., studying 40 children with GHD, found that LS BMD, LS BMAD and TB BMD were significantly lower than normal at baseline and had a significant increase after 2e3 years of GH treatment. Height increased, fat mass decreased (only during the first 6 months of therapy), and lean mass increased continuously [507]. Saggese et al., in a long follow up of 32 children with GHD (56e81 months), demonstrated that height and BMD could reach normal values only with an adequate duration of GH treatment, and suggested that treatment should be continued until the attainment of PBM, and not only of final height [510]. Ho¨gler et al., using DXA, evaluated TB BMC, body composition, and LS and FN vBMD in 20 prepubertal children with GHD (aged 9.423.73 years), treated with GH for 2 years [511]. Since size-corrected TB BMC and LS and FN vBMD did not increase with GH therapy, this study raises the question of whether GH is really effective in increasing vBMD. Hyer et al. observed that not treating CO-GHD patients with GH may lead to reduced bone mass in adulthood [512], but other studies on young adults who had received

477

GH replacement also found low bone mass with respect to controls [513]. These inconclusive results indicate that the optimal regimen for GH replacement therapy in CO-GHD has not yet been defined, while there is strong evidence that GH must be started as soon as possible to obtain good skeletal growth. Unsatisfactory results in the long term may have been due to inadequate GH dose or to the fact that, in most cases, GH treatment was discontinued immediately after final height was reached. How to treat GHD during the transition from adolescence to adulthood, and how to manage GH therapy in adults, are still open questions [514]. Whether the right time to stop GH treatment is the attainment of final height or PBM also remains an unresolved issue [510,514e518]. Fors et al. observed that even after discontinuation of GH therapy in adolescents at or near final height, BMC and BMD continued to increase [515]. Saggese et al., on the contrary, found that a longer treatment might yield greater future benefits, like the attainment of a higher PBM [510], and other authors agree that GH replacement should be continued into early adulthood to achieve both target height and target PBM [512]. Two prospective, multinational, randomized, controlled, 2-year studies were performed on 149 postpubertal patients with CO-GHD. The first study confirmed that continuing GH treatment after the achievement of final height can lead to a significant increase in LS BMC [516], and the second found that all body components, in particular muscle mass, responded positively to prolonged GH treatment [517]. In a longitudinal study by Baroncelli et al., 16 adolescents with GHD were evaluated with DXA, after discontinuation of GH treatment at final height, until they achieved their peak LS BMD, and for 2 years thereafter. At final height, all patients had a vBMD T-score between 0 and 2, and they reached their peak LS vBMD after 1e3 years. The time to reach peak BMD was longer than in controls, and the vBMD value was lower [518]. These results, even if on a very small sample, are a further confirmation that young adults with COGHD who remain GH-deficient after completing statural growth should continue GH treatment to achieve a higher PBM. Of course, safety, clinical response, risk of impaired bone mass accrual and poor muscle strength, must always be carefully weighed to avoid possible complications. Reducing the fracture risk in the long term is the main goal of bone therapy in GHD, since fracture rates are higher among adult patients with GHD compared to the normal population. Wuster et al., analyzing the results from a large pharmacoepidemiological survey of GH-deficient adults, found that the prevalence of fractures was 2.7 times that in the control population [519].

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Other studies confirmed that adults with CO-GHD have a higher fracture risk than the normal population [520,521], and Mazziotti et al. found a higher prevalence of vertebral deformities in adult GH-deficient patients, particularly in those never treated with GH, notwithstanding LS BMD values similar to those of controls [521]. Few data on the fracture risk in GH-deficient children and adolescents are available. Baroncelli et al. evaluated LS BMD and prevalence of fractures in 46 GH-deficient adolescents at final height, and found that the fracture prevalence did not differ from that of healthy controls, but that patients with a history of fractures had a lower LS BMD than those without [522]. A recent study criticized the interpretations of bone densitometry data in children and adults with GHD, and questioned the evidence of a higher than normal fracture risk in CO-GHD. The authors stated that, using appropriate size-corrections, BMD is normal in children and adults with isolated GHD, and concluded that GHD “should not be listed as a cause of osteoporosis in children” [523]. These conclusions, however, should be interpreted with care, considering that most studies had indeed made corrections for size, that the only study with pQCT in children observed a reduced radius CSA and cortical thickness [524], and that there are insufficient data on fractures. GH apart, very few data are available regarding other interventions to prevent bone problems in GHD. A 2-year randomized longitudinal study on 14 prepubertal children with GHD showed that calcium supplementation (1 g/day) combined with GH treatment induced an additional significant increase of TB BMC, TB BMD, leg BMC and BMD (but not lumbar BMD) with respect to matched controls treated with GH alone [525]. PUBERTY AND BONE: AN INTRODUCTION The critical importance of puberty for skeletal growth and development explains why disorders of puberty are considered in this chapter. Sex hormones (estradiol and testosterone) are necessary for the growth spurt at puberty, completion of epiphyseal maturation and bone mass accrual [20,21]. About 25% of the PBM is acquired during the 2 years of peak height velocity and, by age 18, at least 90% of PBM has been acquired [526]. During puberty, the marked increase in height and bone mass is temporally dissociated, and the age of peak BMC accretion velocity (about 14 years in boys and 12.5 years in girls) lags 0.6e0.7 years behind the age of peak height velocity. This dissociation suggests that during the adolescent growth spurt, there is a transient period of relative bone weakness, which may partially explain the increase in fractures seen around the time of peak linear growth [526].

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. Some recent studies investigated the relationship between muscle growth and bone strength, and all recognized that muscle force is an important factor for building bone strength [527,528]. According to Xu et al., however, the view that muscle is the primary source of mechanical force applied to bone may be an oversimplification. External forces (gravitational forces and impact forces during ambulation) are also likely to have a significant influence on bone strength [528]. 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 prepubertal growth. In girls, when estrogen levels rise in puberty, endocortical apposition starts at many skeletal sites, in addition to the bone accrual due to muscle force. Consequently, in the postpubertal, premenopausal phase, girls and women seem to have more bone relative to their mechanical needs than males [28], which may constitute an advantage in view of pregnancy and lactation [529]. When boys reach puberty, the rising levels of testosterone increase muscle mass and force, and this leads to increased bone cross-sectional size through accelerated periosteal apposition, resulting in enlargement of the bone diameter and cortical thickening. These 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 [530]. Macdonald et al. clearly demonstrated the sexual dimorphism, using pQCT to evaluate tibial bone in pre- and earlypubertal boys and girls. Bone strength indexes were greater in pre- and early-pubertal boys than in preand early-pubertal girls, mostly because of greater bone areas in boys. Total BMD at distal tibia was significantly higher in prepubertal boys than in girls, while cortical BMD at midshaft tibia was slightly greater in both subgroups of girls (Fig. 18.4) [531]. DISORDERS OF PUBERTY

Both retardation and acceleration of puberty can severely affect the complex and delicate equilibrium of bone growth and development and, clinically, the first problem is to distinguish between pathological presentations and normal variants of puberty. The pathological conditions include precocious puberty and absence of puberty [532]. Among the normal variants, only constitutional delay of puberty (CDP) has been studied for its consequences on bone. CONSTITUTIONAL DELAY OF PUBERTY In a crosssectional study, TB and LS BMD (DXA) and markers of

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(D)

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FIGURE 18.4 Relationships between muscle cross-sectional area (MCSA) and (A) total bone cross-sectional area (ToA), (B) total density (ToD) and (C) bone strength index (BSI) at the distal tibia (8% site), and (D) cortical area (CoA), (E) cortical density (CoD) and (F) strength strain index (SSI) at the tibial midshaft (50% site) for boys (solid line) and girls (dashed line). )P<0.05; ))P<0.001. At distal tibia: significant positive associations between ToA, BSI, and MCSA in boys and girls, and a weak association between ToD and MCSA in girls only. At tibial midshaft: strong relationships between CoA, SSI and MCSA in boys and girls, and a weak negative relationship between CoD and MCSA in girls only. These models suggest that a 10% increase in MCSA would result in an approximately 69 mm3 (7%) increase in SSI for boys and a 66 mm3 (7%) increase in SSI for girls. (From Macdonald H, Kontulainen S, Petit M, Janssen P, McKay H. Bone strength and its determinants in pre- and early pubertal boys and girls. Bone. 2006;39,598e608.)

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bone turnover were measured in 41 boys with CDP (age 8e18 years). TB BMD, adjusted for bone age, was decreased in 11 boys (27%) and LS BMD in 13 (32%), while bone metabolism markers indicated normal bone turnover [533]. In a retrospective study, Finkelstein et al. reported significant reductions in aBMD at LS and radius in 23 young men with CDP (age 23e29 years), compared to normal controls, and concluded that the timing of puberty is an important determinant of peak BMD in men. Since the peak BMD achieved in young adulthood is a major determinant of BMD in later life, delayed puberty may lead to an increased risk for osteoporotic fractures in advanced age [534]. Two years later, restudying 18 of these young men, the same authors observed that FN BMD was also significantly lower than in controls, and that there were no gains in LS and radius bone mass over that period [535]. On the contrary, Bertelloni et al., studying 21 young men with a history of CDP, and considering that the aBMD value (DXA) may be misleading in the presence of an altered growth pattern, estimated the vBMD and found that, while aBMD was reduced in CDP subjects compared to normal controls, there was no difference in vBMD. Bone metabolism markers and fracture rate were not different in CDP patients and controls [536]. This report of a normal vBMD in CDP patients was however criticized by Finkelstein et al. [537], who reexamined their previous study’s data and confirmed a reduction of BMD in their patients, even after correction for size. In males with CDP, the efficacy of hormonal treatment on bone mass has been the object of only a few studies and its efficacy is doubtful. Bertelloni et al. compared androgen-treated and untreated young men with CDP and did not observe any significant improvement in final height or BMD with HRT [536]. Similarly, Yap et al. found that treated and untreated young males with a history of CDP were not significantly different: both were shorter than healthy controls and had a reduced height-adjusted TB BMC, but LS and femoral vBMD were normal [538]. There are no data on the effect of CDP on bone mineral accrual in otherwise normal females, probably because they come to medical attention less frequently, and are not usually advised to take estrogens to avoid premature epiphyseal fusion and shorter final height. 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. PRECOCIOUS PUBERTY Precocious puberty, most commonly diagnosed in otherwise healthy girls, results in accelerated growth, mineral accrual and skeletal maturation.

Central (“true”) precocious puberty (CPP) and precocious pseudopuberty are distinguished. CPP is determined by a premature activation of the hypothalamic-pituitary-gonadal axis, with the premature release of gonadotropins. It is often idiopathic, but may also be due to hypothalamic-pituitary neoplasms or lesions. CPP is five to ten times more frequent in females, and the prevalence is estimated at 1:5000 to 1:10 000 children [532]. The incidence is on the rise and the action of endocrine disrupting chemicals is strongly suspected [539]. Precocious pseudopuberty is due to an abnormal production of sex hormones by CNS tumors, adrenal glands or other organs, to hereditary disorders (e.g. McCuneeAlbright syndrome) or to other causes [532]. CPP determines an early acceleration of skeletal growth but the attainment of a reduced final height due to early epiphyseal fusion. Data on BMD in this condition are somewhat inconsistent. Compared to age- and gender-matched peers, children with CPP have demonstrated increased aBMD at both LS [540,541] and FN [541], but normal TB BMD [540]. The increased LS aBMD was attributed to advanced skeletal maturation and increased bone size. BMD corrected for skeletal age may be normal [541] or reduced [540]. LS BMAD is normal for chronological age [540]. GnRH analogs (GnRHa) are used to arrest further pubertal development and thus delay epiphyseal fusion in children with CPP. Longitudinal follow ups of males and females treated with GnRHa have demonstrated that height and bone accrual are not impaired by treatment [541e544]. Pasquino et al. studied 87 girls with idiopathic CPP, treated with GnRHa for 4.2  1.6 years and followed for 9.9  2.0 years after discontinuation of treatment. They found that adult height was significantly higher than predicted, hormonal values had returned to normal after 1 year without therapy, and BMD, significantly reduced at discontinuation of treatment, had reached normal values with resumption of gonadal activity [543]. Magiakou et al. evaluated 47 adolescent and young adult women with idiopathic CPP (age 16e32.3 years; 33 treated with GnRHa during childhood, 14 non-treated) after discontinuation of treatment e if any e for at least 1 year, and confirmed that GnRHa treatment has no negative impact on height, BMI, BMD and body composition [544]. Such results were reasserted by a recent consensus statement [545]. ABSENCE OF PUBERTY Absence of puberty may originate at different levels. If the defect is in the hypothalamus or hypophysis (e.g. Kallmann syndrome, neoplasms, traumas, radiotherapy), there are low levels of LH and FSH (hypogonadotropic hypogonadism). If the defect is at the gonadal level (e.g. Turner syndrome, Klinefelter syndrome, radiotherapy, chemotherapy), LH

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and FSH levels are high (hypergonadotropic hypogonadism). In both cases, the level of sex hormones (estradiol or testosterone) is low. Hypogonadism may be complete (absence of all signs of puberty) or incomplete (partial pubertal development) [532]. Most studies on hypogonadal patients have been made in adult men and women, and few data are available for younger patients. In most cases, a decrease in BMD, only partially responding to HRT, was observed. In a recent study with DXA on 25 male children with hypogonadism, Fintini et al. observed significantly lower LS BMC, aBMD and vBMD and lower TB BMC and BMD with respect to normal controls [546]. Similar results were reported on 12 male adolescents with hypogonadotropic hypogonadism: all of them had reduced cortical BMD at radius that was partially reversed with testosterone treatment for 2 years [547]. A reduced LS BMD in young men with hypogonadotropic hypogonadism was also observed after testosterone treatment for about 6 years [548]. Similarly, in 70 untreated hypogonadal young women, reduced LS and hip BMD with respect to healthy controls were already observed at around 18e20 years of age, and the decrease in BMD continued rapidly until 25 years of age, indicating that this condition is a cause of low PBM [549]. TURNER SYNDROME

Turner syndrome (TS) has a prevalence of 50 per 100 000 live-born girls. The main clinical features are reduced adult height, gonadal dysgenesis with consequent deficiency of female sex steroids, and multiple skeletal deformities. TS is associated with an increased morbidity and mortality from cardiovascular diseases, diabetes, cirrhosis, osteoporosis and fractures. Currently, only 15% of postnatal diagnoses are made at birth, a high percentage of cases being discovered during pubertal years (26%) and even in adulthood (38%). For this reason, the most important years for bone development are lost without intervention in many cases. X-ray examination reveals low bone mineralization, thin bone cortex, deformity in bone shape (such as cubitus valgus, shortened fourth metacarpal, and Madelung’s deformity) in 60e90% of TS patients, including young girls [550]. Regarding the bone alterations in TS, all the available data on bone mass and density, and on fractures as well, are derived from studies on relatively small samples, and many intervention trials were not randomized. Reduced BMD has been described at any age, from childhood to middle age. Some of the early studies did not take into account the reduced body size of TS girls when evaluating BMD with DXA. When the appropriate adjustment for size was applied, bone mass was less reduced or normal [551,552]. However, in adult TS women, there is a reduction not only of size-corrected

481

aBMD at LS, FN, and forearm, but also of vBMD [553], and Baena et al. reported a significantly increased relative risk for osteoporosis (RR ¼ 10.1) and fractures (RR ¼ 2.16) [554]. Bechtold et al. used pQCT to evaluate the radial metaphysis and diaphysis in 21 adolescents and young women with TS (age range 16.2e25.4 years), treated during growth with GH, and all but one supplemented with estrogens. They found normal cross-sectional bone size, but reduced BMC and vBMD at both sites of measurement, due to decreased cortical thickness. The trabecular vBMD of the metaphysis, however, was not reduced [555]. Recently, these data were confirmed by another study: in 22 adolescents with TS (mean age 12.7 years), compared with an age-matched group of healthy girls, only cortical vBMD and cortical thickness at the proximal radius were reduced, while trabecular vBMD was normal [556]. It has been hypothesized that the different involvement of cortical and trabecular bone density in both girls and women with TS may indicate a selective cortical bone deficit characteristic of this disease [553,555]. Notwithstanding the more or less normal trabecular BMD, several authors report an increased incidence of fractures in TS patients, beginning in childhood [551,554,557,558]. Most fractures involve the forearm, humerus or clavicle. Vertebral fractures have been described only in older women, but no systematic search has been performed in young women [551,558]. Estrogen deficiency used to be considered the main culprit of low bone density and increased bone fragility, and this was in accordance with longitudinal studies of estrogen-deficient and estrogen-replete adolescents with TS. Patients with spontaneous menstruation have normal BMD [550]. In a 3-year longitudinal study on 21 young women with TS (age 20e40 years), treated with HRT, bone biopsies demonstrated an anabolic effect of estradiol [559]. Prospective, longitudinal studies demonstrated that HRT is able to prevent BMD loss at LS, hip and forearm in adult women with TS [560]. Other studies found the hypothesis of simple estrogen deficiency unsatisfactory. Increased levels of bone resorption markers (CTx and NTx) with unchanged or reduced levels of bone formation markers (osteocalcin, procollagen I N-terminal propeptide) have been reported, suggesting an uncoupling in bone remodeling that could not be explained as a direct effect of estrogen deficiency [553]. Moreover, Zuckerman et al., evaluating BMD at the distal third of the radius and mid-tibial shaft with QUS in 27 young patients with TS (age 21.1  6.3 years), concluded that the lack of correlation between speed of sound (SOS) and age, height and hormonal therapy in TS suggested a primary

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bone defect, rather than an enhanced resorption due to estrogen deficiency [561]. In addition, the different involvement of trabecular bone (relatively normal) and cortical bone (significantly reduced) opened the discussion about the existence of subtler mechanisms. Intrinsic bone defects (skeletal dysmorphogenesis) and haploinsufficiency of the SHOX gene (a gene located on the X-chromosome, coding for a homebox regulatory protein necessary for skeletal growth and development) have been discussed as possible causes of bone metabolic derangements in TS [553,562,563]. Moreover, individuals who have cell mosaicism (45,X/46,XX) with a higher proportion of cells with normal karyotype (46,XX) tend to have higher and close to normal LS BMD with respect to individuals with 45,X cells only [564]. All these data suggest that the low BMD of Turner syndrome may be the complex result of several factors and not only of estrogen deficiency. Some studies considered the other possible factors that could explain the increased fracture rate in TS. Evaluating 177 women with TS (aged 19e60 years), Han et al. found a reduction of LS BMD below 1 T-score in 55% of the women and below 2.5 T-score in 9%. The prevalence of fractures was 32% and that of hearing impairment 84%. The increased fracture risk was independently associated with low BMD and hearing impairment, but hearing impairment was associated with fractures only in the presence of low BMD [565]. Recently, it has been observed that in women with TS, fine motor function and body balance are positively correlated with hearing, which may translate into a higher risk of falls in women with hearing impairment [566]. The altered bone shape and geometry in TS patients have been considered risk factors for fractures. However, a recent study that used DXA to evaluate hip geometry in 58 women with TS, notwithstanding some alterations (shorter hip axis length, and increased neck width versus controls), concluded that the increased risk of proximal femur fractures in TS patients cannot be explained by hip geometry parameters [567]. Regarding the treatment of TS, high-dose GH treatment during childhood succeeded in inducing a relevant gain in height, but had only limited, if any, effect on bone [568e570]. Only GH plus estrogens obtained an increase in both aBMD and vBMD [570]. Treatment for ovarian failure is still discussed. In most girls, puberty must be induced. However, estrogens may interfere with growth, and the age to start estrogen therapy should be accurately individualized. The best treatment regimen with estradiol (dose, duration) is yet to be defined for TS, and long-term studies are lacking. Recently, Nabhan et al. reported that in a small group of 12 girls (aged 14.0  1.7 years), randomly assigned to different estrogen regimens,

transdermal estrogens for 1 year induced faster bone accrual at spine than conjugated oral estrogens [571]. It is generally agreed that starting HRT during adolescence allows the achievement of a normal LS BMD [572]. The current standard is that HRT is not always maintained in adulthood [550], despite the fact that young women with TS stopping HRT develop rapid bone loss, with vertebral fragility fractures and loss of height [573]. In conclusion, considering the increased risk of bone loss (especially in adulthood) and the increased risk of fractures (also in childhood and adolescence), adequate calcium and vitamin D intake, as well as suitable physical activity, is recommended for all girls and women affected by TS. In the absence of spontaneous puberty, girls should receive HRT. Estrogens seem to be important in achieving and maintaining bone mass (especially trabecular bone volume). DXA evaluation is useful, applying adequate corrections for bone size. HYPERPROLACTINEMIA

Hyperprolactinemia is rarely observed in children and adolescents. As in adults, it is most often due to a prolactin-secreting pituitary adenoma (prolactinoma). Less frequently it can appear in other pituitary disorders and in autoimmune diseases (especially SLE). In young patients, hyperprolactinemia can also be a side effect of drugs that reduce dopamine secretion or action, mainly antipsychotic drugs (e.g. risperidone, pimozide, olanzapine, quetiapine) [574,575]. Prolactinomas are the most common hormonesecreting pituitary tumors. They account for about 40% of pituitary adenomas, and the prevalence in the adult population is about 100 per million [575]. Even though prolactinomas have been observed at any age from 2 to 80 years, they are very rare before 18 years. In children and adolescents, the prevalence of all pituitary adenomas is about 1 per million [576], and about 50% of them are prolactinomas [575]. In most patients with prolactinomas, drug therapy with dopamine agonists can effectively control the disease, restoring prolactin levels to normal. In patients with drug-induced hyperprolactinemia, the treatment is based on drug withdrawal or substitution, but estrogen/testosterone replacement therapy could also be considered when appropriate. Hyperprolactinemia stimulates milk production and causes hypogonadotropic hypogonadism. The typical presentation in girls is galactorrhea and primary (in prepubertal girls) or secondary amenorrhea and, in boys, arrested puberty, and more rarely gynecomastia. In pubertal girls menstrual irregularity, amenorrhea and galactorrhea are frequently the first signs. Tumor mass effects (headaches, visual field defects, neurological disorders) may be present in the case of

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macroadenomas. Growth retardation and obesity may also be present [574]. In prepubertal children with hyperprolactinemia, bromocriptine treatment can restore GH secretion, with catch-up growth [577]. In adults, different studies have reported that hyperprolactinemia leads to bone loss and, in the long term, osteoporosis. In adults with prolactinomas, both cortical and trabecular BMD at forearm and LS are decreased [578,579]. In 24 patients with prolactinomas (aged 18e49 years), LS BMD was most affected (Z-score
2 in 20.83% of cases), as would be expected in simple hypogonadism [580]. According to a recent review of 21 studies, there is consistent evidence that subjects with hyperprolactinemia have a 20e30% lower LS BMD than healthy controls. Hypogonadism seems to be the major cause of bone alterations, as some studies have found that: (1) hyperprolactinemic women with normal menses do not have a reduced BMD at LS; (2) women with hyperprolactinemia and oligomenorrhea have an intermediate BMD value between healthy and amenorrheic subjects; and (3) women with hypothalamic amenorrhea and normal prolactin have a lower BMD at LS than hyperprolactinemic women without amenorrhea. Similar results have been reported for men with hyperprolactinemia and hypogonadism. And finally, contrary to some animal studies, prolactin seems to have no direct effect on vitamin D and calcium metabolism in humans [581]. This comprehensive review, however, does not consider the current evidence of a direct effect of prolactin on bone cells, demonstrated by the discovery of prolactin receptors in osteoblasts. An osteoblastinhibiting effect of prolactin has been reported in rats [582], and its direct effect on bone turnover has been confirmed by a recent study on hyperprolactinemic rats with or without ovariectomy. Bone resorption seems to be more influenced than formation, through an increase of RANKL and a decrease of OPG expression by osteoblasts [583]. In most women, drug therapy achieves normalization of prolactinemia and recovery of menstrual function, leading to BMD increase. However, BMD may not normalize even after several years of normal prolactin levels and regular menses [578]. In men, normalization of prolactin levels did not improve osteopenia unless testosterone levels were also normalized [584]. The finding that bone loss may persist after the resolution of hyperprolactinemia raises the question whether hyperprolactinemia may be associated with a permanent increase in fracture risk. According to the recent review by Shibli-Rahhal and Schlechte, there are no reports of increased fractures in men or women with hyperprolactinemic hypogonadism [581]. However, Vestergaard et al. reported that in patients

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with prolactinomas, the fracture risk was significantly increased before diagnosis (RR ¼ 1.6), although not after diagnosis [585]. Bone data for children and adolescents with hyperprolactinemia are very scarce, due to the rarity of the disease at this age and to the lack of controlled studies. Some of the previously cited studies included patients of a wide age range. Observational studies on a few cases have found a reduction in BMD [586]. In adolescents with hyperprolactinemia secondary to antipsychotic treatment, a reduced BMD has been reported [587], especially at trabecular bone (ultradistal radius and LS) [588]. In one of the few longitudinal studies, Colao et al. found significantly lower values of LS and FN BMD in 20 adolescents with hyperprolactinemia compared with gender- and age-matched controls, and the BMD reduction seemed greater in adolescents than in young adults. Treatment with dopamine agonists normalized prolactin levels after 6 months. Serum osteocalcin and urinary cross-linked NTx returned within normal range after 12 months. BMD showed a significant increase, but was not restored to normal after 2 years of treatment [589]. No data are available on the long-term prolactinrelated effects on bone status and fracture risk in adults with a history of childhood hyperprolactinemia. Iatrogenic Causes of Osteoporosis GLUCOCORTICOIDS

Glucocorticoids (GCs) have an essential role in the treatment of many severe diseases, such as autoimmune/inflammatory diseases, asthma, leukemia, organ transplantation, etc. Unfortunately, their long-term use has serious consequences for bone. Even though GC-induced osteoporosis (GIO) has been very well demonstrated in both adult and young patients [217,248,282,288,300,590e597], it remains a seriously underestimated problem, especially in children. Many studies have reported that fractures occur in 30e50% of patients treated with GCs for a prolonged time. Fractures can occur in the first months of therapy and with daily doses as low as 2.5 mg of prednisone equivalents, indicating that there is no “safe dose” of GCs for bone [591,592]. Even inhaled steroids can lead to bone loss and increased fracture risk in the long term [594]. These data refer essentially to adult populations, even though young patients were also included in some studies. In many cases, the bone mass loss induced by GCs overlaps that directly or indirectly caused by the disease itself because of malnutrition, inflammation, immobilization, etc. GCs affect bone metabolism and calcium homeostasis at many levels. They have a direct effect on bone and growth plate cell activity, inhibit intestinal calcium

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18. THE SPECTRUM OF PEDIATRIC OSTEOPOROSIS

FIGURE 18.5 Direct and indirect actions of glucocorticoids on bone, muscle, and growth plate. Ca, calcium; GH, growth hormone; IGF-1, insulin-like growth factor-1; IL, interleukin; M-CSF, macrophage colony-stimulating factor; OPG, osteoprotegerin; PPRAg, peroxisome proliferator-activated protein; PTH, parathyroid hormone; RANKL, receptor activator of nuclear factor kappa B ligand; Wnt, wingless-int.

absorption, hamper renal handling of calcium, and suppress sex hormones and GH secretion (Fig. 18.5). At the cellular level, GCs inhibit osteoblastogenesis and osteoclastogenesis, promote apoptosis of osteoblasts and osteocytes, and prolong the lifespan of osteoclasts, thus disrupting the fine balance between bone resorption and formation. The action of GCs on bone is biphasic. In the early phase, GCs induce a rapid bone loss due to the prolonged osteoclast lifespan. In the late phase, the number of osteoblasts is reduced due to increased apoptosis and reduced osteoblastogenesis, while the osteoclast number remains normal or slightly elevated. The result of these derangements is reduced trabecular width and disruption of trabecular architecture, with decreased bone mass and reduced bone strength [596e598]. In the intestine, GCs inhibit calcium absorption, with effects partially independent of vitamin D. Moreover, fasting hypercalciuria is seen after only 5 days of GC therapy and is due to increased bone resorption and decreased renal tubular calcium reabsorption.

Long-term administration of GCs increases PTH secretion [590], because of the negative calcium balance resulting from the reduced calcium absorption and increased calcium excretion, and PTH stimulates osteoclasts to increase bone resorption. It has been reported that GCs decrease the tonic release of PTH and increase its pulsatile bursts, in addition to enhancing the sensitivity of bone cells to the hormone [592]. GCs reduce pituitary secretion of gonadotropins and adrenal and sex hormone production. Deficiency of sex hormones contributes to bone loss and is responsible for the delayed puberty observed in children on longterm GC treatment. Moreover, GCs inhibit the pituitary secretion of GH in adults and impairs the action of IGF-1 in both adults and children. In children, cortisol has only minor effects on GH secretion, and the observed growth impairment seems more related to a direct action of GCs on their target tissues, as they inhibit the cartilage uptake of sulfate and the synthesis of glucosaminoglycans, and cause disruption in the ultrastructure of chondrocytes and extracellular matrix. These changes are not completely reversible, and GC withdrawal may not

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THE SPECTRUM OF PEDIATRIC OSTEOPOROSIS

ensure that the expected height percentile is reached with the catch-up growth. Important determinants of catch-up growth are the duration and intensity of GC exposure as well as the patient’s age (near-puberty is the most delicate moment). The relationship between the severity of bone loss and the cumulative dose of GCs, the doseeresponse relationship, the existence of a threshold dose of GCs below which bone loss does not occur, and finally the difference in bone involvement related to the way of GC administration (oral, i.v., inhaled, alternate day use, etc.) are still debated. Even if, in general, the use of these drugs is justified by the severity of the underlying disease, the presence of calcium and bone metabolism complications must always be considered, because GCs may prevent the acquisition of an acceptable PBM, determining an increased risk of fractures in later life. This underlines the absolute need to use the minimum effective dose of GCs. Clinically, a precipitous drop in bone mass is observed in the first 6e12 months of therapy, followed by gradual but sustained loss in subsequent years [591,599,600]. In adults, bone loss, measured by DXA, is observed at both LS and FN, and trabecular bone appears to be more sensitive to the deleterious effect of GCs than cortical bone [601]. For this reason, QCT or lateral spine DXA scans may be superior for the assessment of bone mass in GIO than the more commonly used anterior-posterior spine and proximal femur DXA studies [601]. Few data are available on bone mass changes in children and adolescents on long-term GC treatment, and prospective studies are needed to verify both the long-term effects of these drugs on bone mass accrual and the degree of bone recovery after withdrawal. Recently, some studies on bone mass in subjects who had to use GCs during childhood but could later do without them have been published, reporting a persistence of low bone mass with an increased risk for osteoporotic fractures later in life [602,603]. In 82 children (age 4e19 years) with either nephrotic syndrome (N ¼ 56) or SLE (N ¼ 26), regularly followed for up to 12 years (range 3e14 years; mean 7.9  6 years) with DXA measurements of spine BMD, it was observed that GCs influence bone mass in a different way in relation to the age at the start of therapy and to the characteristics of the disease. A full recovery from the underlying disease (such as may happen with nephrotic syndrome) and the suspension of GC therapy before puberty allowed normalization of bone mass. On the contrary, in patients with SLE, who had a normal bone mass at diagnosis, starting GC therapy during puberty caused bone mass loss [288]. GC-induced bone loss appears to be dose dependent, but even low doses affect skeletal metabolism and

485

increase the risk of vertebral and non-vertebral fractures [248,591,592,604,605]. In children, vertebral collapse is more common after a cumulative dose of at least 5 g of prednisone equivalents [248]. In both adults and children, vertebral fractures are often asymptomatic. There are consistent data regarding the fracture risk in adults on GC treatment. A meta-analysis reported a relative risk of 1.91 for all fracture sites, 2.01 for hip, and 2.86 for spine [605]. Much fewer data are available for children. A large study in the UK estimated the fracture risk in over 37 000 children (age 4e17 years) taking oral GCs, even though it was impossible to distinguish between the influence of GC treatment and that of the underlying disease. The fracture risk was increased in children who received four or more courses of oral GCs (adjusted OR for fracture ¼ 1.32), and the risk of humerus fractures was particularly high (OR ¼ 2.17). The risk was also high in children using 30 mg or more of prednisolone each day (OR ¼ 1.24). After stopping GC treatment, the risk of fracture seemed to drop down to that of controls [606]. It is very important to remember that, in several chronic diseases, vertebral fractures may be present in children even prior to prolonged GC exposure [607]. This requires a careful search for fractures before starting GC treatment in order to take adequate measures to avoid a further increase in fracture risk. There are inconsistent data on the use of inhaled GCs and the resultant BMD decrease and fracture risk for both adults and children. In children, inhaled GCs seem to have fewer systemic, including skeletal, effects compared to oral or i.v. therapy, unless they are administered at high doses [594,608]. High doses of inhaled GCs (over 7.5 mg/day of prednisolone equivalents) seem to have a slightly increased risk for fractures at any site in adults (adjusted OR ¼ 1.17) [609]. However, two population-based case-control studies did not find an increased fracture risk for children and adolescents taking inhaled GCs compared with non-exposed peers [610,611]. There are some inconsistent data on other aspects of GC treatment, such as daily versus alternate day use or the use of different GCs. Deflazacort, an oral steroid derivative, seems to have less bone effects than prednisone or methylprednisone in the short term. There are only a few, uncontrolled studies on long-term use, and no conclusions are possible. GC-INDUCED OSTEONECROSIS Another serious bone complication of GC treatment in children is the aseptic necrosis of bone (osteonecrosis) [597]. Osteonecrosis has been reported not only in the long-term therapy with GCs, but also following short courses of high-dose therapy, with an estimated frequency up to 38%. The femoral head is more commonly involved,

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18. THE SPECTRUM OF PEDIATRIC OSTEOPOROSIS

but other large joints may be affected. Joint pain and stiffness are often the earliest symptoms, but lesions are often asymptomatic, and without magnetic resonance imaging the diagnosis may be late or completely missed. Spontaneous improvement or even resolution may rarely occur. A complete description of this complication is beyond the scope of this chapter. OTHER MEDICATIONS

Various drugs exert negative effects on bone cells and bone turnover, and their use is often associated with a reduction in BMD and, in some cases, with fractures. Some of these drugs are also used in children (e.g. methotrexate in ALL and JIA, cyclosporine in transplants, anticonvulsants in epilepsy). It is beyond the scope of this chapter to review this matter in depth. Table 18.4 presents, for each group of substances, a synthesis of what is currently known about their effects on bone, with selected references [138,235,479,612e645]. It must be remembered that in many cases (e.g. chronic use of heparin or immunosuppressive drugs), it can be very difficult to distinguish between the direct effect of a specific drug on bone and the effects due to the primary disease or other concurrent therapies.

GENERAL ASPECTS OF PREVENTION AND TREATMENT The diagnosis of a disease or condition carrying an increased risk of detrimental effects on bone metabolism and bone strength in a child or adolescent demands utmost attention. The current knowledge on the prevention and treatment of bone problems in specific diseases has been presented above. Only the general rules for a rational approach to the prevention and management of pediatric osteoporosis will be summarized here. The basic approach is essentially the same for both the primary and the secondary forms of pediatric osteoporosis, and the first rule is to always think of altered bone metabolism and bone mineral accrual as possible clinical problems. This does not only apply to the secondary osteoporoses, but also to the primary forms like EhlereDanlos or Marfan syndromes, in which other clinical features may be more evident than bone involvement. Of course, the severity and activity of the primary disease should always be taken into account when evaluating the strategies to reduce the risk of bone mass loss and fragility fractures. In all children and adolescents at risk, bone status should be evaluated as soon as possible. Dietary aspects (e.g. calcium and protein intake) and vitamin D status should be assessed, considering the high prevalence of

low calcium intake and vitamin D insufficiency/ deficiency. The determination of baseline densitometric and biochemical values before starting a treatment for the primary disease is recommended, also to distinguish between a pre-existing condition and an adverse drug effect. DXA is currently the preferred densitometric technique [5,6]. General rules for bone density evaluation in pediatric patients have been issued by the International Society for Clinical Densitometry [7], while specific recommendations or guidelines are available for a limited number of diseases. BMD corrections (e.g. BMAD, correction for height) should always be used in pediatric patients to interpret correctly DXA densitometric measures. Changes in BMC and BMD must be evaluated in relation to the patient’s condition (remission, relapse, change in GC dose, etc.) and the response to treatment. Such evaluation is particularly complex, since the effects of therapy must be distinguished from those of growth, puberty, and disease evolution. In the presence of low bone density, or even more so, of fragility fractures, a strict follow up is required, with periodic densitometric measurements and clinical evaluations to monitor bone metabolism parameters, bone pain, changes in kyphosis after vertebral fractures, and ratio of trunk to lower limb length. For most pediatric patients, it seems reasonable to perform densitometric measurements every 2e3 years in the presence of a stable condition, and once a year in the presence of fractures or in high-risk conditions. Since a growing skeleton changes very rapidly, especially during puberty, bone scans may be performed at shorter intervals, such as every 6 months, in special conditions (e.g. after a transplant or in the first phase of GC therapy) [7]. Biochemical bone markers are difficult to interpret in children and adolescents because they reflect both bone modeling (growth) and remodeling (turnover) [4]. Baseline measurement of bone markers before starting a therapy with antiresorptive drugs may be useful, as well as periodical evaluations during the follow up. As in adults, a decrease in bone markers should be expected during BP therapy [645]. Calcium and phosphate levels in plasma and urine should be monitored. They are generally normal in most forms of primary or secondary osteoporosis in children but, if altered, they may reveal the presence of concurrent pathological conditions, such as rickets or hyperparathyroidism. An increase in urinary calcium excretion may reveal hypercalciuria, either idiopathic or related to GC use or prolonged immobilization. Serum levels of PTH and 25OHD should also be monitored. As a rule, control of the primary disease is the best first-line approach to prevent osteoporosis in young

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GENERAL ASPECTS OF PREVENTION AND TREATMENT

TABLE 18.4

Medications (other than Glucocorticoids) known for Negative Effects on Bone Bone loss in children and adolescents

Fractures in children and adolescents

Altered mineralization Increased bone turnover [612]

Yes [138,613,614]

Yes [615,616]

Inhibition of 1a-hydroxylase activity, with decreased production of 1,25OH2D Secondary hyperparathyroidism Decreased number and action of osteoblasts Increased number and action of osteoclasts [617]

Increased bone resorption Decreased bone formation [617]

Few data/ uncertain [618,619]

No data

Warfarin

Inhibition of osteocalcin synthesis Reduced bone formation [620] Teratogenic on fetus

Decreased bone formation Decreased bone resorption [620] Skeletal abnormalities

Yes [621]

No data

Low molecular weight heparin

Decreased number and action of osteoblasts [617]

Decreased bone formation [617]

Few data/ uncertain [618,619]

No data

Methotrexate

Osteoblast inhibition (impaired protein synthesis?) Interference with metabolism/ actions of vitamin C (necessary for proline hydroxylation and collagen synthesis) [622e624]

Decreased bone formation [622e624]

Yes (mainly observed in ALL and solid cancers) [625e627] Bone loss seems dose dependent [235,628,629]

Yes [630]

Calcineurin inhibitors (cyclosporine, tracolimus)

Conflicting studies, uncertain mechanisms

Increased bone resorption [631,632]

No data

No data

Antiretrovirals

Mitochondrial damage Increased osteoclastogenesis Impaired osteoblast function? Alterations in receptor of activated RANK [633,634]

Increased bone resorption Decreased bone formation [633,634]

Yes [635e637]

Yes, reported in studies on bone density [635e637]

Medroxyprogesterone acetate

Central hypogonadism [638]

No data

No data

GnRH analogs

Central hypogonadism [638]

No data

No data

L-thyroxine suppressive therapy

Osteoblast-mediated T3 activation of osteoclasts [479]

No data

No data

Radiotherapy Cranial irradiation

Muscle and bone atrophy Hormonal deficiencies [639]

Yes [640e643]

Yes (also stress fractures) [644,645]

Medication

Proposed mechanism of action on bone

Effect on bone

Antiepileptic drug (phenytoin, phenobarbital, carbamazepine, sodium valproate)

Hepatic induction of CYP450, with increased catabolism of vitamin D Secondary hyperparathyroidism [612]

Heparin

Anticoagulants

Increased bone resorption [638]

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18. THE SPECTRUM OF PEDIATRIC OSTEOPOROSIS

patients. For example, strict GFD is the first (and often the only) therapy needed to ensure bone integrity in celiac disease [320,322,333,334]. Similarly, control of inflammation may reduce both periarticular and systemic bone loss in JIA [243]. Growth retardation, pubertal delay, or hypogonadism, if present, should be corrected with appropriate HRT, also to create a favorable environment for bone growth and development. There is evidence that GCs can negatively influence both secretion and action of GH/IGF-1 and it has been suggested that the anabolic effects of GH and IGF-1 may counteract the GC-related bone loss by enhancing muscle mass accrual and linear growth [646]. Measures should be taken to correct poor nutrition and to ensure the proper intake of essential nutrients, according to the patient’s age, gender and physical characteristics. The adequate availability of calcium, phosphate, proteins and vitamin D is one of the most important environmental factors for bone health, and the advice of a skilled dietician may help. The special disease-dependent needs should also be considered: for example, proteineenergy malnutrition has been reported in 10e50% of adolescents with JIA and a greater caloric intake is needed in CF. To correct insufficient calcium intake, calcium-rich foods should be preferred whenever possible, because this calcium is better absorbed than that of calcium salts [647]. Moreover, calcium-rich foods seem to be safer in the long term, carrying a lower risk of renal stones, an important advantage given the presence of hypercalciuria in many diseases [648]. It should also be noted that most children have a low compliance to calcium salts because of unpleasant gastrointestinal symptoms. Chronically ill young patients are at high risk of vitamin D deficiency [295,308,307,374,381,470,471], because of reduced outdoor activities or because sunlight exposure may need to be avoided (e.g. in SLE). Moreover, hepatic, renal and intestinal diseases as well as several drugs (e.g. GCs, anticonvulsants, heparin, cyclosporine, tacrolimus) may affect the metabolism and function of vitamin D in different ways. In these conditions, there is a high risk of actual vitamin D deficiency, or of inappropriately low levels of circulating vitamin D metabolites, resulting in low calcium absorption, increased PTH secretion (secondary hyperparathyroidism), and increased bone resorption. Regarding vitamin D, a debate has arisen in recent years about the need to re-evaluate the recommended daily allowance (RDA) for children and adolescents. Specific dose-finding trials for the different diseases are lacking, and the RDA for healthy children is also used for children with bone problems. Native vitamin D or calcifediol with or without calcium supplementation

has been used in pediatric patients with JIA, CF, or DMD, either treated with GCs or not [181, 264,265,385,386]. Some positive effects on BMC and BMD have been observed, but the long-term effects, including those on fractures, have not yet been evaluated. The side effects of calcium and vitamin D treatment (hypercalcemia, hypercalciuria) seem to be negligible. The use of calcitriol (1,25-(OH)2 D) or alfacalcidol (1-alpha-hydroxyvitamin D) has not been systematically tested in children, except in those with renal insufficiency. Even for simple calcium and vitamin D supplementation, long-term, prospective, randomized, controlled studies on sufficiently large cohorts are still lacking. Physical activity and weight-bearing exercises are essential for a healthy skeletal growth and the achievement of a high PBM [649,650], and have been shown to favor BMD increase in different pathological conditions. The type, quantity and quality of physical activity must be appropriate for age, gender, and primary disease. There is a threshold of physical activity above which negative effects for bone may appear: the classical example is AN, in which excess of exercise, poor nutrition and hypogonadism act in consert to determine bone loss. When a child has sustained vertebral fractures or repeated long-bone fractures, as often happens in OI or CP-related osteoporosis, and his or her quality and expectancy of life are in jeopardy, the prudent use of a specific bone agent is indicated. BPs are the most widely used antiresorptive drugs, and the only ones used in children and adolescents. They reduce osteoclast-mediated bone resorption, and shift the balance of bone remodeling towards an increase in bone mass. The first systematic use of BPs in children has been for the treatment of OI e to date the only disease on which long-term, prospective, randomized, controlled studies on relatively large numbers of patients have been performed [651]. Subsequently, BPs have been used in many patients with different forms of secondary osteoporosis, e.g. GIO in JIA and other connective tissue diseases [272], cerebral palsy [153,154], CF [355,652], ALL [653]. Cyclical intravenous pamidronate or oral alendronate have been used in most cases, but several other BPs have also been used, including risedronate and zoledronate [88,102,156,206,269,270,445,654]. The current research on BP use in children has been recently reviewed by Bachrach and Ward [655]. Many studies in different diseases have consistently observed a significant BMD increase and reduction of bone pain in children treated with BPs, compared to placebo-treated controls. There are fewer data on fractures, due to the limited number of patients and the limited duration or statistical power of most studies, thus preventing the

PEDIATRIC BONE

SUMMARY AND FUTURE DIRECTIONS

demonstration of the efficacy of BPs in reducing the number of vertebral and appendicular fractures. It should be remembered that, while BP use is now widely accepted in severe cases of pediatric osteoporosis, there are still doubts about using these drugs in children with milder conditions (such as OI with a low fracture rate), with spontaneously resolving illnesses like IJO, or with low bone mass without fractures. The uncertainty arises from the lack of data on the long-term efficacy and safety of these drugs in very young patients, also considering the lack of consistent data on the epidemiology of fragility fractures in such populations, and on the fracture risk related to the attainment of a low PBM. Even though the long-term consequences of BPinduced low bone turnover in children are still unknown, the original concerns about the risks of BP use have not been confirmed after almost 20 years of clinical, radiographic and histological evaluations. Height gain, growth, skeletal maturation, fracture repair, and growth plates seem impaired. The sclerotic lines observed at the distal metaphyses of long bones when BPs are used before epiphyseal closure, seem to have no impact on skeletal growth and maturation. As in adults, BPs are well tolerated. The major side effects of i.v. BP infusion are transient flu-like symptoms and low fever, generally limited to the first administration, which can often be prevented by premedication with acetaminophen (paracetamol). For unknown reasons, in some conditions, mainly in CF, an exaggerated flu-like syndrome may be experienced, for which GCs may be indicated. In younger children, serum calcium levels should be measured before and after each i.v. infusion, as there is a risk of hypocalcemia with possible ECG alteration (prolonged QTc interval) [656]. In infants below 2 years of age with severe OI and pre-existing respiratory problems, acute respiratory distress after the first pamidronate infusion, requiring bronchodilator therapy, has been reported [657]. Oral BPs have occasionally caused esophagitis, fully reversible after withdrawal. Anterior uveitis, scleritis, and transient decrease in lymphocyte count are very rare [655]. BP treatment has recently been associated with a 1.67-year delay in tooth eruption in children with OI [658]. Osteonecrosis of the jaw e a feared complication of BP use in adults e has not been reported in children until now, even with prolonged treatment [655], but a dental evaluation before and during treatment is advisable, especially in children with poor dental health. There is a report on a single case of frank misuse of BPs (high doses of pamidronate for a long time, without any monitoring), ending in severe untoward effects [659]. BPs readily pass the placenta and, in rats, very high doses (much higher than therapeutic doses for humans)

489

are toxic for the fetal skeleton. However, no adverse effects were observed in the children of two pregnant young women, treated with i.v. pamidronate for bone metastases of breast cancer. Regardless, BP use in pregnancy cannot be considered safe, and contraceptive measures must be adopted by young women taking these drugs. The long half-life (years) of BPs in bone also raises concern about the future reproductive health of these patients. BPs must be used in appropriate doses for age and body weight, with regular clinical, biochemical and densitometric assessments. For the time being, it is advisable that only specialists experienced in pediatric bone diseases make the delicate decisions about whether to start BP treatment, the type of drug to be used, the dose, the duration of therapy, the follow-up evaluation, and the criteria for suspension. Other drugs have been recently approved for the treatment of osteoporosis in adults. Teriparatide (recombinant human PTH; hPTH 1e34) and PTH (fulllength PTH; 1e84), used in pulse therapy for up to 24 months, are able to stimulate bone formation in excess of bone resorption. They are used in severe cases of osteoporosis, including GIO, in adult patients. There are no published studies on their efficacy and safety in children affected by primary or secondary osteoporosis, but teriparatide has been used in some children with hypoparathyroidism [660,661]. Utmost caution is recommended, since pharmacological stimulation of bone formation at this age might increase the risk for osteosarcoma (as observed in rat studies) [662]. Denosumab (monoclonal antibody against RANKL) is a new antiresorptive drug used in postmenopausal and GIO in adults. It is not currently used in children. Other therapies, including stem cell and gene therapy, are currently being investigated and may find clinical applications in the near future. Also, systemic transplantation of human adipose-derived stem cells (ASCs) may offer new possibilities for bone repair, through the stimulation of osteoblast differentiation and proliferation and osteoclast differentiation and survival.

SUMMARY AND FUTURE DIRECTIONS Almost 40 years ago, Charles Dent expressed the paradoxical view that senile osteoporosis should be considered as “a pediatric disease”, on the basis that strong bones e the only protection from future osteoporosis e can only be built in childhood. Now we are fully aware that childhood is not only the age for laying the foundations of a healthy and strong skeleton for the future, but is also an age in which such an essential goal may remain out of reach due to

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490 TABLE 18.5

18. THE SPECTRUM OF PEDIATRIC OSTEOPOROSIS

Key Points on Pediatric Osteoporosis

Primary osteoporosis is relatively rare Secondary osteoporosis is increasingly observed in many chronic diseases, particularly in those requiring long-term glucocorticoid therapy All risk factors for osteoporosis must be carefully assessed Correction of such factors may help to attain a higher bone mass, even if the attainment of a normal peak of bone mass cannot be expected in all cases Age, pubertal status, nutrition, physical activity and lifestyle factors must be considered Hormonal deficiencies must be treated The costebenefit ratio of glucocorticoid or other drug treatment of the primary disease must be evaluated Glucocorticoids must be used at the minimum effective doses, for the shortest possible time The simpler, safer measures (e.g. correcting calcium and protein intake, vitamin D supplementation when necessary, regular physical activity) are the first line of osteoporosis treatment in the young Bone-specific agents must be used after careful riskebenefit analysis, when other measures have failed Bisphosphonates are the most extensively studied drugs and their efficacy and safety has been demonstrated in both primary and secondary osteoporoses, also in children and adolescents

a host of genetic disorders and chronic illnesses of childhood that bring about serious bone morbidity. It must be underlined that treating osteoporosis in children and adolescents is not as simple as prescribing a drug on the basis of the pharmacological effects observed in adults. This is a much more complex and difficult task, because at this age many variables interfere with the delicate physiological equilibrium of bone, and the possible consequences in adult life must always be considered. The aims of treating osteoporosis in a growing skeleton are to prevent fractures and disability, relieve pain, maintain locomotor function and sustain satisfactory bone growth and development. Therapeutic interventions must be prudent, beginning with the simplest and safest measures (calcium, vitamin D, physical activity), and must be tailored to the individual patient’s situation and needs, with special attention to his or her quality of life. New bone-specific drugs can only be used after careful riskebenefit analysis, when other measures have failed, and in the presence or risk of severe complications (fractures, prolonged immobilization) (Table 18.5). Recently, much progress has been made in the knowledge of bone biology and of the molecular, genetic and pathogenetic mechanisms of the different forms of primary and secondary osteoporosis affecting children and adolescents, as we have tried to summarize in this chapter.

Research has also offered new insights into the mechanisms of bone development and the factors that favor the achievement of a high PBM. This is not only important to understand the normal process of growth, but also to understand the pathogenesis of bone loss in the elderly. Notwithstanding this progress, there have been only limited advances in the prevention and treatment of bone metabolic diseases in younger patients. Prospective, longitudinal, controlled studies in children are still lacking, and only a few studies have evaluated a sufficient number of patients to reach reliable conclusions. More international collaboration and multicenter studies are needed to recruit large patient cohorts, and to address crucial questions such as the epidemiology of fragility fractures linked to specific diseases, and the consequences and impact of low bone density and fractures during childhood for later adult life. Finally, large prospective, randomized, placebo-controlled trials on the efficacy and safety of BPs and other anti-osteoporotic agents in young patients are also urgently needed. Over the last few years, the pressure from pediatric bone specialists has stimulated the major manufacturers of DXA devices, and some of those of QUS devices, to introduce technical changes and improvements based on the specific requirements of children. The next step is to obtain greater attention from the pharmaceutical companies to develop drugs specifically targeted to fight pediatric osteoporosis, or to implement specific studies with drugs currently used for osteoporosis in adults.

APPENDIX: 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. In the USA, in 2003, 25% of children hospitalized with fractures in the first year of age were actually victims of abuse, and the incidence of fractures attributable to abuse was 36.1 cases per 100 000 [663]. While there is a great deal of medical literature pertaining to the differentiation of physical abuse from accidental trauma, little information is available for distinguishing the abused child from a child with bone fragility, even though some reviews have recently been published [664e666]. 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. It must also be remembered that while disorders characterized by low bone mass make up the majority of bone fragility conditions, increased fragility may occur when bone

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REFERENCES

mass is normal, or pathologically elevated (osteosclerosis). Identifying a child with a bone fragility conditions versus physical abuse requires knowledge of the disorders that predispose to low-trauma fracture [664,665]. Any of the osteoporotic conditions discussed in this chapter may be associated with fractures, but the one most frequently mistaken for abuse is OI. Many parents with children under 2 years of age with fractures and undiagnosed OI have been suspected of child abuse [667]. When the classic features of OI are present, the diagnosis should be obvious. However, when these features are subtle or absent, as may happen with OI types I, atypical OI type IV, and the newer OI forms (types VeVII), the diagnosis may be overlooked and families are then subjected to the demanding and difficult process of an abuse assessment. The vast majority of physically abused children are under 4 years of age, due to the inability to verbally or physically resist harm. Children with 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. The abuser is a related caregiver in 90% of cases. Alcohol and other substance abuse, mental illness, previous history of abuse in childhood, young caregivers, as well as family problems (e.g. social isolation, financial stress, domestic violence, unwanted pregnancies, or other stressful events), are important risk factors 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. 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. Attention should also be paid to the overall status of the child, including growth and nutrition, psychomotor development, hydration and hygiene. The physical examination should 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 incident. Fractures in children who are nonambulatory should raise suspicion, and fractures of certain bones, such as sternum, spinous processes and scapulae, are unusual in accidental injury and disease [666]. Skull fractures are also relatively infrequent in bone fragility conditions, where fractures of the

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extremities predominate. Fractured ribs are rare in accidental injury, but may occur in bone fragility conditions. While lateral rib fractures are sometimes caused by accidental trauma or bone diseases, multiple, bilateral, posteromedial rib fractures are strongly indicative of abuse (squeezing injury). Vigorous pulmonary resuscitation has not been found to cause posteromedial rib fractures in normal children [666]. 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 acetabuli, skull deformity (in the absence of contusions/fractures) and enlarged fontanels point to organic pathology. A complete radiographic skeletal survey, performed by an expert radiologist, familiar with the radiographic characteristics of OI and other bone fragility disorders, is mandatory in the assessment of possible abuse. 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), that occurs as a result of indirect forces when the extremity is pulled, pushed, twisted or when the infant is shaken, is a frequent occurrence in abuse. 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. Additional tests are in accordance with the degree of suspicion for physical abuse versus organic disease. In some instances, it is not possible to diagnose definitively 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.

References [1] Harvey N, Dennison E, Cooper C. Osteoporosis: impact on health and economics. Nat Rev Rheumatol 2010;6:99e105. [2] Holroyd C, Cooper C, Dennison E. Epidemiology of osteoporosis. Best Pract Res Clin Endocrinol Metab 2008;22:671e85. [3] Bianchi ML. Osteoporosis in children and adolescents. Bone 2007;41:486e95. [4] Ju¨rima¨e J. Interpretation and application of bone turnover markers in children and adolescents. Curr Opin Pediatr 2010;22: 494e500. [5] Binkovitz LA, Henwood MJ, Sparke P. Pediatric DXA: technique, interpretation and clinical applications. Pediatr Radiol 2008;38(Suppl. 2):S227e39.

PEDIATRIC BONE

492

18. THE SPECTRUM OF PEDIATRIC OSTEOPOROSIS

[6] Bogunovic L, Doyle SM, Vogiatzi MG. Measurement of bone density in the pediatric population. Curr Opin Pediatr 2009;21: 77e82. [7] Lewiecki EM, Gordon CM, Baim S, et al. International Society for Clinical Densitometry 2007 Adult and Pediatric Official Positions. Bone 2008;43:115e21. [8] Kroger H, Kotaniemi A, Vainio P, Alhava E. Bone densitometry of the spine and femur in children by dual-energy x-ray absorptiometry. Bone Miner 1992;17:75e85. [9] Carter DR, Bouxsein ML, Marcus R. New approaches for interpreting projected bone densitometry data. J Bone Miner Res 1992;7:137e45. [10] Goulding A, Jones I, Taylor RW, Manning PJ, Williams SM. More broken bones: a 4-year double cohort study of young girls with and without distal forearm fractures. J Bone Miner Res 2000;15:2011e8. [11] Skaggs DL, Loro ML, Pitukcheewanont P, Tolo V, Gilsanz V. Increased body weight and decreased radial cross-sectional dimension in girls with forearm fractures. J Bone Miner Res 2001;16:1337e42. [12] Landin L, Nilsson BE. Bone mineral content in children with fractures. Clin Orthop Relat Res 1983;178:292e6. [13] Goulding A, Cannan R, Williams SM, Gold EJ, Taylor RW, Lewis-Barned NJ. Bone mineral density in girls with forearm fractures. J Bone Miner Res 1998;13:143e8. [14] Goulding A, Jones IE, Taylor RW, Williams SM, Manning PJ. Bone mineral density and body composition in boys with distal forearm fractures: a dual-energy X-ray absorptiometry study. J Pediatr 2001;139:509e15. [15] Ma D, Jones G. The association between bone mineral density, metacarpal morphometry and upper limb fractures in children: a population based case-control study. J Clin Endocrinol Metab 2003;88:1486e91. [16] Jones G, Ma D, Cameron F. Bone density interpretation and relevance in Caucasian children aged 9e17 years of age: insights from a population-based fracture study. J Clin Densitom 2006;9: 202e9. [17] Clark EM, Ness AR, Bishop NJ, Tobias JH. Association between bone mass and fractures in children: a prospective cohort study. J Bone Miner Res 2006;21:1489e95. [18] Ferrari SL, Chevalley T, Bonjour JP, Rizzoli R. Childhood fractures are associated with decreased bone mass gain during puberty: an early maker of persistent bone fragility. J Bone Miner Res 2006;21:502e7. [19] Clark EM, Tobias JH, Ness AR. Association between bone density and fractures in children: a systematic review and metaanalysis. Pediatrics 2006;117:291e7. [20] Bass S, Delmas PD, Pearce G, Hendrich E, Tabensky A, Seeman E. The differing tempo of growth in bone size, mass, and density in girls is region-specific. J Clin Invest 1999;104: 795e804. [21] Bradney M, Karlsson MK, Duan Y, Stuckey S, Bass S, Seeman E. Heterogeneity in the growth of the axial and appendicular skeleton in boys: implications for the pathogenesis of bone fragility in men. J Bone Miner Res 2000;15:1871e8. [22] Rauch F, Travers R, Plotkin H, Glorieux FH. The effects of intravenous pamidronate on the bone tissue of children and adolescents with osteogenesis imperfecta. J Clin Invest 2002;110: 1293e9. [23] Glorieux FH, Travers R, Taylor A, et al. Normative data for iliac bone histomorphometry in growing children. Bone 2000;26: 103e9. [24] Rauch F, Schoenau E. The developing bone: slave or master of its cells and molecules? Pediatr Res 2001;50:309e14.

[25] Hernandez CJ, Beaupre GS, Carter DR. A theoretical analysis of the relative influences of peak BMD, age-related bone loss and menopause on the development of osteoporosis. Osteoporos Int 2003;14:843e7. [26] Frost HM. Bone "mass" and the "mechanostat": a proposal. Anat Rec 1987;219:1e9. [27] Frost HM. Bone’s mechanostat: a 2003 update. Anat Rec A Discov Mol Cell Evol Biol 2003;275:1081e101. [28] Schiessl H, Frost HM, Jee WS. Estrogen and bone-muscle strength and mass relationships. Bone 1998;22:1e6. [29] de Vries JI, Visser GH, Prechtl HF. The emergence of fetal behaviour. II. Quantitative aspects. Early Hum Dev 1985;12: 99e120. [30] Schoenau E. From mechanostat theory to development of the "Functional Muscle-Bone-Unit". J Musculoskelet Neuronal Interact 2005;5:232e8. [31] Fricke O, Beccard R, Semler O, Schoenau E. Analyses of muscular mass and function: the impact on bone mineral density and peak muscle mass. Pediatr Nephrol 2010;25: 2393e400. [32] Rittweger J. Ten years muscle-bone hypothesis: What have we learned so far? Almost a Festschrift. J Musculoskelet Neuronal Interact 2008;8:174e8. [33] Hughes JM, Petit MA. Biological underpinnings of Frost’s mechanostat thresholds: the important role of osteocytes. J Musculoskelet Neuronal Interact 2010;10:128e35. [34] Bank RA, Robins SP, Wijmenga C, et al. Defective collagen crosslinking in bone, but not in ligament or cartilage, in Bruck syndrome: indications for a bone-specific telopeptide lysyl hydroxylase on chromosome 17. Proc Natl Acad Sci USA 1999;96:1054e8. [35] Yapicio glu H, Ozcan K, Arikan O, Satar M, Narli N, Ozbek MH. Bruck syndrome: osteogenesis imperfecta and arthrogryposis multiplex congenita. Ann Trop Paediatr 2009;29:159e62. [36] Shaheen R, Al-Owain M, Sakati N, Alzayed ZS, Alkuraya FS. FKBP10 and Bruck syndrome: phenotypic heterogeneity or call for reclassification? Am J Hum Genet 2010;87:306e8. [37] Breslau-Siderius EJ, Engelbert RH, Pals G, van der Sluijs JA. Bruck syndrome: a rare combination of bone fragility and multiple congenital joint contractures. J Pediatr Orthop B 1998;7: 35e8. [38] Hyry M, Lantto J, Myllyharju J. Missense mutations that cause Bruck syndrome affect enzymatic activity, foldng and oligomineralization of lysyl hydroxylase 2. J Biol Chem 2009;284: 30917e24. [39] Andiran N, Alikasifoglu A, Alanay Y, Yordam N. Cyclic pamidronate treatment in Bruck syndrome: proposal of a new modality of treatment. Pediatr Int 2008;50:836e8. [40] Ai M, Heeger S, Bartels CF, Schelling DK, the OsteoporosisPseudoglioma Collaborative Group. Clinical and molecular findings in osteoporosis-pseudoglioma syndrome. Am J Hum Genet 2005;77:741e53. [41] Gong Y, Slee RB, Fukai N, et al. LDL receptor-related protein 5 (LRP5) affects bone accrual and eye development. Cell 2001;107: 513e23. [42] Balemans W, Van Haul W. Minireview: the genetics of lowdensity lipoprotein receptor-related protein 5 in bone: a story of extremes. Endocrinology 2007;148:2622e9. [43] Narumi S, Numakura C, Shiihara T, et al. Various types of LRP5 mutations in four patients with osteoporosis-pseudoglioma syndrome: Identification of a 7.2-kb microdeletion using oligonucleotide tiling microarray. Am J Med Genet 2010;152A: 133e40.

PEDIATRIC BONE

REFERENCES

[44] Marques-Pinheiroa A, Levasseurb R, Cormierd C, et al. Novel LRP5 gene mutation in a patient with osteoporosis-pseudoglioma syndrome. Joint Bone Spine 2010;77:151e3. [45] Lev D, Binson I, Foldes AJ, Watemberg N, Lerman-Sagie T. Decreased bone density in carriers: patients of an Israeli family with osteoporosis-pseudoglioma syndrome. Isr Med Assoc J 2003;5:419e21. [46] Qin M, Hayashi H, Oshima K, Tahira T, Hayashi K, Kondo H. Complexity of the genotype-phenotype correlation in familial exudative vitreoretinopathy with mutations in the LRP5 and/or FZD4 genes. Hum Mutat 2005;26:104e12. [47] Little RD, Carulli JP, Del Mastro RG, et al. A mutation in the LDL receptor-related protein 5 gene results in the autosomal dominant high-bone-mass trait. Am J Hum Genet 2002;70:11e9. [48] Zacharin M, Cundy T. Osteoporosis pseudoglioma syndrome: treatment of spinal osteoporosis with intravenous bisphosphonates. J Pediatr 2000;137:410e5. [49] Streeten EA, McBride D, Puffenberger E, et al. Osteoporosispseudoglioma syndrome: Description of 9 new cases and beneficial response to bisphosphonates. Bone 2008;43:580e4. [50] Barros ER, Dias da Silva MR, Kunii IS, Lazaretti-Castro M. Three years follow-up of pamidronate therapy in two brothers with osteoporosis-pesudoglioma syndrome (OPPG) carrying an LRP5 mutation. J Pediatr Endocrinol Metab 2008;21:811e8. [51] Bayram F, Tanriverdi F, Kurto glu S, et al. Effects of 3 years of intravenous pamidronate treatment on bone markers and bone density in a patient with osteoporosis-pesudoglioma syndrome (OPPG). J Pediatr Endocrinol Metab 2006;19:275e9. [52] Callewaert B, Malfait F, Loeys B, De Paepe A. Ehlers-Danlos syndromes and Marfan syndrome. Best Pract Res Clin Rheumatol 2008;22:165e89. [53] Voermans NC, Knoop H, Bleijenberg G, van Engelen BG. Pain in Ehlers-Danlos syndrome is common, severe, and associated with functional impairment. J Pain Sympt Manag 2010;40: 370e8. [54] Beighton P, De Paepe A, Steinmann B, Tsipouras P, Wenstrup RJ. Ehlers-Danlos syndromes: revised nosology, Villefranche, 1997. Ehlers-Danlos National Foundation (USA) and Ehlers-Danlos Support Group (UK). Am J Med Genet 1998;77:31e7. [55] Schalkwijk J, Zweers MC, Steijlen PM, et al. A recessive form of the Ehlers-Danlos syndrome caused by tenascin-X deficiency. N Engl J Med 2001;345:1167e75. [56] Zweers MC, van Vlijmen-Willems IM, van Kuppevelt TH, et al. Deficiency of tenascin-X causes abnormalities in dermal elastic fiber morphology. J Invest Dermatol 2004;122:885e91. [57] Coelho PC, Santos RA, Gomes JA. Osteoporosis and EhlersDanlos syndrome. Ann Rheum Dis 1994;53:212e3. [58] Deodhar AA, Woolf AD. Ehlers Danlos syndrome and osteoporosis. Ann Rheum Dis 1994;53:841e2. [59] Dolan AL, Arden NK, Grahame R, Spector TD. Assessment of bone in Ehlers Danlos syndrome by ultrasound and densitometry. Ann Rheum Dis 1998;57:630e3. [60] Carbone L, Tylavsky FA, Bush AJ, Koo W, Orwoll E, Cheng S. Bone density in Ehlers-Danlos syndrome. Osteoporos Int 2000;11:388e92. [61] Yen YL, Lin SP, Chen MR, Niu DM. Clinical features of EhlersDanlos syndrome. J Formos Med Assoc 2006;105:475e80. [62] Ho NCY, Tran JR, Bektas A. Marfan’s syndrome. Lancet 2005;366:1978e81. [63] Handford PA, Mayhew M, Brownlee GG. Calcium binding to fibrillin? Nature 1991;353:395. [64] Kohlmeier L, Gasner C, Bachrach LK, Marcus R. The bone mineral status of patients with Marfan syndrome. J Bone Miner Res 1995;10:1550e5.

493

[65] Kivirikko KI. Collagens and their abnormalities in a wide spectrum of diseases. Ann Med 1993;25:113e26. [66] Robinson PN, Arteaga-Solis E, Baldock C, et al. The molecular genetics of Marfan syndrome and related disorders. J Med Genet 2006;43:769e87. [67] Brooke BS, Habashi JP, Judge DP, Patel N, Loeys B, Dietz 3rd HC. Angiotensin II blockade and aortic-root dilation in Marfan’s syndrome. N Engl J Med 2008;358:2787e95. [68] De Paepe A, Devereux RB, Dietz HC, Hennekam RC, Pyeritz RE. Revised diagnostic criteria for the Marfan syndrome. Am J Med Genet 1996;62:417e26. [69] Loeys BL, Dietz HC, Braverman AC, et al. The revised Ghent nosology for the Marfan syndrome. J Med Genet 2010;47: 476e85. [70] Carter N, Duncan E, Wordsworth P. Bone mineral density in adults with Marfan syndrome. Rheumatol (Oxf) 2000;39:307e9. [71] Kohlmeier L, Gasner C, Marcus R. Bone mineral status of women with Marfan syndrome. Am J Med 1993;95:568e72. [72] Tobias JH, Dalzell N, Child AH. Assessment of bone mineral density in women with Marfan syndrome. Br J Rheumatol 1995;34:516e9. [73] Le Parc JM, Plantin P, Jondeau G, Goldschild M, Albert N, Boileau C. Bone mineral density in sixty adults with Marfan syndrome. Osteoporos Int 1999;10:475e9. [74] Giampietro PF, Peterson M, Schneider R, et al. Assessment of bone mineral density in adults and children with Marfan syndrome. Osteoporos Int 2003;14:559e63. [75] Moura B, Tubach F, Sulpice M, et al. Multidisciplinary Marfan Syndrome Clinic Group. Bone mineral density in Marfan syndrome. A large case-control study. Joint Bone Spine 2006;73: 733e5. [76] Grahame R, Pyeritz RE. The Marfan syndrome: joint and skin manifestations are prevalent and correlated. Br J Rheumatol 1995;34:126e31. [77] Mudd SH, Skovby F, Levy HL, et al. The natural history of homocystinuria due to cystathionine beta-synthase deficiency. Am J Hum Genet 1985;37:1e31. [78] Krumdieck CL, Prince CW. Mechanisms of homocysteine toxicity on connective tissues: implications for the morbidity of aging. J Nutr 2000;130:365Se8S. [79] Hubmacher D, Tiedemann K, Bartels R, et al. Modification of the structure and function of fibrillin-1 by homocysteine suggests a potential pathogenetic mechanism in homocystinuria. J Biol Chem 2005;280:34946e55. [80] Kim DJ, Koh JM, Lee O, et al. Homocysteine enhances apoptosis in human bone marrow stromal cells. Bone 2006;39:582e90. [81] Koh JM, Lee YS, Kim YS, et al. Homocysteine enhances bone resorption by stimulation of osteoclast formation and activity through increased intracellular ROS generation. J Bone Miner Res 2006;21:1003e11. [82] Herrmann M, Tami A, Wildemann B, et al. Hyperhomocysteinemia induces a tissue specific accumulation of homocysteine in bone by collagen binding and adversely affects bone. Bone 2009;44:467e75. [83] Azizi ZA, Zamani A, Omrani LR, et al. Effects of hyperhomocysteinemia during the gestational period on ossification in rat embryo. Bone 2010;46:1344e8. [84] Yap S, Naughten E. Homocystinuria due to cystathionine betasynthase deficiency in Ireland: 25 years’ experience of a newborn screened and treated population with reference to clinical outcome and biochemical control. J Inherit Metab Dis 1998;21:738e47. [85] Parrot F, Redonnet-Vernhet I, Lacombe D, Gin H. Osteoporosis in late-diagnosed adult homocystinuric patients. J Inherit Metab Dis 2000;23:338e40.

PEDIATRIC BONE

494

18. THE SPECTRUM OF PEDIATRIC OSTEOPOROSIS

[86] Picker JD, Levy HL. Homocystinuria caused by cystathionine beta-synthase deficiency. In: Pagon RA, Bird TC, Dolan CR, Stephens K, editors. GeneReviews [Internet]. Seattle: University of Washington, Seattle; 2006. p. 1993e2004 [updated 2006 Mar 29]. [87] Gahl WA, Bernardini I, Chen S, Kurtz D, Horvath K. The effect of oral betaine on vertebral body bone density in pyridoxinenon-responsive homocystinuria. J Inher Metab Dis 1998;11: 291e8. [88] Szafra nski T, Pawlak-Bus K, Leszczy nski P. Zoledronic acid in the treatment of secondary osteoporosis due to homocystinuria. Reumatologia 2010;48:133e8. [89] Dent CE, Friedman M. Idiopathic juvenile osteoporosis. Q J Med 1965;34:177e210. [90] Lorenc RS. Idiopathic juvenile osteoporosis. Calcif Tissue Int 2002;70:395e7. [91] Pludowski P, Lebiedowski M, Olszaniecka M, Marowska J, Matusik H, Lorenc RS. Idiopathic juvenile osteoporosis e an analysis of the muscle-bone relationship. Osteoporos Int 2006;17:1681e90. [92] Smith R. Idiopathic juvenile osteoporosis: experience of twentyone patients. Br J Rheumatol 1995;34:68e77. [93] Exner GU, Prader A, Elsasser U, Anliker M. Idiopathic osteoporosis in a three-year-old girl. Follow-up over a period of 6 years by computed tomography bone densitometry (CT). Helv Paediatr Acta 1984;39:517e28. [94] Villaverde V, De Inocencio J, Merino R, Garcia-Consuegra J. Difficulty walking. A presentation of idiopathic juvenile osteoporosis. J Rheumatol 1998;25:173e6. [95] Proszynska K, Wieczorek E, Olszaniecka M, Lorenc RS. Collagen peptides in osteogenesis imperfecta, idiopathic juvenile osteoporosis and Ehlers-Danlos syndrome. Acta Paediatr 1996;85:688e91. [96] Chlebna-Sokol D, Rusinska A. Serum insulin-like growth factor I, bone mineral density and biochemical markers of bone metabolism in children with idiopathic osteoporosis. Endocr Regul 2001;35:201e8. [97] Saggese G, Bertelloni S, Baroncelli GI, Perri G, Calderazzi A. Mineral metabolism and calcitriol therapy in idiopathic juvenile osteoporosis. Am J Dis Child 1991;145:457e62. [98] Rauch F, Travers R, Norman ME, Taylor A, Parfitt AM, Glorieux FH. The bone formation defect in idiopathic juvenile osteoporosis is surface-specific. Bone 2002;31:85e9. [99] Glorieux FH, Norman ME, Travers R, Taylor A. Idiopathic juvenile osteoporosis. In: Christiansen C, Rijs B, editors. Proceedings of the Fourth International Symposium in Osteoporosis and Consensus Development; 1993. p. 200e2. [100] Jackson EC, Strife CF, Tsang RC, Marder HK. Effect of calcitonin replacement therapy in idiopathic juvenile osteoporosis. Am J Dis Child 1988;142:1237e9. [101] Hoekman K, Papapoulos SE, Peters AC, Bijvoet OL. Characteristics and bisphosphonate treatment of a patient with juvenile osteoporosis. J Clin Endocrinol Metab 1985;61:952e6. [102] Shaw NJ, Boivin CM, Crabtree NJ. Intravenous pamidronate in juvenile osteoporosis. Arch Dis Child 2000;83:143e5. [103] Melchior R, Zabel B, Spranger J, Schumacher R. Effective parenteral clodronate treatment of a child with severe juvenile idiopathic osteoporosis. Eur J Pediatr 2005;164:22e7. [104] Koo WW, Chesney RW, Mitchell N. Case report: effect of pregnancy on idiopathic juvenile osteoporosis. Am J Med Sci 1995;309:223e5. [105] Black AJ, Reid R, Reid DM, MacDonald AG, Fraser WD. Effect of pregnancy on bone mineral density and biochemical markers of bone turnover in a patient with juvenile idiopathic osteoporosis. J Bone Miner Res 2003;18:167e71.

[106] Daly RM, Stenevi-Lundgren S, Linden C, Karlsson MK. Muscle determinants of bone mass, geometry and strength in prepubertal girls. Med Sci Sports Exerc 2008;40:1135e41. [107] Gunter K, Baxter-Jones AD, Mirwald RL, et al. Impact exercise increases BMC during growth: an 8-year longitudinal study. J Bone Miner Res 2008;23:986e93. [108] McKay HA, Petit MA, Schutz RW, Prior JC, Barr SI, Khan KM. Augmented trochanteric bone mineral density after modified physical education classes: a randomized school-based exercise intervention study in prepubescent and early pubescent children. J Pediatr 2000;136:156e62. [109] Nurmi-Lawton JA, Baxter-Jones AD, Mirwald RL, et al. Evidence of sustained skeletal benefits from impact-loading exercise in young females: a 3-year longitudinal study. J Bone Miner Res 2004;19:314e22. [110] McKay HA, MacLean L, Petit M, et al. "Bounce at the Bell": a novel program of short bouts of exercise improves proximal femur bone mass in early pubertal children. Br J Sports Med 2005;39:521e6. [111] Zhang P, Hamamura K, Yokota H. A brief review of bone adaptation to unloading. Genomics Proteom Bioinformat 2008;6: 4e7. [112] Krigger KW. Cerebral palsy: an overview. Am Fam Physician 2006;73:91e100. [113] Surveillance of Cerebral Palsy in Europe (SCPE). Prevalence and characteristics of children with cerebral palsy in Europe. Dev Med Child Neurol 2002;44:633e40. [114] Rosenbaum P, Paneth N, Leviton A, et al. A report: the definition and classification of cerebral palsy April 2006. Dev Med Child Neurol Suppl 2007;109:8e14. [115] Mergler S, Evenhuis HM, Boot AM, et al. Epidemiology of low bone mineral density and fractures in children with severe cerebral palsy: a systematic review. Dev Med Child Neurol 2009;51:773e8. [116] Downs J, Bebbington A, Woodhead H, et al. Early determinants of fractures in Rett syndrome. Pediatrics 2008;121:540e6. [117] Leet AI, Shirley ED, Barker C, Launay F, Sponseller PD. Treatment of femur fractures in children with cerebral palsy. J Child Orthop 2009;3:253e8. [118] Brunner R, Doderlein L. Pathological fractures in patients with cerebral palsy. J Pediatr Orthop B 1996;5:232e8. [119] Bischof. Basu D, Pettifor JM. Pathological long-bone fractures in residents with cerebral palsy in a long-term care facility in South Africa. Dev Med Child Neurol 2002;44:119e22. [120] Leet AI, Mesfin A, Pichard C, et al. Fractures in children with cerebral palsy. J Pediatr Orthop 2006;26:624e7. [121] Stevenson RD, Conaway M, Barrington JW, Cuthill SL, Worley G, Henderson RC. Fracture rate in children with cerebral palsy. Pediatr Rehabil 2006;9:396e403. [122] Presedo A, Dabney KW, Miller F. Fractures in patients with cerebral palsy. J Pediatr Orthop 2007;27:147e53. [123] Maruyama K, Nakamura K, Nashimoto M, et al. Bone fracture in physically disabled children attending schools for handicapped children in Japan. Environ Health Prev Med 2010;15: 135e40. [124] Henderson RC, Berglund LM, May R, et al. The relationship between fractures and DXA measures of BMD in the distal femur of children and adolescents with cerebral palsy or muscular dystrophy. J Bone Miner Res 2010;25:520e6. [125] Wilmshurst S, Ward K, Adams JE, Langton CM, Mughal MZ. Mobility status and bone density in cerebral palsy. Arch Dis Child 1996;75:164e5. [126] Tasdemir HA, Buyukavci M, Akcay F, Polat P, Yildiran A, Karakelleoglu C. Bone mineral density in children with cerebral palsy. Pediatr Int 2001;43:157e60.

PEDIATRIC BONE

495

REFERENCES

[127] Henderson RC, Kairalla J, Abbas A, Stevenson RD. Predicting low bone density in children and young adults with quadriplegic cerebral palsy. Dev Med Child Neurol 2004;46:416e9. [128] Cohen M, Lahat E, Bistritzer T, Livne A, Heyman E, Rachmiel M. Evidence-based review of bone strength in children and youth with cerebral palsy. J Child Neurol 2009;24: 959e67. [129] Houlihan CM, Stevenson RD. Bone density in cerebral palsy. Phys Med Rehabil Clin N Am 2009;20:493e508. [130] Chad KE, Bailey DA, McKay HA, Zello GA, Snyder RE. The effect of a weight-bearing physical activity program on bone mineral content and estimated volumetric density in children with spastic cerebral palsy. J Pediatr 1999;135:115e7. [131] Lin PP, Henderson RC. Bone mineralization in the affected extremities of children with spastic hemiplegia. Dev Med Child Neurol 1996;38:782e6. [132] Chad KE, McKay HA, Zello GA, Bailey DA, Faulkner RA, Snyder RE. Body composition in nutritionally adequate ambulatory and non-ambulatory children with cerebral palsy and a healthy reference group. Dev Med Child Neurol 2000;42: 334e9. [133] Ward KA, Caulton JM, Adams JE, Mughal MZ. Perspective: Cerebral palsy as a model of bone development in the absence of postnatal mechanical factors. J Musculoskelet Neuronal Interact 2006;6:154e9. [134] Henderson RC, Lin PP, Greene WB. Bone-mineral density in children and adolescents who have spastic cerebral palsy. J Bone Joint Surg Am 1995;77:1671e81. [135] Henderson RC. Vitamin D levels in noninstitutionalized children with cerebral palsy. J Child Neurol 1997;12:443e7. [136] Hahn TJ, Halstead LR. Anticonvulsant drug-induced osteomalacia: alterations in mineral metabolism and response to vitamin D3 administration. Calcif Tissue Int 1979;27:13e8. [137] Camfield CS, Delvin EE, Camfield PR, Glorieux FH. Normal serum 25-hydroxyvitamin D levels in phenobarbital-treated toddlers. Dev Pharmacol Ther 1983;6:157e61. [138] Rieger-Wettengl G, Tutlewski B, Stabrey A, et al. Analysis of the musculoskeletal system in children and adolescents receiving anticonvulsant monotherapy with valproic acid or carbamazepine. Pediatrics 2001;108:E107. [139] Tjellesen L, Gotfredsen A, Christiansen C. Effect of vitamin D2 and D3 on bone-mineral content in carbamazepine treated epileptic patients. Acta Neurol Scand 1983;68:424e8. [140] Feldkamp J, Becker A, Witte OW, Scharff D, Scherbaum WA. Long-term anticonvulsant therapy leads to low bone mineral density-evidence for direct drug effects of phenytoin and carbamazepine on human osteoblast-like cells. Exp Clin Endocrinol Diabetes 2000;108:37e43. [141] Souverein PC, Webb DJ, Petri H, Weil J, Van Staa TP, Egberts T. Incidence of fractures among epilepsy patients: a populationbased retrospective cohort study in the general practice research database. Epilepsia 2005;46:304e10. [142] Souverein PC, Webb DJ, Weil JG, Van Staa TP, Egberts AC. Use of antiepileptic drugs and risk of fractures: case-control study among patients with epilepsy. Neurology 2006;66:1318e24. [143] Fitzpatrick LA. Pathophysiology of bone loss in patients receiving anticonvulsant therapy. Epilepsy Behav 2004;5 (Suppl. 2):S3eS15. [144] Sheth RD, Wesolowski CA, Jacob JC, et al. Effect of carbamazepine and valproate on bone mineral density. J Pediatr 1995;127:256e62. [145] Sheth RD, Binkley N, Hermann BP. Gender differences in bone mineral density in epilepsy. Epilepsia 2008;49:125e31. [146] Harcke HT, Taylor A, Bachrach S, Miller F, Henderson RC. Lateral femoral scan: an alternative method for assessing bone

[147]

[148]

[149]

[150]

[151]

[152]

[153] [154]

[155]

[156]

[157]

[158]

[159] [160]

[161]

[162]

[163] [164]

[165]

mineral density in children with cerebral palsy. Pediatr Radiol 1998;28:241e6. Caulton JM, Ward KA, Alsop CW, Dunn G, Adams JE, Mughal MZ. A randomised controlled trial of standing programme on bone mineral density in non-ambulant children with cerebral palsy. Arch Dis Child 2004;89:131e5. Dodd KJ, Taylor NF, Damiano DL. A systemic review of the effectiveness of strengthening for people with cerebral palsy. Arch Phys Med Rehabil 2002;83:1157e64. Taylor NF, Dodd KJ, Damiano DL. Progressive resistance exercise in physical therapy: A summary of systematic reviews. Phys Ther 2005;85:1208e23. Ruck J, Chabo G, Rauch F. Vibration treatment in cerebral palsy: A randomized controlled pilot study. J Musculoskelet Neuronal Interact 2010;10:77e83. Stark C, Nikopoulou-Smyrni P, Stabrey A, Semler O, Schoenau E. Effect of a new physiotherapy concept on bone mineral density, muscle force and gross motor function in children with bilateral cerebral palsy. J Musculoskelet Neuronal Interact 2010;10:151e8. Jekovec-Vrhovsek M, Kocijancic A, Prezelj J. Effect of vitamin D and calcium on bone mineral density in children with CP and epilepsy in full-time care. Dev Med Child Neurol 2000;42:403e5. Shaw NJ, White CP, Fraser WD, Rosenbloom L. Osteopenia in cerebral palsy. Arch Dis Child 1994;71:235e8. Henderson R, Lark RK, Kecskemethy HH, Miller F, Harcke HT, Bachrach SJ. Bisphosphonates to treat osteopenia in children with quadriplegic cerebral palsy: a randomized, placebocontrolled clinical trial. J Pediatr 2002;141:644e51. Plotkin H, Coughlin S, Kreikemeier R, Heldt K, Bruzoni M. Low doses of pamidronate to treat osteopenia in children with severe cerebral palsy: a pilot study. Develop Med Child Neurol 2006;48: 709e12. Bachrach SJ, Kecskemethy HH, Harcke HT, Hossain J. Decreased fracture incidence after 1 year of pamidronate treatment in children with spastic quadriplegic cerebral palsy. Developl Med Child Neurol 2010;52:837e42. Ali O, Shim M, Fowler E, et al. Growth hormone therapy improves bone mineral density in children with cerebral palsy: A preliminary pilot study. J Clin Endocrinol Metab 2007;92: 932e7. Sugiyama T, Takaki T, Saito T, Taguchi T. Vitamin K therapy for cortical bone fragility caused by reduced mechanical loading in a child with hemiplegia. J Musculoskelet Neuronal Interact 2007;7:219e23. Deconinck N, Dan B. Pathophysiology of Duchenne muscular dystrophy: current hypotheses. Ped Neurol 2007;36:1e7. Kohler M, Clarenbach CF, Bahler C, Brack T, Russi EW, Bloch KE. Disability and survival in Duchenne muscular dystrophy. J Neurol Neurosurg Psychiatr 2009;80:320e5. Manzur AY, Kuntzer T, Pike M, Swan A. Glucocorticoid corticosteroids for Duchenne muscular dystrophy. Cochrane Database Syst Rev 2004;2:CD003725. Moxley III RT, Pandya S, Ciafaloni E, Fox DJ, Campbell K. Change in natural history of Duchenne muscular dystrophy with long-term corticosteroid treatment: implications for management. J Child Neurol 2010;25:1116e29. Siegel IM. Fractures of long bones in Duchenne muscular dystrophy. J Trauma 1977;17:219e22. So¨derpalm AC, Magnusson P, Ahlander, et al. Low bone mineral density and decreased bone turnover in Duchenne muscular dystrophy. Neuromuscul Disord 2007;17:919e28. Biggar WD, Harris VA, Eliasoph L, Alman B. Long-term benefits of deflazacort treatment for boys with Duchenne muscular

PEDIATRIC BONE

496

[166]

[167]

[168]

[169]

[170]

[171]

[172]

[173]

[174]

[175]

[176]

[177]

[178]

[179]

[180] [181]

[182]

[183] [184]

18. THE SPECTRUM OF PEDIATRIC OSTEOPOROSIS

dystrophy in their second decade. Neuromuscul Disord 2006;16: 249e55. Larson CM, Henderson RC. Bone mineral density and fractures in boys with Duchenne muscular dystrophy. J Pediatr Orthop 2000;20:71e4. Vestergaard P, Glerup H, Steffensen BF, Rejnmark L, Rahbek J, Moseklide L. Fracture risk in patients with muscular dystrophy and spinal muscular atrophy. J Rehabil Med 2001;33:150e5. McDonald DG, Kinali M, Gallagher AC, et al. Fracture prevalence in Duchenne muscular dystrophy. Dev Med Child Neurol 2002;44:695e8. King WM, Ruttencutter R, Nagaraja HN, et al. Orthopedic outcomes of long-term daily corticosteroid treatment in Duchenne muscular dystrophy. Neurology 2007;68:1607e13. Bothwell JE, Gordon KE, Dooley JM, MacSween J, Cummings EA, Salisbury S. Vertebral fractures in boys with Duchenne muscular dystrophy. Clin Pediatr 2003;42:353e6. Chabot G, Alos N, Brousseau Y, et al. Osteoporosis and fractures in children with Duchenne Muscular Dystrophy treated with glucocorticoids: a longitudinal study. J Bone Min Res 2002;17: F419. Crabtree NJ, Roper H, McMurchie H, Shaw NJ. Regional changes in bone area and bone mineral content in boys with Duchenne muscular dystrophy receiving corticosteroid therapy. J Pediatr 2010;156:450e5. Aparicio LF, Jurkovic M, DeLullo J. Decreased bone density in ambulatory patients with Duchenne muscular dystrophy. J Pediatr Orthop 2002;22:179e81. Bianchi ML, Mazzanti A, Galbiati E, et al. Bone mineral density and bone metabolism in Duchenne muscular dystrophy. Osteoporos Int 2003;14:761e7. Montgomery E, Pennington C, Isales CM, Hamrick MW. Muscle-bone interactions in dystrophic-deficient and myostatindeficient mice. Anat Rec 2005;286A:814e22. Ferraris JR, Pasqualini T, Legal S, et al. Effect of deflazacort versus methylprednisone on growth, body composition, lipid profile, and bone mass after renal transplantation. The Deflazacort Study Group. Pediatr Nephrol 2000;14:682e8. Chen YW, Nagaraju K, Bakay M, et al. Early onset of inflammation and later involvement of TGFbeta in Duchenne muscular dystrophy. Neurology 2005;27:826e34. Sun G, Haginoya K, Wu Y, et al. Connective tissue growth factor is overexpressed in muscles of human muscular dystrophy. J Neurol Sci 2008;267:48e56. Bushby K, Finkel R, Birnkrant DJ, et al. for the DMD Care Considerations Working Group. Diagnosis and management of Duchenne muscular dystrophy, part 2: implementation of multidisciplinary care. Lancet Neurol 2010;9:177e89. Davidson ZE, Truby H. A review of nutrition in Duchenne muscular dystrophy. J Hum Nutr Diet 2009;22:383e93. Bianchi ML, Morandi L, Andreucci E, Vai S, Frasunkiewicz J, Cottafava R. Low bone density and bone metabolism alterations in Duchenne muscular dystrophy: response to calcium and vitamin D. treatment. Osteoporos Int 2011;22:529e39. Hawker GA, Ridout R, Harris VA, Chase CC, Fielding LJ, Biggar WD. Alendronate in the treatment of low bone mass in steroid-treated boys with Duchenne’s muscular dystrophy. Arch Phys Med Rehabil 2005;86:284. Takata S, Yasui N. Disuse osteoporosis. J Med Invest 2001;48: 147e56. Zerwekh JE, Ruml LA, Gottschalk F, Pak CY. The effects of twelve weeks of bed rest on bone histology, biochemical markers of bone turnover, and calcium homeostasis in eleven normal subjects. J Bone Miner Res 1998;13:1594e601.

[185] Bloomfield SA. Changes in musculoskeletal structure and function with prolonged bed rest. Med Sci Sports Exerc 1997;29: 197e206. [186] Szalay EA, Harriman D, Eastlund B, Mercer D. Quantifying postoperative bone loss in children. J Pediatr Orthop 2008;28: 320e3. [187] Biering-Sorensen F, Bohr HH, Schaadt OP. Longitudinal study of bone mineral content in the lumbar spine, the forearm and the lower extremities after spinal cord injury. Eur J Clin Invest 1990;20:330e5. [188] Wood DE, Dunkerley AL, Tromans AM. Results from bone mineral density scans in twenty-two complete lesion paraplegics. Spinal Cord 2001;39:145e8. [189] Jones LM, Legge M, Goulding A. Intensive exercise may preserve bone mass of the upper limbs in spinal cord injured males but does not retard demineralisation of the lower body. Spinal Cord 2002;40:230e5. [190] Kiratli BJ. Immobilization osteopenia. In: Marcus R, Feldman D, Kelsey J, editors. 2nd ed. Osteoporosis vol 2, ch 46. Academic Press; 2001. p. 207e27. [191] McDonald CM, Abresch-Meyer AL, Dopler Nelson M, Widman LM. Body mass index and body composition measures by dual x-ray absorptiometry in patients aged 10 to 21 years with spinal cord injury. J Spinal Cord Med 2007;30:S97e104. [192] Lauer R, Johnston TE, Smith BT, Mulcahey MJ, Betz RR, Maurer AH. Bone mineral density of the hip and knee in children with spinal cord injury. J Spinal Cord Med 2007;30:S10e4. [193] Wang Y-C, Wang Y-H, Ting-Fang Shih T, Pan S-L, Huang T-S. Sublesional spinal vertebral bone mineral density correlates with neurological level and body mass index in individuals with chronic complete spinal cord injuries. Spine 2010;35: 958e62. [194] Kannisto M, Alaranta H, Merikanto J, Kroger H, Karkka¨inen J. Bone mineral status after pediatric spinal cord injury. Spinal Cord 1998;36:641e6. [195] Lo¨fvenmark I, Werhagen L, Norrbrink C. Spasticity and bone density after a spinal cord injury. J Rehabil Med 2009;41:1080e4. [196] Giangregorio L, McCartney N. Bone loss and muscle atrophy in spinal cord injury: epidemiology, fracture prediction, and rehabilitation strategies. J Spinal Cord Med 2006;29:489e500. [197] Alekna V, Tamulaitiene M, Sinevicius T, Juocevicius A. Effect of weight bearing activities on bone mineral density in spinal cord injured patients during the period of the first two years. Spinal Cord 2008;46:727e32. [198] Kunkel CF, Scremin AM, Eisenberg B, Garcia JF, Roberts S, Martinez S. Effect of standing on spasticity, contracture, and osteoporosis in paralyzed males. Arch Phys Med Rehabil 1993;74:73e8. [199] Ben M, Harvey L, Denis S, et al. Does 12 weeks of regular standing prevent loss of ankle mobility and bone mineral density in people with recent spinal cord injury? Aust J Phys Ther 2005;51:251e6. [200] Ashe MC, Eng JJ, Krassioukov AV, Warburton DER, Hung C, Tawashy A. Response to functional electrical stimulation cycling in women with spinal cord injuries using dual-energy x-ray absorptiometry and peripheral quantitative computed tomography: A case series. J Spinal Cord Med 2010;33:68e72. [201] Moran de Brito CM, Battistella LR, Saito ET, Sakamoto H. Effect of alendronate on bone mineral density in spinal cord injury patients: a pilot study. Spinal Cord 2005;43:341e8. [202] Nance PW, Schryvers O, Leslie W, Ludwig S, Krahn J, Uebelhart D. Intravenous pamidronate attenuates bone density loss after acute spinal cord injury. Arch Phys Med Rehabil 1999;80:243e51.

PEDIATRIC BONE

497

REFERENCES

[203] Chen B, Mechanick JI, Nierman DM, Stein A. Combined calcitriol-pamidronate therapy for bone hyperresorption in spinal cord injury. J Spinal Cord Med 2001;24:235e40. [204] Mechanick JI, Liu K, Nierman DM, Stein A. Effect of a convenient single 90-mg pamidronate dose on biochemical markers of bone metabolism in patients with acute spinal cord injury. J Spinal Cord Med 2006;29:406e12. [205] Gilchrist NL, Frampton CM, Acland RH, et al. Alendronate prevents bone loss in patients with acute spinal cord injury: A randomized, double-blind, placebo-controlled study. J Clin Endocrinol Metab 2007;92:1385e90. [206] Shapiro J, Smith B, Beck T, et al. Treatment with zoledronic acid ameliorates negative geometric changes in the proximal femur following acute spinal cord injury. Calcif Tissue Int 2007;80: 316e22. [207] Pui CH, Evans WE. Treatment of acute lymphoblastic leukemia. N Engl J Med 2006;354:166e78. [208] Strauss AJ, Su JT, Dalton VM, Gelber RD, Sallan SE, Silverman LB. Bony morbidity in children treated for acute lymphoblastic leukemia. J Clin Oncol 2001;19:3066e72. [209] Ho¨gler W, Wehl G, van Staa T, Meister B, Klein-Franke A, Kropshofer G. Incidence of skeletal complications during treatment of childhood acute lymphoblastic leukemia: comparison of fracture risk with the General Practice Research Database. Pediatr Blood Cancer 2007;48:21e7. [210] Halton JM, Atkinson SA, Fraher L, et al. Altered mineral metabolism and bone mass in children during treatment for acute lymphoblastic leukemia. J Bone Miner Res 1996;11: 1774e83. [211] Lequin MH, van der Sluis IM, van den Heuvel-Eibrink MM, et al. A longitudinal study using tibial ultrasonometry as a bone assessment technique in children with acute lymphoblastic leukaemia. Pediatr Radiol 2003;33:162e7. [212] van der Sluis IM, van den Heuvel-Eibrink MM, Ha¨hlen K, Krenning EP, de Muinck Keizer-Schrama SM. Altered bone mineral density and body composition, and increased fracture risk in childhood acute lymphoblastic leukemia. Pediatrics 2002;141:204e10. [213] Davies JH, Evans BA, Jenney ME, Gregory J-W. Skeletal morbidity in childhood acute lymphoblastic leukaemia. Clin Endocrinol (Oxf) 2005;63:1e9. [214] Strauss AJ, Su JT, Dalton VM, Gelber RD, Sallan SE, Silverman LB. Bony morbidity in children treated for acute lymphoblastic leukemia. J Clin Oncol 2001;19:3066e72. [215] Mussa A, Bertorello N, Porta F, et al. Prospective bone ultrasound patterns during childhood acute lymphoblastic leukemia treatment. Bone 2010;46:1016e20. [216] Hochberg Z. Mechanisms of steroid impairment of growth. Horm Res 2002;58(Suppl. 1):33e8. [217] Halton J, Gaboury I, Grant R, et al. Canadian STOPP Consortium. Advanced vertebral fracture among newly diagnosed children with acute lymphoblastic leukemia: results of the Canadian Steroid-Associated Osteoporosis in the Pediatric Population (STOPP) Research Program. J Bone Miner Res 2009;24:1326e34. [218] Brennan BM, Mughal Z, Roberts SA, et al. Bone mineral density in childhood survivors of acute lymphoblastic leukemia treated without cranial irradiation. J Clin Endocrinol Metab 2005;90: 689e94. [219] Jarfelt M, Fors H, Lannering B, Bjarnason R. Bone mineral density and bone turnover in young adult survivors of childhood acute lymphoblastic leukaemia. Eur J Endocrinol 2006;154: 303e9. [220] Benmiloud S, Steffens M, Beauloye V, et al. Long-term effects on bone mineral density of different therapeutic schemes for acute

[221]

[222]

[223]

[224]

[225]

[226]

[227]

[228]

[229]

[230] [231]

[232] [233]

[234]

[235]

[236]

[237]

[238]

lymphoblastic leukemia or non-Hodgkin lymphoma during childhood. Horm Res Paediatr 2010;74:241e50. Thomas IH, Donohue JE, Ness KK, Dengel DR, Baker KS, Gurney JG. Bone mineral density in young adult survivors of acute lymphoblastic leukemia. Cancer 2008;113:3248e56. Kadan-Lottick N, Marshall JA, Baron AE, Krebs NF, Hambidge KM, Albano E. Normal bone mineral density after treatment for childhood acute lymphoblastic leukemia diagnosed between 1991 and 1998. J Pediatr 2001;138:898e904. van der Sluis IM, van den Heuvel-Eibrink MM, Hahlen K, Krenning EP, de Muinck Keizer-Schrama SM. Bone mineral density, body composition, and height in long-term survivors of acute lymphoblastic leukemia in childhood. Med Pediatr Oncol 2000;35:415e20. Warner JT, Evans WD, Webb DK, Bell W, Gregory JW. Relative osteopenia after treatment for acute lymphoblastic leukemia. Pediatr Res 1999;45:544e51. Kaste SC, Rai SN, Fleming K, McCammon EA, Tylavsky FA, Danish RK. Changes in bone mineral density in survivors of childhood acute lymphoblastic leukemia. Pediatr Blood Cancer 2006;46:77e87. Haddy TB, Mosher RB, Reaman GH. Late effects in long-term survivors after treatment for childhood acute leukemia. Clin Pediatr 2009;48:601e8. Mandel K, Atkinson S, Barr RD, Pencharz P. Skeletal morbidity in childhood acute lymphoblastic leukemia. J Clin Oncol 2004;22:1215e21. Wasilewski-Masker K, Kaste SC, Hudson MM, Esiashvili N, Mattano LA, Meacham LR. Bone mineral density deficits in survivors of childhood cancer: long-term follow-up guidelines and review of the literature. Pediatrics 2008;121:e705e13. Children’s Oncology Group. Long-term follow-up guidelines for survivors of childhood, adolescent and young adult cancers, (2008). Version 3.0. Arcadia, CA: Children’s Oncology Group; October 2008; available on-line: http://www.survivorshipguidelines.org/ pdf/LTFUGuidelines.pdf. Lteif AN, Zimmerman D. Bisphosphonates for treatment of childhood hypercalcemia. Pediatrics 1998;102:990e3. Boudailliez BR, Pautard BJ, Sebert JL, Kremp O, Piussan CX. Leukaemia-associated hypercalcaemia in a 10-year-old boy: effectiveness of aminohydroxypropylidene biphosphonate. Pediatr Nephrol 1990;4:510e1. Sawhney S, Magalha˜es CS. Paediatric rheumatology e a global perspective. Best Pract Res Clin Rheumatol 2006;20:201e21. Romas E, Gillespie MT, Martin TJ. Involvement of receptor activator of NfkappaB ligand and tumor necrosis factor-alpha in bone destruction in rheumatoid arthritis. Bone 2002;30:340e6. Henderson CJ, Specker BL, Sierra RI, Campaigne BN, Lovell DJ. Total-body bone mineral content in non-corticosteroid treated postpubertal females with juvenile rheumatoid arthritis. Frequency of osteopenia and contributing factors. Arthritis Rheum 2000;43:531e40. Bianchi ML, Cimaz R, Galbiati E, Corona F, Cherubini R, Bardare M. Bone mass change during methotrexate treatment in patients with juvenile rheumatoid arthritis. Osteoporosis Int 1999;10:20e5. Uehara R, Suzuki Y, Ichikawa Y. Methotrexate (MTX) inhibits osteoblastic differentiation in vitro: possible mechanism of MTX osteopathy. J Rheumatol 2001;28:251e6. Stein B, Takisawa M, Katz J, et al. Salmon calcitonin prevents cyclosporin-A-induced high turnover bone loss. Endocrinology 1991;129:92e8. Thiebaud D, Krieg MA, Gillard-Berguer D, Jacquet AF, Goy JJ, Burckhardt P. Cyclosporine induces high bone turnover and

PEDIATRIC BONE

498

[239]

[240]

[241]

[242] [243]

[244]

[245]

[246]

[247]

[248]

[249]

[250]

[251]

[252]

[253]

[254]

[255]

18. THE SPECTRUM OF PEDIATRIC OSTEOPOROSIS

may contribute to bone loss after heart transplantation. Eur J Clin Invest 1996;26:549e55. Madsen OR, Sorensen OH, Egsmose C. Bone quality and bone mass as assessed by quantitative ultrasound and dual energy X-ray absorptiometry in women with rheumatoid arthritis: relationship with quadriceps strength. Ann Rheum Dis 2002;61: 325e9. Roth J, Linge M, Tzaribachev N, Schweizer R, KuemmerleDeschner J. Musculoskeletal abnormalities in juvenile idiopathic arthritis e a 4-year longitudinal study. Rheumatology 2007;46: 1180e4. Kotaniemi A, Savolainen A, Kroger H, Kautiainen H, Isomaki H. Weight-bearing physical activity, calcium intake, systemic glucocorticoids, chronic inflammation, and body constitution as determinants of lumbar and femoral bone mineral in juvenile chronic arthritis. Scand J Rheumatol 1999;28: 19e26. Rabinovich CE. Bone metabolism in childhood rheumatic disease. Rheum Dis Clin North Am 2002;28:655e67. Reed AM, Haugen M, Pachman LM, Langman CB. Repair of osteopenia in children with juvenile rheumatoid arthritis. J Pediatr 1993;122:693e6. Pepmueller PH, Cassidy JT, Allen SH, Hillman LS. Bone mineralization and bone mineral metabolism in children with juvenile rheumatoid arthritis. Arthritis Rheum 1996;39:746e57. Mul D, van Suijlekom-Smit LW, ten Cate R, Bekkering WP, de Muinck Keizer-Schrama SM. Bone mineral density and body composition and influencing factors in children with rheumatic diseases treated with corticosteroids. J Pediatr Endocrinol Metab 2002;15:187e92. Pereira RM, Corrente JE, Chahade WH, Yoshinari NH. Evaluation by dual X-ray absorptiometry (DXA) of bone mineral density in children with juvenile chronic arthritis. Clin Exp Rheumatol 1998;16:495e501. Kotaniemi A, Savolainen A, Kautiainen H, Kroger H. Estimation of central osteopenia in children with chronic polyarthritis treated with glucocorticoids. Pediatrics 1993;91:1127e30. Varonos S, Ansell BM, Reeve J. Vertebral collapse in juvenile chronic arthritis: its relationship with glucocorticoid therapy. Calcif Tissue Int 1987;41:75e8. Ma¨enpa¨a¨ H, Savolainen A, Lehto MUK, Belt EA. Multiple stress fractures in a young girl with chronic idiopathic arthritis. Extended case report. Joint Bone Spine 2001;68:432e8. Murray K, Boyle RJ, Woo-London P. Pathological fractures and osteoporosis in a cohort of 103 systemic onset juvenile rheumatoid arthritis. Arthritis Rheum 2000;43(Suppl):S119. Elsasser U, Wilkins B, Hesp R, Thurnham DI, Reeve J, Ansell BM. Bone rarefaction and crash fractures in juvenile chronic arthritis. Arch Dis Child 1982;57:377e80. Burnham JM, Shults J, Weinstein R, Lewis JD, Leonard MB. Childhood onset arthritis is associated with an increased risk of fracture: a population based study using the General Practice Research Database. Ann Rheum Dis 2006;65:1074e9. Njeh CF, Shaw N, Gardner-Medwin JM, Boivin CM, Southwood TR. Use of quantitative ultrasound to assess bone status in children with juvenile arthritis: a pilot study. J Clin Densit 2000;3:251e60. Lettgen B, Neudorf U, Hosse R, Peters S, Reiners C. Bone density in children and adolescents with rheumatic diseases. Preliminary results of selective measurement of trabecular and cortical bone using peripheral computerized tomography. Klin Padiatr 1996;208:114e7. Brik R, Keidar Z, Schapira D, Israel O. Bone mineral density and turnover in children with systemic juvenile chronic arthritis. J Rheumatol 1998;25:990e2.

[256] Lien G, Flatø B, Haugen M, et al. Frequency of osteopenia in adolescents with early-onset juvenile idiopathic arthritis e a long-term outcome study of one hundred five patients. Arth Rheum 2003;48:2214e23. [257] Hillman LS, Cassidy JT, Johnson L, Lee D, Allen S. Vitamin D metabolism and bone mineralization in children with juvenile rheumatoid arthritis. J Pediatr 1994;124:910e6. [258] Reed A, Haugen M, Pachman LM, Langman CB. Abnormalities in serum osteocalcin values in children with chronic rheumatic diseases. J Pediatr 1990;116:574e80. [259] Hopp R, Degan JA, Gallagher JC, Cassidy JT. Estimation of bone mineral density in children with juvenile rheumatoid arthritis. J Rheumatol 1991;18:1235e9. [260] Lurati A, Cimaz R, Gattinara M, et al. Relationship between delayed menarche and bone density in patients affected by juvenile idiopathic arthritis. Reumatismo 2008;60:224e9. [261] Henderson CJ, Cawkwell GD, Specker BLY. Predictors of total body bone mineral density in non-corticosteroid-treated prepubertal children with juvenile rheumatoid arthritis. Arthritis Rheum 1997;40:1967e75. [262] Zak M, Hassager C, Lovell DJY. Assessment of bone mineral density in adults with a history of juvenile chronic arthritis: a cross sectional long-term follow-up study. Arthritis Rheum 1999;42:790e8. [263] French AR, Mason T, Nelson AM, et al. Osteopenia in adults with a history of juvenile rheumatoid arthritis. A population based study. J Rheumatol 2002;29:1065e70. [264] Warady BD, Lindsley CB, Robinson FG, Lukert BP. Effects of nutritional supplementation on bone mineral status of children with rheumatic diseases receiving corticosteroid therapy. J Rheumatol 1994;21:530e5. [265] Reed A, Haugen M, Pachman LM, Langman CB. 25-Hydroxyvitamin D therapy in children with active juvenile rheumatoid arthritis: short-term effects on serum osteocalcin levels and bone mineral density. J Pediatr 1991;119:657e60. [266] Lovell DJ, Glass D, Ranz J, et al. A randomized controlled trial of calcium supplementation to increase bone mineral density in children with juvenile rheumatoid arthritis. Arthr Rheum 2006;54:2235e42. [267] Carrasco R, Lovell DJ, Giannini EH, et al. Biochemical markers of bone turnover associated with calcium supplementation in children with juvenile rheumatoid arthritis: results of a doubleblind, placebo-controlled intervention trial. Arthritis Rheum 2008;58:3932e40. [268] Hillman LS, Cassidy JT, Chanetsa F, Hewett JE, Higgins BJ, Robertson JD. Percent true calcium absorption, mineral metabolism, and bone mass in children with arthritis: effect of supplementation with vitamin D3 and calcium. Arthritis Rheum 2008;58:3255e63. [269] Falcini F, Trapani S, Ermini M, Brandi ML. Intravenous administration of alendronate counteracts the in vivo effects of glucocorticoids on bone remodeling. Calcif Tissue Int 1996;58: 166e9. [270] Lepore L, Pennesi M, Barbi E, Pozzi R. Treatment and prevention of osteoporosis in juvenile chronic arthritis with disodium clodronate. Clin Exp Rheumatol 1991;9(Suppl. 6):33e5. [271] Brumsen C, Hamdy NAT, Papapoulos SE. Long-term effects of bisphosphonates on the growing skeleton: studies of young patients with severe osteoporosis. Medicine (Baltimore) 1997;76: 226e83. [272] Bianchi ML, Cimaz R, Bardare M, et al. Efficacy and safety of alendronate for the treatment of osteoporosis in diffuse connective tissue diseases in children: a prospective multicenter study. Arthritis Rheum 2000;43:1960e6.

PEDIATRIC BONE

499

REFERENCES

[273] Thornton J, Ashcroft DM, Mughal MZ, Elliott RA, O’Neill TW, Symmons D. Systematic review of effectiveness of bisphosphonates in treatment of low bone mineral density and fragility fractures in juvenile idiopathic arthritis. Arch Dis Child 2006;91: 753e61. [274] Touati G, Ruiz JC, Porquet D, Kindermans C, Prewar AM, Czernichow P. Effects on bone metabolism of one year recombinant human growth hormone administration to children with juvenile chronic arthritis undergoing chronic steroid therapy. J Rheumatol 2000;27:1287e93. [275] Rooney M, Davies UM, Reeve J, Preece M, Ansell BM, Woo PM. Bone mineral content and bone mineral metabolism: changes after growth hormone treatment in juvenile chronic arthritis. J Rheumatol 2000;27:1073e81. [276] Simon D, Prieur AM, Quartier P, Charles Ruiz J, Czernichow P. Early recombinant human growth hormone treatment in glucocorticoid-treated children with juvenile idiopathic arthritis: a 3-year randomized study. J Clin Endocrinol Metab 2007;92:2567e73. [277] Bechtold S, Dalla Pozza R, Schwarz HP, Simon D. Effects of growth hormone treatment in juvenile idiopathic arthritis: bone and body composition. Horm Res 2009;72(Suppl. 1):60e4. [278] Siamopoulou A, Challa A, Kapoglou P, Cholevas V, Mavridis AK, Lapatsanis PD. Effects of intranasal salmon calcitonin in juvenile idiopathic arthritis: an observational study. Calcif Tissue Int 2001;69:25e30. [279] Simonini G, Giani T, Stagi S, de Martino M, Falcini F. Bone status over 1 yr of etanercept treatment in juvenile idiopathic arthritis. Rheumatology 2005;44:777e80. [280] Roth J, Bechtold S, Borte G, Dressler F, Girschick HJ, Borte M. Osteoporosis in juvenile idiopathic arthritis e a practical approach to diagnosis and therapy. Eur J Pediatr 2007;166: 775e84. [281] Castro TC, Terreri MT, Szejnfeld VL, et al. Bone mineral density in juvenile systemic lupus erythematosus. Braz J Med Biol Res 2002;35:1159e63. [282] Trapani S, Civinini R, Ermini M, Paci E, Falcini F. Osteoporosis in juvenile systemic lupus erythematosus: a longitudinal study on the effects of steroids on bone mineral density. Rheumatol Int 1998;18:45e9. [283] Ozaki D, Shirai Y, Nakayama Y, Yoshihara K, Huzita T. Multiple fish vertebra deformity in child with systemic lupus erythomatosus: a case report. J Nippon Med Sch 2000;67:271e4. [284] Ellis KJ, Shypailo RJ, Hardin DS, et al. Z score prediction model for assessment of bone mineral content in pediatric diseases. J Bone Miner Res 2001;16:1658e64. [285] Stewart WA, Acott PD, Salisbury SR, Lang B. Bone mineral density in juvenile dermatomyositis: assessment using dual Xray absorptiometry. Arthritis Rheum 2003;48:2294e8. [286] Walters JRF. The role of the intestine in bone homeostasis. Eur J Gastroenterol Hepatol 2003;15:845e9. [287] Shen F, Plamondon MJ, Plamondon P, Gaffen SL. Cytokines link osteoblasts and inflammation: microarray analysis of interleukin-17- and TNF-alpha-induced genes in bone cells. J Leukoc Biol 2005;77:388e99. [288] Bianchi ML. Glucorticoids and bone: some general remarks and some special observations in pediatric patients. Calcif Tissue Int 2002;70:384e90. [289] Søe K, Delaisse´ JM. Glucocorticoids maintain human osteoclasts in the active mode of their resorption cycle. J Bone Miner Res 2010;25:2184e92. [290] Shikhare G, Kugathasan S. Inflammatory bowel disease in children: current trends. J Gastroenterol 2010;45:673e82. [291] Sandhu BK, Fell JME, Beattie RM, Mitton SG, Jenkins H. On behalf of the UK IBD Working Group Guidelines for the

[292] [293]

[294]

[295]

[296]

[297]

[298]

[299]

[300]

[301]

[302]

[303]

[304]

[305]

[306]

[307]

[308]

[309]

management of inflammatory bowel disease (IBD) in children in the United Kingdom. J Pediatr Gastroenterol Nutr 2010;50: S1e13. Engel MA, Neurath MF. New pathophysiological insights and modern treatment of IBD. J Gastrenterol 2010;453:571e83. Sylvester FA, Wyzga N, Hyams JS, Gronowicz GA. Effect of Crohn’s disease on bone metabolism in vitro: a role for interleukin-6. J Bone Miner Res 2002;17:695e702. Varghese S, Wyzga N, Griffiths AM, Sylvester FA. Effects of serum from children with newly diagnosed Crohn disease on primary cultures of rat osteoblasts. J Pediatr Gastroenterol Nutr 2002;35:641e8. Pappa HM, Grand RJ, Gordon CM. Report on the vitamin D status of adult and pediatric patients with inflammatory bowel disease and its significance for bone health and disease. Inflamm Bowel Dis 2006;12:1162e74. Gupta A, Paski S, Issenman R, Webber C. Lumbar spine bone mineral density at diagnosis and during follow-up in children with IBD. J Clin Densitom 2004;7:290e5. Paganelli M, Albanese C, Borrelli O, et al. Inflammation is the main determinant of low bone mineral density in pediatric inflammatory bowel disease. Inflamm Bowel Dis 2007;13: 416e23. Ward LM, Rauch F, Matzinger MA, Benchimol EI, Boland M, Mack DR. Iliac bone histomorphometry in children with newly diagnosed inflammatory bowel disease. Osteoporos Int 2010;21: 331e7. Bechtold S, Alberer M, Arenz T, et al. Reduced muscle mass and bone size in pediatric patients with inflammatory bowel disease. Inflamm Bowel Dis 2010;16:216e25. Walther F, Fusch C, Radke M, Beckert S, Findeisen A. Osteoporosis in pediatric patients suffering from chronic inflammatory bowel disease with and without steroid treatment. J Ped Gastroenterol Nutr 2006;43:42e51. Schmidt S, Mellstro¨m D, Norjavaara E, Sundh SV, Saalman R. Low bone mineral density in children and adolescents with inflammatory bowel disease: A population-based study from Western Sweden. Inflamm Bowel Dis 2009;15:1844e50. Lopes LH, Sdepanian VL, Szejnfeld VL, de Morais MB. Fagundes-Neto U. Risk factors for low bone mineral density in children and adolescents with inflammatory bowel disease. Dig Dis Sci 2008;53:2746e53. Sylvester FA, Wyzga N, Hyams JS, et al. Natural history of bone metabolism and bone mineral density in children with inflammatory bowel disease. Inflamm Bowel Dis 2007;13:42e50. El-Matary W, Sikora S, Spady D. Bone mineral density, vitamin D, and disease activity in children newly diagnosed with inflammatory bowel disease. Dig Dis Sci 2011;56:825e9. Jose FA, Garnett EA, Vittinghoff E, et al. Development of extraintestinal manifestations in pediatric patients with inflammatory bowel disease. Inflamm Bowel Dis 2009;15:63e8. Burnham JM, Shults J, Semeao E, et al. Whole body BMC in pediatric Crohn’s disease: independent effects of altered growth, maturation and body composition. J Bone Miner Res 2004;19:1961e8. Sentongo TA, Semaeo EJ, Stettler N, Piccoli DA, Stallings VA, Zemel BS. Vitamin D status in children, adolescents, and young adults with Crohn disease. Am J Clin Nutr 2002;76:1077e81. Pappa HM, Gordon CM, Saslowsky TM, et al. Vitamin D status in children and young adults with inflammatory bowel disease. Pediatrics 2006;118:1950e61. Bernstein CN, Blanchard JF, Leslie W, Wajda A, Yu BN. The incidence of fracture among patients with inflammatory bowel disease: A population-based cohort study. Ann Int Med 2000;133:795e9.

PEDIATRIC BONE

500

18. THE SPECTRUM OF PEDIATRIC OSTEOPOROSIS

[310] Hill RJ, Brookes DS, Davies PSW. Bones in pediatric Crohn’s disease: a review of fracture risk in children and adults. Inflamm Bowel Dis 2011;17:1223e8. [311] Kappelman MD, Galanko JA, Porter CQ, Sandler RS. Risk of diagnosed fractures in children with inflammatory bowel diseases. Inflamm Bowel Dis 2011;17:1125e30. [312] Persad R, Jaffer I, Issenman RM. The prevalence of long bone fractures in pediatric inflammatory bowel disease. J Pediatr Gastroenterol Nutr 2006;43:597e602. [313] Thearle M, Horlick M, Bilezikian JP, et al. Osteoporosis: an unusual presentation of childhood Crohn’s disease. J Clin Endocrinol Metab 2000;85:2122e6. [314] Semeao EJ, Stallings VA, Peck SN, Piccoli DA. Vertebral compression fractures in pediatric patients with Crohn’s disease. Gastroenterology 1997;112:1710e3. [315] American Gastroenterological Association. American Gastroenterological Association medical position statement: guidelines on osteoporosis in gastrointestinal diseases. Gastroenterology 2003;124:791e4. [316] Heyman MB, Garnett EA, Wojcicki J, et al. Growth hormone treatment for growth failure in pediatric patients with Crohn’s disease. J Pediatr 2008;153:651e8. [317] Thayu M, Leonard MB, Hyams JS, et al. Reach Study Group. Improvement in biomarkers of bone formation during infliximab therapy in pediatric Crohn’s disease: results of the REACH study. Clin Gastroenterol Hepatol 2008;6:1378e84. [318] Dube´ C, Rostom A, Sy R, et al. The prevalence of celiac diseases in average-risk and at-risk Western European populations: a systematic review. Gastroenterology 2005;128:S57e67. [319] Fasano A, Catassi C. Coeliac disease in children. Best Pract Res Clin Gastrenterol 2005;19:467e78. [320] Mora S, Weber G, Barera G, et al. Effect of gluten-free diet on bone mineral content in growing patients with celiac disease. Am J Clin Nutr 1993;57:224e8. [321] Scotta MS, Salvatore S, Salvatoni A, et al. Bone mineralization and body composition in young patients with celiac disease. Am J Gastroenterol 1997;92:1331e4. [322] Barera G, Mora S, Brambilla P, et al. Body composition in children with celiac disease and the effects of a gluten-free diet: a prospective case-control study. Am J Clin Nutr 2000;72:71e5. [323] Sdepanian VJ, De Miranda Carvalho CN, De Morais B, Colugnati FA, Fagundes-Neto U. Bone mineral density of the lumbar spine in children and adolescents with celiac disease on a gluten-free diet in Sao Paulo, Brazil. J Ped Gastroenterol Nutr 2003;37:571e6. [324] Corazza GR, Di Sario A, Sacco G, et al. Subclinical coeliac disease: an anthropometric assessment. J Int Med 1994;236: 183e7. [325] Jansson UHG, Kristiansson B, Magnusson P, Larsson L, Albertsson-Wikland K, Bjarnason R. The decrease of IGF-I, IGFbinding protein-3 and bone alkaline phosphatase isoforms during gluten challenge correlates with small intestinal inflammation in children with coeliac disease. Eur J Endocrinol 2001;144:417e23. [326] Bozzola M, Giovenale D, Bozzola E, et al. Growth hormone deficiency and coeliac disease: an unusual association? Clin Endocrinol 2005;62:372e5. [327] Hansen D, Bennedbaeck FN, Hansen LK, et al. High prevalence of celiac disease in Danish children with type I diabetes mellitus. Acta Paediatr 2001;90:1238e43. [328] Diniz-Santos DR, Brandao FR, Adan L, Moreira A, Vicente EJ, Silva LR. Bone mineralization in young patients with type 1 diabetes mellitus and screening-identified evidence of celiac disease. Dig Dis Sci 2008;53:1240e5.

[329] Hamrick MW, Ferrari SL. Leptin and the sympathetic connection of fat to bone. Osteoporos Int 2008;19:905e12. [330] Ertekin V, Orbak Z, Selimoglu MA, Yildiz L. Serum leptin levels in childhood celiac disease. J Clin Gastroenterol 2006;40:906e9. [331] Olmos M, Antelo M, Vazquez H, Smecuol E, Maurin˜o E, Bai JC. Systematic review and meta-analysis of observational studies on the prevalence of fractures in coeliac disease. Dig Liver Dis 2008;40:46e53. [332] Ludvigsson JF, Michaelsson K, Ekbom A, Montgomery SM. Coeliac disease and the risk of fractures e a general populationbased cohort study. Aliment Pharmacol Ther 2007;25:273e85. [333] Barera G, Beccio S, Proverbio MC, Mora S. Longitudinal changes in bone metabolism and bone mineral content in children with celiac disease during consumption of a gluten-free diet. Am J Clin Nutr 2004;79:148e54. [334] Molteni N, Caraceni MP, Bardella MT, Ortolani S, Gandolini GG, Bianchi P. Bone mineral density in adult celiac patients and the effect of gluten-free diet from childhood. Am J Gastroenterol 1990;85:51e3. [335] Challa A, Moulas A, Cholevas V, Karastergiou E, Lapatsanis D, Tsianos E. Vitamin D metabolites in patients with coeliac disease. Eur J Pediatr 1998;157:262e3. [336] Tau C, Mautalen C, De Rosa S, Roca A, Valenzuela X. Bone mineral density in children with celiac disease. Effect of a gluten-free diet. Eur J Clin Nutr 2006;60:358e63. [337] Motta ME, Faria ME, Silva GA. Prevalence of low bone mineral density in children and adolescents with celiac disease under treatment. Sao Paulo Med J 2009;127:278e82. [338] Cellier C, Flobert C, Cormier C, Roux C, Schmitz J. Severe osteopenia in symptom-free adults with a childhood diagnosis of coeliac disease. Lancet 2000;355:806. [339] Walters RF. Bone mineral density in coeliac disease. Gut 1994;35: 150e1. [340] Valdimarsson T, Lo¨fman O, Toss GM, Stro¨m M. Reversal of osteopenia with diet in adult coeliac disease. Gut 1996;38:322e7. [341] Muzzo S, Burrows R, Burguen˜o M, et al. Effect of calcium and vitamin D supplementation on bone mineral density of celiac children. Nutr Res 2000;20:1241e7. [342] Hepner GW, Jowsey J, Arnaud C, et al. Osteomalacia and celiac disease: response to 25-hydroxyvitamin D. Am J Med 1978;65: 1015e20. [343] Oberhuber G, Caspary WF, Kirchner T, Borchard F, Stolte M. German Society for Pathology Task Force on Gastroenterologic Pathology. Diagnosis of celiac disease and sprue. Recommendations of the German Society for Pathology Task Force on Gastroenterologic Pathology. Pathologe 2001;22:72e81. [344] Hill ID, Dirks MH, Liptak GS, et al. North American Society for Pediatric Gastroenterology, Hepatology and Nutrition. Guideline for the diagnosis and treatment of celiac disease in children: recommendations of the North American Society for Pediatric Gastroenterology, Hepatology and Nutrition. J Pediatr Gastroenterol Nutr 2005;40:1e19. [345] Mehta G, Macek Jr M, Mehta A. on behalf of the European Registry Working Group. Cystic fibrosis across Europe: EuroCareCF analysis of demographic data from 35 countries. J Cyst Fibros 2010;9(suppl. 2):S5e21. [346] King SJ, Topliss DJ, Kotsimbos T, et al. Reduced bone density in cystic fibrosis: deltaF508 mutation is an independent risk factor. Eur Respir J 2005;25:54e61. [347] Aris RM, Guise TA. Cystic fibrosis and bone disease: are we missing a genetic link? Eur Respir J 2005;25:9e11. [348] Dif F, Marty C, Baudoin C, de Vernejoul MC, Levi G. Severe osteopenia in CFTR-null mice. Bone 2004;35:595e603. [349] Pashuck TD, Franz SE, Altman MK, et al. Murine model for cystic fibrosis bone disease demonstrates osteopenia and sex-

PEDIATRIC BONE

REFERENCES

[350]

[351]

[352]

[353]

[354]

[355] [356]

[357] [358]

[359]

[360]

[361]

[362]

[363]

[364]

[365]

[366] [367]

related differences in bone formation. Pediatr Res 2009;65: 311e6. Haston CK, Li W, Li A, Lafleur M, Henderson JE. Persistent osteopenia in adult cystic fibrosis transmembrane conductance regulator-deficient mice. Am J Respir Crit Care Med 2008;177: 309e15. Shead EF, Haworth CS, Condliffe AM, McKeon DJ, Scott MA, Compston JE. Cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in human bone. Thorax 2007;62: 650e1. Shead EF, Haworth CS, Barker H, Bilton D, Compston JE. Osteoclast function, bone turnover and inflammatory cytokines during infective exacerbations of cystic fibrosis. J Cyst Fibros 2010;9:93e8. Le Heron L, Guillaume C, Velard F, et al. Cystic fibrosis transmembrane conductance regulator (CFTR) regulates the production of osteoprotegerin (OPG) and prostaglandin (PG) E2 in human bone. J Cyst Fibros 2010;9:69e72. Bianchi ML, Romano G, Saraifoger S, Costantini D, Limonta C, Colombo C. BMD and body composition in children and young patients affected by cystic fibrosis. J Bone Miner Res 2006;21: 388e96. Haworth CS. Impact of cystic fibrosis on bone health. Curr Opin Pulm Med 2010;16:616e22. Elkin SL, Fairney A, Burnett S, et al. Vertebral deformities and low bone mineral density in adults with cystic fibrosis: a crosssectional study. Osteoporos Int 2001;12:366e72. Bachrach LK, Loutit CW, Moss RB. Osteopenia in adults with cystic fibrosis. Am J Med 1994;96:27e34. Aris RM, Renner JB, Winders AD, et al. Increased rate of fractures and severe kyphosis: sequelae of living into adulthood with cystic fibrosis. Ann Intern Med 1998;128:186e93. Papaioannou A, Kennedy CC, Freitag A, et al. Longitudinal analysis of vertebral fracture and BMD in a Canadian cohort of adult cystic fibrosis patients. BMC Musculoskelet Disord 2008;19:125. Rossini M, Del Marco A, Dal Santo F, et al. Prevalence and correlates of vertebral fractures in adults with cystic fibrosis. Bone 2004;35:771e6. Aris RM, Merkel PA, Bachrach LK, et al. Consensus statement: Guide to bone health and disease in cystic fibrosis. J Clin Endocrinol Metab 2005;90:1888e96. Paccou J, Zeboulon N, Combescure C, Gossec L, Cortet B. The prevalence of osteoporosis, osteopenia, and fractures among adults with cystic fibrosis: a systematic literature review with metaanalysis. Calcif Tissue Int 2010;86:1e7. Conway SP, Oldroyd B, Brownlee KG, Wolfe SP, Truscott JG. A cross-sectional study of bone mineral density in children and adolescents attending a Cystic Fibrosis Centre. J Cyst Fibros 2008;7:469e76. Lucidi V, Bizzarri C, Alghisi F, et al. Bone and body composition analyzed by dual-energy X-ray absorptiometry (DXA) in clinical and nutritional evaluation of young patients with cystic fibrosis: a cross-sectional study. BMC Pediatr 2009;9:61. Rovner AJ, Zemel BS, Leonard MB, Schall JI, Stallings VA. Mild to moderate cystic fibrosis is not associated with increased fracture risk in children and adolescents. J Pediatr 2005;147: 327e31. Henderson RC, Specter BB. Kyphosis and fractures in children and young adults with cystic fibrosis. J Pediatr 1994;125:208e12. Buntain HM, Greer RM, Schluter PJ, et al. Bone mineral density in Australian children, adolescents, and adult with cystic fibrosis: a controlled cross sectional study. Thorax 2004;59: 149e55.

501

[368] Sermet-Gaudelus I, Castanet M, Retsch-Bogart G, Aris RM. Update on cystic fibrosis-related bone disease: a special focus on children. Paediatr Respir Rev 2009;10:134e42. [369] O’Reilly R, Fitzpatrick P, Leen G, Elnazir B, Greally P. Severe bone demineralisation is associated with higher mortality in children with cystic fibrosis. Ir Med J 2009;102:47e9. [370] Douros K, Loukou I, Nicolaidou P, Tzonou A, Doudounakis S. Bone mass density and associated factors in cystic fibrosis patients of young age. J Paediatr Child Health 2008;44:681e5. [371] Kelly A, Schall JI, Stallings VA, Zemel BS. Deficits in bone mineral content in children and adolescents with cystic fibrosis are related to height deficits. J Clin Densitom 2008;11:581e9. [372] Buntain HM, Schluter PJ, Bell SC, et al. Controlled longitudinal study of bone mass accrual in children and adolescents with cystic fibrosis. Thorax 2006;61:146e54. [373] Hardin DS, Arumugam R, Seilheimer DK, LeBlanc A, Ellis KJ. Normal bone mineral density in cystic fibrosis. Arch Dis Child 2001;84:363e8. [374] Grey V, Atkinson S, Drury D, et al. Prevalence of low bone mass and deficiencies of vitamins D and K in pediatric patients with cystic fibrosis from 3 Canadian centers. Pediatrics 2008;122: 1014e20. [375] Cobanoglu N, Atasoy H, Ozcelik U, et al. Relation of bone mineral density with clinical and laboratory parameters in prepubertal children with cystic fibrosis. Pediatr Pulmonol 2009;44: 706e12. [376] Gronowitz E, Mellstro¨m D, Strandvik B. Normal annual increase of BMD during two years in patients with cystic fibrosis. Pediatrics 2004;114:435e42. [377] Schulze KJ, O’Brien KO, Germain-Lee EL, Booth SL, Leonard A, Rosenstein BJ. Calcium kinetics are altered in clinically stable girls with cystic fibrosis. J Clin Endocrinol Metab 2004;89: 3385e91. [378] Haworth CS, Selby PL, Horrocks AW, Mawer EB, Adams JE, Webb AK. A prospective study of change in bone mineral density over one year in adults with cystic fibrosis. Thorax 2002;57:719e23. [379] Pedreira CC, Robert RG, Dalton V, et al. Association of body composition and lung function in children with cystic fibrosis. Pediatr Pulmonol 2005;39:276e80. [380] Frangolias DD, Pare` PD, Kendler DL, et al. Role of exercise and nutrition status on bone mineral density in cystic fibrosis. J Cyst Fibros 2003;2:163e70. [381] Hall WB, Sparks AA, Aris RM. Vitamin D deficiency in cystic fibrosis. Int J Endocrinol 2010. DOI:10.1155/2010/218691. [382] Sermet-Gaudelus I, Nove-Josserand R, Loeille GA, et al. Fe´de´ration franc¸aise des centres de ressource et de compe´tence en mucoviscidose. Recommandations pour la prise en charge de la de´mine´ralisation osseuse dans la mucoviscidose. [Recommendations for the management of bone demineralization in cystic fibrosis]. Arch Pediatr 2008;15:301e12. [383] Arenas-de Larriva MS, Vaquero-Barrios JM, Redel-Montero J, Santos-Luna F. Bone mineral density in lung transplant candidates. Transpl Proc 2010;42:3208e10. [384] Hind K, Truscott JG, Conway SP. Exercise during childhood and adolescence: a prophylaxis against cystic fibrosis-related low bone mineral density? Exercise for bone health in children with cystic fibrosis. J Cyst Fibros 2008;7:270e6. [385] Hillman LS, Cassidy JT, Popescu MF, Hewett JE, Kyger J, Robertson JD. Percent true calcium absorption, mineral metabolism, and bone mineralization in children with cystic fibrosis: effect of supplementation with vitamin D and calcium. Pediatr Pulmonol 2008;43:772e80. [386] Ferguson JH, Chang AB. Vitamin D supplementation for cystic fibrosis. Cochrane Database Syst Rev 2009;7:CD007298.

PEDIATRIC BONE

502

18. THE SPECTRUM OF PEDIATRIC OSTEOPOROSIS

[387] Green DM, Leonard AR, Paranjape SM, Rosenstein BJ, Zeitlin PL, Mogayzel Jr PJ. Transient effectiveness of vitamin D2 therapy in pediatric cystic fibrosis patients. J Cys Fibros 2010;9: 143e9. [388] Nicolaidou P, Stavrinadis I, Loukou I, et al. The effect of vitamin K supplementation on biochemical markers of bone formation in children and adolescents with cystic fibrosis. Eur J Pediatr 2006;165:540e5. [389] Hardin DS, Adams-Huet B, Brown D, et al. Growth hormone treatment improves growth and clinical status in prepubertal children with cystic fibrosis: results of a multicenter randomized controlled trial. J Clin Endocrinol Metab 2006;91:4925e9. [390] Colombo C, Costantini D, Rocchi A, et al. Effects of liver transplantation on the nutritional status of patients with cystic fibrosis. Transpl Int 2005;18:246e55. [391] Mille CA, Golden NH. An introduction to eating disorders: clinical presentation, epidemiology, and prognosis. Nutr Clin Pract 2010;25:110e5. [392] Young JK. Anorexia nervosa and estrogen: current status of the hypothesis. Neuros Biobeh Rev 2010;34:1195e200. [393] Keel PK, Brown TA. Update on course and outcome in eating disorders. Int J Eat Disord 2010;43:195e204. [394] Sim LA, McGovern L, Elamin MB, Swiglo BA, Erwin PJ, Montori VM. Effect on bone health of estrogen preparations in premenopausal women with anorexia nervosa: a systematic review and meta-analyses. Int J Eat Disord 2010;43:218e25. [395] Jayasinghe Y, Grover SR, Zacharin M. Current concepts in bone and reproductive health in adolescents with anorexia nervosa. Br J Obstet Gynaecol 2008;115:304e15. [396] Misra M, Klibanski A. Anorexia nervosa and osteoporosis. Rev Endocr Metab Disord 2006;7:91e9. [397] Mun˜oz MT, Argente J. Anorexia nervosa in female adolescents: endocrine and bone mineral density disturbances. Eur J Endocrinol 2002;147:275e86. [398] Soyka LA, Grinspoon S, Levitsky LL, Herzog DB, Klibanski A. The effects of anorexia nervosa on bone metabolism in female adolescents. J Clin Endocrinol Metab 1999;84:4489e96. [399] Biller BM, Saxe V, Herzog DB, Rosenthal DI, Holzman S, Klibanski A. Mechanisms of osteoporosis in adult and adolescent women with anorexia nervosa. J Clin Endocrinol Metab 1989;68:548e54. [400] Zumoff B, Walsh BT, Katz JL, et al. Subnormal plasma dehydroisoandrosterone to cortisol ratio in anorexia nervosa: a second hormonal parameter of ontogenic regression. J Clin Endocrinol Metab 1983;56:668e72. [401] Legroux-Ge´rot I, Vignau J, Biver E, et al. Anorexia nervosa, osteoporosis and circulating leptin: the missing link. Osteoporos Int 2010;21:1715e22. [402] Utz AL, Lawson EA, Misra M, et al. Peptide YY (PYY) levels and bone mineral density (BMD) in women with anorexia nervosa. Bone 2008;43:135e9. [403] Bachrach LK, Guido D, Katzman D, Litt IF, Marcus R. Decreased bone density in adolescent girls with anorexia nervosa. Pediatrics 1990;86:440e7. [404] Soyka L, Misra M, Frenchman A, et al. Abnormal bone mineral accrual in adolescent girls with anorexia nervosa. J Clin Endocrinol Metab 2002;87:4177e85. [405] Misra M, Aggarwal A, Miller KK, et al. Effects of anorexia nervosa on clinical, hematologic, biochemical, and bone density parameters in community-dwelling adolescent girls. Pediatrics 2004;114:1574e83. [406] Diamanti A, Bizzarri C, Gambarara M, et al. Bone mineral density in adolescent girls with early onset of anorexia nervosa. Clin Nutr 2007;26:329e34.

[407] Bredella MA, Misra M, Miller KK, et al. Distal radius in adolescent girls with anorexia nervosa: trabecular structure analysis with high-resolution flat-panel volume CT. Radiology 2008;249:938e46. [408] Lawson EA, Miller KK, Bredella MA, et al. Hormone predictors of abnormal bone microarchitecture in women with anorexia nervosa. Bone 2010;46:458e63. [409] Grinspoon S, Thomas E, Pitts S, et al. Prevalence and predictive factors for regional osteopenia in women with anorexia nervosa. Ann Intern Med 2000;133:790e4. [410] do Carmo I, Mascarenhas M, Macedo A, et al. A study of bone density change in patients with anorexia nervosa. Eur Eat Disorders Rev 2007;15:457e62. [411] Misra M. Long-term skeletal effects of eating disorders with onset in adolescence. Ann NY Acad Sci 2008;1135:212e8. [412] Miller KK, Lee EE, Lawson EA, et al. Determinants of skeletal loss and recovery in anorexia nervosa. J Clin Endocrinol Metab 2006;91:2931e7. [413] Naessı´n S, Carlstro¨m K, Glant R, Jacobsson H, Linde´n Hirschberg A. Bone mineral density in bulimic women e influence of endocrine factors and previous anorexia. Eur J Endocrinol 2006;155:245e51. [414] Mehler PS, Sabel AL, Watson T, Andersen AE. Risk of osteoporosis in male patients with eating disorders. Int J Eat Disord 2008;41:666e72. [415] Misra M, Katzman DK, Cord J, et al. Bone metabolism in adolescent boys with anorexia nervosa. J Clin Endocrinol Metab 2008;93:3029e36. [416] Weinbrenner T, Zittermann A, Gouni-Berthold I, Stehle P, Berthold HK. Body mass index and disease duration are predictors of disturbed bone turnover in anorexia nervosa. A case-control study. Eur J Clin Nutr 2003;57:1262e7. [417] Mehler PS, MacKenzie TD. Treatment of osteopenia and osteoporosis in anorexia nervosa: a systematic review of the literature. Int J Eat Disord 2009;42:195e201. [418] Oswie˛cimska J, Ziora K, Pluskiewicz W, et al. Skeletal status and laboratory investigations in adolescent girls with anorexia nervosa. Bone 2007;41:103e10. [419] Galusca B, Bossu C, Germain N, et al. Age-related differences in hormonal and nutritional impact on lean anorexia nervosa bone turnover uncoupling. Osteoporos Int 2006;17:888e96. ´ lvaro M, et al. Main[420] Mun˜oz-Calvo MT, Barrios V, Garcı´a De A tained malnutrition produces a progressive decrease in (OPG)/ RANKL ratio and leptin levels in patients with anorexia nervosa. Scand J Clin Lab Invest 2007;67:387e93. [421] Dosta´lova´ I, Kava´kova´ P, Papezova´ H, Domluvilova´ D, Zika´n V, Haluzı´k M. Association of macrophage inhibitory cytokine-1 with nutritional status, body composition and bone mineral density in patients with anorexia nervosa: the influence of partial realimentation. Nutr Metab (Lond) 2010;7:34. [422] Fazeli PK, Bredella MA, Misra M, et al. Preadipocyte factor-1 is associated with marrow adiposity and bone mineral density in women with anorexia nervosa. J Clin Endocrinol Metab 2010;95: 407e13. [423] Vestergaard P, Emborg C, Støving RK, Hagen C, Mosekilde L, Brixen K. Fractures in patients with anorexia nervosa, bulimia nervosa, and other eating disorders e a nation-wide register study. Int J Eat Disord 2002;32:301e8. [424] Lucas AR, Melton 3rd LJ, Crowson CS, O’Fallon WM. Longterm fracture risk among women with anorexia nervosa: a population-based cohort study. Mayo Clin Proc 1999;74:972e7. [425] Miller KK, Grinspoon SK, Ciampa J, Hier J, Herzog D, Klibanski A. Medical findings in outpatients with anorexia nervosa. Arch Intern Med 2005;165:561e6.

PEDIATRIC BONE

REFERENCES

[426] Karlsson MK, Weigall SJ, Duan Y, Seeman E. Bone size and volumetric density in women with anorexia nervosa receiving estrogen replacement therapy and in women recovered from anorexia nervosa. J Clin Endocrinol Metab 2000;85:3177e82. [427] Klibanski A, Biller BM, Schoenfeld DA, Herzog DB, Saxe VC. The effects of estrogen administration on trabecular bone loss in young women with anorexia nervosa. J Clin Endocrinol Metab 1995;80:898e904. [428] Olmos JM, Valero C, Go´mez del Barrio A, et al. Time course of bone loss in patients with anorexia nervosa. Int J Eat Disord 2010;43:537e42. [429] Golden NH, Jacobson MS, Schebendach J, Solanto MV, Hertz SM, Shenker IR. Resumption of menses in anorexia nervosa. Arch Pediatr Adolesc Med 1997;151:16e21. [430] Bass SL, Saxon L, Corral AM, et al. Near normalisation of lumbar spine bone density in young women with osteopenia recovered from adolescent anorexia nervosa: a longitudinal study. J Pediatr Endocrinol Metab 2005;18:897e907. [431] Compston JE, McConachie C, Stott C, et al. Changes in bone mineral density, body composition and biochemical markers of bone turnover during weight gain in adolescents with severe anorexia nervosa: a 1-year prospective study. Osteoporos Int 2006;17:77e84. [432] Stock S, Leichne P, Wong ACK, et al. Ghrelin, peptide YY, glucose dependent insulinotropic polypeptide, and hunger responses to a mixed meal in anorexic, obese and control female adolescents. J Clin Endocrinol Metab 2005;90:2161e8. [433] Schulze UM, Schuler S, Schlamp D, Schneider P. Mehler-Wex C. Bone mineral density in partially recovered early onset anorexic patients e a follow-up investigation. Child Adolesc Psychiatry Ment Health 2010;4:20. [434] DiVasta AD, Feldman HA, Quach AE, Balestrino M, Gordon CM. The effect of bed rest on bone turnover in young women hospitalized for anorexia nervosa: a pilot study. J Clin Endocrinol Metab 2009;94:1650e5. [435] Golden NH, Lanzkowsky L, Schebendach J, Palestro CJ, Jacobson MS, Shenker IR. The effect of estrogen-progestin treatment on bone mineral density in anorexia nervosa. J Pediatr Adolesc Gynecol 2002;15:135e43. [436] Strokosch GR, Friedman AJ, Wu SC, Kamin M. Effects of an oral contraceptive (norgestimate/ethinyl estradiol) on bone mineral density in adolescent females with anorexia nervosa: a doubleblind, placebo-controlled study. J Adolesc Health 2006;39: 819e27. [437] Legroux-Ge´rot I, Vignau J, Collier F, Cortet B. Factors influencing changes in bone mineral density in patients with anorexia nervosa-related osteoporosis: the effect of hormone replacement therapy. Calcif Tissue Int 2008;83:315e23. [438] Grinspoon S, Thomas L, Miller K, Herzog D, Klibanski A. Effects of recombinant human IGF-I and oral contraceptive administration on bone density in anorexia nervosa. J Clin Endocrinol Metab 2002;87:2883e91. [439] Misra M, McGrane J, Mille KK, et al. Effects of rhIGF-1 administration on surrogate markers of bone turnover in adolescents with anorexia nervosa. Bone 2009;45:493e8. [440] Vescovi JD, Jamal SA, De Souza MJ. Strategies to reverse bone loss in women with functional hypothalamic amenorrhea: a systematic review of the literature. Osteoporos Int 2008;19: 465e78. [441] Liu SL, Lebrun CM. Effect of oral contraceptives and hormone replacement therapy on bone mineral density in premenopausal and perimenopausal women: a systematic review. Br J Sports Med 2006;40:11e24. [442] DiVasta AD, Gordon CM. Hormone replacement therapy for the adolescent patient. Ann NY Acad Sci 2008;1135:204e11.

503

[443] Nakahara T, Nagai N, Tanaka M, et al. The effects of bone therapy on tibial bone loss in young women with anorexia nervosa. Int J Eat Disord 2006;39:20e6. [444] Golden NH, Iglesias EA, Jacobson MS, et al. Alendronate for the treatment of osteopenia in anorexia nervosa: a randomized, double-blind, placebo-controlled trial. J Clin Endocrinol Metab 2005;90:3179e85. [445] Miller KK, Grieco KA, Mulder J, et al. Effects of risedronate on bone density in anorexia nervosa. J Clin Endocrinol Metab 2004;89:3903e6. [446] Craig ME, Hattersley A, Donaghue KC. Definition, epidemiology and classification of diabetes in children and adolescents. Pediatr Diab 2009;10(Suppl. 12):3e12. [447] Borchers AT, Uibo R, Gershwin ME. The geoepidemiology of type 1 diabetes. Autoimm Rev 2010;9:A355e65. [448] Fulzele K, Riddle RC, DiGirolamo DJ, et al. Insulin receptor signaling in osteoblasts regulates postnatal bone acquisition and body composition. Cell 2010;142:309e19. [449] Wu K, Schubeck KE, Frost HM, Villanueva A. Haversian bone formation rates determined by a new method in a mastodon, and in human diabetes mellitus and osteoporosis. Calcif Tissue Res 1970;6:204e19. [450] Verhaeghe J, Van Herck E, Visser WJ, et al. Bone and mineral metabolism in BB rats with long-term diabetes: decreased bone turnover and osteoporosis. Diabetes 1990;39:477e82. [451] Retzepi M, Donos N. The effect of diabetes mellitus on osseous healing. Clin Oral Impl Res 2010;21:673e81. [452] Mastrandrea LD, Wactawski-Wende J, Donahue RP, Hovey KM, Clark A, Quattrin T. Young women with type 1 diabetes have lower bone mineral density that persists over time. Diab Care 2008;31:1729e35. [453] Saito M, Marumo K. Collagen cross-links as a determinant of bone quality: a possible explanation for bone fragility in aging, osteoporosis, and diabetes mellitus. Osteoporos Int 2010;21: 195e214. [454] Wiske PS, Wentworth SM, Norton Jr JA, Epstein S, Johnston Jr CC. Evaluation of bone mass and growth in young diabetics. Metabolism 1982;31:848e54. [455] Valerio G, Puente A, Puente AE, del Puente A, Esposito-del Puente A, Buono P, Mozzillo E, Franzese A. The lumbar bone mineral density is affected by long-term poor metabolic control in adolescents with type 1 diabetes mellitus. Horm Res 2002;58: 266e72. [456] Heap J, Murray MA, Miller SC, Jalili T, Moyer-Mileur LJ. Alterations in bone characteristics associated with glycemic control in adolescents with type 1 diabetes mellitus. J Pediatr 2004;144:56e62. [457] Gunczler P, Lanes R, Paoli M, Martinis R, Villaroel O, Weisinger JR. Decreased bone mineral density and bone formation markers shortly after diagnosis of clinical type 1 diabetes mellitus. J Pediatr Endocrinol Metab 2001;14:525e8. [458] Pascual J, Argente J, Lopez MB, et al. Bone mineral density in children and adolescents with diabetes mellitus type 1 of recent onset. Calcif Tissue Int 1998;62:31e5. [459] De Schepper J, Smitz J, Rosseneu S, Bollen P, Louis O. Lumbar spine bone mineral density in diabetic children with recent onset. Horm Res 1998;50:193e6. [460] Brandao FR, Vicente EJ, Daltro CH, Sacramento M, Moreira A, Adan L. Bone metabolism is linked to disease duration and metabolic control in type 1 diabetes mellitus. Diab Res Clin Pract 2007;78:334e9. [461] C ¸ amurdan MO, Cinaz P, Bideci A, Demirel F. Role of hemoglobin A1c, duration and puberty on bone mineral density in diabetic children. Pediatr Int 2007;49:645e51.

PEDIATRIC BONE

504

18. THE SPECTRUM OF PEDIATRIC OSTEOPOROSIS

[462] Heilman K, Zilmer M, Zilmer K, Tillmann V. Lower bone mineral density in children with type 1 diabetes is associated with poor glycemic control and higher serum ICAM-1 and urinary isoprostane levels. J Bone Miner Metab 2009;27: 598e604. [463] Moyer-Mileur LJ, Slater H, Jordan KC, Murray MA. IGF-1 and IGF-binding proteins and bone mass, geometry, and strength: relation to metabolic control in adolescent girls with type 1 diabetes. J Bone Miner Res 2008;23:1884e91. [464] Le´ger J, Marinovic D, Alberti C, et al. Lower bone mineral content in children with type 1 diabetes mellitus is linked to female sex, low insulin-like growth factor type I levels, and high insulin requirement. J Clin Endocrinol Metab 2006;91:3947e53. [465] Va´zquez Ga´meza MA, Marı´n Pe´rez JM, Montoya Garcı´a MJ, et al. Evolucio´n de la masa o´sea durante la infancia y adolescencia en nin˜os con diabetes mellitus tipo 1. Med Clin (Barc) 2008;130:526e30. [466] Saha MT, Sieva¨nen H, Salo MK, Tulokas S, Saha HH. Bone mass and structure in adolescents with type 1 diabetes compared to healthy peers. Osteoporos Int 2009;20:1401e6. [467] Bechtold S, Dirlenbach I, Raile K, Noelle V, Bonfig W, Schwarz HP. Early manifestation of type 1 diabetes in children is a risk factor for changed bone geometry: data using peripheral quantitative computed tomography. Pediatrics 2006;118: e627e34. [468] Bechtold S, Putzker S, Bonfig W, Fuchs O, Dirlenbach I, Schwarz HP. Bone size normalizes with age in children and adolescents with type 1 diabetes. Diab Care 2007;30:2046e50. [469] Miao J, Brismar K, Nyre´n O, Ugarph-Morawski A, Ye W. Elevated hip fracture risk in type 1 diabetic patients. A population-based cohort study in Sweden. Diab Care 2005;28: 2850e5. [470] Greer RM, Rogers MA, Bowling FG, et al. Australian children and adolescents with type 1 diabetes have low vitamin D levels. Med J Aust 2007;187:59e60. [471] Bener A, Alsaied A, Al-Ali M, et al. High prevalence of vitamin D deficiency in type 1 diabetes mellitus and healthy children. Acta Diabetol 2009;46:183e9. [472] Karagu¨zel G, Ozdem S, Boz A, Bircan I, Akc¸urin S. Leptin levels and body composition in children and adolescents with type 1 diabetes. Clin Biochem 2006;39:788e93. [473] Kassem HS, Arabi A, Zantout MS, Azar ST. Negative effect of leptin on bone mass in type 1 diabetes. Acta Diabetol 2008;45: 237e41. [474] Valerio G, Spagnuolo MI, Lombardi F, Spadaro R, Siano M, Franzese A. Physical activity and sports participation in children and adolescents with type 1 diabetes mellitus. Nutr Metab Card Dis 2007;17:376e82. [475] Artz E, Warren-Ulanch J, Becker D, Greenspan S, Freemark M. Seropositivity to celiac antigens in asymptomatic children with type 1 diabetes mellitus: association with weight, height, and bone mineralization. Pediatr Diab 2008;9:277e84. [476] Gogakos AI, Duncan Bassett JH, Williams GR. Thyroid and bone. Arch Biochem Biophys 2010;503:129e36. [477] Agrawal M, Zhu G, Sun L, Zaidi M, Iqbal J. The role of FSH and TSH in bone loss and its clinical relevance. Curr Osteoporos Rep 2010;8:205e11. [478] Zaidi M, Sun L, Davies TF, Abe E. Low TSH triggers bone loss: fact or fiction? Thyroid 2006;16:1075e6. [479] Britto JM, Fenton AJ, Holloway WR, Nicholson GC. Osteoblasts mediate thyroid hormone stimulation of osteoclastic bone resorption. Endocrinology 1994;134:169e76. [480] Saggese G, Bertelloni S, Baroncelli GI. Bone mineralization and calciotropic hormones in children with hyperthyroidism. Effects of methimazole therapy. J Endocrinol Invest 1990;13:587e92.

[481] Siddiqi A, Burrin JM, Noonan K, et al. A longitudinal study of markers of bone turnover in Graves’ disease and their value in predicting bone mineral density. J Clin Endocrinol Metab 1997;82:753e9. [482] Kim CH, Kim HK, Shong YK, Lee KU, Kim GS. Thyroid hormone stimulates basal and interleukin (IL)-1-induced IL-6 production in human bone marrow stromal cells: a possible mediator of thyroid hormone induced bone loss. J Endocrinol 1999;160:97e102. [483] Williamson S, Greene SA. Incidence of thyrotoxicosis in childhood: a national population based study in the UK and Ireland. Clin Endocrinol 2010;72:358e63. [484] Karga H, Papapetrou PD, Korakovouni A, Papandroulaki F, Polymeris A, Pampouras G. Bone mineral density in hyperthyroidism. Clin Endocrinol 2004;61:466e72. [485] Vestergaard P, Mosekilde L. Hyperthyroidism, bone mineral, and fracture risk: a meta-analysis. Thyroid 2003;13:585e93. [486] Mora S, Weber G, Marenzi K, et al. Longitudinal changes of bone density and bone resorption in hyperthyroid girls during treatment. J Bone Min Res 1999;14:1971e7. [487] Mosekilde L, Melsen DF, Bagger JP, Myhrejensen O, Stensen NS. Bone changes in hyperthyroidism: interrelationship between bone morphometry, thyroid function and calcium phosphorus metabolism. Acta Endocrinol 1977;85:515e25. [488] Solomon BL, Wartofsky L, Burman KD. Prevalence of fractures in postmenopausal women with thyroid disease. Thyroid 1993;3:17e23. [489] Lucidarme N, Ruiz JC, Czernichow P, Leger J. Reduced bone mineral density at diagnosis and bone mineral recovery during treatment in children with Graves’ disease. J Pediatr 2000;137: 56e62. [490] Numbenjapon N, Costin G, Gilsanz V, Pitukcheewanont P. Low cortical bone density measured by computed tomography in children and adolescents with untreated hyperthyroidism. J Pediatr 2007;150:527e30. [491] Udayakumar N, Chandrasekaran M, Rasheed MH, Suresh RV, Sivaprakash S. Evaluation of bone mineral density in thyrotoxicosis. Singap Med J 2006;47:947e50. [492] Radetti G, Bona A, Corrias M, et al. Does Graves’ disease during puberty influence adult bone mineral density? Horm Res 2002;58:176e9. [493] Zaidi M, Davies TF, Zallone A, et al. Thyroid-stimulating hormone, thyroid hormones, and bone loss. Curr Osteoporos Rep 2009;7:47e52. [494] Papadimitriou A, Papadimitriou DT, Papadopoulou A, Nicolaidou P, Fretzayas A. Low TSH levels are not associated with osteoporosis in childhood. Eur J Endocrinol 2007;157: 221e3. [495] Kooh SW, Brnjac L, Ehrlich RM, Qureshi R, Krishnan S. Bone mass in children with congenital hypothyroidism treated with thyroxine since birth. J Ped Endocrinol Metab 1996;9:59e62. [496] Leger J, Ruiz JC, Guibourdenche J, Kindermans C, Garabedian M, Czernichow P. Bone mineral density and metabolism in children with congenital hypothyroidism after prolonged 1-thyroxine therapy. Acta Paediatr 1997;86:704e10. [497] Demeester-Mirkine N, Bergmann P, Body JJ, Corvilain J. Calcitonin and bone mass status in congenital hypothyroidism. Calcif Tissue Int 1990;46:222e6. [498] Salerno M, Lettiero T, Esposito-del Puente A, et al. Effect of long-term L-thyroxine treatment on bone mineral density in young adults with congenital hypothyroidism. Eur J Endocrinol 2004;151:689e94. [499] Pitukcheewanont P, Safani D, Gilsanz V, Klein M, Chongpison Y, Costin G. Quantitative computed tomography measurements of bone mineral density in prepubertal children with congenital

PEDIATRIC BONE

REFERENCES

[500]

[501]

[502] [503]

[504]

[505]

[506]

[507]

[508]

[509]

[510]

[511]

[512]

[513]

[514]

[515]

hypothyroidism treated with L-thyroxine. J Pediatr Endocrinol Metab 2004;17:889e93. Demartini Ade A, Kulak CA, Borba VC, et al. Bone mineral density of children and adolescents with congenital hypothyroidism. Arq Bras Endocrinol Metab 2007;51:1084e92. Cannata D, Vijayakumar A, Fierz Y, LeRoith D. The GH/IGF-1 axis in growth and development: new insights derived from animal models. Adv Pediatr 2010;57:331e51. Giustina A, Mazziotti G, Canalis E. Growth hormone, insulinlike growth factors, and the skeleton. End Rev 2008;29:535e59. Lombardi G, Di Somma C, Vuolo L, Guerra E, Scarano E, Colao A. Role of IGF-I on PTH effects on bone. J Endocrinol Invest 2010;33:22e6. Thomas M, Massa G, Craen M, et al. Prevalence and demographic features of childhood growth hormone deficiency in Belgium during the period 1986e2001. Eur J Endocrinol 2004;151:67e72. Murray RD, Adams JE, Shalet SM. A densitometric and morphometric analysis of the skeleton in adults with varying degrees of growth hormone deficiency. J Clin Endocrinol Metab 2006;91:432e8. Kosowicz J, El Ali Z, Ziemnicka K, Sowinski J. Abnormalities in bone mineral density distribution and bone scintigraphy in patients with childhood onset hypopituitarism. J Clin Densitom 2007;10:332e9. Boot AM, Engels MA, Boerma GJ, Krenning EP, De Muinck Keizer-Schrama SM. Changes in bone mineral density, body composition, and lipid metabolism during growth hormone (GH) treatment in children with GH deficiency. J Clin Endocrinol Metab 1997;82:2423e8. Baroncelli GI, Bertelloni S, Ceccarelli C, Saggese G. Measurement of volumetric bone mineral density accurately determines degree of lumbar undermineralization in children with growth hormone deficiency. J Clin Endocrinol Metab 1998;83:3150e4. Darendeliler F, Ranke MB, Bakker B, et al. Bone age progression during the first year of growth hormone therapy in pre-pubertal children with idiopathic growth hormone deficiency, Turner Syndrome or idiopathic short stature, and in short children born small for gestational age: analysis of data from KIGS (Pfizer international growth database). Horm Res 2005;63:40e7. Saggese G, Baroncelli GI, Bertelloni S, Barsanti S. The effect of long-term growth hormone (GH) treatment on bone mineral density in children with GH deficiency. Role of GH in the attainment of peak bone mass. J Clin Endocrinol Metab 1996;81: 3077e83. Ho¨gler W, Briody J, Moore B, Lu PW, Cowell CT. Effect of growth hormone therapy and puberty on bone and body composition in children with idiopathic short stature and growth hormone deficiency IGF. Bone 2005;37:642e50. Hyer SL, Rodin DA, Tobias JH, Leiper A, Nussey SS. Growth hormone deficiency during puberty reduces adult bone mineral density. Arch Dis Child 1992;67:1472e4. Ogle GD, Moore B, Lu PW, Craighead A, Briody JN, Cowell CT. Changes in body composition and bone density after discontinuation of growth hormone therapy in adolescence: an interim report. Acta Paediatr 1994;399(Suppl):3e7. Nguyen VT, Misra M. Transitioning of children with GH deficiency to adult dosing: changes in body composition. Pituitary 2009;12:125e35. Fors H, Bjarnason R, Wire´n L, et al. Currently used growthpromoting treatment of children results in normal bone mass and density. A prospective trial of discontinuing growth hormone treatment in adolescents. Clin Endocrinol 2001;55: 617e24.

505

[516] Shalet SM, Shavrikova E, Cromer M, et al. Effect of growth hormone (GH) treatment on bone in post-pubertal GH deficient (GHD) patients: a 2-year randomized, controlled, dose-ranging study. J Clin Endocrinol Metab 2003;88:4124e9. [517] Attanasio AF, Shavrikova E, Blum WF, et al. the Hypopituitary Developmental Outcome Study Group. Continued growth hormone (GH) treatment after final height is necessary to complete somatic development in childhood-onset GH-deficient patients. J Clin Endocrinol Metab 2004;89:4857e62. [518] Baroncelli GI, Bertelloni S, Sodini F, Saggese G. Longitudinal changes of lumbar bone mineral density (BMD) in patients with GH deficiency after discontinuation of treatment at final height; timing and peak values for lumbar BMD. Clin Endocrinol 2004;60:175e84. [519] Wuster C, Abs R, Bengtsson BA, et al. KIMS Study Group and the KIMS International Board. Pharmacia, Upjohn International Metabolic Database. The influence of growth hormone deficiency, growth hormone replacement therapy, and other aspects of hypopituitarism on fracture rate and bone mineral density. J Bone Miner Res 2001;16:398e405. [520] Bex M, Bouillon R. Growth hormone and bone health. Horn Res 2003;60(suppl. 3):80e6. [521] Mazziotti G, Bianchi A, Bonadonna S, et al. Increased prevalence of radiological spinal deformities in adult patients with GH deficiency: influence of GH replacement therapy. J Bone Miner Res 2006;21:520e8. [522] Baroncelli GI, Bertelloni S, Sodini F, Saggese G. Lumbar bone mineral density at final height and prevalence of fractures in treated children with GH deficiency. J Clin Endocrinol Metab 2002;87:3624e31. [523] Ho¨gler W, Shaw N. Childhood growth hormone deficiency, bone density, structures and fractures: scrutinizing the evidence. Clin Endocrinol (Oxf) 2010;72:281e9. [524] Schweizer R, Martin DD, Schwarze CP, et al. Cortical bone density is normal in prepubertal children with growth hormone (GH) deficiency, but initially decreases during GH replacement due to early bone remodeling. J Clin Endocrinol Metab 2003;88: 5266e72. [525] Zamboni G, Antoniazzi F, Lauriola S, Bertoldo F, Tato` L. Calcium supplementation increases bone mass in GH-deficient prepubertal children during GH replacement. Horm Res 2006;65:223e30. [526] Bailey DA, McKay HA, Minwald RL, Crocker PR, Faulkner RA. A six-year longitudinal study of the relationship of physical activity to bone mineral accrual in growing children: The University of Saskatchewan Bone Mineral Accrual Study. J Bone Miner Res 1999;14:1672e9. [527] Jackowski SA, Faulkner RA, Farthing JP, Kontulainen SA, Beck TJ, Baxter-Jones AD. Peak lean tissue mass accrual precedes changes in bone strength indices at the proximal femur during the pubertal growth spurt. Bone 2009;44:1186e90. [528] Xu L, Nicholson P, Wang Q, Ale´n M, Cheng S. Bone and muscle development during puberty in girls: a seven-year longitudinal study. J Bone Miner Res 2009;24:1693e8. [529] Kovacs CS, Kronenberg HM. Maternal-fetal calcium and bone metabolism during pregnancy, puerperium, and lactation. Endocr Rev 1997;18:832e72. [530] Turner CH, Burr DB. Basic biomechanical measurements of bone: a tutorial. Bone 1993;14:595e608. [531] Macdonald H, Kontulainen S, Petit M, Janssen P, McKay H. Bone strength and its determinants in pre- and early pubertal boys and girls. Bone 2006;39:598e608. [532] Bra¨mswig J, Du¨bbers A. Disorders of pubertal development. Dtsch Arztebl Int 2009;106:295e303.

PEDIATRIC BONE

506

18. THE SPECTRUM OF PEDIATRIC OSTEOPOROSIS

[533] Krupa B, Miazgowski T. Bone mineral density and markers of bone turnover in boys with constitutional delay of growth and puberty. J Clin Endocrinol Metab 2005;90:2828e30. [534] Finkelstein JS, Neer RM, Biller BM, Crawford JD, Klibanski A. Osteopenia in men with a history of delayed puberty. N Engl J Med 1992;326:600e4. [535] Finkelstein JS, Klibanski A, Neer RM. A longitudinal evaluation of bone mineral density in adult men with histories of delayed puberty. J Clin Endocrinol Metab 1996;81:1152e5. [536] Bertelloni S, Baroncelli GI, Ferdeghini M, Perri G, Saggese G. Normal volumetric bone mineral density and bone turnover in young men with histories of constitutional delay of puberty. J Clin Endocrinol Metab 1998;83:4280e3. [537] Finkelstein JS, Klibanski A, Neer RM. Comment on normal volumetric bone mineral density and bone turnover in young men with histories of constitutional delay of puberty. J Clin Endocrinol Metab 1999;84:3400e1. [538] Yap F, Hogler W, Briody J, Moore B, Howman-Giles R, Cowell CT. The skeletal phenotype of men with previous constitutional delay of puberty. J Clin Endocrinol Metab 2004;89: 4306e11. [539] Toppari J, Juul A. Trends in puberty timing in humans and environmental modifiers. Mol Cell Endocrinol 2010;324:39e44. [540] Boot AM, de Muinck Keizer-Schrama S, Pols HA, Krenning EP, Drop SL. Bone mineral density and body composition before and during treatment with gonadotropin-releasing hormone agonist in children with central precocious and early puberty. J Clin Endocrinol Metab 1998;83:370e3. [541] Neely EK, Bachrach LK, Hintz RL, et al. Bone mineral density during treatment of central precocious puberty. J Pediatr 1995;127:819e22. [542] van der Sluis IM, Boot AM, Krenning EP, Drop SL, de Muinck Keizer-Schrama SM. Longitudinal follow-up of bone density and body composition in children with precocious or early puberty before, during and after cessation of GnRH agonist therapy. J Clin Endocrinol Metab 2002;87:506e12. [543] Pasquino AM, Pucarelli I, Accardo F, Demiraj V, Segni M, Di Nardo R. Long-term observation of 87 girls with idiopathic central precocious puberty treated with gonadotropin-releasing hormone analogs: impact on adult height, body mass index, bone mineral content, and reproductive function. J Clin Endocrinol Metab 2008;93:190e5. [544] Magiakou MA, Manousaki D, Papadaki M, et al. The efficacy and safety of gonadotropin-releasing hormone analog treatment in childhood and adolescence: a single center, long-term followup study. J Clin Endocrinol Metab 2010;95:109e17. [545] Carel J-C, Eugster EA, Rogol A, Ghizzoni L, Palmert MR, on behalf of the members of the ESPE-LWPES GnRH Analogs Consensus Conference Group. Consensus statement on the use of gonadotropin-releasing hormone analogs in children. Pediatrics 2009;123:e752e62. [546] Fintini D, Grossi A, Brufani C, et al. Bone mineral density and body composition in male children with hypogonadism. J Endocrinol Invest 2009;32:585e9. [547] Arisaka O, Arisaka M, Nakayama Y, Fujiwara S, Yabuta K. Effect of testosterone on bone density and bone metabolism in adolescent male hypogonadism. Metabolism 1995;44:419e23. [548] De Rosa M, Paesano L, Nuzzo V, et al. Bone mineral density and bone markers in hypogonadotropic and hypergonadotropic hypogonadal men after prolonged testosterone treatment. J Endocrinol Invest 2001;24:246e52. [549] Park KH, Lee SJ, Kim JY, Kim JY, Bai SW, Kim JW. A concomitant decrease in cortical and trabecular bone mass in isolated hypogonadotropic hypogonadism and gonadal dysgenesis. Yonsei Med J 1999;40:444e9.

[550] Hjerrild BE, Mortensen KH, Gravholt CH. Turner syndrome and clinical treatment. Br Med Bull 2008;86:77e93. [551] Ross JL, Long LM, Feuillan P, Cassorla F, Cutler GB. Normal bone density of the wrist and spine and increased wrist fractures in girls with Turner’s syndrome. J Clin Endocrinol Metab 1991;73:355e9. [552] Neely EK, Marcus R, Rosenfeld RG, Bachrach LK. Turner syndrome adolescents receiving growth hormone are not osteopenic. J Clin Endocrinol Metab 1993;76:861e6. [553] Gravholt CH, Lauridsen AL, Brixen K, Mosekilde L, Heickendorff L, Christiansen JS. Marked disproportionality in bone size and mineral, and distinct abnormalities in bone markers and calcitropic hormones in adult Turner syndrome: a cross-sectional study. J Clin Endocrinol Metab 2002;87: 2798e808. [554] Baena N, De Vigan C, Cariati E, et al. Turner syndrome: evaluation of prenatal diagnosis in 19 European registries. Am J Med Genet 2004;129A:16e20. [555] Bechtold S, Rauch F, Noelle V, et al. Musculoskeletal analyses of the forearm in young women with Turner syndrome: a study using peripheral quantitative computed tomography. J Clin Endocrinol Metab 2001;86:5819e23. [556] Holroyd CR, Davies JH, Taylor P, et al. Reduced cortical bone density with normal trabecular bone density in girls with Turner syndrome. Osteoporos Int 2010;21:2093e9. [557] Gravholt CH, Vestergaard P, Hermann AP, Mosekilde L, Brixen K, Christiansen JS. Increased fracture rates in Turner’s syndrome: a nationwide questionnaire survey. Clin Endocrinol (Oxf) 2003;59:89e96. [558] Landin-Wilhelmsen K. Osteoporosis and fractures in Turner syndrome e importance of growth promoting and oestrogen therapy. Clin Endocrinol 1999;51:497e502. [559] Khastgir G, Studd JW, Fox SW, Jones J, Alghband-Zadeh J, Chow JW. A longitudinal study of the effect of subcutaneous estrogen replacement on bone in young women with Turner’s syndrome. J Bone Miner Res 2003;18:925e32. [560] Cleemann L, Hjerrild BE, Lauridsen AL, et al. Long-term hormone replacement therapy preserves bone mineral density in Turner syndrome. Eur J Endocrinol 2009;161:251e7. [561] Zuckerman-Levin N, Yaniv I, Schwartz T, Guttmann H, Hochberg Z. Normal DXA bone mineral density but frail cortical bone in Turner’s syndrome. Clin Endocrinol 2007;67: 60e4. [562] Clement-Jones M, Schiller S, Rao E, et al. The short stature homeobox gene SHOX is involved in skeletal abnormalities in Turner syndrome. Hum Mol Genet 2000;9:695e702. [563] Ross JL, Scott Jr C, Marttila P, et al. Phenotypes associated with SHOX deficiency. J Clin Endocrinol Metab 2001;86:5674e80. [564] El-Mansoury M, Barrena¨s ML, Bryman I, et al. Chromosomal mosaicism mitigates stigmata and cardiovascular risk factors in Turner syndrome. Clin Endocrinol (Oxf) 2007;66:744e51. [565] Han TS, Cadge B, Conway GS. Hearing impairment and low bone mineral density increase the risk of bone fractures in women with Turner’s syndrome. Clin Endocrinol 2006;65: 643e7. [566] El-Mansoury M, Barrena¨s ML, Bryman I, Hanson C, LandinWilhelmsen K. Impaired body balance, fine motor function and hearing in women with Turner syndrome. Clin Endocrinol 2009;71:273e8. [567] Nissen N, Gravholt CH, Abrahamsen B, et al. Disproportional geometry of the proximal femur in patients with Turner syndrome: a cross-sectional study. Clin Endocrinol 2007;67: 897e903. [568] Ari M, Bakalov VK, Hill S, Bondy CA. The effects of growth hormone treatment on bone mineral density and body

PEDIATRIC BONE

REFERENCES

[569]

[570]

[571]

[572]

[573]

[574]

[575] [576]

[577]

[578]

[579]

[580]

[581] [582]

[583]

[584]

[585]

[586]

composition in girls with Turner syndrome. J Clin Endocrinol Metab 2006;91:4302e5. Sas TC, de Muinck Keizer-Schrama SM, Stijnen T, et al. Dutch Advisory Group on Growth Hormone. Bone mineral density assessed by phalangeal radiographic absorptiometry before and during long-term growth hormone treatment in girls with Turner’s syndrome participating in a randomised doseresponse study. Pediatr Res 2001;50:417e22. Bertelloni S, Cinquanta L, Baroncelli GI, Simi P, Rossi S, Saggese G. Volumetric bone mineral density in young women with Turner’s syndrome treated with estrogens or estrogens plus growth hormone. Horm Res 2000;53:72e6. Nabhan ZM, DiMeglio LA, Qi R, Perkins SM, Eugster EA. Conjugated oral versus transdermal estrogen replacement in girls with Turner syndrome: a pilot comparative study. J Clin Endocrinol Metab 2009;94:2009e14. Hogler W, Briody J, Moore B, Garnett S, Lu PW, Cowell CT. Importance of estrogen on bone health in Turner syndrome: a cross-sectional and longitudinal study using dual-energy X-ray absorptiometry. J Clin Endocrinol Metab 2004;89:193e9. Hanton L, Axelrod L, Bakalov V, Bondy CA. The importance of estrogen replacement in young women with Turner syndrome. J Womens Health (Larchmt) 2003;12:971e7. Saranac L, Zivanovic S, Radovanovic Z, Kostic G, Markovic I, Miljkovic P. Hyperprolactinemia: different clinical expression in childhood. Horm Res Paediatr 2010;73:187e92. Fideleff HL, Boquete HR, Sua´rez MG, Azaretzky M. Prolactinoma in children and adolescents. Horm Res 2009;72:197e205. Keil MF, Stratakis CA. Pituitary tumors in childhood: update of diagnosis, treatment and molecular genetics. Expert Rev Neurother 2008;8:563e74. Sakazume S, Obata K, Takahashi E, et al. Bromocriptine treatment of prolactinoma restores growth hormone secretion and causes catch-up growth in a prepubertal child. Eur J Pediatr 2004;163:472e4. Emiliano ABF, Fudge JL. From galactorrhea to osteopenia: rethinking serotonin-prolactin interactions. Neuropsychopharmacology 2004;29:833e46. Naliato EC, Farias ML, Braucks GR, Costa FS, Zylberberg D, Violante AH. Prevalence of osteopenia in men with prolactinoma. J Endocrinol Invest 2005;28:12e7. Bussade I, Naliato EC, Mendonc¸a LM, Violante AH, Farias ML. Decreased bone mineral density in pre-menopause women with prolactinoma. Arq Bras Endocrinol Metab 2007;51:1522e7. Shibli-Rahhal A, Schlechte J. The effects of hyperprolactinemia on bone and fat. Pituitary 2009;12:96e104. Coss D, Yang L, Ku CB, Xu X, Luben RA, Walker AM. Effects of prolactin on osteoblast alkaline phosphatase and bone formation in the developing rat. Am J Physiol Endocrinol Metab 2000;279:E1216e25. Seriwatanachai D, Thongchote K, Charoenphandhu N, et al. Prolactin directly enhances bone turnover by raising osteoblastexpressed receptor activator of nuclear factor B ligand/osteoprotegerin ratio. Bone 2008;42:535e46. Greenspan SL, Oppenheim DS, Klibanski A. Importance of gonadal steroids to bone mass in men with hyperprolactinemic hypogonadism. Ann Intern Med 1989;110:526e31. Vestergaard P, Jørgensen JOL, Hagen C, et al. Fracture risk is increased in patients with GH deficiency or untreated prolactinomas e a case-control study. Clin Endocrinol 2002;56:159e67. Galli-Tsinopoulou A, Nousia-Arvanitakis S, Mitsiakos G, Karamouzis M, Dimitriadis A. Osteopenia in children and adolescents with hyperprolactinemia. J Pediatr Endocrinol Metab 2000;13:439e41.

507

[587] Coelho R, Silva C, Maia A, Prata J, Barros H. Bone mineral density and depression: a community study in women. J Psychosom Res 1999;46:29e35. [588] Calarge CA, Zimmerman B, Xie D, Kuperman S, Schlechte JA. A cross-sectional evaluation of the effect of risperidone and selective serotonin reuptake inhibitors on bone mineral density in boys. J Clin Psychiat 2010;71:338e47. [589] Colao A, Di Somma C, Loche S, et al. Prolactinomas in adolescents: persistent bone loss after 2 years of prolactin normalization. Clin Endocrinol (Oxf) 2000;52:319e27. [590] Lukert BP. Glucocorticoid-induced osteoporosis. South Med J 1992;85:2S48-51. [591] Compston J. Management of glucocorticoid-induced osteoporosis. Nat Rev Rheumatol 2010;6:82e8. [592] Canalis E, Mazziotti G, Giustina A, Bilezikian JP. Glucocorticoid-induced osteoporosis: pathophysiology and therapy. Osteoporos Int 2007;18:1319e28. [593] Naganathan V, Jones G, Nash P, Nicholson G, Eisman J, Sambrook PN. Vertebral fracture risk with long-term corticosteroid therapy: prevalence and relation to age, bone density, and corticosteroid use. Arch Intern Med 2000;160:2917e22. [594] Turpeinen M, Pelkonen AS, Nikander K, et al. Bone mineral density in children treated with daily or periodical inhaled budesonide: the Helsinki Early Intervention Childhood Asthma study. Pediatr Res 2010;68:169e73. [595] Olney RC. Mechanisms of impaired growth: effect of steroids on bone and cartilage. Horm Res 2009;72(Suppl. 1):30e5. [596] Kim HJ. New understanding of glucocorticoid action in bone cells. BMB Rep 2010;43:524e9. [597] Weinstein RS. Glucocorticoids, osteocytes, and skeletal fragility: the role of bone vascularity. Bone 2010;46:564e70. [598] Kondo T, Kitazawa R, Yamaguchi A, Kitazawa S. Dexamethasone promotes osteoclastogenesis by inhibiting osteoprotegerin through multiple levels. J Cell Biochem 2008;103:335e45. [599] Steinbuch M, Youket TE, Cohen S. Oral glucocorticoid use is associated with an increased risk of fracture. Osteoporos Int 2004;15:323e8. [600] Grossman JM, Gordon R, Ranganath VK, et al. American College of Rheumatology 2010 recommendations for the prevention and treatment of glucocorticoid-induced osteoporosis. Arthritis Care Res 2010;62:1515e26. [601] Reid IR, Evans MC, Stapleton J. Lateral spine densitometry is a more sensitive indicator of glucocorticoid-induced bone loss. J Bone Miner Res 1992;7:1221e5. [602] Nysom K, Holm K, Michaelsen KF, Hertz H, Muller J, Mølgaard C. Bone mass after treatment for acute lymphoblastic leukemia in childhood. J Clin Oncol 1998;16:3752e60. [603] Hesseling PB, Hough SF, Nel ED, van Riet FA, Beneke T, Wessls G. Bone mineral density in long-term survivors of childhood cancer. Int J Cancer Suppl. 1998;11:44e7. [604] De Vries F, Bracke M, Leufkens HG, Lammers JW, Cooper C, Van Staa TP. Fracture risk with intermittent high-dose glucocorticoid therapy. Arthritis Rheum 2007;56:208e14. [605] van Staa TP, Leufkens HG, Cooper C. The epidemiology of corticosteroid-induced osteoporosis: a meta-analysis. Osteoporos Int 2002;13:777e87. [606] van Staa TP, Cooper C, Leufkens HG, Bishop N. Children and the risk of fractures caused by oral corticosteroids. J Bone Miner Res 2003;18:913e8. [607] Huber AM, Gaboury I, Cabral DA, et al. Canadian SteroidAssociated Osteoporosis in the Pediatric Population (STOPP) Consortium. Prevalent vertebral fractures among children initiating glucocorticoid therapy for the treatment of rheumatic disorders. Arthritis Care Res 2010;62:516e26.

PEDIATRIC BONE

508

18. THE SPECTRUM OF PEDIATRIC OSTEOPOROSIS

[608] Kelly HW, Van Natta ML, Covar RA, Tonascia J, Green RP, Strunk RC. CAMP Research Group. Effect of long-term corticosteroid use on bone mineral density in children: a prospective longitudinal assessment in the childhood Asthma Management Program (CAMP) study. Pediatrics 2008;122:e53e61. [609] Vestergaard P, Rejnmark L, Mosekilde L. Fracture risk associated with systemic and topical corticosteroids. J Intern Med 2005;257:374e84. [610] van Staa TP, Bishop N, Leufkens HG, Cooper C. Are inhaled corticosteroids associated with an increased risk of fracture in children? Osteoporos Int 2004;15:785e91. [611] Schlienger RG, Jick SS, Meier CR. Inhaled corticosteroids and the risk of fractures in children and adolescents. Pediatrics 2004;114:469e73. [612] Pack AM, Gida lB, Vazquez B. Bone disease associated with antiepileptic drugs. Cleve Clin J Med 2004;71(Suppl. 2):S42e8. [613] Samaniego EA, Sheth RD. Bone consequences of epilepsy and antiepileptic medications. Semin Pediatr Neurol 2007;14: 196e200. [614] Sheth RD, Binkley N, Hermann BP. Progressive bone deficit in epilepsy. Neurology 2008;70:170e6. [615] Sheth RD, Gidal BE, Hermann BP. Pathological fractures in epilepsy. Epilepsy Behav 2006;9:601e5. [616] Gniatkowska-Nowakowska A. Fractures in epilepsy children. Seizure 2010;19:324e5. [617] Rajgopal R, Bear M, Butcher MK, Shaughnessy SG. The effects of heparin and low molecular weight heparins on bone. Thromb Res 2008;122:293e8. [618] Newall F, Johnston L, Ignjatovic V, Monagle P. Unfractionated heparin therapy in infants and children. Pediatrics 2009;123: e510e8. [619] Monagle P, Newall F, Campbell J. Anticoagulation in neonates and children: Pitfalls and dilemmas. Blood Rev 2010;24:151e62. [620] Simon RR, Shaughnessy SG. Effects of anticoagulants on bone. Clin Rev Bone Min Metab 2004;2:151e8. [621] Barnes C, Newall F, Ignjatovic V, et al. Reduced bone density in children on long-term warfarin. Pediatr Res 2005;57:578e81. [622] Wheeler DL, Vander Griend RA, Wronski TJ, Miller GJ, Keith EE, Graves JE. The short- and long-term effects of methotrexate on the rat skeleton. Bone 1995;16:215e21. [623] May KP, Mercill D, McDermott MT, West SG. The effect of methotrexate on mouse bone cells in culture. Arthritis Rheum 1996;39:489e94. [624] Elliot KJ, Millward-Sadler SJ, Wright MO, Robb JE, Wallace WH, Salter DM. Effects of methotrexate on human bone cell responses to mechanical stimulation. Rheumatol (Oxf) 2004;43: 1226e31. [625] Meister B, Gassner I, Streif W, Dengg K, Fink FM. Methotrexate osteopathy in infants with tumors of the central nervous system. Med Pediatr Oncol 1994;23:493e6. [626] Mandel K, Atkinson S, Barr RD, Pencharz P. Skeletal morbidity in childhood acute lymphoblastic leukemia. J Clin Oncol 2004;22:1215e21. [627] Sala A, Barr RD. Osteopenia and cancer in children and adolescents: the fragility of success. Cancer 2007;109:1420e31. [628] Cranney AB, McKendry RJ, Wells GA, et al. The effect of low dose methotrexate on bone density. J Rheumatol 2001;28: 2395e9. [629] Stanisavljevic S, Babcock AL. Fractures in children treated with methotrexate for leukemia. Clin Orthop Relat Res 1977;125: 139e44. [630] Movsowitz C, Epstein S, Fallon M, Ismail F, Thomas S. Cyclosporin-A in vivo produces severe osteopenia in the rat: effect of dose and duration of administration. Endocrinology 1988;123: 2571.

[631] Voggenreiter G, Assenmacher S, Kreuzfelder E, et al. Immunosuppression with FK506 increases bone induction in demineralized isogeneic and xenogeneic bone matrix in the rat. J Bone Miner Res 2000;15:1825e34. [632] Amorosa V, Tebas P. Bone disease and HIV infection. Clin Infect Dis 2006;42:108e14. [633] Stone B, Dockrell D, Bowman C, McCloskey E. HIV and bone disease. Arch Biochem Biophys 2010;503:66e77. [634] Arpadi S, Horlick M, Shane E. Metabolic bone disease in human immunodeficiency virus-infected children. J Clin Endocrinol Metab 2004;89:21e3. [635] Zuccotti G, Vigano` A, Gabiano C, et al. Antiretroviral therapy and bone mineral measurements in HIV-infected youths. Bone 2010;46:1633e8. [636] Kim RJ, Rutstein RM. Impact of antiretroviral therapy on growth, body composition and metabolism in pediatric HIV patients. Pediatr Drugs 2010;12:187e99. [637] Mazziotti G, Canalis E, Giustina A. Drug-induced osteoporosis: mechanisms and clinical implications. Am J Med 2010;123: 877e84. [638] Krasin MJ, Constine LS, Friedman DL, Marks LB. Radiationrelated treatment effects across the age spectrum: differences and similarities or what the old and young can learn from each other. Semin Radiat Oncol 2010;20:21e9. [639] Arikoski P, Komulainen J, Voutilainen R, et al. Reduced bone mineral density in long-term survivors of childhood acute lymphoblastic leukemia. J Pediatr Hematol Oncol 1998;20: 234e40. [640] van der Sluis IM, van den Heuvel-Eibrink MM. Osteoporosis in children with cancer. Pediatr Blood Cancer 2008;50(Suppl. 2): 474e8. [641] Kaste SC. Skeletal toxicities of treatment in children with cancer. Pediatr Blood Cancer 2008;50(Suppl. 2):469e73. [642] Crofton PM. Bone and bone turnover. Endocr Dev 2009;15: 77e100. [643] Bluemke DA, Fishman EK, Scott Jr WW. Skeletal complications of radiation therapy. Radiographics 1994;14:111e21. [644] Wall JE, Kaste SC, Greenwald CA, Jenkins JJ, Douglass EC, Pratt CB. Fractures in children treated with radiotherapy for soft tissue sarcoma. Orthopedics 1996;19:657e64. [645] Cimaz R, Gattorno M, Sormani MP, et al. Changes in markers of bone turnover and inflammatory variables during alendronate therapy in pediatric patients with rheumatic diseases. J Rheumatol 2002;29:1786e92. [646] Mauras N. Can growth hormone counteract the catabolic effects of steroids? Horm Res 2009;72(Suppl. 1):48e54. [647] Bonjour JP, Carrie AL, Ferrari S, et al. Calcium-enriched food and bone mass growth in prepubertal girls: a randomized, doubleblind, placebo controlled trial. J Clin Invest 1997;99:1287e94. [648] Curhan GC, Willett WC, Speizer FE, Spiegelman D, Stampfer MJ. Comparison of dietary calcium with supplemental calcium and other nutrients as factors affecting the risk for kidney stones in women. Ann Intern Med 1997;126:497e504. [649] McKay H, Liu D, Egeli D, Boyd S, Burrows M. Physical activity positively predicts bone architecture and bone strength in adolescent males and females. Acta Paediatr 2011;100:97e101. [650] Nikander R, Sieva¨nen H, Heinonen A, Daly RM, Uusi-Rasi K, Kannus P. Targeted exercise against osteoporosis: A systematic review and meta-analysis for optimising bone strength throughout life. BMC Med 2010;8:47. [651] Phillipi CA, Remmington T, Steiner RD. Bisphosphonate therapy for osteogenesis imperfecta. Cochrane Database Syst Rev 2008;4:CD005088.

PEDIATRIC BONE

REFERENCES

[652] Conwell LS, Chang AB. Bisphosphonates for osteoporosis in people with cystic fibrosis. Cochrane Database Syst Rev 2009;4: CD002010. [653] Lethaby C, Wiernikowski J, Sala A, Naronha M, Webber C, Barr RD. Bisphosphonate therapy for reduced bone mineral density during treatment of acute lymphoblastic leukemia in childhood and adolescence: a report of preliminary experience. J Pediatr Hematol Oncol 2007;29:613e6. [654] Ward L, Tricco AC, Phuong P, et al. Bisphosphonate therapy for children and adolescents with secondary osteoporosis. Cochrane Database Syst Rev 2007;4:CD005324. [655] Bachrach LK, Ward LM. Clinical review: Bisphosphonate use in childhood osteoporosis. J Clin Endocrinol Metab 2009;94:400e9. [656] Rothenbuhler A, Marchand I, Bougne`res P, Linglart A. Risk of corrected QT interval prolongation after pamidronate infusion in children. J Clin Endocrinol Metab 2010;95:3768e70. [657] Munns CF, Rauch F, Mier RJ, Glorieux FH. Respiratory distress with pamidronate treatment in infants with severe osteogenesis imperfecta. Bone 2004;35:231e4. [658] Kamoun-Goldrat A, Ginisty D, Le Merrer M. Effects of bisphosphonates on tooth eruption in children with osteogenesis imperfecta. Eur J Oral Sci 2008;116:195e8. [659] Whyte MP, McAlister WH, Novack DV, Clements KL, Schoenecker PL, Wenkert D. Bisphosphonate-induced osteopetrosis: novel bone modeling defects, metaphyseal osteopenia, and osteosclerosis fractures after drug exposure ceases. J Bone Miner Res 2008;23:1698e707.

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[660] Winer KK, Sinaii N, Peterson D, Sainz Jr B, Cutler Jr GB. Effects of once versus twice-daily parathyroid hormone 1-34 therapy in children with hypoparathyroidism. J Clin Endocrinol Metab 2008;93:3389e95. [661] Sanda S, Schlingmann KP, Newfield RS. Autosomal dominant hypoparathyroidism with severe hypomagnesemia and hypocalcemia, successfully treated with recombinant PTH and continuous subcutaneous magnesium infusion. J Pediatr Endocrinol Metab 2008;21:385e91. [662] Vahle JL, Long GG, Sandusky G, Westmore M, Ma YL, Sato M. Bone neoplasms in F344 rats given teriparatide [rhPTH(1-34)] are dependent on duration of treatment and dose. Toxicol Pathol 2004;32:426e38. [663] Leventhal JM, Martin KD, Asnes AG. Incidence of fractures attributable to abuse in young hospitalized children: results from analysis of a United States database. Pediatrics 2008;122: 599e604. [664] Jenny C, for the Committee on Child Abuse and Neglect. Evaluating infants and young children with multiple fractures. Pediatrics 2006;118:1299e303. [665] Bishop N, Sprigg A, Dalton A. Unexplained fractures in infancy: looking for fragile bones. Arch Dis Child 2007;92:251e6. [666] Dwek JR. The radiographic approach to child abuse. Clin Orthop Relat Res 2011;469:776e89. [667] Shea-Landry GL, Cole DE. Psychosocial aspects of osteogenesis imperfecta. Can Med Assoc J 1986;135:977e81.

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Osteogenesis Imperfecta Francis H Glorieux 1, David Rowe 2 1

Shriners Hospital for Children, and McGill University, Montreal, Canada 2 Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Connecticut, USA

INTRODUCTION Osteogenesis imperfecta (OI) or “brittle bone disease” is characterized by reduced skeletal mass and bone fragility. Over the past recent years, it has evolved from being a collagenopathy caused by mutations in the genes encoding type I collagen, and for which there was no medical treatment to a fascinating heterogeneous group of conditions, caused by numerous different mutations, with variable clinical expression, the prospect of effective symptomatic treatment and exciting prospects for gene therapy. The prevalence of OI is estimated to be 1 in 10 000 births [1]. The prevalence appears to be the same throughout the world [2e5]. Over the years, OI has served as the paradigm for heritable diseases of connective tissue from which advances in molecular diagnosis, mode of inheritance, and new concepts of therapy have been developed. It should continue to play this pivotal role in the future. In the vast majority of cases, mutations within the COL1A1 or COL1A2 genes, encoding type I collagen are responsible for the phenotype, although it is now recognized that mutations in other genes can produce a similar clinical outcome. A comprehensive list of the mutations within type I collagen genes resulting in OI [10] is maintained in an OI mutation database (http://www.le.ac. uk/ge/collagen/). The hallmark of OI is bone brittleness. All other characteristics (blue sclerae, dentinogenesis imperfecta, skin hyperlaxity, joint hypermobility, reduced stature, long bone deformities) are variable, with heterogeneity even in affected members of the same family [6]. Wormian bones are present in the skull in approximately 60% of cases (Fig. 19.1) [7], although they can be present in other conditions, such as progeria, cleidocranial dysplasia, Menkes syndrome, cutis laxa, HajdueCheney syndrome, and pyknodysostosis [8]. Platycephaly due to

Pediatric Bone, Second Edition DOI: 10.1016/B978-0-12-382040-2.10019-X

bone fragility and lack of head control is frequent in infants with severe OI (Fig. 19.2). Affected children suffer recurrent fractures resulting in pain and immobilization, particularly in preschool years. Fracture incidence decreases with age, but the reduced peak bone mass achieved will affect the long-term evolution of the disease particularly in women after menopause.

CLASSIFICATION The severity of OI ranges from mild forms with no deformity, near normal stature, and few fractures to forms that are lethal in the perinatal period. OI was first classified into two forms by Looser in 1906 [9]. 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 after birth or later. OI tarda has also been subdivided into gravis and levis [9]. This classification is no longer in use because it understates the complexity of the condition. The first clinical classification of OI to reflect the spectrum of the disease severity was proposed by Sillence [4,10]. 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, the classification has received wide acceptance. In the original report [4], Sillence et al. classified 154 subjects into four groups that each represents a “type”. Type I includes individuals with bone fragility, blue sclera, and presenile deafness. The majority of the subjects in this group had their first fracture in the preschool years (in five patients, fractures were present

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FIGURE 19.1 Wormian bones are detached portions of the primary ossification centers in adjacent membranous bones. They are suggestive of osteogenesis imperfecta but are not pathognomonic for the condition.

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. Vertebral fractures are common and may lead to mild scoliosis. Dentinogenesis imperfecta (DI) is infrequent. Inheritance was dominant in all cases. Type II is the most severe form resulting in death from respiratory failure in the perinatal period. There is radiographic evidence of multiple intrauterine fractures with crumpled (“accordion-like”) femora and beaded ribs. In most cases, the sclerae were blue. With no evidence for family history, it was initially considered as an autosomal recessive trait. Type III is the most severe form compatible with survival. Fractures may be present in utero and are very common during the growing period, causing severe progressive skeletal deformities. Deformities and frequent fractures often confine these patients to a wheelchair for life. The sclerae are light blue and DI is frequent. All cases in this group were sporadic, suggesting at the time recessive transmission.

FIGURE 19.2 Skull in a severe case of OI. The structure is soft and easily flattened in the back due to the lack of head support.

Type IV is the most clinically diverse group. The clinical picture can vary from severe to mild, with short stature. DI is frequent and sclera is white to grayish. There is evidence for dominant inheritance. In practical terms, this group includes any patients with fragile bones and reduced bone mass that cannot be easily included in one of the three first types. It is from this group that newer types have been segregated. Paterson et al. [11] described 48 subjects who had white sclerae and dominant inheritance, providing a more extensive description of Sillence type IV. They found that there is a wide range for age at first fracture and for total number of fractures. They note the different 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 scleral color, although they mention that several subjects included in type IV had pale blue sclerae. Sillence made it clear that type IV patients may have blue sclerae at a younger age that fades as they grow [12]. In clinical practice, scleral hue has little significance in the diagnosis and classification of OI because blue sclerae 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.

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The mode of inheritance in OI most of the time is dominant, and there is a high incidence of new mutations, regardless of the form of OI. In some families from South Africa and Ireland, a recessive pattern of inheritance has been demonstrated [13,14]. The possibility of a germ cell mosaicism [15] has been proposed to explain cases in families with healthy parents who have more than one child with OI [16,17]. 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 [18]. Thus, there is no clear genotype/phenotype correlate in individuals with OI, and classification should remain clinical. More types were recently added to the four described by Sillence, and they are detailed hereunder. Following Rowe and Shapiro [19], we propose that patients should be described in reference to their severity (from lethal to mild), regardless of the type.

THE WIDENING SPECTRUM OF OI TYPES 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 pulmonary hypoplasia [20] or by central nervous system malformations [21] 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 [22]. Prenatal ultrasound may show shortened and broad limbs, with very low echogenicity and absent acoustic shadow [23], 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 [24]. However, prenatal differential diagnosis between severe and lethal OI is not possible. Differential diagnosis includes chondrodysplasia punctata [25] and other forms of OI. In extremely severe cases, patients can be born dismembered [26]. They may have low birth weight, micro- or macrocephalus, and cataracts [27]. In the majority of cases, they are caused by autosomal dominant new mutations [18,28,29]. Unaffected parents may have more than one child with lethal OI due to germ cell line mosaicism [30]. There may be different clinical forms of lethal OI [31]. 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 collagen gene product causes the disease). The deleterious effect of

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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 GlyeXeY amino acid triplet. A glycine substitution in the helical domain of the collagen a1(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 associate. 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 C-terminal helical and propeptide region results in greater instability and more severe disease, whereas mutations in the mid-helical domain tend to be less severe. However, mutations in the mid-helical 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 [32]. Much less common are mutations that delete a portion of the gene and a number of inframe exons [33] or mutations that insert a segment of intron that remains inframe with the entire transcript [34]. 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 a2(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 of the a2(I) chain or overproduction of the a1(I) chain, 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 [35,36] and an association with osteoporosis due to the sp1 polymorphic alteration in the COL1A1 gene. This variation in disease severity acts in a recessive manner or as a quantitative trait in

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which gene dosage contributes to the severity of bone disease.

Severe OI (Type III) Due to overdevelopment of the head and underdevelopment of the facial 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 vertebral pedicles and posterior rib angulation [37]. They are frequently wheelchair bound, although some are able to walk with canes or a walker. Prenatal diagnosis is sometimes possible using ultrasonography [36]. Long bones are severely deformed, and altered structure of the growth plates lead to a particular “popcorn” appearance of the metaphyses and epiphyses (Fig. 19.3).

Moderate OI (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. As with mild OI, moderate OI has been subdivided into two forms: with and without DI [38].

Mild OI (Type 1) Mild OI typically presents with normal stature and few fractures, mostly during the first 10 years of life. Patients do not present with bowing of the long bones which allows for normal ambulation. It is transmitted as an autosomal dominant trait. Bone density can be very low, with little relationship to clinical severity. Fractures may be present at birth [39], but their incidence decreases 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 [40]. 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. DI can be present, and it has been suggested that this is useful to distinguish discrete forms of mild OI [38]. Early hypoacusia, that may lead to deafness in the third decade of life, is typical of this form of OI. The cause is not clear. Some studies suggest that it is a neuronal syndrome [41], whereas others refer to it as a conductive abnormality [42]. Cardiovascular problems can also be present in these patients, particularly aortic valvular disease [39].

FIGURE 19.3 “Popcorn” appearance of the epiphysis. Severe OI causes distortion of the growth plate, with zones of partially calcified cartilage and broadening of the epiphysis.

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 COL1A1 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 to which one of the COL1A1 alleles underperforms may be one of the determinants of the severity of osteopenia in type I OI. The most frequent cause of diminished activity from a collagen gene is a mutation that introduces a premature stop codon in collagen mRNA [43e45]. This type of mutation leads to rapid destruction of the RNA by a recently described cellular process called nonsense-mediated RNA decay [46e48]. This process appears to be important to prevent 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 [49]. A truncated a1(I) chain produced from a COL1A1 transcript in vitro helps to determine the presence and location of such a stop codon [50]. Otherwise, finding the mutation using a molecular approach is laborious and the mutation can be missed.

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A second cause for underproducing a COL1A1 allele is a mutation that leads to retention of an intron within the mature transcript. Although this is an uncommon cause of type I OI, it has provided insight into the normal pathway for splicing a complex transcript such as collagen [51]. Other causes for diminished transcriptional activity from a collagen gene are extremely rare. Mutations within the 30 untranslated region affecting polyadenylation have been reported, and mutations in the 50 untranslated region are predicted to have a modified phenotype but have not been observed. Finally, a non-functional collagen gene can result from the synthesis of a procollagen chain that is unable to incorporate itself within the triplehelical molecule. Frameshift mutations within the terminal exon of either collagen gene have been identified that lead to the synthesis of full-sized procollagen chains, which are rapidly degraded intracellularly when failing to incorporate into the collagen molecule [52].

Other Clinical Forms of OI Type V OI Some patients with moderate to severe OI develop hyperplastic calluses in long bones that can appear spontaneously or follow fracture or intramedullary rodding (Fig. 19.4A). These patients present with hard, painful, and warm swellings over bones that initially may suggest inflammation or even an osteosarcoma. After a rapid growth period, the size and shape of the callus may remain stable for many years [53], unless a new fracture occurs at the same site. Microscopically, there is increased production of poorly organized extracellular matrix, which is incompletely mineralized [54]. The first description of hyperplastic callus formation in OI was made in 1908 [10]. A number of case reports have been published since [8,55e60]. In a series of 60 patients, 10 (17%) developed hyperplastic callus before age 20 years [61]. In a follow up of 334 patients with OI at the Montreal Shriners, we detected hyperplastic calluses in nine patients (2.6%) (personal communication). Familial occurrence of hyperplastic callus with an autosomal dominant pattern of inheritance has been described [60,62]. These calluses were, in some cases, associated with calcification of the interosseous membrane between radius and ulna and irregular collagen fibril diameter [9,53,63]. Magnetic resonance imaging of the hypercallus is not contributory in the differential diagnosis with osteosarcoma, but computed tomography showing a calcified rim around the lesion associated with the absence of cortical destruction may be useful for ruling out malignancy [64]. It is important to note that, although rare, osteosarcoma may develop in patients with OI [65,66]. The

FIGURE 19.4 OI with hypercallus formation. Individuals with OI with hypercallus formation develop redundant bony formations around fractures (A), which are sometimes confused with osteosarcoma. These patients also present with ossification of the interosseous membrane of the forearm and the leg (B).

hypercallus may also be present in flat bones like the ilium [67]. Glorieux’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 [68]. Matrix lamellae were arranged in a mesh-like fashion, as opposed to the parallel arrangement seen in controls and in patients with mild 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 prevents pronation and supination of the forearm. The membrane between the tibia and fibula may also present with abnormal calcification (see Fig. 19.4B). Of note, there are hyperdense metaphyseal bands under the growth plates of

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the long bones. Their significance is unknown. No patient presented with blue sclerae or DI. Electron microscopy analysis of type V bone showed failure of patches of bone to mineralize [67]. No mutations in the type I collagen genes have been found in this group of patients. No candidate gene has yet been identified. It is inherited as an autosomal dominant trait, with variable penetrance. OI with Mineralization Defect (Type VI) This is a rare form of OI [70], with a prevalence of approximately 6%. Clinically it is undistinguishable from moderate to severe OI. These patients have white sclerae, no DI nor wormian bones. Lumbar spine bone mineral density (BMD) is low, similar to that of moderate OI. Vertebral compression fractures are frequent. The diagnosis is made with an iliac crest bone biopsy, which shows a severe matrix mineralization defect together with a “fish scale” pattern under polarized light. Interestingly, growth plates mineralize normally. Serum parameters of mineral metabolism are normal, except for a 40% increase in alkaline phosphatase levels. There are no mutations in the COL1A1 and COL1A2 genes, and type I collagen protein analysis is normal. Type VI OI is transmitted as a recessive trait. The recent study of a large consanguineous family by homozygosity mapping and next-generation sequencing has allowed the description of loss-of-function mutations in serpin peptidase inhibitor, clade F, member 1 (SERPINF1) in this family and an additional unrelated OI type VI patient. SERPINF1 encodes the pigment epitheliumderived factor (PEDF), a potent anti-angiogenic factor. Thus, loss of PEDF functions represents a novel mechanism for OI and demonstrates its involvement in bone mineralization. Interestingly, PEDF serum levels are undetectable in type VI patients providing an easy disagnostic test (71). In another recent study, truncating mutations in SERPINF1 were found in four unrelated patients with severe OI. Since no histology studies were done in these patients, the possibility that they had type VI OI was not considered [72]. Type VII OI A novel form of OI was discovered in a First Nations community in Quebec [73]. 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 (developing before the age of weight bearing), and osteopenia. Histomorphometrically, bone structure is not different from that of type I OI. It is inherited as an autosomal recessive trait, and the disease locus has been mapped to 3p22-24.1 by linkage analysis [6]. This genomic location excludes COL1A1 and COL1A2 (respectively located in chromosomes 7q and 17q) as candidate genes. It co-localizes

with the gene encoding CRTAP, a cartilage-associated protein involved in the prolyl 3-hydroxylation of type I collagen, a critical step in the processing of the triple helix. In homozygous type VII patients, an intronic mutation in the CRTAP gene causes a 90% reduction in CRTAP mRNA and protein, and the “rhizomelic” form of OI observed in the First Nations community [74]. In some pedigrees where lethal OI was evident, a complete ablation of CRTAP expression correlated with lethal OI [74,75]. These observations underline that a 10% expression of CRTAP makes the difference between survival and early lethality. Other Recessive Forms of OI CRTAP is a critical component of an intracellular complex that controls the folding of the type I collagen triple helix. The intracellular complex is made of CRTAP, LEPRE1 and cyclophilin B (PPIB). It is responsible for a1 (I) Pro986 residue hydroxylation which plays a key role in the folding of the Type I triple helix, and probably in the protein-collagen interactions required for bone formation. Homozygous null mutations in either one of those three genes have been recently associated with an OI phenotype of variable severity(moderate to lethal) [76e77]. Specific chaperones are also involved in the processing (HSP47, encoded by SERPINH1) and secretion (FKBP65, encoded by FKBP10) of the Type I molecule. Mutations affecting the activity of these two proteins have been discovered in recessive, severe forms of OI [78e79]. These findings shed new light on the pathophysiology of OI, and may ultimately lead to novel therapeutic approaches. OSTEOPOROSISePSEUDOGLIOMA SYNDROME

This form of OI also called OI with blindness was first described in 1972 in three families [80]. Subsequently, the syndrome was described in a South African family of Indian stock [81]. 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 USA 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. The genetic defect was mapped to chromosome region 11q12-13, and later it was shown that the defect is in the LRP5 gene, which encodes for the low-density lipoprotein receptor-related protein 5 [82a]. 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

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affected siblings of a consanguineous family [82b]. Two other forms of OI with ocular involvement have been described: one variant with optic atrophy, retinopathy, and severe psychomotor retardation [83] and another with microcephaly and cataracts [27]. OI WITH CRANIOSYNOSTOSIS AND OCULAR PROPTOSIS (COLEeCARPENTER SYNDROME)

Two boys and a girl have been described with this particular form of OI [84,85]. 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. 19.5), and normal intellectual and neurological development (unpublished data). The gene defect is currently unknown. OI WITH CONGENITAL JOINT CONTRACTURES (BRUCK SYNDROME)

This form of OI was first described by Bruck et al. in 1897 in an adult patient [86]. Patients are born with brittle bones, leading to multiple fractures and joint contractures and pterygia (arthrogryposis multiplex congenita) [87,88]. Wormian bones are present in the skull. It appears to be inherited in a recessive fashion [88,90]. In three patients studied, it was not possible to demonstrate any mutations in the COL1A1 and COL1A2 genes [88]. The basic defect in this syndrome was mapped to locus 17p12 (18 cM interval), and a defect in bone specific telopeptidyl hydroxylase encoded by PLOD2 has been identified [9]. This leads to underhydroxylated 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 [89]. This form of the syndrome is now called Bruck Type 2. Bruck Type 1 may occur in association with FKBPIO mutations. The two forms cannot be distinguished clinically [91].

DIFFERENTIAL DIAGNOSIS Frequently, family history, clinical features and biochemical profile are sufficient for diagnosing OI. When feasible, a bone biopsy with histomorphometric analysis is best for making a differential diagnosis and characterizing specific forms. Genetic testing is also

FIGURE 19.5 Severe bony involvement in a patient with ColeeCarpenter 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.

useful, although since it is not always possible to find mutations in the COL1A1 and COL1A2, genes the diagnosis of OI should not only rely on such tests. Currently, two laboratories in the USA 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 and 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 since 80% of bone mineralization in the fetus occurs during the third trimester [92]. Inadequate postnatal management of parenteral or enteral nutrition may also lead to osteopenia. Non-accidental injury (NAI) is one of the most challenging differential diagnoses of OI [93]. Although the social history may be contributory and certain signs are suggestive of NAI [93], such as hand fractures in the non-ambulant child, acromial fractures, fractures of

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the outer end of the clavicle, and spinal, posterior rib, and metaphysial fractures [94, 95], diagnosis is often difficult [96]. Metaphyseal fractures may occur in children with OI but probably only in the presence of obvious bone disease with radiologically abnormal bones [97]. This is complicated by the fact that children with OI may also suffer NAI [98,99]. It is important to note that there are no pathognomonic radiological signs of NAI [100]. Idiopathic juvenile osteoporosis (IJO) is another difficult differential diagnosis (see Chapter 18). IJO is an acquired form of osteoporosis and its main symptoms are vertebral compressions and ensuing scoliosis and disability. If these signs are mild, it is often difficult to make the diagnosis in the absence of 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 The condition appears restricted to cancellous bone with cortical bone hardly touched. This explains why long bone fractures are not frequent [101]. Hypophosphatasia can resemble severe OI clinically, but low levels of serum alkaline phosphatase activity and characteristic alteration of the growth plates make the diagnosis [102] (see Chapter 28). Other differential diagnoses include Cushing’s disease, glucocorticoidinduced osteoporosis, homocystinuria, lysinuric protein intolerance, glycogen storage disease, congenital indifference to pain, calcium deficiency, malabsorption, immobilization, anticonvulsant therapy, and acute lymphoblastic leukemia [103].

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 N-telopeptide 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 [104]. 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 [105]. In severely affected children, hypercalciuria may be present [106], but there is no compromise of renal function [107]. Kidney stones and nephrocalcinosis may also be present [7].

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 predominance for this complication [108]. 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 [109]. Sawin and Menezes [108] 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 [108]. One case of paraplegia occurring in an adolescent girl with OI after chiropractic manipulation has been reported [110]. Reflex sympathetic dystrophy has been described in adults with OI [111]. The cases described in the literature occurred in patients 26e59 years of age. The incidence of this condition in OI patients is not clear [112]. Other neurological manifestations of OI include benign communicating hydrocephalus, macrocephalus, cerebral atrophy [113], usually with no alteration of intellectual status, and idiopathic 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 [114,115]. Hypoacusia is present in approximately 50% of individuals with mild forms of OI, generally only after the third decade of life [116]. However, this problem is probably more prevalent than appreciated because of the lack of proper studies in children. King and Boblechko [9] 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% [117, 118]. With increasing age, the prevalence of hearing impairment in patients with OI may be approximately 100% [119]. Hearing loss may be due to otosclerosis [120, 121], to middle and inner ear pathology [122, 123],

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or a neuronal syndrome [41]. 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 [124]. It has also been recommended that children with OI undergo audiometry at 10 years of age and repeat the study every 3 years thereafter [125]. Stapedectomy has been performed in patients with OI with success [126e128]. 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 [123].

Cardiovascular Involvement There are several published reports of congenital malformations of the heart in children with OI [7,129], but their incidence is probably not higher than that in the unaffected population. In a series of 58 children with OI, four (6.9%) had congenital cardiac malformations [130]. Aortic regurgitation was present in only 2% of patients in another series of affected individuals, whereas aortic root dilatation was present in 12.1% [129]. Dilatation was mild [129,131]. The prevalence of mitral valve prolapse varies from 3.4% [130] to 6.9% [129] in published series, which is not different from the prevalence of mitral valve prolapse in the general population (4e8%) [132]. Others have found that the prevalence of mitral valve prolapse in OI is slightly higher (10%) than in the normal population [131]. These lesions are rarely clinically important [19]. Valve replacement has been performed successfully in patients with OI [133e135]. Epoetin-a has been used to increase hematrocrit preoperatively in mitral valve replacement surgery because of the high risk of perioperative bleeding [136]. Ulnar artery aneurysm has been reported in a patient with OI [137], which may be due to increased weakness of vessel walls that can also produce spontaneous carotidecavernous fistulas [138].

Renal Involvement Hypercalciuria is a common finding in children with OI, being present in 36% of a series of 47 patients [106,107]. 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, 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 [107]. In a series of 58 patients, four patients developed kidney stones and one had papillary calcification without kidney stones. However, it was not clear if these patients were hypercalciuric [130]. One patient was

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described in the literature with chronic renal failure secondary to obstructive uropathy caused by bony pelvic outlet deformities [139].

Endocrine Changes Growth hormone (GH) deficiency is rare in patients with OI. Of 22 children with OI tested by Marini et al. [140], none fulfilled the standard criteria for GH deficiency. Children with OI may present with hypopituitarism [141]. Some patients with OI have a hypermetabolic state, typically reflected by excessive diaphoresis and associated with increased oxygen consumption and elevated thyroxine levels [142]. The cause of this hypermetabolic state is not known. For reasons that are unclear, women with OI have late menarche [143].

Respiratory Problems 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 [144]. Pulmonary hypoplasia has been described in a newborn with lethal OI [23]. Studies of pulmonary function in patients with OI may show different results [144], 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 [20].

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 [145]. Skin of people with OI is stiffer and less elastic than normal skin [146]. Muscle strength is reduced in moderate and severe forms of OI [147,148]. Joint hyperlaxity is common, especially in affected females [149], 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 [150]. Constipation is common, which may be due to severe protusio acetabuli and pelvic deformation in children with severe OI [151]. Treatment of constipation is difficult and frequently frustrating.

Ocular Changes Individuals with mild OI frequently demonstrate blue sclerae and premature arcus corneae. Arcus

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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 [12]. In subjects with mild OI, there is a progressive arcus corneae that can first be seen in some patients in their late teens [152]. Contrary to what is commonly stated, scleral thickness is normal in mild OI, 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 [21]. Scleral collagen fibrils are of normal diameter in mild OI, but there is an increase in electron-dense granular matrix material between collagen fibrils [153]. 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 mild OI [152], as in other types of OI.

Teeth Some individuals with OI have DI (Fig. 19.6) [154]. Although the enamel of the teeth with DI is normal anatomically, it may not attach normally to the dentin [155]. 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 [156]. The severity of DI is not related to the severity of skeletal involvement in the case of OI, 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 [157]. The primary dentition is always more affected than the permanent dentition. Radiographically, the teeth show bulbous crowns with a constriction at the coronaleradicular junction. The roots are shorter and more slender than normal. The pulpal spaces are narrow or obliterated [158]. Subjects with OI do not have an increased susceptibility to cavities

FIGURE 19.6 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.

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. Malocclusion 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 [159]), and the prevalence is 60e80% [157,160]. This complication is more common than DI which has a prevalence of approximately 28% [160]. Patients may require surgical correction of the malocclusion [161]. Changes in the position of the basal bones also may require orthognathic surgery, which has been performed successfully in these patients [162,163]. Unerupted first and second molars are frequent in OI patients in permanent dentition, which is rare in the general population [160]. Other abnormalities include invaginations and hypodontia [164], 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 [165].

Birth and Anesthetic Complications and Life Expectancy There is an increased incidence of breech presentation of the OI fetus at term [166]. A retrospective study on the mode of delivery of children with OI concluded that cesarean delivery does not appear to decrease fracture rate at birth in infants with non-lethal forms of OI, nor does it prolong survival for those with lethal forms [166]. Patients with OI should be considered as high risk for anesthesia [167]. 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 [168]. 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 usually not associated with muscle rigidity and rarely progresses to malignant hyperthermia (MH) [169,170], although a case of MH in OI has been described in the literature [170]. MH is a familial disease, and patients with OI may also be affected, but prophylactic use of dantrolene in these patients is not warranted [169] because MH is considered to be a coincidental occurrence in patients with OI [171]. 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 [169].

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Despite all these potential problems, life expectancy in subjects with non-lethal OI appears to be the same as that for the normal population [172], except in cases of severe OI with respiratory or neurological complications [173].

PATHOPHYSIOLOGY Collagen plays an essential role in forming an interactive network between the cells that make the extracellular matrix [174] and non-collagenous 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.

Formation of Osteoid and Mineralization The impact of the glycine substitution on the structure of the collagen triple-helical structure has been demonstrated by x-ray diffraction [175], nuclear magnetic resonance imaging, and circular dichroism [176e179]. 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 well-defined lateral growth and more fiber disorganization in tissue obtained from OI subjects [180]. 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 [181]. Mutations that interrupt the helix decrease the thermal stability of procollagen molecules and render the molecules more susceptible to proteolytic attack by tissue proteases [182]. This may explain the observation that mutant collagen molecules are not uniformly distributed throughout matrix but are found on the surface of bone [183]. Tissue proteases probably select against the mutant molecules [184], 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 [185,186]. For example, copolymerization of type V collagen within the type I collagen fibril influences the size and structure of the type I collagen fibril [187,188]. Another modifier of collagen fiber size is the incorporation of unprocessed type I procollagen producing another form of EhlerseDanlos syndrome (EDS) that can overlap with features of mild OI. The EDSeOI-like symptoms appear to result from impairing

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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 [189]. Mutations in non-collagenous proteins such as decorin [190], fibromodulin [191,192], and microfibrillin [193] 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. The recent reports of OI associated mutations affecting the activity of the HSP47 and FKBP65 chaperone molecules confirm the importance of those interactions [78,79]. 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 [194e199]. How the helix influences the interaction of non-collagenous proteins and mineral is not fully understood.

Function of the OI Osteoblast The rough endoplasmic reticulum of OI fibroblasts and osteoblasts is grossly dilated [200] and the secretion of fully formed but mutant procollagen is impaired [201,202]. 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 [203]. Recently mutations in SERPINH1 encoding HSP47 have been identified in patients with recessive severe OI [79]. In fact, gene knockout of the HSP47 protein is embryonic lethal. An abnormal type of collagen accumulates [204], suggesting that this chaperone protein plays an essential role in selecting for correctly assembled collagen molecules [205]. The retention of the mutant procollagen molecule also leads to post-translational 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 [204,205] or Oim mice [206] show diminished markers of osteoblastic differentiation, as well as a reduced rate of 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.

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(A)

(B)

(C)

FIGURE 19.7 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.

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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 [207,208]. 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.

Metabolic Activity of OI Bone 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. 19.7). 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 [78] and an increased number of doublelabeled surfaces of normal thickness [209]. In the case of type I OI, the amount of bone formed during a remodeling cycle is decreased compared to controls [209]. It is of note that the occurrence of non-union fractures is increased in children with OI [210], 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 [211e213], 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 non-lethal 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 [214]. The activated osteoblastic lineage can be demonstrated by measuring the content of COL1A1 mRNA 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

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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. 19.8). 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 [214]. 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. The heterozygous Mov 13 mouse is a murine model for mild non-deforming OI. Affected mice demonstrate half of normal levels of COL1A1 mRNA as a consequence of inactivation by a retroviral insertional event. The bones show diminished cortical thickness, which is consistent with human OI, and low levels of procollagen propeptide in blood reflect the low output of the type I collagen-producing cells [215]. Histomorphometry does show increased osteoblast cellularity and boneforming units, and dynamic histomorphometry suggests a decrease in osteoid seams [216]. Excessive

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FIGURE 19.8 Bone formation and resorption 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 resorbed through 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 mass is achieved in early adulthood. In OI, bone resorption is high due to the effort to remove defective matrix, and most of the bone-forming capacity 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, gain in bone mass and somatic growth occur 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 resorption, the deficit in bone formation relative to bone loss will return.

osteoclastic activity does not appear to be present. In both mouse and humans with mild OI, significant skeletal remodeling is apparent upon sexual maturation so that the mechanical properties of the bone are near normal [217,218]. 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 mild 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.

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 [219], including vitamin C [220,221], sodium fluoride

[222e224], magnesium [225,226], and anabolic steroids [227,228]. 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 [229e231]. 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 [232,233]. The use of calcitonin treatment for OI has been abandoned.

Antiresorptive Agents Pamidronate is a second-generation bisphosphonate with a chemical structure based on pyrophosphate, the only naturally occurring inhibitor of bone resorption [234]. The exact mechanism of action of the bisphosphonates remains unclear, although effects on both osteoblasts [235,236] and osteoclasts [237] have been documented. There have been several case reports of treatment of children with OI with bisphosphonates [238e242]. Glorieux and his group administered pamidronate by intermittent intravenous infusion for

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up to 9 years in more than 150 children with severe OI aged 2 months to 18 years. In the first publication [243], 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: 2.6e20.7 months) received pamidronate treatment for 12 months [244]. The drug was administered intravenously in cycles of three 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 65e227% in 1 year. The z score increased significantly, whereas in the control group a significant 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 19.9 and 19.10). 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 [12]. However, despite higher risk of injury due to increased mobility, a marked decrease in fracture

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rate was noted, suggesting a direct effect of therapy. Bisphosphonates, on the other hand, do not appear to interfere with fracture healing [245]. The disappearance of bone pain and decreased fracture incidence may have contributed to greater mobility [246]. Physical activity is an essential factor for the development of the skeletal system [247]. 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 [248]. 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 [244]. In a larger group of patients, height Z scores increased significantly in patients with severe OI and did not change in patients with moderate OI after 1 year of pamidronate therapy. After 4 years of pamidronate therapy, mean height Z-scores increased significantly in children with moderate OI, while patients with mild and severe OI showed non-significant trends of increase [249]. The success of bisphosphonate treatment in patients with Paget’s 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 glycosides [250] and the collagen cross-links pyridinoline and deoxypyridinoline. Plasma alkaline phosphatase activity (as a measure of bone osteoblast activity) also decreases [251,252]. 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 48e72 hours. Delayed osteotomy healing may be present with chronic pamidronate use and it is thus prudent after an osteotomy to withhold treatment until radiological evidence of healing is observed [252a]. 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 [253] 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

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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 in the growing skeleton pamidronate has a twofold 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 patients studied. Biochemistry studies [104] 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 N-telopeptide related to creatinine (uNTX/uCr) decreases by approximately

60e70%. Two to 4 months later, ionized calcium returns to pretreatment levels, and parathyroid hormone concentrations are still above baseline values in patients below 2 years of age. During 4 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 4 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.

FIGURE 19.9 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 metaphyses that each corresponds to a treatment cycle.

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FIGURE 19.9 (continued).

An adequate 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 known. Therefore, this therapy should be administered within strict research protocols. Densitometric studies have shown that gain in BMD levels off after 3e4 years of treatment [253a], but that discontinution

in a growing patient may casuse fractures in the newly formed (untreated) bone. It may thus be prudent to continue therapy at a maintenance dose until epiphyses are fused [253b].

Anabolic Agents Growth hormone, insulin-like growth factor-1, and parathyroid hormone have the potential to increase

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this is another therapeutic setting requiring animal experimentation for concept validation.

Orthopedic Management

FIGURE 19.10 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 on the left.

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 [254,255]. 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 [256]. Reported side effects of GH therapy are arthralgia, myalgia, carpal tunnel syndrome, pseudotumor cerebri, benign intracranial hypertension, slipped capital femoral epiphysis, and transient insulin resistance [257]. 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 [258]. Like all children who are initially started on GH, OI children do experience an initial acceleration of growth rate [105]. Given the underlying physiological basis of OI, it would be surprising if an agent that further stimulates more bone turnover as part of its anabolic action has a long-term 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 combination may be of interest. However,

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 (Fassier, personal communication). For the femora, the extensible rods (DuboweBailey [259,260] or FassiereDuval [261]) 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 non-ambulatory patients with OI are able to walk [262]. The complication rate for DuboweBailey rods ranges from 63.5% [263] to 72% [264] and is approximately 50% for non-elongating 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 [263]. A new elongating rod, called FassiereDuval [261], 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. Postoperative 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 a 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% [71,265]. More than half of the cases of scoliosis are located in the thoracic region, and pectus carinatum and pectus excavatum are common associated findings [266]. These deformities increase with age [265,267], and bracing does not stop the progression of the curve [268]. Thoracic scoliosis of more than 60 has severe adverse effects on pulmonary function in patients with OI [269]. Fusion with bank bone graft [270] and with Keil bone graft without instrumentation [271] has been used for the treatment of severe scoliosis in patients with OI, as has halo gravity traction and posterior spondylodesis with instrumentation [272]. The ideal surgical treatment of severe scoliosis in patients with OI has yet to be determined, and it remains a difficult procedure [273].

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THERAPY

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 [274]. When a patient has a fracture and is immobilized for a certain period of time, the bone density declines dramatically [275]. 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 [276]. 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 [277]. The aim of these programs is to employ children in graded exercise regimens 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 [278]. 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 (Ruck-Gibis and Montpetit, personal communication). Psychosocial aspects are extremely important in the management of patients with OI. Issues regarding self-esteem, sexuality, and peer integration must be addressed to care properly for these patients, particularly during adolescence [279]. 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: • the school must be accessible for handicapped children • the school should have an access ramp, accessible toilets, mobile tables and chairs, and a wheelchair in case of emergency • the school should have an emergency evacuation plan adapted to handicapped children in case of fire

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• some OI children lack balance and need better supervision in school yards or on icy surfaces • OI children should leave 5 min before the end of classes in order to avoid crowds • 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 • the equipment used should be soft (e.g. balls) • the child should rest when he or she is tired • the child should wear ortheses at all times • 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 Therapeutic Options 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 mutation 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 [280]. 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 the bone environment of an individual with OI, the severity of bone disease would be reduced because the high bone turnover and relative inefficiency of bone matrix formation of the bone cells containing the OI mutation provides the opportunity for replacement of the existing matrix by cells that make ample amounts of a normal matrix. The ability of the replacement cells to out compete and perform the endogenous cells is a ideal setting for cell-based therapy of a disease that impairs the effectiveness of the host cells [281,282]. While there are many experimental and practical obstacles to obtaining this end, significant progress is being made to plot a path that has the potential to be clinically applicable. Three of the major challenges will be considered here.

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Source of the Cells for Gene Correction Due to the concerns of long-term immune suppression, cell therapy using allogenic cells from a normal individual is not an attractive option. Instead, cells derived from the affected individual will need to be used in a way that allows for correction of the genetic mutation while still maintaining their ability to regenerate bone when reintroduced into the source individual. Currently, mesenchymal cells derived from bone marrow [283,284] or adipocyte [285] stromal cells are receiving attention as a source of cells for bone repair. The main disadvantage of these cells is their limited capacity for cell division and their subsequent potential for osteogenesis. Because the genetic engineering steps will require selection of a single molecular event and subsequent cell expansion for testing prior to further expansion for therapy, adult somatic cells are unlikely to be satisfactory. With the advent of induced pleuripotential (iPS) human cells, the possibility of unlimited cell division and maintenance of a pleuripotential capacity makes these cells ideal for correction of a genetic defect prior to their subsequent differentiation to cell type specific progenitor cells [286,287]. iPS cells can be developed from skin fibroblasts by delivering a cocktail of four transcription factors that initiate a process that removes the structural features of a differentiated cell back to a cell with a chromatin structure resembling an embryonic stem cell. The concern associated with the use of multiple viral transductions to induce the iPS state are being addressed with the use of non-integrating vectors [288,289], polycistronic transcription factor expressing viral construct flanked by recombination excision sites [290], transfection of RNA encoding the transcription factors [291] or small molecule mimickers of the factors [292]. Thus generating autologous progenitor cells for genetic manipulation should not be an issue, however, directing these cells to become reliable osteoprogenitor cells still remains a challenge for the field. Correction of the Genetic Defect Underlying OI Since the vast majority of the dominantly inherited forms of OI result from the accumulation of a misfolded type I collagen molecule, the initial approach requires inactivation of the allele that is producing the abnormal type I collagen chain. In the previous edition of this chapter, the use of an antiRNA strategy appeared to be the most promising approach [293,294], but this has not proven to be very effective or practicable. Instead, advances in genetic recombineering, i.e. that ability to target a specific sequence with the genome for inserting a gene sequence using the cell’s natural ability to repair minor damage to DNA, has become relevant to human gene therapy. The first demonstration for allele-specific gene inactivation was applied to a Col2A1 mutation

using a small viral vector that carried a gene-inactivating mutation that was targeted to the first intron of the Col2A1 gene [295,296]. The resulting cell clones that were selected for insertion at the targeted site ceased production of the mutant collagen, while those that targeted the normal allele were severely abnormal for collagen production. Thus selection for the desired molecular event is an essential element of any gene inactivation strategy with particular attention to eliminate clonal cell lines that have a second recombination event at another off-target site. A strength of the approach for gene inactivation is that it can be generic for all mutations of either chain, but it has the disadvantage that the resulting cells are haploid insufficient for type I collagen production. Ideally, replacing the activity of the inactivated allele could be a secondary step but the size and complexity of the collagen gene makes this a formidable problem. Other recombinatorial strategies using small oligonucleotides [297,298] or DNA fragments that promote integration [299,300] probably lack the specificity or frequency to be practical for diploid cell applications. A promising new strategy is the targeting of recombination in a region close to the mutation by introducing a double stranded break within the DNA. When the break occurs in the presence of a correcting DNA fragment, it will become incorporated into the host DNA as the cell repairs the DNA break [301]. The key to this approach is the cutting of the DNA at only one site within the entire genome using custom DNA binding sites that have a recognition sequence of 12 or more bases in contrast to the 4e6 base specificity of commonly used restriction enzymes. Two competing strategies are receiving the most attention. Zn finger nucleases are synthetic chimeric peptides that bind to a specific nucleotide sequence (Zn finger) that flank a site where scission is desired. The pair of binding proteins also carry complementary elements of a DNA nuclease that only activates when placed in close proximity [302,303]. Meganucleases are enzymatically active proteins that have been engineered to recognize a specific nucleotide sequence and inactivate an endogenous gene [304,305], but to date have not been used in combination with a recombining fragment for gene correction. It can be anticipated that many variations on the concept of specific DNA cleavage in concert with delivery of a repairing DNA fragment will appear and those which prove to be practical in patient specific iPS cells will eventually lead to correction of the mutation. Delivery of the Corrected Bone Progenitor Cells to the Host Bone Environment A number of publications describing systemic delivery of bone marrow-derived cells have claimed engraftment of osteoblastic cells in both adult rodents

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REFERENCES

and human [306] subjects. In all of these cases, the level of engraftment has been very low, probably too low to have a clinical effect, and the longevity of the engraftment has been limited to a few months [307,308]. Potentially better engraftment has been observed when the correcting cells are administered in the neonatal or late embryonic stage of development, and this approach may circumvent the problem of immune rejection of the implanted cells [309,310]. The evidence for engraftment has been the presence of a histological or fluorescent marker of donor derived cells in close association with the bone surface and, in some cases, with supporting evidence that the engrafted cells express bone-specific genes. However, in all these cases, the markers of donor source do not reflect the lineage of the engrafted cells and it is now clear that cells of the myeloid lineage also reside in close proximity to the bone surface [311]. When a reporter that is active only in differentiated osteoblasts is used to assess engraftment, none is observed whether by direct vascular injection, marrow transplantation [312] or from a parabiotic source [313]. Cells that do populate the bone surface have markers of myeloid cells with many having the morphometry of a mononuclear TRAP positive cell [314]. However, when bone progenitor cells (not fresh bone marrow) are implanted directly into the bone marrow space or within a bone repair defect, donor derived bone is produced as assessed by the host and donor bone-specific gene reporters [312]. The longevity of the transplanted cells can persist over 1 year after engraftment with continued new bone formation. Thus, as the technology currently stands, at least in adult rodent models, engraftment has to be done locally with the expectation that local bone remodeling will disperse the injected progenitor cells to sites of active bone turnover [315]. Another major challenge for patient specific iPS cells to be employed as a source for cell therapy is a preimplantation protocol that will reliably direct the totipotential cells to a progenitor status capable of sustained bone formation after transplantation [316]. Developing convincing preclinical data that human-derived progenitor cells, particular those derived from iPS cells, are capable of ameliorating the severity of bone disease will require developing murine models of OI in an immune compromised background capable of long-term human cell engraftment. While all of the experimental components are now available to develop these protocols, they will require a long-term sustained effort and a highly critical evaluative process to ensure that any procedure that progresses to clinical trial is safe and effective.

References [1] Rauch F, Glorieux FH. Osteogenesis imperpecta. Lancet 2004; 363:1377e85.

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[2] Komai T, Kunii H, Ozaki Y. A note on the genetics of Van der Hoeve’s syndrome, with special reference to a large Japanese kindred. Am J Hum Genet 1956;8:110e9. [3] Heiberg A. Osteogenesis imperfecta in Norway, a clinical and genetic study (Abstract). Clin Genet 1983;23:233. [4] Sillence DO, Senn A, Danks DM. Genetic heterogeneity in osteogenesis imperfecta. J Med Genet 1979;16:101e16. [5] Andersen PE, Hauge M. Osteogenesis imperfecta: a genetic, radiological, and epidemiological study. Clin Genet 1989;36: 250e5. [6] Smith R. Osteogenesis imperfecta: from phenotype to genotype and back again. Int J Exp Pathol 1994;75:233e41. [7] Vetter U, Pontz B, Zauner E, Brenner RE, Spranger J. Osteogenesis imperfecta: a clinical study of the first ten years of life. Calcif Tissue Int 1992;50:36e41. [8] McKusick VA. Mendelian Inheritance in Man. 9th ed. Baltimore: Johns Hopkins University Press; 1990. [9] King JD, Boblechko WP. Osteogenesis imperfecta: An orthopaedic description and surgical review. J Bone Joint Surg 1971; 53B:72e89. [10] Sillence D. Osteogenesis imperfecta: an expanding panorama of variants. Clin Orthop 1981;159:11e25. [11] Paterson CR, McAllion S, Miller R. Osteogenesis imperfecta with dominant inheritance and normal sclerae. J Bone Joint Surg Br 1983;65:35e9. [12] Sillence D, Butler B, Latham M, Barlow K. Natural history of blue sclerae in osteogenesis imperfecta. Am J Med Genet 1993; 45:183e6. [13] Beighton P, Versfeld GA. On the paradoxically high relative prevalence of osteogenesis imperfecta type III in the black population of South Africa. Clin Genet 1985;27:398e401. [14] Williams EM, Nicholls AC, Daw SC, et al. Phenotypical features of an unique Irish family with severe autosomal recessive osteogenesis imperfecta. Clin Genet 1989;35:181e90. [15] Zlotogora J. Germ line mosaicism. Hum Genet 1998;102:381e6. [16] Constantinou-Deltas CD, Ladda RL, Prockop DJ. Somatic cell mosaicism: another source of phenotypic heterogeneity in nuclear families with osteogenesis imperfecta. Am J Med Genet 1993;45:246e221. [17] Wallis GA, Starman BJ, Zinn AB, Byers PH. Variable expression of osteogenesis imperfecta in a nuclear family is explained by somatic mosaicism for a lethal point mutation in the alpha 1(I) gene (COL1A1) of type I collagen in a parent. Am J Hum Genet 1990;46:1034e40. [18] Byers PH, Tsipouras P, Bonadio JF, Starman BJ, Schwartz RC. Perinatal lethal osteogenesis imperfecta (OI type II): a biochemically heterogeneous disorder usually due to new mutations in the genes for type I collagen. Am J Hum Genet 1988;42: 237e48. [19] Rowe DW, Shapiro JR. Osteogenesis imperfecta. In: Avioli LV, Krane SM, editors. Metabolic Bone Disease and Clinically Related Disorders. 3rd ed. San Diego: Academic Press; 1998. p. 651e95. [20] Shapiro JR, Burn VE, Chipman SD, et al. Pulmonary hypoplasia and osteogenesis imperfecta type II with defective synthesis of alpha 1(I) procollagen. Bone 1989;10:165e71. [21] Pauli RM, Gilbert EF. Upper cervical cord compression as cause of death in osteogenesis imperfecta type II. J Pediatr 1986;108:579e81. [22] Andrews M, Amparo EG. In utero clue to congenital lethal osteogenesis imperfecta [Letter]. Am J Roentgenol 1993; 160:212. [23] Brons JT, van der Harten HJ, Wladimiroff JW, et al. Prenatal ultrasonographic diagnosis of osteogenesis imperfecta. Am J Obstet Gynecol 1988;159:176e81.

PEDIATRIC BONE

532

19. OSTEOGENESIS IMPERFECTA

[24] Brown BS. The prenatal ultrasonographic diagnosis of osteogenesis imperfecta lethalis. J Can Assoc Radiol 1984;35:63e6. [25] Sidden CR, Filly RA, Norton ME, Kostiner DR. A case of chondrodysplasia punctata with features of osteogenesis imperfecta type II. J Ultrasound Med 2001;20:699e703. [26] Heller RH, Winn KJ, Heller RM. The prenatal diagnosis of osteogenesis imperfecta congenita. Am J Obstet Gynecol 1975; 121:572e3. [27] Buyse M, Bull MJ. A syndrome of osteogenesis imperfecta, microcephaly, and cataracts. Birth Defects Orig Artic Ser 1978; 14:95e8. [28] Prockop DJ. Mutations in collagen genes. Consequences for rare and common diseases. J Clin Invest 1985;75:783e7. [29] Cole WG, Dalgleish R. Perinatal lethal osteogenesis imperfecta. J Med Genet 1995;32:284e9. [30] Edwards MJ, Wenstrup RJ, Byers PH, Cohn DH. Recurrence of lethal osteogenesis imperfecta due to parental mosaicism for a mutation in the COL1A2 gene of type I collagen. The mosaic parent exhibits phenotypic features of a mild form of the disease. Hum Mutat 1992;1:47e54. [31] Sillence DO, Barlow KK, Garber AP, Hall JG, Rimoin DL. Osteogenesis imperfecta type II delineation of the phenotype with reference to genetic heterogeneity. Am J Med Genet 1984; 17:407e23. [32] Byers PH, Shapiro JR, Rowe DW, David KE, Holbrook KA. Abnormal alpha 2-chain in type I collagen from a patient with a form of osteogenesis imperfecta. J Clin Invest 1983;71:689e97. [33] Mundlos S, Chan D, Weng YM, Sillence DO, Cole WG, Bateman JF. Multiexon deletions in the type I collagen COL1A2 gene in osteogenesis imperfecta type IB. Molecules containing the shortened alpha2(I) chains show differential incorporation into the bone and skin extracellular matrix. J Biol Chem 1996; 271:21068e74. [34] Wang Q, Forlino A, Marini JC. Alternative splicing in COL1A1 mRNA leads to a partial null allele and two in-frame forms with structural defects in non-lethal osteogenesis imperfecta. J Biol Chem 1996;271:28617e23. [35] McBride Jr DJ, Shapiro JR, Dunn MG. Bone geometry and strength measurements in aging mice with the oim mutation. Calcif Tissue Int 1998;62:172e6. [36] Robinson LP, Worthen NJ, Lachman RS, Adomian GE, Rimoin DL. Prenatal diagnosis of osteogenesis imperfecta type III. Prenat Diagn 1987;7:7e15. [37] Versfeld GA, Beighton PH, Katz K, Solomon A. Costovertebral anomalies in osteogenesis imperfecta. J Bone Joint Surg Br 1985;67:602e4. [38] Levin LS, Salinas CF, Jorgenson RJ. Classification of osteogenesis imperfecta by dental characteristics [Letter]. Lancet 1978;1:332e3. [39] Smith R. Osteogenesis imperfecta. Clin Rheum Dis 1986;12: 655e89. [40] Match RM, Corrylos EV. Bilateral avulsion fracture of the triceps tendon insertion from skiing with osteogenesis imperfecta tarda. A case report. Am J Sports Med 1983;11:99e102. [41] Shapiro JR, Pikus A, Weiss G, Rowe DW. Hearing and middle ear function in osteogenesis imperfecta. J Am Med Assoc 1982; 247:2120e6. [42] Pedersen U. Hearing loss in patients with osteogenesis imperfecta. A clinical and audiological study of 201 patients. Scand Audiol 1984;13:67e74. [43] Redford-Badwal DA, Stover ML, Valli M, McKinstry MB, Rowe DW. Nuclear retention of COL1A1 messenger RNA identifies null alleles causing mild osteogenesis imperfecta. J Clin Invest 1996;97:1035e40.

[44] Slayton RL, Deschenes SP, Willing MC. Nonsense mutations in the COL1A1 gene preferentially reduce nuclear levels of mRNA but not hnRNA in osteogenesis imperfecta type I cell strains. Matrix Biol 2000;19:1e9. [45] Willing MC, Deschenes SP, Scott DA, et al. Osteogenesis imperfecta type I: molecular heterogeneity for COL1A1 null alleles of type I collagen. Am J Hum Genet 1994;55:638e47. [46] Serin G, Gersappe A, Black JD, Aronoff R, Maquat LE. Identification and characterization of human orthologues to Saccharomyces cerevisiae Upf2 protein and Upf3 protein (Caenorhabditis elegans SMG-4). Mol Cell Biol 2001;21:209e23. [47] Frischmeyer PA, van Hoof A, O’Donnell K, Guerrerio AL, Parker R, Dietz HC. An mRNA surveillance mechanism that eliminates transcripts lacking termination codons. Science 2002;295:2258e61. [48] Maquat LE. Nonsense-mediated mRNA decay. Curr Biol 2002; 12:R196e7. [49] Medghalchi SM, Frischmeyer PA, Mendell JT, Kelly AG, Lawler AM, Dietz HC. Rent1, a trans-effector of nonsensemediated mRNA decay, is essential for mammalian embryonic viability. Hum Mol Genet 2001;10:99e105. [50] Bateman JF, Freddi S, Lamande SR, et al. Reliable and sensitive detection of premature termination mutations using a protein truncation test designed to overcome problems of nonsensemediated mRNA instability. Hum Mutat 1999;13:311e7. [51] Johnson C, Primorac D, McKinstry M, McNeil J, Rowe D, Lawrence JB. Tracking COL1A1 RNA in osteogenesis imperfecta. Splice-defective transcripts initiate transport from the gene but are retained within the SC35 domain. J Cell Biol 2000; 150:417e32. [52] Willing MC, Pruchno CJ, Byers PH. Molecular heterogeneity in osteogenesis imperfecta type I. Am J Med Genet 199;45:223-7 [53] Banta JV, Schreiber RR, Kulik WJ. Hyperplastic callus formation in osteogenesis imperfecta simulating osteosarcoma. J Bone Joint Surg 1971;53:115e22. [54] Morike M, Windsheimer E, Brenner R, et al. Effects of transforming growth factor beta on cells derived from bone and callus of patients with osteogenesis imperfecta. J Orthop Res 1993;11:564e72. [55] Roberts JB. Bilateral hyperplastic callus formation in osteogenesis imperfecta. J Bone Joint Surg 1976;58:1164e6. [56] Burke TE, Crerand SJ, Dowling F. Hypertrophic callus formation leading to high-output cardiac failure in a patient with osteogenesis imperfecta. J Pediatr Orthop 1988;8:605e8. [57] Burchardt AJ, Wagner AA, Basse P. Hyperplastic callus formation in osteogenesis imperfecta. A case report. Acta Radiol 1994;35:426e8. [58] Azouz EM, Fassier F. Hyperplastic callus formation in OI [Letter; Comment]. Skeletal Radiol 1997;26:744e5. [59] Kutsumi K, Nojima T, Yamashiro K, et al. Hyperplastic callus formation in both femurs in osteogenesis imperfecta [Comments]. Skeletal Radiol 1996;25:384e7. [60] Nakamura K, Kurokawa T, Nagano A, Umeyama T. Familial occurrence of hyperplastic callus in osteogenesis imperfecta. Arch Orthop Trauma Surg 1997;116:500e3. [61] Brenner RE, Schiller B, Pontz BF, et al. Osteogenesis imperfecta in childhood and adolescence. Monatsschr. Kinderheilkd 1993; 141:940e5. [62] Bauze RJ, Smith R, Francis MJ. A new look at osteogenesis imperfecta. A clinical, radiological and biochemical study of forty-two patients. J Bone Joint Surg Br 1975;57:2e12. [63] Stoss H, Pontz B, Vetter U, Karbowski A, Brenner R, Spranger J. Osteogenesis imperfecta and hyperplastic callus formation: light- and electron-microscopic findings. Am J Med Genet 1993;45:260.

PEDIATRIC BONE

REFERENCES

[64] Rieker O, Kreitner KF, Karbowski A. Hyperplastic callus formation in osteogenesis imperfecta: CT and MRI findings. Eur Radiol 1998;8:1137e9. [65] Klenerman L, Ockenden BG, Townsend AC. Osteosarcoma occurring in osteogenesis imperfecta. Report of two cases. J Bone Joint Surg Br 1967;49:314e23. [66] Reid BS, Hubbard JD. Osteosarcoma arising in osteogenesis imperfecta. Pediatr Radiol 1979;8:110e2. [67] Dobrocky I, Seidl G, Grill F. MRI and CT features of hyperplastic callus in osteogenesis imperfecta tarda. Eur Radiol 1999; 9:665e8. [68] Glorieux FH, Rauch F, Plotkin H, et al. Type V osteogenesis imperfecta: A new form of brittle bone disease. J Bone Miner Res 2000;15:1650e8. [69] Jones SJ, Glorieux FH, Travers R, Boyde A. The microscopic structure of bone in normal children and patients with osteogenesis imperfecta: a survey using backscattered electron imaging. Calcif Tissue Int 1999;64:8e17. [70] Glorieux FH, Ward LM, Rauch F, Lalic L, Roughley PJ, Travers R. Osteogenesis imperfecta type VI: a form of brittle bone disease with a mineralization defect. J Bone Miner Res 2002;17:30e8. [71] Homan EP, Rauch F, Grafe I, et al. Mutations in SERPINF1 cause osteogenesis imperfecta type VI. J Bone Miner Res 2011. in press. [72] Becker J, Semler O, Gilissen C, et al. Exome sequencing identifies truncating mutations in human SERPINF1 in autosomalrecessive osteogenesis imperfecta. Am J Hum Genet 2011;88: 368e71. [73] Ward LM, Rauch F, Travers R, et al. Osteogenesis imperfecta type VII: an autosomal recessive form of brittle bone disease. Bone 2002;31:12e8. [74] Morello R, Bertin TK, Chen Y, et al. CRTAP is required for prolyl 3-hydroxylation and mutations cause recessive osteogenesis imperfecta. Cell 2006;127:291e304. [75] Bames AM, Chang W, Morello R, et al. Deficiency of cartilageassociated protein in recessive lethal osteogenesis imperfecta. N Engl J Med 2006;355:2757e64. [76] Baldridge D, Schwarze U, Morello R, et al. CRTAP and DEPRE1 mutations in recessive osteogenesis imperfecta. Hum Mutat 2008;29:1435e42. [77] Bames AM, Carter EM, Cabral WA, et al. Lack of cyclophilin B in osteogenesis imperfecta with normal collagen folding. N Engl J Med 2010;362:521e8. [78] Alanay Y, Avaygan H, Camacho N, et al. Mutations in gene encoding the RER protein FKBP65 cause autosomal-recessive osteogenesis imperfecta. Am J Hum Genet 2010;86:551e9. [79] Christiansen HE, Schwarze U, Pyott SM, et al. Homozygosity for a missense mutation in SERPINH1, which encodes the collagen chaperone protein HSP47, results in severe recessive osteogenesis imperfecta. Am J Hum Genet 2010;86:389e98. [80] Bianchine JW, Briard-Guillemot ML, Maroteaux P, Frezal J, Harrison HE. Generalized osteoporosis with bilateral pseudoglioma e an autosomal recessive disorder of connective tissue: report of three families e review of the literature [Abstract]. Am J Hum Genet 1972;24:34A. [81] Beighton P, Winship I, Behari D. The ocular form of osteogenesis imperfecta: A new autosomal recessive syndrome. Clin Genet 1985;28:69e75. [82a] Gong Y, Slee RB, Fukai N, et al. LDL receptor-related protein 5 (LRP5) affects bone accural and eye development. Cell 2001; 107:513e23. [82b] Teebi AS, Al-Awadi SA, Marafie MJ, Bushnaq RA, Satyanath S. Osteoporosisepseudoglioma syndrome with congenital heart disease: a new association. J Med Genet 1988;25:32e6.

533

[83] al Gazali LI, Sabrinathan K, Nair KG. A syndrome of osteogenesis imperfecta, optic atrophy, retinopathy and severe developmental delay in two sibs of consanguineous parents. Clin Dysmorphol 1994;3:55e62. [84] Cole DE, Carpenter TO. Bone fragility, craniosynostosis, ocular proptosis, hydrocephalus, and distinctive facial features: a newly recognized type of osteogenesis imperfecta. J Pediatr 1987;110:76e80. [85] Amor DJ, Savarirayan R, Schneider AS, Bankier A. New case of ColeeCarpenter syndrome. Am J Med Genet 2000;92:273e7. [86] Blacksin MF, Pletcher BA, David M. Osteogenesis imperfecta with joint contractures: Bruck syndrome. Pediatr Radiol 1998; 28:117e9. [87] McPherson E, Clemens M. Bruck syndrome (osteogenesis imperfecta with congenital joint contractures): review and report on the first North American case. Am J Med Genet 1997; 70:28e31. [88] Leroy JG, Nuytinck L, De Paepe A, et al. Bruck syndrome: neonatal presentation and natural course in three patients. Pediatr Radiol 1998;28:781e9. [89] Bank RA, Robins SP, Wijmenga C, et al. Defective collagen crosslinking in bone, but not in ligament or cartilage, in Bruck Syndrome: indications for a bone-specific telopeptide lysyl hydroxylase on chromosome 17. Proc Natl Acad Sci USA 1999; 96:1054e8. [90] Brady AF, Patton MA. Osteogenesis imperfecta with arthrogryposis multiplex congenita (Bruck syndrome) e evidence for possible autosomal recessive inheritance. Clin Dysmorphol 1997;6:329e36. [91] Kelley BP, Malfait F, Bonafe L, et al. Mutations in FKBP10 cause osteogenesis imperfecta and Bruck syndrome. J Bone Miner Res 2011;26:666e72. [92] Heaney RP, Skillman TG. Calcium metabolism in normal human pregnancy. J Clin Endocrinol Metab 1971;33:661e70. [93] Ablin DS, Greenspan A, Reinhart M, Grix A. Differentiation of child abuse from osteogenesis imperfecta. Am J Roentgenol 1990;154:1035e46. [94] Wright JT, Thornton JB. Osteogenesis imperfecta with dentinogenesis imperfecta: a mistaken case of child abuse. Pediatr Dent 1983;5:207e9. [95] Chapman S, Hall CM. Non-accidental injury or brittle bones. Pediatr Radiol 1997;27:106e10. [96] Ablin DS, Sane SM. Non-accidental injury: confusion with temporary brittle bone disease and mild osteogenesis imperfecta. Pediatr Radiol 1997;27:111e3. [97] Astley R. Metaphyseal fractures in osteogenesis imperfecta. Br J Radiol 1979;52:441e3. [98] Knight DJ, Bennet GC. Nonaccidental injury in osteogenesis imperfecta: a case report. J Pediatr Orthop 1990;10:542e4. [99] Westcott H. The abuse of disabled children: a review of the literature. Child Care Health Dev 1991;17:243e58. [100] Gahagan S, Rimsza ME. Child abuse or osteogenesis imperfecta: how can we tell? Pediatrics 1991;88:987e92. [101] Rauch F, Travers R, Norman ME, Taylor A, Parfitt AM, Glorieux FH. Deficient bone formation in idiopatic juvenile osteoporosis: a histomorphometric study of cancellous iliac bone. J Bone Miner Res 2000;15:957e63. [102] Whyte MP. Hypophosphatasia. In: Favus MJ, editor. Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. 4th ed. Philadelphia: Lippincott Willians & Wilkins; 1999. p. 337e9. [103] Shapiro FD, de Levai L, Bramson RT, Preffer FI. A boy with vertebral compression fractures e pre B-cell acute lymphoblastic leukemia (precursor B-lymphoblastic leukemia). N Engl J Med 2000;343:1168e76.

PEDIATRIC BONE

534

19. OSTEOGENESIS IMPERFECTA

[104] Rauch F, Plotkin H, Travers R, et al. Osteogenesis imperfecta types I, III and IV: Effect of pamidronate therapy on bone and mineral metabolism. J Clin Endocrinol Metab 2003;88:986e92. [105] Viera NE, Goans RE, Weiss GH, Hopkins E, Marini JC, Yergey AL. Calcium kinetics in children with osteogenesis imperfecta type III and IV: pre- and post-growth hormone therapy. Calcified Tissue Int 2000;67:97e100. [106] Chines A, Petersen DJ, Schranck FW, Whyte MP. Hypercalciuria in children severely affected with osteogenesis imperfecta. J Pediatr 1991;119:51e7. [107] Chines A, Boniface A, McAlister W, Whyte M. Hypercalciuria in osteogenesis imperfecta: A follow-up study to assess renal effects. Bone 1995;16:333e9. [108] Sawin PD, Menezes AH. Basilar invagination in osteogenesis imperfecta and related osteochondrodysplasias: medical and surgical management. J Neurosurg 1997;86:950e60. [109] Chamas LR, Marini JC. Communicating hydrocephalus, basilar invagination, and other neurologic features in osteogenesis imperfecta. Neurology 1993;43:2603e8. [110] Ziv I, Rang M, Hoffman HJ. Paraplegia in osteogenesis imperfecta. A case report. J Bone Joint Surg Br 1983;65:184e5. [111] Karras D, Karargiris G, Vassilopoulos D, Karatzetzos C. Reflex sympathetic dystrophy syndrome and osteogenesis imperfecta. A report and review of the literature. J Rheumatol 1993;20: 162e4. [112] Bouvier M, Colson F, Noel E, Tebib JG, Felman C. Two new case-reports of reflex sympathetic dystrophy syndrome in patients with osteogenesis imperfecta. Review of the literature [Letter]. Rev Rhum Engl Ed 1997;64:202e4. [113] Brooks ML, Gall C, Wang AM, Schick R, Rumbaugh CL. Osteogenesis imperfecta associated with basilar impression and cerebral atrophy: a case report. Comput Med Maging Graphics 1989;13:363e7. [114] Verkh Z, Russell M, Miller CA. Osteogenesis imperfecta type II: microvascular changes in the CNS. Clin Neuropathol 1995;14: 154e8. [115] Emery SC, Karpinski NC, Hansen L, Masliah E. Abnormalities in central nervous system development in osteogenesis imperfecta type II. Pediatr Dev Pathol 1999;2:124e30. [116] Garretsen AJ, Cremers CW, Huygen PL. Hearing loss (in nonoperated ears) in relation to age in osteogenesis imperfecta type I. Ann Otol Rhinol Laryngol 1997;106:575e82. [117] Verstreken M, Claes J, Van de Heyning PH. Osteogenesis imperfecta and hearing loss. Acta Otorhinolaryngol Belg 1996; 50:91e8. [118] Pedersen U. Osteogenesis imperfecta clinical features, hearing loss and stapedectomy. Biochemical, osteodensitometric, corneometric and histological aspects in comparison with otosclerosis. Acta Otolaryngol (Stockholm) Suppl 1985;415: 1e36. [119] Paterson CR, Monk EA, McAllion SJ. How common is hearing impairment in osteogenesis imperfecta? J Laryngol Otol 2001; 115:280e2. [120] Tabor EK, Curtin HD, Hirsch BE, May M. Osteogenesis imperfecta tarda: appearance of the temporal bones at CT. Radiology 1990;175:181e3. [121] Ziyeh S, Berger R, Reisner K. MRI-visible pericochlear lesion in osteogenesis imperfecta type I. Eur Radiol 2000;10:1675e7. [122] Brosnan M, Burns H, Jahn AF, Hawke M. Surgery and histopathology of the stapes in osteogenesis imperfecta tarda. Arch Otolaryngol 1977;103:294e8. [123] Bergstrom L. Fragile bones and fragile ears. Clin Orthop 1981; 159:58e63. [124] Vallejo Valdezate LA, Martin Gil J, Yamacan MJ, Martin Gil FJ, Gil Carcedo LM, Herrero Laso JL. Aportacion al estudio de la

[125]

[126] [127]

[128]

[129]

[130]

[131] [132] [133]

[134]

[135] [136]

[137] [138]

[139]

[140]

[141]

[142] [143]

[144]

[145] [146] [147]

otosclerosis y del sindrome de Van der Hoeve. Acta Otorrinolaringol Esp 2001;52:85e93. Kuurila K, Grenman R, Johansson R, Kaitila I. Hearing loss in children with osteogenesis imperfecta. Eur J Pediatr 2000;159: 515e9. Cremers C, Garretsen T. Stapes surgery in osteogenesis imperfecta. Am J Otol 1989;10:474e6. Dieler R, Muller J, Helms J. Stapes surgery in osteogenesis imperfecta patients. Eur Arch Otorhinolaryngol 1997;254: 120e7. Ferekidis E, Stavroulaki P, Vossinakis I, Yiotakis J, Manolopoulos L, Adamopoulos G. Stapedotomy in osteogenesis imperfecta patients. J Laryngol Otol 2000;114:424e8. Hortrop J, Tsipouras P, Hanley JA, Maron BJ, Shapiro JR. Cardiovascular involvement in osteogenesis imperfecta. Circulation 1986;73:54e61. Vetter U, Maierhofer B, Muller M, et al. Osteogenesis imperfecta in childhood: Cardiac and renal manifestations. Eur J Pediatr 1989;149:184e7. White NJ, Winearls CG, Smith R. Cardiovascular abnormalities in osteogenesis imperfecta. Am Heart J 1983;106:1416e20. Levy D, Savage D. Prevalence and clinical features of mitral valve prolapse. Am Heart J 1987;113:1281e90. Wood SJ, Thomas J, Braimbridge MV. Mitral valve disease and open heart surgery in osteogenesis imperfecta tarda. Br Heart J 1973;35:103e6. Weisinger B, Glassman E, Spencer FC, Berger A. Successful aortic valve replacement for aortic regurgitation associated with osteogenesis imperfecta. Br Heart J 1975;37:475e7. Stein D, Kloster FE. Valvular heart disease in osteogenesis imperfecta. Am Heart J 1977;94:637e41. Podesta A, Crivellari R, Dottori V, Parodi E, Passerone G. Anaemia, osteogenesis imperfecta and valve diseases. The preoperative treatment with epoetin-alpha to increase haematocrit and haemoglobin levels in patients with high risk of perioperative bleeding. Minerva Cardiol 2000;48:323e7. Moore JB, Zook EG, Kinkead LR. Ulnar artery aneurysm in osteogenesis imperfecta. Hand 1983;15:91e5. de Campos JM, Ferro MO, Burzaco JA, Boixados JR. Spontaneous carotidecavernous fistula in osteogenesis imperfecta. J Neurosurg 1982;56:590e3. Butani L, Rosekrans JA, Morgenstern BZ, Milliner DS. An unusual renal complication in a patient with osteogenesis imperfecta. Am J Kidney Dis 1995;25:489e91. Marini JC, Bordenick S, Heavner G, et al. The growth hormone and somatomedin axis in short children with osteogenesis imperfecta. J Clin Endocrinol Metab 1993;76:251e6. Scott CI, Mengel MC, Lawrence GD, Schultz KT, Edgar PJ. Osteogenesis imperfecta and hypopituitarism in two unrelated males. Birth Defects Orig Artic Ser 1971;7:259e62. Cropp GJ, Myers DN. Physiological evidence of hypermetabolism in osteogenesis imperfecta. Pediatrics 1972;49:375e91. Allanson JE, Hall JG. Obstetric and gynecologic problems in women with chondrodystrophies. Obstet Gynecol 1986;67: 74e8. Falvo KA, Klain DB, Krauss AN, Root L, Auld PA. Pulmonary function studies in osteogenesis imperfecta. Am Rev Respir Dis 1973;108:1258e60. Evensen SA, Myhre L, Stormorken H. Haemostatic studies in osteogenesis imperfecta. Scand J Haematol 1984;33:177e9. Hansen B, Jemec GBE. The mechanical properties of skin in osteogenesis imperfecta. Arch Dermatol 2002;138:909e11. Engelbert RHH, van der Graaf Y, van Empelen R, Beemer FA, Helders PJM. Osteogenesis imperfecta in childhood: impairment and disability. Pediatrics 1997;99:E3.

PEDIATRIC BONE

REFERENCES

[148] Plotkin H, Montpetit K, Cloutier S, et al. Gain in BMD and grip strength after one year of pamidronate treatment in 132 children with osteogenesis imperfecta. J Bone Mine Res 2000;S1:S484. [149] Wordsworth P, Ogilvie D, Smith R, Sykes B. Joint mobility with particular reference to racial variation and inherited connective tissue disorders. Br J Rheumatol 1987;26:9e12. [150] Spence PA, Cohen Z, Salerno TA. Strangulated diaphragmatic hernia in a patient with osteogenesis imperfecta. Can Med Assoc J 1984;131:1369e70. [151] Lee JH, Gamble JG, Moore RE, Rinsky LA. Gastrointestinal problems in patients who have type-III osteogenesis imperfecta. J Bone Joint Surg Am 1995;77:1352e6. [152] Mackey DA, Buttery RG, Wise GM, Denton MJ. Description of the X-linked megalocornea with identification of the gene locus. Arch Ophthal 1991;109:829e33. [153] Pedersen U, Bramsen T. Central corneal thickness in osteogenesis imperfecta and otosclerosis. J Otorhinolaryngol Relat Spec 1984;46:38e41. [154] Lukinmaa PL, Ranta H, Ranta K, Kaitila I. Dental findings in osteogenesis imperfecta: I. Occurrence and expression of type I dentinogenesis imperfecta. J Craniofacial Genet Dev Biol 1987; 7:115e25. [155] Lindau BM, Dietz W, Hoyer I, Lundgren T, Storhaug K, Noren JG. Morphology of dental enamel and dentineeenamel junction in osteogenesis imperfecta. In. J Pediatr Dent 1999;9: 13e21. [156] Levin LS. The dentition in the osteogenesis imperfecta syndromes. Clin Orthop 1981;159:64e74. [157] Schwartz S, Tsipouras P. Oral findings in osteogenesis imperfecta. Oral Surg Oral Med Oral Pathol 1984;57:161e7. [158] Petersen K, Wetzel WE. Recent findings in classification of osteogenesis imperfecta by means of existing dental symptoms. ASDC J Dentistry Child 1998;65:305e9. 354. [159] Johnson D, Tinanoff N. Malocclusion. In: Behrman RE, Kliegman RM, editors. Nelson Textbook of Pediatrics. 16th ed. Philadelphia: W.B. Saunders; 2000. p. 1110e1. [160] O’Connell AC, Marini JC. Evaluation of oral problems in an osteogenesis imperfecta population. Oral Surg Oral Med Oral Pathol Oral Radiol Endodont 1999;87:189e96. [161] Whitestone BW, Chapnick P. Correction of mandibular prognathism in osteogenesis imperfecta tarda. A case report. J Can Dental Assoc 1986;52:853e6. [162] Ormiston IW, Tideman H. Orthognathic surgery in osteogenesis imperfecta: a case report with management considerations. J Craniomaxillofac Surg 1995;23:261e5. [163] Bell RB, White RPJ. Osteogenesis imperfecta and orthognatic surgery: case report with long-term follow-up. Int J Adult Orthodon Orthognath 2000;15:171e8. [164] Lukinmaa PL, Ranta H, Ranta K, Kaitila I, Hietanen J. Dental findings in osteogenesis imperfecta: II. Dysplastic and other developmental defects. J Craniofacial Genet Dev Biol 1987;7: 127e35. [165] Stephen LXG, Beighton P. Dental management of severe dentinogenesis imperfecta in a mild form of osteogenesis imperfecta. J Clin Pediatric Dent 2002;26:131e6. [166] Cubert R, Cheng EY, Mack S, Pepin MG, Byers PH. Osteogenesis imperfecta: mode of delivery and neonatal outcome. Obstet Gynecol 2001;97:66e9. [167] Paily BT. The patient with osteogenesis imperfecta. In: Frost EAM, editor. Preanesthetic Assessment. McMahon Publishing Group; 1996. p. 301e16. [168] Cho E, Dayan SS, Marx GF. Anaesthesia in a parturient with osteogenesis imperfecta. Br J Anaesth 1992;68:422e3. [169] Ryan CA, Al-Ghamdi AS, Gayle M, Finer NN. Osteogenesis imperfecta and hyperthermia. Anesth Analg 1989;68:811e4.

535

[170] Rampton AJ, Kelly DA, Shanahan EC, Ingram GS. Occurrence of malignant hyperpyrexia in a patient with osteogenesis imperfecta. Br J Anaesth 1984;56:1443e6. [171] Brownell AKW. Malignant hyperthermia: relationship to other diseases. Br J Anaesth 1988;60:303e8. [172] Paterson CR, Ogston SA, Henry RM. Life expectancy in osteogenesis imperfecta. Br Med J 1996;312:351. [173] McAllion SJ, Paterson CR. Causes of death in osteogenesis imperfecta. J Clin Pathol 1996;49:627e30. [174] Di Lullo GA, Sweeney SM, Korkko J, Ala-Kokko L, San Antonio JD. Mapping the ligand-binding sites and diseaseassociated mutations on the most abundant protein in the human type I collagen. J Biol Chem 2002;277:4223e31. [175] Beck K, Chan VC, Shenoy N, Kirkpatrick A, Ramshaw JA, Brodsky B. Destabilization of osteogenesis imperfecta collagen-like model peptides correlates with the identity of the residue replacing glycine. Proc Natl Acad Sci USA 2000; 97:4273e8. [176] Baum J, Brodsky B. Folding of peptide models of collagen and misfolding in disease. Curr Opin Struct Biol 1999;9:122e8. [177] Liu X, Kim S, Dai QH, Brodsky B, Baum J. Nuclear magnetic resonance shows asymmetric loss of triple helix in peptides modeling a collagen mutation in brittle bone disease. Biochemistry 1998;37:15528e33. [178] Melacini G, Bonvin AM, Goodman M, Boelens R, Kaptein R. Hydration dynamics of the collagen triple helix by NMR. J Mol Biol 2000;300:1041e9. [179] Buevich A, Baum J. Nuclear magnetic resonance characterization of peptide models of collagen-folding diseases. Philos Trans R Soc London B Biol Sci 2001;356:159e68. [180] McBride Jr DJ, Choe V, Shapiro JR, Brodsky B. Altered collagen structure in mouse tail tendon lacking the alpha 2(I) chain. J Mol Biol 1997;270:275e84. [181] Eyden B, Tzaphlidou M. Structural variations of collagen in normal and pathological tissues: role of electron microscopy. Micron 2001;32:287e300. [182] Bachinger HP, Morris NP, Davis JM. Thermal stability and folding of the collagen triple helix and the effects of mutations in osteogenesis imperfecta on the triple helix of type I collagen. Am J Med Genet 1993;45:152e62. [183] Bank RA, Tekoppele JM, Janus GJM, et al. Pyridinium crosslinks in bone of patients with osteogenesis imperfecta: evidence of a normal intrafibrillar collagen packing. J Bone Miner Res 2000;15:1330e6. [184] Bateman JF, Golub SB. Deposition and selective degradation of structurally-abnormal type I collagen in a collagen matrix produced by osteogenesis imperfecta fibroblasts in vitro. Matrix Biol 1994;14:251e62. [185] Christiansen DL, Huang EK, Silver FH. Assembly of type I collagen: fusion of fibril subunits and the influence of fibril diameter on mechanical properties. Matrix Biol 2000;19: 409e20. [186] Ottani V, Raspanti M, Rugged A. Collagen structure and functional implications. Micron 2001;32:251e60. [187] Kpreos KE, Birk D, Trinkaus-Randall V, Hartmann DJ, Sonenshein GE. Type V collagen regulates the assembly of collagen fibrils in cultures of bovine vascular smooth muscle cells. J Cell Biochem 2000;80:146e55. [188] Mizuno K, Adachi E, Imamura Y, Katsumata O, Hayashi T. The fibril structure of type V collagen triple-helical domain. Micron 2001;32:317e313. [189] Holmes DF, Watson RB, Chapman JA, Kadler KE. Enzymic control of collagen fibril shape. J Mol Biol 1996;261:93e7. [190] Danielson KG, Baribault H, Holmes DF, Graham H, Kadler KE, Iozzo RV. Targeted disruption of decorin leads to abnormal

PEDIATRIC BONE

536

[191]

[192]

[193]

[194]

[195]

[196]

[197]

[198]

[199]

[200]

[201]

[202]

[203]

[204]

[205]

[206]

[207]

19. OSTEOGENESIS IMPERFECTA

collagen fibril morphology and skin fragility. J Cell Biol 1997; 136:729e43. Ezura Y, Chakravarti S, Oldberg A, Chervoneva I, Birk DE. Differential expression of lumican and fibromodulin regulate collagen fibrillogenesis in developing mouse tendons. J Cell Biol 2000;151:779e88. Svensson L, Aszodi A, Reinholt FP, Fassler R, Heinegard D, Oldberg A. Fibromodulin-null mice have abnormal collagen fibrils, tissue organization, and altered lumican deposition in tendon. J Biol Chem 1999;274:9636e47. Kielty CM, Raghunath M, Siracusa D, et al. The Tight skin mouse: Demonstration of mutant fibrillin-1 production and assembly into abnormal microfibrils. J Cell Biol 1998;140: 1159e66. Camacho NP, Rinnerthaler S, Paschalis EP, Mendelsohn R, Boskey AL, Fratzl P. Complementary information on bone ultrastructure from scanning small angle X-ray scattering and Fourier-transform infrared microspectroscopy. Bone 1999;25: 287e93. Landis WJ, Hodgens KJ, Song MJ, et al. Mineralization of collagen may occur on fibril surfaces: evidence from conventional and high-voltage electron microscopy and threedimensional imaging. J Struct Biol 1996;117:24e35. Landis WJ, Hodgens KJ, Arena J, Song MJ, McEwen BF. Structural relations between collagen and mineral in bone as determined by high voltage electron microscopic tomography. Microsc Res Tech 1996;33:192e202. Cassella JP, Barrie PJ, Garrington N, Ali SY. A Fourier transform infrared spectroscopic and solid-state NMR study of bone mineral in osteogenesis imperfecta. J Bone Miner Metab 2000; 18:291e6. Cassella JP, Pereira R, Khillan JS, Prockop DJ, Garrington N, Ali SY. An ultrastructural, microanalytical, and spectroscopic study of bone from a transgenic mouse with a COL1.A1 proalpha-1 mutation. Bone 1994;15:611e9. Fratzl P, Paris O, Klaushofer K, Landis WJ. Bone mineralization in an osteogenesis imperfecta mouse model studied by smallangle X-ray scattering. J Clin Invest 1996;97:396e402. Lamande SR, Chessler SD, Golub SB, et al. Endoplasmic reticulum-mediated quality control of type I collagen production by cells from osteogenesis imperfecta patients with mutations in the pro alpha 1 (I) chain carboxyl-terminal propeptide which impair subunit assembly. J Biol Chem 1995;270:8642e9. Fitzgerald F, Lamande SR, Bateman JF. Proteasomal degradation of unassembled mutant type I collagen pro-alphal(I) chains. J Biol Chem 1999;274:27392e8. Lamande SR, Bateman JF. Procollagen folding and assembly: the role of endoplasmic reticulum enzymes and molecular chaperones. Semin Cell Dev Biol 1999;10:455e64. Kojima T, Miyaishi O, Saga S, Ishiguro N, Tsutsui Y, Iwata H. The retention of abnormal type I procollagen and correlated expression of HSP 47 in fibroblasts from a patient with lethal osteogenesis imperfecta. J Pathol 1998;184:212e8. Nagai N, Hosokawa M, Itohara S, et al. Embryonic lethality of molecular chaperone hsp47 knockout mice is associated with defects in collagen biosynthesis. J Cell Biol 2000;150:1499e506. Thomson CA, Ananthanarayanan VS. Structure-function studies on hsp47: pH-dependent inhibition of collagen fibril formation in vitro. Biochem J 2000;349:877e83. Fedarko NS, D’Avis P, Frazier CR, et al. Cell proliferation of human fibroblasts and osteoblasts in osteogenesis imperfecta: Influence of age. J Bone Miner Res 1995;10:1705e12. Wenstrup RJ, Witte DP, Florer JB. Abnormal differentiation in MC3T3eE1 preosteoblasts expressing a dominant-negative type I collagen mutation. Connect Tissue Res 1996;35:249e57.

[208] Wenstrup RJ, Fowlkes JL, Witte DP, Florer JB. Discordant expression of osteoblast markers in MC3T3eE1 eels that synthesize a high turnover matrix. J Biol Chem 1996;271: 10271e6. [209] Rauch F, Travers R, Parfitt AM, Glorieux FH. Static and dynamic bone histomorphometry in children with osteogenesis imperfecta. Bone 2000;26:581e9. [210] Gamble JG, Rinsky LA, Strudwick J, Bleck EE. Non-union of fractures in children who have osteogenesis imperfecta. J Bone Joint Surg 1988;70:439e43. [211] Brenner RE, Vetter U, Bollen AM, Morike M, Eyre DR. Bone resorption assessed by immunoassay of urinary cross-linked collagen peptides in patients with osteogenesis imperfecta. J Bone Miner Res 1994;9:993e7. [212] Goans RE, Abrams SA, Vieira NE, Marini JC, Perez MD, Yergey AL. A three-hour measurement to evaluate bone calcium turnover. Bone 1995;16:33e8. [213] Lund AM, Hansen M, Kollerup G, Juul A, Teisner B, Skovby F. Collagen-derived markers of bone metabolism in osteogenesis imperfecta. Acta Paediatr 1998;87:1131e7. [214] Kalajzic I, Terzic J, Rumboldt Z, et al. Osteoblastic response to the defective matrix in the osteogenesis imperfecta murine (oim) mouse. Endocrinology 2002;143:1594e601. [215] Minisola S, Piccioni AL, Rosso R, et al. Reduced serum levels of carboxy-terminal propeptide of human type I procollagen in a family with type I-A osteogenesis imperfecta. Metabolism 1994;43:1261e5. [216] McCarthy EF, Earnest K, Rossiter K, Shapiro J. Bone histomorphometry in adults with type IA osteogenesis imperfecta. Clin Orthop 1997;336:254e62. [217] Bonadio J, Jepsen KJ, Mansoura MK, Jaenisch R, Kuhn JL, Goldstein SA. A murine skeletal adaptation that significantly increases cortical bone mechanical properties. Implications for human skeletal fragility. J Clin Invest 1993;92:1697e705. [218] Jepsen KJ, Schafner MB, Kuhn JL, Goulet RW, Bonadio J, Goldstein SA. Type I collagen mutation alters the strength and fatigue behavior of Mov13 cortical tissue. J Biomech 1997;30: 1141e7. [219] Albright JA. Systemic treatment of osteogenesis imperfecta. Clin Orthop 1981;159:88e96. [220] Winterfeldt EA, Eyring EJ, Vivian VM. Ascorbic-acid treatment for osteogenesis imperfecta. Lancet 1970;760:1347e8. [221] Kurz D, Eyring EJ. Effects of vitamin C on osteogenesis imperfecta. Pediatrics 1974;54:56e61. [222] Albright JA, Grunt JA. Studies of patients with osteogenesis imperfecta. J Bone Joint Surg 1971;53:1415e25. [223] Bilginturan N, Ozsoylu S. The results of sodium fluoride treatment in osteogenesis imperfecta. Turk J Pediatr 1966;8: 129e42. [224] Aeschlimann MI, Grunt JA, Crigler Jr JF. Effects of sodium fluoride on the clinical course and metabolic balance of an infant with osteogenesis imperfecta congenita. Metabolism 1966;15:905e14. [225] Solomons CC, Styner J. Osteogenesis imperfecta: Effect of magnesium administration on pyrophosphate metabolism. Calcif Tissue Res 1969;3:318e26. [226] Granda JL, Falvo KA, Bullough PG. Pyrophosphate levels and magnesium oxide therapy in osteogenesis imperfecta. Clin Orthop 1977;126:228e31. [227] Cattell HS, Clayton B. Failure of anabolic steroids in the therapy of osteogenesis imperfecta: a clinical, metabolic and biochemical study. J Bone Joint Surg Am 1968;50:123e41. [228] Castells S. New approaches to treatment of osteogenesis imperfecta. Clin Orthop 1973;93:239e49.

PEDIATRIC BONE

537

REFERENCES

[229] Rosenberg E, Lang R, Boisseau V, Rojanasathit S, Avioli LV. Effect of long-term calcitonin therapy on the clinical course of osteogenesis imperfecta. J Clin Endocrinol Metab 1977;44: 346e55. [230] Gedikoglu O, Laleli Y, Aydinli U. Synthetic salmon calcitonin therapy in osteogenesis imperfecta. Hacettepe Med J 1986;19: 141e9. [231] Rebelo I, da Silva LP, Blanco JC, Monteiro ME, Ferreira NC. Effects of synthetic salmon calcitonin therapy in children with osteogenesis imperfecta. J Int Med Res 1989;17:401e5. [232] August GP, Shapiro J, Hung W. Calcitonin therapy of children with osteogenesis imperfecta. J Pediatr 1977;91:1001e5. [233] Pedersen U, Charles P, Hansen HH, Elbrond O. Lack of effects of human calcitonin in osteogenesis imperfecta. Acta Orthop Scand 1985;56:260e4. [234] Rodan GA, Balena R. Bisphosphonates in the treatment of metabolic bone diseases. Ann Med 1993;25:373e8. [235] Plotkin LI, Parfitt AM, Manolagas SC, Bellido T. Prevention of osteocyte and osteoblast apoptosis by bisphosphonates and calcitonin. J Clin Invest 1999;104:1363e74. [236] Mathov I, Plotkin LI, Sgarlata CL, Leoni J, Bellido T. Extracellular signal-regulated kinases and calcium channels are involved in the proliferative effect of bisphosphonates on osteoblastic cells in vitro. J Bone Miner Res 2001;16:2050e5. [237] Hughes DE, Wright KR, Uy HL, et al. Bisphosphonates promote apoptosis in murine osteoclasts in vitro and in vivo. J Bone Miner Res 1995;10:1478e87. [238] Devogelaer JP, Malghem J, Maldague B, Nagant de Deuxchaisnes C. Radiological manifestations of bisphosphonate treatment with apd in a child suffering from osteogenesis imperfecta. Skeletal Radiol 1987;16:360e3. [239] Landsmeer-Beker EA, Massa GG, Maaswinkel-Mooy PD, van de Kamp JJ, Papapoulos SE. Treatment of osteogenesis imperfecta with the bisphosphonate olpadronate (dimethylaminohydroxypropylidene bisphosphonate). Eur J Pediatr 1997; 156:792e4. [240] Astrom E, Soderhall S. Beneficial effect of bisphosphonate during five years of treatment of severe osteogenesis imperfecta. Acta Paediatr 1998;87:64e8. [241] Astrom E, Soderhall S. Beneficial effect of long term intravenous bisphosphonate treatment of osteogenesis imperfecta. Arch Dis Child 2002;86:356e64. [242] Bembi B, Parma A, Bottega M, et al. Intravenous pamidronate treatment in osteogenesis imperfecta. J Pediatr 1997;131: 622e5. [243] Glorieux FH, Bishop NJ, Plotkin H, Chabot G, Lanoue G, Travers R. Cyclic administration of pamidronate in children with severe osteogenesis imperfecta. N Engl J Med 1998;339: 947e52. [244] Plotkin H, Rauch F, Bishop N, et al. Pamidronate treatment of severe osteogenesis imperfecta in children under three years of age. J Clin Endocrinol Metab 2000;85:1846e50. [245] Fleisch H. Can bisphosphonates be given to patients with fractures? J Bone Miner Res 2001;16:437e40. [246] Plotkin H, Gibis J, Glorieux FH. Pamidronate treatment improves gross motor function and growth in children with osteogenesis imperfecta. Bone 2001;28(5S):578. [247] Frost HM. Vital biomechanics: Proposed general concepts for skeletal adaptations to mechanical usage. Calcif Tissue Int 1988;42:145e56. [248] Lepola V, Hannuniemi R, Kippo K, Lauren L, Jalovaara P, Vaananen H. Long-term effects of clodronate on growing rat bone. Bone 1996;18:191e6. [249] Zeitlin L, Rauch F, Plotkin H, Glorieux FH. Height and weight development during four years of therapy with cyclical

[250]

[251]

[252a]

[252b]

[253a]

[253b]

[254]

[255]

[256]

[257]

[258]

[259] [260]

[261]

[262]

[263]

[264]

[265]

[266] [267]

intravenous pamidronate in children and adolescents with osteogenesis imperfecta types I, III and IV. Pediatrics 2003;111: 630e6. Krane SM, Kantrowitz FG, Byrne M, Pinnell SR, Singer FR. Urinary excretion of hydroxylysine and its glycosides as an index of collagen degradation. J Clin Invest 1977;59:819e27. Wimalawansa SJ, Gunasekera RD. Pamidronate is effective for Paget’s disease of bone refractory to conventional therapy. Calcif Tissue Int 1993;53:237e41. Munns CFJ, Rauch F, Zeitlin L, et al. Delayed osteotomy but not fracture healing in pediatric osteogenesis imperfecta patients receiving pamidronate. J Bone Miner Res 2004;19:1e8. Chakravarty K, Merry P, Scott DG. A single infusion of bisphosphonate AHPrBP in the treatment of Paget’s disease of bone. J Rheumatol 1994;21:2118e21. Rauch F, Travers R, Glorieux FH. . Pamidronate in children with osteogenesis imperfecta: Histomorphometric effects of long-term therapy. J Clin Endocrinol Metab 2006;91:511e6. Rauch F, Cornibert S, Cheung M, et al. Long-bone changes after pamidronate discontinuation in children and adolescents with osteogenesis imperfecta. Bone 2007;40:821e7. Kinugasa A, Fujita K, Inoue F, Kodo N, Sawada T. A case of osteogenesis imperfecta type 1A with an increased frequency of bone fracture after growth hormone therapy. Acta Pediatr Scand 1991;379(Suppl.):559e60. Kodama H, Kubota K, Abe T. Osteogenesis imperfecta: are fractures and growth hormone treatment linked? [Letter]. J Pediatr 1998;132:559e60. Antoniazzi F, Bertoldo F, Mottes M, et al. Growth hormone treatment in osteogenesis imperfecta with quantitative defect of type I collagen synthesis. J Pediatr 1996;129:432e9. Gharib H, Saenger PH, Zimmerman D. AACE clinical practice guidelines for growth hormone use in adults and children. Endocrine Pract 1998;4:165e73. Wright NM. Just taller or more bone? The impact of growth hormone on osteogenesis imperfecta and idiopathic juvenile osteoporosis. J Pediatr Endocrinol Metab 2000; 13(Suppl. 2):999e1002. Bailey RW. Further clinical experience with the extensible nail. Clin Orthop 1981;159:171e6. Bailey RW, Dubow HI. Evolution of the concept of an extensible nail accommodating to normal longitudinal bone growth: Clinical considerations and implications. Clin Orthop 1981;159: 157e70. Fassier F, Duval P, Dujovne A. Experience with the FassierDuval telescopic system in the treatment of osteogenesis imperfecta. Proceedings of the Annual Meeting of the American Orthopedic Surgeons. New Orleans, Louisiana 2003;4:598. Nicholas RW, James P. Telescoping intramedullary stabilization of the lower extremities for severe osteogenesis imperfecta. J Pediatr Orthop 1990;10:219e23. Jerosch J, Mazzotti I, Tomasevic M. Complications after treatment of patients with osteogenesis imperfecta with a BaileyDubow rod. Arch Orthop Trauma Surg 1998;117:240e5. Porat S, Heller E, Seidman DS, Meyer S. Functional results of operation in osteogenesis imperfecta: Elongating and nonelongating rods. J Pediatr Orthop 1991;11:200e3. Falvo KA, Root L, Bullough PG. Osteogenesis imperfecta: Clinical evaluation and management. J Bone Joint Surg 1974;56: 783e93. Renshaw TS, Cook RS, Albright JA. Scoliosis in osteogenesis imperfecta. Clin Orthop 1979;145:163e7. Benson DR, Donaldson DH, Millar EA. The spine in osteogenesis imperfecta. J Bone Joint Surg 1978;60:925e9.

PEDIATRIC BONE

538

19. OSTEOGENESIS IMPERFECTA

[268] Yong-Hing K, MacEwen GD. Scoliosis associated with osteogenesis imperfecta. J Bone Joint Surg Br 1982;64:36e43. [269] Widmann RF, Bitan FD, Laplaza J, Burke SW, DiMaio MF, Schneider R. Spinal deformity, pulmonary compromise, and quality of life in osteogenesis imperfecta. Spine 1999;24:1673e8. [270] Benson DR, Newman DC. The spine and surgical treatment in osteogenesis imperfecta. Clin Orthop 1981;159:147e53. [271] Livesley PJ, Webb PJ. Spinal fusion in situ in osteogenesis imperfecta. Int Orthoped 1996;20:43e6. [272] Janus GJM, Finidori G, Engelbert RHH, Pouliquen M, Pruijs JEH. Operative treatment of severe scoliosis in osteogenesis imperfecta: results of 20 patients after halo traction and posterior spondylodesis with instrumentation. Eur Spine J 2000;9:486e91. [273] Sperry K. Fatal intraoperative hemorrhage during spinal fusion surgery for osteogenesis imperfecta. Am J Forensic Med Pathol 1989;10:54e9. [274] Binder H, Hawks L, Graybill G, Gerber NL, Weintrob JC. Osteogenesis imperfecta: rehabilitation approach with infants and young children. Arch Phys Med Rehab 1984;65:537e41. [275] Daci E, Van Cromphaut S, Bouillon R. Mechanisms influencing bone metabolism in chronic illness. Horm Res 2002;58(Suppl 1):44e51. [276] Ruck-Gibis J, Plotkin H, Hanley J, Wood-Duphinee S. Reliability of the gross motor function measure for children with osteogenesis imperfecta. Pediatr Phys Ther 2001;13:10e7. [277] Binder H, Conway A, Gerber LH. Rehabilitation approaches to children with osteogenesis imperfecta: A ten-year experience. Arch Phys Med Rehab 1993;74:386e90. [278] Binder H, Conway A, Hason S, et al. Comprehensive rehabilitation of the child with osteogenesis imperfecta. Am J Med Genet 1993;45:265e9. [279] Cole DE. Psychosocial aspects of osteogenesis imperfecta: an update. Am J Med Genet 1993;45:207e11. [280] Cabral W, Marini JC. High proportion of mutant osteoblasts is compatible with normal skeletal function in mosaic carriers of osteogenesis imperfecta. Am J Hum Genet 2004;74:752e60. [281] Otsu M, Sugamura K, Candotti F. In vivo competitive studies between normal and common gamma chain-defective bone marrow cells: implications for gene therapy. Hum Gene Ther 2000;11:2051e6. [282] Roberts C, Kean L, Archer D, Balkan C, Hsu LL. Murine and math models for the level of stable mixed chimerism to cure beta-thalassemia by nonmyeloablative bone marrow transplantation. Ann NY Acad Sci 2005;1054:423e8. [283] Titorencu I, Jinga VV, Constantinescu E, et al. Proliferation, differentiation and characterization of osteoblasts from human BM mesenchymal cells. Cytotherapy 2007;9:682e96. [284] Yin D, Wang Z, Gao Q, et al. Determination of the fate and contribution of ex vivo expanded human bone marrow stem and progenitor cells for bone formation by 2.3ColGFP. Mol Ther 2009;17:1967e78. [285] Lin G, Garcia M, Ning H, et al. Defining stem and progenitor cells within adipose tissue. Stem Cells Dev 2008;17:1053e63. [286] Takahashi KTK, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007;131:861e72. [287] Yu JVM, Smuga-Otto K, Antosiewicz-Bourget J, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 2007;318:1917e20. [288] Woltjen K, Michael IP, Mohseni P, et al. piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature 2009;458:766e70. [289] Montserrat N, Garreta E, Gonzalez F, et al. Simple generation of human induced pluripotent stem cells using poly-{beta}-

[290]

[291]

[292]

[293]

[294]

[295]

[296]

[297]

[298]

[299]

[300]

[301]

[302]

[303]

[304]

[305]

[306]

[307]

amino esters as the non-viral gene delivery system. J Biol Chem 2011;286:12417e28. Gonzalez F, Barragan Monasterio M, Tiscornia G, et al. Generation of mouse-induced pluripotent stem cells by transient expression of a single nonviral polycistronic vector. Proc Natl Acad Sci USA 2009;106:8918e22. Yakubov E, Rechavi G, Rozenblatt S, Givol D. Reprogramming of human fibroblasts to pluripotent stem cells using mRNA of four transcription factors. Biochem Biophys Res Commun 2010;394:189e93. Lyssiotis CA, Foreman RK, Staerk J, et al. Reprogramming of murine fibroblasts to induced pluripotent stem cells with chemical complementation of Klf4. Proc Natl Acad Sci USA 2009;106:8912e7. Millington-Ward S, McMahon HP, Allen D, et al. RNAi of COL1A1 in mesenchymal progenitor cells. Eur J Hum Genet 2004;12:864e6. Lindahl K, Rubin CJ, Kindmark A, Ljunggren O. Allele dependent silencing of COL1A2 using small interfering RNAs. Int J Med Sci 2008;5:361e5. Chamberlain JR, Schwarze U, Wang PR, et al. Gene targeting in stem cells from individuals with osteogenesis imperfecta. Science 2004;303:1198e201. Chamberlain JR, Deyle DR, Schwarze U, et al. Gene targeting of mutant COL1A2 alleles in mesenchymal stem cells from individuals with osteogenesis imperfecta. Mol Ther 2008;16: 187e93. Liu Y, Nairn RS, Vasquez KM. Targeted gene conversion induced by triplex-directed psoralen interstrand crosslinks in mammalian cells. Nucleic Acids Res 2009;37:6378e88. Lonkar P, Kim KH, Kuan JY, et al. Targeted correction of a thalassemia-associated beta-globin mutation induced by pseudo-complementary peptide nucleic acids. Nucleic Acids Res 2009;37:3635e44. Sakurai K, Shimoji M, Tahimic CG, et al. Efficient integration of transgenes into a defined locus in human embryonic stem cells. Nucleic Acids Res 2010;38:e96. Tarnowski M, Szydlo A, Aniol J, et al. Optimization of genetic engineering and homologous recombination of collagen type I genes in rat bone marrow mesenchymal stem cells (MSC). Cell Reprogram 2010;12:275e82. Tichy ED, Pillai R, Deng L, et al. Mouse embryonic stem cells, but not somatic cells, predominantly use homologous recombination to repair double-strand DNA breaks. Stem Cells Dev 2010;19:1699e711. Remy S, Tesson L, Menoret S, Usal C, Scharenberg AM, Anegon I. Zinc-finger nucleases: a powerful tool for genetic engineering of animals. Transgenic Res 2010;19:363e71. Handel EM, Cathomen T. Zinc-finger nuclease based genome surgery: it’s all about specificity. Curr Gene Ther 2011;11: 28e37. Cabaniols JP, Ouvry C, Lamamy V, et al. Meganuclease-driven targeted integration in CHO-K1 cells for the fast generation of HTS-compatible cell-based assays. J Biomol Screen 2010;15: 956e67. Munoz IG, Prieto J, Subramanian S, et al. Molecular basis of engineered meganuclease targeting of the endogenous human RAG1 locus. Nucleic Acids Res 2011;39:729e43. Horwitz EM, Prockop DJ, Fitzpatrick LA, et al. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med 1999;5:309e13. Dominici M, Marino R, Rasini V, et al. Donor cell-derived osteopoiesis originates from a self-renewing stem cell with

PEDIATRIC BONE

REFERENCES

[308]

[309]

[310]

[311]

a limited regenerative contribution after transplantation. Blood 2008;111:4386e91. Kumagai K, Vasanji A, Drazba JA, Butler RS, Muschler GF. Circulating cells with osteogenic potential are physiologically mobilized into the fracture healing site in the parabiotic mice model. J Orthop Res 2008;26:165e75. Panaroni C, Gioia R, Lupi A, et al. In utero transplantation of adult bone marrow decreases perinatal lethality and rescues the bone phenotype in the knockin murine model for classical, dominant osteogenesis imperfecta. Blood 2009;114:459e68. Vanleene M, Saldanha Z, Cloyd KL, et al. Transplantation of human fetal blood stem cells in the osteogenesis imperfecta mouse leads to improvement in multiscale tissue properties. Blood 2011;117:1053e60. Chang MK, Raggatt LJ, Alexander KA, et al. Osteal tissue macrophages are intercalated throughout human and mouse bone lining tissues and regulate osteoblast function in vitro and in vivo. J Immunol 2008;181:1232e44.

539

[312] Wang L, Liu Y, Kalajzic Z, Jiang X, Rowe DW. Heterogeneity of engrafted bone-lining cells after systemic and local transplantation. Blood 2005;106:3650e7. [313] Boban I, Barisic-Dujmovic T, Clark SH. Parabiosis model does not show presence of circulating osteoprogenitor cells. Genesis 2010;48:171e82. [314] Boban I, Jacquin C, Prior K, et al. The 3.6 kb DNA fragment from the rat Col1a1 gene promoter drives the expression of genes in both osteoblast and osteoclast lineage cells. Bone 2006; 39:1302e12. [315] Li F, Wang X, Niyibizi C. Bone marrow stromal cells contribute to bone formation following infusion into femoral cavities of a mouse model of osteogenesis imperfecta. Bone 2010;47:546e55. [316] Larsen KH, Frederiksen CM, Burns JS, Abdallah BM, Kassem M. Identifying a molecular phenotype for bone marrow stromal cells with in vivo bone forming capacity. J Bone Miner Res 2009;25:796e808.

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Sclerosing Bone Dysplasias Bram Perdu 1, Geert Mortier 1, Filip Vanhoenacker 2, Wim Van Hul 1 1

Department of Medical Genetics, University and University Hospital Antwerp, Belgium 2 Department of Radiology, University Hospital of Antwerp, Belgium

INTRODUCTION

NEONATAL OSTEOSCLEROTIC DYSPLASIAS

Osteoporosis is by far the most common and known condition characterized by an abnormal bone mineral density. At the other end of the spectrum, an extended group of rare conditions exist, the so-called sclerosing bone dysplasias, all characterized by too much bone tissue. This is a very heterogeneous group both at clinical, radiological and molecular-genetic level. This is not unexpected considering the many variations that can occur either in the age of onset, the affected skeletal sites or the underlying pathogenic mechanisms. Increased bone mass can be due to an impaired bone resorption mechanism or by an increased bone anabolism. In the latter, this can involve the mechanism of either endochondral or intramembranous bone formation or even a combination of both. Depending on the disturbed mechanism, sclerotic bones can be due to thickening of cortical or trabecular bone. Clinically, an increased bone mass can have direct effects on the skeleton itself with deformities and, depending on the architecture and the microscopic structure of the bone formed, a decreased or increased fracture rate. Furthermore, secondary effect on neighboring tissues can result in pain, contractures, nerve palsy etc. In the last decades, for many of these diseases, underlying genetic defects have been identified which, in many cases, provide novel insights into the pathogenesis of these conditions. Although alternative classifications could also be considered, we used for this mainly descriptive review the classification as defined by the Nosology and Classification Working Group of the International Skeletal Dysplasia Society in 2006 [1].

Pediatric Bone, Second Edition DOI: 10.1016/B978-0-12-382040-2.10020-6

Blomstrand Dysplasia Blomstrand dysplasia is a very severe disorder being lethal during pregnancy or in the neonatal period [2]. The pregnancy can be complicated by polyhydramnios and hydrops may be apparent on prenatal ultrasound. Midface hypoplasia with short nose, depressed nasal bridge and protruding eyes are characteristic facial features. The limbs are short and broad with relatively normal sized hands and feet. An advanced skeletal maturation is an important radiographic feature of the condition. The carpal, tarsal and even hyoid bones are prematurely ossified. The long bones are short and wide with club-shaped ends and some bowing may be present. The ribs are short and thick, giving the thorax a long and narrow appearance [3]. Blomstrand dysplasia is an autosomal recessive condition due to loss-of-function mutations in the gene encoding parathyroid hormone (PTH)/PTH related peptide receptor-1 (PTHR1) [4]. Impairment of this signaling pathway results in abnormal endochondral bone formation.

Desmosterolosis and Raine Dysplasia Desmosterolosis is a very rare autosomal recessive metabolic disorder characterized by multiple congenital anomalies and elevated levels of the cholesterol precursor desmosterol. Patients have macrocephaly, hypoplastic nasal bridge, thick alveolar ridges, cleft palate, short limbs and generalized osteosclerosis [5]. Biochemically, there is an accumulation of desmosterol

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in several tissues and a lack of cholesterol. It is caused by deficiency of 3-beta-hydroxysterol-delta-24-reductase (DHCR24) [6]. There is a clinical overlap with Raine dysplasia but, in the latter, no accumulation of desmosterol is present. In 1989, Raine and colleagues described this lethal disorder characterized by microcephaly, hypoplastic nose, cleft palate, exophthalmos and diffuse osteosclerosis [7]. Nearly all patients have died shortly after birth from lung hypoplasia. Radiologically, a generalized increased bone density is present, with extensive periosteal thickening (“cloaking”) of mandible, clavicles, scapula, long bones and ribs but their size is normal. The ribs can show vertical lines suggestive of fractures, but no real fractures are found. The cranial vault is sclerotic with wide cranial sutures. The vertebral bodies are usually normal but sclerotic. Bone histology shows no abnormality of cartilage and growth plates suggesting normal endochondral ossification. There is a periosteal accumulation of an amorphous ground substance-like, alcian blue-positive mucoid material. Osteoblasts and fibroblasts are normal [8]. Raine dysplasia has recently been shown to be caused by homozygous or compound heterozygous mutations in the FAM20C gene which encodes a secreted protein with calcium-binding properties [9].

Caffey Disease This condition is also known as infantile cortical hyperostosis. It was originally described by Ro¨ske in 1930, and afterwards in 1945 by Caffey and Silverman [10,11]. The disorder is characterized by acute inflammation with painful soft-tissue swelling mainly involving the jaws and cheeks, the clavicula and the long bones. This is often accompanied by mild fever. Besides the classical mild and autosomal dominant form of Caffey disease, a severe prenatal form is described characterized by polyhydramnios and more generalized hyperostotic bone involvement. This severe form frequently results in intrauterine or early neonatal death and has most likely an autosomal recessive inheritance [12]. Finally, also sporadic forms do occur. In some of these instances, a paraproteinemia in the pregnant mother has been noted. Radiographically, the diaphyses of the long tubular bones, mandible, and clavicles show subperiosteal new bone formation. The onset of the disease in the classical form is typically before 5 months of age with resolution of the symptoms before 3 years of age. The most frequently affected bone is the mandible with sometimes involvement of the clavicles, tibia, ulna, femur, rib, humerus, maxilla, and fibula [13]. New periosteal bone formation, appearing most often during the ninth week, undergoes resolution slowly. Though complete clinical resolution takes place within 3 to 30 months,

radiographic evidence may persist for many years. Bowing of the tibia as well as inequality of the leg lengths are quite common. Most of the cases are sporadic, but some families show autosomal dominant inheritance with incomplete penetrance and variable expression [14,15]. In a large family with autosomal dominant Caffey disease, Gensure et al. identified a mutation in COL1A1, resulting in an arginine to cysteine substitution (p.R836C) [16]. The same mutation was found in affected individuals from two other unrelated families but so far no mutations have been identified in the prenatal severe forms of Caffey disease.

DISEASES WITHOUT MAJOR CHANGES IN BONE SHAPE Osteopetrosis The osteopetroses are a heterogeneous group of sclerosing bone dysplasias, marked by the inability of osteoclasts to resorb bone due to defects in the osteoclastogenesis or the acidification of the extracellular compartment (Fig. 20.1). General histological hallmarks of the osteopetroses are remnants of unresorbed cartilage in the mature bone which also explains the reduced strength of the bones in these patients. The different subforms of osteopetrosis are classified on the basis of inheritance, age of onset, severity, secondary clinical features and the number of osteoclasts. Severe Neonatal or Infantile Osteopetrosis This disorder is characterized by increased density of nearly all bones and a range of clinical complications that occur due to failure of resorption of the primary spongiosa and its resultant persistence. These include anemia, hepatosplenomegaly, blindness, deafness, facial paralysis, and osteomyelitis [18]. Increased bone density has been found by fetal x-ray examination at 24 weeks of gestation. If untreated, children usually die in their first decade as a consequence of recurrent infections. After allogenic bone marrow transplantation, the chances for survival are much better, although a considerable number of patients still die in early childhood [18]. Major radiological findings include a homogeneous increase in bone density with lack of corticomedullary differentiation and loss of trabecular structure, metaphyseal undermodeling (“Erlenmeyer flask shaped bones”) and lucent bands at the metaphyseal ends in the long bones (Fig. 20.2). Bone biopsy specimens show a higher number of osteoclasts, suggesting a normal differentiation but affected osteoclast functioning.

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FIGURE 20.1 The osteopetrosis genes in the functioning osteoclast. RANKL and M-CSF binding to their receptors induce the transcription of specific genes that are essential for osteoclastogenesis. The OPG/RANKL ratio is an important factor for osteoclast differentiation. Osteoclasts are large multinucleated cells formed by cellular fusion of their mononuclear precursors and are responsible for the resorption of bone. When bone resorption is induced, osteoclasts migrate and attach to the site of resorption, become highly polarized, and form a ruffled border. The ruffled border is formed through fusion of a large number of acidic vesicles with the bone-facing membrane and is encircled by an actin-rich sealing zone which mediates attachment to the extracellular mineralized matrix. The resorption is largely dependent on this secretion of protons and proteolytic enzymes. Proteolytic enzymes and protons are secreted peripherally into the resorption lacuna. Debris is centrally endocytosed at the ruffled border and secreted at the basolateral side of the osteoclast. Genes involved in human osteopetrosis are indicated in bold. (Modified figure from Perdu et al. [17].)

Defects in three genes are known to impair osteoclast functioning and cause malignant recessive osteopetrosis. The majority of mutations (z50%) thus far are located in the TCIRG1 gene encoding for a subunit of the vacuolar Hþ-ATPase (V-ATPase) that transports the generated protons across the ruffled border into the resorption lacuna [19]. Less frequent are loss-of-function mutations in CLCN7, needed for the maintenance of electroneutrality, and OSTM1 that serves as a b-subunit for CLCN7 [20,21]. In contrast to the patients with mutations in the TCIRG1 gene, these patients also suffer from retinal atrophy and neurodegeneration. Human stem cell transplantation (HSCT) will not fully cure these patients, because this procedure has no effect on the nervous system. Currently, not all malignant cases can be explained by mutations in any of these genes highlighting

the existence of, at least one, other gene involved in this severe form of osteopetrosis [22]. Intermediate Osteopetrosis Intermediate osteopetrosis can be differentiated from the malignant form because the outcome is less severe and the life expectancy is higher. The disease may be associated with short stature, osteomyelitis, dental problems and a higher fracture risk [23]. Patients with intermediate autosomal recessive osteopetrosis display the typical radiological features of the other forms, including generalized sclerosis and widened metaphyses. Defects in two genes are known to cause this form of osteopetrosis: CLCN7 and PLEKHM1 [24,25]. Mutations in CLCN7 not only account for severe osteopetrosis, they are also responsible for intermediate

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FIGURE 20.2 Neonatal skeletal radiographs of a patient with infantile osteopetrosis. (A) Lateral view of the skull. Generalized increased bone density of the skull bones is seen. (B) The anteroposterior view of the pelvis shows bone-within-bone appearance of the femora and iliac bones. Radiolucent bands and splaying of the distal femoral metaphyses are visible. (C) Anteroposterior view of the left hand with superposition of adult fingers fixing the baby’s hand. Note bone-within-bone appearance of the metacarpals and proximal phalanges, as well as splaying and radiolucency of the metaphyses of the tubular bones.

osteopetrosis and even dominant osteopetrosis (discussed further below). The CLCN7 protein is a dimeric transporter and, in the dominant form, it is believed that patients have an assortment of normal and mutant dimers, the latter supposed to be inefficient in Cl transport. It is believed that homozygosity for a complete loss-of-function mutation of CLCN7 results in severe osteopetrosis, whereas the intermediate form is due to mutations that only mildly reduce Cl conductance [26]. Finally, we demonstrated that mutations in the PLEKHM1 gene underlie an intermediate type of human osteopetrosis in two members of the same family [25]. The clinical outcome in this family is mild. The oldest patient suffers from Erlenmeyer flask deformities of the distal femora and a chondrolysis of the hip. Her younger brother has not yet developed clinical features. The PLEKHM1 protein is involved in osteoclastic vesicular transport, although the exact function is not yet known. Currently, the molecular genetic causes of a number of cases with an intermediate form of osteopetrosis still remain undefined. Osteopetrosis with Renal Tubular Acidosis This disease is also known as GuibaudeVainsel syndrome or marble brain disease. Clinical manifestations include short stature (2 SD to 4 SD), cerebral calcifications (basal ganglia and periventricular white matter), mental retardation, visual impairment, mixed renal tubular acidosis, osteomalacia, extramedullary hematopoiesis, hepatosplenomegaly, pancytopenia, and sensorineural hearing loss [27]. Usually, there are no hematological manifestations of the disease. Recently, a patient with this form of osteopetrosis was diagnosed with primary pulmonary hypertension. Most of the

patients have a combination of proximal and distal renal tubular acidosis. Major radiographic findings include a thick sclerotic skull base, a poor pneumatization of the paranasal sinuses and mastoids, generalized sclerosis, defective metaphyseal modeling with and without fractures, poor corticomedullary differentiation, transverse striations in metaphyses and “sandwich” vertebrae. Loss-of-function mutations in the carbonic anhydrase II (CAII) gene have been shown to be responsible for this form of osteopetrosis [28]. This enzyme forms protons essential for the acidification and resorption of the extracellular matrix. Autosomal Dominant Osteopetrosis Type II Autosomal dominant osteopetrosis has been reported with a prevalence estimated up to 5.5:100 000 [29]. Bollerslev divided these into two types based on clinical and radiological characteristics [29]. Afterwards, this was confirmed at the molecular genetic level with the identification of LRP5 mutations in the type I as discussed below. Type II is also known as AlberseScho¨nberg disease. Onset of clinical and radiological manifestations usually occurs in late childhood or adolescence, although earlier onset has been reported. Clinical manifestations can include anemia and hepatosplenomegaly, poor dentition, defects of vision and hearing and osteomyelitis of maxilla and mandible. Patients with this form of osteopetrosis also have an increased fracture risk. The most affected bone is the femur, but fractures in any long bone and in the vertebral arches can occur. Hip osteoarthritis and lumbar scoliosis may develop in adulthood. These patients display a generalized sclerosis, predominantly at the vertebral endplates (rugger-jersey spine), the iliac wings (Fig. 20.3A, B and C) and the skull

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FIGURE 20.3 Osteopetrosis type II. (A) Plain radiograph of the abdomen. Note the presence of “endobones (bone-within-bone)” at the iliac bones and proximal femora. Sclerosis is seen at the vertebral end-plates (rugger-jersey spine). The latter is better seen on coronal reformatted CT scan of the abdomen. (B) and (C) Coronal reformatted CT scan of the abdomen (bone window) confirming sclerosis at the pelvic bones and the vertebral end-plates (rugger-jersey spine). (D) Plain radiograph of the distal femur and knee (same patient as A, B and C), showing the “bonewithin-bone” aspect.

base. The long bones show often a “bone-within- bone” aspect and patchy sclerosis (Fig. 20.3D). Most cases of this dominant osteopetrosis have been associated with heterozygous missense mutations causing a dominant negative effect on the CLCN7 protein that acts as a homodimer [26].

Osteoclast-Poor Osteopetrosis A small subset of cases, the so-called osteoclast-poor osteopetroses, is characterized by a reduced number of osteoclasts indicating an impaired osteoclast differentiation. First, X-linked osteopetrosis, anhydrotic ectodermal dysplasia, and immunodeficiency (OLEDAID) shows

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a broad phenotypic spectrum in affected boys: all suffer from immunodeficiency, and most of them have anhydrotic ectodermal dysplasia, whereas osteopetrosis, lymphedema, and hemangioma are rather variable features. OLEDAID is caused by hypomorphic mutations in the nuclear factor kB essential modulator (NEMO), an upstream signaling protein of NFkB, a key regulator of osteoclastogenesis [30]. Second, Sobacchi and colleagues reported six patients with autosomal recessive osteopetrosis whose biopsy specimens lacked osteoclasts [31]. In comparison with the osteoclast-rich forms of severe osteopetrosis, the disease progression in these patients is slower. Mutations in the TNFSF11 gene encoding RANKL were found to be causative. Binding of RANKL to its receptor, RANK, is essential for the commitment of osteoclast precursors to differentiate into mature osteoclasts. HSCT will probably not improve bone remodeling due to the absence of the right microenvironment for osteoclastogenesis. These patients might benefit from early RANKL administration or bone marrow mesenchymal stem cell transplantation in order to restore osteoclastogenesis, but so far none of these treatments has been reported. Finally, mutations in the TNFRSF11A gene encoding RANK were found to underlie severe autosomal recessive osteopetrosis in eight patients of seven unrelated families [32]. These patients suffered from increased bone density, anemia, severe narrowing of the optical foramina, hypogammaglobulinemia and three of these patients died at young age. HSCT has proven to be a possible cure in these cases. Conclusion on the Osteopetroses The osteopetroses are clinically and radiologically a heterogeneous group but with a shared underlying

mechanism of impaired bone resorption. Molecular and genetic studies in the last decade confirmed this heterogeneity as illustrated in Figure 20.1. In some cases, the impaired bone resorption is due to reduced osteoclastogenesis while in other forms it is really intrinsic to the functioning of the osteoclast. This has also implications to the efficiency of bone marrow transplantation which can only work in cases where the underlying genetic defect involves the osteoclast. Molecular genetic testing is therefore definitely recommended before bone marrow transplantation is performed.

Pycnodysostosis Maroteaux and Lamy, and Andre´n and colleagues introduced the name pycnodysostosis (the Greek word pyknos meaning “thick, stocky”) for a condition characterized by a short-limb type of dwarfism. Clinical features include dolichocephaly, persistence of open fontanels, small face, often with hypoplastic maxilla and bulging eyes, parrot-like nose, receding chin, hypoplastic mandibular angle and dental abnormalities [33,34]. Bone fragility is increased; over 70% of affected individuals have multiple fractures during their lifetime. Because of shortness of the extremities, adult height is reduced to 135e160 cm. About 125 cases have been reported. The most characteristic radiological findings include generalized sclerosis, persistence of cranial sutures, thin and dense calvaria, obtuse angle of the mandibula, and osteolysis of the distal phalanges and acromial ends of the clavicles (Fig. 20.4). Using densitometry studies, Karkabi and colleagues showed that the increased bone density mainly resides within the trabecular bone and not the cortical bone [35].

FIGURE 20.4 Pycnodysostosis. (A) Plain radiograph of the hands. Generalized sclerosis of the tubular bones, with relative preservation of the medullary canal. (B) Lateral radiograph of the mandible showing the characteristic obtuse mandibular angle.

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Microscopic studies of the involved bones have shown reduction in osteoclastic and osteoblastic activity and the bone has been found to be sclerotic. Electron microscopic studies have demonstrated that osteoclasts have diminished or even inactive secretory functions [36]. Pycnodysostosis has an autosomal recessive inheritance pattern. Gelb et al. found mutations in the cysteine protease gene Cathepsin K (CTSK) [37]. This is a lysosomal protease with high expression in osteoclasts, which secretes the enzyme for bone matrix degradation. Hence, pycnodysostosis is a lysosomal disorder caused by defective tissue-specific expression of cathepsin K. Twelve different mutations, spread throughout the whole gene have been reported [38]. CTSK is responsible for the degradation of collagen type 1 at low pH. Patients’ osteoclasts are polarized as normal and able to demineralize the matrix underneath the ruffled border. Collagen degradation, however, does not occur and these cells are found to contain many cytoplasmatic vacuoles filled with undegraded collagen.

Osteopoikilosis and Melorheostosis Osteopoikilosis, literally meaning “spotted bones”, is a benign condition characterized by osteosclerotic foci that occur in the epimetaphyseal regions of long bones (Fig. 20.5A). They are usually found in the shoulders, wrists, knees, ankles, pelvis, and scapula. It is usually found incidentally since patients are often asymptomatic. Sometimes it is associated with skin manifestations and, in such cases, it is called BuschkeeOllendorff syndrome [39]. The skin lesions represent disseminated

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connective tissue nevi with both elastic-type nevi (juvenile elastoma) and collagen-type nevi (dermatofibrosis lenticularis disseminata) being described [40]. In a few patients with osteopoikilosis melorheostosis can also be present [41]. The latter condition is characterized by linear hyperostosis of the cortex of long bones reminiscent of dripping candle wax (“rheos” means flowing) (Fig. 20.5B,C). These bony lesions are often accompanied by abnormalities of adjacent soft tissues, such as joint contractures, sclerodermatous skin lesions, muscle atrophy, hemangiomas, and lymphedema [42]. Radiographically, the hyperostotic spots in osteopoikilosis show symmetric but unequal distribution in different parts of the skeleton (see Fig. 20.5A). They represent loci of old remodelled bone with lamellar structure, either connected to adjacent trabeculae of spongy bone or attached to the subchondral cortex. In melorheostosis, the lesions are usually asymmetric: they may involve only one limb or correspond to a particular sclerotome. Osteopoikilosis and BuschkeeOllendorff syndrome are segregating in an autosomal dominant manner with considerable intrafamilial variation. Hellemans et al. identified heterozygous loss-of-function mutations in the gene encoding LEMD3 [40]. LEMD3 is an integral nuclear membrane protein with the capacity to inhibit BMP and TGFb signalling. Melorheostosis is mostly seen as an isolated feature, however, there seems to be an increased prevalence for this very rare condition in families with osteopoikilosis or BuschkeeOllendorff syndrome. It was hypothesized that a somatic mutation in the LEMD3 gene may cause

FIGURE 20.5 (A) Osteopoikilosis. Plain radiograph of the hand. Multiple foci of sclerosis are seen within the carpal bones and the epiphyses of the distal radius and ulna and the tubular bones of the hand. (B) Melorheostosis. Plain radiograph of the left hip. Focal sclerosis of the femoral head, neck and proximal diaphysis, with thickening of the cortex. (C) CT scan of left hip. Extensive intramuscular soft-tissue ossification is seen as well as cortical thickening of the anterior aspect of the femoral neck.

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sporadic melorheostosis. However, no experimental evidence to support this hypothesis could be obtained so far [40,42]. Hellemans et al. took skin biopsy samples from two affected individuals, one from an elastic-type nevus in a person with BuschkeeOllendorff syndrome and the second from a hard scleroderma-like lesion in an individual with melorheostosis, but no somatic mutations (second hits) in the LEMD3 gene could be identified [40].

Osteopathia Striata with Cranial Sclerosis (OSCS) In 1924, Voorhoeve was the first to describe a syndrome of cranial sclerosis with osteopathia striata of which currently over 100 cases have been reported [43]. In males, this entity is usually associated with fetal or neonatal lethality, due to severe heart defects and/or gastrointestinal malformations, and is often accompanied by bilateral fibula aplasia [44]. In females, the disease is mostly much milder with macrocephaly, cleft palate and mild learning disabilities. At birth, the cranium is usually biparietally enlarged. This is frequently mildly progressive so that adult head circumference is often 60e65 cm. There is frontal bossing, the nasal bridge is broad, and the eyes appear wide set [45]. On radiographs, there is evidence for sclerosis of the long bones and skull, and longitudinal striations mainly in the metaphyses of the long bones, pelvis, and scapulae [46] (Fig. 20.6). However, the striations are in general only seen in female patients. Osteopathia striata with cranial sclerosis is an X-linked dominant sclerosing bone dysplasia with a more severe presentation in males [47]. Recently, Jenkins et al. were able to identify mutations in the gene encoding WTX (Wilms tumor on the X-chromosome), a repressor for Wnt signaling, as the cause of X-linked OSCS which we confirmed in a large set of families and patients [48]. WTX encodes a 1135 amino-acid protein but with two splice forms, WTXS1 and the shorter WTXS2 resulting from excluding residues 50e326. Both isoforms retain the ability to bind b-catenin, but only WTXS1 is localized to the plasma membrane, and is therefore important for the suppression of WNT signaling [48]. The wide phenotypic spectrum of OSCS cannot be explained solely by the type of WTX gene defect as illustrated by the highly variable expression of the disease within one family [49]. Nonrandom X-inactivation in OSCS could provide an explanation for the clinical variation as seen in females. For males, Jenkins and colleagues suggested a possible genotypeephenotype correlation that relates the position of the mutations in WTX with survival but our data could not confirm this [49]. The involvement of a WNT-inhibitor in a sclerosing bone dysplasia is not unexpected since several lines of evidence indicated

FIGURE 20.6 Osteopathia striata. Plain radiograph of the right knee. Note typical dense linear striations in the (sub)metaphyseal regions of the distal femur and proximal tibia.

the essential role of this pathway in bone formation. Mutations in other regulators of this pathway were identified in other sclerosing bone dysplasias including the endosteal hyperostoses (see below). The remarkable finding of longitudinal striations in female patients suggests the presence of a mixture of affected and nonaffected osteoblasts in the growth plate, due to random X-inactivation. Unexpectedly, a male patient with longitudinal striations was reported recently but DNA sequencing revealed mosaicism for a WTX mutation in this patient [50].

INCREASED BONE DENSITY GROUP WITH METAPHYSEAL AND/OR DIAPHYSEAL INVOLVEMENT Craniometaphyseal Dysplasia Craniometaphyseal dysplasia (CMD) is characterized by abnormal modeling of the long bones, resulting in metaphyseal flaring in combination with hyperostosis and sclerosis of the cranial bones [51]. The facial abnormalities with a thick bony wedge over the bridge of the nose and glabella, are usually the first signs of the disease. About 80 cases have been reported. Bony

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overgrowth and sclerosis of the skull base cause variable compression of cranial nerves, resulting in combined hearing loss (VIII), impaired vision (II) and facial nerve paralysis (VII). Hypertelorism is a frequent feature, and nystagmus is common. Bony alterations in the temporal bone and pyramid produce mixed hearing loss that becomes evident in childhood in about onehalf of the cases. Chronic otitis media is common. Hyperostosis and sclerosis involve the frontal and occipital portions of the calvaria, skull base and, less often, mandible. The ribs are wide and dense. Long bones have a club-shaped metaphyseal widening which may be minimal during the first years of life. Cortical hyperostosis of the diaphyses is noted in infancy, but usually disappears with age. Both an autosomal dominant form and a severe recessive form have been reported with the former being the most common. The recessive form has been mapped to 6q21-q22 but the genetic defect is still unknown [52]. The dominant form is caused by mutations in the progressive ankylosis gene (ANKH), a transmembrane protein that transports intracellular pyrophosphate to the extracellular milieu [53,54]. Recent work by Kirsch and colleagues suggests that ANKH is a positive regulator of differentiation events towards a mature osteoblastic phenotype [55]. ANKH mutations are also associated with familial chrondrocalcinosis which sometimes cosegregates with craniometaphyseal dysplasia [56].

Progressive Diaphyseal Dysplasia (CamuratieEngelmann Disease) This condition was originally reported by Cockayne in 1920 [57], and later defined by Camurati in 1922 and by Engelmann in 1929 and currently more than 200 cases have been reported [57e59]. CamuratieEngelmann disease or progressive diaphyseal dysplasia is a sclerotic and hyperostotic disorder of bone. The clinical and radiological aspects of the disease were reviewed by Janssens et al [60]. The most common clinical findings include bone pain, easy fatiguability, a waddling gait, generalized neuromuscular weakness, delayed ambulation and reduced subcutaneous fat and muscular mass. There is a large variability in the severity, with some cases being asymptomatic while, in others, the disease is severely disabling. Symptoms can manifest from the age of 3 but the mean age for full development of the disorder is about 15e20 years. Some patients exhibit frontal bossing, exophthalmos, papilledema, epiphora, optic atrophy, and headache. Mixed hearing loss has been noted in 5e50% of patients and sometimes diminishes after decompression procedures. Corticosteroid therapy has proven to be effective in many cases, reducing pain, muscle weakness and joint contractures, and improving exercise tolerance but obviously long-

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term glucocorticoid treatment is not advisable because of unfavorable side effects. The radiographic hallmark is a symmetrical but irregular cortical thickening mainly of the diaphyses of the long tubular bones with narrowing of the medullary cavities (Fig. 20.7). The metaphyses are rarely involved and the epiphyses are not. The base of the skull and calvaria are often sclerotic. The vertebrae can also be affected but, in some cases, they are reported to be osteopenic. The scintigraphic changes are also striking. The disease is inherited in an autosomal dominant mode with reduced penetrance. Anticipation was reported in some families but later on no molecular support for this could be obtained. Missense mutations in the latency associated peptide of TGFB1, the gene on chromosome 19q13.1-q13.3 encoding transforming growth factor b-1 (TGFb1) have been identified [61,62]. These mutations cause a decreased stability of the binding of the latency-associated peptide to mature TGFb1, thereby causing an increased activity of the growth factor. Genetic heterogeneity cannot be excluded as no TGFb1 mutations were found in some cases [63].

Oculodento-Osseous Dysplasia (ODOD) Oculodento-osseous dysplasia, also known as oculodentodigital syndrome, is characterized by a typical facial appearance with short palpebral fissures and a thin, long nose with hypoplastic alae and thin nostrils. Ocular anomalies include microcornea, microphthalmia (rather rare) and eccentric pupil. The teeth may be small

FIGURE 20.7 CamuratieEngelmann disease. Plain radiograph of the left forearm. Cortical sclerosis of the diaphyses of the radius and ulna, causing narrowing of the medullary canal.

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and have enamel hypoplasia. The characteristic digital malformation is complete syndactyly of the fourth and fifth fingers and associated camptodactyly is a common finding [64]. ODOD is usually inherited as an autosomal dominant condition. Paznekas et al. identified mutations in the connexin-43 gene (GJA1) which may cause misassembly of channels or alter channel conduction properties [65]. In a later study, Paznekas et al. reviewed the known mutations in GJA1 as well as the phenotypic information available on 177 affected individuals from 54 genotyped families [66]. The characteristic facies was seen in 92% of the families, with ocular, dental, and digital manifestations present in 68%, 70%, and 80% of the families, respectively. Neurologic manifestations were seen in 30% of families, conductive hearing loss in 26%, and poor hair growth in 26%. Because of phenotypic variability, even within families, and since mutations were equitably distributed throughout the protein, no clear genotype/phenotype correlations could be established. A few cases are reported with a putative autosomal recessive mode of inheritance but it is assumed this might represent homozygosity for a mild form of ODOD [67]. In some of these patients, a homozygous loss-of-function mutation was found in the connexin43 gene [68].

Trichodento-Osseous Dysplasia (TDO) This condition was first described by Robinson and Miller in 1966 and is characterized by amelogenesis imperfecta, taurodontism and curly hair [69]. In some cases, mild increase in bone density, particularly in the skull, has been noted [70]. Both keratin and enamel are defective as fingernails show either laminated splitting of the superficial layers or thick cornification. Unique kinky/curly hair at birth was reported in 85% of affected individuals. The curly hair phenotype is retained after infancy in 46% of affected individuals. Taurodontism is present in most cases but with variable expression [71]. Radiologically, in some patients, sclerosis and thickening of the calvaria is seen with long bones that showed subtle undertubulation but no sclerosis [70]. Thick cranial bones, lack of visible pneumatization of the mastoid processes, and/or obliteration of the calvarial diploe¨ was seen in the majority of affected persons. Price et al. identified a 4-bp deletion in human DLX3 causing a frameshift and premature termination codon, thus resulting in a functionally altered DLX3 protein [72]. It was also concluded that the variable clinical phenotype observed in these families that share a common mutation might reflect genetic heterogeneity at other epigenetic loci or contributing environmental factors or both.

Juvenile Paget’s Disease This condition was first described by Sorrel and Legrand-Lambling in 1938 [73]. It is also known as osteoectasia with hyperphosphatasia or familial hyperphosphatasemia [74]. The syndrome is characterized by fever, bone pain, and swelling of the extremities during the first year of life. Later on, an increased fracture rate, enlargement of the calvaria as well as bending of the bones of the extremities occur. Progressive mixed hearing loss is often observed. Muscle weakness retards walking and running and often a wheelchair is needed. Adult patients are short with a height varying between 120 and 154 cm [75]. Biochemically, extremely high serum alkaline phosphatase levels are reported. Radiographic examination of the skull reveals changes (“cotton ball patches”) similar to those seen in classical Paget’s disease. Long bones exhibit bending and generalized cortical widening due to broadening of the diaphyseal areas of tubular bones. The bone trabeculation is coarse and bone density diminished. Short bones are involved to a lesser degree, mostly on the endosteal side. There is mild flattening of vertebral bodies. The facial bones are in general not involved [76]. The condition has an autosomal recessive inheritance. Homozygous loss of function mutations in the TNFRSF11B gene have been identified in most cases but genetic heterogeneity cannot be excluded [77]. TNFRSF11B encodes osteoprotegerin (OPG), a secreted glycoprotein of the tumor necrosis factor receptor superfamily that regulates bone resorption by acting as a decoy receptor to the RANKL protein. This protein binds to its receptor RANK at the (pre-)osteoclast and in this way induces NFkB signaling, a key pathway for osteoclastogenesis and bone resorption. The lack of OPG induces increased bone resorption consecutively compensated by accelerated bone formation. This increased bone turnover results in the formation of bone tissue of inferior quality.

Endosteal Hyperostoses or Craniotubular Hyperostoses This is a group of conditions mainly characterized by an increased cortical thickness. As for some bones a narrowed medullary canal or disappearance of diploe¨ were reported, they were called the endosteal hyperostoses. However, detailed radiological survey of Van Buchem patients indicated that besides an increased outer diameter of the tibia there was also an increased inner diameter [78]. Furthermore, in older patients, periosteal bone spikes can be detected. This all indicates an increased periosteal bone formation rather than endosteal. Based on this and the major involvement of skull and long tubular bones, we previously suggested to name these disorders the craniotubular hyperostoses [79].

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Sclerosteosis and Van Buchem Disease Sclerosteosis was described as early as 1929 by Hirsch but later on defined as sclerosteosis by Hansen [80,81]. The disorder, reviewed by Beighton in 1988, is characterized by hyperostosis of the calvaria, mandible, clavicles, pelvis and long bones [82]. The typical facies is characterized by frontal bossing, hypertelorism, and broad flat nasal root. The mandible is prognathic, broadened and squared, and dental malocclusion is frequent. In most cases, mixed hearing loss appears in childhood. Head circumference is enlarged and facial nerve paralysis is common in adulthood. There is increased intracranial pressure in 80% of cases which can result in sudden death due to impaction of the medulla in the foramen magnum. Visual loss is a frequent complication. Affected individuals are usually tall (height is over 180 cm in 70% of patients). Partial or complete cutaneous syndactyly of the index and middle fingers occurs often as well as radial deviation of the distal phalanx of the index fingers and dysplastic nails. On radiographs, there is hyperostosis of the calvaria, base of the skull and mandible. The tubular bones lack diaphyseal constriction and show endosteal thickening (Fig. 20.8A). The index finger may have no middle phalanx or only a small triangular bone (delta phalanx), producing radial deviation. Osseous syndactyly may involve the second and third fingers. Marked thickening

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and sclerosis of the skull bones and narrowing of the internal auditory canals can be seen (Fig. 20.8B). Sclerosteosis is always considered to be an autosomal recessive trait, although radiological examination of several heterozygotes suggests an autosomal partial dominant inheritance. Close to 100 cases have been reported, mostly South Africans of Dutch ancestry [83]. Its frequency in Afrikaners has been estimated to be about 1/60 000. Sclerosteosis is caused by homozygous mutations of the SOST gene encoding sclerostin located on chromosome 17q12-q21 [84,85]. So far, four different loss-of-function mutations in SOST have been reported in patients with sclerosteosis. Very recently, we found the first missense mutation in this gene substituting one of the cystein residues [86]. Because of the presence of a cystein knot motif in sclerostin and the homology with members of the gremlin-DAN gene family, sclerostin was initially suggested to be a BMP-inhibitor. However, functional studies indicated that it is more likely to act as an inhibitor of Wnt-signaling, an essential pathway for bone anabolism [87]. This was partially based on the fact that it can bind the LDL receptor related protein (LRP)-5 protein. This protein acts as a co-receptor for Wnt molecules and turned out to be involved in autosomal dominant forms of craniotubular hyperostoses (see below). Very recently, we were able to obtain further support for this pathogenic mechanism

FIGURE 20.8 Van Buchem’s disease. (A) Plain radiograph of the left hand. Endosteal thickening of the diaphyseal cortices of the tubular bones. Note also modeling defect of the metacarpals and phalanges. (B) CT scan at the level of the posterior fossa. Marked thickening and sclerosis of the skull bones and narrowing of the internal auditory canals.

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by the identification of two missense mutations in LRP4 in sclerosteosis patients which was shown to have a facilitator function on SOST’s Wnt-inhibitory capacities. Again, the missense mutations disrupt the binding with sclerostin and therefore result in increased Wntsignaling [88]. Van Buchem disease, first described in 1955, is marked by a similar but milder phenotype than sclerosteosis. Most cases are of Dutch ancestry and related to each other, originating from a small ethnic isolate in The Netherlands that used to be a small island until the 1940s. Patients are of normal height and do not show syndactyly. The radiographic abnormalities are very similar compared to sclerosteosis (see Fig. 20.8). Both rare bone disorders are allelic conditions with a genetic defect on chromosome 17q12-q21 [89,90]. However, no mutation was found in the SOST gene of these patients, but we and others have shown that individuals with Van Buchem disease harbor a 52-kb deletion downstream of the SOST gene without evidence of an intragenic mutation in the gene itself [85,91]. Afterwards, it was shown that within this deleted region is located an enhancer that drives the expression of the SOST gene [92]. Autosomal Dominant Craniotubular Hyperostoses Besides the autosomal recessive conditions sclerosteosis and Van Buchem disease, patients have been described with a similar phenotype segregating in an autosomal dominant mode [93]. Their craniofacial phenotype is characterized by frontal prominence, hypertelorism and a square jaw. Sometimes a torus palatinus is present. Based on this, the diagnoses of Worth disease, autosomal dominant osteosclerosis or endosteal hyperostosis have been reported in the literature. Furthermore, in two unrelated kindreds from Caucasian origin, this was defined as the “high bone mass” phenotype [94,95]. Genetic analysis revealed the same missense mutation (G171V) in the low-density lipoprotein receptor-related protein 5 (LRP5) gene in both families [96]. The influence of this protein on bone density was also supported by revealing loss-of-function mutations in this gene underlying the osteoporosis-pseudoglioma syndrome [97]. In the following years, patients initially diagnosed with endosteal hyperostosis, Worth disease, osteosclerosis, or even autosomal dominant osteopetrosis type I, were shown to carry a heterozygous missense mutation in LRP5. These mutations all clustered in the first b-propeller domain, situated in the extracellular part of the protein [98]. The bone phenotype, with dense bones and cortical hyperostosis mainly affecting the cranial and tubular bones, is similar in all patients described (Fig. 20.9). Functional studies revealed that all the missense mutations impaired the binding between LRP5 and the extracellular Wnt-antagonists sclerostin

FIGURE 20.9 Osteopetrosis type I. Plain radiograph of the distal femur showing uniform sclerosis at the diaphysis, metaphysis and epiphysis.

and dickkopf 1 [99,100]. The precise mechanism by which Wnt signaling increases bone formation seems to play a role at different levels. Canonical Wnt signaling promotes osteoblast differentiation from mesenchymal stem cells, while it inhibits both chondrocyte and adipocyte differentiation. Wnt signalling enhances osteoblast proliferation and activation (mineralization) and suppresses osteoblast apoptosis [101]. This pathway also interferes with osteoclastogenesis by increasing the OPG/RANKL ratio [102,103]. Recently, Yadav and colleagues reported evidence that LRP5 can also act in a b-catenin-independent manner demonstrating that LRP5 controls bone formation by inhibiting serotonin synthesis in enterochromaffin cells in the duodenum [104]. Gut- but not osteoblast-specific LRP5 regulates the expression of tryptophan hydroxylase 1 (Tph1), the rate-limiting biosynthetic enzyme for serotonin in enterochromaffin cells. High peripheral gut-derived serotonin levels inhibit osteoblast proliferation through the 5-HTreceptor (Htr1b) on the osteoblasts. As these discoveries conflict with the common belief that LRP5 mutations directly effect the osteoblast, further studies will be needed to unveil LRP5’s role in the regulation of bone mass.

LenzeMajewski Hyperostotic Dysplasia The syndrome was first reported by Braham in 1969 and later on by Lenz and Majewski in 1974 and 2000

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[105e107] and is characterized by large head, characteristic facies, loose skin, mental retardation, and skeletal anomalies. Gorlin and Whitley reviewed 11 cases [108]. The head appears disproportionately enlarged with large fontanels and widely separated, late-closing sutures while the sizes of trunk and limbs are reduced. The ears are large and floppy. Hypertelorism is usually present. Cryptorchidism has been a uniform finding in affected males and the anus may be anteriorly displaced. Inguinal hernia is common. The digits are hyperflexible and proximal interdigital webbing of the fingers is frequently observed. All children have been mentally retarded with IQs between 20 and 40. Sensorineural hearing loss is frequent. The skin is thin, loose, wrinkled, and atrophic with veins that can be prominent. Radiographic features include progressive sclerosis of the skull base, mandible, maxilla, orbital roofs and vertebrae. Skeletal maturation is often retarded. The clavicles and ribs are broad. The middle phalanges are short during infancy and may fuse with proximal phalanges during childhood resulting in proximal symphalangism. The long bones exhibit meta- and diaphyseal undermodeling and midshaft cortical thickening. All cases reported so far are isolated. The genetic defect is unknown.

families suggesting an autosomal recessive mode of inheritance [113,114]. Genevieve et al. identified mutations in the TBXAS1 gene, which encodes thromboxane synthase (TXAS), an enzyme of the arachidonic acid cascade [115]. Platelets from subjects with GHDD showed a specific deficit in arachidonic acid-produced aggregation. Furthermore, in primary cultured osteoblasts, an effect was reported on the expression of TNFSF11 and TNFRSF11B encoding RANKL and osteoprotegerin (OPG) respectively. As these proteins are key players in osteoclastogenesis and bone resorption, this might explain the effect of the mutations on bone homeostasis.

CONCLUSIONS As illustrated, the underlying genetic defects for many of the sclerosing bone dysplasias have been identified in recent years. Besides applications towards diagnostic testing and genetic counseling, these findings elucidated the involvement of several pathways related to bone resorption or bone formation and contributed towards the current understanding of bone metabolism and homeostasis.

References

Pyle Disease This is a mild disease with only a few clinical findings including genu valgum, joint pain and muscular weakness. The disorder is radiographically characterized by metaphyseal flaring of the tubular bones, giving the typical “Erlenmeyer flask appearance” to femora and tibiae. Hyperostosis of the cranial vault is mild to moderate in contrast to craniometaphyseal dysplasia where the cranial sclerosis is more pronounced [109]. Pyle disease is an autosomal recessive disorder. The underlying genetic defect remains unidentified [110].

Ghosal Syndrome or Ghosal Hematodiaphyseal Dysplasia (GHDD) Ghosal syndrome was first described in 1988 by Ghosal et al. and is characterized by severe anemia, leukopenia, and thrombocytopenia [111]. Radiologically, it presents with wide medullary cavities in the long bones and discrete cortical hyperostosis [112]. There are striking similarities with CamuratieEngelmann syndrome except that both the diaphyses and metaphyses are affected. The smooth surface of the long bones indicates that there is no periosteal but only endosteal hyperostosis. Bone marrow is hypocellular causing the hematological problems that differentiate the disorder from CamuratieEngelmann syndrome. Parental consanguinity was present in several

[1] Superti-Furga A, Unger S. Nosology and classification of genetic skeletal disorders: 2006 revision. Am J Med Genet A 2007;143:1e18. [2] Blomstrand S, Claesson I, Save-Soderbergh J. A case of lethal congenital dwarfism with accelerated skeletal maturation. Pediatr Radiol 1985;15:141e3. [3] Loshkajian A, Roume J, Stanescu V, Delezoide AL, Stampf F, Maroteaux P. Familial Blomstrand chondrodysplasia with advanced skeletal maturation: further delineation. Am J Med Genet 1997;71:283e8. [4] Jobert AS, Zhang P, Couvineau A, et al. Absence of functional receptors for parathyroid hormone and parathyroid hormonerelated peptide in Blomstrand chondrodysplasia. J Clin Invest 1998;102:34e40. [5] FitzPatrick DR, Keeling JW, Evans MJ, et al. Clinical phenotype of desmosterolosis. Am J Med Genet 1998;75:145e52. [6] Waterham HR, Koster J, Romeijn GJ, et al. Mutations in the 3beta-hydroxysterol delta24-reductase gene cause desmosterolosis, an autosomal recessive disorder of cholesterol biosynthesis. Am J Hum Genet 2001;69:685e94. [7] Raine J, Winter RM, Davey A, Tucker SM. Unknown syndrome: microcephaly, hypoplastic nose, exophthalmos, gum hyperplasia, cleft palate, low set ears, and osteosclerosis. J Med Genet 1989;26:786e8. [8] Kan AE, Kozlowski K. New distinct lethal osteosclerotic bone dysplasia (Raine syndrome). Am J Med Genet 1992;43:860e4. [9] Simpson MA, Hsu R, Keir LS, et al. Mutations in FAM20C are associated with lethal osteosclerotic bone dysplasia (Raine syndrome), highlighting a crucial molecule in bone development. Am J Hum Genet 2007;81:906e12. [10] Ro¨ske G. Eine eigenartige knochenerkrankung im sa¨uglingsalter. Monatsschr Kinderheilkd 1930;47:385e400.

PEDIATRIC BONE

554

20. SCLEROSING BONE DYSPLASIAS

[11] Caffey J, Silverman WA. Infantile cortical hyperostosis. J Pediatr 1945;48:739e53. [12] Turnpenny PD, Davidson R, Stockdale EJ, Tolmie JL, Sutton AM. Severe prenatal infantile cortical hyperostosis (Caffey’s disease). Clin Dysmorphol 1993;2:81e6. [13] Holman GH. Infantile cortical hyperostosis: a review. Q Rev Pediatr 1962;17:24e31. [14] Maclachlan AK, Gerrard JW, Houston CS, Ives EJ. Familial infantile cortical hyperostosis in a large Canadian family. Can Med Assoc J 1984;130:1172e4. [15] Saul RA, Lee WH, Stevenson RE. Caffey’s disease revisited. Further evidence for autosomal dominant inheritance with incomplete penetrance. Am J Dis Child 1982;136:55e60. [16] Gensure RC, Makitie O, Barclay C, et al. A novel COL1A1 mutation in infantile cortical hyperostosis (Caffey disease) expands the spectrum of collagen-related disorders. J Clin Invest 2005;115:1250e7. [17] Perdu B, Van Hul W. Sclerosing bone disorders: too much of a good thing. Crit Rev Eukaryot Gene Expr 2010;20:195e212. [18] Gerritsen EJ, Vossen JM, van Loo IH, et al. Autosomal recessive osteopetrosis: variability of findings at diagnosis and during the natural course. Pediatrics 1994;93:247e53. [19] Frattini A, Orchard PJ, Sobacchi C, et al. Defects in TCIRG1 subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive osteopetrosis. Nat Genet 2000;25:343e6. [20] Kornak U, Kasper D, Bosl MR, et al. Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man. Cell 2001;104:205e15. [21] Chalhoub N, Benachenhou N, Rajapurohitam V, et al. Greylethal mutation induces severe malignant autosomal recessive osteopetrosis in mouse and human. Nat Med 2003;9:399e406. [22] Balemans W, Van Wesenbeeck L, Van Hul W. A clinical and molecular overview of the human osteopetroses. Calcif Tissue Int 2005;77:263e74. [23] Kahler SG, Burns JA, Aylsworth AS. A mild autosomal recessive form of osteopetrosis. Am J Med Genet 1984;17:451e64. [24] Frattini A, Pangrazio A, Susani L, et al. Chloride channel ClCN7 mutations are responsible for severe recessive, dominant, and intermediate osteopetrosis. J Bone Miner Res 2003;18:1740e7. [25] Van Wesenbeeck L, Odgren PR, Coxon FP, et al. Involvement of PLEKHM1 in osteoclastic vesicular transport and osteopetrosis in incisors absent rats and humans. J Clin Invest 2007;117:919e30. [26] Cleiren E, Benichou O, Van Hul E, et al. Albers-Schonberg disease (autosomal dominant osteopetrosis, type II) results from mutations in the ClCN7 chloride channel gene. Hum Mol Genet 2001;10:2861e7. [27] Ohlsson A, Cumming WA, Paul A, Sly WS. Carbonic anhydrase II deficiency syndrome: recessive osteopetrosis with renal tubular acidosis and cerebral calcification. Pediatrics 1986;77:371e81. [28] Sly WS, Hewett-Emmett D, Whyte MP, Yu YS, Tashian RE. 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 1983;80:2752e6. [29] Bollerslev J, Andersen Jr PE. Radiological, biochemical and hereditary evidence of two types of autosomal dominant osteopetrosis. Bone 1988;9:7e13. [30] Doffinger R, Smahi A, Bessia C, et al. X-linked anhidrotic ectodermal dysplasia with immunodeficiency is caused by impaired NF-kappaB signaling. Nat Genet 2001;27:277e85. [31] Sobacchi C, Frattini A, Guerrini MM, et al. Osteoclast-poor human osteopetrosis due to mutations in the gene encoding RANKL. Nat Genet 2007;39:960e2.

[32] Guerrini MM, Sobacchi C, Cassani B, et al. Human osteoclast-poor osteopetrosis with hypogammaglobulinemia due to TNFRSF11A (RANK) mutations. Am J Hum Genet 2008;83:64e76. [33] Maroteaux P, Lamy M. [Pyknodysostosis.]. Presse Med 1962;70:999e1002. [34] Andren L, Dymling JF, Hogeman KE, Wendeberg B. Osteopetrosis acro-osteolytica. A syndrome of osteopetrosis, acro-osteolysis and open sutures of the skull. Acta Chir Scand 1962;124:496e507. [35] Karkabi S, Reis ND, Linn S, et al. Pyknodysostosis: imaging and laboratory observations. Calcif Tissue Int 1993;53:170e3. [36] Soto RJ, Mautalen CA. Pycnodysostosis: Metabolic and histologic studies. Birth Defects 1969;5:109e16. [37] Gelb BD, Shi GP, Chapman HA, Desnick RJ. Pycnodysostosis, a lysosomal disease caused by cathepsin K deficiency. Science 1996;273:1236e8. [38] Bromme D, Okamoto K, Wang BB, Biroc S. Human cathepsin O2, a matrix protein-degrading cysteine protease expressed in osteoclasts. Functional expression of human cathepsin O2 in Spodoptera frugiperda and characterization of the enzyme. J Biol Chem 1996;271:2126e32. [39] Giro MG, Duvic M, Smith LT, et al. Buschke-Ollendorff syndrome associated with elevated elastin production by affected skin fibroblasts in culture. J Invest Dermatol 1992;99:129e37. [40] Hellemans J, Preobrazhenska O, Willaert A, et al. Loss-offunction mutations in LEMD3 result in osteopoikilosis, Buschke-Ollendorff syndrome and melorheostosis. Nat Genet 2004;36:1213e8. [41] Debeer P, Pykels E, Lammens J, Devriendt K, Fryns JP. Melorheostosis in a family with autosomal dominant osteopoikilosis: report of a third family. Am J Med Genet A 2003;119A:188e93. [42] Zhang Y, Castori M, Ferranti G, Paradisi M, Wordsworth BP. Novel and recurrent germline LEMD3 mutations causing Buschke-Ollendorff syndrome and osteopoikilosis but not isolated melorheostosis. Clin Genet 2009;75:556e61. [43] Voorhoeve N. L’image radiologique non encore de´crite d’une anomalie du squelette. Acta Radiol 1924;3(407). [44] Rott HD, Krieg P, Rutschle H, Kraus C. Multiple malformations in a male and maternal osteopathia strata with cranial sclerosis (OSCS). Genet Couns 2003;14:281e8. [45] Konig R, Dukiet C, Dorries A, Zabel B, Fuchs S. Osteopathia striata with cranial sclerosis: variable expressivity in a four generation pedigree. Am J Med Genet 1996;63:68e73. [46] Savarirayan R, Nance J, Morris L, Haan E, Couper R. Osteopathia striata with cranial sclerosis: highly variable phenotypic expression within a family. Clin Genet 1997;52:199e205. [47] Pellegrino JE, McDonald-McGinn DM, Schneider A, Markowitz RI, Zackai EH. Further clinical delineation and increased morbidity in males with osteopathia striata with cranial sclerosis: an X-linked disorder? Am J Med Genet 1997;70:159e65. [48] Jenkins ZA, van Kogelenberg M, Morgan T, et al. Germline mutations in WTX cause a sclerosing skeletal dysplasia but do not predispose to tumorigenesis. Nat Genet 2009;41:95e100. [49] Perdu B, de Freitas F, Frints SG, et al. Osteopathia striata with cranial sclerosis due to WTX gene defect. J Bone Miner Res 2009;25:82e90. [50] Joseph DJ, Ichikawa S, Econs MJ. Mosaicism in osteopathia striata with cranial sclerosis. J Clin Endocrinol Metab 2010;95:1506e7. [51] Beighton P. Craniometaphyseal dysplasia (CMD), autosomal dominant form. J Med Genet 1995;32:370e4. [52] Iughetti P, Alonso LG, Wilcox W, Alonso N, Passos-Bueno MR. Mapping of the autosomal recessive (AR) craniometaphyseal dysplasia locus to chromosome region 6q21-22 and confirmation of genetic heterogeneity for mild AR spondylocostal dysplasia. Am J Med Genet 2000;95:482e91.

PEDIATRIC BONE

REFERENCES

[53] Nurnberg P, Thiele H, Chandler D, et al. Heterozygous mutations in ANKH, the human ortholog of the mouse progressive ankylosis gene, result in craniometaphyseal dysplasia. Nat Genet 2001;28:37e41. [54] Reichenberger E, Tiziani V, Watanabe S, et al. Autosomal dominant craniometaphyseal dysplasia is caused by mutations in the transmembrane protein ANK. Am J Hum Genet 2001;68:1321e6. [55] Kirsch T, Kim HJ, Winkles JA. Progressive ankylosis gene (ank) regulates osteoblast differentiation. Cells Tissues Organs 2009;189:158e62. [56] Baynam G, Goldblatt J, Schofield L. Craniometaphyseal dysplasia and chondrocalcinosis cosegregating in a family with an ANKH mutation. Am J Med Genet A 2009;149A:1331e3. [57] Cockeyane EA. Case for diagnosis. Proc R Soc Med 1920;13:132e6. [58] Camaruti M. Di un raro do osteite simmetrica ereditaria degli art inferiori. Clin Organi Mov 1922;6:622e65. [59] Engelmann G. Ein Fall von Osteopathia hyperostotica (sclerosis) multiplex infantiles. Fortschr Roentgenol 1929;39:1101e16. [60] Janssens K, Vanhoenacker F, Bonduelle M, et al. CamuratiEngelmann disease: review of the clinical, radiological, and molecular data of 24 families and implications for diagnosis and treatment. J Med Genet 2006;43:1e11. [61] Janssens K, Gershoni-Baruch R, Guanabens N, et al. Mutations in the gene encoding the latency-associated peptide of TGF-beta 1 cause Camurati-Engelmann disease. Nat Genet 2000;26:273e5. [62] Kinoshita A, Saito T, Tomita H, et al. Domain-specific mutations in TGFB1 result in Camurati-Engelmann disease. Nat Genet 2000;26:19e20. [63] Nishimura G, Nishimura H, Tanaka Y, et al. Camurati-Engelmann disease type II: progressive diaphyseal dysplasia with striations of the bones. Am J Med Genet 2002;107:5e11. [64] Judisch GF, Martin-Casals A, Hanson JW, Olin WH. Oculodentodigital dysplasia. Four new reports and a literature review. Arch Ophthalmol 1979;97:878e84. [65] Paznekas WA, Boyadjiev SA, Shapiro RE, et al. Connexin 43 (GJA1) mutations cause the pleiotropic phenotype of oculodentodigital dysplasia. Am J Hum Genet 2003;72:408e18. [66] Paznekas WA, Karczeski B, Vermeer S, et al. GJA1 mutations, variants, and connexin 43 dysfunction as it relates to the oculodentodigital dysplasia phenotype. Hum Mutat 2009;30:724e33. [67] Beighton P, Hamersma H, Raad M. Oculodento-osseous dysplasia: heterogeneity or variable expression? Clin Genet 1979;16:169e77. [68] Richardson RJ, Joss S, Tomkin S, Ahmed M, Sheridan E, Dixon MJ. A nonsense mutation in the first transmembrane domain of connexin 43 underlies autosomal recessive oculodentodigital syndrome. J Med Genet 2006;43:e37. [69] Robinson GC, Miller JR. Hereditary enamel hypoplasia: its association with characteristic hair structure. Pediatrics 1966;37:498e502. [70] Lichtenstein J, Warson R, Jorgenson R, Dorst JP, McKusick VA. The tricho-dento-osseous (TDO) syndrome. Am J Hum Genet 1972;24:569e82. [71] Wright JT, Kula K, Hall K, Simmons JH, Hart TC. Analysis of the tricho-dento-osseous syndrome genotype and phenotype. Am J Med Genet 1997;72:197e204. [72] Price JA, Bowden DW, Wright JT, Pettenati MJ, Hart TC. Identification of a mutation in DLX3 associated with tricho-dentoosseous (TDO) syndrome. Hum Mol Genet 1998;7:563e9. [73] Sorrel ME, Legrand-Lambling M. Dystrophie osseuse ge´ne´ralise´e. Paris Bull Soc pe´diat 1938;36:89e92. [74] Dunn V, Condon VR, Rallison ML. Familial hyperphosphatasemia: diagnosis in early infancy and response to human thyrocalcitonin therapy. Am J Roentgenol 1979;132:541e5.

555

[75] Einhorn TA, Vigorita VJ, Teitcher JB. Hyperphosphatasemia in an adult. Clinical, roentgenographic, and histomorphometric findings and comparison to classical Paget’s disease. Clin Orthop Relat Res 1986;(204):253e60. [76] Bakwin H, Golden A, Fox S. Familial osteoectasia with macrocranium. Am J Roentgenol Radium Ther Nucl Med 1964;91:609e17. [77] Cundy T, Hegde M, Naot D, et al. A mutation in the gene TNFRSF11B encoding osteoprotegerin causes an idiopathic hyperphosphatasia phenotype. Hum Mol Genet 2002;11:2119e27. [78] Vanhoenacker FM, De Beuckeleer LH, Van Hul W, et al. Sclerosing bone dysplasias: genetic and radioclinical features. Eur Radiol 2000;10:1423e33. [79] Johnson ML, Harnish K, Nusse R, Van Hul W. LRP5 and Wnt signaling: a union made for bone. J Bone Miner Res 2004;19:1749e57. [80] Hirsch IS. Generalized osteitis fibrosa. Radiology 1929;13: 44e84. [81] Hansen HG. Sklerosteose. Handbuch Kinderheilk 1967;6:351e5. [82] Beighton P. Sclerosteosis. J Med Genet 1988;25:200e3. [83] Beighton P. Heterozygous manifestations in the heritable disorders of the skeleton. Pediatr Radiol 1997;27:397e401. [84] Balemans W, Ebeling M, Patel N, et al. Increased bone density in sclerosteosis is due to the deficiency of a novel secreted protein (SOST). Hum Mol Genet 2001;10:537e43. [85] Brunkow ME, Gardner JC, Van Ness J, et al. Bone dysplasia sclerosteosis results from loss of the SOST gene product, a novel cystine knot-containing protein. Am J Hum Genet 2001;68:577e89. [86] Piters E, Culha C, Moester M, et al. First missense mutation in the SOST gene causing sclerosteosis by loss of sclerostin function. Hum Mutat 2010;31:E1526e43. [87] van Bezooijen RL, ten Dijke P, Papapoulos SE, Lowik CW. SOST/sclerostin, an osteocyte-derived negative regulator of bone formation. Cytokine Growth Factor Rev 2005;16:319e27. [88] Leupin O, Piters E, Boudin E, et al. Bone overgrowth-associated mutations in LRP4 impair sclerostin-facilitator function. J Biol Chem 2011. submitted. [89] Van Hul W, Balemans W, Van Hul E, et al. Van Buchem disease (hyperostosis corticalis generalisata) maps to chromosome 17q12-q21. Am J Hum Genet 1998;62:391e9. [90] Balemans W, Van Den Ende J, Freire Paes-Alves A, et al. Localization of the gene for sclerosteosis to the van Buchem disease-gene region on chromosome 17q12-q21. Am J Hum Genet 1999;64:1661e9. [91] Balemans W, Patel N, Ebeling M, et al. Identification of a 52 kb deletion downstream of the SOST gene in patients with van Buchem disease. J Med Genet 2002;39:91e7. [92] Loots GG, Kneissel M, Keller H, et al. Genomic deletion of a long-range bone enhancer misregulates sclerostin in Van Buchem disease. Genome Res 2005;15:928e35. [93] Johnston Jr CC, Lavy N, Lord T, Vellios F, Merritt AD, Deiss Jr WP. Osteopetrosis. A clinical, genetic, metabolic, and morphologic study of the dominantly inherited, benign form. Medicine (Baltimore) 1968;47:149e67. [94] Johnson ML, Gong G, Kimberling W, Recker SM, Kimmel DB, Recker RB. Linkage of a gene causing high bone mass to human chromosome 11 (11q12-13). Am J Hum Genet 1997;60:1326e32. [95] Boyden LM, Mao J, Belsky J, et al. High bone density due to a mutation in LDL-receptor-related protein 5. N Engl J Med 2002;346:1513e21. [96] Little RD, Carulli JP, Del Mastro RG, et al. A mutation in the LDL receptor-related protein 5 gene results in the autosomal dominant high-bone-mass trait. Am J Hum Genet 2002;70:11e9.

PEDIATRIC BONE

556

20. SCLEROSING BONE DYSPLASIAS

[97] Gong Y, Slee RB, Fukai N, et al. LDL receptor-related protein 5 (LRP5) affects bone accrual and eye development. Cell 2001;107:513e23. [98] Van Wesenbeeck L, Cleiren E, Gram J, et al. Six novel missense mutations in the LDL receptor-related protein 5 (LRP5) gene in different conditions with an increased bone density. Am J Hum Genet 2003;72:763e71. [99] Semenov M, Tamai K, He X. SOST is a ligand for LRP5/LRP6 and a Wnt signaling inhibitor. J Biol Chem 2005;280(29):26770e5. [100] Balemans W, Cleiren E, Siebers U, Horst J, Van Hul W. A generalized skeletal hyperostosis in two siblings caused by a novel mutation in the SOST gene. Bone 2005;36:943e7. [101] Kubota T, Michigami T, Ozono K. Wnt signaling in bone metabolism. J Bone Miner Metab 2009;27:265e71. [102] Spencer GJ, Utting JC, Etheridge SL, Arnett TR, Genever PG. Wnt signalling in osteoblasts regulates expression of the receptor activator of NFkappaB ligand and inhibits osteoclastogenesis in vitro. J Cell Sci 2006;119:1283e96. [103] Kubota T, Michigami T, Sakaguchi N, et al. Lrp6 hypomorphic mutation affects bone mass through bone resorption in mice and impairs interaction with Mesd. J Bone Miner Res 2008;23:1661e71. [104] Yadav VK, Ryu JH, Suda N, et al. Lrp5 controls bone formation by inhibiting serotonin synthesis in the duodenum. Cell 2008; 135:825e37. [105] Braham RL. Multiple congenital abnormalities with diaphyseal dysplasia (Camurati-Engelmann’s syndrome). Report of a case. Oral Surg Oral Med Oral Pathol 1969;27:20e6.

[106] Lenz WD, Majewski F. A generalized disorder of the connective tissues with progeria, choanal atresia, symphalangism, hypoplasia of dentine and craniodiaphyseal hypostosis. Birth Defects Orig Artic Ser 1974;10:133e6. [107] Majewski F. Lenz-Majewski hyperostotic dwarfism: reexamination of the original patient. Am J Med Genet 2000;93: 335e8. [108] Gorlin RJ, Whitley CB. Lenz-Majewski syndrome. Radiology 1983;149:129e31. [109] Gorlin RJ, Koszalka MF, Spranger J. Pyle’s disease (familial metaphyseal dysplasia). A presentation of two cases and argument for its separation from craniometaphyseal dysplasia. J Bone Joint Surg Am 1970;52:347e54. [110] Beighton P. Pyle disease (metaphyseal dysplasia). J Med Genet. 1987;24:321e4. [111] Ghosal SP, Mukherjee AK, Mukherjee D, Ghosh AK. Diaphyseal dysplasia associated with anemia. J Pediatr 1988;113: 49e57. [112] Gumruk F, Besim A, Altay C. Ghosal haemato-diaphyseal dysplasia: a new disorder. Eur J Pediatr 1993;152:218e21. [113] Ozsoylu S. High-dose intravenous methylprednisolone for Kasabach-Merritt syndrome. Am J Hematol 1989;31: 219e20. [114] Isidor B, Dagoneau N, Huber C, et al. A gene responsible for Ghosal hemato-diaphyseal dysplasia maps to chromosome 7q33-34. Hum Genet 2007;121:269e73. [115] Genevieve D, Proulle V, Isidor B, et al. Thromboxane synthase mutations in an increased bone density disorder (Ghosal syndrome). Nat Genet 2008;40:284e6.

PEDIATRIC BONE

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Parathyroid Disorders Murat Bastepe 1, Harald Ju¨ppner 2, Rajesh V. Thakker 3 1

Endocrine Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA, 2 Endocrine Unit and Pediatric Nephrology Unit, Department of Medicine and Pediatrics, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA 3 Academic Endocrine Unit, Nuffield Department of Medicine, Oxford Centre for Diabetes, Endocrinology and Metabolism (OCDEM), Churchill Hospital, Headington, Oxford, UK

INTRODUCTION 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 upon the prevailing extracellular calcium ion concentration. Hypercalcemic or hypocalcemic disorders can be classified according to whether they arise from an excess or deficiency of PTH, a defect in the receptor for PTH (i.e. the PTH/PTHrP receptor), or insensitivity to PTH caused by defects downstream of the PTH/PTHrP receptor. Recent advances in studying 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. 21.1). 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, will be reviewed in this chapter.

PTH GENE STRUCTURE AND FUNCTION The PTH gene is located on chromosome 11p15 and consists of three exons, which are separated by two introns [2,3]. Exon 1 of the PTH gene is 85 bp in length and is untranslated (Fig. 21.2), whereas exons 2 and 3 encode the 115 amino acid pre-proPTH peptide. Exon

Pediatric Bone, Second Edition DOI: 10.1016/B978-0-12-382040-2.10021-8

2 is 90 bp in length and encodes the initiation (ATG) codon, the pre-hormone sequence and part of the prohormone sequence. Exon 3 is 612 bp in size and encodes the remainder of the prohormone sequence, the mature PTH peptide and the 30 untranslated region [3]. The 50 regulatory sequence of the human PTH gene contains a vitamin D response element 125 bp upstream of the transcription start site, which downregulates PTH mRNA transcription in response to vitamin D receptor binding [5,6]. PTH gene transcription (as well as PTH peptide secretion) is also dependent upon the extracellular calcium and phosphate [7e10] concentration, although the presence of specific upstream “calciumor phosphate-response element(s)” has not yet been demonstrated [11,12]. The secretion of mature PTH, an 84 amino acid peptide, from the parathyroid chief cell is regulated through a G protein-coupled calciumsensing receptor, which is also expressed in renal tubules, and in numerous other tissues, albeit at lower abundance. PTH mRNA is first translated into a preproPTH peptide. The “pre” sequence consists of a 25 amino acid signal peptide (leader sequence), which is responsible for directing the nascent peptide into the endoplasmic reticulum to be packaged for secretion from the cell [13]. The “pro” sequence is six amino acids in length and, although its function is less well defined than that of the “pre” sequence, it is also essential for correct PTH processing and secretion [13]. After the mature PTH peptide comprising 84 amino acids is secreted from the parathyroid cell, it is cleared from the circulation with a short half-life of about 2 minutes, via non-saturable hepatic uptake and renal excretion. The PTH gene shares significant homology with the gene encoding PTH-related peptide (PTHrP; also known

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PARATHYROID CELL Ca2+ CaSR Gq/11 PLC

Gi

PIP2

Gain-of-function

X

FBHH, NSHP

ADHH

X

MELAS, KSS, MTPD5

X

Kenny-Caffey, Sanjad-Sakati

X

Parathyroid tumors eg. MEN1, HPT-JT, CCND1, Rb Hypoparathyroidism

AC

ATP cAMP

DAG + IP3 PKC

Loss-of-function

TBCE

↑ [Ca2+]i Proto-oncogenes and tumour -suppressor genes

X

PTH Transcription factors eg. GATA3, GCM2, AIRE

PTHrP

DiGeorge Syndrome, HDR, APECED Hypoparathyroidism

Hypoparathyroidism

X

Blomstrand’s lethal chondrodysplasia

Jansen’s metaphyseal chondrodysplasia

X

Pseudohypoparathyroidism

McCune-Albright syndrome

X

FBHH, NSHPT

ADHH

PTH PTH/PTHrP receptor

Gq/11 PLC

X

Gs AC

PIP2

IP3 + DAG

ATP cAMP

CaSR

TARGET CELL (eg. kidney, bone or cartilage) FIGURE 21.1

Schematic representation of some of the components involved in calcium homeostasis. Alterations in extracellular calcium are detected by the calcium-sensing receptor (CaSR), which is a 1078 amino acid G-protein coupled receptor. The PTH/PTHrP receptor, which mediates the actions of PTH and PTHrP, is also a G-protein coupled receptor. Thus, Ca2þ and PTH and PTHrP involve G protein-coupled signaling pathways and interaction with their specific receptors can lead to activation of Gs, Gi, and Gq/11, respectively. 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/11, stimulates PLC, which catalyzes the hydrolysis of the phosphoinositide (PIP2) to inositol triphosphate (IP3), which increases intracellular calcium, and diacylglycerol (DAG), which activates PKC. These proximal signals modulate downstream pathways, which result in specific physiological effects. Mutations in several genes, which encode proteins in these pathways, have been identified in specific disorders of calcium homeostasis (see Table 21.1). (Adapted from [1].)

as PTH-related hormone, PTHrH) [14,15]. Both peptides mediate their actions through a common receptor [15], albeit with different signaling characteristics (for details see Chapter 6). This PTH/PTHrP receptor, also termed PTH1R (see Fig. 21.1), is a member of a subgroup of G protein-coupled receptors, and its gene is located on chromosome 3p21-p24 [16,17]. The PTH1R receptor is closely related to PTH2R (the gene for PTH2R is located on chromosome 2q33), which binds with high affinity

the tuberoinfundibular peptide comprising 39 amino acids (TIP39) [18]. The human, but not the rodent receptor PTH2R is also activated by PTH [19,20]. Interestingly, the PTH1R too binds TIP39 but the peptide acts as an antagonist at this receptor [21,22]. The biological roles of PTH2R have been explored incompletely, but it was shown to have roles in nociception and male fertility [23e25]; it is only expressed in a few tissues, including the hypothalamus, pancreas, placenta,

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FIGURE 21.2 Schematic representation of the PTH gene, PTH mRNA and PTH peptide. The PTH gene consists of 3 exons and 2 introns; the peptide is encoded by exons 2 and 3. The PTH peptide is synthesized as a precursor, which contains a pre- and a pro-sequence. The mature PTH peptide, which contains 84 amino acids, and larger carboxy-terminal PTH fragments are secreted from the parathyroid cell. (Adapted from [4].)

and testis [19,26]. In contrast, the PTH1R shows a much broader expression profile, with highest expression in kidney and bone, where it mediates the endocrine actions of PTH. However, the most abundant expression of the PTH/PTHrP receptor occurs in chondrocytes of the metaphyseal growth plate where it mediates predominantly the autocrine/paracrine actions of PTHrP [15]. Mutations involving the genes that encode PTH, the calcium-sensing receptor, the PTH/PTHrP receptor, and Gsa all affect the regulation of calcium homeostasis and thus can be associated with genetic disorders characterized by hypercalcemia or hypocalcemia (Table 21.1).

HYPOCALCEMIC DISORDERS Hypoparathyroidism

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shown that the mutant PTH is trapped intracellularly, predominantly in the endoplasmic reticulum (ER), which is toxic for the cells and leads to apoptosis [28]. In kindreds with autosomal recessive isolated hypoparathyroidism, different PTH gene abnormalities have been identified [4,30e31]. In one family an abnormality involving a donor splice site at the exon 2eintron 2 boundary was identified [4]. This mutation involved a single base transition (G/C) at position 1 of intron 2 and an assessment of the effects of this alteration in the invariant gt dinucleotide of the 50 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 (see Fig. 21.2), which are required respectively for the commencement of PTH mRNA translation and for the translocation of the PTH peptide. 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 [31]. This mutation alters the e3 position of the pre-pro-PTH protein cleavage site [32]. The presence of the mutant proline at this position is likely to disrupt cleavage of the preproPTH that would be subsequently degraded in the rough endoplasmic reticulum (RER) and PTH would not be available. X-Linked Recessive Hypoparathyroidism

Hypoparathyroidism may occur as part of a pluriglandular autoimmune disorder or as a complex congenital defect, as in the example of the DiGeorge syndrome. In addition, hypoparathyroidism may develop as a solitary endocrinopathy and this has been called isolated or idiopathic hypoparathyroidism. Familial occurrences of isolated hypoparathyroidism with autosomal dominant, autosomal recessive, and X-linked recessive inheritances have been established. Parathyroid Hormone (PTH) Gene Abnormalities DNA sequence analysis of the PTH gene (see Fig. 21.2) from one patient with autosomal dominant isolated hypoparathyroidism has revealed a single base substitution (T/C) in exon 2 [27], which resulted in the substitution of arginine (CGT) for cysteine (TGT) in the signal peptide. The presence of this changed amino acid in the midst of the hydrophobic core of the signal peptide impeded the processing of the mutant preproPTH, as demonstrated by in vitro studies. These 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 [27,28]. Studies using transfected HEK293 cells have

X-linked recessive hypoparathyroidism has been reported in two multigenerational kindreds from Missouri, USA [33,34]. 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 [35]. Relatedness of the two kindreds has been established by demonstrating an identical mitochondrial DNA sequence, which is inherited via the maternal lineage, in affected males from the two families [36]. Studies utilizing X-linked polymorphic markers in these families localized the mutant gene to chromosome Xq26-q27 [37]. Further characterization of this genomic region led to the identification of a deletioneinsertion involving chromosomes Xq27 and 2p25 [38]. This deletioneinsertion is located approximately 67 kb downstream of SOX3, and hence it is likely to exert a positional effect on SOX3 expression. Moreover, SOX3 was shown to be expressed in the developing parathyroids of mouse embryos indicating that SOX3 may have a role during embryonic development of the parathyroid glands [38]. SOX3 belongs to a family of genes that encode high-mobility group (HMG) box transcription factors and is related to SRY, the sex-determining gene on the Y chromosome. The mouse homolog is expressed in the prestreak

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TABLE 21.1

Parathyroid Diseases and Chromosomal Locations of the Mutated Genes

Metabolic abnormality

Disease

Inheritance

Gene product

Chromosomal location

Hypocalcemia

Isolated hypoparathyroidism

Autosomal dominant

PTH, GCMB

11p15*, 6p24.2

Autosomal recessive

PTH, GCMB

11p15*, 6p24.2

X-linked recessive

Unknown (SOX3)

Xq27.1

Hypocalcemic hypercalciuria

Autosomal dominant

CaSR1

3q21.1

Hypoparathyroidism associated with polyglandular autoimmune syndrome (APECED)

Autosomal recessive

AIRE

21q22.3

Hypoparathyroidism associated with KearnseSayre and MELAS

Maternal

Mitochondrial genome

DiGeorge

Autosomal dominant

TBX1

22q11.2/10p14

HDR syndrome

Autosomal dominant

GATA3

10p14

Blomstrand lethal chondrodysplasia

Autosomal recessive

PTHR1

3p21.3

KenneyeCaffey/Sanjad-Sakati

Autosomal dominant/ recessive

TBCE

1q42.3

Barakat

Autosomal recessive y

Unknown

?

Lymphedema

Autosomal recessive

Unknown

?

Unknown

?

GNAS exons 1-13

20q13.3

Deletions in STX16 or GNAS

20q13.3

patUPD20 or unknown

20

GNAS exons 1-13

20q13.3

Hypoparathyroidism associated with complex congenital syndromes

Nephropathy, nerve deafness

Autosomal dominant

Pseudohypoparathyroidism (type Ia)

Autosomal dominant

y

Maternally inherited Pseudohypoparathyroidism (type Ib)

Autosomal dominant Maternally inherited Sporadic

Progressive osseous hyperplasia (POH) Plate-like osteoma cutis Hypercalcemia

Multiple endocrine neoplasia type 1

Autosomal dominant

MENIN

11q13

Multiple endocrine neoplasia type 2

Autosomal dominant

RET

10q11.2

Hereditary hyperparathyroidism and jaw tumors (HPT-JT)

Autosomal dominant

PARAFIBROMIN

1q21-31

Sporadic hyperparathyroidism

Sporadic

PRAD1/CCND1

11q13

Retinoblastoma

Familial benign hypercalcemia

FBH3q FBH19p FBHOk

Neonatal severe hyperparathyroidism (NSHPT)

13q14 Unknown

1p32-pter

Autosomal dominant Autosomal dominant Autosomal dominant

CaSR Unknown Unknown

3q21.1 19p13 19q13

Autosomal recessive

CaSR

3q21.1

Autosomal dominant Jansen’s disease

Autosomal dominant

PTHR1

3p21.3

Williams syndrome

Autosomal dominant

Elastin (and other genes)

7q11.23

* Mutations of PTH gene identified only in some families; y Most likely inheritance shown; MELAS: Mitochondrial encephalopathy, stroke-like episodes and lactic acidosis; ? Location not known

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embryo and subsequently in the developing central nervous system (CNS) that includes the region of the ventral diencephalons, which induces development of the anterior pituitary and gives rise to the hypothalamus, the olfactory placodes, and the parathyroids [38e41]. The location of the deletioneinsertion z67 kb downstream of SOX3 in X-linked recessive hypoparathyroid patients is likely to result in altered SOX3 expression, as SOX3 expression has been reported to be sensitive to position effects caused by X-chromosome abnormalities [42]. Indeed, reporter-construct studies of the mouse Sox3 gene have demonstrated the presence of both 50 and 30 regulatory elements [43] and thus it is possible that the deletioneinsertion in the X-linked recessive hypoparathyroid patients may have a position effect on SOX3 expression, and parathyroid development from the pharyngeal pouches. 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-candidiasisectodermal dystrophy (APECED) syndrome or the polyglandular autoimmune type 1 syndrome (APS1) [44]. This disorder has a high incidence in Finland, and a genetic analysis of Finnish families indicated autosomal recessive inheritance of the disorder [45]. 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 [46]. Linkage studies of Finnish families mapped the APECED gene to chromosome 21q22.3 [47]. Further positional cloning studies led to the isolation of a novel gene. This gene, referred to as AIRE (autoimmune regulator), 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 [48]. In the APECED families, a number of different mutations throughout the coding exons of AIRE have been reported [49e52], although a codon 257 (Arg/Stop) mutation was the predominant abnormality in 82% of the Finnish families [48]. AIRE has been shown to regulate the elimination of organ-specific T cells in the thymus, and thus APECED is likely to be caused by a failure of this specialized mechanism for deleting forbidden T cells, and establishing immunological tolerance [53]. Patients with APS1 may also develop other autoimmune disorders in association with organ-specific autoantibodies, which are similar to those in patients with non-APS1 forms of the disease. Examples of such autoantibodies and related diseases are GAD6S autoantibodies in diabetes mellitus type 1A and 21hydroxylase autoantibodies in Addison’s disease.

561

Patients with APS1 may also develop autoantibodies that react with specific autoantigens that are not found in non-APS1 pateints, and examples of this are autoantibodies to type 1 inferferon w, which are present in all APS1 patients [54], and to NACHT leucine-rich-repeatprotein 5 (NALP5), which is a parathyroid-specific autoantibody present in 49% of patients with APS1associated hypoparathyroidism [55]. NALP proteins are essential components of the inflammasone and activate the innate immune system in different inflammatory and autoimmune disorders, such as vitiligo, which involves NALP1, and gout, which involves NALP3 [56]. The precise role of NALP5 in APS1-associated hypoparathyroidism remains to be elucidated. Mitochondrial Disorders Associated with Hypoparathyroidism Hypoparathyroidism has been reported to occur in two disorders associated with mitochondrial dysfunction: the KearnseSayre syndrome (KSS) and the MELAS syndrome. KSS is characterized by progressive external ophthalmoplegia and pigmentary retinopathy before the age of 20 years, and 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 conditions. Both the KSS and MELAS syndromes have been reported to occur with insulindependent diabetes mellitus and hypoparathyroidism [57,58]. A point mutation in the mitochondrial gene tRNA leucine (UUR) has been reported in one patient with the MELAS syndrome who also suffered from hypoparathyroidism and diabetes mellitus [59]. A large deletion, consisting of 6903 base pairs and involving 39% of the mitochondrial genome, has been reported in another patient who suffered from KSS, hypoparathyroidism and sensorineural deafness [58]. The role of these mitochondrial mutations in the etiology of hypoparathyroidism remains to be further elucidated. DiGeorge Syndrome Patients with the DiGeorge syndrome (DGS1) typically suffer from hypoparathyroidism, immunodeficiency, congenital heart defects, and deformities of the ear, nose and mouth. The disorder arises from 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 DGS1 are sporadic but an autosomal dominant inheritance of DGS1 has been observed and an association between the syndrome and an unbalanced translocation and deletions involving 22q11.2 have also been reported [60]. In some patients, deletions of another

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locus on chromosome 10p have been observed in association with DGS2 [61]. Mapping studies of the DGS1 deleted region on chromosome 22q11.2 have defined a 250e3000 kb critical region [62,63] that contained approximately 30 genes. Studies of DGS1 patients have reported deletions of several of the genes (e.g. rnex40, nex2.2 e nex 3, UDFIL and TBX1) from the critical region [60,64e66] and studies of transgenic mice deleted for such genes (e.g. Udf1l, Hira and Tbx1) have revealed developmental abnormalities of the pharyngeal arches [67e69]. However, point mutations in DGS1 patients have only been detected in the TBX1 gene [70] and TBX1 is now considered to be the gene causing DGS1 [71]. TBX1 is a DNA binding transcriptional factor of the T-Box family that is known to have an important role in vertebrate and invertebrate organogenesis and pattern formation. The TBX1 gene is deleted in z96% of all DGS1 patients. Moreover, DNA sequence analysis of unrelated DGS1 patients, who did not have deletions of chromosome 22q11.2, revealed the occurrence of three heterozygous point mutations [70]. One of these mutations resulted in a frameshift with a premature truncation, while the other two were missense mutations (Phe148Tyr and Gly310Ser). All of these patients had the complete pharyngeal phenotype but did not have mental retardation or learning difficulties. Interestingly, transgenic mice with deletion of Tbx1 have a phenotype that is similar to that of DGS1 patients [69]. Thus, Tbx1 null mutant-mice (/) had all the developmental anomalies of DGS1 (i.e. thymic and parathyroid hypoplasia; abnormal facial structures and cleft palate; skeletal defects; and cardiac outflow tract abnormalities), while Tbx1 haploinsufficiency in mutant mice (þ/) was associated only with defects of the fourth branchial pouch (i.e. cardiac outflow tract abnormalities). The basis of the phenotypic differences between DGS1 patients, who are heterozygous, and the transgenic þ/ mice remains to be elucidated. It is plausible that Tbx1 dosage, together with the downstream genes that are regulated by Tbx1 could provide an explanation, but the roles of these putative genes in DGS1 are uncertain. Some patients may have a late onset DGS1 and these develop symptomatic hypocalcemia in childhood or during adolescence with only subtle phenotypic abnormalities [72]. These late-onset DGS1 patients have similar microdeletions in the 22q11 region. It is of interest to note that the age of diagnosis in the families of the three DGS1 patients with inactivating Tbx1 mutations ranged from 7 to 46 years, which is in keeping with late onset DGS1 [70]. Hypoparathyroidism (glial cells missing B) Glial cells missing B (GCMB), which is the human ortholog of the Drosophilia Gcm gene and of the mouse

Gcm2 gene, is expressed exclusively in the parathyroid glands, suggesting a specific regulatory role during parathyroid gland development [73e75]. In order to investigate this, mice that are “null” for Gcm2 have been generated by homologous recombination. Mice heterozygous (þ/) 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 [75]. However, despite their lack of parathyroid glands, Gcm2 deficient (Gcm2/) mice did not have undetectable serum PTH levels. In fact, PTH levels were indistinguishable from those of normal (þ/þ, wild-type) mice. However, this concentration of endogenous PTH in the Gcm2/ mice appeared insufficient to correct the hypocalcemia. Only the administration of exogenous PTH fully corrected the changes in the mineral ion metabolism [75], indicating that Gcm2/ mice have a normal response to PTH and are not resistant to this hormone. The auxillary source of PTH was determined by combined expression and ablation studies, which revealed a cluster of PTH-expressing cells under the thymic capsule in both the Gcm2/ and wild-type (þ/þ) mice. These thymic PTH-producing cells also expressed the calcium-sensing receptor (CaSR) and long-term treatment of the Gcm2-null mice with 1,25(OH)2 vitamin D3 restored the serum calcium concentrations to normal and reduced the serum PTH levels, thereby indicating that the thymic production of PTH can be downregulated. However, it appears that the thymic production of PTH cannot be sufficiently upregulated fully to correct the hypocalcemia in the Gcm2-deficient mice. This absence of upregulation of PTH expression would be consistent with a very limited number of thymic PTH-producing cell clusters, which is vastly different from the number of hormone producing cells in a normal parathyroid gland. The development of the thymic PTH-producing cells likely involves Gcm1, which is the other mouse ortholog of Drosophila Gcm [73]. Gcm1 expression, which could not be detected in parathyroid glands, co-localized with PTH expression in the thymus [75]. The specific role of Gcm2 in the development of the parathyroids from the third pharyngeal pouch, has been further investigated by studying the expression of the Hoxa3-Pax1/9-Eya1 transcription factor and Sonic hedgehogeBone morphogenetic protein 4 (Shh-Bmp4) signaling networks [74]. These studies revealed that Gcm2/ embryos at day 12.0 d.p.c. have a parathyroid-specific domain, but that this parathyroid domain undergoes coordinated programmed cell death (apoptosis) by 12.5 d.p.c. in the Gcm2-null mouse embryos [74]. Moreover, the expression of the transcription factors Hoxa3, Pax1, Pax9, Eya1 and Tbx1, as well as Shh and Bmp4 was normal

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HYPOCALCEMIC DISORDERS

in the third pharyngeal pouch of these Gcm2/ mouse embryos. These findings indicate that the Hoxa3-Pax1/ 9-Eya transcription factor cascade, the transcription factor Tbx1 and the Shh-Bmp4 signaling network, all act upstream of Gcm2 [74]. Moreover, these studies have revealed that Gcm2 has a role in promoting differentiation and survival of parathyroid cells in the developing embryo [74]. Studies of patients with isolated hypoparathyroidism have shown that GCMB mutations are associated with autosomal recessive and dominant forms of the disease [76e80]. Thus, a homozygous intragenic deletion of GCMB has been identified in a patient with autosomal recessive hypoparathyroidism [76]. In other hypoparathyroid patients, homozygous missense mutations Arg47Leu and Arg110Trp, and a nonsense mutation Arg39Stop, as well as a frameshifting deletion, I298fsx307, have been reported [77,80]. Haplotype analysis has established that the Arg47Leu and the I298fsx307 mutations arose in families from the Indian subcontinent as ancient founders, or recurrent de novo mutations [80]. Functional analysis using subcellular localization studies, electrophoretic mobility shift

A

DNA-binding Domain 21

460 |

Transactivation Domain 1 174

470 |

480 |

assays (EMSA) and luciferase reporter assays have demonstrated that the Arg39Stop mutant failed to localize to the nucleus; the Arg476Leu and Arg110Trp mutants both lost DNA binding ability, while the I298fsx307 mutant had reduced transactivation ability [80]. Three-dimensional modeling of the GCMB DNAbinding domain revealed that the Arg110 residue is likely important for the structural integrity of helix 2, which forms part of the GCMB/DNA binding interface. In addition, heterozygous GCMB mutations, which consist of single nucleotide deletions (c1389delT and c1399delC) that introduce frame-shifts and premature truncations, have been identified in two unrelated families with autosomal dominant hypoparathyroidism [78,79] (Fig. 21.3). Both of these mutations were shown, by using a GCMB-associated luciferase reporter, to inhibit the action of the wild-type transcription factor, thereby indicating that these GCMB mutants have dominant-negative properties [78]. However, it is important to note that, in one study, z33% of probands with hypoparathyroidism do not have mutations involving the GCMB or PTH genes, thereby indicating further genetic heterogeneity and

Inhibitory Domain

263

490 |

Transactivation Domain 2 352

500 |

428

510 |

506

520 |

WT TVAIPHEPVS SRTDEAETWD VCLSGLGSAV SYSDRVGPFF TYNNEDF mutA TVAIPTSQFP LGQMKQRLGM CVCLGWAPQS VTQTEWVPSL PTTMRIFERQ SRGHNSSVHA GRRQGNVKWQ mutB TVAIPHEQFP LGQMKQRLGM CVCLGWAPQS VTQTEWVPSL PTTMRIFERQ SRGHNSSVHA GRRQGNVKWQ * p<0.05

B

* p<0.05 GCMB WT GCMB WT GCMB WT + Mutant B GCMB WT + Mutant A Mutant B Mutant A Vector

Vector

FIGURE 21.3 Heterozygous GCMB mutations can be a cause of autosomal dominant, isolated hypoparathyroidism. Panel A: Domain structure of human GCMB and carboxyl-terminal amino acid sequence of wild-type human GCMB (WT), and of the mutants identified in families A (mutA) and B (mutB). Both single nucleotide deletions lead to a frameshift and translation of 65 novel amino acids after proline 464 and of 63 novel amino acids after glutamic acid 466, respectively. Novel amino acids are in italics and underlined. Numbering of amino acids above. Panel B: Luciferase reporter assay using chicken fibroblast DF-1 cells. Cells were co-transfected with plasmids encoding wild-type human GCMB (GCMB WT), combined with either the c.1389delT mutant identified in family A (left), the c.1399delC mutant identified in family B (right), or empty vector; luciferase activity obtained with empty plasmid was defined as 1. (from [1]).

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the involvement of other genes [80]. Another study suggested that even fewer hypoparathyroid patients carry GCMB mutations [81]. Hypoparathyroidism, Deafness and Renal Anomalies (HDR) Syndrome The combined inheritance of hypoparathyroidism, deafness and renal dysplasia (HDR) as an autosomal dominant trait was reported in one family in 1992 [82]. Patients had asymptomatic hypocalcemia with undetectable or inappropriately normal serum concentrations of PTH, and normal brisk increases in plasma cAMP in response to the infusion of PTH. The patients also had bilateral, symmetrical, sensorineural deafness involving all frequencies. The 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 [82]. However, cytogenetic abnormalities involving chromosome 10p14-10pter have been identified in two unrelated patients with laboratory and clinical features that were consistent with HDR. These two patients suffered from hypoparathyroidism, deafness and growth and mental retardation; one patient also had a solitary dysplastic kidney with vesico-ureteric reflux and a uterus bicornis unicollis [83] and the other patient, who had a complex reciprocal, insertional translocation of chromosomes 10p and 8q, had cartilaginous exostoses [84]. Neither of these patients had immunodeficiency or heart defects, which are key features of DGS2 (see above), and further studies defined two non-overlapping regions; thus, the DGS2 region was located on 10p13-14 and HDR on 10p1410pter. Deletion mapping studies in two other HDR patients further defined a critical 200 kb region that contained GATA3 [85], which belongs to a family of zinc-finger transcription factors that are involved in vertebrae embryonic development. DNA sequence analysis in other HDR patients identified mutations that resulted in a haploinsufficiency and loss of GATA3 function [85e88]. GATA3 has two zinc-fingers, and the C-terminal finger (ZnF2) binds DNA, while the N-terminal finger (ZnF1) stabilizes this DNA binding and interacts with other zinc finger proteins, such as the Friends of GATA (FOG) [89]. HDR-associated mutations involving GATA3 ZnF2 or the adjacent basic amino acids were found to result in a loss of DNA binding, while those involving ZnF1 either led to a loss of interaction with FOG2 ZnFs or altered DNA binding affinity [87,88,90]. These findings are consistent with the proposed 3-dimensional model of GATA3 ZnF1, which has separate DNA and protein binding surfaces [87,88,91]. Thus, the HDR-associated GATA3 mutations can be subdivided into two broad classes,

which depend upon whether they disrupt ZnF1 or ZnF2, and their subsequent effects on interactions with FOG2 and altered DNA binding, respectively. The majority (>75%) of these HDR associated mutations are predicted to result in truncated forms of the GATA3 protein. Each proband and family will generally have its own unique mutation and there appears to be no correlation with the underlying genetic defect and the phenotypic variation, e.g. the presence or absence of renal dysplasia. Over 90% of patients with two or three of the major clinical features of the HDR syndrome, i.e. hypoparathyroidisim, deafness or renal abnormalities, have a GATA3 mutation [88]. The remaining 10% of HDR of patients, who do not have a GATA3 mutation of the coding region, may harbor mutations in the regulatory sequences flanking the GATA3 gene, or else they may represent heterogeneity. The phenotypes of HDR patients with GATA3 mutations appear to be similar to those without GATA3 mutations [88]. 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 humans and mice, and this suggests that GATA3 may have a more complex role. Indeed, studies of mice that are deleted for a Gata3 allele (Gata3þ/), or both Gata3 alleles (Gata3/) have revealed important roles for Gata3 in the development of the brain, spinal cord, peripheral auditory system, T cells, fetal liver hematopoiesis and urogenital system [92]. Gata3þ/ mice are viable, appear to be normal when fed a diet with a high calcium and vitamin D content, have a normal life span, and are fertile [92]. However, upon challenge with a diet low in calcium and vitamin D, Gata3þ/ mice, when compared to Gata3þ/þ mice, had a higher mortality, lower plasma concentrations of calcium and PTH and smaller parathyroid glands that had a reduced proliferation rate [93]. Moreover, Gata3þ/ embryos were found to have a smaller parathyroide thymus primordial that had fewer cells expressing Gcm2. In addition, through the use of EMSAs, luciferase reporter, and chromatin immunoprecipitation assays, GATA3 was shown to bind specifically to a functional double GATA motif within the GCMB promoter [93]. Thus, GATA3 is critical for the differentiation and survival of parathyroid progenitor cells and forms, with Gcm2 (GCMB), part of a transcriptional cascade in parathyroid development and function [93]. Gata3þ/ mice also have hearing loss that is associated with cochlear abnormalities, which consist of a significant progressive morphological degeneration that starts with the outer hair cells at the apex and

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eventually involves all the inner hair cells, pillar cells and nerve fibers [94,95]. These studies have shown that hearing loss in Gata3 haploinsufficiency commences in the early postnatal period and is progressive through adulthood, and that it is peripheral in origin and is predominantly due to malfunctioning of the outer hair cells of the cochlea [94,95]. Gata3/  mice are embryonically lethal as null embryos die between 11 to 12 d.p.c. [92]. Examination of these Gata3/ embryos revealed a variety of abnormalities that included massive internal bleeding, resulting in anemia, marked growth retardation, severe deformities of the brain and spinal cord, a hypopigmented retina, gross aberrations in fetal liver hematopoiesis, a total block of T-cell differentiation, and a retarded or missing lower jaw area [92,96]. These Gata3/ mice had an anatomically normal sympathetic nervous system, yet the sympathetic ganglia lacked tyrosine hydroxylase and dopamine beta-hydroxylase, which are key enzymes that convert tyrosine to L-DOPA, and dopamine to noradrenaline, respectively, in the catecholamine synthesis pathway. Thus, the Gata3/ mice lacked noradrenaline in the sympathetic neurons, and this was contributing to the early embryonic lethality [96]. Feeding of catecholamine intermediates to the pregnant dams, helped partially to rescue the Gata3/ embryos to 12.5 to 16.5 d.p.c. The older, pharmacologically rescued Gata3/ embryos showed abnormalities that could not be detected in the untreated mice [96]. These late embryonic defects included thymic hypoplasia, a thin walled ventricular septum, a poorly developed mandible, other developmental defects in structures derived from the cephalic neural crest cells, renal hypoplasia, a failure to form the metanephros, and an aberrant elongation of the nephric duct along the anteroposterior axis of the embryo [96,97]. The nephric duct defect, consisting of abnormal morphogenesis and guidance in the developing kidney, was characterized by the loss of Ret expression, an essential component of the glialderived-nerve-factor (GDNF) signaling pathway involved in ureteric bud formation and nephric duct guidance [97]. Thus, Gata3 has a role in the differentiation of multiple cell lineages during embryogenesis as well as being a key regulator of nephric duct morphogenesis and guidance of the nephric duct in its caudal extension in the pro/mesonephric kidney [96,97]. However, it is important to note that HDR patients with GATA3 haploinsufficiency do not have immune deficiency, and this suggests that the immune abnormalities observed in some patients with 10p deletions are most likely to be caused by other genes on 10p. Similarly, the facial dysmorphism, growth and development delay, commonly seen in patients with larger 10p deletions were absent in

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the HDR patients with GATA3 mutations, further indicating that these features were likely due to other genes on 10p [85]. These studies of HDR patients clearly indicate an important role for GATA3 in parathyroid development and in the etiology of hypoparathyroidism. KenneyeCaffey, SanjadeSakati and KirkeRichardson Syndromes Hypoparathyroidism has been reported to occur in over 50% of patients with the KenneyeCaffey syndrome which is associated with short stature, osteosclerosis and cortical thickening of the long bones, delayed closure of the anterior fontanel, basal ganglia calcification, nanophthalmos and hyperopia [98]. Parathyroid tissue could not be found in a detailed post-mortem examination of one patient [99], suggesting that hypoparathyroidism may be due to an embryological defect of parathyroid development. In the KirkeRichardson and SanjadeSakati syndromes, which are similar, hypoparathyroidism is associated with severe growth failure and dysmorphic features [100,101]. This has been reported in patients of Middle Eastern origin [100e102]. Consanguinity was noted in the majority of the families, indicating that this syndrome is inherited as an autosomal recessive disorder. Homozygosity and linkage disequilibrium studies located this gene to chromosome 1q42-q43 [101]. Molecular genetic investigations have identified that mutations of the Tubulin-specific chaperone (TBCE) are associated with the KenneyeCaffey and SanjadeSakati syndromes [103]. TBCE encodes one of several chaperone proteins required for the proper folding of a-tubulin subunits and the formation of aeb tubulin heterodimers (see Fig. 21.1) [103]. 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 (see Table 21.1). An association of hypoparathyroidism, renal insufficiency and developmental delay has been reported in one Asian family in whom autosomal recessive inheritance of the disorder was established; an analysis of the PTH gene in this family revealed no abnormalities [30]. The occurrence of hypoparathyroidism, nerve deafness and a steroid-resistant nephrosis leading to renal failure, which has been referred to as the Barakat syndrome [104], has been reported in four brothers from one family, and an association of hypoparathyroidism with congenital lymphedema, nephropathy, mitral valve prolapse and brachytelephalangy has been observed in two brothers from another family [105]. Molecular genetic studies have not been reported from these two families.

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Calcium-Sensing Receptor (CaSR) Abnormalities Autosomal Dominant Hypocalcemia with Hypercalciuria CaSR mutations that result in a loss of function are associated with familial benign (hypocalciuric) hypercalcemia and it has therefore been postulated that CaSR mutations that result in a gain of function may lead to hypocalcemia with hypercalciuria [106,107]. Investigation of kindreds with autosomal dominant forms of hypocalcemia have led to the identification of such CaSR mutations [106,107]. 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 [106,107], which is different from the findings in other disorders caused by activating mutations in different G-protein coupled receptors. Bartter Syndrome Type V Bartter syndrome is a heterozygous group of autosomal recessive disorders of electrolyte homeostasis characterized by hypokalemic alkalosis, renal salt wasting that may lead to hypotension, hyper-reninemic hyperaldosteronism, increased urinary prostaglandin excretion, and hypercalciuria with nephrocalcinosis [108]. Mutations of several ion transporters and channels have been associated with Bartter syndrome, and five types are now recognized [108]. Thus, type 1 is due to mutations involving the bumetamide-sensitive sodium-potassium-chloride co-transporter (NKCC2 or SLC12A2); type II is due to mutations of the outwardly rectifying renal potassium channel (ROMK); type III is due to mutations of the voltage-gated chloride channel (CLC-Kb); type IV is due to mutations of Barttin, which is a beta subunit that is required for trafficking of CLCKb and CLC-Ka, and this form is also associated with deafness as Barttin, CLC-Ka and CLC-Kb are also expressed in the marginal cells of the scala media of the inner ear that secrete potassium ion-rich endolymph; and type V is due to activating mutations of the CaSR. Patients with Bartter syndrome type V have the classical features of the syndrome, i.e. hypokalemic metabolic alkalosis, hyper-reninemia and hyperaldosteronism [109,110]. In addition, they develop hypocalcemia, which may be symptomatic and lead to carpo-pedal spasm, and an elevated fractional excretion of calcium that may be associated with nephrocalcinosis [109,110]. Such patients have been reported to carry heterozygous gain-of-function CaSR mutations, and in vitro functional

expression of these mutations has revealed a more severe set point abnormality for the receptor than that found in patients with ADHH [109, 110]. This suggests that the additional features occurring in Bartter syndrome type V, but not in ADHH, are due to severe gain-of-function mutations of the CaSR [108]. Autoimmune Acquired Hypoparathyroidism (AH) Twenty percent of patients who had acquired hypoparathyroidism (AH) in association with autoimmune hypothyroidism were found to have autoantibodies to the extracellular domain of the CaSR [111,112]. The CaSR autoantibodies did not persist for long; 72% of patients who had AH for less than 5 years had detectable CaSR autoantibodies; whereas only 14% of patients with AH for more than 5 years had such autoantibodies [112]. The majority of the patients who had CaSR autoantibodies were females, a finding that is similar to that found in other autoantibody-mediated diseases. Indeed, a few AH patients have also had features of autoimmune polyglandular syndrome type 1 [APS1]. These findings establish that the CaSR is an autoantigen in AH [111,112].

Pseudohypoparathyroidism (PHP) and GNAS The term pseudohypoparathyroidism (PHP) has been introduced to describe patients with hypocalcemia and hyperphosphatemia due to PTH-resistance rather than PTH-deficiency [113]. Affected individuals show partial or complete resistance to biologically active, exogenous PTH as demonstrated by a lack of increase in urinary cyclic AMP and urinary phosphate excretion; this condition is referred to as PHP type I [114e116]. This form of PHP can be associated with resistance to other hormones, such as thyroid stimulating hormone (TSH) [117,118], gonadotropins [119], growth hormone releasing hormone [120,121], and calcitonin [122] and characteristic physical stigmata, now collectively termed Albrights’ hereditary osteodystrophy (AHO) [113]. In that case, the condition is referred to as PHP type Ia. The features of AHO typically include obesity, short stature, brachydactyly, subcutaneous ectopic ossification, and mild mental retardation. Brachydactyly often involves the distal phalanx of the thumb, as well as the fourth and fifth metacarpal bones, but other metacarpals and metatarsals can also be shortened [123]. The latter syndrome is associated with heterozygous inactivating mutations in GNAS located on chromosome 20q13.3. The GNAS gene gives rise to at least five differentlyspliced mRNAs, including the alpha-subunit of the stimulatory G protein (Gsa). Mutations in exons 1 through 13 of GNAS lead to an z50% reduction in Gsa activity/protein, explaining, at least partially, the resistance towards PTH and other hormones that

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mediate their actions through G protein-coupled receptors [114e116]. A similar decrease in the Gsa activity/ protein is also found in patients with pseudo-pseudohypoparathyroidism (PPHP), who lack any endocrine abnormalities, including resistance to PTH, but have a similar physical appearance as individuals with PHP-Ia. Most AHO features characteristic of PHP-Ia are present in PPHP patients, but recent studies have revealed that obesity and cognitive impairment are associated with PHP-Ia significantly more frequently than with PPHP [124,125]. Because the same genetic defect causes related but distinct clinical findings, mutations in the Gsa-specific exons of GNAS have been thought to be necessary, but not sufficient to explain fully either PHP-Ia or PPHP [114e116,126e129). In fact, a retrospective analysis of numerous previously published cases with either PHP-Ia or PPHP has indicated that both disorders are typically found within the same kindred, but never within the same sibship [130]. Furthermore, hormonal resistance is paternally imprinted, i.e. PHPIa occurs only if the defective gene is inherited from a female affected by either PHP-Ia or PPHP; PPHP occurs only if the defective gene is inherited from a male affected by either of the two disorders [130,131]. Observations consistent with these findings in humans have been made in mice that are heterozygous for the disruption of exon 2 of the GNAS gene [132]. 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 for endocrine abnormalities. Moreover, mice that carried the disrupted allele on their maternal chromosome had undetectable Gsa protein in the renal cortex, but not in many other tissues [132]. Similar parent-of-origin specific findings have also been demonstrated for mice in which GNAS exon 1 is disrupted [133]. Tissue- or cell-specific Gsa expression is thus almost certainly involved in the pathogenesis of PHP-Ia and PPHP, and also provides a reasonable explanation for the finding that heterozygous GNAS mutations result in a dominant phenotype. Mutations in patients with PHP-Ia are spread throughout most of the Gsa-coding exons of GNAS. Thus far, more than 35 different mutations have been identified, and a 4-bp deletion in exon 7 is the most frequently reported mutation [116,129]. No direct phenotypeegenotype correlation has been established for patients with PHP-Ia, except for a missense mutation identified in two unrelated boys with pseudohypoparathyroidism type Ia and testotoxicosis [134]. This Ala-to-Ser substitution at codon 366 generates a temperature-sensitive Gsa mutant, which is constitutively active at cooler temperatures due to accelerated GDP release, but results in a loss of function at the

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ambient body temperature due to thermolability [134]. In addition, another Gsa mutant carrying AlaVal-Asp-Thr amino acid repeats in the guanine nucleotide-binding domain has been identified in patients with PHP-Ia accompanied by neonatal diarrhea [135]. This mutant is unstable and localized to the cytosol, but it shows constitutive activity due to rapid GDP release and decelerated GTP hydrolase activity. While the PHP-Ia phenotype is consistent with the instability and the cytoplasmic localization of this Gsa mutant, the diarrhea phenotype observed during the neonatal period results from its plasma membrane localization in the intestinal epithelium [135]. Alternative splicing of GNAS exon 3 results in a short and a long variant of Gsa [136e138]. To date, only a single inactivating mutation within exon 3 has been discovered in a familial case of PHP-Ia, leading to the deficiency of the long but not the short Gsa variant [139]. Although erythrocyte Gsa activity was reduced by only z25% in the proband, the patient had both AHO and biochemical features consistent with PTH and TSH resistance. It is possible that the clinical presentation in this case depends largely on the relative abundance of the short and the long Gsa variants in different tissues, although the phenotype could also reflect, to some extent, the subtle differences observed between the cellular activities of these two Gsa variants [140e146]. Heterozygous inactivating mutations of the Gsa coding GNAS exons or reduced Gsa levels have also been identified in some patients with progressive osseous heteroplasia (POH), which is a congenital disorder of ectopic bone formation that, unlike the ossification in AHO, affects deep connective tissue and skeletal muscle [147]. Ectopic bone in POH (as well as AHO) is formed through intramembranous ossification. This is different from the mechanism of bone formation underlying fibrodysplasia ossificans progressiva (FOP), in which the ectopic ossification entails endochondral mechanisms. Nevertheless, the distribution and the severity of heterotopic ossifications in some POH cases can be similar to those in FOP cases [148]. A variant of POH, severe plate-like osteoma cutis, has been associated with a mutation found in several patients with PHP-Ia and PPHP, although no other “AHO-like” feature or hormone resistance was documented [147]. A POH patient with a unique exon 1 mutation was reported, who also had mild brachydactyly but lacked any additional features [149]. POH was also present in another patient, who presented with PHP-Ia and showed reduced Gsa levels [149]. A study of several familial POH cases with Gsa mutations revealed an interesting, paternal-exclusive inheritance pattern [150]. In a large three-generation kindred, affected

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individuals who inherited from their father the 4-bp deletion in GNAS exon 7, which was previously found in patients with PHP-Ia and PPHP [116,129], demonstrated extraskeletal ossification without developmental and hormonal abnormalities. In contrast, the affected children of female disease-gene carriers had clinical features of AHO. Several other cases of POH carrying GNAS mutations have been reported [148,151e158]. In most, but not all, cases, the GNAS mutation is located on the paternal allele, but there seems to be no striking genotypeephenotype correlation regarding the location of the mutation [148,154]. It remains to be determined whether as-yet-undefined genes that modify the actions of Gsa and/or changes in the activities of other GNAS products (see below), could explain the variation in the degree of extraskeletal ossification among patients with the same mutations. The GNAS gene is complex, in that alternative promoter use and splicing results in several different mRNAs (Fig. 21.4). Some of these transcripts are derived from either the paternal or the maternal allele, while others show bi-allelic expression. It thus appears likely that this complexity of the GNAS gene contributes to the unique phenotypic abnormalities in patients with PHP. Gsa is encoded by exons 1 through 13 of the GNAS gene and mediates the biological functions of a large variety of G protein-coupled receptors, including

the PTH1R. The Gsa transcript shows a non-imprinted expression profile in most tissues [159e162]. Nonetheless, its expression appears to take place only from the maternal allele in several tissues including thyroid [163e165], pituitary [166], and ovary [165]. As described above, mouse studies have also identified the renal proximal tubule as an important tissue in which Gsa is expressed predominantly from the maternal allele [132,133], although the analysis of human fetal tissues indicated bi-allelic Gsa expression at the renal cortex [167]. It is important to note that the tissues in which Gsa expression is monoallelic correlate well with those that exhibit resistance to their respective hormones. A second transcript, XLas, comprises a novel first exon (XL) that splices onto exons 2 through 13. The encoded z92 kDA protein is identical to Gsa in its carboxyl-terminal portion [168] and it is capable of functioning as a stimulatory G protein [169,170]. The mRNA encoding XLas is found at numerous sites, particularly high concentrations were identified in endocrine and neuroendocrine cells [168,171], and in all investigated tissues, it appears to be transcribed only from the paternal allele [159,160,162]. From the same XLas mRNA, an additional protein with an electrophoretic mobility of z48 kDA appears to be also translated using a second open reading frame; this protein, termed ALEX (for alternative gene product encoded by XL-exon), does

GNAS CH3

STX16 4A

4 3

4.4 kb

-

-

XL

A/B

CH3

CH3

pat

NESP55

5

3 kb

-

2 1

CH3

1

2-13

mat

4.2 kb 4.0 kb/4.7 kb

Gsα coding mutations

FIGURE 21.4 Mutations identified in patients with PHP-Ia or autosomal dominant PHP Ib (AD-PHP-Ib). A variety of loss-of-function mutations located in one of 13 exons encoding Gsa are found in patients with PHP-Ia. Note that those located in exons 2e13 also affect XLas protein, as well as the other GNAS transcripts that use exons 2e13. The most frequent mutation causing AD-PHP-Ib is a 3-kb deletion within STX16, a gene located more than 200 kb upstream of GNAS. Another deletion in the same gene overlapping with the 3-kb deletion has also been reported, and both STX16 deletions are predicted to disrupt a cis-acting control element of GNAS necessary for the establishment or maintenance of the imprint mark located at exon A/B. In some AD-PHP-Ib kindreds, methylation changes involve multiple GNAS DMRs. In three such kindreds, the disease is caused by maternally inherited deletions that remove the NESP55 DMR completely or partially. The overlapping region points to a cis-acting element that controls imprinting of the entire maternal GNAS allele. Boxes and connecting lines indicate exons and introns, respectively. STX16 exons and GNAS exons 2 through 13 are shown as single rectangles for simplicity. Paternal (pat) and maternal (mat) methylation (CH3) and parental origin of transcription (arrows) are indicated. The dotted arrow depicts the tissue-specific silencing of the paternal Gsa transcription. Deletions identified in patients with AD-PHP-Ib are shown by gray horizontal bars.

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not share any sequence identity with Gas or XLas [172]. At least two variants of XLas have been described. XLasN1 [171] is a C-terminally truncated variant analogous to Gsa-N1 [173], and XXLas is an N-terminally extended variant that includes all the sequence of XLas [174,175]. Mice in which the paternal XL exon is disrupted through targeted homologous recombination (i.e. the knockout of XLas, ALEX, XLas-N1 and XXLas) demonstrate poor adaptation to feeding after birth and defects in glucose and energy metabolism [176]. A similar phenotype has also been reported in three children with large deletions of the paternal GNAS allele [177,178]. The third transcript, NESP55 [179], is expressed from the maternal allele only [160,162] and encoded by yet another exon of the GNAS gene that is located upstream of exon XL and the Gsa-specific exons 1 through 13. NESP55, which is a chromogranin-like neuroendocrine secretory protein [179], shares no amino acid sequence homology with either XLas or Gsa, but its mRNA contains Gsaespecific exons 2e13 in the 30 non-coding region. Ablation of the Nesp55 protein in mice leads to an alteration of behavioral reactivity to novel environments [180], while premature truncation of its transcript is associated with loss of imprinting at the downstream differentially methylated regions of the maternal GNAS allele [181]. Another transcript with broad paternal-specific expression is termed A/B (also known as 10 or 1A), which uses a unique promoter and first exon located z2.5 kb upstream of Gsa [182e184]. The A/B transcript too shares exons 2 through 13 with the Gsa transcript but it is uncertain whether the former is translated into a protein. Another spliced transcript reads from the opposite strand of the GNAS gene, and is hence termed the antisense transcript (AS). As the A/B transcript, the AS transcript is thought to be non-coding [185,186]. Consistent with the parent-specific expression profiles of its individual transcripts, GNAS shows allele-specific methylation. While the promoter of Gsa lacks methylation, promoters of the transcripts with parent-specific expression are methylated on the inactive, silenced allele. Studies in mice have identified the differentially methylated regions encompassing the promoters of the A/B and antisense transcripts as female germ-line imprint marks [183,187] and, furthermore, implicated these two transcripts in the regulation of imprinting at this gene locus [188e190]. Because of the complexity of the GNAS gene and because of the use of different, allele- and strand-specific promoters, it appears plausible that mutations in the Gsa-specific exons 2 through 13 can affect not only the functional properties of Gsa, but also those of XLas, XXLas, NESP55, and the A/B transcript. Mutations in the exons encoding Gsa have not been detected in PHP type Ib (PHP-Ib), a disorder in which

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affected individuals typically show PTH-resistant hypocalcemia and hyperphosphatemia, but lack developmental defects and additional endocrine abnormalities [114e116]. Furthermore, individuals with PHP-Ib frequently show a normal osseous response to PTH, or even biochemical and radiological evidence for increased bone turnover and osteoclastic bone resorption, indicating that the PTH-dependent actions on osteoblasts are not impaired [114,115,191,192]. Studies of the PTH/PTHrP receptor gene and mRNA in PHPIb patients have failed to identify mutations [193e197]. In one study, however, a single amino acid deletion in the carboxyl-terminal region of the PTH/PTHrP receptor, del382Ile, has been demonstrated in three siblings with isolated PTH-resistance [198]. This mutation appears to uncouple Gsa from the PTH/PTHrP receptor only, leaving the function of several other Gscoupled receptors intact [198], although a subsequent study using a different cell line showed that this mutant is uncoupled from other receptors, as well [199]. It may thus represent 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 to the isolated PTH-resistance. A genome-wide scan in four unrelated kindreds, on the other hand, mapped the PHP-Ib locus to chromosome 20q13.3, which contains the GNAS gene [200]. In this study, it was furthermore 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 PPHP. Investigation of the methylation status of GNAS has revealed in most PHPIb patients a uniform loss of methylation at the exon A/B differentially methylated region [201,202]. Further investigations of kindreds with PHP-Ib have led to the identification of a 3-kb deletion located z220 kb upstream of GNAS within the neighboring STX16 locus, the gene encoding syntaxin-16 [203]. Flanked by two direct repeats, this microdeletion is a frequent cause of the autosomal dominant form of PHP-Ib (AD-PHP-Ib), which is associated with an isolated loss of A/B methylation. The 3-kb microdeletion, as well as 4.4-kb microdeletion that overlaps with the latter [204], causes PHP-Ib and loss of A/B methylation only after maternal inheritance. Since STX16 appears to be a non-imprinted gene [204], it is likely that the mutations identified at this locus disrupt a cis-acting element controlling imprinting of the A/B differentially methylated region. Methylation defects of GNAS are found in many sporadic and some familial PHP-Ib patients and can involve not only the A/B differentially methylated region but also the other differentially methylated regions of this locus [201,202,205]. Paternal uniparental

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disomy involving chromosome 20 has rarely been identified as a cause of sporadic PHP-Ib [206e208]. Due to this genetic aberration, these patients display a gain of methylation at the NESP55 differentially methylated region and a loss of methylation at all the maternally methylated GNAS differentially methylated regions, i.e. exon A/B, exon XL, and the promoter of the antisense transcript. In two unrelated PHP-Ib kindreds, in whom affected individuals show broad GNAS methylation defects, microdeletions that remove the entire NESP55 differentially methylated region have been identified [209]. The deleted region also includes exons 3 and 4 of the antisense transcript, which are located within the same differentially methylated region. These microdeletions lead to PHP-Ib and loss of all maternal GNAS imprints upon maternal inheritance only. More recently, a microdeletion that removes antisense exons 3 and 4 but not the exon that encodes NESP55 has been identified in another PHP-Ib kindred with broad GNAS methylation defects [210]. The patients, who inherited the latter microdeletion (but not the deletion involving NESP55) from their mothers, show a partial gain of NESP55 methylation in addition to a loss of all maternal imprints. The deletion appears to reduce the abundance of the antisense transcript on the paternal allele and, interestingly, the family members who inherit the deletion paternally show a partial loss of NESP55 methylation and a partial gain of A/B methylation. Some recent investigations have revealed an interesting overlap between the clinical and/or molecular features of PHP-Ia and PHP-Ib patients. It is now well established that TSH resistance can be present not only in patients with PHP-Ia but also in patients with PHPIb, although the resistance to TSH is clinically milder in the latter patients [163,202,206]. This finding is consistent with the monoallelic expression of Gsa in the thyroid tissue [163e165]. Note that the range of hormone resistance in PHP-Ib does not seem directly to reflect the tissue-specific monoallelic Gsa expression, as resistance to GHRH could not be demonstrated in PHP-Ib [211]. On the other hand, AHO features, which had been typically associated with inactivating mutations affecting the Gsa-coding GNAS exons (i.e. patients with PHP-Ia and PPHP), were documented in some patients who have, in lieu of coding Gsa mutations, methylation abnormalities of the GNAS locus that are commonly found in patients with PHP-Ib [212e215]. This finding suggests that, at least in some cases, Gsa expression is monoallelic in more tissues than currently recognized. Clinical management of patients with PHP type I targets the treatment of each endocrine abnormality individually. Calcium supplements and calcitriol are given to maintain serum calcium levels within the normal range. This treatment should also aim to prevent

long-term elevations of serum PTH, which can otherwise lead to hyperparathyroid bone disease and osteitis fibrosa cystica due to increased bone resorption [192]. Hypothyroidism is treated with thyroid replacement therapy. For growth hormone deficiency, the effect of growth hormone replacement has recently been investigated in a small group of prepubertal PHP-Ia patients, concluding that the treatment is potentially effective but has to be initiated as early as possible [216].

Blomstrand’s disease Blomstrand’s chondrodysplasia is an autosomal recessive human disorder characterized by early lethality, dramatically advanced bone maturation and accelerated chondrocyte differentiation [217]. Affected infants are typically born to consanguineous healthy parents (only in one instance did unrelated healthy parents have two affected offspring) [218e222], show pronounced hyperdensity of the entire skeleton (Fig. 21.5) and markedly advanced ossification. Particularly, the long bones are extremely short and poorly modeled. Homozygous loss-of-function mutations in the gene encoding the PTH/PTHrP receptor have been identified as the most likely cause of Blomstrand’s disease. The identified mutations include a proline-toleucine point mutation at residue 132 (P132L) [223e225], a homozygous single nucleotide deletion in exon EL2 [226], a 27-bp insertion between exon M4 and EL2 [227], a nonsense mutation at residue 104 [225], and a homozygous nucleotide change (intron M4þ27C>T) creating an aberrant splice site [225]. In one case, a heterozygous nucleotide exchange in exon M5 of the maternal PTH/PTHrP receptor allele was demonstrated, introducing a novel splice acceptor site and thus leading to the synthesis of a receptor mutant that does not mediate, despite seemingly normal cell surface expression, the actions of PTH or PTHrP (Fig. 21.6); the patient’s paternal PTH/PTHrP receptor allele is, for yet unknown reasons, only poorly expressed [228]. As in Jansen’s disease (see below), 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 affected infants show, besides the striking skeletal defects, abnormalities in other organs including secondary hyperplasia of the parathyroid glands, presumably due to hypocalcemia. In addition, analysis of fetuses with Blomstrand’s disease has revealed abnormal breast development and tooth impaction, highlighting the involvement of the PTH/PTHrP receptor in the normal development of breast and tooth [229]. Depending on the severity of the disease, it has been proposed that Blomstrand chondrodysplasia be

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27-bp ins

P132L

373-383

I458R

1-bp del

H223R

R104X

extracellular

T410P T410R

intracellular

FIGURE 21.6

Schematic representation of the human PTH/ PTHrP receptor. The approximate locations of heterozygous missense mutations that lead in patients with Jansen’s disease to constitutive receptor activation 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.

A

B

C

D FIGURE 21.5 Radiological findings in a patient with Blomstrand’s disease. 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. (From [219] with permission.)

molecular exploration of these genes has provided important novel insights into the pathogenesis of different forms of hyperparathyroidism (Fig. 21.7). 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 proto-oncogene, and a single mutant allele may affect the phenotype of the cell; these genes may also be referred to as dominant oncogenes (Fig. 21.7A). The mutant versions, i.e. the oncogene, which are usually excessively or inappropriately active, may arise because of point mutations, gene amplifications, or chromosomal translocations. Tumor suppressor genes, also referred to as recessive oncogenes or anti-oncogenes, normally inhibit cell

classified as type I and type II [230]. In type I, which is the more severe type, these mutations appear to abolish PTH/PTHrP receptor activity and/or expression completely, whereas in type II the mutations are associated with some residual receptor activity, such as the P132L and the intron M4þ27C>T mutations [225]. Interestingly, heterozygous PTH/PTHrP receptor mutations, which may have a dominant negative effect, were shown to be associated with familial, non-syndromic primary failure of tooth eruption [231]. FIGURE 21.7 Schematic illustration of the molecular defects that

HYPERCALCEMIC DISEASES Similar to the 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

can lead to the development of parathyroid tumors. Panel A: a somatic mutation (point mutation or translocation) affecting a proto-oncogene (for example, PRAD1 or RET) results in a growth advantage of single parathyroid cell and thus its clonal expansion. Panel B: an inherited single point mutation or deletion affecting a tumor-suppressor gene (first hit) makes the parathyroid cell suseptible to a second, somatic “hit” (point mutation or deletion, i.e. LOH), which then leads to the clonal expansion of a single cell.

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proliferation, while 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 arises by point mutations, or alternatively, by deletions that can involve substantial genomic portions or a whole chromosome. Larger deletions may be detected by cytogenetic methods, by Southern blot analysis, or by PCRbased analysis of polymorphic markers. Typically, genomic DNA from the patient’s tumor cells lack, in comparison to genomic DNA from other cells (e.g. lymphocytes), certain chromosomal regions, a finding referred to as loss of heterozygosity (LOH) (Fig. 21.7B). Since in order to transform a normal cell to a tumor cell, both alleles of the tumor-suppressor gene must be inactivated, the finding of LOH suggests a point mutation in the other allele. For all these somatic mutations, a single point mutation or a deletion provides 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, or as part of inherited tumor syndromes [232], such as the multiple endocrine neoplasias (MEN) or hereditary hyperparathyroidism with jaw tumors (HPT-JT) [233], or in response to chronic overstimulation as in uremic hyperparathyroidism [234]. 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 (see Fig. 21.6A). Furthermore, in a significant number of patients, LOH has been documented for one of various chromosomal loci that comprise different tumor suppressor 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 which affects only one allele of a gene encoding an anti-oncogene (see Fig. 21.6B). Subsequently, a somatic mutation or deletion affecting the second allele occurs in a single parathyroid cell and this mutation leads, because of the resulting growth advantage, to its monoclonal expansion and thus the development of a parathyroid tumor. Examples of this 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 and the retinoblastoma (Rb) gene, as well as a yet undefined gene located on chromosome 1p. PRAD1 and PTH Genes Investigations of the PTH gene in sporadic parathyroid adenomas have revealed abnormally sized restriction fragment length polymorphisms (RFLPs) with a DNA probe for the 50 part of the PTH gene in some adenomas [235], indicating disruption of the gene. Further studies of the tumor DNA demonstrated that the first exon of the PTH gene (see Fig. 21.2) was separated from the fragments containing the second and third exons, and that a rearrangement had occurred juxtaposing the 50 PTH regulatory elements with “new” non-PTH DNA [236]. This rearrangement was not found in the DNA from the peripheral leukocytes of the patients, thereby indicating that it represented a somatic event and not an inherited germ-line mutation. Investigation of this rearranged DNA sequence localized it to chromosome 11q13, and detailed analysis revealed that it was highly conserved in different species and expressed in normal parathyroids and in parathyroid adenomas. The protein expressed as a result of this rearrangement, which was designated PRAD1, was demonstrated to encode a 295 amino acid member of the cyclin-D family of cell-cycle regulatory proteins. Cyclins have been initially characterized in the dividing cells of budding yeast where they controlled the G1 to S transition of the cell cycle and in marine molluscs where they regulated the mitotic phase (M-phase) of the cell cycle [237]. Cyclins are also present in man and have an important role in regulating many stages of cell cycle progression. Thus, PRAD1, which encodes a novel cyclin referred to as cyclin D1 (CCND1), is an important cell cycle regulator, and overexpression of PRAD1 may be an important event in the development of at least 15% of sporadic parathyroid adenomas [238]. Interestingly, >66% of the transgenic mice overexpressing PRAD1 under the control of a mammary tissue specific promoter have been found to develop breast carcinoma in adult life [239]. Furthermore, expression of this protooncogene under the control of the 50 regulatory region of the PTH gene has provided a good model for primary hyperparathyroidism, as the results included abnormal parathyroid cell proliferation, and mild-to-moderate chronic hyperparathyroidism with altered PTH response to variations in serum calcium concentration [240]. Taken together, these findings in transgenic animals provide further evidence for the conclusion that PRAD1 can be involved in the development of a significant number of parathyroid adenomas.

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In addition to the rearrangement of the PTH gene in some parathyroid adenomas, a nonsense mutation, Arg83Stop (note that 31 amino acids of the pre-pro sequence precede the mature peptide), of the PTH gene that occurred in association with LOH of the PTH locus has been reported in a parathyroid adenoma [241]. The patient, who had presented with hypercalcemia and an undetectable serum PTH concentration, showed heterozygosity in the peripheral-blood leukocytes with wild-type and mutant alleles, but the parathyroid adenoma has a loss of the wild-type allele and retention of the mutant (Arg83Stop) allele, which predicts that tumor secretes only a PTH peptide that is truncated after the 52nd amino acid. Following removal of the parathryoid adenoma, normocalcemia was restored. These findings demonstrate that PTH nonsense mutations, which result in truncated forms of PTH, may be associated with parathyroid adenoma, and that endogenously produced N-terminal PTH fragments can be biologically active [241].

TABLE 21.2

The Multiple Endocrine Neoplasia (MEN) Syndromes, their Characteristic Tumors and Associated Genetic Abnormalities.

Type (chromosomal location) MEN1 (11q13)

Tumors Parathyroids Pancreatic islets Gastrinoma Insulinoma Glucagonoma VIPoma PPoma Pituitary (anterior) Prolactinoma Somatotrophinoma Corticotrophinoma Non-functioning Associated tumors Adrenal cortical Carcinoid Lipoma Angiofibromas Collagenomas

The MEN1 Gene Multiple endocrine neoplasia type 1 (MEN1) is characterized by the combined occurrence of tumors of the parathyroids, pancreatic islet cells and anterior pituitary (Table 21.2) [232,242]. Parathyroid tumors occur in 95% of MEN1 patients, and the resulting hypercalcemia is the first manifestation of MEN1 in about 90% of patients. Pancreatic islet cell tumors occur in 40% of MEN1 patients and gastrinomas, leading to the ZollingereEllison syndrome, are the most common type and also the 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, which may also occur in MEN1, include adrenal cortical tumors, carcinoid tumors, lipomas, angiofibromas and collagenomas [242,243]. The gene causing MEN1 was localized to a <300 kb region on chromosome 11q13 by genetic mapping studies that investigated MEN1 associated tumors for loss of heterozygosity (LOH) and by segregation studies in MEN1 families [244]. The results of these studies, which were consistent with Knudson’s model for tumor development, indicated that the MEN1 gene represented a putative tumor suppressor gene (see Fig. 21.6B). Characterization of genes from this region led to the identification of the MEN1 gene [245,246], which consists of 10 exons that encode a novel 610 amino acid protein, referred to as “MENIN”. The majority (>80%) of the germ-line MEN1 mutations in the families are inactivating, and are consistent with its role as a tumor suppressor gene. These mutations are diverse in their types and z25% are nonsense, z45% are deletions, z15% are insertions, <5% are

MEN2 (10 cen-10q.11.2) MEN2a

Medullary thyroid carcinoma (MTC) Pheochromocytoma Parathyroid

MTC-only

Medullary thyroid carcinoma (MTC)

MEN2b

Medullary thyroid carcinoma (MTC) Pheochromocytoma Associated abnormalities: Mucosal neuromas Marfanoid habitus Medullated corneal nerve fibers Megacolon

Autosomal dominant inheritance of the MEN syndromes has been established.

donor-splice mutations and z10% are missense mutations [244]. In addition, the MEN1 mutations are scattered throughout the 1830 bp coding region of the gene with no evidence for clustering. Any correlation between the location of the MEN1 germ-line mutations and the clinical manifestations of the disorder appears to be absent [247]. Tumors from MEN1 patients and non-MEN1 patients have been observed to harbor the germ-line mutation together with a somatic LOH involving chromosome 11q13 or small intragenic deletions, as expected from Knudson’s model and the proposed role of the MEN1 gene as a tumor suppressor [248e256]. The role of the MEN1 gene in the etiology of familial isolated hyperparathyroidism (FIHP) has also been investigated, and germ-line MEN1 mutations have been reported in 29 families with FIHP [257e260]. The sole occurrence of parathyroid tumors in these families is remarkable and the mechanisms

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that determine the altered phenotypic expressions of these mutations remain to be authenticated. The function of MENIN has been investigated by identifying its interactions with other proteins, and by under- or overexpression in in vitro studies. MENIN has no homology to any known proteins or sequence motifs other than three nuclear localization signals (NLSs) in its C-terminal segment. Subcellular localization studies have shown that MENIN is predominantly a nuclear protein in non-dividing cells, but in dividing cells it is found in the cytoplasm. MENIN has been shown to interact with a number of proteins that are involved in transcriptional regulation, genome stability, cell division and proliferation [257]. The functional role of MENIN as a tumor suppressor also has been investigated and studies in human fibroblasts have revealed that MENIN acts as a repressor of telomerase activity via hTERT (a protein component of telomerase) [261]. Furthermore, overexpression of MENIN in the human endocrine pancreatic tumor cell line (BON1) resulted in an inhibition of cell growth [262] that was accompanied by upregulation of JunD expression but downregulation of delta-like protein 1/preadipocyte factor-1, proliferating cell nuclear antigen, and QM/Jif-1, which is a negative regulator of c-Jun [262]. These findings of growth suppression by MENIN were observed in other cell types. Thus, expression of MENIN in the RAS-transformed NIH3T3 cells partially suppressed the RAS-mediated tumor phenotype in vitro and in vivo [263]. Overexpression of MENIN in CHO-IR cells also suppressed insulin-induced AP-1 transactivation, and this was accompanied by an inhibition of c-Fos induction at the transcriptional level [264]. Furthermore, MENIN re-expression in Men1-deficient mouse Leydig tumor cell lines, induced cell cycle arrest and apoptosis [265]. In contrast, depletion of MENIN in human fibroblasts resulted in their immortalization [261]. Thus, MENIN appears to have a large number of functions through interactions with proteins, and these mediate alterations in cell proliferation. The MEN2 Gene (c-ret) MEN2 describes the association (see Table 21.2) of medullary thyroid carcinoma (MTC), pheochromocytomas, and parathyroid tumors [232,244]. Three clinical variants of MEN2 are recognized e MEN2a, MEN2b and MTC-only. MEN2a is the most common variant, and the 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-10q11.2, a region containing the c-ret proto-oncogene, which encodes a tyrosine kinase receptor with cadherin-like and cysteine-rich extracellular domains, and a tyrosine kinase intracellular domain [266,267]. 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, the codon 634 mutations do not appear to be present in sporadic non-MEN2a parathyroid adenomas [268,269]. MTConly 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 proto-oncogene 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 [267,270]. Hyperparathyroidism-Jaw Tumor (HPT-JT) Syndrome Gene The HPT-JT syndrome is an autosomal dominant disorder characterized by the development of parathyroid adenomas and carcinomas, and fibro-osseous jaw tumors [271,272]. In addition, some patients may also develop uterine tumors and renal abnormalities, which include Wilms’ tumors, renal cysts, renal hamartomas, renal cortical adenomas, and papillary renal cell carcinomas [273]. Other tumors, including pancreatic adenocarcinomas, testicular mixed germ cell tumors with a major seminoma component, and Hurthle cell thyroid adenomas have also been reported in some patients [233,273]. It is important to note that the parathyroid tumors may occur in isolation and without any evidence of jaw tumors, and this may cause confusion with other hereditary hypercalcemic disorders such as MEN1, familial benign hypercalcemia (FBH), which is also referred to as familial hypocalciuric hypercalcemia (FHH), and familial isolated hyper parathyroidism (FIHP) [274]. HPT-JT can be distinguished from FBH, as in FBH serum calcium levels are elevated during the early neonatal or infantile period, whereas in HPT-JT, such elevations are uncommon in the first decade. In addition, HPT-JT patients, unlike

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FBH patients, will have associated hypercalciuria. The distinction between HPT-JT patients and MEN1 patients, who have only developed the usual first manifestation of hypercalcemia (>90% of patients), is more difficult and is likely to be influenced by operative and histological findings, and by the occurrence of other characteristic lesions in each disorder. It is important to note that HPT-JT patients will usually have single adenomas or a carcinoma, while MEN1 patients will often have multiglandular parathyroid disease. The distinction between FIHP and HPT-JT in the absence of jaw tumors is difficult but important as HPT-JT patients may be at a higher risk of developing parathyroid carcinomas [275e277]. These distinctions may be helped by the identification of additional features, and a search for jaw tumors, renal, pancreatic, thyroid and testicular abnormalities may help to identify HPT-JT patients. The jaw tumors in HPT-JT are different from the brown tumors observed in some patients with primary hyperparathyroidism, and do not resolve after parathyroidectomy [274]. Indeed ossifying fibromas of the jaw are an important distinguishing feature of HPT-JT from FIHP, and the occurrence of these may occasionally precede the development of hypercalcemia in HPT-JT patients by several decades. The gene causing HPT-JT is located on chromosome 1q31.2, and consists of 17 exons that encode a ubiquitously expressed and evolutionary conserved 531 amino acid protein, designated PARAFIBROMIN [233,278]. This gene is also referred to as HRPT2 (i.e. hyperparathyroidism type 2). HRPT2 mutations associated with HPT-JT are scattered throughout the 1593 bp coding region, with the majority (>80%) predicting a functional loss through premature truncation. A genotypeephenotype correlation was not apparent from these analyses [273,278e280]. The observation of LOH involving the chromosome 1q21.32 region in HPT-JT associated tumors indicated that PARAFIBROMIN may be acting as a tumor suppressor, consistent with Knudson’s two hit hypothesis [273,274,278e280]. This was supported by the observations of germ-line and somatic HRPT2 mutations in HPT-JT associated tumors [273,278e281]. Similar germ-line and somatic HRPT2 mutations have also been found in sporadic parathyroid carcinomas and the frequency of such mutations is high, ranging from 67% to 100% [279,282]; however, the frequency of HRPT2 mutations in sporadic parathyroid adenomas is low at 0% to 4%, indicating that HRPT2 mutations likely confer an aggressive growth potential to the parathyroid cells [278e280,282,283]. HRPT2 mutations and allelic imbalances have also been identified in sporadic renal tumors [284], and a loss or downregulation of HRPT2 expression has been reported in both breast and gastric cancers [285,286]. These studies indicate that

575

HRPT2 and its encoded protein, PARAFIBROMIN play a critical role in inherited and sporadic parathyroid cancers as well as other non-hereditary solid tumors. The role of PARAFIBROMIN, which is predominantly a nuclear protein with a monopartite NLS [287], was not readily apparent as it has no homologies to known proteins. However, the approximate 200 amino acids of the C-terminal domain shared over 25% sequence identity with the yeast Cdc73 protein which is a component of the yeast polymerase-associated factor 1 (PAF1) complex, a key transcriptional regulatory complex that interacts directly with RNA polymerase II [288,289]. Studies of the PAF1 complex in yeast and Drosophila as well as in mammalian cells, have revealed that PARAFIBROMIN, as part of the PAF1 complex, is a mediator of the key transcriptional events of histone modification, chromatin remodeling, initiation and elongation, and the Wnt/beta-catenin signaling pathway [288e290]. Studies of a mouse deleted for Hrpt2 have revealed that Hrpt2 expression and the PARAFIBROMIN/PAF complex directly regulate genes, e.g. H19, IgF1, Ifg3, Igfbp4, Hmga1, Hmga2 and Hmga3, that are involved in cell growth and apoptosis [291]. LRP5 and the Wnt/b-catenin Pathway Aberrant Wnt/b-catenin signaling with an accumulation of b-catenin in the cytoplasm and nucleus is associated with several types of tumor development, e.g. adenomatous polyposis coli and colorectal cancer. Investigations of this pathway in parathyroid tumors have revealed that b-catenin accumulation occurs in parathyroid adenomas and in parathyroid tumors associated with chronic renal failure [292]. In addition, a protein-stabilizing mutation, Ser37Ala, in exon 3 of bcatenin was detected in over 7% of parathyroid adenomas, but not parathyroid tumors of chronic renal failure, from Swedish patients [293], but not North American [294] or Japanese [295] patients. The Ser37Ala b-catenin mutations were homozygous in the parathyroid adenomas, which had a higher expression of bcatenin and the non-phosphorylated active form of b-catenin [296]. In addition, MYC which is a direct target of the Wnt/b-catenin signaling pathway in colorectal cancer cells and critical mediator of the early stages of intestinal neoplasia, was also overexpressed, and the stable activity of endogenous b-catenin was found to be necessary for MYC and cyclin D1 expression [292]. Stability of b-catenin is regulated by Wnt ligands, which bind to the cell-surface frizzled receptors and LRP5 and LRP6 co-receptors that alter phosphorylation of several intracellular second messengers and consequently accumulation of non-phosphorylated b-catenin. Investigation of the Wnt-signaling pathway in parathyroid tumors revealed over 85% of adenomas and 100% of tumors

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from chronic renal failure patients to have a shorter LRP5 transcript, which contained an in-frame deletion of 142 amino acids (residues 666 to 809) that encompassed the third YWTD b-propeller domain between the second and third epidermal growth factor repeats [293]. This internally truncated LRP5 receptor activated b-catenin signaling in parathyroid tumors, by a mechanism that may involve an impaired inhibitory action of the Wnt antagonist DKK1 [293]. The parathyroid tumors expressing the internally truncated LRP5 receptor did not harbor the b-catenin stabilizing mutation, Ser37Ala, and those that had the stabilizing b-catenin mutation did not express the truncated LRP5 receptor [293]. Thus, it seems that the presence of the stabilizing b-catenin mutation and the expression of the truncated LRP5 receptor are mutually exclusive. However, these studies demonstrate an important role for the Wnt b-catenin signaling pathway in parathyroid tumorigenesis. Rb Gene The Rb gene, which is a tumor suppressor gene [297] located on chromosome 13q14, 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 [298], and was accompanied by abnormal staining patterns for the Rb protein in 50% of the parathyroid carcinomas but in none of the parathyroid adenomas [298]. These results demonstrate an important role for the Rb gene in the development of parathyroid carcinomas, and may be of help in the histological distinction of parathyroid adenoma from carcinoma [298]. However, the findings of extensive deletions of the long arm of chromosome 13 (including the Rb locus) in some parathyroid adenomas and carcinomas [299], and similar findings in pituitary carcinomas [300] suggest that other tumor suppressor genes on chromosome 13q may also have a role in the development of such tumors. Retinoblastoma-interacting Zing Finger Protein Gene on Chromosome 1p Loss of heterozygosity studies have revealed allelic loss of chromosome 1p32-pter in 40% of sporadic parathyroid adenomas [301]. This region, estimated to be about 110 cM, and equivalent to about 110 million base pairs (Mbp) of DNA, was subsequently narrowed to an interval of approximately 4 cM, i.e. about 4 Mbp [302]. Investigations of one candidate gene, retinoblastomainteracting zinc-finger protein 1 (RIZ1), have revealed that over 25% of parathyroid tumors had LOH of the RIZ1 locus and that over 35% of parathyroid tumors had hypermethylation of the RIZ1 promoter region [303]. Moreover, the RIZ1 promoter hypermethylation

was related to LOH in these tumors indicating that these two events may represent the “two hits” (see Fig. 21.7B) required for tumor development in Knudson’s hypothesis for tumorigenesis [303]. Non-syndromic Familial Isolated Hyperparathyroidism Syndromes Familial isolated hyperparathyroidism (FIHP) may represent an incomplete manifestation of a syndromic form such as MEN1, FHH, or HPT-JT [259,260,278,304]. To date, 29 MEN1, nine HRPT2 and five CaSR germline mutations have been reported in FIHP kindreds [260]. However, it is important to note that the genetic etiology of non-syndromic FIHP in the majority of families remains to be elucidated [305,306]. Thus, studies of 32 kindreds with non-syndromic FIHP for mutations of the MEN1, CaSR and HRPT2 genes, have revealed that only one family harbored a germ-line mutation, and this involved the HRPT2 gene that encodes PARAFIBROMIN [305,306]. However, studies of 10 other FIHP kindreds have indicated that another locus, referred to as HRPT3, on chromosome 2p13.3-p14, is likely to be involved in the etiology of non-syndromic FIHP [307]. Thus, the genes and their underlying abnormalities that lead to non-syndromic FIHP remain to be identified. 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 has initially suggested that the autonomous parathyroid tissue might have undergone hyperplastic change and therefore be polyclonal 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 [235]. In addition, LOH involving several loci on chromosome Xp11 was detected in one of these parathyroid tumors, thereby suggesting the involvement of a tumor suppressor gene from this region in the pathogenesis of such tumors [235]. Interestingly, none of the parathyroid tumors from these patients with chronic renal failure had LOH involving loci from chromosome 11q13. 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 e MEN1 and PRAD1 e located on chromosome 11q13. In addition, parathyroid tumors from patients with chronic renal failure have been shown to accumulate b-catenin, and

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to have a truncated form of the LRP5 receptor, which lacks 142 amino acids [292].

Disorders of the Calcium-Sensing Receptor (CaSR) Three hypercalcemic disorders due to mutations of the calcium-sensing receptor (CaSR) have been reported [304] and these are familial benign hypercalcemia (FBH), which is also referred to as familial hypocalciuric hypercalcemia (FHH), neonatal severe hyperparathyroidism (NSHPT) and autoimmune hypocalciuric hypercalcemia. Mutational analyses of the human CaSR, which is a G-protein coupled receptor located on chromosome 3q13-q21 [308], have revealed different mutations, that result in a loss of function of the CaSR in patients with FBH and NSHPT [309e314]. Many of these mutations cluster around the aspartate- and glutamate-rich regions (codons 39e300) within the extracellular domain of the receptor, and this has been proposed to contain low affinity calcium-binding sites, based on similarities to that of calsequestrin, in which the ligand-binding pockets also contain negatively charged amino acid residues [308]. Approximately two-thirds of the FBH kindreds investigated have been found to 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 [309,312e315]. NSHPT occurring in the offspring of consanguineous FBH families has been shown to be due to homozygous CaSR mutations [309e312,315]. However, some patients with sporadic neonatal hyperparathyroidism have been reported to be associated with de-novo heterozygous CaSR mutations [313,314], thereby suggesting that factors other than mutant gene dosage [312], for example, the degree of set-point abnormality, the sensitivity of bone to PTH and the maternal extracellular calcium concentration,

may also all play a role in the phenotypic expression of a CaSR mutation in the neonate. The remaining one-third of FBH families in whom a mutation within the coding region of the CaSR has not been demonstrated may either have 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 is located on chromosome 19p and is referred to as FBH19p [316]. Studies of another FBH kindred from Oklahoma that also suffered from progressive elevations in PTH, hypophosphatemia and osteomalacia [317,318] demonstrated that this variant, designated FBHOk, was linked to chromosome 19q13 [319]. These three FBH loci located on chromosomes 3q, 19p and 19q have also been referred to as FBH (or FHH) types 1, 2 and 3, respectively [319]. Some patients who have the clinical features of FHH but not CaSR mutations may have autoimmune hypocalciuric hypercalcemic (AHH). Four patients with AHH who all had other autoimmune manifestations have been reported [111]. Thus, three patients had anti-thyroid antibodies, and one had sprue with anti-gliadin and antiendomyseal antibodies. These patients were shown to have circulating antibodies to the extracellular domain of the CaSR, and these antibodies stimulated PTH release from dispersed human parathyroid cells in vitro, probably by inhibiting the activation of the CaSR by extracellular calcium [111]. Thus, AHH is a condition of abnormal extracellular calcium sensing that should be considered in FHH patients who do not have CaSR mutations.

Jansen’s Disease and Related Disorders Jansen’s disease (see Figs 21.6 and 21.8) is an autosomal dominant disease that is characterized by shortlimbed dwarfism caused by an abnormal regulation of chondrocyte proliferation and differentiation in the metaphyseal growth plate, and an associated severe hypercalcemia and hypophosphatemia, despite normal FIGURE 21.8 Hand radiographs of the patient first described by Jansen, at age 10 (left) and 44 (right). (From [320] with permission.)

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or undetectable serum levels of PTH or PTHrP [320e323]. These abnormalities are caused by mutations of the PTH/PTHrP receptor that lead to constitutive PTH- and PTHrP-independent receptor activation. Three different mutations of the PTH/PTHrP receptor have been identified, and these involve codon 223 (His/Arg), codon 410 (Thr/Pro or Arg), and codon 458 (Ile/Arg) [324e328] (see Fig. 21.6). Expression of the mutant receptors in COS-7 cells resulted in constitutive, ligand independent accumulation of cAMP, while the basal accumulation of inositol phosphates was not increased. Since the PTH/PTHrP receptor is most abundantly expressed in 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 further by observations in mice expressing the human PTH/ PTHrP receptor with the H223R mutation under the control of the rat a1(II) collagen promoter [329]. This promoter targeted expression of the mutant receptor to the layer of proliferative chondrocytes, delayed their differentiation into hypertrophic cells, and led, at least in animals with multiple copies of the transgene, to a mild impairment in growth of long bones. 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.

Ollier’s Disease and Eiken Syndrome Another PTH/PTHrP receptor mutation (c.448C>T predicting p.R150C) has been identified in some patients with enchondromatosis (Ollier’s disease), a familial disorder with autosomal dominant inheritance which is characterized by multiple benign cartilage tumors, and a predisposition to malignant osteosarcoma [330]. Although a subsequent study of a larger group of patients could not detect the same mutation or identify any other mutations within the gene encoding PTH/PTHrP receptor [331], a more recent study discovered three novel heterozygous mutations in patients with Ollier’s disease. Two of the mutations (p.G121E, p.A122T) were present only in enchondromas, and the third mutation (p.R255H) in both enchondroma and leukocyte DNA [332]. A recessive mutation in the same gene was also identified as a cause of Eiken syndrome, which is a rare autosomal recessive skeletal dysplasia [333]. This mutation leads to the truncation of the PTH/PTHrP receptor at its cytoplasmic C-terminal tail. It thus appears that different mutations within the PTH/PTHrP receptor gene can lead to divergent skeletal phenotypes.

Brachydactyly Type-E and Delayed Tooth Eruption Defects in the gene encoding PTHrP (PTHLH; PTHlike hormone;) have been recently identified as a cause of brachydactyly type-E (BDE). A family with autosomal dominant BDE has been investigated, and the affected individuals were found to carry a t(8;12)(q13;p11.2) translocation with one of the breakpoints (BPs) upstream of PTHLH on chromosome 12p11.2 [334]. This genetic defect resulted in the disruption of a cis-acting element regulating the expression of PTHrP. In a more recent study describing a large kindred with BDE, short stature, and learning disabilities, a 900 kb microdeletion comprising PTHLH was discovered [335]. In the same study, investigations of additional patients with BDE and short stature revealed distinct heterozygous mutations within PTHLH, including two missense (L44P and L60P), a nonstop (X178WextX(*)54), and a nonsense (K120X) mutation. The L60P mutation appears to cause a PTHrP mutant with diminished activity, as determined by the analysis of alkaline phosphatase activity in chicken micromass cultures overexpressing this mutant vs. wild-type PTHrP [335].

Williams Syndrome Williams syndrome is an autosomal dominant disorder characterized by supravalvular aortic stenosis, elfin-like facies, psychomotor retardation and infantile hypercalcemia [336]. The underlying abnormality of calcium metabolism remains unknown but abnormal 1,25-dihydroxy vitamin D3 metabolism or decreased calcitonin production have been implicated, although none has been consistently demonstrated. Studies have demonstrated hemizygosity at the elastin locus on chromosome 7q11.23 in over 90% of patients with the classical Williams phenotype [337e339] and only one patient had a cytogenetically identifiable deletion, thereby indicating that the syndrome is usually due to a microdeletion of 7q11.23 [339]. Interestingly, ablation of the elastin gene in mice results in vascular abnormalities similar to those observed in patients with Williams syndrome [340]. However, the microdeletions that have been reported involve also another gene, designated LIM-kinase, that is expressed in the central nervous system [341]. The calcitonin receptor gene, which is located on chromosome 7q21, is not involved in the deletions found in Williams syndrome, and is therefore unlikely to be implicated in the hypercalcemia of such children [342]. While the involvement of the elastin and LIM-kinase genes in the deletions of Williams syndrome patients can explain the respective cardiovascular and neurological features of this disorder, it seems

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likely that another, an as-yet-uncharacterized gene located within this contiguously deleted region, is likely to explain the abnormalities of calcium metabolism.

CONCLUDING REMARKS Considerable advances were made over the past few years in identifying key proteins that are involved, either 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 furthermore 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 in further defining the biological role(s) of known proteins, 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 in the techniques to sequence entire genomes or exomes, will continue to provide important insights into the regulation of blood calcium and phosphate.

Acknowledgments RVT is grateful to the Medical Research Council and Wellcome Trust (UK) for support. HJ is supported by grants from the NIH, NIDDK (R37DK46718 and PO1DK11794). MB is supported by grants from NIH, NIDDK (DK073911) and the March of Dimes Foundation. We are grateful to Tracey Walker for her help in preparing the manuscript and expert secretarial assistance.

References [1] Thakker R, Bringhurst F, Ju¨ppner H. Genetics disorders of calcium homeostasis caused by abnormal regulation of parathyroid hormone secretion or responsiveness. In: DeGroot L, Jameson J, editors. Endocrinology. Philadelphia: W.B. Saunders Company; 2010. [2] Naylor SL, Sakaguchi AY, Szoka P, et al. Human parathyroid hormone gene (PTH) is on short arm of chromosome 11. Somatic Cell Genet 1983;9:609e16. [3] Vasicek T, McDevitt BE, Freeman MW, Potts Jr JT, Rich A, Kronenberg HM. Nucleotide sequence of genomic DNA encoding human parathyroid hormone. Proc Natl Acad Sci USA 1983;80:2127e31. [4] Parkinson D, Thakker R. A donor splice site mutation in the parathyroid hormone gene is associated with autosomal recessive hypoparathyroidism. Nature Genet 1992;1:149e53. [5] Okazaki T, Igarashi T, Kronenberg HM. 5’-Flanking region of the parathyroid hormone gene mediates negative regulation by 1,25(OH)2vitamin D3. J Biol Chem 1989;263:2203e8.

579

[6] Demay MB, Kiernan MS, DeLuca HF, Kronenberg HM. Sequences in the human parathyroid hormone gene that bind the 1,25-dihydroxyvitamin D3 receptor and mediate transcriptional repression in response to 1,25-dihydroxyvitamin D3. Proc Natl Acad Sci USA 1992;89:8097e101. [7] Naveh-Many T, Rahaminov R, Livini N, Silver J. Parathyroid cell proliferation in normal and chronic renal failure in rats. The effects of calcium, phosphate, and vitamin D. J Clin Invest 1995;96:1786e93. [8] Almaden Y, Canalejo A, Hernandez A, et al. Direct effect of phosphorus on PTH secretion from whole rat parathyroid glands in vitro. J Bone Miner Res 1996;11:970e6. [9] Silver J, Kronenberg HM. Parathyroid hormone e molecular biology and regulation. In: Bilezikian JP, Raisz LG, Rodan GA, editors. Principles of Bone Biology. New York: Academic Press; 1996. p. 325e46. [10] Slatopolsky E, Finch J, Denda M, et al. Phosphorus restriction prevents parathyroid gland growth. High phosphorus directly stimulates PTH secretion in vitro. J Clin Invest 1996;97:2534e40. [11] Russell J, Lettieri D, Sherwood LM. Direct regulation by calcium of cytoplasmic messenger ribonucleic acid coding for pre-proparathyroid hormone in isolated bovine parathyroid cells. J Clin Invest 1983;72:1851e5. [12] Naveh-Many T, Friedlaender MM, Mayer H, Silver J. Calcium regulates parathyroid hormone messenger ribonucleic acid (mRNA), but not calcitonin mRNA in vivo in the rat. Dominant role of 1,25-dihydroxyvitamin D. Endocrinology 1989;125: 275e80. [13] Kemper B, Habener JF, Mulligan RC, Potts Jr JT, Rich A. Preproparathyroid hormone: a direct translation product of parathyroid messenger RNA. Proc Natl Acad Sci USA 1974;71: 3731e5. [14] Broadus AE, Stewart AF. Parathyroid hormone-related protein: Structure, processing, and physiological actions. In: Bilezikian JP, Levine MA, Marcus R, editors. The Parathyroids Basic and Clinical Concepts. New York: Raven Press; 1994. p. p.259e94. [15] Gardella TJ, Ju¨ppner H, Brown EM, Kronenberg HM, Potts Jr JT. Parathyroid hormone and parathyroid hormonerelated peptide in the regulation of calcium homeostasis and bone development. In: DeGroot LJ, Jameson JL, editors. Endocrinology. 6th ed. Philadelphia: W.B. Saunders Company; 2010. p. 1040e73. [16] Gelbert L, Schipani E, Ju¨ppner H, et al. Chromosomal location of the parathyroid hormone/parathyroid hormone-related protein receptor gene to human chromosome 3p21.2-p24.2. J Clin Endocrinol Metab 1994;79:1046e8. [17] Pausova Z, Bourdon J, Clayton D, et al. Cloning of a parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR) cDNA from a rat osteosarcoma (UMR106) cell line: chromosomal assignment of the gene in the human, mouse, and rat genomes. Genomics 1994;20:20e6. [18] Usdin TB, Hoare SRJ, Wang T, Mezey E, Kowalak JA. Tip39: a new neuropeptide and PTH2-receptor agonist from hypothalamus. Nat Neurosci 1999;2:941e3. [19] Usdin TB, Gruber C, Bonner TI. Identification and functional expression of a receptor selectively recognizing parathyroid hormone, the PTH2 receptor. J Biol Chem 1995;270:15455e8. [20] Gardella TJ, Luck MD, Jensen GS, Usdin TB, Ju¨ppner H. Converting parathyroid hormone-related peptide (PTHrP) into a potent PTH-2 receptor agonist. J Biol Chem 1996;271: 19888e93. [21] Hoare SR, Clark JA, Usdin TB. Molecular determinants of tuberoinfundibular peptide of 39 residues (TIP39) selectivity for the parathyroid hormone (PTH) 2 receptor: N-terminal

PEDIATRIC BONE

580

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32] [33] [34]

[35]

[36]

[37]

[38]

[39]

21. PARATHYROID DISORDERS

truncation of TIP39 reverses PTH2 receptor/PTH1 receptor binding selectivity. J Biol Chem 2000;275:27274e83. Jonsson KB, John MR, Gensure RC, Gardella TJ, Ju¨ppner H. Tuberoinfundibular peptide 39 binds to the parathyroid hormone (PTH)/PTH-related peptide receptor, but functions as an antagonist. Endocrinology 2001;142:704e9. Usdin TB, Paciga M, Riordan T, et al. Tuberoinfundibular peptide of 39 residues is required for germ cell development. Endocrinology 2008;149:4292e300. Dimitrov EL, Petrus E, Usdin TB. Tuberoinfundibular peptide of 39 residues (TIP39) signaling modulates acute and tonic nociception. Exp Neurol 2010;226:68e83. Dobolyi A, Palkovits M, Usdin TB. The TIP39-PTH2 receptor system: unique peptidergic cell groups in the brainstem and their interactions with central regulatory mechanisms. Prog Neurobiol 2010;90:29e59. Usdin TB, Bonner TI, Harta G, Mezey E. Distribution of PTH-2 receptor messenger RNA in rat. Endocrinology 1996;137: 4285e97. Arnold A, Horst SA, Gardella TJ, Baba H, Levine MA, Kronenberg HM. Mutation of the signal peptide-encoding region of the preproparathyroid hormone gene in familial isolated hypoparathyroidism. J Clin Invest 1990;86:1084e7. Karaplis AC, Lim SK, Baba H, Arnold A, Kronenberg HM. Inefficient membrane targeting, translocation, and proteolytic processing by signal peptidase of a mutant preproparathyroid hormone protein. J Biol Chem 1995;270:1629e35. Datta R, Waheed A, Shah GN, Sly WS. Signal sequence mutation in autosomal dominant form of hypoparathyroidism induces apoptosis that is corrected by a chemical chaperone. Proc Natl Acad Sci USA 2007;104:19989e94. Parkinson DB, Shaw NJ, Himsworth RL, Thakker RV. Parathyroid hormone gene analysis in autosomal hypoparathyroidism using an intragenic tetranucleotide (AAAT)n polymorphism. Hum Genet 1993;91:281e4. Sunthornthepvarakul T, Churesigaew S, Ngowngarmratana S. A novel mutation of the signal peptide of the preproparathyroid hormone gene associated with autosomal recessive familial isolated hypoparathyroidism. J Clin Endocrinol Metab 1999;84: 3792e6. Von Heijne G. Patterns of amino acids near signal-sequence cleavage sites. Eur J Biochem 1983;133:17e21. Peden V. True idiopathic hypoparathyroidism as a sex-linked recessive trait. Am J Human Genet 1960;12:323e37. Whyte M, Weldon V. Idiopathic hypoparathyroidism presenting with seizures during infancy: x-linked recessive inheritance in a large Missouri kindred. J Pediatr 1981;99:628e31. Whyte M, Kim G, Kosanovich M. Absence of parathyroid tissue in sex-linked recessive hypoparathyroidism. J Paediatr 1986;109:915. Mumm S, Whyte MP, Thakker RV, Buetow KH, Schlessinger D. mtDNA analysis shows common ancestry in two kindreds with X-linked recessive hypoparathyroidism and reveals a heteroplasmic silent mutation. Am J Hum Genet 1997;60:153e9. Thakker RV, Davies KE, Whyte MP, Wooding C, O’Riordan JL. Mapping the gene causing X-linked recessive idiopathic hypoparathyroidism to Xq26-Xq27 by linkage studies. J Clin Invest 1990;86:40e5. Bowl M, Nesbit M, Harding B, et al. An interstitial deletioninsertion involving chromosomes 2p25.3 and Xq27.1, near SOX3, causes X-linked recessive hypoparathyroidism. J Clin Invest 2005;115:2822e31. Solomon NM, Ross SA, Morgan T, et al. Array comparative genomic hybridisation analysis of boys with X linked

[40]

[41]

[42]

[43]

[44]

[45]

[46] [47]

[48] [49]

[50]

[51]

[52]

[53]

[54]

[55]

[56]

[57]

[58]

hypopituitarism identifies a 3.9 Mb duplicated critical region at Xq27 containing SOX3. J Med Genet 2004;41:669e78. Rizzoti K, Brunelli S, Carmignac D, Thomas PQ, Robinson IC, Lovell-Badge R. SOX3 is required during the formation of the hypothalamo-pituitary axis. Nat Genet 2004;36:247e55. Collignon J, Sockanathan S, Hacker A, et al. A comparison of the properties of Sox-3 with Sry and two related genes, Sox-1 and Sox-2. Development 1996;122:509e20. Kleinjan DA, van Heyningen V. Long-range control of gene expression: emerging mechanisms and disruption in disease. Am J Hum Genet 2005;76:8e32. Brunelli S, Silva Casey E, Bell D, Harland R, Lovell-Badge R. Expression of Sox3 throughout the developing central nervous system is dependent on the combined action of discrete, evolutionarily conserved regulatory elements. Genesis 2003;36: 12e24. Ahonen P, Myllarniemi S, Sipila I, Perheentupa J. Clinical variation of autoimmune polyendocrinopathy-candidiasisectodermal dystrophy (APECED) in a series of 68 patients. N Engl J Med 1990;322:1829e36. Ahonen P. Autoimmune polyendocrinopathyecandidosise ectodermal dystrophy (APECED): autosomal recessive inheritance. Clin Genet 1985;27:535e42. Zlotogora J, Shapiro MS. Polyglandular autoimmune syndrome type I among Iranian Jews. J Med Genet 1992;29:824e6. Aaltonen J, Bjorses P, Sandkuijl L, Perheentupa J, Peltonen L. An autosomal locus causing autoimmune disease: autoimmune polyglandular disease type I assigned to chromosome 21. Nat Genet 1994;8:83e7. Nagamine K, Peterson P, Scott HS, et al. Positional cloning of the APECED gene. Nat Genet 1997;17:393e8. Cihakova D, Trebusak K, Heino M, et al. Novel AIRE mutations and P450 cytochrome autoantibodies in Central and Eastern European patients with APECED. Hum Mutat 2001;18:225e32. Heino M, Peterson P, Kudoh J, et al. APECED mutations in the autoimmune regulator (AIRE) gene. Hum Mutat 2001;18: 205e11. Ishii T, Suzuki Y, Ando N, Matsuo N, Ogata T. Novel mutations of the autoimmune regulator gene in two siblings with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. J Clin Endocrinol Metab 2000;85:2922e6. Bjorses P, Halonen M, Palvimo JJ, et al. Mutations in the AIRE gene: effects on subcellular location and transactivation function of the autoimmune polyendocrinopathy-candidiasisectodermal dystrophy protein. Am J Hum Genet 2000;66: 378e92. Liston A, Lesage S, Wilson J, Peltonen L, Goodnow CC. Aire regulates negative selection of organ-specific T cells. Nat Immunol 2003;4:350e4. Meager A, Visvalingam K, Peterson P, et al. Anti-interferon autoantibodies in autoimmune polyendocrinopathy syndrome type 1. PLoS Med 2006;3:e289. Alimohammadi M, Bjorklund P, Hallgren A, et al. Autoimmune polyendocrine syndrome type 1 and NALP5, a parathyroid autoantigen. N Engl J Med 2008;358:1018e28. Eisenbarth SC, Colegio OR, O’Connor W, Sutterwala FS, Flavell RA. Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants. Nature 2008;453:1122e6. Moraes CT, DiMauro S, Zeviani M, et al. Mitochondrial DNA deletions in progressive external ophthalmoplegia and KearnsSayre syndrome. N Engl J Med 1989;320:1293e9. Zupanc ML, Moraes CT, Shanske S, Langman CB, Ciafaloni E, DiMauro S. Deletion of mitochondrial DNA in patients with

PEDIATRIC BONE

581

REFERENCES

[59]

[60]

[61]

[62]

[63] [64] [65]

[66]

[67]

[68]

[69]

[70] [71] [72]

[73]

[74]

[75]

[76]

[77]

[78]

[79]

combined features of Kearns-Sayre and MELAS syndromes. Ann Neurol 1991;29:680e3. Morten KJ, Cooper JM, Brown GK, Lake BD, Pike D, Poulton J. A new point mutation associated with mitochondrial encephalomyopathy. Hum Mol Genet 1993;2:2081e7. Scambler PJ, Carey AH, Wyse RK, et al. Microdeletions within 22q11 associated with sporadic and familial DiGeorge syndrome. Genomics 1991;10:201e6. Monaco G, Pignata C, Rossi E, Mascellaro O, Cocozza S, Ciccimarra F. DiGeorge anomaly associated with 10p deletion. Am J Med Genet 1991;39:215e6. Gong W, Emanuel BS, Collins J, et al. A transcription map of the DiGeorge and velo-cardio-facial syndrome minimal critical region on 22q11. Hum Mol Genet 1996;5:789e800. Scambler PJ. The 22q11 deletion syndromes. Hum Mol Genet 2000;9:2421e6. Augusseau S, Jouk S, Jalbert P, Prieur M. DiGeorge syndrome and 22q11 rearrangements. Hum Genet 1986;74:206. Budarf ML, Collins J, Gong W, et al. Cloning a balanced translocation associated with DiGeorge syndrome and identification of a disrupted candidate gene. Nat Genet 1995;10: 269e78. Yamagishi H, Garg V, Matsuoka R, Thomas T, Srivastava D. A molecular pathway revealing a genetic basis for human cardiac and craniofacial defects. Science 1999;283:1158e61. Lindsay EA, Botta A, Jurecic V, et al. Congenital heart disease in mice deficient for the DiGeorge syndrome region. Nature 1999;401:379e83. Magnaghi P, Roberts C, Lorain S, Lipinski M, Scambler PJ. HIRA, a mammalian homologue of Saccharomyces cerevisiae transcriptional co-repressors, interacts with Pax3. Nat Genet 1998;20:74e7. Jerome LA, Papaioannou VE. DiGeorge syndrome phenotype in mice mutant for the T-box gene, Tbx1. Nat Genet 2001;27: 286e91. Yagi H, Furutani Y, Hamada H, et al. Role of TBX1 in human del22q11.2 syndrome. Lancet 2003;362:1366e73. Baldini A. DiGeorge’s syndrome: a gene at last. Lancet 2003;362: 1342e3. Scire G, Dallapiccola B, Iannetti P, et al. Hypoparathyroidism as the major manifestation in two patients with 22q11 deletions. Am J Med Genet 1994;52:478e82. Kim J, Jones B, Zock C, et al. Isolation and characterization of mammalian homologs of the Drosophila gene glial cells missing. Proc Natl Acad Sci USA 1998;95:12364e9. Liu Z, Yu S, Manley NR. Gcm2 is required for the differentiation and survival of parathyroid precursor cells in the parathyroid/ thymus primordia. Dev Biol 2007;305:333e46. Gu¨nther T, Chen ZF, Kim J, et al. Genetic ablation of parathyroid glands reveals another source of parathyroid hormone. Nature 2000;406:199e203. Ding C, Buckingham B, Levine MA. Familial isolated hypoparathyroidism caused by a mutation in the gene for the transcription factor GCMB. J Clin Invest. 200;108:1215e20. Baumber L, Tufarelli C, Patel S, et al. Identification of a novel mutation disrupting the DNA binding activity of GCM2 in autosomal recessive familial isolated hypoparathyroidism. J Med Genet 2005;42:443e8. Mannstadt M, Bertrand G, Muresan M, et al. Dominant-negative GCMB mutations cause an autosomal dominant form of hypoparathyroidism. J Clin Endocrinol Metab 2008;93:3568e76. Canaff L, Zhou X, Mosesova I, Cole DE, Hendy GN. Glial Cells Missing-2 (GCM2) transactivates the calcium-sensing receptor gene: effect of a dominant-negative GCM2 mutant associated

[80]

[81]

[82]

[83] [84]

[85]

[86]

[87]

[88]

[89]

[90]

[91] [92]

[93]

[94]

[95]

[96]

[97]

with autosomal dominant hypoparathyroidism. Hum Mutat 2009;30:85e92. Bowl MR, Mirczuk SM, Grigorieva IV, et al. Identification and characterization of novel parathyroid-specific transcription factor Glial Cells Missing Homolog B (GCMB) mutations in eight families with autosomal recessive hypoparathyroidism. Hum Mol Genet 2010;19:2028e38. Maret A, Ding C, Kornfield SL, Levine MA. Analysis of the GCM2 gene in isolated hypoparathyroidism: a molecular and biochemical study. J Clin Endocrinol Metab 2008;93:1426e32. Bilous R, Murty G, Parkinson D, et al. Autosomal dominant familial hypoparathyroidism, sensineural deafness and renal dysplasia. N Engl J Med 1992;327:1069e84. Fryns J, Muelenaeve AD, Bergue HV. Distal 10p deletion syndrome. Ann Genet 1981;24:189e90. Van Esch H, Groenen P, Daw S, et al. Partial DiGeorge syndrome in two patients with a 10p rearrangement. Clin Genet 1999;55:269e76. Van Esch H, Groenen P, Nesbit MA, et al. GATA3 haploinsufficiency causes human HDR syndrome. Nature 2000;406: 419e22. Muroya K, Hasegawa T, Ito Y, et al. GATA3 abnormalities and the phenotypic spectrum of HDR syndrome. J Med Genet 2001;38:374e80. Nesbit MA, Bowl MR, Harding B, et al. Characterization of GATA3 mutations in the hypoparathyroidism, deafness, and renal dysplasia (HDR) syndrome. J Biol Chem 2004;279: 22624e34. Ali A, Christie PT, Grigorieva IV, et al. Functional characterization of GATA3 mutations causing the hypoparathyroidismdeafness-renal (HDR) dysplasia syndrome: insight into mechanisms of DNA binding by the GATA3 transcription factor. Hum Mol Genet 2007;16:265e75. Tsang AP, Visvader JE, Turner CA, et al. FOG, a multitype zinc finger protein, acts as a cofactor for transcription factor GATA-1 in erythroid and megakaryocytic differentiation. Cell 1997;90: 109e19. Zahirieh A, Nesbit MA, Ali A, et al. Functional analysis of a novel GATA3 mutation in a family with the hypoparathyroidism, deafness, and renal dysplasia syndrome. J Clin Endocrinol Metab 2005;90:2445e50. Dai YS, Markham BE. p300 Functions as a coactivator of transcription factor GATA-4. J Biol Chem 2001;276:37178e85. Pandolfi PP, Roth ME, Karis A, et al. Targeted disruption of the GATA3 gene causes severe abnormalities in the nervous system and in fetal liver haematopoiesis. Nat Genet 1995;11:40e4. Grigorieva IV, Mirczuk S, Gaynor KU, et al. Gata3-deficient mice develop parathyroid abnormalities due to dysregulation of the parathyroid-specific transcription factor Gcm2. J Clin Invest 2010;120:2144e55. van der Wees J, van Looij MA, de Ruiter MM, et al. Hearing loss following Gata3 haploinsufficiency is caused by cochlear disorder. Neurobiol Dis 2004;16:169e78. van Looij MA, van der Burg H, van der Giessen RS, et al. GATA3 haploinsufficiency causes a rapid deterioration of distortion product otoacoustic emissions (DPOAEs) in mice. Neurobiol Dis 2005;20:890e7. Lim KC, Lakshmanan G, Crawford SE, Gu Y, Grosveld F, Engel JD. Gata3 loss leads to embryonic lethality due to noradrenaline deficiency of the sympathetic nervous system. Nat Genet 2000;25:209e12. Grote D, Souabni A, Busslinger M, Bouchard M. Pax 2/8regulated Gata 3 expression is necessary for morphogenesis and guidance of the nephric duct in the developing kidney. Development 2006;133:53e61.

PEDIATRIC BONE

582

21. PARATHYROID DISORDERS

[98] Franceschini P, Testa A, Bogetti G, et al. Kenny-Caffey syndrome in two sibs born to consanguineous parents: evidence for an autosomal recessive variant. Am J Med Genet 1992;42: 112e6. [99] Boynton JR, Pheasant TR, Johnson BL, Levin DB, Streeten BW. Ocular findings in Kenny’s syndrome. Arch Ophthalmol 1979;97:896e900. [100] Richardson RJ, Kirk JM. Short stature, mental retardation, and hypoparathyroidism: a new syndrome. Arch Dis Child 1990;65: 1113e7. [101] Parvari R, Hershkovitz E, Kanis A, et al. Homozygosity and linkage-disequilibrium mapping of the syndrome of congenital hypoparathyroidism, growth and mental retardation, and dysmorphism to a 1-cM interval on chromosome 1q42-43. Am J Hum Genet 1998;63:163e9. [102] Sanjad SA, Sakati NA, Abu-Osba YK, Kaddoura R, Milner RD. A new syndrome of congenital hypoparathyroidism, severe growth failure, and dysmorphic features. Arch Dis Child 1991;66:193e6. [103] Parvari R, Hershkovitz E, Grossman N, et al. Mutation of TBCE causes hypoparathyroidism-retardation-dysmorphism and autosomal recessive Kenny-Caffey syndrome. Nat Genet 2002;32:448e52. [104] Barakat AY, D’Albora JB, Martin MM, Jose PA. Familial nephrosis, nerve deafness, and hypoparathyroidism. J Pediatr 1977;91:61e4. [105] Dahlberg PJ, Borer WZ, Newcomer KL, Yutuc WR. Autosomal or X-linked recessive syndrome of congenital lymphedema, hypoparathyroidism, nephropathy, prolapsing mitral valve, and brachytelephalangy. Am J Med Genet 1983;16:99e104. [106] Pearce SH, Williamson C, Kifor O, et al. A familial syndrome of hypocalcemia with hypercalciuria due to mutations in the calcium-sensing receptor [see comments]. N Engl J Med 1996;335:1115e22. [107] Baron J, Winer K, Yanovski J, et al. Mutations in the Ca2þ sensing receptor cause autosomal dominant and sporadic hypoparathyroidism. Hum Mol Genet 1996;5:601e6. [108] Hebert SC. Bartter syndrome. Curr Opin Nephrol Hypertens 2003;12:527e32. [109] Watanabe S, Fukumoto S, Chang H, et al. Association between activating mutations of calcium-sensing receptor and Bartter’s syndrome. Lancet 2002;360:692e4. [110] Vargas-Poussou R, Huang C, Hulin P, et al. Functional characterization of a calcium-sensing receptor mutation in severe autosomal dominant hypocalcemia with a Bartter-like syndrome. J Am Soc Nephrol 2002;13:2259e66. [111] Kifor O, Moore Jr FD, Delaney M, et al. A syndrome of hypocalciuric hypercalcemia caused by autoantibodies directed at the calcium-sensing receptor. J Clin Endocrinol Metab 2003;88: 60e72. [112] Li Y, Song YH, Rais N, et al. Autoantibodies to the extracellular domain of the calcium sensing receptor in patients with acquired hypoparathyroidism. J Clin Invest 1996;97:910e4. [113] Albright F, Burnett CH, Smith PH, Parson W. Pseudohypoparathyroidism e an example of "Seabright-Bantam syndrome". Endocrinology 1942;30:922e32. [114] van Dop C. Pseudohypoparathyroidism: clinical and molecular aspects. Semin Nephrol 1989;9:168e78. [115] Levine M. Hypoparathyroidism and pseudohypoparathyroidism. In: DeGroot LJ, Jameson J, editors. Endocrinology. 4th ed. Philadelphia: W.B. Saunders; 2000. p. 1133e53. [116] Weinstein L, Yu S, Warner D, Liu J. Endocrine manifestations of stimulatory G protein alpha-subunit mutations and the role of genomic imprinting. Endocr Rev 2001;22:675e705.

[117] Levine MA, Downs Jr RW, Moses AM, et al. Resistance to multiple hormones in patients with pseudohypoparathyroidism. Association with deficient activity of guanine nucleotide regulatory protein. Am J Med 1983;74:545e56. [118] Mallet E, Carayon P, Amr S, et al. Coupling defect of thyrotropin receptor and adenylate cyclase in a pseudohypoparathyroid patient. J Clin Endocrinol Metab 1982;54:1028e32. [119] Wolfsdorf JI, Rosenfield RL, Fang VS, Kobayashi R, Razdan AK, Kim MH. Partial gonadotrophin-resistance in pseudohypoparathyroidism. Acta Endocrinol (Copenh) 1978;88:321e8. [120] Mantovani G, Maghnie M, Weber G, et al. Growth hormonereleasing hormone resistance in pseudohypoparathyroidism type ia: new evidence for imprinting of the Gs alpha gene. J Clin Endocrinol Metab 2003;88:4070e4. [121] Germain-Lee EL, Groman J, Crane JL, Jan de Beur SM, Levine MA. Growth hormone deficiency in pseudohypoparathyroidism type 1a: another manifestation of multihormone resistance. J Clin Endocrinol Metab 2003;88:4059e69. [122] Vlaeminck-Guillem V, D’Herbomez M, Pigny P, et al. Pseudohypoparathyroidism Ia and hypercalcitoninemia. J Clin Endocrinol Metab 2001;86:3091e6. [123] Poznanski AK, Werder EA, Giedion A, Martin A, Shaw H. The pattern of shortening of the bones of the hand in PHP and PPHP e A comparison with brachydactyly E, Turner syndrome, and acrodysostosis. Radiology 1977;123:707e18. [124] Long DN, McGuire S, Levine MA, Weinstein LS, GermainLee EL. Body mass index differences in pseudohypoparathyroidism type 1a versus pseudopseudohypoparathyroidism may implicate paternal imprinting of Galpha(s) in the development of human obesity. J Clin Endocrinol Metab 2007;92:1073e9. [125] Mouallem M, Shaharabany M, Weintrob N, et al. Cognitive impairment is prevalent in pseudohypoparathyroidism type Ia, but not in pseudopseudohypoparathyroidism: possible cerebral imprinting of Gsalpha. Clin Endocrinol (Oxf) 2008;68:233e9. [126] Schuster V, Eschenhagen T, Kruse K, Gierschik P, Kreth HW. Endocrine and molecular biological studies in a German family with Albright hereditary osteodystrophy. Eur J Pediatr 1993;152:185e9. [127] Miric A, Vechio JD, Levine MA. Heterogeneous mutations in the gene encoding the a-subunit of the stimulatory G protein of adenylyl cyclase in Albright hereditary osteodystrophy. J Clin Endocrinol Metab 1993;76:1560e8. [128] Weinstein LS, Gejman PV, Friedman E, et al. Mutations of the Gs asubunit gene in Albright hereditary osteodystrophy detected by denaturing gradient gel electrophoresis. Proc Natl Acad Sci USA 1990;87:8287e90. [129] Ahrens W, Hiort O, Staedt P, Kirschner T, Marschke C, Kruse K. Analysis of the GNAS1 gene in Albright’s hereditary osteodystrophy. J Clin Endocrinol Metab 2001;86:4630e4. [130] Davies AJ, Hughes HE. Imprinting in Albright’s hereditary osteodystrophy. J Med Genet 1993;30:101e3. [131] Wilson LC, Oude-Luttikhuis MEM, Clayton PT, Fraser WD, Trembath RC. Parental origin of Gsa gene mutations in Albright’s hereditary osteodystrophy. J Med Genet 1994;31: 835e9. [132] Yu S, Yu D, Lee E, Eckhaus M, et al. Variable and tissue-specific hormone resistance in heterotrimeric Gs protein alpha-subunit (Gsa) knockout mice is due to tissue-specific imprinting of the Gsa gene. Proc Natl Acad Sci USA 1998;95:8715e20. [133] Germain-Lee EL, Schwindinger W, Crane JL, et al. A mouse model of Albright hereditary osteodystrophy generated by targeted disruption of exon 1 of the Gnas gene. Endocrinology 2005;146:4697e709.

PEDIATRIC BONE

REFERENCES

[134] Iiri T, Herzmark P, Nakamoto JM, van Dop C, Bourne HR. Rapid GDP release from Gs in patients with gain and loss of function. Nature 1994;371:164e8. [135] Makita N, Sato J, Rondard P, et al. Human G(salpha) mutant causes pseudohypoparathyroidism type Ia/neonatal diarrhea, a potential cell-specific role of the palmitoylation cycle. Proc Natl Acad Sci USA 2007;104:17424e9. [136] Bray P, Carter A, Simons C, et al. Human cDNA clones for four species of G alpha s signal transduction protein. Proc Natl Acad Sci USA 1986;83:8893e7. [137] Kozasa T, Itoh H, Tsukamoto T, Kaziro Y. Isolation and characterization of the human Gsa gene. Proc Natl Acad Sci USA 1988;85:2081e5. [138] Robishaw JD, Smigel MD, Gilman AG. Molecular basis for two forms of the G protein that stimulates Adenylate cyclase. J Biol Chem 1986;261:9587e90. [139] Thiele S, Werner R, Ahrens W, et al. A disruptive mutation in exon 3 of the GNAS gene with Albright hereditary osteodystrophy, normocalcemic pseudohypoparathyroidism, and selective long transcript variant Gsalpha-L deficiency. J Clin Endocrinol Metab 2007;92:1764e8. [140] Sternweis PC, Northup JK, Smigel MD, Gilman AG. The regulatory component of adenylate cyclase. Purification and properties. J Biol Chem 1981;256:11517e26. [141] Walseth TF, Zhang HJ, Olson LK, Schroeder WA, Robertson RP. Increase in Gs and cyclic AMP generation in HIT cells. Evidence that the 45-kDa alpha-subunit of Gs has greater functional activity than the 52-kDa alpha-subunit. J Biol Chem 1989;264: 21106e11. [142] Graziano MP, Freissmuth M, Gilman AG. Expression of Gs alpha in Escherichia coli. Purification and properties of two forms of the protein. J Biol Chem 1989;264:409e18. [143] Seifert R, Wenzel-Seifert K, Lee TW, Gether U, Sanders-Bush E, Kobilka BK. Different effects of Gsalpha splice variants on beta2-adrenoreceptor-mediated signaling. The Beta2-adrenoreceptor coupled to the long splice variant of Gsalpha has properties of a constitutively active receptor. J Biol Chem 1998;273:5109e16. [144] Kvapil P, Novotny J, Svoboda P, Ransnas LA. The short and long forms of the alpha subunit of the stimulatory guaninenucleotide-binding protein are unequally redistributed during ()-isoproterenol-mediated desensitization of intact S49 lymphoma cells. Eur J Biochem 1994;226:193e9. [145] el Jamali A, Rachdaoui N, Jacquemin C, Correze C. Long-term effect of forskolin on the activation of adenylyl cyclase in astrocytes. J Neurochem 1996;67:2532e9. [146] Bourgeois C, Duc-Goiran P, Robert B, Mondon F, Ferre F. G protein expression in human fetoplacental vascularization. Functional evidence for Gs alpha and Gi alpha subunits. J Mol Cell Cardiol 1996;28:1009e12. 1. [147] Yeh GL, Mathur S, Wivel A, et al. GNAS1 mutation and Cbfa1 misexpression in a child with severe congenital platelike osteoma cutis. J Bone Miner Res 2000;15:2063e73. [148] Lebrun M, Richard N, Abeguile G, et al. Progressive osseous heteroplasia: a model for the imprinting effects of GNAS inactivating mutations in humans. J Clin Endocrinol Metab 2010. [149] Eddy MC, Jan de Beur SM, Yandow SM, et al. Deficiency of the alpha-subunit of the stimulatory G protein and severe extraskeletal ossification. J Bone Miner Res 2000;15:2074e83. [150] Shore E, Ahn J, Jan de Beur S, et al. Paternally-inherited inactivating mutations of the GNAS1 gene in progressive osseous heteroplasia. New Engl J Med 2002;346:99e106. [151] Schimmel RJ, Pasmans SG, Xu M, et al. GNAS-associated disorders of cutaneous ossification: two different clinical presentations. Bone 2010;46:868e72.

583

[152] Goto M, Mabe H, Nishimura G, Katsumata N. Progressive osseous heteroplasia caused by a novel nonsense mutation in the GNAS1 gene. J Pediatr Endocrinol Metab. 23:303e9. [153] Kumagai K, Motomura K, Egashira M, et al. A case of progressive osseous heteroplasia: a first case in Japan. Skeletal Radiol 2008;37:563e7. [154] Adegbite NS, Xu M, Kaplan FS, Shore EM, Pignolo RJ. Diagnostic and mutational spectrum of progressive osseous heteroplasia (POH) and other forms of GNAS-based heterotopic ossification. Am J Med Genet A 2008;146A:1788e96. [155] Gelfand IM, Hub RS, Shore EM, Kaplan FS, Dimeglio LA. Progressive osseous heteroplasia-like heterotopic ossification in a male infant with pseudohypoparathyroidism type Ia: a case report. Bon 2007;40:1425e8. [156] Chan I, Hamada T, Hardman C, McGrath JA, Child FJ. Progressive osseous heteroplasia resulting from a new mutation in the GNAS1 gene. Clin Exp Dermatol 2004;29:77e80. [157] Faust RA, Shore EM, Stevens CE, et al. Progressive osseous heteroplasia in the face of a child. Am J Med Genet A 2003;118A:71e5. [158] Ahmed SF, Barr DG, Bonthron DT. GNAS1 mutations and progressive osseous heteroplasia. N Engl J Med 2002;346: 1669e71. [159] Hayward B, Kamiya M, Strain L, et al. The human GNAS1 gene is imprinted and encodes distinct paternally and biallelically expressed G proteins. Proc Natl Acad Sci USA 1998;95: 10038e43. [160] Hayward BE, Moran V, Strain L, Bonthron DT. Bidirectional imprinting of a single gene: GNAS1 encodes maternally, paternally, and biallelically derived proteins. Proc Natl Acad Sci USA 1998;95:15475e80. [161] Campbell R, Gosden CM, Bonthron DT. Parental origin of transcription from the human GNAS1 gene. J Med Genet 1994;31:607e14. [162] Peters J, Wroe SF, Wells CA, et al. A cluster of oppositely imprinted transcripts at the Gnas locus in the distal imprinting region of mouse chromosome 2. Proc Natl Acad Sci USA 1999;96:3830e5. [163] Liu J, Erlichman B, Weinstein L. The stimulatory G protein alpha-subunit Gs alpha is imprinted in human thyroid glands: implications for thyroid function in pseudohypoparathyroidism types 1A and 1B. J Clin Endocrinol Metab 2003;88:4336e41. [164] Germain-Lee EL, Ding CL, Deng Z, et al. Paternal imprinting of Galpha(s) in the human thyroid as the basis of TSH resistance in pseudohypoparathyroidism type 1a. Biochem Biophys Res Commun 2002;296:67e72. [165] Mantovani G, Ballare E, Giammona E, Beck-Peccoz P, Spada A. The gsalpha gene: predominant maternal origin of transcription in human thyroid gland and gonads. J Clin Endocrinol Metab 2002;87:4736e40. [166] Hayward B, Barlier A, Korbonits M, et al. Imprinting of the G(s) alpha gene GNAS1 in the pathogenesis of acromegaly. J Clin Invest 2001;107:R31e6. [167] Zheng H, Radeva G, McCann JA, Hendy GN, Goodyer CG. Gas transcripts are biallelically expressed in the human kidney cortex: implications for pseudohypoparathyroidism type Ib. J Clin Endocrinol Metab 2001;86:4627e9. [168] Kehlenbach RH, Matthey J, Huttner WB. XLas is a new type of G protein. Nature 1994;372:804e9. [169] Klemke M, Pasolli H, Kehlenbach R, Offermanns S, Schultz G, Huttner W. Characterization of the extra-large G protein alphasubunit XLalphas. II. Signal transduction properties. J Biol Chem 2000;275:33633e40. [170] Bastepe M, Gunes Y, Perez-Villamil B, Hunzelman JL, Weinstein LS, Ju¨ppner H. Receptor-mediated adenylyl cyclase

PEDIATRIC BONE

584

[171]

[172]

[173]

[174]

[175]

[176]

[177]

[178]

[179]

[180]

[181]

[182]

[183]

[184]

[185] [186]

[187]

21. PARATHYROID DISORDERS

activation through XLas, the extra-large variant of the stimulatory G protein alpha subunit. Mol Endocrinol 2002;16:1912e9. Pasolli H, Klemke M, Kehlenbach R, Wang Y, Huttner W. Characterization of the extra-large G protein alpha-subunit XLalphas. I. Tissue distribution and subcellular localization. J Biol Chem 2000;275:33622e32. Klemke M, Kehlenbach RH, Huttner WB. Two overlapping reading frames in a single exon encode interacting proteins e a novel way of gene usage. Embo J 2001;20:3849e60. Crawford JA, Mutchler KJ, Sullivan BE, Lanigan TM, Clark MS, Russo AF. Neural expression of a novel alternatively spliced and polyadenylated Gs alpha transcript. J Biol Chem 1993;268: 9879e85. Abramowitz J, Grenet D, Birnbaumer M, Torres HN, Birnbaumer L. XLalphas, the extra-long form of the alphasubunit of the Gs G protein, is significantly longer than suspected, and so is its companion Alex. Proc Natl Acad Sci USA 2004;101:8366e71. Aydin C, Aytan N, Mahon MJ, et al. Extralarge XL(alpha)s (XXL(alpha)s), a variant of stimulatory G protein alpha-subunit (Gs(alpha)), is a distinct, membrane-anchored GNAS product that can mimic Gs(alpha). Endocrinology 2009;150:3567e75. Plagge A, Gordon E, Dean W, et al. The imprinted signaling protein XL alpha s is required for postnatal adaptation to feeding. Nat Genet 2004;36:818e26. Aldred M, Aftimos S, Hall C, et al. Constitutional deletion of chromosome 20q in two patients affected with albright hereditary osteodystrophy. Am J Med Genet Suppl 2002;113:167e72. Genevieve D, Sanlaville D, Faivre L, et al. Paternal deletion of the GNAS imprinted locus (including Gnasxl) in two girls presenting with severe pre- and post-natal growth retardation and intractable feeding difficulties. Eur J Hum Genet 2005;13: 1033e9. Ischia R, Lovisetti-Scamihorn P, Hogue-Angeletti R, Wolkersdorfer M, Winkler H, Fischer-Colbrie R. Molecular cloning and characterization of NESP55, a novel chromograninlike precursor of a peptide with 5-HT1B receptor antagonist activity. J Biol Chem 1997;272:11657e62. Plagge A, Isles A, Gordon E, et al. Imprinted Nesp55 influences behavioral reactivity to novel environments. Mol Cell Biol 2005;25:3019e26. Chotalia M, Smallwood SA, Ruf N, et al. Transcription is required for establishment of germline methylation marks at imprinted genes. Genes Dev 2009;23:105e17. Swaroop A, Agarwal N, Gruen JR, Bick D, Weissman SM. Differential expression of novel Gs alpha signal transduction protein cDNA species. Nucleic Acids Res 1991;19:4725e9. Liu J, Yu S, Litman D, Chen W, Weinstein L. Identification of a methylation imprint mark within the mouse gnas locus. Mol Cell Biol 2000;20:5808e17. Ishikawa Y, Bianchi C, Nadal-Ginard B, Homcy CJ. Alternative promoter and 5’ exon generate a novel Gsa mRNA. J Biol Chem 1990;265:8458e62. Hayward B, Bonthron D. An imprinted antisense transcript at the human GNAS1 locus. Hum Mol Genet 2000;9:835e41. Wroe SF, Kelsey G, Skinner JA, et al. An imprinted transcript, antisense to Nesp, adds complexity to the cluster of imprinted genes at the mouse Gnas locus. Proc Natl Acad Sci USA 2000;97: 3342e6. Coombes C, Arnaud P, Gordon E, et al. Epigenetic properties and identification of an imprint mark in the Nesp-Gnasxl domain of the mouse Gnas imprinted locus. Mol Cell Biol 2003;23:5475e88.

[188] Williamson C, Turner M, Ball S, et al. Identification of an imprinting control region affecting the expression of all transcripts in the Gnas cluster. Nat Genet 2006;38:350e5. [189] Williamson CM, Ball ST, Nottingham WT, et al. A cis-acting control region is required exclusively for the tissue-specific imprinting of Gnas. Nat Genet 2004;36:894e9. [190] Liu J, Chen M, Deng C, et al. Identification of the control region for tissue-specific imprinting of the stimulatory G protein alpha-subunit. Proc Natl Acad Sci USA 2005;102:5513e8. [191] Murray T, Gomez Rao E, Wong MM, et al. Pseudohypoparathyroidism with osteitis fibrosa cystica: direct demonstration of skeletal responsiveness to parathyroid hormone in cells cultured from bone. J Bone Miner Res 1993;8:83e91. [192] Farfel Z. Pseudohypohyperparathyroidism-pseudohypoparathyroidism type Ib. J Bone Miner Res 1999;14:1016. [193] Schipani E, Weinstein LS, Bergwitz C, et al. Pseudohypoparathyroidism type Ib is not caused by mutations in the coding exons of the human parathyroid hormone (PTH)/PTH-related peptide receptor gene. J Clin Endocrinol Metab 1995;80: 1611e21. [194] Suarez F, Lebrun JJ, Lecossier D, Escoubet B, Coureau C, Silve C. Expression and modulation of the parathyroid hormone (PTH)/PTH-related peptide receptor messenger ribonucleic acid in skin fibroblasts from patients with type Ib pseudohypoparathyroidism. J Clin Endocrinol Metab 1995;80: 965e70. [195] Fukumoto S, Suzawa M, Takeuchi Y, et al. Absence of mutations in parathyroid hormone (PTH)/PTH-related protein receptor complementary deoxyribonucleic acid in patients with pseudohypoparathyroidism type Ib. J Clin Endocrinol Metab 1996;81:2554e8. [196] Bettoun JD, Minagawa M, Kwan MY, et al. Cloning and characterization of the promoter regions of the human parathyroid hormone (PTH)/PTH-related peptide receptor gene: analysis of deoxyribonucleic acid from normal subjects and patients with pseudohypoparathyroidism type Ib. J Clin Endocrinol Metab 1997;82:1031e40. [197] Minagawa M, Watanabe T, Kohno Y, et al. Analysis of the P3 promoter of the human parathyroid hormone (PTH)/PTHrelated peptide receptor gene in pseudohypoparathyroidism type 1b. J Clin Endocrinol Metab 2001;86:1394e7. [198] Wu WI, Schwindinger WF, Aparicio LF, Levine MA. Selective resistance to parathyroid hormone caused by a novel uncoupling mutation in the carboxyl terminus of Gas: A cause of pseudohypoparathyroidism type Ib. J Biol Chem 2001;276: 165e71. [199] Linglart A, Mahon MJ, Kerachian MA, et al. Coding GNAS mutations leading to hormone resistance impair in vitro agonist- and cholera toxin-induced adenosine cyclic 3’,5’monophosphate formation mediated by human XLalphas. Endocrinology 2006;147:2253e62. [200] Ju¨ppner H, Schipani E, Bastepe M, et al. The gene responsible for pseudohypoparathyroidism type Ib is paternally imprinted and maps in four unrelated kindreds to chromosome 20q13.3. Proc Natl Acad Sci USA 1998;95:11798e803. [201] Liu J, Litman D, Rosenberg M, Yu S, Biesecker L, Weinstein L. A GNAS1 imprinting defect in pseudohypoparathyroidism type IB. J Clin Invest 2000;106:1167e74. [202] Bastepe M, Pincus JE, Sugimoto T, et al. Positional dissociation between the genetic mutation responsible for pseudohypoparathyroidism type Ib and the associated methylation defect at exon A/B: evidence for a long-range regulatory element within the imprinted GNAS1 locus. Hum Mol Genet 2001;10:1231e41. [203] Bastepe M, Fro¨hlich LF, Hendy GN, et al. Autosomal dominant pseudohypoparathyroidism type Ib is associated with

PEDIATRIC BONE

REFERENCES

[204]

[205]

[206]

[207]

[208]

[209]

[210]

[211]

[212]

[213]

[214]

[215]

[216]

[217]

[218]

a heterozygous microdeletion that likely disrupts a putative imprinting control element of GNAS. J Clin Invest 2003;112: 1255e63. Linglart A, Gensure RC, Olney RC, Ju¨ppner H, Bastepe M. A novel STX16 deletion in autosomal dominant pseudohypoparathyroidism type Ib redefines the boundaries of a cis-acting imprinting control element of GNAS. Am J Hum Genet 2005;76: 804e14. Linglart A, Bastepe M, Ju¨ppner H. Similar clinical and laboratory findings in patients with symptomatic autosomal dominant and sporadic pseudohypoparathyroidism type Ib despite different epigenetic changes at the GNAS locus. Clin Endocrinol (Oxf) 2007;67:822e31. Bastepe M, Lane AH, Ju¨ppner H. Paternal uniparental isodisomy of chromosome 20q (patUPD20q) e and the resulting changes in GNAS1 methylation e as a plausible cause of pseudohypoparathyroidism. Am J Hum Genet 2001;68:1283e9. Bastepe M, Altug-Teber O, Agarwal C, Oberfield SE, Bonin M, Ju¨ppner H. Paternal uniparental isodisomy of the entire chromosome 20 as a molecular cause of pseudohypoparathyroidism type Ib (PHP-Ib). Bone 2011. Fernandez-Rebollo E, Lecumberri B, Garin I, et al. New mechanisms involved in paternal 20q disomy associated with pseudohypoparathyroidism. Eur J Endocrinol 2011;163:953e62. Bastepe M, Fro¨hlich LF, Linglart A, et al. Deletion of the NESP55 differentially methylated region causes loss of maternal GNAS imprints and pseudohypoparathyroidism type Ib. Nat Genet 2005;37:25e7. Chillambhi S, Turan S, Hwang DY, Chen HC, Ju¨ppner H, Bastepe M. Deletion of the noncoding GNAS antisense transcript causes pseudohypoparathyroidism type Ib and biparental defects of GNAS methylation in cis. J Clin Endocrinol Metab 2010. Mantovani G, Bondioni S, Linglart A, et al. Genetic analysis and evaluation of resistance to thyrotropin and growth hormonereleasing hormone in pseudohypoparathyroidism type Ib. J Clin Endocrinol Metab 2007;92:3738e42. de Nanclares GP, Fernandez-Rebollo E, Santin I, et al. Epigenetic defects of GNAS in patients with pseudohypoparathyroidism and mild features of Albright’s hereditary osteodystrophy. J Clin Endocrinol Metab 2007;92:2370e3. Mariot V, Maupetit-Mehouas S, Sinding C, Kottler ML, Linglart A. A maternal epimutation of GNAS leads to Albright osteodystrophy and parathyroid hormone resistance. J Clin Endocrinol Metab 2008;93:661e5. Unluturk U, Harmanci A, Babaoglu M, et al. Molecular diagnosis and clinical characterization of pseudohypoparathyroidism type-Ib in a patient with mild Albright’s hereditary osteodystrophy-like features, epileptic seizures, and defective renal handling of uric acid. Am J Med Sci 2008;336:84e90. Mantovani G, de Sanctis L, Barbieri AM, et al. Pseudohypoparathyroidism and GNAS epigenetic defects: clinical evaluation of albright hereditary osteodystrophy and molecular analysis in 40 patients. J Clin Endocrinol Metab 2010;95:651e8. Mantovani G, Ferrante E, Giavoli C, et al. Recombinant human GH replacement therapy in children with pseudohypoparathyroidism type Ia: first study on the effect on growth. J Clin Endocrinol Metab 2010;95:5011e7. Blomstrand S, Clae¨sson I, Sa¨ve-So¨derbergh J. A case of lethal congenital dwarfism with accelerated skeletal maturation. Pediatr Radiol 1985;15:141e3. Young ID, Zuccollo JM, Broderick NJ. A lethal skeletal dysplasia with generalised sclerosis and advanced skeletal maturation: Blomstrand chondrodysplasia. J Med Genet 1993;30:155e7.

585

[219] Leroy JG, Keersmaeckers G, Coppens M, Dumon JE, Roels H. Blomstrand lethal chondrodysplasia. Am J Med Genet 1996;63: 84e9. [220] Loshkajian A, Roume J, Stanescu V, Delezoide AL, Stampf F, Maroteaux P. Familial Blomstrand chondrodysplasia with advanced skeletal maturation: further delineation. Am J Med Genet 1997;71:283e8. [221] den Hollander NS, van der Harten HJ, Vermeij-Keers C, Niermeijer MF, Wladimiroff JW. First-trimester diagnosis of Blomstrand lethal osteochondrodysplasia. Am J Med Genet 1997;73:345e50. [222] Oostra RJ, Baljet B, Dijkstra PF, Hennekam RCM. Congenital anomalies in the teratological collection of museum Vrolik in Amsterdam, The Netherlands. II: Skeletal dysplasia. Am J Med Genet 1998;77:116e34. [223] Zhang P, Jobert AS, Couvineau A, Silve C. A homozygous inactivating mutation in the parathyroid hormone/parathyroid hormone-related peptide receptor causing Blomstrand chondrodysplasia. J Clin Endocrinol Metab 1998;83:3365e8. [224] Karaplis AC, Bin He MT, et al. Inactivating mutation in the human parathyroid hormone receptor type 1 gene in Blomstrand chondrodysplasia. Endocrinology 1998;139:5255e8. [225] Hoogendam J, Farih-Sips H, Wynaendts LC, Lowik CW, Wit JM, Karperien M. Novel mutations in the parathyroid hormone (PTH)/PTH-related peptide receptor type 1 causing Blomstrand osteochondrodysplasia types I and II. J Clin Endocrinol Metab 2007;92:1088e95. [226] Karperien MC, van der Harten HJ, van Schooten R, et al. A frame-shift mutation in the type I parathyroid hormone/ parathyroid hormone-related peptide receptor causing Blomstrand lethal osteochondrodysplasia. J Clin Endocrinol Metab 1999;84:3713e20. [227] Karperien M, Sips H, Harten Hvd, et al. Novel mutations in the type I PTH/PTHrP receptor causing Blomstrand lethal osteochondrodysplasia (abstract). J Bone Mineral Res 2001; 16(Suppl. 1):S549. [228] Jobert AS, Zhang P, Couvineau A, et al. Absence of functional receptors parathyroid hormone and parathyroid hormonerelated peptide in Blomstrand chondrodysplasia. J Clin Invest 1998;102:34e40. [229] Wysolmerski JJ, Cormier S, Philbrick W, et al. Absence of functional type 1 PTH/PTHrP receptors in humans is associated with abnormal breast development and tooth impactation. J Clin Endocrinol Metab 2001;86:1788e94. [230] Oostra R, van der Harten J, Rijnders W, Scott R, Young M, Trump D. Blomstrand osteochondrodysplasia: three novel cases and histological evidence for heterogeneity. Virchows Arch 2000;436:28e35. [231] Decker E, Stellzig-Eisenhauer A, Fiebig BS, et al. PTHR1 loss-offunction mutations in familial, nonsyndromic primary failure of tooth eruption. Am J Hum Genet 2008;83:781e6. [232] Thakker RV. The molecular genetics of the multiple endocrine neoplasia syndromes. Clin Endocrinol (Oxf) 1993;38:1e14. [233] Szabo J, Heath B, Hill VM, et al. Hereditary hyperparathyroidism-jaw tumor syndrome: the endocrine tumor gene HRPT2 maps to chromosome 1q21-q31. Am J Hum Genet 1995;56:944e50. [234] Arnold A, Brown MF, Urena P, Gaz RD, Sarfati E, Drueke TB. Monoclonality of parathyroid tumors in chronic renal failure and in primary parathyroid hyperplasia. J Clin Invest 1995;95: 2047e53. [235] Arnold A, Kim HG, Gaz RD, et al. Molecular cloning and chromosomal mapping of DNA rearranged with the parathyroid hormone gene in a parathyroid adenoma. J Clin Invest 1989;83:2034e40.

PEDIATRIC BONE

586

21. PARATHYROID DISORDERS

[236] Motokura T, Bloom T, Kim HG, et al. A BCL1-linked candidate oncogene which is rearranged in parathyroid tumors encodes a novel cyclin. Nature 1991;350:512e5. [237] Nurse P. Universal control mechanism regulating onset of M-phase. Nature 1990;344:503e8. [238] Hsi ED, Zukerberg LR, Yang WI, Arnold A. Cyclin D1/PRAD1 expression in parathyroid adenomas: an immunohistochemical study. J Clin Endocrinol Metab 1996;81:1736e9. [239] Wang TC, Cardiff RD, Zukerberg L, Lees E, Arnold A, Schmidt EV. Mammary hyperplasia and carcinoma in MMTVcyclin D1 transgenic mice. Nature 1994;369:669e71. [240] Imanishi Y, Hosokawa Y, Yoshimoto K, et al. Primary hyperparathyroidism caused by parathyroid-targeted overexpression of cyclin D1 in transgenic mice. J Clin Invest 2001;107:1093e102. [241] Au AY, McDonald K, Gill A, et al. PTH mutation with primary hyperparathyroidism and undetectable intact PTH. N Engl J Med 2008;359:1184e6. [242] Trump D, Farren B, Wooding C, et al. Clinical studies of multiple endocrine neoplasia type 1 (MEN1). Q J Med 1996;89: 653e69. [243] Marx S. Multiple Endocrine Neoplasia type 1. In: Vogelstein B, Kinzler K, editors. The Genetic Basis of Human Cancer. New York: McGraw Hill; 1998. p. 489e506. [244] Thakker RV. Multiple endocrine neoplasiaesyndromes of the twentieth century. J Clin Endocrinol Metab 1998;83:2617e20. [245] Chandrasekharappa SC, Guru SC, Manickam P, et al. Positional cloning of the gene for multiple endocrine neoplasia-type 1. Science 1997;276:404e7. [246] Lemmens I, Van de Ven WJ, Kas K, et al. Identification of the multiple endocrine neoplasia type 1 (MEN1) gene. The European Consortium on MEN1. Hum Mol Genet 1997;6:1177e83. [247] Bassett JH, Forbes SA, Pannett AA, et al. Characterization of mutations in patients with multiple endocrine neoplasia type 1. Am J Hum Genet 1998;62:232e44. [248] Teh BT, Kytola S, Farnebo F, et al. Mutation analysis of the MEN1 gene in multiple endocrine neoplasia type 1, familial acromegaly and familial isolated hyperparathyroidism. J Clin Endocrinol Metab 1998;83:2621e6. [249] Heppner C, Kester MB, Agarwal SK, et al. Somatic mutation of the MEN1 gene in parathyroid tumours. Nat Genet 1997;16: 375e8. [250] Zhuang Z, Vortmeyer AO, Pack S, et al. Somatic mutations of the MEN1 tumor suppressor gene in sporadic gastrinomas and insulinomas. Cancer Res 1997;57:4682e6. [251] Zhuang Z, Ezzat SZ, Vortmeyer AO, et al. Mutations of the MEN1 tumor suppressor gene in pituitary tumors. Cancer Res 1997;57:5446e51. [252] Prezant TR, Levine J, Melmed S. Molecular characterization of the men1 tumor suppressor gene in sporadic pituitary tumors. J Clin Endocrinol Metab 1998;83:1388e91. [253] Debelenko LV, Brambilla E, Agarwal SK et al. Identification of MEN1 gene mutations in sporadic carcinoid tumors of the lung. Hum Mol Genet 6:2285e90. [254] Vortmeyer AO, Boni R, Pak E, Pack S, Zhuang Z. 1998 Multiple endocrine neoplasia 1 gene alterations in MEN1-associated and sporadic lipomas. J Natl Cancer Inst 1997;90:398e9. [255] Farnebo F, Teh BT, Kytola S, et al. Alterations of the MEN1 gene in sporadic parathyroid tumors. J Clin Endocrinol Metab 1998;83:2627e30. [256] Carling T, Correa P, Hessman O, et al. Parathyroid MEN1 gene mutations in relation to clinical characteristics of nonfamilial primary hyperparathyroidism. J Clin Endocrinol Metab 1998;83: 2960e3.

[257] Lemos MC, Thakker RV. Multiple endocrine neoplasia type 1 (MEN1): analysis of 1336 mutations reported in the first decade following identification of the gene. Hum Mutat 2008;29:22e32. [258] Teh BT, Esapa CT, Houlston R, et al. A family with isolated hyperparathyroidism segregating a missense MEN1 mutation and showing loss of the wild-type alleles in the parathyroid tumors. Am J Hum Genet 1998;63:1544e9. [259] Pannett AA, Kennedy AM, Turner JJ, et al. Multiple endocrine neoplasia type 1 (MEN1) germline mutations in familial isolated primary hyperparathyroidism. Clin Endocrinol (Oxf) 2003;58:639e46. [260] Hannan FM, Nesbit MA, Christie PT, et al. Familial isolated primary hyperparathyroidism caused by mutations of the MEN1 gene. Nat Clin Pract Endocrinol Metab 2008;4:53e8. [261] Lin SY, Elledge SJ. Multiple tumor suppressor pathways negatively regulate telomerase. Cell 2003;113:881e9. [262] Stalberg P, Grimfjard P, Santesson M, et al. Transfection of the multiple endocrine neoplasia type 1 gene to a human endocrine pancreatic tumor cell line inhibits cell growth and affects expression of JunD, delta-like protein 1/preadipocyte factor-1, proliferating cell nuclear antigen, and QM/Jif-1. J Clin Endocrinol Metab 2004;89:2326e37. [263] Kim YS, Burns AL, Goldsmith PK, et al. Stable overexpression of MEN1 suppresses tumorigenicity of RAS. Oncogene 1999;18: 5936e42. [264] Yumita W, Ikeo Y, Yamauchi K, Sakurai A, Hashizume K. Suppression of insulin-induced AP-1 transactivation by menin accompanies inhibition of c-Fos induction. Int J Cancer 2003;103:738e44. [265] Hussein N, Casse H, Fontaniere S, et al. Reconstituted expression of menin in Men1-deficient mouse Leydig tumour cells induces cell cycle arrest and apoptosis. Eur J Cancer 2007;43: 402e14. [266] Mulligan LM, Kwok JBJ, Healey CS. Germ-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A. Nature 1993;363:458e60. [267] Mulligan LM, Ponder BA. Genetic basis of endocrine disease: multiple endocrine neoplasia type 2. J Clin Endocrinol Metab 1995;80:1989e95. [268] Pausova Janicic, Konrad. Analysis of the RET proto-oncogene in sporadic parathyroid tumors. J Bone Miner Res 1994;9:S151. [269] Padberg BC, Schroder S, Jochum W, et al. Absence of RET protooncogene point mutations in sporadic hyperplastic and neoplastic lesions of the parathyroid gland. Am J Pathol 1995;147:1600e7. [270] Heshmati HM, Gharib H, Khosla S, et al. Genetic testing in medullary thyroid carcinoma syndromes: mutation types and clinical significance. Mayo Clin Proc 1997;72:430e6. [271] Kennett S, Pollick H. Jaw lesions in familial hyperparathyroidism. Oral Surg Oral Med Oral Pathol 1971;31:502e10. [272] Jackson CE, Norum RA, Boyd SB, et al. Hereditary hyperparathyroidism and multiple ossifying jaw fibromas: a clinically and genetically distinct syndrome. Surgery 1990;108:1006e12. discussion 1012e13. [273] Bradley KJ, Hobbs MR, Buley ID, et al. Uterine tumours are a phenotypic manifestation of the hyperparathyroidism-jaw tumour syndrome. J Intern Med 2005;257:18e26. [274] Cavaco BM, Barros L, Pannett AA, et al. The hyperparathyroidism-jaw tumour syndrome in a Portuguese kindred. Q J Med 2001;94:213e22. [275] Wassif WS, Moniz CF, Friedman E, et al. Familial isolated hyperparathyroidism: a distinct genetic entity with an increased risk of parathyroid cancer. J Clin Endocrinol Metab 1993;77: 1485e9.

PEDIATRIC BONE

587

REFERENCES

[276] Williamson C, Cavaco BM, Jauch A, et al. Mapping the gene causing hereditary primary hyperparathyroidism in a Portuguese kindred to chromosome 1q22-q31. J Bone Miner Res 1999;14:230e9. [277] Weinstein LS, Simonds WF. HRPT2, a marker of parathyroid cancer. N Engl J Med 2003;349:1691e2. [278] Carpten JD, Robbins CM, Villablanca A, et al. HRPT2, encoding parafibromin, is mutated in hyperparathyroidism-jaw tumor syndrome. Nat Genet 2002;32:676e80. [279] Shattuck TM, Valimaki S, Obara T, et al. Somatic and germ-line mutations of the HRPT2 gene in sporadic parathyroid carcinoma. N Engl J Med 2003;349:1722e9. [280] Howell VM, Haven CJ, Kahnoski K, et al. HRPT2 mutations are associated with malignancy in sporadic parathyroid tumours. J Med Genet 2003;40:657e63. [281] Bradley KJ, Cavaco BM, Bowl MR, et al. Parafibromin mutations in hereditary hyperparathyroidism syndromes and parathyroid tumours. Clin Endocrinol (Oxf) 2006;64:299e306. [282] Cetani F, Pardi E, Ambrogini E, et al. Genetic analyses in familial isolated hyperparathyroidism: implication for clinical assessment and surgical management. Clin Endocrinol (Oxf) 2006;64:146e52. [283] Krebs LJ, Shattuck TM, Arnold A. HRPT2 mutational analysis of typical sporadic parathyroid adenomas. J Clin Endocrinol Metab 2005;90:5015e7. [284] Zhao J, Yart A, Frigerio S, et al. Sporadic human renal tumors display frequent allelic imbalances and novel mutations of the HRPT2 gene. Oncogene 2007;26:3440e9. [285] Zheng HC, Takahashi H, Li XH, et al. Downregulated parafibromin expression is a promising marker for pathogenesis, invasion, metastasis and prognosis of gastric carcinomas. Virchows Arch 2008;452:147e55. [286] Selvarajan S, Sii LH, Lee A, et al. Parafibromin expression in breast cancer: a novel marker for prognostication? J Clin Pathol 2008;61:64e7. [287] Bradley KJ, Bowl MR, Williams SE, et al. Parafibromin is a nuclear protein with a functional monopartite nuclear localization signal. Oncogene 2007;26:1213e21. [288] Rozenblatt-Rosen O, Hughes CM, Nannepaga SJ, et al. The parafibromin tumor suppressor protein is part of a human Paf1 complex. Mol Cell Biol 2005;25:612e20. [289] Yart A, Gstaiger M, Wirbelauer C, et al. The HRPT2 tumor suppressor gene product parafibromin associates with human PAF1 and RNA polymerase II. Mol Cell Biol 2005;25:5052e60. [290] Mosimann C, Hausmann G, Basler K. Parafibromin/Hyrax activates Wnt/Wg target gene transcription by direct association with beta-catenin/Armadillo. Cell 2006;125:327e41. [291] Wang P, Bowl MR, Bender S, et al. Parafibromin, a component of the human PAF complex, regulates growth factors and is required for embryonic development and survival in adult mice. Mol Cell Biol 2008;28:2930e40. ˚ kerstrom G, Westin G. Accumulation of non[292] Bjorklund P, A phosphorylated beta-catenin and c-myc in primary and uremic secondary hyperparathyroid tumors. J Clin Endocrinol Metab 2007;92:338e44. ˚ kerstrom G, Westin G. An LRP5 receptor with [293] Bjorklund P, A internal deletion in hyperparathyroid tumors with implications for deregulated WNT/beta-catenin signaling. PLoS Med 2007;4: e328. [294] Costa-Guda J, Arnold A. Absence of stabilizing mutations of beta-catenin encoded by CTNNB1 exon 3 in a large series of sporadic parathyroid adenomas. J Clin Endocrinol Metab 2007;92:1564e6. [295] Ikeda S, Ishizaki Y, Shimizu Y, et al. Immunohistochemistry of cyclin D1 and beta-catenin, and mutational analysis of exon 3 of

[296]

[297] [298]

[299]

[300]

[301]

[302]

[303]

[304] [305]

[306]

[307]

[308]

[309]

[310]

[311]

[312]

[313]

beta-catenin gene in parathyroid adenomas. Int J Oncol 2002;20: 463e6. ˚ kerstrom G, Westin G. Stabilizing Bjorklund P, Lindberg D, A mutation of CTNNB1/beta-catenin and protein accumulation analyzed in a large series of parathyroid tumors of Swedish patients. Mol Cancer 2008;7:53. Weinberg RA. Tumor suppressor genes. Science 1991;254: 1138e46. Cryns VL, Thor A, Xu HJ, et al. Loss of the retinoblastoma tumor suppressor gene in parathryoid carcinoma. N Engl J Med 1994;330:757e61. Pearce SH, Trump D, Wooding C, Sheppard MN, Clayton RN, Thakker RV. Loss of heterozygosity studies at the retinoblastoma and breast cancer susceptibility (BRCA2) loci in pituitary, parathyroid, pancreatic and carcinoid tumours. Clin Endocrinol (Oxf) 1996;45:195e200. Pei L, Melmed S, Scheithauer B, Kovacs K, Benedict WF, Prager D. Frequent loss of heterozygosity at the retinoblastoma susceptibility gene (RB) locus in aggressive pituitary tumors: evidence for a chromosome 13 tumor suppressor gene other than RB. Cancer Res 1995;55:1613e6. Cryns VL, Yi SM, Tahara H, Gaz RD, Arnold A. Frequent loss of chromosomes arm 1p DNA in parathyroid adenomas. Genes Chromosomes Cancer 1995;13:9e17. Williamson C, Pannett AA, Pang JT, et al. Localisation of a gene causing endocrine neoplasia to a 4 cM region on chromosome 1p35-p36. J Med Genet 1997;34:617e9. Carling T, Du Y, Fang W, Correa P, Huang S. Intragenic allelic loss and promoter hypermethylation of the RIZ1 tumor suppressor gene in parathyroid tumors and pheochromocytomas. Surgery 2003;134:932e9. discussion 939e40. Brown EM, MacLeod RJ. Extracellular calcium sensing and extracellular calcium signaling. Physiol Rev 2001;81:239e97. Simonds WF, James-Newton LA, Agarwal SK, et al. Familial isolated hyperparathyroidism: clinical and genetic characteristics of 36 kindreds. Medicine (Baltimore) 2002;81:1e26. Simonds WF, Robbins CM, Agarwal SK, Hendy GN, Carpten JD, Marx SJ. Familial isolated hyperparathyroidism is rarely caused by germline mutation in HRPT2, the gene for the hyperparathyroidism-jaw tumor syndrome. J Clin Endocrinol Metab 2004;89:96e102. Warner JV, Nyholt DR, Busfield F, et al. Familial isolated hyperparathyroidism is linked to a 1.7 Mb region on chromosome 2p13.3-14. J Med Genet 2006;43:e12. Brown EM, Gamba G, Riccardi D, et al. Cloning and characterization of an extracellular Ca2þ-sensing receptor from bovine parathyroid. Nature 1993;366:575e80. Pollak MR, Brown EM, WuChou YH, et al. Mutations in the human Ca2þ-sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Cell 1993;75:1297e303. Chou YH, Pollak MR, Brandi ML, et al. Mutations in the human Ca(2þ)-sensing-receptor gene that cause familial hypocalciuric hypercalcemia. Am J Hum Genet 1995;56:1075e9. Janicic N, Pausova Z, Cole DE, Hendy GN. Insertion of an Alu sequence in the Ca(2þ)-sensing receptor gene in familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Am J Hum Genet 1995;56:880e6. Pollak MR, Chou YH, Marx SJ, et al. Familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Effects of mutant gene dosage on phenotype. J Clin Invest 1994;93:1108e12. Pearce S, Trump D, Wooding C, et al. Calcium-sensing receptor mutations in familial benign hypercalcaemia and neonatal hyperparathyroidism. J Clin Invest 1995;96:2683e92.

PEDIATRIC BONE

588

21. PARATHYROID DISORDERS

[314] Bai M, Pearce SH, Kifor O, et al. In vivo and in vitro characterization of neonatal hyperparathyroidism resulting from a de novo, heterozygous mutation in the Ca2þ-sensing receptor gene: normal maternal calcium homeostasis as a cause of secondary hyperparathyroidism in familial benign hypocalciuric hypercalcemia. J Clin Invest 1997;99:88e96. [315] Brown EM. Extracellular Ca2þ sensing, regulation of parathyroid cell function, and role of Ca2þ and other ions as extracellular (first) messengers. Physiol Rev 1991;71:371e411. [316] Heath III H, Jackson CE, Otterrud B, Leppert MF. Genetic linkage analysis in familial benign (hypocalciuric) hypercalcemia: evidence for locus heterogeneity. Am J Hum Genet 1993;53:193e200. [317] McMurtry CT, Schranck FW, Walkenhorst D, et al. Significant developmental elevation in serum parathyroid hormone levels in a large kindred with familial benign (hypocalciuric) hypercalcemia. Am J Med 1992;93:247e58. [318] Trump D, Whyte MP, Wooding C, et al. Linkage studies in a kindred from Oklahoma, with familial benign (hypocalciuric) hypercalcaemia (FBH) and developmental elevations in serum parathyroid hormone levels, indicate a third locus for FBH. Hum Genet 1995;96:183e7. [319] Lloyd SE, Pannett AA, Dixon PH, Whyte MP, Thakker RV. Localization of familial benign hypercalcemia, Oklahoma variant (FBHOk), to chromosome 19q13. Am J Hum Genet 1999;64:189e95. [320] De Haas WHD, De Boer W, Griffioen F. Metaphysial dysostosis. A late follow-up of the first reported case. J Bone Joint Surg 1969;51B:290e9. [321] Frame B, Poznanski AK. Conditions that may be confused with rickets. In: DeLuca HF, Anast CS, editors. Pediatric Diseases Related to Calcium. New York: Elsevier; 1980. p. 269e89. [322] Rao DS, Frame B, Reynolds WA, Parfitt AM. Hypercalcemia in metaphyseal chondrodysplasia of Jansen (MCD): an enigma. In: Norman AW, Schaefer K, von Herrath D, et al., editors. Vitamin D, Basic Research and its Clinical Application. Berlin: Walter de Gruyter; 1979. p. 1173e6. [323] Kruse K, Schu¨z C. Calcium metabolism in the Jansen type of metaphyseal dysplasia. Eur J Pediatr 1993;152:912e5. [324] Schipani E, Kruse K, Ju¨ppner H. A constitutively active mutant PTH-PTHrP receptor in Jansen-type metaphyseal chondrodysplasia. Science 1995;268:98e100. [325] Minagawa M, Arakawa K, Minamitani K, Yasuda T, Niimi H. Jansen-type metaphyseal chondrodysplasia: analysis of PTH/ PTH-related protein receptor messenger RNA by the reverse transcription-polymerase chain method. Endocrine J 1997;44: 493e9. [326] Schipani E, Langman CB, Hunzelman J, et al. A novel PTH/ PTHrP receptor mutation in Jansen’s metaphyseal chondrodysplasia. J Clin Endocrinol Metab 1999;84:3052e7. [327] Schipani E, Langman CB, Parfitt AM, et al. Constitutively activated receptors for parathyroid hormone and parathyroid

[328]

[329]

[330]

[331]

[332]

[333]

[334]

[335]

[336] [337]

[338]

[339]

[340] [341] [342]

hormone-related peptide in Jansen’s metaphyseal chondrodysplasia. N Engl J Med 1996;335:708e14. Bastepe M, Raas-Rothschild A, Silver J, Weissman I, Ju¨ppner H, Gillis D. A form of Jansen’s metaphyseal chondrodysplasia with limited metabolic and skeletal abnormalities is caused by a novel activating PTH/PTHrP receptor mutation. J Clin Endocrinol Metab 2004;89:3595e600. Schipani E, Lanske B, Hunzelman J, et al. Targeted expression of constitutively active PTH/PTHrP receptors delays endochondral bone formation and rescues PTHrP-less mice. Proc Natl Acad Sci USA 1997;94:13689e94. Hopyan S, Gokgoz N, Poon R, et al. A mutant type I PTH/ PTHrP receptor in enchondromatosis. Nat Genet 2002;30: 306e10. Rozeman L, Sangiorgi L, Briaire-de Bruijn I, et al. Enchondromatosis (Ollier disease, Maffucci syndrome) is not caused by the PTHR1 mutation p.R150C. Hum Mutat 2004;24:466e73. Couvineau A, Wouters V, Bertrand G, et al. PTHR1 mutations associated with Ollier disease result in receptor loss of function. Hum Mol Genet 2008;17:2766e75. Duchatelet S, Ostergaard E, Cortes D, Lemainque A, Julier C. Recessive mutations in PTHR1 cause contrasting skeletal dysplasias in Eiken and Blomstrand syndromes. Hum Mol Genet 2005;14:1e5. Maass PG, Wirth J, Aydin A, et al. A cis-regulatory site downregulates PTHLH in translocation t(8;12)(q13;p11.2) and leads to Brachydactyly Type E. Hum Mol Genet 2010;19:848e60. Klopocki E, Hennig BP, Dathe K, et al. Deletion and point mutations of PTHLH cause brachydactyly type E. Am J Hum Genet 2010;86:434e9. Pober BR. Williams-Beuren syndrome. N Engl J Med 2010;362: 239e52. Ewart AK, Morris CA, Atkinson DL, et al. Hemizygosity at the elastin locus in a developmental disorder, Williams syndrome. Nature Genet 1993;5:11e6. Nickerson E, Greenberg F, Keating MT, McCaskill C, Shaffer LG. Deletions of the elastin gene at 7q11.23 occur in approximately 90% of patients with Williams syndrome. Am J Hum Genet 1995;56:1156e61. Lowery MC, Morris CA, Ewart A, et al. Strong correlation of elastin deletions, detected by FISH, with Williams syndrome: evaluation of 235 patients. Am J Hum Genet 1995;57:49e53. Li D, Brooke B, Davis E, et al. Elastin is an essential determinant of arterial morphogenesis. Nature 1998;393:276e80. Tassabehji M, Metcalfe K, Fergusson WD, et al. LIM-kinase deleted in Williams syndrome. Nat Genet 1996;13:272e3. Perez Jurado LA, Li X, Francke U. The human calcitonin receptor gene (CALCR) at 7q21.3 is outside the deletion associated with the Williams syndrome. Cytogenet Cell Genet 1995;70:246e9.

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Fibrous Dysplasia Paolo Bianco 1, Shlomo Wientroub 2 1

Department of Molecular Medicine, Sapienza University, Rome, Italy 2 Department of Pediatric Orthopedic Surgery, Dana Children’s Hospital, Tel Aviv Medical School, Tel Aviv, Israel

INTRODUCTION Recognition of fibrous dysplasia as a distinct skeletal disease is commonly attributed to the description of an osteitis fibrosa 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 osteitis fibrosa, 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 osteitis fibrosa 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 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 the disease, remain the most common, the most severe, the least understood, and the least treatable. Today, the McCuneeAlbright syndrome (OMIM #174800) eponym is kept alive mainly by virtue of its popular use among endocrinologists to refer to what is

Pediatric Bone, Second Edition DOI: 10.1016/B978-0-12-382040-2.10022-X

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 non-osteogenic (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 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

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that fibrous dysplasia of bone (OMIM174800) is a genetic, non-inherited disease of bone and bone marrow caused by activating mutations of the GNAS gene, which encodes the a-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 Gsa. In bone and marrow, the mutation affects cells of the osteogenic lineage at various stages of maturation [10e13], 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. 22.1) (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 semiological 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 pathologic or fatigue fractures. Coxa vara may result from fatigue fracture in the femoral neck or non-ad unguem 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 upper third of the femur (the shepherd’s crook

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FIGURE 22.1

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 section.)

deformity) (Figs. 22.2 and 22.3), 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 weight-bearing bones, even in the absence of fractures and may, in these cases, solely reflect the malacic compliance of the abnormal bone (Fig. 22.4).

Radiographic Features 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

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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. 22.5). In lateral projections, the contour of calvarial bones appears fuzzy and hairy due to excess bone formation resulting in a thicker than normal but non-compact bone (conceptually akin to, but 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. 22.6). This appearance may be altered by superimposed secondary changes, thus generating complex pictures significantly different from baseline (Fig. 22.7). 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, or conspicuous patches of densely mineralized material appearing in the context of the ground glass background, may represent a non-osseous 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. 22.8). 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

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FIGURE 22.2

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.

and promote additional expansion and thinning of the bone contour.

Associated Extraskeletal Lesions Endocrine and non-endocrine extraskeletal changes may be associated with bone lesions. Hyperfunctional endocrinopathies and skin pigmentation are associated with polyostotic FD in McCuneeAlbright 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 Gsa, which mediates the transduction of signals generated by heptatransmembrane domain receptors (thyroid stimulating hormone [TSH], adrenocorticotrophic hormone [ACTH], gonadotropin, and growth hormone releasing hormone [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 than in males [19e23]. 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. Sexual precocity can be observed in boys, and is often associated with macroorchidism, microlithiasis, and Leydig cell hyperplasia [25e31]. Growth hormone (GH) excess is also common in patients with FD. However, somatotroph adenomas are detectable in only a minority of patients with FD and GH excess [32], indicating that either nontumor-forming hyperplasia or cell hypersecretion without hyperplasia may be the most common effect of GNAS mutations in the pituitary. This is in agreement with the observation that gspþ somatotroph adenomas not associated with FD are usually smaller than gsp adenomas [33]. Cases of gigantism are rare [34e38], whereas acromegalic traits (which are untimely in growing individuals and young adults) [39,40] may occur simultaneously with the native facies fibrodysplastica 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 GNAS mutation in the adrenal and the development of multinodular adrenocortical hyperplasia. It appears to subside spontaneously with time [41e47].

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FIGURE 22.3 Severe shepherd’s crook deformity of the femur, with superimposed cystic changes.

Neonatal/infantile Cushing’s syndrome caused by GNAS 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 GNAS mutation (i.e. the kind of mutation common in FD) was demonstrated [48], whereas Q227 mutations are more common [49]. Thyroid hyperfunction is more common than GH excess and may be caused either by goiter (more commonly) or by welldefined adenoma [50]. Cases of thyroid malignancies are sporadic [51]. All endocrinopathies associating with FD, and even major physiological changes in the hormonal climate such as pregnancy [52], 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.

FIGURE 22.4 Bowing of non-weight-bearing bones: humerus of a 24-year-old patient with severe polyostotic FD and hypophosphatemia.

Cutaneous pigmented macules (Fig. 22.9) are commonly referred to as cafe´ 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, melanocyte-stimulating hormone (MSH)-independent synthesis and transfer of melanin by GNAS-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 [53].

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FIGURE 22.5

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 details 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.

The association of skeletal myxomas with FD was first recognized by Henschen and Fallon [54]. 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 [55,56]. They may occur in the same limb segment as FD lesions but tend to appear after the establishment of an FD lesion [57e61]. GNAS mutations have been demonstrated in skeletal myxomas occurring in conjunction with FD [62e65], but they have also been demonstrated in its absence [66] with significant frequency [67]. Interestingly, Gsa is a negative regulator of myogenic differentiation [68], and histological pictures indistinguishable from skeletal myxomas have also been observed within FD bone as local changes in the fibrous tissue. A “myxofibrous” liposclerosing (benign) tumor of bone seems associated with GNAS mutations and may represent a variant of monostotic FD [69e71]. Cholestatic liver disease has been seen in neonates and infants [41,72e74] 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 [22,75], and the spectrum of extraskeletal disorders associated with fibrous dysplasia continues to expand [76e88], although a true link with GNAS mutations in many instances remains to be determined. Changes directly dependent on the local expression of mutated Gsa may, nonetheless, be broader than currently appreciated. Renal phosphate wasting and hypophosphatemia with low levels of 1,25(OH)2D3 occur in variable degrees in approximately half of patients with FD/MAS [89], 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 [90]. Direct involvement of the mutated Gsa in the proximal tubule of the kidney as a contributing cause of phosphate wasting has not been conclusively ruled out [91]. However, direct evidence has now clarified that a humoral factor, produced in the FD tissue, is the cause of FD-associated renal phosphate wasting, similar to oncogenic osteomalacia as previously surmised [89,92]. Fibroblast growth factor 23 (FGF-23), which causes

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FIGURE 22.7 Complex and diffuse FD lesions of tibia and fibula. Note the marked bowing, multiple lytic lesions, and focal cystic and sclerotic changes.

FIGURE 22.6 FD lesion of the femur showing the typical ground glass appearance in the upper third and rarefaction of the ground glass appearance in the mid-diaphysis. The inset shows a detail of the diaphyseal cortical bone, demonstrating scalloping and internal erosion (arrows).

oncogenic osteomalacia when produced by so-called phosphaturic tumors [93], is in fact expressed in FD tissue, and is elevated in the serum of FD patients with extensive FD involvement of the skeleton and renal phosphate wasting [94]. This is consistent with the finding that mutated stromal cells from FD lesions produce FGF-23 apparently at the same level as normal stromal cells, indicating that a mass effect underlies the overproduction of FGF-23 in FD lesions as well as within the entire skeleton of FD patients. FGF-23 levels, nonetheless, can also be high and cause phosphate wasting in isolated FD lesions [95], suggesting that additional determinants (e.g. age of lesions) may be involved. Within FD tissue, FGF-23 is expressed throughout the osteogenic lineage, i.e. in osteoblasts, osteocytes, and

stromal cells within the fibrous stroma, as well as in vascular walls [94]. These data provided the first indication that the skeleton is actually an endocrine organ regulating phosphate metabolism by acting on the kidney. Whereas skeletal lesions are believed to be mostly synchronous, the combination of skeletal and extraskeletal diseases may develop synchronously or metachronously. In a series of 32 patients with MAS [96], 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. In a subsequent larger series, the vast majority of craniofacial bone lesions were established by 4 years of age, limb lesions by 14 years of age, and axial skeleton lesions by 16 years of age [97]. 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 [98]. These rare cases are the only ones in which adult metabolic remodeling, rather than growth and modeling of

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apparent bone lesions) may signify the existence of age-specific syndromic associations.

MOLECULAR GENETICS The GNAS Gene and its Products

FIGURE 22.8

Non-deformed femur of a 50-year-old patient with FD. The lesional areas are outlined by a typical rind (arrows).

FIGURE 22.9

Cafe´ 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. (See color plate section.)

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 [99]. 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

Originally described as a 20-kilobase gene containing 13 exons and 12 introns [100], human GNAS is an imprinted complex locus located at chromosome 20ql3. Multiple promoters and at least five alternative first exons, spliced onto the common set of exons 2e13, give rise to a complex family of transcripts e Gsa, XLas, NESP and 1A (alternatively termed A/B) [101e108]. The XLas transcript is expressed in neuroendocrine cells and, to a lesser extent, in other tissues and cell types such as brain, pancreas, adrenal gland, heart, kidney, and adipose tissue [109e112]. XLas protein shares with Gsa the entire carboxyl-terminal region [108], and displays some Gsa-like properties in vitro [113e115]. However, knockout of XLas in the mouse suggests divergent roles for XLas and Gsa in energy and fat metabolism [111,112]. NESP expression is mostly restricted to neuroendocrine tissues in which the transcript is translated into a chromogranin-like protein, NESP-55. The NESP coding sequence is limited to the first exon and the resulting protein is unrelated to either Gsa or XLas [116]. 1A mRNA is ubiquitously expressed, but not translated into a functional protein and its function remains as yet unclear. In addition to Gsa, NESP, XLas, 1A and splice variants thereof, other less-well known GNAS transcripts, such as an antisense transcript, NESPAS [117], and others have been identified, which add further complexity to the locus [117e120]. Following transcription, each GNAS transcript is alternatively spliced to generate long and short isoforms, plus or minus exon 3. In addition, insertion of CAG (coding for serine) by the use of two alternative splice acceptors (TG instead of the consensus AG) three base pairs upstream of exon 4 generates additional splice variants [121e123]. For Gsa, the following four major isoforms, all functionally active, can be formed: Gsa 1 (long), Gsa 2 (short), Gsa 3 (long þ Ser), and Gsa 4 (short þ Ser)]. Gsa 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 [124]. Gsa splicing isoforms have been the subject of multiple studies. The long and the short Gsa mRNAs are translated into different molecular weight proteins, the distribution and relative ratio of which vary in different developmental and metabolic conditions [125e127]. The role of different Gsa isoforms in disease

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is 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, post-translational modifications, membrane trafficking, association with receptors and the bg-subunit and, finally, in their interactions with adenylyl cyclase [128e131]. The expression of GNAS products is epigenetically regulated according to a complex pattern of imprinting [132]. XLas and 1A are paternally transcribed and their promoters are methylated on the maternal allele. In contrast, the origin of NESP is exclusively maternal and its promoter is methylated on the paternal allele [133,134]. The Gsa promoter is unmethylated on both alleles. However, recent studies identified an imprinting mark around the 1A promoter, which is thought to suppress the expression of Gsa from the paternal allele in a tissue-specific manner [135]. Partial imprinting of Gsa has also been observed in the human renal proximal tubule, anterior pituitary and thyroid [136e138], which has clinical implications in patients carrying loss-offunction mutations of GNAS.

GNAS Transcripts in Skeletal Progenitors Short and long forms of Gsa are expressed at similar levels in cultured skeletal progenitor cells [139], but their relative abundance changes remarkably during in vitro differentiation (unpublished observations). In normal fetal and postnatal bone, as well as in FD tissue, Gsa transcripts are biallelic in origin and the amount of the transcript generated from each allele is, overall, similar [140]. Analysis of individual skeletal clonogenic progenitors isolated in vitro, conversely, reveals that the transcriptional activity of the two Gs alleles may be significantly different [139] in each resulting clone. Each allele can be selectively expressed or conversely, almost completely silenced, in a random fashion, independent of parental origin of each allele. This pattern of regulation of expression of the two Gsa alleles has not been reported in other systems and its mechanism remains to be clarified. However, it can neither be referred to as imprinting, which specifically implies methylation and true silencing of maternal or paternal genes [141] nor as allelic expression imbalance, which is an inherited trait and therefore consistent in different cells from the same patient [142]. XLas, 1A and NESP are also expressed in skeletal progenitors, although the expression of NESP seems to be restricted to a small subset of clones. While NESP and 1A transcripts retain the monoallelic origin observed in other tissues, XLas displays, in cultured

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skeletal progenitors, a biallelic pattern of transcription never previously reported in human cells [139]. In summary, the overall imprinting status of the gene in skeletal progenitor cells seems to be different from that observed in other human tissues. In addition, multiple GNAS transcripts are expressed in skeletal stem cells and undifferentiated progenitors, but the GNAS transcript portfolio is remarkably heterogeneous among different clonogenic cells, even beyond the variable allelic origin of Gsa itself. Further studies should elucidate how this complexity is modulated during differentiation events.

Causative Mutations Activating missense mutations of the GNAS gene associated with fibrous dysplasia consist of single base substitutions at codon R201 in exon 8 (Fig. 22.10). R201C and R201H mutations of the GNAS gene were first identified, along with mutations at codon Q227, in isolated endocrine tumors [143e147]. Hence, the mutated gene was designated the gsp oncogene [144]. However, this label is used with caution since there is no evidence that GNAS mutations are in and of themselves sufficient for transformation. Most of the endocrine tumors associated with GNAS activating mutations are benign, and there is no evidence of a causative role of GNAS 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 [148]. Excessive cAMP-induced protein kinase A (PKA) activation, the putative mediator of the transforming effect, may even counter, rather than promote, cell transformation in certain systems [149]. 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 [150] and in cells grown in culture from fibrous dysplastic bone [11] e a 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 [12,151,152]. Thus, all forms of fibrous dysplasia, all forms of FD-associated hyperfunctional endocrinopathy, and some sporadic, isolated

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FIGURE 22.10 (A) Mutations at codon 201 demonstrated by sequencing of the relevant PCR-amplifled 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]. (See color plate section.)

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 [153,154] and R01S and R201L mutations have been reported in patients with isolated endocrine tumors in the absence of skeletal disease [137,155]. 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 non-polar), 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 [154].

Mutability of the R201 Codon 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 (50 CGT-30 ) 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 CG/TG and CG/CA transitions, respectively. These two transitions of CG

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dinucleotides represent 32% of all point mutations known to cause human disease, a 12% higher frequency than that predicted from random expectations [156]. 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 methylationedeamination 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 [156]. The non-random nature of CG mutations might be used to predict the rate of mutation and even the prevalence of human disease [156], which would be of direct clinical relevance for FD, for which no epidemiological assessments have been performed.

Functional Consequences of the GNAS1 Mutations Heterotrimeric G proteins couple receptors for a variety of extracellular signals to intracellular effectors and consist of three different polypeptides [157e159]. The a-subunits bind guanine nucleotides with high affinity and specificity, whereas the b and g peptides are non-covalently associated with one another in a functional dimer subunit. The abg 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 Gsa, the heterotrimer dissociates from the plasma membrane, and the GTP-binding a-subunit dissociates from the bgsubunit. GTP-binding Gsa binds and activates adenylyl cyclase (AC), thus generating cAMP. AC remains active only as long as it is bound to activated Gsa. The amount of time that this association exists is dependent on the rate at which Gsa-bound GTP is hydrolyzed to GDP by the intrinsic GTPase activity of Gsa. Hydrolysis of GTP has been found to rely primarily on Q227 and R201 [159], 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 [160]. 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 [161]. R201 (or a homologous residue) is conserved in homologous regions in all proteins with GTPase activity, further highlighting its importance [160]. Mutations of Q227 and R201 (in endocrine tumors and fibrous dysplasia) reduce the kcat of the GTPase activity inherent to Gsa [160]. Q227 mutations are not

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uncommon in isolated endocrine tumors, but have only recently been identified as very rare causes of fibrous dysplasia as well [162]. Prolonged binding of GTP to mutated Gsa induces a constitutive activation of adenylate cyclase resulting in enhanced cAMP production. In cells transfected with wild-type or R201C mutated Gsa, immunofluorescence studies document that although Gsa shuttles between the plasma membrane and the cytoplasm depending on activation/inactivation cycles induced by receptor stimulation, mutated Gsa is stably localized in the cytoplasm with a diffuse distribution [163]. 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 Gsa, and also with the inference that a cAMP-dependent pathway(s) may mediate many of the phenotypic effects of the mutation in bone. However, the abnormal cAMP response induced by the mutated Gsa may be subject to significant modulation. In many systems, cAMP may act as a gating mechanism, of which the net effect on cellular physiology may be highly diversified [164]. Furthermore, concurrent upregulation of phosphodiesterases induced by cAMP may buffer the effects of the constitutive activity of AC in some systems, resulting in normal or nearnormal levels of cAMP. Basal levels of cAMP may be similar in gspþ or gsp pituitary tumors [165]. However, the increase in cAMP concentration induced by specific phosphodiesterase blockade is markedly higher in gspþ tumors compared to gsp lesions, and a sevenfold higher level of phosphodiesterase activity has been detected in human pituitary GH-secreting adenomas [165]. Likewise, either the basal cAMP levels or the mitogenic response induced by mutated Gsa require blockade of phosphodiesterases in order to be demonstrated [166,167]. Similar effects are observed in human osteogenic progenitors in culture after stable transduction with mutated Gsa using lentiviral vectors [168]. In these systems, marked and selective upregulation of mRNAs and activity of specific PDE isoforms rapidly follows expression of the mutated protein, affecting cAMP basal levels but also responses to certain differentiative stimuli. Thus, the presence of an activating mutation of Gsa does not necessarily translate into constitutively high levels of cAMP under all conditions, and a suitable context of extracellular stimuli and intracellular permissive or adaptive conditions (many of which remain to be dissected and may in theory include post-translational 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 Gsa may be upor downregulated in mutated cells [155,169].

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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 of the lines of Blaschko, which represent directions of dorsoventral migration of embryonic cells [170]. 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 non-uniform 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 [170] to postulate, long before the recognition of the causative GNAS 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 germ line (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. It should be noted, however, that embryonic lethality of the germ-line-transmitted mutation has not been directly proven, and that alternative explanations for the observed lack of inheritance in

humans obviously exist, e.g. a reduced viability of mutated gametes.

Site and Time of Origin of the Mutated Clone 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. While entertained widely, this assumption, based on clinical observation, has never been proven directly, as it could be for example through proper animal models. However, two considerations suggest a more complex picture and a less stringent correlation of extent of disease with embryonic time of mutation, than is assumed in Happle’s prediction. With the exception of monostotic FD, the vast majority of FD mutations must in fact arise in a very narrow developmental window, regardless of the ultimate extent of disease. The first consideration is related to the pluripotency of the originally mutated embryonic cell, which is implied by the nature and distribution of the mutated cells in the postnatal organism. In most forms of McCuneeAlbright syndrome and polyostotic FD, mutated cells are comprised within derivatives of different germ layers, implying the origin of the mutated clone from a mutational event occurring prior to gastrulation. Of note, this not only applies to clinical phenotypes in which organs of different general nature and functions are clinically affected (e.g. thyroid, bone and skin), but also to the highly common forms of polyostotic disease of limited extent. For example, the two most common sites of skeletal involvement, the femur and the craniofacial bones, are derived from different germ layers (lateral mesoderm the femur, ectoderm of the neural crests the facial bones). Therefore, the mutation underlying an otherwise limited disease that involves these two sites, and the mutation underlying a severe McCuneeAlbright phenotype with multiorgan involvement, must both occur prior to gastrulation. It is safe to state that with the exception of truly monostotic forms of the disease, the vast majority of FD/MAS patients carry mutated cells in derivatives of different embryonic germ layers, and therefore the mutation causing their disease must have occurred prior to gastrulation. The second consideration is related to the dependency of the causative mutations on methylation of a CpG dinucleotide. The two mutations that account for nearly all cases of FD/MAS (R201C and R201H) can both be brought about by methylation of a CpG dinucleotide in

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codon 201, in the sense (R201C) or antisense (R201H) strand, followed by deamination and base transition (C/T in the sense or antisense strand, resulting in C/T or G/A in the sense strand, and dependent amino acid substitutions, respectively) [171]. Both mutations are thus dependent on methylation. In the embryo, following two distinct phases of active and passive demethylation of the genome that follow fertilization, de novo methylation begins at the stage of 8-cell embryo, and in the blastocyst, differential methylation establishes a major differentiative distinction between the pluripotent cells of the inner cell mass (where methylation is high) and the trophectoderm (where methylation is low). Taken together, the two considerations lead to postulate that, in the vast majority of individuals, mutation must arise between the 8-cell embryo stage and gastrulation. Not before, when there is no de novo methylation, and not after, when there is no pluripotency left [171].

Survival and Adaptation If so, then the marked variability in the severity (extent) of the human disease may arise from differential growth or survival of the mutated clone. Major differences in proliferation or apoptotic demise would suffice to account for clinical variability of phenotype. Alternatively, one could postulate that the disease genotype can exist in tissues, organ, and perhaps individuals, in a non-pathogenic, silent form. Longitudinal observation of development and growth of lesions in humans do not dispel this possibility. Embryonic development and postnatal growth occur normally in virtually all individuals and skeletal segments that will eventually develop a postnatal FD lesion. In patients with MAS, tissues and organs that clearly carry the disease genotype, such as heart or digestive tract, may not show a clinically expressive phenotype [7,75], suggesting that adaptive mechanisms may exist that contribute to the tuning of clinical expression of the disease phenotype. For example, allelic variation in genes that encode known modulators of cAMP levels such as phosphodiesterase, or even mere changes over time in PDE expression and activity, could contribute to adaptive responses to the disease gene and perhaps to the failure over time of such responses. These questions are not relevant simply for their speculative appeal, but also because they may conceal important lessons on how to tackle the adverse effects of the disease gene in cell and tissue. Addressing these questions is however difficult on clinical grounds alone, and requires the development of proper animal models.

Allelic Expression Differential methylation between maternal and paternal alleles occurs within CpG islands of some

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autosomal genes [172]. The resulting repression of transcription from the methylated allele determines the maternal or paternal epigenotype, and imprinted genes are involved in certain human diseases [172,173]. A tissue-specific pattern of imprinting of Gsa 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-of-function mutations of Gsa [106,174,175]. Tissuespecific imprinting with monoallelic expression of Gsa was then demonstrated for certain tissues (kidney and brown and white adipose tissue) in the mouse [176,177]. Recently, the first direct evidence for tissuespecific imprinting of Gsa in humans was obtained. Only the maternal allele appears to be expressed in the normal pituitary, and in 21 of 22 gspþ GH-secreting adenomas, the mutation was demonstrated in the maternal allele [137]. Although predicted by clinical observation in patients with pseudohypoparathyroidism type la and inferred from direct evidence in the mouse, Gsa imprinting in the human kidney has not been demonstrated to date and, in fact, biallelic expression was observed in immature human kidneys in one study [178]. It is conceivable that Gsa imprinting in the human kidney is developmentally (temporally) regulated [101] 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 Gsa alleles are transcribed as a result [137]. Taken together, these data could imply that imprinting of Gsa may be acquired or lost during normal development or abnormal growth of tissues. In principle, tissue-specific imprinting of Gsa, 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. In keeping with the prediction, MAS patients with GH-secreting adenomas have been shown to carry maternal allele mutations [179]. Although Gsa is biallelically expressed in bone [140,180], and in clonogenic skeletal progenitors ex vivo [139], 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 [181]. Thus, in bone, as in any organ, Gsa might still be imprinted in specific histological structures and still be seen as biallelically expressed if assayed in whole tissues or organs, or in cultured cells. Regardless of

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imprinting, the observed, marked variation of allelic expression of Gsa in individual clonogenic skeletal progenitors can itself influence expression of the disease phenotype.

PATHOLOGY Modeling of FD Bone 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 (Fig. 22.11) [16,182], is established through abnormal modeling of a growing skeletal site but may involve remodeling of an established bony structure such as a femoral cortex. Depending on the specific

FIGURE 22.11 (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. 22.16). (Photographs courtesy of Professor Alan Boyde, University College London.)

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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.

Deposition and Internal Structure of FD Bone 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 [10,183,184]. The bone trabeculae resulting from FD bone formation are woven in structure. Detection of lamellar trabeculae within FD indicates their origin from resorption of pre-existing 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 [10,183]. 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. 22.12). 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 [183].

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

FIGURE 22.12 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 section.)

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 [152,185]. 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, readily reveals that FD bone is undermineralized more

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than it is reduced in mass (Fig. 22.13). 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 of water and hydrophilic non-collagenous proteins (including the putative mineral nucleator bone sialoprotein [BSP]) and relatively lower content of collagen [186,187]. 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 [185], 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 [188], also show that the degree of mineralization attained even in mineralized portions of the FD bone is lower than normal [185], 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 [189]. Overproduction of FGF-23 in extensive skeletal lesions, renal phosphate wasting, hypophosphatemia, and low levels of 1,25(OH)2D3 [185] 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. A marked undermineralization within FD lesions is regularly observed in cases in which no significant derangement of phosphate metabolism is seen. The likely, additional, local rather than systemic mechanisms of impaired mineralization of FD bone remain to be clarified. The possibility that additional humoral factors are involved also warrants consideration [190].

Remodeling of FD Bone Internal remodeling and resorption of FD bone gives rise to bizarre trabecular shapes (C-shaped and

FIGURE 22.13 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 methacrylate 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 section.)

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S-shaped trabeculae e so-called “Chinese writing” or “alphabet soup” patterns (Fig. 22.14) [16,18]. These patterns are more commonly seen in long-standing FD lesions, in which multiple cycles of remodeling have occurred. Hence, they are more common in the axial and limb bones than in craniofacial bones [182], in which histological patterns produced by modeling prevail. Lesions of gnathic bones, for example, often exhibit a unique pattern, termed hyperosteocytic [183], 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. FD

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bone undergoes extensive tunneling resorption (resorption 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 [191]. 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

FIGURE 22.14 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. (See color plate section.)

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in FD bone than age-matched reference values and correlate strictly with urinary pyridinium cross-links. Furthermore, they correlate with levels of circulating PTH [185]. 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. 22.15). In these cases, tunneling resorption is more obvious and extensive than usual, and large clusters of osteoclasts and mononuclear TRAP-positive 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 (see Fig. 22.14). Both arterial capillaries and venous sinusoids are increased in number per unit volume of tissue, and ectatic capillaries engorged with

FIGURE 22.15 Images from a 24-year-old patient with polyostotic FD and hypophosphatemia, 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 section.)

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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 post-hemorrhagic aneurysmal bone cysts secondarily engraft on FD lesions [192e194]. 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 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. 22.16) (inappropriately referred to as either aneurysmal bone cysts or mucoceles) [195e197] 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 arteriovenous shunts generated by vascular remodeling of the unusually rich local vascularity [198].

Growth of Lesions Examination of the edges of expanding lesions demonstrates a marked increase in vascularity, loose perivascular edema of tissues, and two distinct changes

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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 a-smooth muscle actin [199]. In both respects, the endosteal fibrosis of FD closely resembles that observed in hyperparathyroidism [200,201]. 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 Gsa is expressed at high levels [10].

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 Gsa 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 [202]. 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 [203,204] 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 Other Fibro-osseous Lesions FIGURE 22.16 Hemorrhagic pseudocyst (pc) with air-fluid level in the maxillary sinus secondary to FD of the maxillary bone. Compare with Figure 22.11.

Assessment of GNAS mutations should be useful to define unequivocally other bone lesions whose distinction from FD is vague or uncertain. It is now clear that

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the lesion called osteofibrous dysplasia (ossifying fibroma of long bones or Campanacci’s lesion [18,205]), 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 GNAS mutations [206]. Likewise, ossifying and cemento-ossifying fibroma of jawbone, often referred to as part of a common spectrum of benign fibroosseous lesions that include FD [207], can be distinguished from FD based on the absence of GNAS mutations. Nonetheless, true mutation-positive FD of jawbone may share significant histological features with cemento-ossifying fibroma, including the formation of cementum and 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 non-typical cellularity and may coalesce to form large masses of mineralized non-osseous material, which can be prominent on plain radiographs [18]. Non-ossifying 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 non-ossifying fibroma (NOF) when there is radiographic evidence of cortical involvement. NOF-like changes reflect post-hemorrhagic reactions in FD. It is important to remember that multiple NOFs occur along with skin pigmentation and various extraskeletal disorders in JaffeeCampanacci syndrome (JCS) [208], 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 GNAS mutations in JCS as a potential cause. Interestingly, many, if not all, fibrous or fibro-osseous 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 pre-existing 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 [209]. 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. Analysis of more recent series confirms the occurrence of malignant change independent of prior irradiation [210], although radiation therapy does greatly enhance the risk of transformation in FD [211]. Sarcoma may also complicate the Mazabraud’s syndrome [212e214]. Different types of clonal chromosomal aberrations (t (6;11), þ2, rearrangements involving chromosome band 12p13, and others) [215e218] have been observed in FD and used to argue for a true neoplastic nature of FD. Evidence for clonality in FD lesions has also been obtained using analysis of polymorphisms at the androgen receptor gene [219,220]. Although the contention for a true neoplastic nature of FD lesions made on these bases may be overstated, these observations may represent the not infrequent detection of a phase of clonal evolution of FD. Occasionally, and depending on the nature of the associated chromosomal changes, clonal evolution 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 commonly arise in the craniofacial skeleton, usually behave aggressively, and have a poor prognosis [221,222]. Osteogenic sarcoma is the most common type of tumor, followed by chondrosarcoma, fibrosarcoma, and malignant fibrous histiocytoma [223e233]. Rarely, angiosarcoma has been reported as well [234]. 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 GNAS mutation with physiologically significant impact. Likewise, the rare occurrence of malignant change in pregnancy [235] recalls the hormonal responsiveness of FD itself. 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 [236,237]. 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 GNAS mutation was demonstrated in one case of FD-like osteogenic sarcoma [237], which in fact complicates the use of mutation analysis for differential diagnosis between FD and LGCOS but suggests that FD

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may evolve into a tumor retaining resemblance to the original FD lesion.

PATHOGENESIS Expression of Gsa in the Bone/Bone Marrow Environment In some tissues and organs of patients with PFD or MAS, the presence of mutated cells may remain pathologically and clinically silent. Although Gsa 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 GNAS-mutated cells. Immunolocalization and in situ hybridization studies have shown that in normal bone, Gsa 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 Gsa, whereas the “fibroblastic cells” filling the marrow spaces (which are preosteoblastic in phenotype) exhibit comparatively lower levels of Gsa mRNA and protein. The increased levels of Gsa signal in mature osteoblasts compared to less mature osteogenic cells may reflect either the increase in total RNA associated with osteoblast maturation [238] or a specific upregulation of the Gsa mRNA transcription. In either case, the net effect is a higher expression of Gsa protein in mature osteoblasts involved in the deposition of new bone matrix. Hence, it is logical to assume that mutated Gsa is also expressed at a comparatively high level in mature osteoblasts. Given the increased AC-stimulating activity of mutated Gsa, the maturation process of osteoblasts amplifies the effect of carrying a mutated Gsa, 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 Gsa 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 cAMP-mediated 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 non-mutated ones on a per cell basis. The higher

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resorptive activity observed in FD bone in most cases cannot easily be envisioned as a direct, cell-autonomous effect of Gsa mutation in osteoclasts and rather postulates an effect of enhanced osteoclast differentiation mediated by osteogenic cells. Consistent with this interpretation, in osteoprogenitor cells transduced with lentiviral vectors directing the expression of Gsa R201C, a potent upregulation of RANK-L and a concurrent downregulation of osteoprotegerin is observed [168]. The sustained expression of Gsa 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 [239e241]; hence, the expression of mutated Gsa 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 [199] may be consistent with this view. This raises the question of the relative merit of adrenergic signaling in microvascular cells vs signaling from osteotropic hormones like PTH in the triggering of FD lesion development. Of note, osteoprogenitor cells in human [239] and mouse [242] are associated with the microvascular walls, and at least in the mouse, they may be controlled by adrenergic signaling [242].

Nature of the FD Fibrous Tissue 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) [243] in the normal human bone marrow, and like stromal osteoprogenitors ex vivo [239], they are noted for an incomplete osteogenic phenotype and express alkaline phosphatase and certain non-collagenous proteins of bone. Some of the abnormal stromal cells also express the pericyte marker a-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. a-Smooth muscle actin [200] and ALP activity [201] 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 [240,244]. Assessment of the frequency of clonogenic cells in the FD marrow stroma

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compared to normal stroma shows a several-fold enrichment in clonogenic cells in FD. Cbfal, a pivotal transcription factor regulating osteogenic differentiation [245], is expressed in mutated stromal colonies like in normal clones of marrow-derived fibroblasts [246]. 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 [247]. The absence of hematopoiesis in the FD marrow suggests 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 [248], consistent with the known effects of Gsa activity, cholera toxin, and cAMP on the adipose conversion in a variety of fibroblastic/preadipocytic cell systems [249,250]. Normal stromal osteoprogenitors transduced with mutated Gsa lose the ability to differentiate into adipocytes in vitro, when conventional differentiation cocktails including the phosphodiesterase inhibitor,

IBMX, are used, but not when adipogenesis is induced in the absence of IBMX [168]. 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 [240]. Under the same experimental conditions, FD cells generate abnormal woven bone but fail to establish a hematopoietic microenvironment and to give rise to adipocytes (Fig. 22.17) [13].

Downstream Effects of Excess cAMP in FD Osteogenic Cells Downstream effects of excess cAMP production in bone cells have only partially been characterized. In cells grown in culture from FD lesions, higher levels of cAMP are observed compared to those of controls [9]. It was initially shown that in situ FD cells express high levels of c-fos [251]. Fibrous changes and abnormal osteogenesis are observed in mice overexpressing c-fos [252], which further supports a role for it as one of the mediators of the FD phenotype. Furthermore, c-fos is regulated

FIGURE 22.17 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). Undecalcifled methyl-metacrylate embedding; Goldner’s stain, hac, hydroxyapatite carrier. (See color plate section.)

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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 Gsa produce sustained levels of IL-6 [253], and enhanced production of IL-6 in human FD cells has been shown in three studies [9,254,255]. 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 [255]. However, both stromal/ osteoblastic cells and non-osteoblastic cells appear to express IL-6 in FD tissue in situ. Analysis of production of IL-6 by clonal strains of PTH-stimulated 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 [255]. 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 Gsa 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 [256]. 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 [184]. 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 db-cAMP, 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 [201], exhibit a retracted morphology. In a variety of cell systems, disruption of actin cytoskeletal

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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 [257e260]. This suggests that inordinate levels of endogenous cAMP in mutated osteogenic cells may cause enhanced matrix degradation. Data obtained using clonal strains of human mutated cells in an in vitro collagen degradation assay support this suggestion [246]. 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 non-collagenous proteins, such as BSP and osteopontin [187]. In situ hybridization and immunolocalization data show that in FD bone formed de novo, these proteins and 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). After de novo transduction with mutated Gsa, normal human osteoprogenitors express an altered pattern of expression of non-collagenous proteins, noted for a blunted upregulation of osteocalcin after induction of osteogenic differentiation [168]. 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, or with de novo transduced osteoprogenitors [168], 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 non-mutated cells coexisting in the tissue and in the non-clonal 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 non-growing bone but orders of magnitude lower than those at sites of physiological skeletal growth. Interestingly, the highest

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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 Gsa [199].

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 non-mutated 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 [261]. Ectopic transplantation of mosaic strains of mutated and non-mutated 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 surprisingly high frequencies of apoptotic osteoblasts in FD bone [262], 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 non-osteoclastic degradation of FD osteoid indicate that FD bone is a non-viable tissue deposited by non-viable cells, which is not surprising in view of the contended celllethal 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 non-skeletal 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 conventional “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 [262]. 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 both earlier and more recent observations [263e265]. 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

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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 fibro-osseous 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 [266,267], antithyroidal agents are used 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 ophthalmological and ENTevaluation 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 [268], evidence of renal phosphate wasting, hypophosphatemia, and low levels of 1,25 (OH)2D3 must be specifically sought at initial evaluation, corrected even if overt radiographic evidence of rickets or osteomalacia are missing, and monitored over time.

Mutation Analysis in Clinical Practice A number of methods have been developed to determine the presence of mutations at codon R201 in clinical material [7,13,152,153]. Direct sequencing of the relevant

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PCR amplification product of genomic DNA extracted from tissue is the simplest method. Because of the variable proportion of mutated to non-mutated 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 [162,269e272], and techniques based on peptide nucleic acid (PNA) clamping seem particularly apt to detect low levels of mutation load in a sample. 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 only recently been detected in FD [162], but are known to occur in isolated 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 [273], 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. Pathological fracture is one of the main orthopedic concerns, and the fracture history in FD patients is clearly inscribed into the more general history of lesion development in a defined time window [274]. Loss of femoral neck angle is one crucial determinant of outcome in FD patients [275]. Particularly in MAS patients with extensive disease, conservative treatment of femoral fractures seems to be discouraged, and internal fixation with intramedullary nails may be

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the best option [276]. Extensive lesions in a limb bone may require surgery for correction of deformity or fracture non-union [277e279], although the frequency of the latter is probably overestimated in the literature [280]. Internal fixation using an intramedullary nail or an external plate is used in these cases, but the nature of the FD bone can make 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 cases. Thus, an adequate understanding of the tissue and cell biology of FD lesions is important for improving the effectiveness of surgery. Involvement of the spine and scoliosis associated or not with spinal FD are emerging concerns for orthopedic surgeons [281e283]. 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 [284e286].

Bisphosphonates Intravenous pamidronate has been used in open studies for the medical treatment of FD both in adults and in children and adolescents [287e293]. 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 [294e296] and considered as possible evidence of improved bone quality. The ethical need for medical intervention in a disease that is occasionally devastating justifies, in the face of proven safety, an attempt to treat. Conclusive evidence on a direct beneficial effect on the abnormal bone structure in FD is missing, and evidence suggests that in fact FD histology may remain essentially unaffected by bisphosphonate treatment [297]. Likewise, reports indicate that lesions may continue to expand in spite of treatment [298]. 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 may be the concurrent administration of phosphorus and vitamin D supplements [299]. A second

caveat concerns the effect of bisphosphonates in FD lesions with a predominant sclerotic nature, such as those in the craniofacial skeleton, although isolated observations are encouraging [300e302]. A more general question that remains open is whether reducing remodeling of FD bone per se should be necessarily seen as a longterm 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 age-related differences in the disease biology would allow identification of the correct time frames 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 [303e305] 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 [306], a type of posttranslational modification critical to a variety of GTP binding proteins. Gsa is palmitoylated [307], whereas the g-subunit is prenylated [308e310]. Palmitoylation and depalmitoylation regulate membrane association and redistribution to the cytosol of Gsa [307], and turnover of palmitate is greatly enhanced in mutated Gsa [311]. Prenylation of the bg-subunit affects its affinity for Gsa and stabilizes and enhances the effects of palmitoylation with respect to membrane anchorage of Gsa [312]. Additional GTP binding proteins that are prenylated interfere with downstream effects of cAMP, such as cytoskeletal remodeling. Analysis of the effects of bisphosphonates on mutated osteogenic cells from FD thus appears warranted.

Strategies for Innovative Treatment 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

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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 Gsa activity. However, important gaps remain in our understanding of the cell biology of the mutated bone cells. First, whereas recognition of the pathogenetic role of osteogenic cells and their progenitors is important, knowledge of the effects of mutation on the diverse members of the osteogenic lineage is needed, as is the precise definition of the whole spectrum of cells potentially involved that reside in the bone environment and are linked to the osteogenic lineage, including vascular wall cells and adipocytes. Second, it is imperative to understand the downstream effects of the mutation in greater detail, and their evolution over time. Adaptation mechanisms, and trigger of stable metabolic changes downstream of cAMP can be crucial to devise treatment strategies. To this end, one needs model systems that are less variable and more easily controlled than primary cultures of cells derived from clinical FD lesions. Models in which normal cells are stably transduced with the causative mutation [168] are likely to speed up progress in this area. 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 [13,313] 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 non-skeletal effects of the mutation. A separate avenue may relate to the viability of the mutated clone and a way to promote selectively 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) 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

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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 ex vivo for use in local transplantation in conjunction with a suitable biomaterial. Based on extensive preclinical (and limited clinical) experience with engineering of bone tissue using marrow-derived progenitor cells [314], satisfactory regeneration of bone may be expected from these procedures. However, regrowth of lesional tissue (as happens with bone grafts) at the site of transplantation cannot be ruled out. FD causing mutations are dominant gain-of-function mutations in an essential and ubiquitously expressed gene. They thus represent perhaps the worst-case scenario (the most difficult challenge) for gene therapy. The possibility to silence genes through RNA interference and the ease with which this is combined with stable lentiviral transduction of cells open however an interesting avenue. Recently, stable and effective, selective silencing of the mutated Gsa has been obtained in vitro in human osteoprogenitor cells, followed by reversion of the fundamental cell phenotype of the disease [168]. These data have provided preliminary proof-ofprinciple for a gene therapy approach in FD. They remain removed from a clinical application in the near future, but pursuit of this avenue may along the way elucidate crucial aspects of the cell biology of the disease, while refinement of cell transduction strategies may circumvent many of the hurdles presently associated with the use of viral vectors in the clinic. Major efforts are required in both clinical and basic areas of research before one or more rational and effective therapeutic options become available. The rarity of the disease is a major limiting step to the acquisition of the necessary knowledge and requires coordination of efforts worldwide. As Di George [315] noted, FD is “a rare disorder, yes, an unimportant one, never,” given its devastating consequences.

Acknowledgments We are indebted to Dr Mara Riminucci (Sapienza University of Rome, Italy) for extensive discussions, critical reading of the manuscript, and liberal sharing of data and observations. We acknowledge insightful discussions with Professor Alan Boyde (University College London), who also generously provided the photographs in Figure 22.11. His contributions and those of Drs Sergei Kuznetsov, Larry Fisher,

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Michael T. Collins, and Kenn Holmbeck (NIDCR, NIH NIDCR, NIH) and of Dr Alessandro Corsi, Stefania Piersanti, Isabella Saggio (Sapienza University of Rome) to work mentioned in this chapter are also acknowledged. Published and unpublished personal investigations mentioned in this chapter were supported in part by Telethon Grants E.519 and E.1029, GGP04263 and GGP09227 to PB.

[15]

[16] [17]

[18] [19]

References [1] Albright F, Butler AM, Hampton AO, Smith P. Syndrome characterized by osteitis fibrosa disseminata, areas of pigmentation and endocrine dysfunction with precocious puberty in females. N Engl J Med 1937;216:727e46. [2] Albright F, Scoville B, Sulkowitch HW. Syndrome characterized by osteitis fibrosa disseminata, areas of pigmentation, and a gonadal dysfunction: further observations including the report of two more cases. Endocrinology 1938;22:411e21. [3] McCune DJ, Bruch H. Progress in pediatrics: Osteodystrophia fibrosa. Am J Dis Child 1937;54:806e48. [4] von Recklinghausen F. Die fibrose order deformierende Ostitis, die Osteomalazie und die osteoplastische Karzinose in ihren gegenseitigen Beziehungen. Festschrift fur Rudolf Virchow. Berlin: Reiner; 1891. [5] Lichtenstein L. Polyostotic fibrous dysplasia. Arch Surg 1938;36:874e9. [6] Lichtenstein L, Jaffe HL. Fibrous dysplasia of bone: a condition affecting one, several or many bones, the graver cases of which may present abnormal pigmentation of skin, premature sexual development, hyperthyroidism and still other extraskeletal abnormalities. Arch Pathol 1942;33:777e97. [7] Weinstein LS, Shenker A, Gejman PV, Merino MJ, Friedman E, Spiegel AM. Activating mutations of the stimulatory G protein in the McCuneeAlbright syndrome. N Engl J Med 1991;325:1688e95. [8] Schwindinger WF, Francomano CA, Levine MA. Identification of a mutation in the gene encoding the alpha subunit of the stimulatory G protein of adenylyl cyclase in McCuneeAlbright syndrome. Proc Natl Acad Sci USA 1992;89:5152e6. [9] Yamamoto T, Ozono K, Kasayama S, et al. Increased IL-6production by cells isolated from the fibrous bone dysplasia tissues in patients with McCuneeAlbright syndrome. J Clin Invest 1996;98:30e5. [10] Riminucci M, Fisher LW, Shenker A, Spiegel AM, Bianco P, Gehron Robey P. Fibrous dysplasia of bone in the McCuneeAlbright syndrome: abnormalities in bone formation. Am J Pathol 1997;151:1587e600. [11] Shenker A, Chanson P, Weinstein LS, et al. Osteoblastic cells derived from isolated lesions of fibrous dysplasia contain activating somatic mutations of the Gs alpha gene. Hum Mol Genet 1995;4:1675e6. [12] Marie PJ, de Pollak C, Chanson P, Lomri A. Increased proliferation of osteoblastic cells expressing the activating Gs alpha mutation in monostotic and polyostotic fibrous dysplasia. Am J Pathol 1997;150:1059e69. [13] Bianco P, Kuznetsov S, Riminucci M, Fisher LW, Spiegel AM, Gehron Robey P. Reproduction of human fibrous dysplasia of bone in immunocompromised mice by transplanted mosaics of normal and Gs-alpha mutated skeletal progenitor cells. J Clin Invest 1998;101:1737e44. [14] Cole DEC, Fraser FC, Glorieux FH, et al. Panostotic fibrous dysplasia: a congenital disorder of bone with unusual facial

[20]

[21]

[22] [23] [24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

appearance, bone fragility, hyperphosphatasemia, and hypophosphatemia. Am J Med Genet 1983;14:725e35. Ascenzi A. Physiological relationship and pathological interferences between bone tissue and marrow. In: Bourne GH, editor. The Biochemistry and Physiology of Bone. 2nd ed. New York: Academic Press; 1976. p. 403e45. MacMahon HE. Albright’s syndrome e thirty years later. Pathol Annu 1971;6:81e146. Dorfman HD, Ishida T, Tsuneyoshi M. Exophytic variant of fibrous dysplasia (fibrous dysplasia protuberans). Hum Pathol 1994;25:1234e7. Dorfman HD, Czerniak B. Bone Tumors. St Louis: Mosby; 1997. Comite F, Shawker TH, Pescovitz OH, Loriaux DL, Cutler Jr GB. Cyclical ovarian function resistant to treatment with an analogue of luteinizing hormone releasing hormone in McCuneeAlbright syndrome. N Engl J Med 1984;311:1032e6. Foster CM, Feuillan P, Padmanabhan V, et al. Ovarian function in girls with McCuneeAlbright syndrome. Pediatr Res 1986;20:859e63. Foster CM, Levine Ross J, Shawker T, et al. Absence of pubertal gonadotropin secretion in girls with McCuneeAlbright syndrome. J Clin Endo Metab 1984;58:1161e5. Feuillan PP. McCuneeAlbright syndrome. Curr Ther Endocrinol Metab 1994;5:205e9. Feuillan PP. McCuneeAlbright syndrome. Curr Ther Endocrinol Metab 1997;6:235e9. Pienkowski C, Lumbroso S, Bieth E, Sultan C, Rochiccioli P, Tauber M. Recurrent ovarian cyst and mutation of the Gs alpha gene in ovarian cyst fluid cells: what is the link with McCuneeAlbright syndrome? Acta Paediatr 1997;86:1019e21. Arrigo T, Pirazzoli P, De Sanctis L, et al. McCuneeAlbright syndrome in a boy may present with a monolateral macroorchidism as an early and isolated clinical manifestation. Horm Res 2006;65:114e9. Chakrabarti S, Marshall I. Another unusual presentation of McCune Albright syndrome with fibrous dysplasia, unilateral testicular enlargement, and testicular microlithiasis. J Pediatr Endocrinol Metab 2010;23:513e5. De Luca F, Mitchell V, Wasniewska M, et al. Regulation of spermatogenesis in McCuneeAlbright syndrome: lessons from a 15-year follow-up. Eur J Endocrinol 2008;158:921e7. Mamkin I, Philibert P, Anhalt H, Ten S, Sultan C. Unusual phenotypical variations in a boy with McCuneeAlbright syndrome. Horm Res Paediatr 2010;73:215e22. Rey RA, Venara M, Coutant R, et al. Unexpected mosaicism of R201H-GNAS1 mutant-bearing cells in the testes underlie macroorchidism without sexual precocity in McCuneeAlbright syndrome. Hum Mol Genet 2006;15:3538e43. Savas Erdeve S, Balta H, Balta Z, Dallar Y. Testicular microlithiasis and McCuneeAlbright syndrome. J Pediatr 2006;148:422. Author reply 422e3. Wasniewska M, De Luca F, Bertelloni S, et al. Testicular microlithiasis: an unreported feature of McCuneeAlbright syndrome in males. J Pediatr 2004;145:670e2. Akintoye SO, Chebli C, Booher S, et al. Characterization of gspmediated growth hormone excess in the context of McCunee Albright syndrome. J Clin Endocrinol Metab 2002;87:5104e12. Barlier A, Gunz G, Zamora AJ, et al. Prognostic and therapeutic consequences of Gs alpha mutations in somatotroph adenomas. J Clin Endocrinol Metab 1998;83:1604e10. Dotsch J, Kiess W, Hanze J, et al. Gs alpha mutation at codon 201 in pituitary adenoma causing gigantism in a 6-year-old boy with McCuneeAlbright syndrome. J Clin Endocrinol Metab 1996;81:3839e42.

PEDIATRIC BONE

REFERENCES

[35] Nakagawa H, Nagasaka A, Sugiura T, et al. Gigantism associated with McCuneeAlbright’s syndrome. Horm Metab Res 1985;17:522e7. [36] Pacini F, Perri G, Bagnolesi P, Cilotti A, Pinchera A. McCuneeAlbright syndrome with gigantism and hyperprolactinemia. J Endocrinol Invest 1987;10:417e20. [37] Polychronakos C, Tsoukas G, Ducharme JR, Letarte J, Collu R. Gigantism and hyperprolactinemia in polyostotic fibrous dysplasia (McCuneeAlbright syndrome). J Endocrinol Invest 1982;5:323e6. [38] Queirolo S, Gallarotti F, Capuano E, Garre ML, Spaziante R, Di Battista E. Gigantism with pituitary macroadenoma: an unusual variant of McCuneeAlbright syndrome. J Pediatr Endocrinol Metab 2009;22:177e9. [39] Chanson P, Dib A, Visot A, Derome PJ. McCuneeAlbright syndrome and acromegaly: clinical studies and responses to treatment in five cases. Eur J Endocrinol 1994;131:229e34. [40] Daly BD, Chow CC, Cockram CS. Unusual manifestations of craniofacial fibrous dysplasia: clinical, endocrinological and computed tomographic features. Postgrad Med J 1994;70:10e6. [41] Boston BA, Mandel S, LaFranchi S, Bliziotes M. Activating mutation in the stimulatory guanine nucleotide-binding protein in an infant with Cushing’s syndrome and nodular adrenal hyperplasia. J Clin Endocrinol Metab 1994;79:890e3. [42] Danon M, Robboy SJ, Kim S, Scully R, Crawford JD. Cushing syndrome, sexual precocity, and polyostotic fibrous dysplasia (Albright syndrome) in infancy. J Pediatr 1975;87:917e21. [43] Kirk JMW, Brain CE, Carson DJ, Hyde JC, Grant DB. Cushing’s syndrome caused by nodular adrenal hyperplasia in children with McCuneeAlbright syndrome. J Pediatr 1999;134:789e92. [44] Brown RJ, Kelly MH, Collins MT. Cushing syndrome in the McCuneeAlbright syndrome. J Clin Endocrinol Metab 2010;95:1508e15. [45] Paris F, Philibert P, Lumbroso S, Servant N, Kalfa N, Sultan C. Isolated Cushing’s syndrome: an unusual presentation of McCuneeAlbright syndrome in the neonatal period. Horm Res 2009;72:315e9. [46] Gillis D, Rosler A, Hannon TS, Koplewitz BZ, Hirsch HJ. Prolonged remission of severe Cushing syndrome without adrenalectomy in an infant with McCuneeAlbright syndrome. J Pediatr 2008;152:882e4. 884. [47] Fragoso MC, Domenice S, Latronico AC, et al. Cushing’s syndrome secondary to adrenocorticotropin-independent macronodular adrenocortical hyperplasia due to activating mutations of GNAS1 gene. J Clin Endocrinol Metab 2003;88:2147e51. [48] Riminucci M, Collins M, Lala R, et al. An R201H activating mutation of the GNAS1 (Gs alpha) gene in a corticotroph pituitary adenoma. Mol Pathol 2002;55:58e60. [49] Williamson EA, Ince PG, Harrison D, Kendall-Taylor P, Harris PE. G-protein mutations in human pituitary adrenocorticotrophic hormone-secreting adenomas. Eur J Clin Invest 1995;25:128e31. [50] Mastorakos G, Mitsiades NS, Doufas AG, Koutras DA. Hyperthyroidism in McCuneeAlbright syndrome with a review of thyroid abnormalities sixty years after the first report. Thyroid 1997;7:433e9. [51] Collins MT, Sarlis NJ, Merino MJ, et al. Thyroid carcinoma in the McCuneeAlbright syndrome: contributory role of activating Gs alpha mutations. J Clin Endocrinol Metab 2003;88:4413e7. [52] Kaplan FS, Fallon MD, Boden SD, Schmidt R, Senior M, Haddad JG. Estrogen receptors in bone in a patient with polyostotic fibrous dysplasia (McCuneeAlbright syndrome). N Engl J Med 1988;319:421e5.

617

[53] Kim IS, Kim ER, Nam HJ, et al. Activating mutation of GS alpha in McCuneeAlbright syndrome causes skin pigmentation by tyrosinase gene activation on affected melanocytes. Horm Res 1999;52:235e40. [54] Henschen F, Fallon H. Ostitis fibrosa mit multiplen tumores in der umgebenden muskulature. Verh Dtsch Ges Pathol 1926;21:93e7. [55] Mazabraud A, Girard J. Un cas particulier de dysplasis fibreuse a localisations osseuses et tendineuses. Rev Rhum 1957;34:652e9. [56] Mazabraud A, Semat P, Rose R. A propos de l’association de fibromixomes des tissus mous a la dysplasie fibreuse de l’os. Press Med 1967;75:2223e8. [57] Aoki T, Kouho H, Hisaoka M, Hashimoto H, Nakata H, Sakai A. Intramuscular myxoma with fibrous dysplasia: a report of two cases with a review of the literature. Pathol Int 1995;45:165e71. [58] Cabral CE, Guedes P, Fonseca T, Rezende JF, Cruz Junior LC, Smith J. Polyostotic fibrous dysplasia associated with intramuscular myxomas: Mazabraud’s syndrome. Skeletal Radiol 1998;27:278e82. [59] Szendroi M, Rahoty P, Antal I, Kiss J. Fibrous dysplasia associated with intramuscular myxoma (Mazabraud’s syndrome): a long-term follow-up of three cases. J Cancer Res Clin Oncol 1998;124:401e6. [60] Thomachot B, Daumen-Legre V, Pham T, Acquaviva PC, Lafforgue P. Fibrous dysplasia with intramuscular myxoma (Mazabraud’s syndrome). Report of a case and review of the literature. Rev Rhum Engl Ed 1999;66:180e3. [61] Wirth WA, Leavitt D, Enzinger FM. Multiple intramuscular myxomas: another extraskeletal manifestation of fibrous dysplasia. Cancer 1971;27:1167e73. [62] Calisir C, Inan U, Yavas US, Isiksoy S, Kaya T. Mazabraud’s syndrome coexisting with a uterine tumor resembling an ovarian sex cord tumor (UTROSCT): a case report. Korean J Radiol 2007;8:438e42. [63] Endo M, Kawai A, Kobayashi E, et al. Solitary intramuscular myxoma with monostotic fibrous dysplasia as a rare variant of Mazabraud’s syndrome. Skeletal Radiol 2007;36:523e9. [64] Santos CT, Choo CT, Loh AH. Orbital fibrous dysplasia with soft tissue hamartoma e a variant of Mazabraud’s syndrome. Orbit 2008;27:207e9. [65] Zoccali C, Teori G, Prencipe U, Erba F. Mazabraud’s syndrome: a new case and review of the literature. Int Orthop 2009;33:605e10. [66] Okamoto S, Hisaoka M, Ushijima M, Nakahara S, Toyoshima S, Hashimoto H. Activating Gs(alpha) mutation in intramuscular myxomas with and without fibrous dysplasia of bone. Virchows Arch 2000;437:133e7. [67] Delaney D, Diss TC, Presneau N, et al. GNAS1 mutations occur more commonly than previously thought in intramuscular myxoma. Mod Pathol 2009;22:718e24. [68] Tsai CC, Saffitz JE, Billadello JJ. Expression of the Gs protein alpha-subunit disrupts the normal program of differentiation in cultured murine myogenic cells. J Clin Invest 1997;99:67e76. [69] Corsi A, De Maio F, Ippolito E, et al. Monostotic fibrous dysplasia of the proximal femur and liposclerosing myxofibrous tumor: which one is which? J Bone Miner Res 2006;21:1955e8. [70] Heim-Hall JM, Williams RP. Liposclerosing myxofibrous tumour: a traumatized variant of fibrous dysplasia? Report of four cases and review of the literature. Histopathology 2004;45:369e76. [71] Matsuba A, Ogose A, Tokunaga K, et al. Activating Gs alpha mutation at the Arg201 codon in liposclerosing myxofibrous tumor. Hum Pathol 2003;34:1204e9.

PEDIATRIC BONE

618

22. FIBROUS DYSPLASIA

[72] Silva ES, Lumbroso S, Medina M, Gillerot Y, Sultan C, Sokal EM. Demonstration of McCuneeAlbright mutations in the liver of children with high gammaGT progressive cholestasis. J Hepatol 2000;32:154e8. [73] El-Rifai N, Lumbroso S, Cartigny M, Weill J, Sultan C, Gottrand F. Infant cholestasis in McCuneeAlbright syndrome. Acta Paediatr 2004;93:141. [74] Lumbroso S, Paris F, Sultan C. Activating Gsalpha mutations: analysis of 113 patients with signs of McCuneeAlbright syndrome e a European Collaborative Study. J Clin Endocrinol Metab 2004;89:2107e13. [75] Shenker A, Weinstein LS, Moran A, et al. Severe endocrine and nonendocrine manifestations of the McCuneeAlbright syndrome associated with activating mutations of stimulatory G protein GS. J Pediatr 1993;123:509e18. [76] Bajpai A, Greenway A, Zacharin M. Platelet dysfunction and increased bleeding tendency in McCuneeAlbright syndrome. J Pediatr 2008;153:287e9. [77] Belcastro V, Parnetti L, Prontera P, Donti E, Rossi A, Tambasco N. Hot water epilepsy and McCuneeAlbright syndrome: a case report. Seizure 2009;18:161e2. [78] Cabanillas M, Aneiros A, Monteagudo B, Santos-Garcia D, Suarez-Amor O, Ramirez-Santos A. Epidermal nevus syndrome associated with polyostotic fibrous dysplasia, CNS lipoma, and aplasia cutis. Dermatol Online J 2009;15:7. [79] Chen ST, Fan PC, Hwu WL, Wu MH. Fibrous dysplasia in a child with mitochondrial A8344G mutation. J Child Neurol 2008;23:1447e50. [80] Gasparetto EL, de Carvalho Neto A, Bruck I, Antoniuk S. Tuberous sclerosis and fibrous dysplasia. Am J Neuroradiol 2003;24:835e7. [81] Ghosal N, Furtado SV, Santosh V, Sridhar M, Hegde AS. Coexisting fibrous dysplasia and atypical lymphoplasmacyte-rich meningioma. Neuropathology 2007;27:269e72. [82] Huston TL, Simmons RM. Ductal carcinoma in situ in a 27-yearold woman with McCuneeAlbright syndrome. Breast J 2004;10:440e2. [83] Kate A, Gothi D, Joshi JM. Marfan syndrome with multiseptate pneumothorax and mandibular fibrous dysplasia. Lung India 2009;26:146e8. [84] Narayan RL, Maldjian PD. Restrictive lung disease and cor pulmonale secondary to polyostotic fibrous dysplasia. Int J Cardiol 2009;131:e48e50. [85] Ohata Y, Yamamoto T, Mori I, et al. Severe arterial hypertension: a possible complication of McCuneeAlbright syndrome. Eur J Pediatr 2009;168:871e6. [86] Ozcan-Kara P, Mahmoudian B, Erbas B, Erbas T. McCunee Albright syndrome associated with acromegaly and bipolar affective disorder. Eur J Intern Med 2007;18:600e2. [87] Roman R, Johnson MC, Codner E, Boric MA, aVila A, Cassorla F. Activating GNAS1 gene mutations in patients with premature thelarche. J Pediatr 2004;145:218e22. [88] Tasar M, Ors F, Yetiser S, Ugurel MS, Ucoz T. Multiple globoid meningiomas associated with craniomandibular fibrous dysplasia: case report. Clin Imaging 2004;28:20e2. [89] Collins MT, Chebli C, Jones J, et al. Renal phosphate wasting in fibrous dysplasia of bone is part of a generalized renal tubular dysfunction similar to that seen in tumor-induced osteomalacia. J Bone Miner Res 2001;16:806e13. [90] Dent CE, Gertner JM. Hypophosphataemic osteomalacia in fibrous dysplasia. Q J Med 1976;45:411e20. [91] Zung A, Chalew SA, Schwindinger WF, et al. Urinary cyclic adenosine 30 ,50 -monophosphate response in McCuneeAlbright syndrome: clinical evidence for altered renal adenylate cyclase activity. J Clin Endocrinol Metab 1995;80:3576e81.

[92] Yamamoto T, Miyamoto K, Ozono K, et al. Hypophosphatemic rickets accompanying McCuneeAlbright syndrome: evidence that a humoral factor causes hypophosphatemia. J Bone Miner Metab 2001;19:287e95. [93] White KE, Jonsson KB, Karn G, et al. The autosomal dominant hypophosphatemic rickets (ADHR) gene is a secreted polypeptide overexpressed by tumors that cause phosphate wasting. J Clin Endocrinol Metab 2001;86:497e500. [94] Riminucci M, Collins MT, Fedarko NS, et al. FGF-23 in fibrous dysplasia of bone and its relationship to renal phosphate wasting. J Clin Invest 2003;112:683e92. [95] Kobayashi K, Imanishi Y, Koshiyama H, et al. Expression of FGF23 is correlated with serum phosphate level in isolated fibrous dysplasia. Life Sci 2006;78:2295e301. [96] de Sanctis C, Lala R, Matarazzo P, et al. McCuneeAlbright syndrome: a longitudinal clinical study of 32 patients. J Pediatr Endocrinol Metab 1999;12:817e26. [97] Hart ES, Kelly MH, Brillante B, et al. Onset, progression, and plateau of skeletal lesions in fibrous dysplasia and the relationship to functional outcome. J Bone Miner Res 2007;22:1468e74. [98] Malchoff CD, Reardon G, MacGillivray DC, Yamase H, Rogol AD, Malchoff DM. An unusual presentation of McCuneeAlbright syndrome confirmed by an activating mutation of the Gs alpha-subunit from a bone lesion. J Clin Endocrinol Metab 1994;78:803e6. [99] Hall MB, Sclar AG, Gardner DF. Albright’s syndrome with reactivation of fibrous dysplasia secondary to pituitary adenoma and further complicated by osteogenic sarcoma. Oral Surg 1984;57:616e9. [100] Kozasa T, Itoh H, Tsukamoto T, Kaziro Y. Isolation and characterization of the human Gs alpha gene. Proc Natl Acad Sci USA 1988;85:2081e5. [101] Weinstein LS. The stimulatory G protein alpha-subunit gene: mutations and imprinting lead to complex phenotypes. J Clin Endocrinol Metab 2001;86:4662e6. [102] Rao VV, Schnittger S, Hansmann I. G protein Gs alpha (GNAS 1), the probable candidate gene for Albright hereditary osteodystrophy, is assigned to human chromosome 20q12-q13.2. Genomics 1991;10:257e61. [103] Hayward BE, Kamiya M, Strain L, et al. The human GNAS1 gene is imprinted and encodes distinct paternally and biallelically expressed G proteins. Proc Natl Acad Sci USA 1998;95:10038e43. [104] Hayward BE, Moran V, Strain L, Bonthron DT. Bidirectional imprinting of a single gene: GNAS1 encodes maternally, paternally, and biallelically derived proteins. Proc Natl Acad Sci USA 1998;95:15475e80. [105] Bastepe M. The GNAS Locus: quintessential complex gene encoding Gsalpha, XLalphas, and other imprinted transcripts. Curr Genomics 2007;8:398e414. [106] Weinstein LS, Yu S. The role of genomic imprinting of Galpha in the pathogenesis of Albright hereditary osteodystrophy. Trends Endocrinol Metab 1999;10:81e5. [107] Swaroop A, Agarwal N, Gruen JR, Bick D, Weissman SM. Differential expression of novel Gs alpha signal transduction protein cDNA species. Nucleic Acids Res 1991;19:4725e9. [108] Kehlenbach RH, Matthey J, Huttner WB. XL alpha s is a new type of G protein. Nature 1994;372:804e9. [109] Pasolli HA, Huttner WB. Expression of the extra-large G protein alpha-subunit XLalphas in neuroepithelial cells and young neurons during development of the rat nervous system. Neurosci Lett 2001;301:119e22. [110] Pasolli HA, Klemke M, Kehlenbach RH, Wang Y, Huttner WB. Characterization of the extra-large G protein alpha-subunit

PEDIATRIC BONE

REFERENCES

[111]

[112]

[113]

[114]

[115]

[116]

[117]

[118]

[119]

[120]

[121] [122]

[123]

[124]

[125]

[126]

XLalphas. I. Tissue distribution and subcellular localization. J Biol Chem 2000;275:33622e32. Plagge A, Gordon E, Dean W, et al. The imprinted signaling protein XL alpha s is required for postnatal adaptation to feeding. Nat Genet 2004;36:818e26. Plagge A, Kelsey G, Germain-Lee EL. Physiological functions of the imprinted Gnas locus and its protein variants Galpha(s) and XLalpha(s) in human and mouse. J Endocrinol 2008;196:193e214. Bastepe M, Gunes Y, Perez-Villamil B, Hunzelman J, Weinstein LS, Juppner H. Receptor-mediated adenylyl cyclase activation through XLalpha(s), the extra-large variant of the stimulatory G protein alpha-subunit. Mol Endocrinol 2002;16:1912e9. Klemke M, Pasolli HA, Kehlenbach RH, Offermanns S, Schultz G, Huttner WB. Characterization of the extra-large G protein alpha-subunit XLalphas. II. Signal transduction properties. J Biol Chem 2000;275:33633e40. Linglart A, Mahon MJ, Kerachian MA, et al. Coding GNAS mutations leading to hormone resistance impair in vitro agonist- and cholera toxin-induced adenosine cyclic 30 ,50 monophosphate formation mediated by human XLalphas. Endocrinology 2006;147:2253e62. Ischia R, Lovisetti-Scamihorn P, Hogue-Angeletti R, Wolkersdorfer M, Winkler H, Fischer-Colbrie R. Molecular cloning and characterization of NESP55, a novel chromograninlike precursor of a peptide with 5-HT1B receptor antagonist activity. J Biol Chem 1997;272:11657e62. Wroe SF, Kelsey G, Skinner JA, et al. An imprinted transcript, antisense to Nesp, adds complexity to the cluster of imprinted genes at the mouse Gnas locus. Proc Natl Acad Sci USA 2000;97:3342e6. Freson K, Jaeken J, Van Helvoirt M, et al. Functional polymorphisms in the paternally expressed XLalphas and its cofactor ALEX decrease their mutual interaction and enhance receptor-mediated cAMP formation. Hum Mol Genet 2003;12:1121e30. Klemke M, Kehlenbach RH, Huttner WB. Two overlapping reading frames in a single exon encode interacting proteins e a novel way of gene usage. Embo J 2001;20:3849e60. Holmes R, Williamson C, Peters J, Denny P, Wells C. A comprehensive transcript map of the mouse Gnas imprinted complex. Genome Res 2003;13:1410e5. Granneman JG, Bannon MJ. Splicing pattern of Gs alpha mRNA in human and rat brain. J Neurochem 1991;57:1019e23. Bray P, Carter A, Simons C, et al. Human cDNA clones for four species of G alpha s signal transduction protein. Proc Natl Acad Sci USA 1986;83:8893e7. Robishaw JD, Russell DW, Harris BA, Smigel MD, Gilman AG. Deduced primary structure of the alpha subunit of the GTPbinding stimulatory protein of adenylate cyclase. Proc Natl Acad Sci USA 1986;83:1251e5. Novotny J, Svoboda P. The long (Gs(alpha)-L) and short (Gs(alpha)-S) variants of the stimulatory guanine nucleotidebinding protein. Do they behave in an identical way? J Mol Endocrinol 1998;20:163e73. Frey UH, Nuckel H, Dobrev D, et al. Quantification of G protein Gaalphas subunit splice variants in different human tissues and cells using pyrosequencing. Gene Expr 2005;12:69e81. Ihnatovych I, Hejnova L, Kostrnova A, Mares P, Svoboda P, Novotny J. Maturation of rat brain is accompanied by differential expression of the long and short splice variants of G(s) alpha protein: identification of cytosolic forms of G(s)alpha. J Neurochem 2001;79:88e97.

619

[127] van der Vuurst H, Hendriks M, Lapetina EG, van Willigen G, Akkerman JW. Maturation of megakaryoblastic cells is accompanied by upregulation of G(s)alpha-L subtype and increased cAMP accumulation. Thromb Haemost 1998;79:1014e20. [128] Novotny J, Svoboda P. The long (Gs(alpha)-L) and short (Gs(alpha)-S) variants of the stimulatory guanine nucleotidebinding protein. Do they behave in an identical way? J Mol Endocrinol 1998;20:163e73. [129] Kvapil P, Novotny J, Svoboda P, Ransnas LA. The short and long forms of the alpha subunit of the stimulatory guaninenucleotide-binding protein are unequally redistributed during ()-isoproterenol-mediated desensitization of intact S49 lymphoma cells. Eur J Biochem 1994;226:193e9. [130] Unson CG, Wu CR, Sakmar TP, Merrifield RB. Selective stabilization of the high affinity binding conformation of glucagon receptor by the long splice variant of Galpha(s). J Biol Chem 2000;275:21631e8. [131] Wenzel-Seifert K, Kelley MT, Buschauer A, Seifert R. Similar apparent constitutive activity of human histamine H(2)receptor fused to long and short splice variants of G(salpha). J Pharmacol Exp Ther 2001;299:1013e20. [132] Reik W, Walter J. Genomic imprinting: parental influence on the genome. Nat Rev Genet 2001;2:21e32. [133] Hayward BE, Kamiya M, Strain L, et al. The human GNAS1 gene is imprinted and encodes distinct paternally and biallelically expressed G proteins. Proc Natl Acad Sci USA 1998;95:10038e43. [134] Hayward BE, Moran V, Strain L, Bonthron DT. Bidirectional imprinting of a single gene: GNAS1 encodes maternally, paternally, and biallelically derived proteins. Proc Natl Acad Sci USA 1998;95:15475e80. [135] Liu J, Chen M, Deng C, et al. Identification of the control region for tissue-specific imprinting of the stimulatory G protein alpha-subunit. Proc Natl Acad Sci USA 2005;102:5513e8. [136] Germain-Lee EL, Ding CL, Deng Z, et al. Paternal imprinting of Galpha(s) in the human thyroid as the basis of TSH resistance in pseudohypoparathyroidism type 1a. Biochem Biophys Res Commun 2002;296:67e72. [137] Hayward BE, Barlier A, Korbonits M, et al. Imprinting of the G(s)alpha gene GNAS1 in the pathogenesis of acromegaly. J Clin Invest 2001;107:R31e6. [138] Mantovani G, Ballare E, Giammona E, Beck-Peccoz P, Spada A. The gsalpha gene: predominant maternal origin of transcription in human thyroid gland and gonads. J Clin Endocrinol Metab 2002;87:4736e40. [139] Michienzi S, Cherman N, Holmbeck K, et al. GNAS transcripts in skeletal progenitors: evidence for random asymmetric allelic expression of Gs alpha. Hum Mol Genet 2007;16:1921e30. [140] Mantovani G, Bondioni S, Locatelli M, et al. Biallelic expression of the Gsalpha gene in human bone and adipose tissue. J Clin Endocrinol Metab 2004;89:6316e9. [141] Constancia M, Pickard B, Kelsey G, Reik W. Imprinting mechanisms. Genome Res 1998;8:881e900. [142] Yan H, Zhou W. Allelic variations in gene expression. Curr Opin Oncol 2004;16:39e43. [143] Vallar L, Spada A, Giannattasio G. Altered Gs and adenylate cyclase activity in human GH secreting pituitary adenomas. Nature 1987;330:566e8. [144] Landis CA, Masters SB, Spada A, Pace AM, Bourne HR, Vallar L. GTPase inhibiting mutations activate the alpha chain of Gs and stimulate adenylyl cyclase in human pituitary tumours. Nature 1989;340:692e6. [145] Lyons J, Landis CA, Harsh G, et al. Two G protein oncogenes in human endocrine tumors. Science 1990;249:655e9.

PEDIATRIC BONE

620

22. FIBROUS DYSPLASIA

[146] Suarez HG, du Villard JA, Caillou B, Schlumberger M, Parmentier C, Monier R. gsp mutations in human thyroid tumours. Oncogene 1991;6:677e9. [147] Spada A, Lania A, Ballare E. G protein abnormalities in pituitary adenomas. Mol Cell Endocrinol 1998;142:1e14. [148] Ivan M. An amphotropic retroviral vector expressing a mutant gsp oncogene: effects on human thyroid cells in vitro. J Clin Endocrinol Metab 1997;82:2702e9. [149] Chen J, Iyengar R. Suppression of Ras-induced transformation of NIH 3T3 cells by activated G alpha s. Science 1994;263:1278e81. [150] Shenker A, Weinstein LS, Sweet DE, Spiegel AM. An activating Gs alpha mutation is present in fibrous dysplasia of bone in the McCuneeAlbright syndrome. J Clin Endocrinol Metab 1994;79:750e5. [151] Alman BA, Greel DA, Wolfe HJ. Activating mutations of Gs protein in monostotic fibrous lesions of bone. J Orthop Res 1996;14:311e5. [152] Bianco P, Riminucci M, Majolagbe A, et al. Mutations of the GNAS1 gene, stromal cell dysfunction, and osteomalacic changes in non-McCuneeAlbright fibrous dysplasia of bone. J Bone Miner Res 2000;15:120e8. [153] Candeliere GA, Roughley PJ, Glorieux FH. Polymerase chain reaction-based technique for the selective enrichment and analysis of mosaic arg201 mutations in G alpha s from patients with fibrous dysplasia of bone. Bone 1997;21:201e6. [154] Riminucci M, Fisher LW, Majolagbe A, et al. A novel GNAS1 mutation, R201G, in McCuneeAlbright syndrome. J Bone Miner Res 1999;14:1987e9. [155] Gorelov VN, Dumon K, Barteneva NS, Palm D, Roher HD, Goretzki PE. Overexpression of Gs alpha subunit in thyroid tumors bearing a mutated Gs alpha gene. J Cancer Res Clin Oncol 1995;121:219e24. [156] Cooper DN, Krawczak M. The mutational spectrum of single base-pair substitutions causing human genetic disease: patterns and predictions. Hum Genet 1990;85:55e74. [157] Bourne HR. How receptors talk to trimeric G proteins. Curr Opin Cell Biol 1997;9:134e42. [158] Spiegel AM. Inborn errors of signal transduction: mutations in G proteins and G protein-coupled receptors as a cause of disease. J Inherit Metab Dis 1997;20:113e21. [159] Spiegel AM. The molecular basis of disorders caused by defects in G proteins. Horm Res 1997;47:89e96. [160] Bourne HR, Landis CA, Masters SB. Hydrolysis of GTP by the alpha-chain of Gs and other GTP binding proteins. Proteins 1989;6:222e30. [161] Cassel D, Selinger Z. Mechanism of adenylate cyclase activation by cholera toxin: inhibition of GTP hydrolysis at the regulatory site. Proc Natl Acad Sci USA 1977;74:3307e11. [162] Idowu BD, Al-Adnani M, O’Donnell P, et al. A sensitive mutation-specific screening technique for GNAS1 mutations in cases of fibrous dysplasia: the first report of a codon 227 mutation in bone. Histopathology 2007;50:691e704. [163] Wedegaertner PB, Bourne HR, von Zastrow M. Activationinduced subcellular redistribution of Gs alpha. Mol Biol Cell 1996;7:1225e33. [164] Iyengar R. Gating by cyclic AMP: expanded role for an old signaling pathway. Science 1996;271:461e3. [165] Lania A, Persani L, Ballare E, Mantovani S, Losa M, Spada A. Constitutively active Gs alpha is associated with an increased phosphodiesterase activity in human growth hormonesecreting adenomas. J Clin Endocrinol Metab 1998:1624e8. [166] Nemoz G, Sette C, Hess M, Muca C, Conti M. Activation of cyclic nucleotide phosphodiesterases in FRTL-5 thyroid cells

[167]

[168]

[169]

[170] [171] [172] [173]

[174]

[175]

[176]

[177]

[178]

[179]

[180]

[181]

[182] [183]

[184]

[185]

expressing a constitutively active Gs alpha. Mol Endocrinol 1995;9:1279e87. Zachary I, Masters SB, Bourne HR. Increased mitogenic responsiveness of Swiss 3T3 cells expressing constitutively active Gs alpha. Biochem Biophys Res Commun 1990;168:1184e93. Piersanti S, Remoli C, Saggio I, et al. Transfer, analysis, and reversion of the fibrous dysplasia cellular phenotype in human skeletal progenitors. J Bone Miner Res 2010;25:1103e16. Ballare E, Mantovani S, Lania A, Di Blasio AM, Vallar L, Spada A. Activating mutations of the Gs alpha gene are associated with low levels of Gs alpha protein in growth hormonesecreting tumors. J Clin Endocrinol Metab 1998;83:4386e90. Happle R. The McCuneeAlbright syndrome: a lethal gene surviving by mosaicism. Clin Genet 1986;29:321e4. Riminucci M, Saggio I, Robey PG, Bianco P. Fibrous dysplasia as a stem cell disease. J Bone Miner Res 2006;21(Suppl 2):P125e31. Bartolomei MS, Tilghman SM. Genomic imprinting in mammals. Annu Rev Genet 1997;31:493e525. Liu J, Yu S, Litman D, Chen W, Weinstein LS. Identification of a methylation imprint mark within the mouse Gnas locus. Mol Cell Biol 2000;20:5808e17. Weinstein LS, Chen M, Xie T, Liu J. Genetic diseases associated with heterotrimeric G proteins. Trends Pharmacol Sci 2006;27:260e6. Weinstein LS, Liu J, Sakamoto A, Xie T, Chen M. Minireview: GNAS: normal and abnormal functions. Endocrinology 2004;145:5459e64. Yu S, Gavrilova O, Chen H, et al. Paternal versus maternal transmission of a stimulatory G-protein alpha subunit knockout produces opposite effects on energy metabolism. J Clin Invest 2000;105:615e23. Yu S, Yu D, Lee E, et al. Variable and tissue-specific hormone resistance in heterotrimeric Gs protein alpha-subunit (Gsalpha) knockout mice is due to tissue-specific imprinting of the gsalpha gene. Proc Natl Acad Sci USA 1998;95:8715e20. Zheng H, Radeva G, McCann JA, Hendry GN, Goodyer CG. G alpha s transcripts are biallelically expressed in human kidney cortex: implications for pseudohypoparathyroidism type Ib. J Clin Endocrinol Metab 2001;86:4627e9. Mantovani G, Bondioni S, Lania AG, et al. Parental origin of Gsalpha mutations in the McCuneeAlbright syndrome and in isolated endocrine tumors. J Clin Endocrinol Metab 2004;89:3007e9. Campbell R, Gosden CM, Bonthron DT. Parental origin of transcription from the human GNAS1 gene. J Med Genet 1994;31:607e14. Weinstein LS, Yu S, Ecelbarger CA. Variable imprinting of the heterotrimeric G protein G(s) alpha-subunit within different segments of the nephron. Am J Physiol Renal Physiol 2000;278:F507e14. Harris WH, Dudley HR, Barry RJ. The natural history of fibrous dysplasia. J Bone Joint Surg 1962;44A:207e33. Riminucci M, Liu B, Corsi A, et al. The histopathology of fibrous dysplasia of bone in patients with activating mutations of the Gs alpha gene: site-specific patterns and recurrent histological hallmarks. J Pathol 1999;187:249e58. Miller SS, Wolf AM, Arnaud CD. Bone cells in culture: morphologic transformation by hormones. Science 1976;192:1340e3. Corsi A, Collins MT, Riminucci M, et al. Osteomalacic and hyperparathyroid changes in fibrous dysplasia of bone: core biopsy studies and clinical correlations. J Bone Miner Res 2003;18:1235e46.

PEDIATRIC BONE

REFERENCES

[186] Bianco P. Structure and mineralization of bone. In: Bonucci E, editor. Mineralization in Biological Systems. Boca Raton: CRC Press; 1992. p. 243e68. [187] Gehron Robey P, Bianco P. The cellular biology and molecular biochemistry of bone formation. In: Coe, Favus, editors. Disorders of Bone and Mineral Metabolism. 2nd edn. New York: Raven Press; 2002. p. 199e226. [188] Boyde A, Maconnachie E, Reid SA, Delling G, Mundy GR. Scanning electron microscopy in bone pathology: review of methods, potential and applications. Scan. Electron Microsc 1986;(Pt 4):1537e54. [189] Boyde A, Travers R, Glorieux FH, Jones SJ. The mineralization density of iliac crest bone from children with osteogenesis imperfecta. Calcif Tissue Int 1999;64:185e90. [190] Yamamoto T. Clinical approach to clarifying the mechanism of abnormal bone metabolism in McCuneeAlbright syndrome. J Bone Miner Metab 2006;24:7e10. [191] Delling G. Bone morphology in primary hyperparathyroidism e a qualitative and quantitative study of 391 cases. Appl Pathol 1987;5:147e59. [192] Arden RL, Bahu SJ, Lucas DR. Mandibular aneurysmal bone cyst associated with fibrous dysplasia. Otolaryngol Head Neck Surg 1997;117:S153e6. [193] Bandiera S, Bacchini P, Bertoni F. Secondary aneurysmal bone cyst simulating malignant transformation in fibrous dysplasia. Orthopedics 2000;23:1205e7. [194] Burd TA, Lowry KJ, Stokesbary SJ, Allen IC. Aneurysmal bone cyst associated with fibrous dysplasia. Orthopedics 2001;24:1087e9. [195] Suzuki F, Fukuda S, Yagi K, Chida E, Inuyama Y. A rare aneurysmal bone cyst of the maxillary sinus: a case report. Auris Nasus Larynx 2001;28(Suppl):S131e7. [196] Dowler JG, Sanders MD, Brown PM. Bilateral sudden visual loss due to sphenoid mucocele in Albright’s syndrome. Br J Ophthalmol 1995;79:503e4. [197] Hirabayashi S, Kagawa K, Ohkubo E, Sugawara Y, Sakurai A. Craniofacial fibrous dysplasia with rapidly increasing proptosis due to a mucocele behind the globe. Ann Plast Surg 1998;40:182e5. [198] Fischer JA, Bollinger A, Lichtlen P, Wellauer J. Fibrous dysplasia of the bone and high cardiac output. Am J Med 1970;49:140e6. [199] Riminucci M, Corsi A, Kuznetsov S, Licci S, Gehron Robey P, Bianco P. Angiogenesis and expression of alpha-sm actin in fibrous dysplasia of bone. Bone 2001;28(s):S124. [200] Schmitt-Graff A, Skalli O, Gabbiani G. Alpha-smooth muscle actin is expressed in a subset of bone marrow stromal cells in normal and pathological conditions. Virchows Arch B Cell Pathol Incl Mol Pathol 1989;57:291e302. [201] Bianco P, Bonucci E. Endosteal surfaces in hyperparathyroidism: an enzyme cytochemical study on low-temperatureprocessed, glycol-methacrylate-embedded bone biopsies. Virchows Arch A Pathol Anat Histopathol 1991;419:425e31. [202] Schipani E, Lanske B, Hunzelman J, et al. 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 1997;94:13689e94. [203] Hermann G, Klein M, Abdelwahab IF, Kenan S. Fibrocartilaginous dysplasia. Skeletal Radiol 1996;25:509e11. [204] Choi IH, Kim CJ, Cho TJ, et al. Focal fibrocartilaginous dysplasia of long bones: report of eight additional cases and literature review. J Pediatr Orthop 2000;20:421e7.

621

[205] Park YK, Unni KK, McLeod RA, Pritchard DJ. Osteofibrous dysplasia: clinicopathologic study of 80 cases. Hum Pathol 1993;24:1339e47. [206] Sakamoto A, Oda Y, Iwamoto Y, Tsuneyoshi M. A comparative study of fibrous dysplasia and osteofibrous dysplasia with regard to Gsalpha mutation at the Arg201 codon: polymerase chain reaction-restriction fragment length polymorphism analysis of paraffin- embedded tissues. J Mol Diagn 2000;2:67e72. [207] Voytek TM, Ro JY, Edeiken J, Ayala AG. Fibrous dysplasia and cemento-ossifying fibroma. A histologic spectrum. Am J Surg Pathol 1995;19:775e81. [208] Campanacci M, Laus M, Boriani S. Multiple non-ossifying fibromata with extraskeletal anomalies: a new syndrome? J Bone Joint Surg 1983;65B:627e32. [209] Ruggieri P, Sim FH, Bond JR, Unni KK. Malignancies in fibrous dysplasia. Cancer 1994;73:1411e24. [210] Hoshi M, Matsumoto S, Manabe J, et al. Malignant change secondary to fibrous dysplasia. Int J Clin Oncol 2006;11:229e35. [211] Hansen MR, Moffat JC. Osteosarcoma of the skull base after radiation therapy in a patient with McCuneeAlbright syndrome: Case report. Skull Base 2003;13:79e83. [212] Jhala DN, Eltoum I, Carroll AJ, et al. Osteosarcoma in a patient with McCuneeAlbright syndrome and Mazabraud’s syndrome: a case report emphasizing the cytological and cytogenetic findings. Hum Pathol 2003;34:1354e7. [213] Nguyen BD, Ram PC. Mazabraud’s syndrome with sarcomatous transformation: scintigraphic and radiologic imaging. Clin Nucl Med 2005;30:829e30. [214] Crawford EA, Brooks JS, Ogilvie CM. Osteosarcoma of the proximal part of the radius in Mazabraud syndrome. A case report. J Bone Joint Surg Am 2009;91:955e60. [215] Dal Cin P, Bertoni F, Bacchini P, Hagemeijer A, Van den Berghe H. Fibrous dysplasia and the short arm of chromosome 12. Histopathology 1999;34:279e80. [216] Dal Cin P, Sciot R, Brys P, et al. Recurrent chromosome aberrations in fibrous dysplasia of the bone: a report of the CHAMP study group. CHromosomes And MorPhology. Cancer Genet Cytogenet 2000;122:30e2. [217] Dal Cin P, Sciot R, Speleman F, et al. Chromosome aberrations in fibrous dysplasia. Cancer Genet Cytogenet 1994;77:114e7. [218] Mertens F, Albert A, Heim S, et al. Clonal structural chromosome aberrations in fibrous dysplasia. Genes Chromosomes Cancer 1994;11:271e2. [219] Gong L, Zhang W, Su Q. Clonal status of fibrous dysplasia. Pathology 2008;40:392e5. [220] Mikami M, Koizumi H, Ishii M, Nakajima H. The identification of monoclonality in fibrous dysplasia by methylation-specific polymerase chain reaction for the human androgen receptor gene. Virchows Arch 2004;444:56e60. [221] Kim GT, Lee JK, Choi BJ, Kim J, Han SH, Kwon YD. Malignant transformation of monostotic fibrous dysplasia in the mandible. J Craniofac Surg 2010;21:601e3. [222] Beuerlein ME, Schuller DE, DeYoung BR. Maxillary malignant mesenchymoma and massive fibrous dysplasia. Arch Otolaryngol Head Neck Surg 1997;123:106e9. [223] Bohay RN, Daley T. Osteosarcoma and fibrous dysplasia: radiographic features in the differential diagnosis: a case report. J Can Dent Assoc 1993;59:931e4. [224] Harris NL, Eilert RE, Davino N, Ruyle S, Edwardson M, Wilson V. Osteogenic sarcoma arising from bony regenerate following Ilizarov femoral lengthening through fibrous dysplasia. J Pediatr Orthop 1994;14:123e9. [225] Lopez-Ben R, Pitt MJ, Jaffe KA, Siegal GP. Osteosarcoma in a patient with McCuneeAlbright syndrome and Mazabraud’s syndrome. Skeletal Radiol 1999;28:522e6.

PEDIATRIC BONE

622

22. FIBROUS DYSPLASIA

[226] Ruggieri P, Sim FH, Bond JR, Unni KK. Osteosarcoma in a patient with polyostotic fibrous dysplasia and Albright’s syndrome. Orthopedics 1995;18:71e5. [227] Blanco P, Schaeverbeke T, Baillet L, Lequen L, Bannwarth B, Dehais J. Chondrosarcoma in a patient with McCuneeAlbright syndrome. In: Rev Rhum Engl, editor. Report of a case, 66; 1999. p. 177e9. [228] Heller AJ, DiNardo LJ, Massey D. Fibrous dysplasia, chondrosarcoma, and McCuneeAlbright syndrome. Am J Otolaryngol 2001;22:297e301. [229] Cheng MH, Chen YR. Malignant fibrous histiocytoma degeneration in a patient with facial fibrous dysplasia. Ann Plast Surg 1997;39:638e42. [230] Ohmori K, Matsui H, Kanamori M, Yudoh K, Yasuda T, Terahata S. Malignant fibrous histiocytoma secondary to fibrous dysplasia. A case report. Int Orthop 1996;20:385e8. [231] Doganavsargil B, Argin M, Kececi B, Sezak M, Sanli UA, Oztop F. Secondary osteosarcoma arising in fibrous dysplasia, case report. Arch Orthop Trauma Surg 2009;129:439e44. [232] Fu CJ, Hsu CY, Shih TT, Wu MZ. Monostotic fibrous dysplasia of the thoracic spine with malignant transformation. J Formos Med Assoc 2004;103:711e4. [233] Reis C, Genden EM, Bederson JB, Som PM. A rare spontaneous osteosarcoma of the calvarium in a patient with long-standing fibrous dysplasia: CT and MR findings. Br J Radiol 2008;81:e31e4. [234] Fukuroku J, Kusuzaki K, Murata H, et al. Two cases of secondary angiosarcoma arising from fibrous dysplasia. Anticancer Res 1999;19:4451e7. [235] Kanazawa I, Yamauchi M, Yano S, et al. Osteosarcoma in a pregnant patient with McCuneeAlbright syndrome. Bone 2009;45:603e8. [236] Steiner GC, Forest M, Vacher-Lavenu MC. Ultrastructure of low-grade intraosseous osteosarcoma of bone: a comparative study with fibrous dysplasia and parosteal osteosarcoma. Ultrastruct Pathol 2006;30:293e9. [237] Pollandt K, Engels C, Kaiser E, Werner M, Delling G. Gsalpha gene mutations in monostotic fibrous dysplasia of bone and fibrous dysplasia-like low-grade central osteosarcoma. Virchows Arch 2001;439:170e5. [238] Owen M. Uptake of [3H]uridine into precursor pools and RNA in osteogenic cells. J Cell Sci 1967;2:39e49. [239] Sacchetti B, Funari A, Michienzi S, et al. Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell 2007;131:324e36. [240] Bianco P, Robey PG, Simmons PJ. Mesenchymal stem cells: revisiting history, concepts, and assays. Cell Stem Cell 2008;2:313e9. [241] Bianco P, Riminucci M, Gronthos S, Robey PG. Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells 2001;19:180e92. [242] Mendez-Ferrer S, Michurina TV, Ferraro F, et al. Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature 2010;466:829e34. [243] Bianco P, Riminucci M, Kuznetsov S, Robey PG. Multipotential cells in the bone marrow stroma: regulation in the context of organ physiology. Crit Rev Eukaryot Gene Expr 1999;9:159e73. [244] Bianco P, Gehron Robey P. Marrow stromal stem cells. J Clin Invest 2000;105:1663e8. [245] Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsenty G. Osf2/ Cbfa1: a transcriptional activator of osteoblast differentiation [see comments]. Cell 1997;89:747e54. [246] Riminucci M, Yamada S, Festuccia C, et al. Collagenolytic properties of GNAS1 mutated osteogenic cells from fibrous

[247] [248]

[249]

[250]

[251]

[252]

[253]

[254]

[255]

[256]

[257]

[258]

[259]

[260]

[261]

[262]

[263]

[264]

dysplasia and their in vivo correlate. J Bone Min Res 2000;15(s):S212 (Abstr.). Bianco P, Gehron Robey P. Diseases of bone and the stromal cell lineage. J Bone Min Res 1999;14. Bianco P, Costantini M, Dearden LC, Bonucci E. Alkaline phosphatase positive precursors of adipocytes in the human bone marrow. Br J Haematol 1988;68:401e3. Watkins DC, Rapiejko PJ, Ros M, Wang H, Malbon CC. G protein mRNA levels during adipocyte differentiation. Biochem Biophys Res Commun 1989;165:929e33. Wang H, Watkins DC, Malbon CC. Antisense oligodeoxynucleotides to Gs protein alpha subunit sequence accelerate differentiation of fibroblasts to adipocytes. Nature 1992;358:334e7. Candeliere GA, Glorieux FH, Prud’Homme J, St.-Arnaud R. Increased expression of the c-fos proto-oncogene in bone from patients with fibrous dysplasia. N Engl J Med 1995;332:1546e51. Ruther U, Garber C, Komitowski D, Muller R, Wagner EF. Deregulated c-fos expression interferes with normal bone development in trasngenic mice. Nature 1987;325:412e6. Motomura T, Kasayama S, Takagi M, et al. Increased interleukin-6 production in mouse osteoblastic MC3T3-E1 cells expressing activating mutant of the stimulatory G protein. J Bone Miner Res 1998;13:1084e91. Stanton RP, Hobson GM, Montgomery BE, Moses PA, SmithKirwin SM, Funanage VL. Glucocorticoids decrease interleukin6 levels and induce mineralization of cultured osteogenic cells from children with fibrous dysplasia. J Bone Miner Res 1999;14:1104e10. Riminucci M, Kuznetsov SA, Cherman N, Corsi A, Bianco P, Gehron Robey P. Osteoclastogenesis in fibrous dysplasia of bone: in situ and in vitro analysis of IL-6 expression. Bone 2003;33:434e42. Fraser WD, Walsh CA, M.A.B, et al. Parathyroid hormonerelated protein in the aetiology of fibrous dysplasia of bone in the McCune Albright syndrome. Clin Endocrinol (Oxf) 2000;53:621e8. Warshafsky B, Aubin JE, Heersche JN. Cytoskeleton rearrangements during calcitonin-induced changes in osteoclast motility in vitro. Bone 1985;6:179e85. Werb Z, Hembry RM, Murphy G, Aggeler J. Commitment to expression of the metalloendopeptidase collagenase and stromelysin: relationship of inducing events to changes in cytoskeletal architecture. J Cell Biol 1986;102:697e702. Teti A, Farina AR, Villanova I, et al. Activation of MMP-2 by human GCT23 giant cell tumor cells induced by osteopontin, bone sialoprotein and GRGDSP peptides is RGD and cell shape dependent. Int J Cancer 1998;77:82e93. Overall CM, Sodek J. Concanavalin A produces a matrixdegradative phenotype in human fibroblasts. J Biol Chem 1990;265:21141e51. Kitoh H, Yamada Y, Nogami H. Different genotype of periosteal and endosteal cells of a patient with polyostotic fibrous dysplasia. J Med Genet 1999;36:724e5. Kuznetsov SA, Cherman N, Riminucci M, Collins MT, Robey PG, Bianco P. Age-dependent demise of GNAS-mutated skeletal stem cells and "normalization" of fibrous dysplasia of bone. J Bone Miner Res 2008;23:1731e40. Sissons HA, Malcolm AJ. Fibrous dysplasia of bone: case report with autopsy study 80 years after the original clinical recognition of the bone lesions. Skeletal Radiol 1997;26:177e83. Alvares LC, Capelozza AL, Cardoso CL, Lima MC, Fleury RN, Damante JH. Monostotic fibrous dysplasia: a 23-year follow-up

PEDIATRIC BONE

REFERENCES

[265] [266]

[267]

[268] [269]

[270]

[271]

[272]

[273] [274]

[275]

[276]

[277]

[278] [279]

[280]

[281]

[282]

of a patient with spontaneous bone remodeling. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;107:229e34. Chanson P, Salenave S, Orcel P. McCuneeAlbright syndrome in adulthood. Pediatr Endocrinol Rev 2007;4(Suppl 4):453e62. Feuillan PP, Foster CM, Pescovitz OH, et al. Treatment of precocious puberty in the McCuneeAlbright syndrome with the aromatase inhibitor testolactone. N Engl J Med 1986;315:1115e9. Feuillan PP, Jones J, Cutler Jr GB. Long-term testolactone therapy for precocious puberty in girls with the McCuneeAlbright syndrome. J Clin Endocrinol Metab 1993;77:647e51. Chapurlat R, Meunier PJ. Fibrous dysplasia of bone. Baillie`re’s Clin Rheum 2000;14:385e98. Imanaka M, Iida K, Nishizawa H, et al. McCune-Albright syndrome with acromegaly and fibrous dysplasia associated with the GNAS gene mutation identified by sensitive PNAclamping method. Intern Med 2007;46:1577e83. Kalfa N, Philibert P, Audran F, et al. Searching for somatic mutations in McCuneeAlbright syndrome: a comparative study of the peptidic nucleic acid versus the nested PCR method based on 148 DNA samples. Eur J Endocrinol 2006;155:839e43. Karadag A, Riminucci M, Bianco P, et al. A novel technique based on a PNA hybridization probe and FRET principle for quantification of mutant genotype in fibrous dysplasia/ McCuneeAlbright syndrome. Nucleic Acids Res 2004;32:e63. Lietman SA, Ding C, Levine MA. A highly sensitive polymerase chain reaction method detects activating mutations of the GNAS gene in peripheral blood cells in McCuneeAlbright syndrome or isolated fibrous dysplasia. J Bone Joint Surg Am 2005;87:2489e94. Guille JT, Kumar SJ, MacEwen GD. Fibrous dysplasia of the proximal part of the femur. J Bone Joint Surg 1998;80A:648e58. Leet AI, Chebli C, Kushner H, et al. Fracture incidence in polyostotic fibrous dysplasia and the McCuneeAlbright syndrome. J Bone Miner Res 2004;19:571e7. Leet AI, Wientroub S, Kushner H, et al. The correlation of specific orthopaedic features of polyostotic fibrous dysplasia with functional outcome scores in children. J Bone Joint Surg Am 2006;88:818e23. Ippolito E, Bray EW, Corsi A, et al. Natural history and treatment of fibrous dysplasia of bone: a multicenter clinicopathologic study promoted by the European Pediatric Orthopaedic Society. J Pediatr Orthop B 2003;12:155e77. Andrisano A, Soncini G, Calderoni PP, Stilli S. Critical review of infantile fibrous dysplasia: surgical treatment. J Ped Orthop 1991;11:478e81. Stephenson RB, London MD, Hankin FM, Kaufer H. Fibrous dysplasia. J Bone Joint Surg 1987;69A:400e9. Keijser LC, Van Tienen TG, Schreuder HW, Lemmens JA, Pruszczynski M, Veth RP. Fibrous dysplasia of bone: management and outcome of 20 cases. J Surg Oncol 2001;76:157e66. discussion 167e8. Freeman BH, Bray EW, Meyer LC. Multiple osteotomies with Zyckel nail fixation for polyostotic fibrous dysplasia involving the proximal part of the femur. J Bone Joint Surg 1987;69A:691e8. Leet AI, Magur E, Lee JS, Wientroub S, Robey PG, Collins MT. Fibrous dysplasia in the spine: prevalence of lesions and association with scoliosis. J Bone Joint Surg Am 2004;86A:531e7. Mancini F, Corsi A, De Maio F, Riminucci M, Ippolito E. Scoliosis and spine involvement in fibrous dysplasia of bone. Eur Spine J 2009;18:196e202.

623

[283] Schoenfeld AJ, Koplin SA, Garcia R, et al. Monostotic fibrous dysplasia of the spine: a report of seven cases. J Bone Joint Surg Am 2010;92:984e8. [284] Lee JS, FitzGibbon E, Butman JA, et al. Normal vision despite narrowing of the optic canal in fibrous dysplasia. N Engl J Med 2002;347:1670e6. [285] Cutler CM, Lee JS, Butman JA, et al. Long-term outcome of optic nerve encasement and optic nerve decompression in patients with fibrous dysplasia: risk factors for blindness and safety of observation. Neurosurgery 2006;59:1011e7. discussion 1017e8. [286] Riminucci M, Collins MT, Jane J, Lin KY. Craniofacial fibrous dysplasia. In: Jane J, Lin KY, editors. Textbook of Craniofacial Surgery. Orlando-Philadelphia: WB Saunders; 2001. p. 366e81. [287] Chapurlat RD, Delmas PD, Liens D, Meunier PJ. Long-term effects of intravenous pamidronate in fibrous dysplasia of bone. J Bone Miner Res 1997;12:1746e52. [288] Devogelaer JP. Treatment of bone diseases with bisphosphonates, excluding osteoporosis. Curr Opin Rheumatol 2000;12:331e5. [289] Lala R, Matarazzo P, Bertelloni S, Buzi F, Rigon F, de Sanctis C. Pamidronate treatment of bone fibrous dysplasia in nine children with McCuneeAlbright syndrome. Acta Paediatr 2000;89:188e93. [290] Lane JM, Khan SN, O’Connor WJ, et al. Bisphosphonate therapy in fibrous dysplasia. Clin Orthop 2001:6e12. [291] Weinstein RS. Long-term aminobisphosphonate treatment of fibrous dysplasia: spectacular increase in bone density. J Bone Miner Res 1997;12:1314e5. [292] Pfeilschifter J, Ziegler R. [Effect of pamidronate on clinical symptoms and bone metabolism in fibrous dysplasia and McCuneeAlbright syndrome]. Med Klin 1998;93:352e9. [293] Liens D, Delmas PD, Meunier PJ. Long-term effects of intravenous pamidronate in fibrous dysplasia of bone. Lancet 1994;343:953e4. [294] Chapurlat RD, Hugueny P, Delmas PD, Meunier PJ. Treatment of fibrous dysplasia of bone with intravenous pamidronate: long-term effectiveness and evaluation of predictors of response to treatment. Bone 2004;35:235e42. [295] Kos M, Luczak K, Godzinski J, Klempous J. Treatment of monostotic fibrous dysplasia with pamidronate. J Craniomaxillofac Surg 2004;32:10e5. [296] Lala R, Matarazzo P, Andreo M, et al. Bisphosphonate treatment of bone fibrous dysplasia in McCuneeAlbright syndrome. J Pediatr Endocrinol Metab 2006;19(Suppl 2):583e93. [297] Plotkin H, Rauch F, Zeitlin L, Munns C, Travers R, Glorieux FH. Effect of pamidronate treatment in children with polyostotic fibrous dysplasia of bone. J Clin Endocrinol Metab 2003;88:4569e75. [298] Chan B, Zacharin M. Pamidronate treatment of polyostotic fibrous dysplasia: failure to prevent expansion of dysplastic lesions during childhood. J Pediatr Endocrinol Metab 2006;19:75e80. [299] Chapurlat RD. Medical therapy in adults with fibrous dysplasia of bone. J Bone Miner Res 2006;21(Suppl 2):P114e9. [300] Chao K, Katznelson L. Use of high-dose oral bisphosphonate therapy for symptomatic fibrous dysplasia of the skull. J Neurosurg 2008;109:889e92. [301] Mansoori LS, Catel CP, Rothman MS. Bisphosphonate treatment in polyostotic fibrous dysplasia of the cranium: case report and literature review. Endocr Pract 2010;21:1e14. [302] Makitie AA, Tornwall J, Makitie O. Bisphosphonate treatment in craniofacial fibrous dysplasia e a case report and review of the literature. Clin Rheumatol 2008;27:809e12.

PEDIATRIC BONE

624

22. FIBROUS DYSPLASIA

[303] Glorieux FH. Bisphosphonate therapy for severe osteogenesis imperfecta. J Pediatr Endocrinol Metab 2000;13(Suppl 2): 989e92. [304] Glorieux FH, Bishop NJ, Plotkin H, Chabot G, Lanoue G, Travers R. Cyclic administration of pamidronate in children with severe osteogenesis imperfecta. N Engl J Med 1998;339:947e52. [305] Plotkin H, Rauch F, Bishop NJ, et al. Pamidronate treatment of severe osteogenesis imperfecta in children under 3 years of age. J Clin Endocrinol Metab 2000;85:1846e50. [306] Luckman SP, Hughes DE, Coxon FP, Graham R, Russell G. Rogers MJ. Nitrogen-containing bisphosphonates inhibit the mevalonate pathway and prevent post-translational prenylation of GTP-binding proteins, including Ras. J Bone Miner Res 1998;13:581e9. [307] Wedegaertner PB, Chu DH, Wilson PT, Levis MJ, Bourne HR. Palmitoylation is required for signaling functions and membrane attachment of Gq alpha and Gs alpha. J Biol Chem 1993;268:25001e8. [308] Iniguez-Lluhi JA, Simon MI, Robishaw JD, Gilman AG. G protein beta gamma subunits synthesized in Sf9 cells.

[309]

[310] [311] [312]

[313] [314] [315]

Functional characterization and the significance of prenylation of gamma. J Biol Chem 1992;267:23409e17. Muntz KH, Sternweis PC, Gilman AG, Mumby SM. Influence of gamma subunit prenylation on association of guanine nucleotide-binding regulatory proteins with membranes. Mol Biol Cell 1992;3:49e61. Fukada Y. Prenylation and carboxylmethylation of G-protein gamma subunit. Methods Enzymol 1995;250:91e105. Wedegaertner PB, Bourne HR. Activation and depalmitoylation of Gs alpha. Cell 1994;77:1063e70. Iiri T, Backlund Jr PS, Jones TL, Wedegaertner PB, Bourne HR. Reciprocal regulation of Gs alpha by palmitate and the beta gamma subunit. Proc Natl Acad Sci USA 1996;93:14592e7. Bianco P, Robey PG. An animal model of fibrous dysplasia. Mol Med Today 1999;5:322e3. Bianco P, Gehron Robey P. Stem cells in tissue engineering. Nature 2001. Di George AM. Albright syndrome: is it coming of age? J Pediatr 1975;87:1018e20.

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C H A P T E R

23

Nutritional Rickets John M. Pettifor MRC Mineral Metabolism Research Unit, Department of Paediatrics, University of the Witwatersrand and Chris Hani Baragwanath Hospital, Johannesburg, South Africa

AN OVERVIEW OF RICKETS Introduction 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 in Europe provided detailed and accurate descriptions of the clinical features of the disease in excellent treatises in the middle of the 17th century [1]. Several centuries later, at the turn of the 20th century, rickets was an almost universal finding at autopsies conducted on children dying during the winter months in northern Europe. Even during summer, the prevalence of rickets remained high. Paleopathological studies from the UK have indicated that compared to the high prevalence in children in the 18th and 19th century, rickets was uncommon in Medieval populations [2]. In the USA in the second decade of the 20th century, Hess found that rickets was pervasive among African-American infants living in New York (quoted by [3]). 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 at the present time, and there has been an increase in the incidence of the disease in a number of countries in which it had almost been totally eradicated. By early in the 20th century, it had been established that nutritional rickets could be prevented by ultraviolet light irradiation, sunlight or the provision of cod liver oil [3]. In the 1920s, vitamin D was isolated and its role in the pathogenesis of rickets was established [4,5]. These discoveries provided cheap and effective means for prevention and treatment. Over the last 40 years, considerable strides have been made in our understanding of the metabolism and functions of vitamin D (for reviews see [6e8]) (see Chapters 6, 7 and 8) and of the factors influencing mineral and

Pediatric Bone, Second Edition DOI: 10.1016/B978-0-12-382040-2.10023-1

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 non-nutritional causes of rickets, which were originally grouped together under the term of vitamin D resistant rickets, have now been divided into a number of different etiologies based on a much clearer understanding of the various pathogenetic mechanisms. In this chapter, the general features of rickets will be reviewed and a classification of the various causes of the disease will be presented. Finally, nutritional rickets will be discussed in detail.

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, while once the growth plates have fused and growth has ceased, only features of osteomalacia are found [9]. Although it might be considered pedantic to distinguish between rickets and osteomalacia, the structures and cells involved are different (see Chapter 2). 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 type X. Mineralization of the cartilage matrix takes place initially in the matrix vesicles, which act as a nidus for further mineralization to occur. In osteomalacia, it is the osteoblast and its extracellular product, osteoid, rich

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Copyright Ó 2012 Elsevier Inc. All rights reserved.

626

23. NUTRITIONAL RICKETS

TABLE 23.1

A Classification of the Causes of Rickets Based on the Primary Pathogenetic Mechanisms. Not all the Causes of Rickets have been Included in the Table Inhibition of mineralization

Calciopenic

Phosphopenic

Alterations of vitamin D metabolism: Vitamin D deficiency Increased catabolism of 25-OHD Decreased production of 1,25-(OH)2D End-organ resistance to vitamin D Dietary calcium deficiency

Dietary phosphorus deficiency Impaired intestinal phosphate absorption Increased renal phosphate loss associated with FGF-23 abnormalities: X-linked hypophosphatemic rickets [14] Autosomal dominant hypophosphatemic rickets [15] Autosomal recessive hypophosphatemic rickets [16] Tumor-associated rickets Neurocutaneous syndromes Polyostotic fibrous dysplasia Increased renal phosphate loss unassociated with FGF-23: Fanconi syndrome Distal renal tubular acidosis

in collagen type I and other non-collagenous proteins such as osteocalcin, which are affected. These two target organs may respond differently to rachitic factors and to various therapeutic regimens [10]. There is evidence to indicate that apoptosis of the chondrocyte of the hypertrophied zone of the growth plate is responsive to extracellular phosphate concentrations, with hypophosphatemia delaying or preventing apoptosis, resulting in the typical growth plate features of vitamin D deficiency rickets [11]. At the osteoid seam, on the other hand, the osteoblast is dependent on alkaline phosphatase activity to optimize phosphate concentrations necessary for mineralization [10]. As an example of possible evidence of differences in response at the growth plate and the osteoid surface to serum phosphate concentrations, in X-linked hypophosphataemic 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

TABLE 23.2

Hereditary hypophosphatasia Aluminum toxicity Fluoride toxicity First generation bisphosphonates

osteomalacic component on the trabecular bone surfaces, as bone biopsies may show continued osteomalacia when the growth plate shows no radiological features of rickets [12].

Classification of Rickets The causes of rickets may be divided 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 different factors, including the presence of normal concentrations of both calcium and phosphorus [13], and of alkaline phosphatase. Thus, the broad divisions are based on whether or not the primary defect results in hypocalcemia (calciopenic rickets), hypophosphatemia (phosphopenic rickets) or direct inhibition of mineralization (Table 23.1). The list is not complete, as isolated and unusual descriptions

Typical Biochemical Features of the Various Categories of Rickets

Calciopenic

Phosphopenic

Hypocalcemia

Hypophosphatemia

Hyperparathyroidism

Normal serum calcium Normal PTH

Hypocalciuria

Increased TmP/GFR in the dietary and intestinal causes Decreased TmP/GFR in the renal causes

PEDIATRIC BONE

Impaired mineralization May have normal serum calcium and phosphorus conc.

NUTRITIONAL RICKETS

of rickets have not been included. The next six chapters (Chapters 24 to 29) will 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 which help in establishing the pathogenesis of the disease in an individual child. In the calciopenic forms of rickets, the typical biochemical changes include hypocalcemia and hyperparathyroidism, while in the phosphopenic form, hypophosphatemia with normal parathyroid hormone concentrations are characteristic and in many situations is associated with elevated fibroblast growth factor 23 (FGF-23) concentrations [17] (Table 23.2).

NUTRITIONAL RICKETS An Historical Perspective The first accurate descriptions of nutritional rickets were published by Whistler in Leyden in 1645 and by 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 2.5 years. Glisson considered rickets to be a new disease, as it had not been previously described in books on the diseases of children. Of interest is the comment that at that time rickets was more common in infants of wellto-do than of poor families [18,19]. The reason for this was probably that children of well-to-do parents were not encouraged to play out of doors, while the children of poor families spent time outside with their mothers, who were working on farms, and thus received adequate sunlight exposure. With the onset of the industrial revolution, and the movement of large numbers of people from the rural farming areas to rapidly developing, overcrowded and squalid towns and cities, the disease became more prevalent in the urban poor [18,20]. During the 19th century, several studies in England [18], Scotland [21] 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; in Vienna after World War I, for instance, rickets was a major public health problem among children [22]. Prior to the 20th century, the pathogenesis of nutritional rickets was generally thought to be related to poor hygiene, poor diets and bad air, even though Trousseau in Paris in the middle of the 19th century had suggested that osteomalacia was adult rickets, and that rickets was caused by nutritional problems and a lack of sunlight. Further he had proposed the use of cod-liver

627

oil and prolonged breastfeeding for the treatment of the disease [23], although Hess later reported that rickets developed in breastfed infants but that cod-liver oil was important in preventing and treating the disease [3]. It is of interest to note that fish oils, especially codliver oil, had been used for the treatment of rickets as early as the 18th century. However, it was not until the second decade of the 20th century that sunlight and cod-liver oil were accepted as effective forms of treatment. At that time, work by McCollum and Park in the USA and Mellanby in England among others highlighted the importance of ultraviolet irradiation and the role of vitamin D in the pathogenesis of rickets [24]. Chick, who worked in Austria immediately after World War I, gives a vivid description of the effect of either cod-liver oil or ultraviolet light on the healing of rickets in young infants [22]. With the general acceptance of the critical role vitamin D deficiency plays 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. Further, 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 USA and Canada. In the UK, the excessive fortification of a number of foods (dried milk powders and cereals) in the early 1950s was considered to be a possible cause for 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 [25]. 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 (MIM ID 194050) have mutations in the elastin (ELN) gene [26]. Over the last 30 years, concern has been expressed by researchers in a number of developed countries 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 will be discussed further later in this chapter.

The Epidemiology of Nutritional Rickets Vitamin D Deficiency Vitamin D deficiency is generally considered to be the primary cause of nutritional rickets in most communities. Thus, nutritional rickets and/or osteomalacia are diseases with their peak prevalence at the two extremes of life, the young and the elderly, to a large extent as

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23. NUTRITIONAL RICKETS

a result of these two age groups’ lack of independent mobility and thus their inability to spend sufficient time out of doors to obtain adequate skin exposure to sunlight. Although traditionally grouped with the vitamins, vitamin D is more appropriately considered to be a prohormone, as it needs to be metabolized to 1,25dihydroxyvitamin D (the hormone) mainly in the kidney before inducing its physiological effects. Further, 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 [27,28]. 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-dehydrocholesterol under the influence of ultraviolet light [29]. The subject of vitamin D biology is covered in detail in Chapter 8. In this section of the current chapter we will concentrate 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 ultraviolet-B (UV-B) radiation (290e315 nm). Previtamin D then undergoes thermal isomerization to vitamin D3 (cholecalciferol) within the skin. This process is relatively slow taking some 36e48 hours to equilibrate at body temperature [30]. 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 isomerization of previtamin D to vitamin D prevents rapid surges of vitamin D being released into the circulation when an individual is exposed to sunlight. Further, 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 a similar degradation of vitamin D to suprasterols and 5,6-trans-vitamin D occurs [31]. There are no reports of vitamin D toxicity resulting from excessive sunlight exposure, except in disease states, such as sarcoidosis. Factors that play important roles in influencing the amount of vitamin D formed in the skin are listed in Table 23.3. By and large, the most important factors influencing the vitamin D status of individuals are the duration of time spent out-of-doors, the amount of skin exposed to UV-B radiation, altitude, the solar zenith angle, which inversely influences the amount of UV-B

TABLE 23.3 Factors Influencing the Amount of Vitamin D Produced in the Skin The amount of UV-B reaching the earth • The latitude of the country • The season of the year • Atmospheric pollution • The average sunshine hours/day The amount of vitamin D formed in the skin • 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

reaching the earth [32], and the melanin concentration in the skin [33,34]. Studies conducted in North America have shown that sunlight during the winter months in Boston (42
N) [33] and Edmonton (52
N) is unable to synthesize vitamin D from 7-dehydrocholesterol. In the southern hemisphere, similar studies conducted in Johannesburg (26
S) and Cape Town (32
S) showed that vitamin D synthesis was significantly reduced during the winter months in Cape Town when compared to Johannesburg [35] and in two cities in Argentina at 34
S and 55
S respectively, similar seasonal trends were seen with a prolonged ‘vitamin D winter’ in the southern most city of Ushuaia [36]. Melanin pigmentation plays a limiting role in dark skinned populations living in countries in northern latitudes, where the amount of UV-B radiation reaching the earth is not optimal [33]. The peak prevalence of vitamin D deficiency rickets in children is between 3 and 24 months of age [37e39]. Infants less 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 [40] (see Chapters 10 and 24). It is unclear whether vitamin D itself crosses the placenta to any extent; however, as 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 fall rapidly in the neonatal period unless an exogenous source of vitamin D is provided. In one study of breastfed infants conducted during the winter months, serum concentrations of 25-OHD had fallen to very low levels by 6 weeks of age in babies whose mothers or they themselves were not supplemented with vitamin D [41], but this is not a universal finding [42]. Evidence that the maternal transfer of vitamin D metabolites to the fetus is protective against vitamin D

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NUTRITIONAL RICKETS

deficiency and rickets 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 [43,44]. Prior to the era of vitamin D fortified infant milk formulas, breastfed infants were less likely to develop nutritional rickets than those who were fed breast milk substitutes, despite the vitamin D activity of breast milk being low. It has been suggested that the lower prevalence of rickets in breastfed infants was due to the fact that breast milk contained considerable quantities of water-soluble vitamin D sulfate (9e22 mg/L), which was thought to be biologically active [45], however, these findings have not been confirmed [46]. More recently, the low 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 30e80 IU of vitamin D activity per liter of breast milk under normal circumstances [47]. It appears that the majority of the vitamin D activity in breast milk is made up by the concentration of the parent compound, vitamin D, itself [48]. As the vitamin D concentration in most persons is very low, the vitamin D content of breast milk is similarly low. Thus, if an infant consumes about 600e700 ml of breast milk daily, the vitamin D intake from this source will generally be less than 40 IU, which is insufficient to maintain the normal vitamin D status of the infant. As mentioned above, the vitamin D content of breast milk is dependent on the maternal vitamin D status [48], and can be increased by improving the mother’s daily intake of vitamin D, thus improving vitamin D status in the suckling infant [41,48e50]. A recent study in which lactating mothers were supplemented with vitamin D (6400 IU/ day) found 25-OHD concentrations in their breastfed infants to be similar to those achieved by supplementing infants of non-supplemented mothers with 300 IU/day (mean 25-OHD 46 ng/mL (115 nmol/L) vs 43 ng/mL (107.5 nmol/L) respectively) [51]. However, in situations where mothers or infants are not vitamin D supplemented, serum 25-OHD levels in the breastfed infant mirror skin exposure to sunlight rather than breast milk vitamin D concentrations [52], emphasizing the importance of sunlight exposure in the maintenance of vitamin D sufficiency in the breastfed infant. The same researchers [53] have shown 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 was as a result of decreased vitamin D synthesis in the skin consequent on either decreased sun exposure or the increased melanin pigmentation. The reduced vitamin D activity might contribute to the higher prevalence of nutritional rickets in breastfed African-American infants than their white peers (see below). They have also estimated that in order to

629

maintain vitamin D sufficiency in breastfed infants during the summer months in Cincinnati (latitude 39
N), infants need to be exposed to sunlight for 30 minutes/week if wearing only a diaper, or for 2 hours/week if fully clothed but not wearing a hat [54]. It has been estimated that Caucasian adults would require about 3e6 minutes in the sun with 25% of the body surface exposed to synthesize 400 IU of vitamin D in Miami, Florida, USA (25.8
N) [55] It should be noted, however, that due to the zenith angle of the sun in areas greater than 35
N or S of the equator, no skin formation of vitamin D occurs during the winter months of the year [33]. The possible explanation for the reported lower incidence of rickets in breastfed infants compared to those fed unfortified cow’s milk preparations [56] may relate to the more physiological calcium:phosphorus ratio in breast milk. The calcium:phosphorus ratio in breast milk is approximately 2:1 while in unmodified cow’s milk preparations it is approximately 1:1. 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 breastfeeding becoming a risk factor in the pathogenesis of nutritional rickets [37,57e59]. Thus vitamin D deficiency is more common in infants who are breastfed, do not receive vitamin D supplements and are 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 fell dramatically in developed countries. This occurred at a time when exclusive breastfeeding 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 23.4). For example, in 1968 in Glasgow in the UK, it was estimated that some 9% of young children had radiological features of rickets [60], while in 1996 in England between 20 and 34% of Asian 2-year-old children had 25-OHD concentrations below 25 nmol/L [70]. In 2001, in Tibet, 66% of children older than 2 years of age had clinical features of rickets [63]. It should be noted that rickets is a problem not only in temperate climates or northern latitudes, such as in Tibet or the UK, but also in tropical and subtropical countries such as Nigeria. Extensive reviews of nutritional rickets in the world including the tropics have been provided [71e73]. In a number of the tropical and subtropical countries the pathogenesis of the rickets appears to be classic vitamin D deficiency related to overcrowding in urban communities or social, religious

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630 TABLE 23.4

23. NUTRITIONAL RICKETS

The Prevalence of Rickets in a Number of Countries

Country

Year

Prevalence

Sample

Reference

UK

1968

9% radiological changes

12e24 months random sample

[60]

1976

45% biochemical changes

9e16 years Asian schoolchildren

[61]

South Africa

1969

17% clinical features

3e8 months immunization attendance during winter

[62]

Tibet

1994

66% clinical features

>24 months representative sample

[63]

China

1977e83

41% clinical features 3.7% with radiological and biochemical changes

<3 years of age community sample

[64]

Greece

1968

15% biochemical changes

<12 months

[65]

Nigeria

1998

9% clinical features

6e36 months community sample

[66]

Turkey

1994

10%

<36 months community sample

[67]

Iran

1975

15%

<5 years consecutive x-rays

[38]

Yemen

1997

50%

<5 years admitted with pneumonia

[68]

Ethiopia

1997

42% radiological changes

<5 years admitted with pneumonia

[69]

or clothing customs, such as purdah, which preclude adequate sunlight exposure [74]. In the Middle East, vitamin D deficiency is a major problem in young infants [75,76] and children in the teenage years, especially among girls in the latter age group [77]. In Kuwait, poor maternal education, lack of exposure to sunlight and a poor weaning diet with prolonged breastfeeding were factors which have been incriminated in the pathogenesis of the disease [39]. Similar contributing factors appear to be responsible in Iran and Saudi Arabia as well [59], and in Somalia and Ethiopia, besides the contributing factors mentioned above, malnutrition is strongly associated with rickets [78,79]. Low serum levels of 25-hydroxyvitamin D are characteristic of mothers of rachitic infants in the Middle East [80], and these contribute to the low vitamin D status of the breastfed infants [75,76]. 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 co-workers [81] have suggested that there might be a genetic element in some families. A third of

their infants with rickets had persistent amino-aciduria after the rickets had healed and many of their parents had renal tubular abnormalities manifesting as phosphaturia and increased a-amino-nitrogen excretion. Further, 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 [59,82], although this finding is not supported by all studies [67] and probably reflects social, religious and environmental factors which might play roles in predisposing one sex over the other. Further support for a possible genetic predisposition to the development of rickets comes from a recent study from Nigeria, in which it was reported that over a third of older children (3e6 years of age) with nutritional rickets had associated features of incomplete distal renal tubular acidosis [83]. This study did not differentiate those with vitamin D deficiency from those with presumed dietary calcium deficiency. Gene polymorphisms have also been suggested to play a role in predisposing an individual to the development of rickets. Conflicting evidence around the role of vitamin D receptor polymorphisms might play in predisposing

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NUTRITIONAL RICKETS

children to vitamin D deficiency rickets has been found. A study in Turkey suggested that the vitamin D receptor (VDR) gene ‘A’(Apa1) allele was more common in children with rickets than those without, while there were no differences in the frequencies of Fok1 or Taq1 alleles between the two groups [84], In another study from Turkey, the F allele was found to be more frequent in children with rickets [85], while in the same study, Egyptian children with and without rickets had similar allele frequencies, but the number of subjects in each group was small [85]. In an Indian study, the frequencies of VDR Bsm1 and Fok1 alleles were similar in subjects with and without rickets, as were the frequencies of PTH gene single nucleotide polymorphisms (SNPs) (BstB1 and Dral1) [86]. A similar lack of association between VDR polymorphisms and the propensity to develop rickets was found in a study conducted in Mongolia, where the prevalence of rickets is high [87]. Recently, studies have also looked at genetic influences on circulating 25-OHD concentrations. In a study of Caucasian adults, SNPs in the cytochrome P450 hydroxylase (CYP2R1) and vitamin D binding protein (GC) genes were found to influence 25-OHD levels [88], and thus may influence an individual’s propensity to vitamin D deficiency and rickets. In a systematic review, McGrath and co-workers reported a SNP each in CYP27B1 and VDR that might influence 25-OHD concentrations besides those mentioned above [89]. An interesting small study from Australia has suggested that the presence of the F allele in the VDR gene was associated with lower birth weight in those mothers who were vitamin D deficient [90]. Many of the studies reported above are limited by the relatively small number of subjects in both the patient and control groups. Further studies are required before definitive conclusions can be drawn on the role of gene variations in the predisposition for rickets in children. Over the last 30 or so years, a number of reports have appeared suggesting that there has been an upsurge of rickets among minority groups and communities in several developed countries [57,73,91]. In the USA, attention was drawn some 25 years ago to the association of rickets with prolonged unsupplemented breastfeeding, strict vegetarianism in mothers and fad diets [57]. The vast majority of infants were African-American [58]. 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 breastfeed; there is a belief that breast milk contains all the nutrients a young infant requires for normal growth and development; the increased skin pigmentation reduces cutaneous vitamin D production in situations of marginal UV radiation, and finally, there is increasing concern about the risk of skin cancer as a result of sunlight

631

exposure. In the light of these factors, the American Academy of Pediatrics recommends a minimum intake of 200 IU/day vitamin D beginning within the first 2 months of life [92] and continuing throughout childhood and adolescence. It is not only in the USA where increased melanin pigmentation places infants and toddlers at increased risk from rickets. The disease has been reported in young children of dark skinned immigrants in Australia [93,94], New Zealand [95], Canada [37] and a number of European countries [96,97], furthermore, in the UK, rickets and vitamin D deficiency remain major problems in the Indian/Pakistani/ Bangladeshi (Asian) community [70,98,99]. In the Netherlands, children from Morocco and Turkey have lower 25-OHD levels and higher parathyroid hormone (PTH) values than Caucasian children [100]. These biochemical perturbations were considered to be due to differences in skin pigmentation and calcium intakes. In Canada, it was recently estimated that 89% of infants and young children with rickets had intermediate or darker skin and the majority lived in the northern parts of the country [37]. A number of researchers have highlighted the role of macrobiotic or vegetarian diets in exacerbating vitamin D deficiency and the development of rickets [101e103]. 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 (see below). The Induction of Vitamin D Deficiency by Low Dietary Calcium Intakes In the UK, the Asian communities (Pakistani/Indian/ Bangladeshi) are particularly at risk for developing rickets, not only during the infant and toddler age groups, but also throughout childhood, adolescence [104] and into adulthood [105,106]. It is clear that the majority of affected children have darker skins but conflicting evidence exists as to whether or not those of African descent are less prone to the disease than those of Asian origin [107,108]. Attention was drawn to the problem among Asian immigrants in the 1960s [60,109] and since that time a large number of studies has been conducted to determine the pathogenesis and the best methods of management. There is good evidence that vitamin D deficiency is an essential component of the pathogenesis [73,110,111], however, other factors, which exacerbate the development of vitamin D deficiency, appear to be involved [21,112]. The factors include vegetarianism [113,114], poor calcium and high phytate contents of the diet [115e117], and extensive skin coverage by clothing [118]. In 1989, Clements [112] proposed that the pathogenesis of Asian rickets was related to the induction of vitamin D deficiency as a result of the effects of the

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632

23. NUTRITIONAL RICKETS

low dietary calcium content and its poor bioavailability on the catabolism of vitamin D and its metabolites (Fig. 23.1). Studies in rats have shown that dietary calcium deficiency or the addition of phytate to the diet reduces the half-life of circulating 25-OHD, without altering the metabolic clearance of 1,25-(OH)2D [119]. This is achieved by an increase in the inactivation of 25-OHD by the liver through the promotion of the hepatic conversion of vitamin D to polar inactivate 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 [120,121] have shown 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 suggest, unlike Clements and co-workers, that this most 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 25-OHD are similar in humans to those demonstrated in rats and can be shown to occur within 24 hours of the

administration of 1,25-(OH)2D [122]. Furthermore, intestinal malabsorption syndromes and high fiber diets have been shown to reduce the plasma half-life of 25-OHD [123,124]. Thus it is suggested that the high prevalence of rickets in the Asian population in the UK 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, the latter causing increased catabolism of 25-OHD, resulting in a shortened half-life and resultant vitamin D deficiency (Fig. 23.1) [112]. The interaction of poor vitamin D status with low calcium bioavailability in inducing vitamin D deficiency and rickets probably does not only occur in the Asian community in the UK, but may also play a role in many situations where calcium intakes are poor and vitamin D status marginal, as may be the situation in, for example, African-Americans [125] and Egyptians [85]. In fact, this combination of factors may be a more common cause of rickets in the 21st century than isolated vitamin D deficiency. This mechanism may also explain the relatively high prevalence of rickets during adolescence, as it is during this period that calcium requirements increase to meet the demands of the rapidly growing skeleton [126e129]. Dietary Calcium Deficiency

s

FIGURE 23.1 The pathogenesis of rickets in Asian children in the UK.

Although nutritional rickets has generally been considered to be associated with vitamin D deficiency, a number of studies from developing countries over the past 25 years 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 [130,131], however, until recently, the typically low calcium content of staple diets in many developing countries has been considered to cause few problems [132e134]. Since the 1970s, studies suggest that this is not the case. Children described with dietary calcium deficiency rickets in studies conducted in South Africa [135,136], Nigeria [137e139], and, more recently, in Bangladesh [140,141] are typically older than the infants and toddlers who are at risk of developing vitamin D deficiency rickets. In South Africa, the children were aged between 4 and 16 years, while the mean age of those in Nigeria was about 4 years and in Bangladesh it was 5.5 years. The diets are exceptionally low in calcium with estimated calcium intakes being approximately 150e200 mg/day. Further compromising calcium absorption is the high phytate content of the

PEDIATRIC BONE

NUTRITIONAL RICKETS

typically cereal-based diet (corn or rice), although a recent study from Nigeria in which phytase was used to reduce the phytate content of corn porridge failed to show any difference in intestinal calcium absorption between those fed the phytase-treated porridge and the control porridge (calcium absorption 50.4 þ 17.8% vs 44.5 þ 17.3% respectively). The phytate content of the corn porridge was reduced by 26% by phytase, which was thought possibly to be too small a reduction to affect absorption [142]. Characteristic of the diet is the absence of dairy products [143,144]. It is of interest that reports from Bangladesh suggest that the disease is of recent origin (over the past 30 years) in that country [140,145]. 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 fallen. The biochemical features of the disease will be discussed later in the chapter, however, a striking hallmark of dietary calcium deficiency rickets described in the three developing countries is the finding of circulating levels of 25-OHD greater than 10e12 ng/mL (25e30 nmol/L) in most patients, thus excluding vitamin D deficiency as the primary factor in the pathogenesis of the disease. Although there is general consensus among the researchers in these areas that low dietary calcium intakes play a major role in the pathogenesis of the disease as it responds rapidly to calcium supplements, it is possible that there might be other contributing factors in some areas. The studies conducted by Thacher et al. [138,144] 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 frequency of vitamin D receptor alleles has been found in subjects compared to community controls [146]. Further work in this area is needed to establish the factors responsible for making some children on low calcium diets susceptible to rickets. Over the last 30 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.

The Clinical Presentation of Rickets Rickets presents clinically as a consequence of hypocalcemia, of bone abnormalities associated with rickets

633

and the accompanying osteomalacia, or of the effects of vitamin D deficiency on other systems, such as the muscular or immune systems. Further, the presentation differs depending on the age of the child and on the bones that are subjected to weight bearing or are under bending stress. Nearly 50 years ago, Fraser and co-workers proposed that vitamin D deficiency progressed and evolved through three stages based on the clinical, biochemical and radiological features as it became progressively more severe [147]. Although there is considerable overlap between the three stages and stage 1 may not be seen in all patients with rickets, the concept remains clinically useful and helps in understanding the pathogenesis of the disease. 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 young infants less than 6 months of age. They may present with apnoeic episodes [148], convulsions [149,150], tetany [151], stridor or cardiac failure [152]. 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 [153,154]. Late neonatal hypocalcemia occurs more commonly in neonates who are vitamin D deficient as a result of maternal deficiency [155e157]. Maternal subclinical vitamin D deficiency may also result in impaired infant growth [158e160], impaired neonatal cardiac function [161] and enamel hypoplasia [151]. During stage 2, hypocalcemia improves to near normal or normal levels and the bony changes of rickets start to become apparent. In stage 3, the clinical features of rickets become progressively more severe and hypocalcemia may once again become symptomatic as the homeostatic mechanisms attempting to maintain normocalcemia fail. The initial bony changes of rickets are 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 later two sites are often difficult to assess unless severe. As the disease becomes more prolonged and severe, deformities of the appendicular skeleton take place at sites affected by weight bearing or stress. These 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

PEDIATRIC BONE

634

23. NUTRITIONAL RICKETS

child starts to stand and walk, the physiological bowing around the knees becomes accentuated and bowlegs (genu varum) become prominent. Bowlegs is 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 e the so-called “wind-swept” deformity. In longstanding 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 labors once adulthood is reached [162]. 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 longstanding 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. 23.2). As the ribs progressively soften, so 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. 23.2). 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 as a result of severe muscular hypotonia, but vertebral body collapse may also occur. Rickets is associated with delayed closure of the cranial fontanels, with the anterior fontanel being excessively large for age. The skull develops the “hot cross bun” appearance due to frontal and parietal bossing. In the infant, the presence of craniotabes (softening of the skull bones behind the ear over the occipital region) is considered to be very suggestive of rickets, especially in the young infant [164,165], however, it may be a normal feature in infants 3 months of age or younger [166,167]. Eruption of primary dentition is delayed and if the mother had been vitamin D deficient during pregnancy, the primary dentition may show signs of enamel hypoplasia [151]. Asymmetry of the skull (plageocephaly) develops as a result of the softness of the cranial vault and the hypotonia, which is so 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 [168]. Pseudotumor cerebri and cataracts

FIGURE 23.2

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 [163]).

have been described in a young infant with severe rickets and hypocalcemia [169]. Besides the bony deformities, vitamin D deficiency rickets is characterized by a delay in gross motor milestones as a result of the 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 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 in climbing stairs or getting out of chairs. Despite the hypotonia, deep tendon reflexes may be brisk. The pathogenesis of the hypotonia is thought to be the result of a direct effect of vitamin D deficiency [170] or hypophosphatemia [171] on muscle function. Cardiac abnormalities including dilated cardiomyopathy [172], electrocardiographic changes and left ventricular dysfunction [173]

PEDIATRIC BONE

635

NUTRITIONAL RICKETS

have also been described in children with vitamin D deficiency. These abnormalities improve on treatment and are thought to be as a consequence of hypocalcemia. Profuse sweating is also described as a feature of severe rickets in young children, and 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 [67,174,175]. Many reported series of children with rickets have been derived from hospital admission data and it is noted that the majority of children (excluding young infants who are frequently admitted for hypocalcemic symptoms) were not admitted because of rickets but rather because of respiratory or gastrointestinal infections [176e178]. The results of a case-control study of children admitted with pneumonia to a hospital in Ethiopia emphasized the importance of rickets as a predisposing factor (rickets being 13 times more common in children with pneumonia than controls) [69]. Although in this study 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 the Yemen, found that rickets together with anemia and malnutrition were associated with an increased risk of dying [68,179]. The mechanisms by which vitamin D deficiency causes an increase in infection risk are probably a combination of both 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 adequately to clear the lung of pathogens and mucus. The role of vitamin D, or more specifically 1,25(OH)2D, in immune modulation is now well described [180]. A number of abnormalities of the innate and cell-mediated immune systems have been described in vitamin D deficiency, including impaired production of the antimicrobial peptide, cathelicidin [181] and stimulation of Th 1-associated cytokine production [182], among many other effects on the immune system. Other hematological abnormalities reported in children with rickets include the von JackscheLuzet syndrome, which is associated with anemia, thrombocytopenia, leukocytosis, myeloid metaplasia and hepatosplenomegaly [183,184]. Myelofibrosis has also been described in longstanding vitamin D deficiency [185,186]. In several studies, an association has been found between the presence of iron deficiency anemia and vitamin D deficiency [187,188]. 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 [189], to reduce fetal tibial diaphyseal bone growth [190] and tooth enamel formation. Further, there is evidence that both intrauterine and postnatal growth might be affected [159,191]. Using fetal ultrasound, another group of researchers was unable to confirm the effect of maternal vitamin D insufficiency on fetal bone growth in length but did find an increase in metaphyseal splaying and cross-sectional area of the fetal femur as early as 19 weeks’ gestation [192]. The clinical features of rickets due to dietary calcium deficiency are similar to those described in 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 23.5, the features are more often related to limb deformities and growth plate enlargement, rather than of craniotabes, delayed motor milestones, and open fontanelles. Further, hypotonia and muscle weakness appear to be less prominent in dietary calcium deficiency. In studies in South Africa, the lack of the latter two 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 23.5

The Prevalence of Various Presenting Symptoms and Signs in Children Aged 18 Months and Older with Radiological Rickets in Nigeria [193] Prevalence (%) N [ 278

Characteristic Symptoms

Signs

PEDIATRIC BONE

Weakness

65

Leg pain when walking

60

Excessive falling

58

Unable to walk

11

Previous fracture

9

Enlarged costochondral junctions

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

636

23. NUTRITIONAL RICKETS

The classical picture of a child with severe active rickets is not difficult to diagnose, however, the 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 23.4, which assessed the prevalence of rickets in a community, were based on clinical features alone. Depending on the clinical features used for diagnosis it is highly possible that the prevalence of active rickets is overestimated, as not only are the features likely to be not specific for rickets but even less so for active disease. In a study from Bangladesh, of 78 children with physical features of rickets, only eight (10.2%) had radiographic features of rickets and an elevated alkaline phosphatase concentration [194]. 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 [195]. Even the use of biochemical tests, such as an alkaline phosphatase level, to help confirm the diagnosis is problematic [195]. Strand and co-workers assessed the specificity of clinical evidence of rickets to assess active rickets in northern China. Although clinical features were present in 41% of infants, only 3.7% had active rickets when assessed by clinical signs, radiology and elevated alkaline phosphatase values. If clinical and radiological features were used with 25(OH)D values, the prevalence was 21% [196]. In a recent study conducted in Nigeria of over 700 children greater than 18 months of age, who were referred because of leg deformities and not being able to walk and who were suspected of having rickets, various clinical features (see Table 23.5) were assessed for sensitivity and specificity to diagnose active rickets against radiological confirmation [193]. The following features were found to be independently predictive of active rickets: age less than 5 years, height for age less than 2 SD below the mean, leg pain during walking, wrist enlargement, and costochondral enlargement. Three or more of these clinical features together accurately identified 87% of the children who had active rickets, and less than three features accurately classified 76% of the children as not having active rickets. Although these findings suggest reasonable sensitivity, 24% of children with less than three signs were incorrectly classified as not having active rickets. It should be noted that the children in this study were likely to have been suffering from dietary calcium deficiency rickets and thus were older than typical children with vitamin D deficiency rickets. As a result, these findings might not be applicable to infants/young children with vitamin D deficiency. A similar study was conducted in a group of younger subjects (3e30 months) attending an outpatient clinic in South Africa, using a clinical scoring system which

included the presence or absence of craniotabes, frontal and parietal bossing, thickened costochondral junctions, Harrison’s sulcus and thickened wrists [165]. At that time, vitamin D deficiency rickets was common in the community and over 30% of children were diagnosed radiologically and biochemically to have rickets. The researchers found that enlargement of the costochondral junctions was the most reliable clinical sign, followed by enlargement of the wrists. Over 12 months of age, delayed closure of the fontanel was a useful sign. However, their conclusion was that rickets could not be diagnosed or excluded with certainty using clinical signs alone, as 31% of the infants diagnosed clinically as having rickets did not have biochemical or radiological evidence of the disease, and 13% of infants with no clinical signs have 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 between 3 and 12 months of age [167]. In infants between 3 and 6 months of age, 40% of those with craniotabes had radiological evidence of rickets, while in the older group aged 7e12 months, 60% with craniotabes had rickets. In a control group, none of the infants aged 3e6 months, who did not have craniotabes, had rickets, while 35% of those between 7 and 12 months without craniotabes had radiological evidence of rickets. Thus the finding of craniotabes in older infants is more suggestive of rickets than in younger infants, despite some studies suggesting that craniotabes at birth is a feature of vitamin D deficiency [164]. The conclusion from these various studies is that it is unlikely that a single set of clinical signs or symptoms will be able to be used to diagnose active rickets at all ages. Some of the reasons include: some of the signs are age dependent; mild/early rickets is difficult to diagnose clinically; and the usefulness of the clinical signs will depend on the prevalence of rickets in the community. Furthermore, once healed, the clinical features of rickets such as the widening of the wrist and leg deformities may take months or years to correct.

The Biochemical Changes Associated with Nutritional Rickets The classic biochemical abnormalities of nutritional rickets, whether due to vitamin D deficiency or dietary calcium lack, relate to the fact that the basic defect is an inability to maintain normal calcium homeostasis. Thus, as described by Fraser and co-workers [147], the initial biochemical abnormality is hypocalcemia. As discussed earlier, this early phase in vitamin D deficiency may not be clinically apparent as it may be transient and relatively mild, however, it is enough to induce

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NUTRITIONAL RICKETS

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, which is thought to be largely responsible for the development of the characteristic abnormalities at the growth plate [10]. The other effects of increased parathyroid activity on the renal tubules include decreased urinary calcium loss, increased cyclic AMP excretion, generalized amino aciduria [197], and increased bicarbonate loss. Although stage 1 vitamin D deficiency is considered to be associated with normal PTH levels [147,150], other workers [198] have found elevated PTH concentrations and increased urinary cyclic AMP excretion. This should not be surprising as the three stages of vitamin D deficiency are a continuum, thus as stage 1 merges with stage 2, elevated PTH levels would be expected (Fig. 23.3) [200]. Hypocalcemia (low ionized calcium concentration) is the hallmark of calciopenic rickets; nevertheless, many children with rickets due either to vitamin D or dietary calcium deficiency have total calcium values within the normal range. In the majority of cases these are at the lower end of normal. On treatment, the values rise and then stabilize at a new steady state, suggesting that for the individual child the low “normal” values

FIGURE 23.3 The progression of biochemical changes that occur in vitamin D deficiency (reproduced with permission from Arnaud et al. [199]).

637

are not normal. As discussed earlier, symptomatic hypocalcemia may occur in both the early and late stages of the disease. In the early stage, a lack of PTH response to hypocalcemia has been suggested as the mechanism, although Kruse provides evidence for PTH resistance [198]. In the more severe forms of the disease, an inability to maintain calcium homeostasis probably results from a failure adequately to mobilize mineral from bone [201]. 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 [198,202e204]. Normal or elevated levels of serum phosphate have also been described in children suffering from rickets due to dietary calcium deficiency [135,205,206]. In those children, in whom it has been studied, the tubular reabsorption of phosphate is high and is unresponsive to infusions of PTH (JM Pettifor, unpublished results). A similar finding has been noted in adolescent vitamin D deficient children with hyperphosphatemia [204]. Correction of the associated hypocalcemia restores the renal responsiveness to PTH and serum phosphate concentrations return to normal (JM 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 as, in the vast majority of cases, values are generally elevated [165], but it does lack specificity [195]. In a recent study assessing alkaline phosphatase as a screening test for rickets in unselected breastfed infants aged 6e15 months not receiving vitamin D supplements and living in Seattle, USA, it was found that 13.4% of 246 infants had values above the upper limit of the reference values. Of these 33 children, 18 had radiographs and rickets was noted in four, all of whom had values more than double the upper limit of normal. Thus, using a cut off of approximately double the normal upper limit, the specificity for rickets was 97.4% and the positive predictive value 40.0%. The authors suggest that alkaline phosphatase might be a useful screening test for rickets in breastfed children not receiving vitamin D supplements [207]. As 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. Further, other factors such as growth rates, the stage of puberty and the nutritional status of the child [71] may influence the results. It is

PEDIATRIC BONE

638

23. NUTRITIONAL RICKETS

unclear why alkaline phosphatase values are elevated in rickets as osteoblastic activity is decreased on bone histology [9]. The measurement of urinary hydroxyproline excretion is also problematic, as the collection of complete 24 hour urine samples is difficult in children, there is considerable fluctuation in daily urinary excretion, and the amount of hydroxyproline excreted in the urine is not only a reflection of bone collagen breakdown, but also of collagen breakdown elsewhere in the body and from that ingested in food. Nevertheless, hydroxyproline excretion is consistently increased in children with vitamin D deficiency rickets reflecting the increased bone resorption associated with secondary hyperparathyroidism [198]. Over the past 30 years, a number of new markers of bone formation and resorption have been described and are readily measured in either serum or urine [208,209]. They have been measured in normal children and in those with metabolic bone diseases [210e213]. The markers of bone formation include bone specific alkaline phosphatase (bone ALP), osteocalcin (OC) and carboxy- and N-terminal propeptides of type I procollagen (PICP and PINP, respectively), while the markers of bone resorption include pyridinoline (PYD) and deoxypyridinoline (DPD) cross-links of collagen, the crosslinked carboxyterminal 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 [211]. Further, 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 [212e214]. As is to be expected with secondary hyperparathyroidism and increased bone resorption, serum CTX and urine NTX levels are elevated prior to treatment in children with vitamin D deficiency rickets [213]. With treatment, values rise initially and then fall to the normal range within 4e6 weeks. Markers of bone formation show divergent patterns: alkaline phosphatase values fall from elevated levels to normal values within 8 weeks; serum PICP levels are only slightly elevated on admission, rise significantly over the first 2 weeks of treatment and then return slowly to normal values by 8 weeks; osteocalcin levels, on the other hand, are significantly lower than normal on admission, rise sharply in the first 2 weeks and then fall to normal values by 6 weeks. The discordance between alkaline phosphatase and osteocalcin values in rickets has been noted by a number of researchers [214,215], 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 [213]. Osteocalcin levels in rickets are in keeping with the histological evidence of decreased osteoblastic activity [9]. A number of bone turnover markers have been measured in children with dietary calcium deficiency rickets [212,214,216]. Scariano and co-workers [212] have reported that CTX and OC values are elevated in Nigerian children with presumed dietary calcium deficiency, however, the elevated OC levels prior to treatment have not been confirmed in other studies [137,214]. The Nigerian workers have also reported a rise in NTX and PTH values with calcium treatment [216]. Despite a number of markers being available to measure bone turnover in children with rickets, serum alkaline phosphatase probably still represents the cheapest and most appropriate way of following the response to therapy in a child with nutritional rickets [213]. 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 normal levels should be are being debated at present [217e221]. In children, the range in normal pediatric populations has been found to be between 12 and 50 ng/mL (30e125 nmol/L) [52,54,222,223]. 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 <12 ng/mL (<30 nmol/L) [223e226] and in many cases <5 ng/mL (<12.5 nmol/L) [110,198]. In subclinical rickets, similar values have been found [227], although in another study higher values were documented [224], but the very mild radiological rickets in this group of children had 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 rickets may be associated with a range of 25-OHD levels, as 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, 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, while “vitamin D insufficiency” has been reserved for serum 25-OHD concentrations which are associated with perturbations of calcium homeostasis, but without osteomalacia or rickets [219].

PEDIATRIC BONE

NUTRITIONAL RICKETS

A number of studies have documented changes in serum PTH and/or calcium concentrations in association with seasonal fluctuations in serum 25-OHD levels [127,228e231]. In two of these studies, serum 25-OHD concentrations of z30 [230] and z40 nmol/L [231] (z12 and z16 ng/mL) were calculated as the levels below which serum PTH started to rise. A similar value of 30 nmol/L was found to be the cut-point below which neonates developed hypocalcemia and secondary hyperparathyroidism [189]. Using a different technique to assess the desirable 25-OHD concentration in prepubertal children, Docio and co-workers [232] estimated the level to lie between 12 and 20 ng/mL (30 and 50 nmol/L). Although at the current time there is still considerable debate around the 25-OHD concentrations to be used to define vitamin D sufficiency, insufficiency and deficiency, the majority of researchers in the pediatric field currently define vitamin D deficiency as <10 or <12 ng/mL (25 or 30 nmol/L), vitamin D insufficiency as >10 and <20 ng/mL (>25 and <50 nmol/L) and vitamin D sufficiency as >20 ng/mL (50 nmol/L) [233e236]. However, some authors suggest that the ideal serum concentration in children should be higher (>25e30 ng/mL) [237]. Little is known of the effect that differing calcium intakes and changing calcium demands associated with changing growth rates might have on the serum 25-OHD concentration required to maintain normal calcium homeostasis. The studies by Clements have highlighted the role that low dietary calcium content and high dietary phytate play on inducing vitamin D deficiency and rickets [112,119] and, recently, Thacher and co-workers have suggested that low dietary calcium intakes might increase vitamin D requirements in otherwise healthy children [238]. 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 [198,223,225,239e241]. The finding of normal or elevated levels of 1,25(OH)2D in children with florid rickets has led some investigators to postulate that other metabolites, besides 1,25-(OH)2D, might be necessary to prevent the development of rickets and osteomalacia [240,242,243]. Certainly, there is evidence that 24,25-dihydroxyvitamin D (24,25-(OH)2D) may be necessary for normal fetal bone development [244] and fracture repair [245], but there are few data to suggest that vitamin D metabolites other than 1,25-(OH)2D are necessary for normal calcium homeostasis. Another interpretation of the normal or elevated levels of 1,25-(OH)2D seen in some children with florid rickets is that these values are actually inappropriately low for the degree of secondary hyperparathyroidism present [198,241]. A plausible explanation for the differing 1,25-(OH)2D concentrations

639

in various studies is provided by Arnaud [200] and Kruse [198] (see Fig. 23.3). In the early stages of vitamin D deficiency (stage 1), serum 1,25-(OH)2D concentrations fall to levels insufficient to maintain intestinal calcium absorption at the required level for normocalcemia and growth, thus ionized calcium values fall and secondary hyperparathyroidism develops, increasing 1a-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 [198], which increases intestinal calcium absorption and bone resorption partially correcting the hypocalcemia (stage 2). As the substrate (25-OHD) for 1,25-(OH)2D production continues to decline, a stage is reached when 25OHD concentrations fall to such a level that 1,25(OH)2D concentrations are unable to be maintained and metabolite levels fall. That the 1,25-(OH)2D levels are inappropriately low for the degree of secondary hyperparathyroidism is evidenced by the rapid rise in levels induced by small doses of vitamin D [239]. From the discussion, it is apparent that the diagnosis of vitamin D deficiency should be based on the measurement of circulating levels of 25-OHD and not 1,25-(OH)2D . Serum 24,25-(OH)2D 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)2D levels are low or undetectable [223,225,239,241,246], as would be 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)2D. It is these relatively normal 25-OHD and elevated 1,25-(OH)2D concentrations in association with evidence of enhanced intestinal fractional calcium absorption [247], and the biochemical and radiological response to an improved calcium intake which distinguish dietary calcium deficiency from vitamin D deficiency rickets [248,249]. Mean serum concentrations of 25-OHD are reported to be lower than those of controls but above the vitamin D deficient range in children suffering from dietary calcium deficiency rickets in a number of studies conducted in Nigeria, Bangladesh and South Africa [135,137,140,144,250] (Table 23.6). Mean values are typically above the level of 12 ng/mL (30 nmol/L), which has been suggested as the lower limit of vitamin D sufficiency in children (see above). In only one small study of ten rachitic children in Nigeria was the mean 25-OHD level suggestive of vitamin D insufficiency [251]. The reason for the poorer vitamin D status in rachitic children than controls has not been well studied. In Nigeria, neither socio-cultural factors nor lack of exposure to sunlight appear to be responsible [144,252]. A possible explanation may be

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640 TABLE 23.6

23. NUTRITIONAL RICKETS

Comparison of Rachitic and Control Subjects in a Nigerian Study of Children with Dietary Calcium Deficiency

Variable

Rachitic subjects N [ 123

Control subjects N [ 123

P value

Age (months)

46 (34,63)a

42 (25,70)

0.14

Age at stopping breast feeding (months)

16.0  5.3b

17.3  4.5

0.04

Daily calcium intake (mg)

217  88

214  77

0.6

Serum Ca (mmol/L)

1.93  0.22

2.24  0.15

<0.0001

Serum Pi (mmol/L)

1.67  0.61

1.92  0.59

0.0017

Alkaline phosphatase (U/l)

707 (545,1021)

235 (196,278)

<0.0001

25-OHD (nmol/L)

32 (22,40)

50 (42,62)

<0.0001

1,25-(OH)2D (pmol/L)

322  96

278  91

0.0007

PTH (pmol/L)

20 (13,31)

12 (11,16)

0.006

a

Median and 25th and 75th percentiles. mean  standard deviation. Adapted from [144]. b

found in the fact that 1,25-(OH)2D concentrations are higher in rachitic subjects than in controls (Table 23.6), and thus could be responsible for increased catabolism of circulating 25-OHD with a resultant decrease in serum levels [112]. Support for this hypothesis comes from the results of a randomized controlled trial in which children with active rickets were treated for 6 months with vitamin D, calcium supplements or both. In the group receiving calcium supplements alone (1000 mg Ca daily), mean serum 25-OHD concentrations rose from 16 ng/mL (40 nmol/L) to 21 ng/mL (52.5 nmol/L), while 1,25-(OH)2D levels fell from 130 pg/mL (312 pmol/L) to 109 pg/mL (262 pmol/L) [138], possibly indicating a decrease in 25-OHD catabolism associated with the fall in 1,25-(OH)2D. As mentioned above, serum 1,25-(OH)2D concentrations are typically elevated in children suffering from dietary calcium deficiency rickets [137,139,140,144,253]. Not only are the values two- to threefold higher than reference values derived from children living in developed countries but, in the majority of studies, they are also significantly higher than age-matched controls from the same communities as the subjects [144] (see Table 23.6). It is presumed that 1,25-(OH)2D levels rise in children with dietary calcium deficiency in response to the habitually low dietary calcium content (approximately 150e200 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 [254], there are many rachitic children with PTH values within the normal range [137,139,144,212,251,253]. In these

children, it is unclear what the stimulus for increasing 1,25-(OH)2D production is or 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)2D concentrations [255,256], thus PTH values would be expected to be lower in dietary calcium deficiency rickets than those found in vitamin D deficiency rickets, but it is difficult to explain values well within the normal range. Further studies are required to elucidate possible mechanisms. Recent studies in children with dietary calcium deficiency rickets in Nigeria have highlighted the early response in vitamin D metabolites to a vitamin D bolus [238,254]. As expected, 25-OHD concentrations rose to a maximum on day 3, but unexpectedly the already elevated 1,25-(OH)2D concentrations almost doubled from a mean of 184 pg/mL to 349 pg/mL over the same period and then fell to initial values by day 14 [254]. A similar pattern was seen in rachitic children whether the bolus given was vitamin D2 or D3 [238,257]. The rise in 1,25-(OH)2D was highly correlated with the initial 25-OHD concentrations [257]. Of interest was the finding that these changes in 1,25-(OH)2D were not seen in control children from the same community despite the expected improvement in 25-OHD concentrations following the bolus of vitamin D, suggesting that in children with dietary calcium deficiency rickets the relatively normal 25-OHD concentrations in the rachitic children are insufficient to meet the calcium demands of these children [238]. Stable isotopes of calcium have been used to assess the fractional calcium absorption in children with dietary calcium deficiency rickets in Nigeria, as it would be expected that intestinal calcium absorption would

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be maximal given their low dietary calcium intakes (approximately 150e200 mg/day) and their markedly elevated concentrations of 1,25-(OH)2D [258]. Combining the results of several studies, untreated rachitic children had elevated but similar fractional calcium absorption as control children (61  22% in both groups) with very similar calcium intakes (181  70 mg/day vs 227  103 mg/day respectively) [259]. Following 2 weeks of treatment of the rachitic children with calcium supplements (600 mg/day), fractional calcium absorption had fallen from a mean of 72% to 57% [247], indicating that intestinal calcium absorption is responsive to changes in calcium intake in these children. In these rachitic children, fractional calcium absorption was not related to serum 25-OHD or 1,25(OH)2D concentrations, despite several children having 25-OHD levels below 10 ng/mL (Fig. 23.4) [257,259]. In a recent study of rachitic children in The Gambia, in whom dietary calcium deficiency was suspected, Prentice and co-workers found markedly elevated concentrations of serum FGF-23 [260]. The vitamin D profile of the children was similar to that reported in Nigerian children with lower 25-OHD and higher 1,25(OH)2D concentrations than age-matched controls, however, mean FGF-23 levels were some sevenfold higher than controls, and 74% had elevated concentrations. Plasma phosphorus concentrations were inversely related to FGF-23 levels. Although the pathogenesis of the elevated FGF-23 levels is unclear at present, the authors have proposed that the elevation is driven by chronically low dietary calcium intakes, which increase PTH and 1,25-(OH)2D levels, which in turn increase FGF-23 levels with a resultant increase in urinary

FIGURE 23.4 The relationship between 25(OH)D concentrations in children with active dietary calcium deficiency rickets and intestinal fractional calcium absorption measure by the dual staple isotope technique. Fractional absorption is generally higher both in children with active rickets and controls compared to children on more adequate calcium intakes (reproduced with permission from Thacher and Abrams [259]).

641

FIGURE 23.5

Proposed mechanism for the elevation of FGF-23 concentrations in The Gambian children with rickets (reproduced from [260] with permission from Elsevier).

phosphate excretion and thus a fall in serum phosphorus concentrations (Fig. 23.5) [260]. This intriguing finding needs to be confirmed in other studies.

The Radiological Diagnosis of Rickets As discussed earlier in the section on the definition of rickets, several different aspects make up the disease and manifest radiologically. First and most prominently in children, there is the failure of endochondral calcification manifesting at the growth plates, secondly, there is the failure of or delay in mineralization of preformed osteoid at the sites of bone turnover and of intramembranous mineralization (osteomalacia) and, thirdly, there are 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 co-workers [224] is a loss of the well-demarcated 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 (Fig. 23.6) [261]. At the wrist, the distance between the distal radial metaphysis and the radial epiphysis is never greater than 1 mm in normal children [262]. 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

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642

(A)

23. NUTRITIONAL RICKETS

(B)

FIGURE 23.6

Radiographic features of vitamin D deficiency in an 18-month-old child. (A) The wrist shows marked widening of the growth plates at the distal radius and ulna with apparent soft tissue swelling. The distal metaphyses are splayed, cupped and frayed. The development of the distal radius epiphysis is delayed. The trabecular structure of the metaphysis is coarsened and the cortices are ill-defined. (B) Radiographs of both knees in the same child show similar features as those described for the upper limbs.

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 appear frayed with spur formation (Fig. 23.6). 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 to diagnose early rickets. In the older child, the bones around the knee are better suited as 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 over-diagnosis of the disease unless suspicious features are confirmed by biochemical or other radiographic evidence [227]. Once long bone epiphyses fuse during adolescence, the diagnosis of rickets becomes 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 [263]. 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 the Looser’s zone (pseudofracture), which is a translucent

strip of unmineralized osteoid running perpendicularly through the bone cortex (Fig. 23.7). 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 [262]. The pathogenesis of Looser’s zones is unclear, although they are thought to occur at sites of skeletal stress. As a considerable extent 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 longstanding rickets and result from bending of the shafts of the softened osteomalacic long bones or because of 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 genu valgum (knock knees) (see above). Longstanding and severe rickets may also be associated with pelvic deformities (triradiate deformity) and protrusio acetabulae. The femoral neck may also be affected with coxa vara being more common than coxa valga [264]. 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 to structural deformities. Features of secondary hyperparathyroidism are often subtle in children with nutritional rickets. The increase

PEDIATRIC BONE

NUTRITIONAL RICKETS

643

FIGURE 23.7 A Looser’s zone in 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.

the hands of children diagnosed with dietary calcium deficiency have a loss of waisting 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. Thacher and co-workers developed and published a scoring system [265] for the changes at the wrist and knee that has been found to be useful in assessing the response to therapy [138]. The degree of severity at the wrist is graded out of four and at the knee out of six, giving a maximum score of 10 for a child with severe rickets (Fig. 23.8 and Table 23.7). 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 are provided [266]. 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. 23.9). This is followed by a gradual filling in of the undermineralized metaphysis. Gradually, the coarse trabecular pattern at the metaphyses becomes finer and the trabeculae appear 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, radiographic evidence of healing may be seen within a month, but complete healing of the metaphyseal and growth plate changes may take several more months. The long bone deformities may improve gradually over several years as modeling reshapes the bones to the more normal stresses.

in bone turnover associated with elevated PTH levels aggravates the generalized osteopenia, which is so 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 sacro-iliac 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 if carefully looked for. 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 as the pathogenesis of the bone disease is similar in both situations. Nevertheless, many of the radiographs of

FIGURE 23.8 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. [265]).

PEDIATRIC BONE

644 TABLE 23.7

23. NUTRITIONAL RICKETS

Ten-Point Radiographic Scoring Method for the Assessment of the Severity of Rickets

Wrista e 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  2 points ¼ 4 points possible Kneea e 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:

B:

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

Multiplier

Portion of growth plate affected

0.5

1 condyle or plateau

1

2 condyles or plateaus

2 bones  1 point  3 points ¼ 6 points possible Total: 10 points possible a

Score the worst knee and the worst wrist. Reproduced with permission from [265].

The Treatment and Prevention of Rickets As was discussed in the section on the epidemiology of nutritional rickets, the disease is caused by a spectrum of conditions with vitamin D deficiency at one end of the spectrum and dietary calcium deficiency at the other. In between the two extremes are varying combinations of relative vitamin D insufficiency and low dietary calcium intake. Treatment

FIGURE 23.9 Radiograph of the wrist showing features of early healing. Note the irregular sclerotic band at the distal end of the metaphyses and periosteal new bone formation along the cortex of the ulna.

For more than a century, the beneficial effects of sunlight on the healing of rickets have been known, although not generally accepted. More recently, Dent and co-workers [267] showed that vitamin D deficiency rickets in an Asian child could be very effectively cured by ultraviolet irradiation to the whole body for a period of 2 weeks. Similarly, significant improvements in vitamin D status and biochemical abnormalities are seen during the summer months in communities living in areas of high latitude [268]. Holick estimates that a young adult exposed to a whole body dose of sunlight, which causes minimal erythema, receives the equivalent of an oral dose of 10 000 IU of vitamin D3 [32]. Thus, children with vitamin D deficiency rickets can be treated effectively by exposing them to adequate amounts of ultraviolet irradiation. However, treatment of vitamin D deficiency rickets is probably more effectively managed by the oral

PEDIATRIC BONE

NUTRITIONAL RICKETS

administration of small doses of vitamin D2 or D3. There is little evidence in humans to suggest that the response to either form of vitamin D differs [238,269], however, a study has shown that vitamin D3 is more effective than vitamin D2 in raising 25-OHD concentrations [270e272]. Stanbury et al. [239], using small daily doses of vitamin D (200e450 IU), were able to show in subjects with vitamin D deficiency that 1,25-(OH)2D levels rose to values some fivefold higher than normal within 7e10 days and that 25-OHD concentrations also rose over the same period. Furthermore, over 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 vitamin D [273]. Recovery is speeded up by the use of larger doses and it is recommended that doses of between 5000 and 15 000 IU/day be used for a period of 4e8 weeks. On these doses, serum calcium, phosphorus and PTH values rapidly return to normal within 2e3 weeks [198], although 1,25-(OH)2D and alkaline phosphatase levels remain elevated for several months [223,239]. Many clinicians provide a calcium supplement of between 500 and 1500 mg/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 [198,274]. Although this is probably unnecessary in children who have calcium intakes near the daily recommended intake (DRI) or recommended daily allowance (RDA) for age (600e1000 mg/day), in children who are vegetarian or have low calcium intakes for other reasons, calcium supplementation would be prudent. All children, who have symptomatic hypocalcemia, should receive oral calcium supplements and, if necessary, be given calcium as calcium gluconate (1e2 mL/kg of a 10% solution) in a slow intravenous infusion. A single large dose of vitamin D (200 000e600 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 as the response is delayed when vitamin D is given intramuscularly [184]. However, in a study comparing the treatment of rachitic children between 6 months and 3 years of age using a single dose of either vitamin D3 200 000 IU orally or intramuscularly, no difference in response was found between the two forms of therapy [275]. An oral dose of 600 000 IU vitamin D results in a rapid improvement in the biochemical abnormalities within 4e7 days, and radiographic evidence of healing within 2 weeks in the majority of patients [154,274]. 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

645

such as are outlined above, are used for the treatment of vitamin D deficiency. In a study comparing the change in bone mass in rachitic children treated with either a single dose of vitamin D 600 000 IU or 20 000 IU for 30 days, no difference in response was found [276]. Smaller doses are also effective. In a study from the Middle East, a single intramuscular injection of 10 000 IU/kg resulted in the normalization of biochemistry within 1 month and complete radiological healing in 95% of the infants with rickets by 3 months [277]. The above methods of treatment are considered by some authors to have an advantage over the smaller daily dose [184,274], as compliance is assured in the single dose method, which is not the case in the daily dose regimen [82]. In a number of countries, preparations of vitamin D are not readily available in concentrations appropriate for treatment of rickets, thus, the active metabolites of vitamin D, such as alphacalcidiol, have been used for treatment. A recent report has documented an apparent failure of healing of rickets in children treated with alphacalcidiol, which subsequently responded rapidly to vitamin D therapy [278]. Not only is treatment with alphacalcidiol expensive, but it does not restore the vitamin D status of deficient children. It has been customary to treat children with rickets in developing countries on vitamin D combined occasionally with calcium supplements. This is an acceptable policy in those countries in which it is known that the pathogenesis of rickets is related to vitamin D deficiency rather than dietary calcium lack. Such countries probably include China, and countries of the Middle East and Arabian Gulf, however, this form of therapy is probably not ideal for patients outside the infant and toddler age groups 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 (1000 mg/day) for 6 months are recommended [135,137,138,251,266]. In a randomized, controlled trial, the addition of vitamin D (600 000 IU 3 monthly) to calcium supplements did not produce significantly better results as far as the completeness or speed of response is concerned [138]. Vitamin D therapy alone, on the other hand, was significantly less effective, although an improvement in biochemical and radiological evidence of rickets did occur. The 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) (TD Thacher, personal communication), however, no data are available to compare the efficacy of these treatments

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646

23. NUTRITIONAL RICKETS

with those used in the randomized, controlled trial mentioned above. Prevention of Nutritional Rickets Effective and cheap means of preventing vitamin D deficiency exist, yet nutritional rickets remains a public health problem in a number of countries, as outlined at the beginning of this chapter. There are probably a number of reasons for this; one major reason in developed countries though, is that the individuals or communities at risk are often members of minority groups, which may not have integrated well into the larger community, or who have dietary patterns which might be considered alternative by the majority of the population. In developing countries, rickets often forms part of the much broader problem of overcrowding, poverty, undernutrition and poor primary health care facilities, thus making its eradication difficult as it may not be seen as a public health priority, when there are so many other needs. Both the UK and North America have recently reassessed the dietary requirements of vitamin D. The UK continues to recommend a reference nutrient intake (RNI) of 8.5 mg/day (340 IU/day) for infants less than 6 months of age and 7 mg/day (280 IU/day) for children between 6 months and 3 years [279]. The report highlights the point that unless adequate sun exposure is ensured, many infants and young children will not maintain an adequate vitamin D status, and supports the recommendation of an earlier Department of Health Report on Weaning and Weaning Diet that routine vitamin D supplementation would provide an effective safety net for groups at risk. An RNI is not set for older children because the diet is not the major source of vitamin D in older children, reliance being made on adequate sunlight exposure. The Institute of Medicine of the National Academies of Science in 2011 [280] has recommended an adequate intake (AI) for infants and a recommended dietary allowance (RDA) for children from 0e18 years of 400 IU (10 mg) e 600 IU (15 mg)/day. Few dietary sources contain adequate amounts of vitamin D for humans to maintain vitamin D sufficiency without a vitamin D supplement, ingesting vitamin D fortified foods such as infant milk formulas, or being exposed to ultraviolet irradiation (see above). Thus, in countries or communities where adequate ultraviolet exposure is difficult to achieve because of the latitude, or because of social or religious custom or personal attributes, such as infirmity, increased melanin pigmentation, or overcrowding, vitamin D supplementation should be considered. The exclusively breastfed 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 care-giver. In the USA, the American Academy of Pediatrics now recommends the universal supplementation of all infants, including those breastfeeding, and that all infants, children and adolescents should have an intake of vitamin D 200 IU/day beginning in the first 2 months of life [234,281] which, in formula fed infants, may be provided by the formula itself. The Drugs and Therapeutics Committee of the Lawson Wilkins Pediatric Endocrine Society has recently suggested that 200 IU vitamin D may be insufficient in many situations, such as extremes of latitude, and increased skin pigmentation [282]. Certainly, in countries where vitamin D deficiency rickets is still common among breastfed infants such as in China and Tibet, routine supplementation should be mandatory. Results of a study conducted in China showed that a vitamin D supplement of 400 IU/day to infants for 6 months from birth maintained serum 25-OHD levels in a more normal range than supplements of 100 or 200 IU/day [226]. In most countries, infant milk formulas are fortified with vitamin D at a level of 400 IU/L or quart when reconstituted, thus, if an infant drinks z600 ml/day, he/she will receive over 200 IU from that source alone. There is thus no reason for the artificially fed infant to be vitamin D supplemented, however, if given, a supplement of 400 IU would not produce vitamin D toxicity [217]. The best method of providing the vitamin D supplement to those children, who require it is controversial [283]. As with any medication that requires a daily dose, long-term compliance may be a problem [82]. In a number of central European countries, “stosstherapie” has been used to prevent vitamin D deficiency in young children. The dose, usually 600 000 IU, is administered every 3e5 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 [284]. Since that time, further studies using intermittent large doses to prevent vitamin D deficiency have been conducted [285]. The researchers recommended that 2.5 mg (100 000 IU) vitamin D administered 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 in with the immunization program in countries where vitamin D deficiency in infants remains a major public health problem (e.g. Middle East, Tibet or Mongolia). In many developed countries, one or more foods are vitamin D fortified. For example, in the USA, 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, within the community. In the UK, margarine and other fat spreads and some cereals are

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REFERENCES

fortified, however, this fortification does not prevent the high prevalence of vitamin D deficiency in the Asian community, as 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 the fortification of chapatti flour at 6000 IU/kg [286] as a cheap means of preventing vitamin D deficiency in the Asian community. This fortification raised 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 a proportion of the community. As has been discussed earlier, there is now widespread concern about the high prevalence of vitamin D deficiency among pregnant and lactating mothers, resulting in poor vitamin D stores in the newborn infant [287,288]. A number of studies have assessed the vitamin D requirements of these women and most indicate that vitamin D 400 IU/day is insufficient to raise circulating 25(OH)D concentrations into the normal range [289]. Vitamin D supplementation of 1000e2000 IU/day has been recommended. Of concern is the evidence that vitamin D 800e1000 IU/day may be insufficient to bring 25(OH)D concentrations into the sufficiency range [48,290,291]. The prevention of dietary calcium deficiency rickets is a major problem as the disease occurs in developing countries and is associated with the lack of food variety in the diet of families and, in particular, children suffering from the disease. The diet of these families is based on calcium poor staples (maize [corn], 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 out of reach of most families because of cost. Trials are currently underway in Nigeria to determine whether local calcium-rich foods (powdered whole fish or limestone) added to the diet will be culturally acceptable and reduce the incidence of rickets in young children (TD Thacher, personal communication). In South Africa, a daily calcium supplement of 500 mg increased serum calcium values and lowered alkaline phosphatase values over a period of 3 months in children attending school in a community in which biochemical evidence of dietary calcium deficiency was prevalent [292].

References [1] O’Riordan JL. Rickets in the 17th century. J Bone Miner Res 2006;21:1506e10.

647

[2] Mays S, Brickley M, Ives R. Skeletal manifestations of rickets in infants and young children in a historic population from England. Am J Phys Anthropol 2005. [3] Rajakumar K, Thomas SB. Reemerging nutritional rickets: a historical perspective. Arch Pediatr Adolesc Med 2005;159:335e41. [4] McCollum EV, Simmonds N, Shipley PG, Park EA. Studies on experimental rickets. XII. Is there a substance other than fatsoluble A associated with certain fats which plays an important role in bone development? J Biol Chem 1922;50:5e30. [5] Steenbock H, Black A. Fat soluble vitamins. XXIII The induction of growth-promoting and calcifying properties in fats and their unsaponifiable constituents by exposure to light. J Biol Chem 1925;64:263e98. [6] Chun RF, Adams JS, Hewison M. Back to the future: a new look at ‘old’ vitamin D. J Endocrinol 2008;198:261e9. [7] Bouillon R, Carmeliet G, Verlinden L, et al. Vitamin D and human health: lessons from vitamin D receptor null mice. Endocr Rev 2008;29:726e76. [8] Norman AW. From vitamin D to hormone D: fundamentals of the vitamin D endocrine system essential for good health. Am J Clin Nutr 2008;88:491Se9. [9] Frame B, Parfitt AM. Osteomalacia: current concepts. Ann Intern Med 1978;89:966e82. [10] Tiosano D, Hochberg Z. Hypophosphatemia: the common denominator of all rickets. J Bone Miner Metab 2009;27:392e401. [11] Sabbagh Y, Carpenter TO, Demay MB. Hypophosphatemia leads to rickets by impairing caspase-mediated apoptosis of hypertrophic chondrocytes. Proc Natl Acad Sci USA 2005;102:9637e42. [12] Glorieux FH, Marie PJ, Pettifor JM, Delvin EE. Bone response to phosphate salts, ergocalciferol and calcitriol in hypophosphatemic vitamin-D resistant rickets. N Engl J Med 1980;303:1023e31. [13] Demay MB, Sabbagh Y, Carpenter TO. Calcium and vitamin D: what is known about the effects on growing bone. Pediatrics 2007;119(Suppl. 2):S141e4. [14] Dixon PH, Christie PT, Wooding C, et al. Mutational analysis of PHEX gene in X-linked hypophosphatemia. J Clin Endocrinol Metab 1998;83:3615e23. [15] White KE, Evans WE, O’Riordan JL, et al. Autosomal dominant hypophosphataemic rickets is associated with mutations in FGF23. Nat Genet 2000;26:345e8. [16] Feng JQ, Ward LM, Liu S, et al. Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism. Nat Genet 2006;38:1310e5. [17] Pettifor JM. What’s new in hypophosphataemic rickets? Eur J Pediatr 2008;167:493e9. [18] Gibbs D. Rickets and the crippled child: an historical perspective. J R Soc Med 1994;87:729e32. [19] Dunn PM. Francis Glisson (1597e1677) and the “discovery” of rickets. Arch Dis Child Fetal Neonatal Ed 1998;78:F154e5. [20] Stephen JML. Epidemiological and dietary aspects of rickets and osteomalacia. Proc Nutr Soc 1975;34:131e8. [21] Arneil GC. Nutritional rickets in children in Glasgow. Proc Nutr Soc 1975;34:101e9. [22] Chick DH. Study of rickets in Vienna 1919e1922. Med Hist 1976;20:41e51. [23] Dunn PM. Professor Armand Trousseau (1801e67) and the treatment of rickets. Arch Dis Child Fetal Neonatal Ed 1999;80:F155e7. [24] Rafter GW. Elmer McCollum and the disappearance of rickets. Perspect Biol Med 1987;30:527e34. [25] British Paediatric Association. Infantile hypercalcaemia, nutritional rickets, and infantile scurvy in Great Britain. Br Med J 1964;1:1659e61.

PEDIATRIC BONE

648

23. NUTRITIONAL RICKETS

[26] Perez Jurado LA, Peoples R, Kaplan P, Hamel BC, Francke U. Molecular definition of the chromosome 7 deletion in Williams syndrome and parent-of-origin effects on growth. Am J Hum Genet 1996;59:781e92. [27] Dusso AS, Brown AJ, Slatopolsky E. Vitamin D. Am J Physiol Renal Physiol 2005;289:F8e28. [28] Norman AW. From vitamin D to hormone D: fundamentals of the vitamin D endocrine system essential for good health. Am J Clin Nutr 2008;88:491Se9S. [29] Rajakumar K, Greenspan SL, Thomas SB, Holick MF. SOLAR ultraviolet radiation and vitamin D: a historical perspective. Am J Public Health 2007;97:1746e54. [30] Holick MF, MacLaughlin JA, Clark MB, et al. Photosynthesis of previtamin D3 in human skin and the physiologic consequences. Science 1980;210:203e5. [31] Holick MF, MacLaughlin JA, Doppelt SH. Regulation of cutaneous previtamin D3 photosynthesis in man: skin pigmentation is not an essential regulator. Science 1981;211:590e3. [32] Holick MF. Photosynthesis, metabolism, and biologic actions of vitamin D. In: Glorieux FH, editor. Rickets. New York: Nestec Ltd., Vevey/Raven Press; 1991. p. 1e20. [33] Holick MF, Chen TC, Lu Z, Sauter E. Vitamin D and skin physiology: a D-lightful story. J Bone Miner Res 2007;22:V28e33. [34] Kimlin MG. Geographic location and vitamin D synthesis. Molec Aspects Med 2008;29:453e61. [35] Pettifor JM, Moodley GP, Hough FS, et al. The effect of season and latitude on in vitro vitamin D formation by sunlight in South Africa. S Afr Med J 1996;86:1270e2. [36] Ladizesky M, Lu Z, Oliveri B, et al. Solar ultraviolet B radiation and photoproduction of vitamin D3 in central and southern areas of Argentina. J Bone Miner Res 1995;10:545e9. [37] Ward LM, Gaboury I, Ladhani M, Zlotkin S. Vitamin D-deficiency rickets among children in Canada. Can Med Assoc J 2007;177:161e6. [38] Salimpour R. Rickets in Tehran. Arch Dis Child 1975;50:63e6. [39] Molla AM, Badawi MH, Al-Yaish S, Sharma P, el-Salam RS, Molla AM. Risk factors for nutritional rickets in children in Kuwait. Pediatr Int 2000;42:280e4. [40] Kovacs CS. Vitamin D in pregnancy and lactation: maternal, fetal, and neonatal outcomes from human and animal studies. Am J Clin Nutr 2008;88:520Se8S. [41] Rothberg AD, Pettifor JM, Cohen DF, Sonnendecker EWW, Ross FP. Maternaleinfant vitamin D relationships during breast-feeding. J Pediatr 1982;101:500e3. [42] Roberts CC, Chan GM, Folland D, Rayburn C, Jackson R. Adequate bone mineralization in breast-fed infants. J Pediatr 1981;99:192e6. [43] Anatoliotaki M, Tsilimigaki A, Tsekoura T, Schinaki A, Stefanaki S, Nikolaidou P. Congenital rickets due to maternal vitamin D deficiency in a sunny island of Greece. Acta Paediatr 2003;92:389e91. [44] Mohapatra A, Sankaranarayanan K, Kadam SS, Binoy S, Kanbur WA, Mondkar JA. Congenital rickets. J Trop Pediatr 2003;49:126e7. [45] Lakdawala DR, Widdowson EM. Vitamin-D in human milk. Lancet 1977;1:167e8. [46] Hollis BW, Roos BA, Draper HH, Lambert PW. Occurrence of vitamin D sulfate in human milk whey. J Nutr 1981;111:384e90. [47] Hollis BW. Vitamin D requirement during pregnancy and lactation. J Bone Miner Res 2007;22:V39e44. [48] Taylor SN, Wagner CL, Hollis BW. Vitamin D supplementation during lactation to support infant and mother. J Am Coll Nutr 2008;27:690e701.

[49] Ala-Houhala M. 25-hydroxyvitamin D levels during breastfeeding with or without maternal or infantile supplementation of vitamin D. J Pediatr Gastroenterol Nutr 1985;4:220e6. [50] Ala-Houhala M, Koskinen T, Terho A, Kiovula T, Visakorpi J. Maternal compared with infant vitamin D supplementation. Arch Dis Child 1986;61:1159e63. [51] Wagner CL, Hulsey TC, Fanning D, Ebeling M, Hollis BW. High-dose vitamin D3 supplementation in a cohort of breastfeeding mothers and their infants: a 6-month follow-up pilot study. Breastfeed Med 2006;1:59e70. [52] Specker BL, Tsang RC. Cyclical serum 25-hydroxyvitamin D concentrations paralleling sunshine exposure in exclusively breast-fed infants. J Pediatr 1987;110:744e7. [53] Specker BL, Tsang RC, Hollis BW. Effect of race and diet on human-milk vitamin D and 25-hydroxyvitamin D. Am J Dis Child 1985;139:1134e7. [54] Specker BL, Valanis B, Hertzberg V, Edwards N, Tsang RC. Sunshine exposure and serum 25-hydroxyvitamin D concentrations in exclusively breast-fed infants. J Pediatr 1985;107:372e6. [55] Terushkin V, Bender A, Psaty EL, Engelsen O, Wang SQ, Halpern AC. Estimated equivalency of vitamin D production from natural sun exposure versus oral vitamin D supplementation across seasons at two US latitudes. J Am Acad Dermatol 2010;62:929.e1e9. [56] Dancaster CP, Jackson WPU. Studies in rickets in the Cape Peninsula II. Aetiology. S Afr Med J 1961;35:890e4. [57] Weisberg P, Scanlon KS, Li R, Cogswell ME. Nutritional rickets among children in the United States: review of cases reported between 1986 and 2003. Am J Clin Nutr 2004;80:1697Se705S. [58] Edidin DV, Levitsky LL, Schey W, Dumbuvic N, Campos A. Resurgence of nutritional rickets associated with breast-feeding and special dietary practices. Pediatrics 1980;65:232e5. [59] Al-Atawi MS, Al-Alwan IA, Al-Mutair AN, Tamim HM, Al-Jurayyan NA. Epidemiology of nutritional rickets in children. Saudi J Kidney Dis Transpl 2009;20:260e5. [60] Richards IDG, Sweet EM, Arneil GC. Infantile rickets persists in Glasgow. Lancet 1968;i:803e5. [61] Ford JA, McIntosh WB, Butterfield R, et al. Clinical and subclinical vitamin D deficiency in Bradford children. Arch Dis Child 1976;51:939e43. [62] Robertson I. Survey of clinical rickets in the infant population in Cape Town 1967e1968. S Afr Med J 1969;43:1072e6. [63] Harris NS, Crawford PB, Yangzom Y, Pinzo L, Gyaltsen P, Hudes M. Nutritional and health status of Tibetan children living at high altitudes. N Engl J Med 2001;344:341e7. [64] Zhou H. Rickets in China. In: Glorieux FH, editor. Rickets. New York: Nestec, Vevey, Raven Press; 1991. p. 253e61. [65] Lapatsanis P, Deliyanni V, Doxiadis S. Vitamin D deficiency rickets in Greece. J Pediatr 1968;73:195e202. [66] Pfitzner MA, Thacher TD, Pettifor JM, et al. Absence of vitamin D deficiency in young Nigerian children. J Pediatr 1998;133:740e4. [67] Beser E, Cakmakci T. Factors affecting the morbidity of vitamin D deficiency rickets and primary protection. East Afr Med J 1994;71:358e62. [68] Banajeh SM, al Sunbali NN, al Sanahani SH. Clinical characteristics and outcome of children aged under 5 years hospitalized with severe pneumonia in Yemen. Ann Trop Paediatr 1997;17:321e6. [69] Muhe L, Luiseged S, Mason KE, Simoes EAF. Case-control study of the role of nutritional rickets in the risk of developing pneumonia in Ethiopian children. Lancet 1997;349:1801e4. [70] Lawson M, Thomas M. Vitamin D concentrations in Asian children aged 2 years living in England: population survey. Br Med J 1999;318:28.

PEDIATRIC BONE

REFERENCES

[71] Bhattacharyya AK. Nutritional rickets in the tropics. In: Simopoulos AP, editor. Nutrtional Triggers for Health and in Disease. Basel: Karger; 1992. p. 140e97. [72] Thacher TD, Fischer PR, Strand MA, Pettifor JM. Nutritional rickets around the world: causes and future directions. Ann Trop Paediatr 2006;26:1e16. [73] Prentice A. Vitamin D deficiency: a global perspective. Nutr Rev 2008;66:S153e64. [74] Arabi A, El Rassi R, El-Hajj Fuleihan G. Hypovitaminosis D in developing countries e prevalence, risk factors and outcomes. Nat Rev Endocrinol 2010;6:550e61. [75] Dawodu A, Agarwal M, Sankarankutty M, Hardy D, Kochiyil J, Badrinath P. Higher prevalence of vitamin D deficiency in mothers of rachitic than nonrachitic children. J Pediatr 2005;147:109e11. [76] Kazemi A, Sharifi F, Jaffe CA, Mousavinasab N. High prevalence of vitamin D deficiency among pregnant women and their newborns in an Iranian population. J Women Health 2009;18:835e9. [77] Rabbani A, Alavian SM, Motlagh ME, et al. Vitamin D insufficiency among children and adolescents living in Tehran. Iran. J Trop Pediatr 2009;55:189e91. [78] el Hag AI, Karrar ZA. Nutritional vitamin D deficiency rickets in Sudanese children. Ann Trop Paediatr 1995;15:69e76. [79] Wondale Y, Shiferaw F, Lulseged S. A systematic review of nutritional rickets in Ethiopia: status and prospects. Ethiop Med J 2005;43:203e10. [80] Molla AM, Al Badawi M, Hammoud MS, et al. Vitamin D status of mothers and their neonates in Kuwait. Pediatr Int 2005;47:649e52. [81] Doxiadis S, Angelis C, Karatzas P, Vrettos C, Lapatsanis P. Genetic aspects of nutritional rickets. Arch Dis Child 1976;51:83e90. [82] Lubani MM, Al-Shab TS, Al-Saleh QA, et al. Vitamin-D-deficiency rickets in Kuwait: the prevalence of a preventable disease. Ann Trop Paediatr 1989;3:134e9. [83] Oduwole AO, Giwa OS, Arogundade RA. Relationship between rickets and incomplete distal renal tubular acidosis in children. Ital J Pediatr 2010;36:54. [84] Bora G, Ozkan B, yangac-Erden D, Erdem-Yurter H, Coskun T. Vitamin D receptor gene polymorphisms in Turkish children with vitamin D deficient rickets. Turk J Pediatr 2008;50:30e3. [85] Baroncelli GI, Bereket A, El Kholy M, et al. Rickets in the Middle East: role of environment and genetic predisposition. J Clin Endocrinol Metab 2008;93:1743e50. [86] Ray D, Goswami R, Gupta N, Tomar N, Singh N, Sreenivas V. Predisposition to vitamin D deficiency osteomalacia and rickets in females is linked to their 25(OH)D and calcium intake rather than vitamin D receptor gene polymorphism. Clin Endocrinol (Oxf) 2009;71:334e40. [87] Kaneko A, Urnaa V, Nakamura K, et al. T Vitamin D receptor polymorphism among rickets children in Mongolia. J Epidemiol 2007;17:25e9. [88] Bu FX, Armas L, Lappe J, et al. Comprehensive association analysis of nine candidate genes with serum 25-hydroxy vitamin D levels among healthy Caucasian subjects. Hum Genet 2010:1e8. [89] McGrath JJ, Saha S, Burne THJ, Eyles DW. A systematic review of the association between common single nucleotide polymorphisms and 25-hydroxyvitamin D concentrations. J Steroid Biochem Molec Biol 2010;121:471e7. [90] Morley R, Carlin JB, Pasco JA, Wark JD, Ponsonby AL. Maternal 25-hydroxyvitamin D concentration and offspring birth size: effect modification by infant VDR genotype. Eur J Clin Nutr 2009;63:802e4.

649

[91] Robinson PD, Hogler W, Craig ME, et al. The re-emerging burden of rickets: A decade of experience from Sydney. Arch Dis Child 2006;91:564e8. [92] Gartner LM, Greer FR. Section on Breastfeeding and Committee on Nutrition. Prevention of rickets and vitamin D deficiency: new guidelines for vitamin D intake. Pediatrics 2003;111:908e10. [93] Robinson PD, Hogler W, Craig ME, et al. The re-emerging burden of rickets: A decade of experience from Sydney. Arch Dis Child 2006;91:564e8. [94] Nozza JM, Rodda CP. Vitamin D deficiency in mothers of infants with rickets. Med J Aust 2001;175:253e5. [95] Blok BH, Grant CC, McNeil AR, Reid IR. Characteristics of children with florid vitamin D deficient rickets in the Auckland region in 1998. NZ Med J 2000;113:374e6. [96] Garabedian M, Ben-Mekhbi H. Is vitamin D-deficiency rickets a public health problem in France and Algeria? In: Glorieux FH, editor. Rickets. New York: Nestec, Vevey, Raven Press; 1991. p. 215e21. [97] Markestad T, Elzouki AY. Vitamin D-deficiency rickets in Northern Europe and Libya. In: Glorieux FH, editor. Rickets. New York: Nestec, Vevey, Raven Press; 1991. p. 203e13. [98] Ladhani S, Srinivasan L, Buchanan C, Allgrove J. Presentation of vitamin D deficiency. Arch Dis Child 2004;89:781e4. [99] Iqbal SJ, Kaddam I, Wassif W, Nichol F, Walls J. Continuing clinically severe vitamin D deficiency in Asians in the UK (Leicester). Postgrad Med J 1994;70:708e14. [100] Meulmeester JF, van den Berg H, Wedel M, Boshuis PG, Hulshof KFAM, Luyken R. Vitamin D status, parathyroid hormone and sunlight in Turkish, Moroccan and Caucasian children in The Netherlands. Eur J Clin Nutr 1990;44:461e70. [101] Dagnelie PC, Vergote FJVRA, van Staveren WA, van den Berg H, Dingjan PG, Hautvast JGAJ. High prevalence of rickets in infants on macrobiotic diets. Am J Clin Nutr 1990;51:202e8. [102] Hellebostad M, Markestad T, Halvorsen KS. Vitamin D deficiency rickets and vitamin B12 deficiency in vegetarian children. Acta Paediatr Scand 1985;74:191e5. [103] Carvalho NF, Kenney RD, Carrington PH, Hall DE. Severe nutritional deficiencies in toddlers resulting from health food milk alternatives. Pediatrics 2001;107:E46. [104] Das G, Crocombe S, McGrath M, Berry J, Mughal Z. Hypovitaminosis D among healthy adolescent girls attending an inner city school. Arch Dis Child 2006;91:569e72. [105] Dunnigan MG, Henderson JB, Hole DJ, Barbara ME, Berry JL. Meat consumption reduces the risk of nutritional rickets and osteomalacia. Br J Nutr 2005;94:983e91. [106] Shaw NJ, Pal BR. Vitamin D deficiency in UK Asian families: activating a new concern. Arch Dis Child 2002;86:147e9. [107] Callaghan AL, Moy RJ, Booth IW, Debelle GD, Shaw NJ. Incidence of symptomatic vitamin D deficiency. Arch Dis Child 2006;91:606e7. [108] Ford L, Graham V, Wall A, Berg J. Vitamin D concentrations in an UK inner-city multicultural outpatient population. Ann Clin Biochem 2006;43:468e73. [109] Richards IDG, Hamilton FMW, Taylor EC, Sweet EM, Bremner E, Price H. A search for sub-clinical rickets in Glasgow children. Scot Med J 1968;13:297e305. [110] Preece MA, Ford JA, McIntosh WB, Dunnigan MG, Tomlinson S, O’Riordan JLH. Vitamin-D deficiency among Asian immigrants to Britain. Lancet 1973;i:907e10. [111] Hodgkin P, Kay GH, Hine PM, Lumb GA, Stanbury SW. Vitamin-D deficiency in Asians at home and in Britain. Lancet 1973;ii:167e72. [112] Clements MR. The problem of rickets in UK Asians. J Hum Nutr Diet 1989;2:105e16.

PEDIATRIC BONE

650

23. NUTRITIONAL RICKETS

[113] Henderson JB, Dunnigan MG, McIntosh WB, Abdul-Motaal A, Hole D. Asian osteomalacia is determined by dietary factors when exposure to ultraviolet radiation is restricted: a risk factor model. Q J Med 1990;76:923e33. [114] Henderson JB, Dunnigan MG, McIntosh WB, AbdulMotaal AA, Gettinby G, Glekin BM. The importance of limited exposure to ultraviolet radiation and dietary factors in the aetiology of Asian rickets: a risk-factor model. Q J Med 1987;63:413e25. [115] Dunnigan MG, Childs WC, Smith CM, McIntosh WB, Ford JA. The relative roles of ultra-violet deprivation and diet in the aetiology of Asian rickets. Scot Med J 1975;20:217e8. [116] Robertson I, Ford JA, McIntosh WB, Dunnigan MG. The role of cereals in the aetiology of nutritional rickets: the lesson of the Irish National Nutrition Survey 1943e8. Br J Nutr 1981;45:17e22. [117] Polanska N, Wills MR. Factors contributing to osteomalacia in the elderly and in the Asian communities in the United Kingdom. J Hum Nutr 1976;30:371e6. [118] O’Hara-May J, Widdowson EM. Diets and living conditions of Asian boys in Coventry with and without signs of rickets. Br J Nutr 1976;36:23e36. [119] Clements MR, Johnson L, Fraser DR. A new mechanism for induced vitamin D deficiency in calcium deprivation. Nature 1987;325:62e5. [120] Halloran BP, Bikle DD, Levens MJ, Castro ME, Globus RK, Holton E. Chronic 1,25-dihydroxyvitamin D3 administration in the rat reduces serum concentration of 25-hydroxyvitamin D by increasing metabolic clearance rate. J Clin Invest 1986;78:622e8. [121] Halloran BP, Castro ME. Vitamin D kinetics in vivo: effect of 1,25-dihydroxyvitamin D administration. Am J Physiol 1989;256:E686e91. [122] Clements MR, Davies M, Hayes ME, Hickey CD, Lumb GA, Mawer EB, Adams PH. The role of 1,25-dihydroxyvitamin D in the mechanism of acquired vitamin D deficiency. Clin Endocrinol 1992;37:17e27. [123] Batchelor AJ, Watson G, Compston JE. Changes in plasma halflife and clearance of 3H-25-hydroxyvitamin D3 in patients with intestinal malabsorption. Gut 1982;23:1068e71. [124] Batchelor AJ, Compston JE. Reduced plasma half-life of radiolabelled 25-hydroxyvitamin D3 in subjects receiving a high-fibre diet. Br J Nutr 1983;49:213e6. [125] DeLucia MC, Mitnick ME, Carpenter TO. Nutritional rickets with normal circulating 25-hydroxyvitamin D: a call for reexamining the role of dietary calcium intake in North American infants. J Clin Endocrinol Metab 2003;88:3539e45. [126] Cashman KD. Vitamin D in childhood and adolescence. Postgrad Med J 2007;83:230e5. [127] Du X, Greenfield H, Fraser DR, Ge K, Trube A, Wang Y. Vitamin D deficiency and associated factors in adolescent girls in Beijing. Am J Clin Nutr 2001;74:494e500. [128] Moncrieff MW, Lunt HRW, Arthur LJH. Nutritional rickets at puberty. Arch Dis Child 1973;48:221e4. [129] Narchi H, El Jamil M, Kulaylat N. Symptomatic rickets in adolescence. Arch Dis Child 2001;84:501e3. [130] Maltz HE, Fish MB, Holliday MA. Calcium deficiency rickets and the renal response to calcium infusion. Pediatrics 1970;46:865e70. [131] Kooh SW, Fraser D, Reilly BJ, Hamilton JR, Gall D, Bell L. Rickets due to calcium deficiency. N Engl J Med 1977;297:1264e6. [132] Walker ARP. Does a low intake of calcium cause or promote the development of rickets? Am J Clin Nutr 1953;3:114e20. [133] Walker ARP. The human requirement of calcium: should low intakes be supplemented? Am J Clin Nutr 1972;25:518e30. [134] Irwin MI, Kienholz EW. A conspectus of research on calcium requirements of man. J Nutr 1973;103:1020e95.

[135] Pettifor JM, Ross P, Wang J, Moodley G, Couper-Smith J. Rickets in children of rural origin in South Africa: is low dietary calcium a factor? J Pediatr 1978;92:320e4. [136] Marie PJ, Pettifor JM, Ross FP, Glorieux FH. Histological osteomalacia due to dietary calcium deficiency in children. N Engl J Med 1982;307:584e8. [137] Okonofua F, Gill DS, Alabi ZO, Thomas M, Bell JL, Dandona P. Rickets in Nigerian children: a consequence of calcium malnutrition. Metabolism 1991;40:209e13. [138] Thacher TD, Fischer PR, Pettifor JM, et al. A comparison of calcium, vitamin D, or both for nutritional rickets in Nigerian children. N Engl J Med 1999;341:563e8. [139] Oginni LM, Worsfold M, Oyelami OA, Sharp CA, Powell DE, Davie MW. Etiology of rickets in Nigerian children. J Pediatr 1996;128:692e4. [140] Fischer PR, Rahman A, Cimma JP, et al. Nutritional rickets without vitamin D deficiency in Bangladesh. J Trop Pediatr 1999;45:291e3. [141] Craviari T, Pettifor JM, Thacher TD, Meisner C, Arnaud J, Fischer PR. Rickets: an overview and future directions, with special reference to Bangladesh. A summary of the Rickets Convergence Group meeting, Dhaka, 26-27. J Health Popul Nutr January 2006;2008;26:112e21. [142] Thacher TD, Aliu O, Griffin IJ, et al. Meals and dephytinization affect calcium and zinc absorption in Nigerian children with rickets. J Nutr 2009;139:926e32. [143] Eyberg C, Pettifor JM, Moodley G. Dietary calcium intake in rural black South African children. The relationship between calcium intake and calcium nutritional status. Hum Nutr Clin Nutr 1986;40C:69e74. [144] Thacher TD, Fischer PR, Pettifor JM, Lawson JO, Isichei C, Chan GM. Case-control study of factors associated with nutritional rickets in Nigerian children. J Pediatr 2000;137:367e73. [145] Craviari T, Pettifor JM, Thacher TD, Meisner C, Arnaud J, Fischer PR. the Rickets Convergence Group. Rickets: An overview and future directions, with special reference to Bangladesh. J Health Popul Nutr 2008;26:112e21. [146] Fischer PR, Thacher TD, Pettifor JM, Jorde LB, Eccleshall TR, Feldman D. Vitamin D receptor polymorphisms and nutritional rickets in Nigerian children. J Bone Miner Res 2000;15:2206e10. [147] Fraser D, Kooh SW, Scriver CR. Hyperparathyroidism as the cause of hyperaminoaciduria and phosphaturia in human vitamin D deficiency. Pediat Res 1967;1:425e35. [148] Buchanan N, Pettifor JM, Cane RD, Bill PLA. Infantile apnoea due to profound hypocalcaemia associated with vitamin D deficiency. S Afr Med J 1978;53:766e7. [149] Ashraf A, Mick G, Atchison J, Petrey B, Abdullatif H, McCormick K. Prevalence of hypovitaminosis D in early infantile hypocalcemia. J Pediatr Endocrinol Metab 2006;19:1025e31. [150] Bonnici F. Functional hypoparathyroidism in infantile hypocalcaemic stage I vitamin D deficiency rickets. S Afr Med J 1978;54:611e2. [151] Purvis RJ, Barrie MWJ, MacKay GS, et al. Enamel hypoplasia of the teeth associated with neonatal tetany: a manifestation of maternal vitamin-D deficiency. Lancet 1973;ii:811e4. [152] Maiya S, Sullivan I, Allgrove J, et al. Hypocalcaemia and vitamin D deficiency: an important, but preventable, cause of life-threatening infant heart failure. Heart 2008;94:581e4. [153] Park EA. The influence of severe illness on rickets. Arch Dis Child 1954;29:369e80. [154] Harrison HE, Harrison HC. Rickets and osteomalacia. In: Disorders of Calcium and Phosphate Metabolism in Childhood and Adolescence. Philadelphia: W.B.Saunders; 1979. p. 141e256.

PEDIATRIC BONE

REFERENCES

[155] Shenoy SD, Swift P, Cody D, Iqbal J. Maternal vitamin D deficiency, refractory neonatal hypocalcaemia, and nutritional rickets. Arch Dis Child 2005;90:437e8. [156] Teaema FH, Al AK. Nineteen cases of symptomatic neonatal hypocalcemia secondary to vitamin D deficiency: a 2-year study. J Trop Pediatr 2010;56:108e10. [157] Camadoo L, Tibbott R, Isaza F. Maternal vitamin D deficiency associated with neonatal hypocalcaemic convulsions. Nutr J 2007;6:23. [158] Mulligan ML, Felton SK, Riek AE, Bernal-Mizrachi C. Implications of vitamin D deficiency in pregnancy and lactation. Am J Obstet Gynecol 2010;202:429.e1e9. [159] Brooke OG, Brown IR, Bone CD, et al. Vitamin D supplements in pregnant Asian women: effects on calcium status and fetal growth. Br Med J 1980;280:751e4. [160] Morley R, Carlin JB, Pasco JA, Wark JD. Maternal 25-hydroxyvitamin D and parathyroid hormone concentrations and offspring birth size. J Clin Endocrinol Metab 2006;91:906e12. [161] Brown J, Nunez S, Russell M, Spurney C. Hypocalcemic rickets and dilated cardiomyopathy: case reports and review of literature. Pediatr Cardiol 2009;30:818e23. [162] Maxwell JP. Further studies in adult rickets (osteomalacia) and foetal rickets. Proc Roy Soc Med 1934;28:265e300. [163] Pettifor JM, Daniels ED. Vitamin D deficiency and nutritional rickets in children. In: Feldman D, Glorieux FH, Pike JW, editors. Vitamin D. San Diego: Academic Press; 1997. p. 663e78. [164] Yorifuji J, Yorifuji T, Tachibana K, et al. Craniotabes in normal newborns: the earliest sign of subclinical vitamin D deficiency. J Clin Endocrinol Metab 2008;93:1784e8. [165] Opie WH, Muller CJB, Kamfer H. The diagnosis of vitamin D deficiency rickets. Pediatr Radiol 1975;3:105e10. [166] Pettifor JM, Pentopoulos M, Moodley GP, Isdale JM, Ross FP. Is craniotabes a pathognomonic sign of rickets in 3-month-old infants? S Afr Med J 1984;65:549e51. [167] Dancaster CP, Jackson WPU. Studies in rickets in the Cape Peninsula 1. Cranial softening in a coloured population and its relationship to the radiological and biochemical changes of rickets. S Afr Med J 1960;34:776e80. [168] Reilly BJ, Leeming JM, Fraser D. Craniosynostosis in the rachitic spectrum. J Pediatr 1964;64:396e405. [169] Hochman HI, Mejlszenkier JD. Cataracts and pseudotumor cerebri in an infant with vitamin D-deficiency rickets. J Pediatr 1977;90:252e4. [170] Hamilton B. Vitamin D and human skeletal muscle. Scand J Med Sci Sports 2010;20:182e90. [171] Schubert L, DeLuca HF. Hypophosphatemia is responsible for skeletal muscle weakness of vitamin D deficiency. Arch Biochem Biophys 2010;500:157e61. [172] Abdullah M, Bigras JL, McCrindle BW. Dilated cardiomyopathy as a first sign of nutritional vitamin D deficiency rickets in infancy. Can J Cardiol 1999;15:699e701. [173] Uysal S, Kalayci AG, Baysal K. Cardiac functions in children with vitamin D deficiency rickets. Pediatr Cardiol 1999;20:283e6. [174] Mansbach JM, Camargo Jr CA. Respiratory viruses in bronchiolitis and their link to recurrent wheezing and asthma. Clin Lab Med 2009;29:741e55. [175] Walker VP, Modlin RL. The vitamin D connection to pediatric infections and immune function. Pediatr Res 2009;65:106Re13R. [176] Tezer H, Siklar Z, Dallar Y, Dogankoc S. Early and severe presentation of vitamin D deficiency and nutritional rickets among hospitalized infants and the effective factors. Turk J Pediatr 2009;51:110e5. [177] Al-Atawi MS, Al-Alwan IA, Al-Mutair AN, Tamim HM, AlJurayyan NA. Epidemiology of nutritional rickets in children. Saudi J Kidney Dis Transpl 2009;20:260e5.

651

[178] Najada AS, Habashneh MS, Khader M. The frequency of nutritional rickets among hospitalized infants and its relation to respiratory diseases. J Trop Pediatr 2004;50:364e8. [179] Banajeh SM. Nutritional rickets and vitamin D deficiencye association with the outcomes of childhood very severe pneumonia: a prospective cohort study. Pediatr Pulmonol 2009;44. 1207e1.5. [180] Bikle DD. Vitamin D and the immune system: role in protection against bacterial infection. Curr Opin Nephrol Hypertens 2008;17:348e52. [181] Liu PT, Stenger S, Li H, et al. Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response. Science 2006;311:1770e3. [182] Lange NE, Litonjua A, Hawrylowicz CM, Weiss S. Vitamin D, the immune system and asthma. Expert Rev Clin Immunol 2009;5:693e702. [183] Yetgin S, Ozoylu S. Myeloid metaplasia in vitamin D deficiency rickets. Scand J Haematol 1982;28:180e5. [184] David L. Common vitamin D-deficiency rickets. In: Glorieux FH, editor. Rickets. New York: Nestec, Vevey, Raven Press; 1991. p. 107e22. [185] Balasubramanian S, Varadharajan R, Ganesh R, Shivbalan S. Myelofibrosis and vitamin D deficient ricketsea rare association. Indian Pediatr 2005;42:482e4. [186] Kamien B, Harris L. Twin troubles e rickets causing myelofibrosis. J Paediatr Child Health 2007;43:573e5. [187] Grindulis H, Scott PH, Belton NR, Wharton BA. Combined deficiency of iron and vitamin D in Asian toddlers. Arch Dis Child 1986;61:843e8. [188] Heldenberg D, Tenenbaum G, Weisman Y. Effect of iron on serum 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D concentrations. Am J Clin Nutr 1992;56:533e6. [189] Zeghoud F, Vervel C, Guillozo H, Walrant-Debray O, Boutignon H, Garabedian M. Subclinical vitamin D deficiency in neonates: definition and response to vitamin D supplements. Am J Clin Nutr 1997;65:771e8. [190] Viljakainen HT, Saarnio E, Hytinantti T, et al. Maternal vitamin D status determines bone variables in the newborn. J Clin Endocrinol Metab 2010;95:1749e57. [191] Bodnar LM, Catov JM, Zmuda JM, et al. Maternal serum 25hydroxyvitamin D concentrations are associated with small-forgestational age births in white women. J Nutr 2010;140:999e1006. [192] Mahon P, Harvey N, Crozier S, et al. Low maternal vitamin D status and fetal bone development: cohort study. J Bone Miner Res 2009;25:14e9. [193] Thacher TD, Fischer PR, Pettifor JM. The usefulness of clinical features to identify active rickets. Ann Trop Paediatr 2002;22:229e37. [194] Kabir ML, Rahman M, Talukder K, et al. Rickets among children of a coastal area of Bangladesh. Mymensingh Med J 2004;13:53e8. [195] Editorial. Diagnosis of nutritional rickets. Lancet 1971;ii:28e9. [196] Strand MA, Perry J, Jin M, et al. Diagnosis of rickets and reassessment of prevalence among rural children in northern China. Pediatr Int 2007;49:202e9. [197] Muldowney FP, Freaney R, McGeeney D. Renal tubular acidosis and amino-aciduria in osteomalacia of dietary or intestinal origin. Q J Med 1968;37:517e39. [198] Kruse K. Pathophysiology of calcium metabolism in children with vitamin D-deficiency rickets. J Pediatr 1995;126:736e41. [199] Arnaud SB, Arnaud CD, Bordier P, Goldsmith RS, Flueck JA. The interrelationships between vitamin D and parathyroid hormone in disorders of mineral metabolism in man. In: Norman AW, Schaefer K, Grigoleit HG, von Herrath D, Ritz E,

PEDIATRIC BONE

652

[200]

[201] [202]

[203]

[204]

[205] [206]

[207]

[208] [209]

[210]

[211]

[212]

[213]

[214]

[215]

[216]

[217] [218]

23. NUTRITIONAL RICKETS

editors. Vitamin D and Problems of Uremic Bone Disease. New York: Walter de Gruyter; 1975. p. 397e416. Arnaud CD. Parathyroid hormone and its role in the pathophysiology of the common forms of rickets and osteomalacia. In: Glorieux FH, editor. Rickets. New York: Nestec, Vevey, Raven Press; 1991. p. 47e61. Arnstein AR, Frame B, Frost HM. Recent progress in osteomalacia and rickets. Ann Intern Med 1967;67:1296e330. Taitz LS, de Lacy CD. Parathyroid function in vitamin D deficiency rickets II. The relationship of parathyroid function to bone changes and incidence of tetany in vitamin D deficiency rickets in South African bantu infants. Pediatrics 1962;30:884e92. Lumb GA, Stanbury SW. Parathyroid function in human vitamin D deficiency and vitamin D deficiency in primary hyperparathyroidism. Am J Med 1974;56:833e9. Stanbury SW, Torkington P, Lumb GA, Adams PH, De Silva P, Taylor CM. Asian rickets and osteomalacia: patterns of parathyroid response in vitamin D deficiency. Proc Nutr Soc 1975;34:111e7. Ekanem EE, Bassey DE, Eyong M. Nutritional rickets in Calabar, Nigeria. Ann Trop Paediatr 1995;15:303e6. Thacher TD, Ighogboja SI, Fischer PR. Rickets without vitamin D deficiency in Nigerian children. Ambulatory Child Health 1997;3:56e64. Taylor JA, Richter M, Done S, Feldman KW. The utility of alkaline phosphatase measurement as a screening test for rickets in breast-fed infants and toddlers: a study from the Puget Sound Pediatric Research Network. Clin Pediatr 2010. OnlineFirst:doi: 10.1177/0009922810376993. Delmas PD. Biochemical markers of bone turnover. J Bone Miner Res 1993;8:S549e55. Calvo S, Eyre DR, Gundberg CM. Molecular basis and clinical application of biological markers of bone turnover. Endocr Rev 1996;17:333e68. de Ridder CM, Delemarre-van de Waal HA. Clinical utility of markers of bone turnover in children and adolescents. Curr Opin Pediatr 1998;10:441e8. Szule P, Seeman E, Delmas PD. Biochemical measurements of bone turnover in children and adolescents. Osteoporos Int 2000;11:281e94. Scariano JK, Walter EA, Glew RH, et al. Serum levels of the pyridinoline crosslinked carboxyterminal telopeptide of type I collagen (ICTP) and osteocalcin in rachitic children in Nigeria. Clin Biochem 1995;28:541e5. Baroncelli GI, Bertelloni S, Ceccarelli C, Amato V, Saggese G. Bone turnover in children with vitamin D deficiency rickets before and during treatment. Acta Paediatr 2000;89:513e8. Daniels ED, Pettifor JM, Moodley GP. Serum osteocalcin has limited usefulness as a diagnostic marker for rickets. Eur J Pediatr 2000;159:730e3. Greig F, Casas J, Castells S. Changes in plasma osteocalcin concentrations during treatment of rickets. J Pediatr 1989;114:820e3. Scariano JK, Vanderjagt DJ, Thacher T, Isichei CO, Hollis BW, Glew RH. Calcium supplements increase the serum levels of crosslinked N-telopeptides of bone collagen and parathyroid hormone in rachitic Nigerian children. Clin Biochem 1998;31:421e7. Vieth R. Vitamin D supplementation, 25-hydroxyvitamin D concentrations, and safety. Am J Clin Nutr 1999;69:842e56. Pettifor JM. What is the optimal 25(OH)D level for bone in children? Vitamin D endocrine system: structural, biological, genetic and clinical aspects. Proceedings of the 11th Workshop on vitamin D. Riverside; 2000. p. 903e7.

[219] Heaney RP. Vitamin D: how much do we need, and how much is too much? Osteoporos Int 2000;11:553e5. [220] Norman AW, Bouillon R. Vitamin D nutritional policy needs a vision for the future. Proc Soc Exp Biol Med 2010;235:1034e45. [221] Henry HL, Bouillon R, Norman AW, et al. 14th Vitamin D Workshop consensus on vitamin D nutritional guidelines. J Steroid Biochem Mol Biol 2010;121:4e6. [222] Haddad JG, Chyu KJ. Competitive protein binding radioassay for 25-hydroxycholecalciferol. J Clin Endocrinol 1971;33:992e5. [223] Markestad T, Halvorsen S, Seeger Halvorsen K, Aksnes L, Aarskog D. Plasma concentrations of vitamin D metabolites before and during treatment of vitamin D deficiency rickets in children. Acta Paediatr Scand 1984;73:225e31. [224] Goel KM, Sweet EM, Logan RW, Warren JM, Arneil GC, Shanks RA. Florid and subclinical rickets among immigrant children in Glasgow. Lancet 1976;i:1141e5. [225] Garabedian M, Vainsel M, Mallet E, et al. Circulating vitamin D metabolite concentrations in children with nutritional rickets. J Pediatr 1983;103:381e6. [226] Specker BL, Ho ML, Oestreich A, et al. Prospective study of vitamin D supplementation and rickets in China. J Pediatr 1992;120:733e9. [227] Pettifor JM, Isdale JM, Sahakian J, Hansen JDL. Diagnosis of subclinical rickets. Arch Dis Child 1980;55:155e7. [228] Olivieri MB, Ladizesky M, Mautalen CA, Alonso A, Martinez L. Seasonal variations of 25 hydroxyvitamin D and parathyroid hormone in Ushuaia (Argentina), the southernmost city of the world. Bone Miner 1993;20:99e108. [229] Guillemant J, Cabrol S, Allemandou A, Peres G, Guillemant S. Vitamin D-dependent seasonal variation of PTH in growing male adolescents. Bone 1995;17:513e6. [230] Guillemant J, Le HT, Maria A, Allemandou A, Peres G, Guillemant S. Wintertime vitamin D deficiency in male adolescents: effect on parathyroid function and response to vitamin D3 supplements. Osteoporos Int 2001;12:875e9. [231] Outila TA, Karkkainen MU, Lamberg-Allardt CJ. Vitamin D status affects serum parathyroid hormone concentrations during winter in female adolescents: associations with forearm bone mineral density. Am J Clin Nutr 2001;74:206e10. [232] Docio S, Riancho JA, Perez A, Olmos JM, Amado JA, GonzalesMacias J. Seasonal deficiency of vitamin D in children: a potential target for osteoporosis-preventing strategies? J Bone Miner Res 1998;13:544e8. [233] Pearce SH, Cheetham TD. Diagnosis and management of vitamin D deficiency. Br Med J 2010;340:5664. [234] Wagner CL, Greer FR. The Section on Breastfeeding and Committee on Nutrition. Prevention of Rickets and Vitamin D Deficiency in Infants, Children, and Adolescents. Pediatrics 2008;122:1142e52. [235] Gordon CM, Williams AL, Feldman HA, et al. Treatment of hypovitaminosis D in infants and toddlers. J Clin Endocrinol Metab 2008;93:2716e21. [236] Munns C, Zacharin MR, Rodda CP, et al. Prevention and treatment of infant and childhood vitamin D deficiency in Australia and New Zealand: a consensus statement. Med J Aust 2006;185:268e72. [237] Hochberg Z, Bereket A, Davenport M, et al. Consensus development for the supplementation of vitamin D in childhood and adolescence. Horm Res 2002;58:39e51. [238] Thacher TD, Fischer PR, Obadofin MO, Levine MA, Singh RJ, Pettifor JM. Comparison of metabolism of vitamins D2 and D3 in children with nutritional rickets. J Bone Miner Res 2010;25:1988e95. [239] Stanbury SW, Taylor CM, Lumb GA, et al. Formation of vitamin D metabolites following correction of human vitamin D

PEDIATRIC BONE

REFERENCES

[240]

[241]

[242] [243]

[244]

[245]

[246]

[247]

[248] [249]

[250]

[251]

[252] [253]

[254]

[255] [256]

[257]

[258] [259]

[260]

deficiency: observations in patients with nutritional osteomalacia. Miner Electrolyte Metab 1981;5:212e27. Eastwood JB, de Wardener HE, Gray RW, Lemann Jr JR. Normal plasma-1,25-(OH)2-vitamin-D concentrations in nutritional osteomalacia. Lancet 1979;i:1377e8. Chesney RW, Zimmerman J, Hamstra A, DeLuca HF, Mazess RB. Vitamin D metabolite concentrations in vitamin D deficiency. Am J Dis Child 1981;135:1025e8. Rosen JF, Chesney RW. Circulating calcitriol concentrations in health and disease. J Pediatr 1983;103:1e17. Rasmussen H, Baron R, Broadus A, DeFronzo R, Lang R, Horst R. 1,25(OH)2D3 is not the only D metabolite involved in the pathogenesis of osteomalacia. Am J Med 1980;69:360e8. St-Arnaud R, Messerlian S, Moir JM, Omdahl JL, Glorieux FH. The 25-hydroxyvitamin D 1-alpha-hydroxylase gene maps to the pseudovitamin D-deficiency rickets (PDDR) disease locus. J Bone Miner Res 1997;12:1552e9. St-Arnaud R. CYP24A1-deficient mice as a tool to uncover a biological activity for vitamin D metabolites hydroxylated at position 24. J Steroid Biochem Mol Biol 2010;121:254e6. NGuyen TM, Guillozo H, Garabedian M, Mallet E, Balsan S. Serum concentrations of 24,25-dihydroxyvitamin D in normal children and in children with rickets. Pediat Res 1979;13:973e6. Oramasionwu GE, Thacher TD, Pam SD, Pettifor JM, Abrams SA. Adaptation of calcium absorption during treatment of nutritional rickets in Nigerian children. Br J Nutr 2008;100:387e92. Pettifor JM. Dietary calcium deficiency. In: Glorieux FH, editor. Rickets. New York: Nestec, Vevey, Raven Press; 1991. p. 123e43. Fischer PR, Thacher TD, Pettifor JM. Pediatric vitamin D and calcium nutrition in developing countries. Rev Endocr Metab Disord 2008;9:181e92. Oginni LM, Worsfold M, Sharp CA, Oyelami OA, Powell DE, Davie MW. Plasma osteocalcin in healthy Nigerian children and in children with calcium-deficiency rickets. Calcif Tissue Int 1996;59:424e7. Thacher T, Glew RH, Isichei C, et al. Rickets in Nigerian children: response to calcium supplementation. J Trop Pediatr 1999;45:202e7. Akpede GO, Omotara BA, Ambe JP. Rickets and deprivation: a Nigerian study. J R Soc Health 1999;119:216e22. Pettifor JM, Ross FP, Travers R, Glorieux FH, DeLuca HF. Dietary calcium deficiency: a syndrome associated with bone deformities and elevated serum 1,25-dihydroxyvitamin D concentrations. Metab Bone Rel Res 1981;2:301e5. Thacher TD, Fischer PR, Isichei CO, Pettifor JM. Early response to vitamin D2 in children with calcium deficiency rickets. J Pediatr 2006;149:840e4. Pocotte SL, Ehrenstein G, Fitzpatrick LA. Regulation of parathyroid hormone secretion. Endocr Rev 1991;12:291e301. Marks KH, Kilav R, Naveh-Many T, Silver J. Calcium, phosphate, vitamin D, and the parathyroid. Pediatr Nephrol 1996;10:364e7. Thacher TD, Obadofin MO, O’Brien KO, Abrams SA. The effect of vitamin D2 and vitamin D3 on intestinal calcium absorption in Nigerian children with rickets. J Clin Endocrinol Metab 2009;94:3314e21. Graff M, Thacher TD, Fischer PR, et al. Calcium absorption in Nigerian children with rickets. Am J Clin Nutr 2004;80:1415e21. Thacher TD, Abrams SA. Relationship of calcium absorption with 25(OH)D and calcium intake in children with rickets. Nutr Rev 2010;68:682e8. Prentice A, Ceesay M, Nigdikar S, Allen SJ, Pettifor JM. FGF23 is elevated in Gambian children with rickets. Bone 2008;42:788e97.

653

[261] Oestreich AE, Ahmad BS. The periphysis and its effect on the metaphysis. II. Application to rickets and other abnormalities. Skeletal Radiol 1993;22:115e9. [262] Steinbach HL, Noetzli M. Roentgen appearance of the skeleton in osteomalacia and rickets. Am J Roentgen 1964;91:955e72. [263] Hunter GJ, Schneidau A, Hunter JV, Chapman M. Rickets in adolescence. Clin Radiol 1984;35:419e21. [264] Adams JE. Radiology of rickets and osteomalacia. In: Feldman D, Pike JW, Glorieux FH, editors. Vitamin D. 2nd ed. San Diego: Elsevier Academic Press; 2005. p. 967e94. [265] Thacher TD, Fischer PR, Pettifor JM, Lawson JO, Manaster BJ, Reading JC. Radiographic scoring method for the assessment of the severity of nutritional rickets. J Trop Pediatr 2000;46:132e9. [266] Oginni LM, Sharp CA, Worsfold M, Badru OS, Davie MW. Healing of rickets after calcium supplementation. Lancet 1999;353:296e7. [267] Dent CE, Round JM, Rowe DJF, Stamp TCB. Effect of chapattis and ultraviolet irradiation on nutritional rickets in an Indian immigrant. Lancet 1973;i:1282e4. [268] Gupta MM, Round JM, Stamp TCB. Spontaneous cure of vitamin-D deficency in Asians during summer in Britain. Lancet 1974;i:586e8. [269] Holick MF, Biancuzzo RM, Chen TC, et al. Vitamin D2 is as effective as vitamin D3 in maintaining circulating concentrations of 25-hydroxyvitamin D. J Clin Endocrinol Metab 2008;93:677e81. [270] Houghton LA, Vieth R. The case against ergocalciferol (vitamin D2) as a vitamin supplement. Am J Clin Nutr 2006;84:694e7. [271] Trang HM, Cole DEC, Rubin LA, Pierratos A, Siu S, Vieth R. Evidence that vitamin D3 increases serum 25-hydroxyvitamin D more efficiently than does vitamin D2. Am J Clin Nutr 1998;68:854e8. [272] Armas LAG, Hollis BW, Heaney RP. Vitamin D2 is much less effective than vitamin D3 in humans. J Clin Endocrinol Metab 2004;89:5387e91. [273] Venkataraman PS, Tsang RC, Buckley DD, Ho M, Steichen JJ. Elevation of serum 1,25-dihydroxyvitamin D in response to physiologic doses of vitamin D in vitamin D-deficient infants. J Pediatr 1983;103:416e9. [274] Shah BR, Finberg L. Single-day therapy for nutritional vitamin Ddeficiency rickets: a preferred method. J Pediatr 1994;125:487e90. [275] Billoo AG, Murtaza G, Memon MA, Khaskheli SA, Iqbal K, Rao MH. Comparison of oral versus injectable vitamin-d for the treatment of nutritional vitamin-d deficiency rickets. J Coll Physicians Surg Pak 2009;19:428e31. [276] Akcam M, Yildiz M, Yilmaz A, Artan R. Bone mineral density in response to two different regimes in rickets. Indian Pediatr 2006;43:423e7. [277] Soliman AT, El-Dabbagh M, Adel A, Ali MA, Aziz Bedair EM, ElAlaily RK. Clinical responses to a mega-dose of vitamin D3 in infants and toddlers with vitamin D deficiency rickets. J Trop Pediatr 2009. fmp040. [278] Rajah J, Abdel-Wareth L, Haq A. Failure of alphacalcidol (1alpha-hydroxyvitamin D3) in treating nutritional rickets and the biochemical response to ergocalciferol. J Steroid Biochem Mol Biol 2010;121:273e6. [279] Department of Health. Nutrition and Bone Health: with Particular Reference to Calcium and Vitamin D. London: Her Majesty’s Stationery Office; 1998. p.115. [280] Institute of Medicine. Dietary reference intakes for calcium and vitamin D. Washington: National Academies Press; 2011. [281] Gartner LM, Greer FR. Prevention of rickets and vitamin D deficiency: new guidelines for vitamin D intake. Pediatrics 2003;111:908e10. [282] Misra M, Pacaud D, Petryk A, Collett-Solberg PF, Kappy M, on behalf of the Drug and Therapeutics Committee of the Lawson

PEDIATRIC BONE

654

[283] [284]

[285]

[286] [287]

23. NUTRITIONAL RICKETS

Wilkins Pediatric Endocrine Society. Vitamin D deficiency in children and its management: review of current knowledge and recommendations. Pediatrics 2008;122:398e417. Holick MF. Vitamin D deficiency. N Engl J Med 2007;357:266e81. Markestad T, Hesse V, Siebenhuner M, et al. Intermittent highdose vitamin D prophylaxis during infancy: effect on vitamin D metabolites, calcium, and phosphorus. Am J Clin Nutr 1987;46:652e8. Zeghoud F, Ben-Mekhbi H, Djeghri N, Garabedian M. Vitamin D prophylaxis during infancy: comparison of the long-term effects of three intermittent doses (15, 5, or 2.5 mg) on 25hydroxyvitamin D concentrations. Am J Clin Nutr 1994;60:393e6. Pietrek J, Preece MA, Windo J, et al. Prevention of vitamin-D deficiency in Asians. Lancet 1976;i:1145e8. Holmes VA, Barnes MS, Alexander HD, McFaul P, Wallace JMW. Vitamin D deficiency and insufficiency in pregnant women: a longitudinal study. Br J Nutr 2009;102:876e81.

[288] McCullough ML. Vitamin D deficiency in pregnancy: bringing the issues to light. J Nutr 2007;137:305e6. [289] Ginde AA, Sullivan AF, Mansbach JM, Camargo Jr CA. Vitamin D insufficiency in pregnant and nonpregnant women of childbearing age in the United States. Am J Obstet Gynecol 2010;202:436.e1e8. [290] Saadi HF, Dawodu A, Afandi BO, Zayed R, Benedict S, Nagelkerke N. Efficacy of daily and monthly high-dose calciferol in vitamin D-deficient nulliparous and lactating women. Am J Clin Nutr 2007;85:1565e71. [291] Yu CK, Sykes L, Sethi M, Teoh TG, Robinson S. Vitamin D deficiency and supplementation during pregnancy. Clin Endocrinol (Oxf) 2009;70:685e90. [292] Pettifor JM. Vitamin D deficiency and nutritional rickets in children. In: Feldman D, Pike JW, Glorieux FH, editors. Vitamin D. 2nd ed. San Diego: Elsevier Academic Press; 2005. p. 1065e83.

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Metabolic Bone Disease in the Neonatal Period and its Later Sequelae Nick Bishop 1, Mary Fewtrell 2, Nicholas C Harvey 3 1

2

Academic Unit of Child Health, University of Sheffield, Sheffield Childrens Hospital, Sheffield, UK Childhood Nutrition Research Centre, UCL Institute of Child Health, London, UK 3 The MRC Lifecourse Epidemiology Unit, University of Southampton, Southampton General Hospital, Southampton, UK

EARLY LIFE INFLUENCES ON SKELETAL HEALTH AND DEVELOPMENT An individual’s genetic inheritance has historically been thought to be the main determinant of adult bone mass [1]. Over the last 20 years, however, evidence has accrued that environmental factors, operating during periods of developmental plasticity in early development, may also contribute significantly via modulation of physiological systems and gene expression resulting in altered postnatal skeletal development [2,3]. Data emerging from this area of research suggest that prevention of osteoporotic fracture, for which reduced adult bone mass is a major risk factor, should be addressed throughout the lifecourse perhaps even from conception. Osteoporosis is the commonest bone disorder in Western populations. It is characterized by low bone mass and microarchitectural deterioration of bone tissue, which lead to increased bone fragility and the potentially devastating consequences of fragility fracture [4]. It has been estimated that at age 50 the remaining lifetime risk of fracture at the wrist, spine or hip is 39% among women and 13% among men [5]. The number of fractures sustained is likely to rise substantially over coming years, as fracture rates seem to be rising in many parts of the world, and elderly people are the fastest growing age group worldwide. Even if age-adjusted incidence rates for hip fracture increase by only 1% per year, the estimated number of hip fractures worldwide will rise from 1.7 million in 1990 to 8.2 million in 2050 [5]. Osteoporotic fracture also has a huge economic impact, the annual cost to the USA has been estimated as $20 billion, and to the European Union as $30 billion [6].

Pediatric Bone, Second Edition DOI: 10.1016/B978-0-12-382040-2.10024-3

An individual’s bone mass (a composite measure of bone size and volumetric density) appears to track through childhood and adolescence to reach a peak in early adulthood (termed peak bone mass) [7e9] in association with tracking of body size. Therefore, an individual tends to stay in the same position in the overall distribution relative to peers throughout growth. Bone mass then declines into older age, with an accelerated rate of decline after the female menopause. Recent studies have suggested that peak bone mass is at least as important a predictor of osteoporosis risk as is rate of bone loss [10]. Thus optimization of peak bone mass, by improving the early life environment, is likely to be an important target for policies aimed at reducing future risk of osteoporotic fracture in old age.

SKELETAL DEVELOPMENT AND MINERAL ACCRETION IN UTERO During fetal life, plasma levels of calcium and phosphorus are higher than those seen 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 metabolites of vitamin D, 25(OH) D and 1,25(OH)2D [11,12]. Of the active metabolites, 25(OH)D crosses the placenta, whereas 1,25(OH)2D does not, but is produced in the placenta itself. Umbilical cord 25(OH)D concentration is thought to reflect the degree of maternal vitamin D sufficiency, with concentrations greater than 20 ng/mL (50 nmol/L) being regarded as adequate [13,14].

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For adequate bone development to occur, the human fetus requires approximately 30 g of calcium during gestation, actively transported across the placenta. To supply the demand for calcium, the mother increases both calcium absorption from the gut, and bone resorption. There is a net positive calcium flux from the maternal to fetal circulation across the placenta. A miniature version of the skeleton is laid down in the embryonic period, and primary ossification centers form in the vertebrae and long bones between the 8th and 12th weeks, but it is not until the third trimester that the bulk of mineralization occurs [15]. The mechanism of regulation of this process is poorly understood in humans, but it is thought that both PTH and parathyroid hormone-related peptide (PTHrP) have important and complementary roles [16]. In the rat, 1,25(OH)2 D has been shown to influence placental calcium flux [17], although in a mouse model, lack of VDR did not significantly affect placental calcium transport or skeletal mineralization [18]. Recent work (described below) has suggested that vitamin D may be important in the human situation. The main determinant of skeletal mineralization in utero appears to be the fetal plasma calcium concentration [16], and this is mainly influenced by fetal PTH activity, even though this is set at a low level throughout gestation. Lack of parathyroids in fetal mice leads to low fetal calcium levels and decreased skeletal mineralization [16]. PTH does not cross the placenta, and maternal hypo- and hyperparathyroidism appear to affect the fetus via decreasing or increasing the calcium load presented to the fetal circulation. In humans, maternal hyperparathyroidism may lead to stillbirth or neonatal hypocalcemia [19], secondary to suppression of fetal PTH. Maternal hypoparathyroidism leads to increased levels of fetal PTH via fetal parathyroid hyperplasia, and generalized skeletal demineralization [19]. The action of PTH seems to include increasing calcium resorption from the fetal kidney, and possibly bone, to increase calcium concentration. PTH does not seem to influence placental calcium transfer, as injection of PTH into thyroparathyroidectomized fetal sheep does not increase placental calcium flux, in contrast to injection of mid-regions of PTHrP [20]. Thus, there is increasing evidence that PTHrP is the major determinant of placental calcium transport in animals, and that levels of PTHrP are increased in response to a low fetal plasma calcium level [16,21,22]. PTHrP appears to be produced in the parathyroid glands in some species, but not in others, and this may explain the differing responses of mice and sheep to its removal [20]. This procedure influences placental calcium transport in the latter but not in the former [20,22]. The role of PTHrP is less well characterized in human pregnancy, but

may be produced in the placenta, and is present in high concentrations in breast milk [19,23]. It is unclear how the actions of fetal PTH and PTHrP interact with the molecular apparatus in the placenta: placental calcium transfer occurs in the syncytiotrophoblast and proceeds through a sequence of events consisting of facilitated apical entry through a calcium transport channel, cytosolic diffusion of calcium bound to calbindin and, finally, basolateral extrusion of calcium ions through a plasma membrane calcium-dependent ATPase [24]. This last group of transport channels includes four individual isoforms (PMCA 1e4). These have been previously demonstrated in human placenta, as well as in fetal skeletal muscle and brain. PMCA 1 and 4 are present in most tissues while PMCA 2 and 3 are found in more specialized cell types [25]. One study in the rat has suggested that a two- to threefold increase in PMCA gene expression is associated with a 72-fold increase in calcium transport across the placenta during late gestation [26]. The regulation of this process is as yet unknown, but at least one of the isoforms of PMCA has been shown to be regulated by vitamin D [27], and in some animals, but not others, 1,25(OH)2D appears necessary for maintenance of the maternofetal calcium gradient [17]. Recent work in human subjects has shown that the level of mRNA expression of an active placental calcium transporter (PMCA 3), thought to be situated on the basal membrane of the placenta, is positively correlated with whole body bone mineral content (BMC) in the offspring at birth [28]. These observations may suggest a possible mechanism for the influence of maternal vitamin D status on placental calcium transport and intrauterine bone mineral accrual. In addition to its effects on placental calcium transport, PTHrP influences linear bone growth by acting on prehypertrophic chondrocytes in the fetal growth plate to inhibit differentiation to hypertrophic chondrocytes, and both over- and underexpression [29,30] of PTHrP or its receptor are associated with short-limbed dwarfism in animals and humans [31e35]. Additionally, there is evidence that PTH and PTHrP differentially affect mineralization of cortical and trabecular bone [36,37]. Thus both fetal PTH and PTHrP activity contribute to fetal plasma calcium concentration, but the action of PTH appears to predominate. It is unclear in human pregnancy how 25(OH)D or 1,25(OH)2D are involved in this process.

Placental Supply versus Genetic Inheritance: Twin Studies One powerful study design with which to examine the relative contribution of genetic inheritance and placental nutrient supply is the twin study. In a UK study of 445 monozygotic (MZ) and 966 dizygotic (DZ)

PEDIATRIC BONE

PHYSIOLOGICAL CHANGES IN MINERAL HOMEOSTASIS AT BIRTH

twins, at a mean age of 47 years, birth weight was found positively to predict BMC and bone mineral density (BMD) [38] at the hip. The MZ twins, despite being genetically identical, had greater intra-pair variability in birth weight and subsequent bone mass as adults than DZ twins. Two-thirds of the MZ twins will have shared a placenta; a common consequence of the resulting competition for placental blood flow is one large and one small, albeit genetically identical, twin. The greater intra-pair differences in bone mass as adults for the MZ than the DZ twins suggest that these early influences have long-term implications.

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 falls rapidly, reaching a nadir of 1.0e1.2 mmol/L by around 16 hours of age. The rapid fall 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(OH)2D. Paradoxically, in the face of a falling calcium supply, plasma calcitonin rises rapidly after birth in infants. The mechanism responsible for this surge is as yet 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 hours after birth and is greater in preterm infants than in term small for gestational age (SGA) infants [39e41]. The surge can be ameliorated in preterm infants by the provision of large supplements (2 mmol/kg/day) of calcium [42]. PTH concentrations rise immediately after birth as plasma calcium falls and increase several-fold over the first 24 to 48 hours [43]. 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 30e90 minutes of the infusion commencing [44]. 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 RANK-ligand by osteoblast lineage cells in response to both PTH and 1,25(OH)2D [45]. It is likely that in newborn infants similar mechanisms are employed to maintain calcium homeostasis. There is at present no indication that tumor necrosis factor a (TNF-a) or interleukin 6 (IL-6) play any role in osteoclast activation in the immediate postnatal period. The recruitment, activation and fusion of

657

osteoclastic precursors, and their subsequent activity in response to osteoblast-derived 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 insufficiency [46], indicating the importance of having both PTH and 1,25(OH)2D 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 at present 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 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 [47]. 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 9e10). Anderson [47] has hypothesized that at the physiological pH of the bone mineralization front, alkaline phosphatase will function 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 calciumephosphate crystals can occur. Electron microscope pictures have 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

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658

24. METABOLIC BONE DISEASE IN THE NEONATAL PERIOD AND ITS LATER SEQUELAE

as foci for further crystallization [48]. 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 [49]. Figure 24.1 shows how the processes described above might link together around the time of birth and provides a framework against which to set what is known about metabolic disease in premature infants.

PRETERM DELIVERY AND BONE METABOLISM An infant born before 37 weeks of completed pregnancy is by definition preterm. With modern neonatal intensive care, survival is possible after as little as 23 weeks 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’ gestation, gaining 15e20 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’ 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 [50]. 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 baby can survive only 4e5 days starvation, compared to 1 month for a term baby and 3 months for a healthy adult. 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’ gestation and term, with estimated daily fetal accretion rates of 2.6e3.2 mmol/kg/ day for calcium and 2.1e2.5 mmol/kg/day for phosphorus [15]. There is an early peak of accretion at 27e28 weeks’ gestation, followed by a slight fall, and then an exponential rise, with a second peak at 37e38 weeks’ gestation [51] (calculated from the data of Ziegler 1976 [52]; Fig. 24.2). Calcium and phosphorus are accreted in an almost constant molar ratio of 1.2e1.3:1 over the whole period. Preterm infants are thus born during an extremely rapid phase of mineral accretion and have low skeletal mineral stores at birth compared to a term infant that has built up large stores of mineral 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 a number of ways that renders them susceptible to mineral deficiency and metabolic bone disease: • born during a phase of rapid growth and mineral accretion • poor early nutrition/mineral intake • frequently ill during the neonatal period (respiratory distress syndrome, sepsis, necrotizing enterocolitis) • lack of movement due to sedation or paralysis associated with ventilatory support. Moyer-Mileur [53] found that a daily 5e10 minute 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 • frequent use of drugs that may alter bone mineralization (e.g. steroids, 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 [6,21e25,54e58]. Although markers of bone turnover appear to return to normal values when treatment is stopped [58], the long-term effects of this early suppression are unknown.

Historical Perspective on “Rickets of Prematurity” The problems associated with mineral metabolism in preterm infants were in fact identified as early as 1919 [59]. Benjamin et al. [60] 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 [61] 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 [62], working in Oslo, 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

PEDIATRIC BONE

659

PRETERM DELIVERY AND BONE METABOLISM

High fetal blood calcium, phosphate, calcitonin. Low PTH, 1,25(OH)2 D High mineral accretion rate

In the uterus:

Loss of transplacental calcium supply

First 48 hours:

Maternal vitamin D Exercise

Continued bone mineral accretion

Calcitonin surge

Hypocalcemia

Renal effects of parathormone

Increased production of 1,25(OH)2 D

Bone effects of parathormone

Ca reabsorption PO4 excretion

Bone effects of 1,25(OH) 2 D

Bone resorption

Matrix degradation

Release of calcium, phosphate

Urinary loss of phosphate

Bone mineral accretion

Matrix vesicles, local factors

Nutrient supply

Production of bone matrix

New bone formation; growth, remodeling and mineralization

FIGURE 24.1

Events influencing skeletal homeostasis around the time of birth in term infants.

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24. METABOLIC BONE DISEASE IN THE NEONATAL PERIOD AND ITS LATER SEQUELAE

Mineral accretion (mmol/kg/day)

4

Calcium Phosphorus

3

2

1

20

30 Gestation (weeks)

40

FIGURE 24.2 Accretion rates for calcium and phosphorus during fetal life.

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. Mean plasma phosphorus was lowest in the infants fed human milk, highest in the group receiving cow’s milk and intermediate for 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, rickety rosary) was reported. The mean length gain was 0.116 cm/day for the group fed cow’s milk, versus 0.104 cm/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 4weekly) were also reported. Initially, there was metaphyseal rarefaction, with progressive radiological osteoporosis spreading from the metaphyseal zone towards the midpoint of the diaphysis; later there was generalized osteoporosis from the age of 10e13 weeks. Periosteal double contours were seen earlier in the infants fed either cow’s milk or supplemented human milk than human milk alone (113 or 105 days vs 210 days), and interpreted as the first sign of improved mineralization, since such findings are also observed in cases of healing rickets.

Lewin, in 1971 [63], reported four cases of infants born at 27e32 weeks’ gestation and weighing 680e1300 g who were fed formula thought to contain sufficient vitamin D for normal bone growth and mineralization. However, all four infants showed some radiological evidence of rachitic changes at 2e4 months postnatal age. The mineral content of the milk (Enfalac liquid, Mead Johnson Laboratories) was not stated; the calculated vitamin D intakes for the infants averaged 100e150 IU/day of vitamin D. 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 [64] reported a further two cases of “rickets” in preterm infants receiving vitaminsupplemented adapted cow’s milk-based formula. Clinical signs e craniotabes and swollen costochondral junctions e were noted in both infants at 12e16 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 raised 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 step forward allowing for the first time precise in vivo quantification of the loss of bone mineral during the first few weeks of postnatal life [65e67]. Photon absorptiometry became a widely used tool in this context, and facilitated work directed not only at understanding the predisposing factors, but also 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. [68] reported calcium and phosphorus balances in preterm infants receiving either human milk or standard infant formula, and 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

PEDIATRIC BONE

SKELETAL HEALTH AND METABOLIC BONE DISEASE IN PRETERM INFANTS

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 180e200 ml/kg/day, human milk provides only 21e28 mg/kg/day (0.7e0.9 mmol/kg/day) of phosphorus and 51e68 mg/kg/day (1.25e1.7 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 (3 mmol/kg/day) of calcium required 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 which 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 by serial urinary calcium/phosphate ratios. This test can be performed on single, untimed samples, and should be less than 1 by 3 weeks of age if the infant is phosphate replete (calcium and phosphate in mmol). A later (4e6 weeks of age) feature of disturbed mineral metabolism is a raised 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 later [69]. 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 over the period of acute illness, growth accelerates, and with it, the likelihood of mineral deficiency unless an adequate mineral supply is ensured. Although, historically, plain radiographs have been used to assess the degree of osteopenia as well as looking 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 initially of single photon

661

absorptiometry (SPA) and subsequently dual energy x-ray absorptiometry (DXA) 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 AND METABOLIC BONE DISEASE 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 [70,71]. 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 DXA, there has 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. [72] measured radial 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 [67] reported similar findings; BMC in preterm infants remained fairly static despite overall body and bone growth. James et al. [73] 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 it was not simply a reflection of smaller body size. Lyon et al. [70] measured the bone mineral content of the distal radius (mixed cortical and trabecular bone) weekly in 15 preterm infants of less than 30 weeks’ gestation using an “in-house” DXA instrument with contemporaneous

PEDIATRIC BONE

662

24. METABOLIC BONE DISEASE IN THE NEONATAL PERIOD AND ITS LATER SEQUELAE

measurement of an aluminum step wedge. Mineral content rose during the first week of life but then fell before increasing slowly from 6 weeks of age, and was poor compared to a fetus at the equivalent postconceptual age. Measured mineral intakes in these infants were considerably below calculated intrauterine requirements. Pohlandt [74,75] 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. examined the effect of intrauterine growth retardation on BMC in both term and preterm infants and found that, while term small for gestational age (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 the result of protracted intrauterine malnutrition [76]. In contrast, Pohlandt and Mathers [75] 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. [77] also found lower whole body BMC in SGA term and preterm infants using dual photon absorptiometry (DPA). 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. [78] found that bone mineralization in low birth weight infants fed a term infant formula lagged significantly behind intrauterine mineralization rates [72], and that this was avoided by using a nutrient-enriched preterm infant formula [78] or by supplementing the formula with calcium, phosphorus and vitamin D [79]. Pittard et al. [80] 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 at the same age. Chan et al. [81] studied radial BMC in 36 preterm infants (<1.6 kg) who were fed: (1) preterm formula (117 mg calcium and 58.8 mg phosphorus/100 kcal); (2) the same formula with extra phosphorus (82 mg/ 100 kcal); (3) the same formula with extra calcium (140 mg/100 kcal) and phosphorus (82 mg/100 kcal); (4) human milk. BMC was similar in all three formula groups, but only groups (1) and (3) attained the in utero mineral accretion rate. BMC in infants fed human milk was significantly lower than in the formula groups. Venkataramen and Blick [82] 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 composition of some of the milks available for use in preterm infants is shown in Table 24.1. Greer [88] measured radial BMC over 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 [65] compared the BMC of the humerus in 50 preterm infants with birth weight <1.6 kg, who were randomized to three different dietary regimens in the neonatal period: (1) unsupplemented human milk (40e50 mg/kg/day calcium and 23e30 mg/kg/day phosphorus); (2) human milk mixed with a high mineral-containing formula (130 mg/kg/day calcium and 68 mg/kg/day phosphorus); (3) human milk mixed with a powdered fortifier (160 mg/kg/day calcium and 90 mg/kg/day phosphorus). After hospital discharge, infants were either breastfed or received a standard term infant formula. While receiving the randomized diet, infants fed human milk with formula supplementation had significantly higher serum phosphorus and lower serum alkaline phosphatase concentrations than those fed unsupplemented human milk. However, at the equivalent of term, there were no differences in BMC between the three randomized groups, nor any effect of post-discharge diet. Pettifor et al. [89] compared radial BMC in very low birth weight (1e1.5 kg) infants randomly assigned to receive unsupplemented human milk or human milk with a breast-milk fortifier until they reached 1.8 kg in weight. 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. More recently, Lapillone et al. [90] measured whole body BMC in 25 very low birth weight infants (<1.5 kg 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 100 mg/kg/day calcium and 72 mg/kg/day phosphorus e higher than in the earlier studies. Wauben et al. [91] 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 (calcium

PEDIATRIC BONE

663

SKELETAL HEALTH AND METABOLIC BONE DISEASE IN PRETERM INFANTS

TABLE 24.1

Nutrient Content of Low-birthweight Formulas Available in the UK (per 100 ml)

Composition per 100 ml mature human milk

Aptamil DHSSa

Nutriprem 1 Macy et alb

SMA gold (Milupa)

Preterm** (C&G)

Prem 1 (Wyeth)

Protein* (g)

1.34

1.45

2.5

2.5

2.2

Fat (g)

4.2

3.8

4.4

4.4

4.4

50.1

52

1.8

1.8

2.2

Total LCP (mg)

41

41

43

Dha (mg)

20

20

17

AA (mg)

15

15

26

Macronutrients

Saturates (g)

Carbohydrate (g) Total

7.0

7.0

7.6

7.6

8.4

Sugars

7.0

7.0

6.8

6.8

4.5

kcal

70

68

80

80

82

kJ

293

285

335

335

343

Calcium

35

33

120

100

101

Chloride

43

43

68

68

67

Magnesium

2.8

4.0

7.9

7.9

8.2

Phosphorus

15

15

66

66

61

Potassium

60

55

82

82

74

Sodium

15

15

50

50

44

Copper

39

40

80

80

90

Iodine

7

7

25

25

10

Iron

76

150

1400

1400

1400

Manganese

ND

0.7

10

10

4.8

Zinc

295

530

900

900

800

88

91

315

315

202

A retinol (mg)

60

53

180

180

185

B1 thiamin (mg)

16

16

140

140

140

Energy

Minerals (mg)

Trace elements (mg)

Osmolarity (mosmol/L) Vitamins

B2 riboflavin (mg)

31

42.6

200

200

200

B6 pyridoxine (mg)

6

11

120

120

120

B12 cyanocobalamin (mg)

0.01

trace

0.27

0.27

0.19

Biotin (mg)

0.76

0.4

3.0

3.0

2.4

Folic acid (mg)

5.2

0.18

28

28

29

Niacin (mg)

230

172

2400

2400

2400

C ascorbic acid (mg)

3.8

4.3

13

13

15

SD cholecalciferol (mg)

0.01

0.01

3.0

3.0

3.4 (Continued)

PEDIATRIC BONE

664 TABLE 24.1

24. METABOLIC BONE DISEASE IN THE NEONATAL PERIOD AND ITS LATER SEQUELAE

Nutrient Content of Low-birthweight Formulas Available in the UK (per 100 ml)dcont’d

Composition per 100 ml mature human milk

Aptamil DHSSa

Nutriprem 1 Macy et alb

SMA gold (Milupa)

Preterm** (C&G)

Prem 1 (Wyeth)

ED-X-tocopherol (mg)

0.35

0.56

3.0

3.0

3.3

K phytomenadione (mg)

ND

1.7

6.0

6.0

6.3

Carnitine

ND

ND

1.8

1.8

2.6

Choline

ND

9

13

13

15

Inositol

ND

39

40

40

30

Taurine

4.8

ND

5.5

5.5

5.7

3.2

3.2

Other (mg)

Nucleotides a

DHSS Reports on Health and Social Subjects [83e85]; [86,87]; * Total nitrogen 3 6.38; ** manufacturer’s information ND, not stated. LCP, long chain polyunsaturated fatty acids.

b

130 mg/kg/day and phosphorus 104 mg/kg/day), human milk with a multinutrient fortifier (calcium 108 mg/kg/day and phosphorus 101 mg/kg/day) or a preterm infant formula (calcium 102 mg/kg/day and phosphorus 68 mg/kg/day). BMC was not significantly different between the groups and was within the normal range of term infants at birth, both when expressed as absolute values and as a function of length or lean body mass. Faerk et al. [92] measured whole body mineral content in preterm infants at 36 weeks’ postconception and found no significant difference between those randomized to receive 200 ml/kg/day of human milk with phosphate, fortified human milk or a preterm formula. However, all infants had lower BMC (both absolute and per kg) than expected for a term infant at birth. There is thus a general impression 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 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. Growth-retarded infants may have low BMC compared to AGA infants, although the differences frequently disappear when BMC is related to body size rather than gestation.

More recently, quantitative ultrasound (QUS) has also been tested as a method of assessing bone health in preterm infants. This technique measures either the attenuation or speed of sound (SOS) through the appendicular skeleton; machines are available for performing measurements of the tibia and radius even in the smallest preterm infant. QUS has the advantage of being simple to perform at the bedside and does not involve radiation. However, there is still uncertainty about what exactly is being measured in terms of bone quality or strength. McDevitt et al. made longitudinal QUS measurements in 18 preterm infants and found that baseline SOS correlated with gestational age, but fell with time [93]. Similarly, Fewtrell et al. measured 99 preterm infants, 75 of whom were followed longitudinally [94]. Baseline SOS was positively related to gestational age, and fell with postnatal age. However, in multivariate analyses, there was no evidence that biochemical evidence of mineral deficiency or metabolic bone disease predicted the fall in SOS, and baseline SOS measurements did not predict the subsequent development of high plasma alkaline phosphatase or a low plasma phosphate. Thus, it is not yet clear whether QUS has a role in monitoring bone health in the neonatal unit setting, and at present it remains essentially a research tool. Bone Turnover A number of investigators have measured markers of bone turnover in preterm infants over 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 additional note, the colorimetric assays used for urinary creatinine estimations may be affected by urinary excretion of

PEDIATRIC BONE

SKELETAL HEALTH AND METABOLIC BONE DISEASE IN PRETERM INFANTS

conjugated bilirubin and its photo-isomers as will occur in jaundiced infants receiving phototherapy. The picric acid method of creatinine estimation is not affected by bilirubin compounds. Tissue non-specific alkaline phosphatase, usually referred to simply as alkaline phosphatase, is the most widely reported marker. However, the interpretation of values varies as widely as the normal ranges quoted for individual assays. The buffer systems used in the various kits and systems available mean that the upper limit of the normal range quoted for adults can vary from 130 IU/L to 950 IU/L. 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 as the value obtained at any one time is a composite of the modeling activity in the metaphyseal region close to the growth plate and under the periosteum as well as the remodeling activity occurring in the diaphysis of individual bones. At or close to the time of delivery, alkaline phosphatase activity is relatively low implying that bone formation activity is also low; rather 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 while 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, alkaline phosphatase activity rose over the first 4e6 weeks of life and then plateaued [95]. In infants receiving diets low in mineral (mothers’ own expressed breast milk or donor breast milk, neither supplemented in any way), ALP then rose further. In the studies of Beyers [96], increased ALP and urinary hydroxyproline excretion were associated with endosteal resorption and a thinning of the humeral cortex as assessed by magnification radiogrammetry. Bhandari [97] 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 fell after birth in association with changes in weight while BSALP increased. These changes did not correlate with changes in serum OC. By contrast, Crofton and colleagues [98] 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 gain in weight, length, and lower leg length and with bone mineral

665

content, measured weekly over the first 10 weeks 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, while ICTP decreased. Markers reached a plateau during weeks 4e10. 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 perhaps be viewed in the 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 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 dose-dependent fashion, but the degree of depression varied widely between infants. Seibold-Weiger [99] 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 (IGF-1) concentrations. Shiff et al. [100] 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, then continued with a more gradual increase until week 10. ICTP levels increased during the first week then gradually decreased during follow up. These results are consistent with increased bone formation in the first 3 months of postnatal life. Gfatter [101] 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. There is thus general agreement that markers of both formation and resorption are significantly higher in

PEDIATRIC BONE

666

24. METABOLIC BONE DISEASE IN THE NEONATAL PERIOD AND ITS LATER SEQUELAE

preterm infants than in those born at term, that concentrations show an inverse relationship with gestational age and with birth weight and that levels rise 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 have so far appeared to be transient, but longer term follow up is required to substantiate this and to examine whether there are any longer term consequences for bone growth or turnover.

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 [95]. 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 mineral, protein and energy.

Fractures There are case reports of fractures occurring in preterm infants prior to discharge from hospital. In a recent series, 7% of babies weighing <1000 g at birth had fractures; none survived to leave the neonatal unit [102]. Fractures may be of the ribs, possibly in association with physiotherapy, and at the ends of the long bones at the metaphysealediaphyseal junction. In this latter situation, the fractures have been associated with three point bending forces occurring during the insertion of peripheral or central venous access lines [103]. There are no reports of metaphyseal corner or buckethandle fractures, suggestive of twisting and pulling or shearing lateral forces occurring during this initial period of hospitalization. These observations would fit with the model proposed by Beyers [96] 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 might however occur in those infants with persistently elevated serum PTH as a result of cortical erosions. The factors most commonly identified as being associated with fractures in premature infants <28 weeks’ gestation either during the period of hospitalization or after discharge but within the first 6 months of life are: conjugated hyperbilirubinemia; prolonged (>21 days) intravenous feeding; bronchopulmonary dysplasia; furosemide use [104]. In combination, these factors identify sick, often immobilized infants with a parlous nutritional state; understanding these risks may however ameliorate the likelihood of fractures.

Bone Mass

Discharge to 2 Years Linear Growth In a large randomized trial of diet during the early neonatal period in preterm infants, those who had

In contrast to the large number of studies of bone mineralization during the first few weeks of life prior to full-term, there are fewer reports of outcome during infancy. Salle et al. [105] measured lumbar spine BMC in 49 preterm infants over the first year of life at 41, 89, 184 and 365 days, and compared the results to those of 22 full-term infants at 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 kg body weight using the data provided by the authors, there is little difference between term and preterm children, particularly beyond 6 months of age. Koo et al. [106] studied 74 preterm infants weighing around 1 kg at birth. Radial BMC was measured at 5 weeks, 14 weeks, 26 weeks, 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 compared to those without, although all infants had BMC values below expected levels. No information was given on diet in this study. Congdon [107] 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 46e71 weeks postconceptual age (6e31 weeks post-term), 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. McDevitt studied preterm infants after discharge using QUS, and reported catch-up in SOS values in most infants by 6 months [93]. This catch-up was independent of postnatal growth, and was greatest in infants with the lowest SOS at term. Other investigators have examined the influence of diet during the post-discharge period on bone

PEDIATRIC BONE

SKELETAL HEALTH AND METABOLIC BONE DISEASE IN PRETERM INFANTS

mineralization. Chan and Mileur [66] 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). Breastfed infants received their mother’s milk with supplemental calcium while in 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 the breastfed 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. [108] studied 17 very low birth weight (VLBW) 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 post-hospitalization period (weeks 10e25). After 25 weeks, infants were weaned onto solids and some of the breastfed infants received formula milk. However, differences in BMC remained at 1 year of age, independent of current weight. The BMC for formula-fed preterm infants was within the reference range for term infants, while that for the breast-fed infants was low. Breastfed 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 breastfed and formula-fed groups and within the range reported for term infants in both groups. Thus, infants fed human milk during the neonatal period had demonstrated catch-up in bone mineralization by the age of 2 years. Bishop et al. [109] 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 post-discharge 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 of age, independently of body size (which was also higher in the follow-on formula group). Wauben et al. [91] reported that preterm infants who were breastfed after discharge had significantly lower bone mass at 6 months post-term than infants fed a standard term formula, even after adjustment for body size. Brunton and colleagues [110] studied 60 preterm infants with bronchopulmonary dysplasia randomized to either nutrient-enriched formula or standard formula during the post-discharge period, measuring both radial

667

and total body bone mass. At 3 months corrected age infants fed enriched formula attained greater length, radial bone mineral content and greater lean mass. The male infants in the enriched formula group had greater whole body bone mineral content than did male infants in the standard formula group. Collectively, these studies suggest that post-discharge nutrition in vulnerable infants might affect bone mass, although with the exception of the study by Abrams, 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 post-discharge 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 are undergoing regular blood sampling for clinical purposes. Fractures In Koo’s prospective study [106], fractures did occur after discharge from hospital but no further fractures appeared after 6 months corrected postnatal age. Amir [111] reported clinically apparent fractures in 1.2% of preterm infants between the 24th and 160th day of postnatal life. The true incidence of fractures in this population following hospital discharge is likely to remain unknown. Rib fractures are difficult to detect clinically as single fractures may be splinted by adjoining ribs. The finding of apparently unexplained fractures, typically seen on a chest x-ray taken during an episode of respiratory illness, will usually initiate child protection proceedings. The difficulty in identifying prospectively 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.

2 Years Onwards Linear Growth Several studies have shown that preterm infants remain shorter than their term peers during childhood. In an analysis of factors predicting height in children born preterm at 9e12 years, Fewtrell et al. [112] found the only factor from the neonatal period that was associated with childhood height was biochemical evidence of metabolic bone disease during the neonatal

PEDIATRIC BONE

668

24. METABOLIC BONE DISEASE IN THE NEONATAL PERIOD AND ITS LATER SEQUELAE

period. Children with evidence of this condition were, on average, 0.12 standard deviations shorter than those without, suggesting that asymptomatic bone disease may be associated with stunting of linear growth for at least a decade. However, at later follow up there was no persisting effect of early metabolic bone disease on final height [113]. In a related analysis, children born preterm who had shown the greatest catch-up in height during childhood had the highest bone mass at 9e12 years of age [112] 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. [114] 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 to 5 months were below 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 fell 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 e details obtained from the clinical notes and by maternal recall) had no effect on BMC. Hori et al. [115] measured lumbar spine BMC in 21 preterm children at 3e4 years of age using DXA, compared to 16 term children aged 4 years. Preterm infants had been fed a mixture of human milk plus term formula (gestation >30 weeks) or preterm formula (gestation <30 weeks). Eleven of the preterm infants had previous BMC measurements at the equivalent of term showing 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 kg body weight. Helin et al. [116] measured radial BMC using SPA in 75 children born prematurely at age 4e16 years compared to 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. [117] reported lumbar spine BMC and BMD measurements in 38 preterm children at 6e7 years of age. All had received banked human milk supplemented with 100e105 mg/100 kcal calcium and 60e70 mg/100 kcal 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 6e7 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 which 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 6e7 years, after adjusting for current weight. This suggests that preterm children who showed the greatest increase in weight between one year and 6e7 years had the highest BMC and, therefore, that childhood growth may affect later bone mineralization. Bowden [118] measured bone mass using DXA in 46 ex-preterm infants at 8 years of age. BMC was significantly lower at all skeletal sites than for age-matched term children; however, differences disappeared when adjusted for the smaller body size of the preterm group. Backstrom [119] randomized 70 preterm infants to receive either 500 IU or 100 IU 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 age 9e11 years no differences were seen between the groups. Bishop [120] studied 54 children who were born preterm and randomly assigned to diet during the neonatal period at 5 years of age using SPA. 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 on 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. 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 5 years later. Two possible explanations were advanced for these findings. First, the low early mineral intakes might “program” bone cells to be conservative with mineral, 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. Two hundred and forty-three children from the same original preterm cohort were studied using DXA at 9e11 years of age [121]. Compared to their term peers, these children had lower lumbar spine, hip and whole body bone mass, although this was commensurate

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669

RECOMMENDATIONS

with their smaller body size. No effect of early randomized diet on bone mass was found at this age. Most recently, 230 members of this cohort were studied in early adult life (age 20 years) [113]. Compared to population reference data, these subjects were significantly shorter with lower lumbar spine BMD in early adult life; and these deficits were greatest in subjects born SGA with birthweight <1250 g. However, there was no significant effect of randomized neonatal diet on peak bone mass (measured by DXA). In non-randomized analyses, the proportion of human milk in the neonatal diet was found to be a positive predictor of whole body bone mass in early adult life; subjects with greater intakes of human milk had larger bones with a proportionate increase in mineral content, although there was no association between human milk intake and later weight or height. In this study, in accordance with practice at the time, human milk was fed without mineral supplements. Thus, as observed in the 5-year follow up of this cohort, it appears that a lower early mineral and nutrient intake may paradoxically be associated with higher later bone mass. In further analyses, there was no apparent association between estimated neonatal mineral intakes of these subjects and their bone mass in early adult life, and the observed beneficial effect of human milk may be most plausibly explained as a “non-nutrient” effect, for example due to one of the many growth factors or hormones present in human milk. Bone Turnover Hori et al. [115] reported higher plasma osteocalcin concentrations at 3e4 years in children born preterm compared to those born at term. In our large randomized trial [121] children randomized to the least adequate diets during the neonatal period (both in overall nutrients and in minerals) had significantly higher plasma osteocalcin at 9e11 years than those fed the more optimal preterm formula, suggesting that early diet may influence later bone formation. Urinary TABLE 24.2

deoxypyridinoline concentrations were not significantly different between groups; nevertheless, it seems likely that the raised serum osteocalcin may represent increased bone turnover, since bone mass was not increased. No persisting effect of neonatal diet on bone turnover in early adult life was observed at later follow up of this cohort [113]. Fractures Dahlenberg et al. [122] 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 towards an earlier presentation of fractures (age less than 2 years) in infants born at less than 32 weeks’ gestation. Bowden [118] found that fractures were less common by 8 years in a group of 46 ex-preterm infants than in age-matched peers. Fewtrell et al. also found that similar proportions of children born term and preterm had sustained fractures by 12 years of age [112] and by early adult life [113].

RECOMMENDATIONS Mineral and Vitamin D Intake Current recommendations for mineral intakes in preterm infants made by Expert Groups [123,124] focus on meeting in-utero accretion for these minerals (Table 24.2). It is generally accepted that a calcium retention level ranging from 60 to 90 mg/kg/day ensures appropriate mineralization and decreases the risk of fracture and clinical symptoms of osteopenia. This figure could be met by an intake of 100e160 mg/kg/day of highly bioavailable calcium salts; however, with less bioavailable salts a higher intake would be needed. Contemporary recommendations for mineral intake are generally significantly higher than the intakes achieved by infants in the research studies reported in this chapter, particularly older studies with the longest follow up. The fact

Recommended Enteral Intakes for Calcium, Phosphorus and Vitamin D per kg per day

Nutrient

Unit

ESPGHAN

Tsangb <1000 g

Vitamin D

IU

800e1600

100e308

115e364

75e270

Calcium

mg

110e130

67e169

77e200

123e185

Phosphorus

mg

55e80

40e108

46e127

82e109

Ca:P

mg:mg

NS

NS

NS

1.7e2.0:1

a

a

[124]; [123]; c [125]. LSRO ¼ Life Sciences Research Office (LSRO) of the American Society for Nutritional Sciences. b

PEDIATRIC BONE

Tsangb >1000 g

LSROc (Preterm)

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24. METABOLIC BONE DISEASE IN THE NEONATAL PERIOD AND ITS LATER SEQUELAE

that no apparent detrimental long-term effects were apparent in young adults who experienced very low neonatal mineral intakes raises the issue of whether the current high recommended intakes are actually necessary, especially since the majority of the subjects suffered no clinically apparent short-term problems related to bone development. Meeting current recommendations for mineral intake is recognized to be particularly difficult when the infant is receiving parenteral nutrition, due to solubility issues with calcium and phosphorus salts, particularly in settings where organic phosphate salts are not available such as the USA. The use of calcium gluconate in parenteral nutrition solutions is also known to result in exposure of the infant to aluminum, which leaches from glass storage vials. Aluminum accumulates in the body under conditions where the gastrointestinal tract is bypassed and renal function is compromised e both situations seen in preterm infants receiving parenteral nutrition. In a randomized trial in preterm infants receiving either standard aluminum solutions or specially prepared lowaluminum parenteral nutrition solution, in which calcium gluconate was replaced by calcium chloride, we showed that infants fed the low-aluminum solution had significantly higher scores on the Bayley mental development index at 18 months corrected age [126]. More recently, at age 15 years, we reported higher lumbar spine BMD in these subjects [127]. Furthermore, subjects exposed to aluminum concentrations above the median during the neonatal period had significantly lower hip bone mineral content. Our findings of neurotoxicity and bone toxicity are plausible given experience in adults and data from animal models. Together, these findings suggest that it would be advisable to reduce the exposure of preterm infants to aluminum via parenteral feeding solutions. Indeed, the US Food and Drug Administration (FDA) have recommended that daily aluminum intakes greater than 4e5 mg/kg/day should be avoided in infants weighing <3 kg, although this is almost impossible using available solutions [128,129]. Avoiding the use of calcium gluconate solutions stored in small volume glass vials, as recently recommended by the UK Medicine and Healthcare products Regulatory Agency (MHRA) [130], would be the single most important step to reduce exposure. Replacing calcium gluconate solutions with alternatives, and the use of organic phosphate salts are other solutions. Current recommendations for vitamin D intake in preterm infants generally reflect custom and practice rather than evidence. Most neonatologists give a minimum of 400 IU per day to preterm infants. A randomized trial comparing 200e400 IU versus 960 IU per day in preterm infants reported no significant

difference in bone mineralization between the groups at 3 or 6 months [131]. However, given the fact that many mothers are known to have suboptimal vitamin D status, and increasing recognition of the wider actions of vitamin D, some experts consider that a higher dose of vitamin D is advisable. This area needs further research.

Type of Milk There is evidence that the use of human milk may have long-term benefits for bone health in preterm infants (in addition to proven benefits for several other outcomes). However, human milk cannot meet the mineral requirements of this population and therefore requires supplementation with phosphorus as a minimum in the short term. Supplementation should be titrated to maintain plasma phosphorus >1.8 mmol/L. The issue of mineral supplementation of the breastfed preterm infant after hospital discharge is unresolved but important, particularly in the context of earlier discharge and higher breastfeeding rates. It is unlikely that unsupplemented human milk can meet mineral accretion requirements for an infant who is around 34 weeks’ gestation at discharge e especially if the infant has acquired nutrient deficits during the early postnatal period. Ideally, these infants should be carefully monitored after discharge, and supplementation offered if plasma phosphate concentrations fall below 1.8 mmol/L. In the absence of human milk, or when insufficient is available to meet full enteral requirements, a preterm infant formula should be used. Term infant formulas contain insufficient mineral (as well as other nutrients) for preterm infants.

NEONATAL HYPOCALCEMIA Neonatal hypocalcemia most commonly arises as a result of poor maternal vitamin D status [132]. Less commonly, it can be due to maternal disease such as hyperparathyroidism leading to suppression of fetal parathyroid gland activity [133], iatrogenic factors such as the administration of magnesium as a tocolytic agent [134,135], or occur because of abnormalities of the development or function of the infant’s own parathyroid glands [136,137]. The typical presentation is with jitteriness; if untreated, tetany and convulsions may ensue. Administration of vitamin D in France was associated with a reduction in the incidence of hypocalcemia [138]. In infants of severely vitamin D deficient mothers who are then breastfed without supplemental vitamin D, cardiomyopathy may occur [139].

PEDIATRIC BONE

SPECIFIC ENVIRONMENTAL EXPOSURES IN UTERO

TERM INFANTS: EARLY GROWTH AND LATER BONE DEVELOPMENT The first indications of a link between an adverse intrauterine environment, resulting in low birth weight, and reduced bone mass in postnatal life came from studies in which older adults, for whom birth records existed, underwent assessment of bone mass by DXA. Thus, in a study of 21-year-old women, around peak bone mass, BMC at the lumbar spine and femoral neck were predicted by their weight at 1 year old, independent of adult weight and body mass index [140]. The maintenance of this association into older age (60e75 years) was demonstrated in the Hertfordshire cohort [141]; work in this group of elderly men and women has also enabled elucidation of the persisting contributions of birth weight, weight at 1 year and adult weight to adult bone mass in conditional analyses [142]. Similar findings have come from the USA, Australia, Sweden and the Netherlands [140]. The use of novel techniques such as hip strength analysis from DXA scans of the proximal femur [143], and pQCT of the distal tibia [144], in the Hertfordshire cohort, have demonstrated that poor early growth is associated with a narrower proximal femur and reduced strength at the distal tibia, both of which outcomes will predispose an individual to an increased risk of osteoporotic fracture. The relevance of these findings in terms of fracture has been demonstrated in a Finnish cohort of elderly men and women, for whom birth records were linked to hospital discharge data [145]. Here, poor childhood growth was associated with an increased risk of adult hip fracture; additionally, people who had hip fractures were more likely to be shorter at birth but of average height by age 7 years, suggesting that hip fracture risk may be particularly elevated among children in whom the growth of the skeletal envelope had been forced ahead of its capacity to mineralize.

SPECIFIC ENVIRONMENTAL EXPOSURES IN UTERO Maternal Body Build, Diet, Lifestyle Results from an initial small UK mothereoffspring cohort suggested that babies of mothers who were poorly nourished, smoked and took vigorous exercise in late pregnancy were born with reduced bone mass, measured by DXA, compared with babies of other mothers [146]. These findings have been replicated in a larger cohort of mothers and their offspring from the Southampton Women’s Survey (UK); additionally greater maternal parity, thinness and vigorous physical activity in late pregnancy were associated with narrower

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bones in the offspring at birth [147]. The quality of the maternal dietary pattern during late pregnancy (e.g. whether a large proportion of the diet consists of foods such as brown bread, fruit and vegetables) predicted offspring bone size at 9 years in a further mothere offspring study [147].

Maternal Vitamin D and Calcium Nutrition However, the specific micronutrient that appears to have the greatest individual contribution, certainly for the developed world, is vitamin D. In a study of white Caucasian women in the south of the UK, 31% of the mothers had insufficient and 18% had deficient circulating concentrations of 25(OH)D during late pregnancy (11e20 and <11 mg/L respectively) [148]. Lower concentrations of serum 25(OH)D in mothers during late pregnancy were associated with reduced whole-body BMC and BMD in children at age 9 years, independently of social class, diet, and body size. Estimated exposure to ultraviolet B radiation during late pregnancy and the maternal use of vitamin D supplements both predicted maternal 25(OH)D concentration, and childhood bone mass. Adjunctive evidence supporting a role for maternal vitamin D status was obtained in the Southampton Women’s Survey, where maternal vitamin D concentrations correlated positively with neonatal bone mass [149]. A more recent study using the Avon Longitudinal Study of Parents and Children [150] demonstrated a positive association between ambient ultraviolet B radiation in pregnancy and offspring BMC at 9 years old, further supporting the notion that maternal vitamin D status is an important determinant of offspring bone development. In the Southampton study, the effect of maternal 25(OH)D status appeared to be partly mediated via concentrations of venous umbilical cord calcium, suggesting that placental calcium transfer to the fetus may be a critical step in these associations. In the developing world, vitamin D status may be adequate and here the limiting factor may be maternal calcium intake. Thus, in a mothereoffspring cohort in Pune, India [151], children of mothers who had a higher frequency of intake of calcium-rich foods during pregnancy had higher total and lumbar spine BMC and areal BMD, independent of parental size and bone composition.

Biochemical Markers in Umbilical Venous Cord Blood: Leptin and IFG-1 Both leptin and IGF-1 influence bone development; recent studies have demonstrated positive associations between concentrations of leptin [152] and IGF-1 [153] in umbilical cord venous blood and bone mass at birth

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in the offspring when assessed by DXA. There appeared to be a suggestion of size/density disparity, with leptin concentrations positively predicting volumetric density of the baby’s skeleton, whereas concentrations of IGF-1 were primarily associated with bone size. Developing chondrocytes have been found to express IGF-1 mRNA, and it has been shown to stimulate their proliferation [154]. The placenta acts as a barrier between the maternal and fetal IGF systems, with fetal IGF predominantly produced in the fetal liver. Leptin is a hormone that is best known for its role in fat metabolism in adults, but laboratory-based studies have shown that it has a positive effect on mesenchymal cell differentiation into osteoblasts [155]. Leptin receptors have been found on osteoblasts, chondrocytes and bone marrow stromal cells. Leptin is produced in the feto-placental circulation by fetal fat, heart, liver, muscle, and interestingly the placenta itself [156,157]. Consistent with these previous findings, in a recent study, growth velocity of fetal abdominal circumference, which is a composite measure of adiposity and liver volume, positively predicted estimated volumetric bone density at 4 years [158]. Finally, profiles of circulating growth hormone (GH) and cortisol were compared with bone density and birth records in subjects from the Hertfordshire cohort study [159e161]. Here, weight at 1 year positively correlated with median GH concentration and negatively with cortisol concentration at age 61e72 years, suggesting a “memory” of a difficult intrauterine or early life environment. Furthermore, the profiles of these two hormones were found to be determinants of prospectively determined bone loss rate, suggesting a physiological outcome of this “memory”.

POTENTIAL MECHANISMS: DEVELOPMENTAL PLASTICITY, GENEeENVIRONMENT INTERACTIONS AND EPIGENETICS There is a strong biological basis for a model of disease pathogenesis in which a single genotype can give rise to several different phenotypes, allowing the organism to adapt future generations to prevailing environmental conditions: this phenomenon is termed developmental plasticity [162]. There are striking illustrations of such phenomena in the natural world: for example, the thickness of the neonatal meadow vole’s coat is determined by the amount of light the mother is exposed to prenatally [163]. In this way the vole offspring is adapted for the season into which it is born. Experimentalists have repeatedly demonstrated that alterations to the diet of pregnant animals can produce lasting changes in the offspring’s physiology and metabolism [164]. The key mechanistic point here is that of an

interaction between genes and environment. Thus, in the Hertfordshire cohort study, there was no significant association between either vitamin D receptor (VDR) genotype and adult BMD in the cohort as a whole [141]. However, the relationship between lumbar spine BMD and VDR varied according to birth weight. After adjusting for age, gender and adult weight, individuals in the lowest third for birth weight had a higher spine BMD if they were of the BB genotype. People of the same genotype who were in the highest third for birth weight had a lower spine BMD compared to those of bb genotype in the same birth weight category. Similar interactions between birth weight and genome were observed for the growth hormone gene [165]. Data emerging from animal and human studies suggest that epigenetic modification of gene expression may be the central phenomenon in mediating the early life origins of adult disease. Epigenetics refers to changes in phenotype or gene expression caused by mechanisms other than changes in the underlying DNA sequence [166]. Each cell in the body is said to acquire a unique “epigenetic signature” reflecting the genotype, developmental history and environmental influences upon the cell [167]. These changes then influence cell function, and so the phenotype of the organism. The two most studied forms of epigenetic marking are DNA methylation and histone modification. DNA methylation involves the addition of a methyl group to cytosine residues at the carbon-5 position of CpG dinucleotides. DNA methylation is generally associated with gene repression, either by decreased binding of transcription factors or by attracting methyl-CpG-binding proteins that act as transcriptional repressors [168,169]. Histone modification refers to post-translational modification of histone tails, such that the conformation of packaging into chromatin is altered, allowing, or blocking gene expression. Widespread methylation occurs during two major points in development: gametogenesis and preimplantation [167]. As the new embryo implants, new methylation patterns become established via de novo methylation by the activities of DNA methyltransferases. From this baseline level of methylation epigenetic mechanisms are then thought to influence a range of processes, including the commitment of cells to develop a particular lineage [167], and adaptations the organism makes in response to its environment [170]. There are ample data confirming that alterations to maternal diet during pregnancy may lead to alteration of epigenetic marking in the offspring [171e173]. The glucocorticoid system provides a possible exemplar in terms of human bone development. Thus rats fed a reduced protein intake during pregnancy have offspring with reduced expression of a DNA methyl

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transferase (Dnmt 1) [171] in the liver. Dnmt1 is involved in maintaining patterns of DNA methylation through mitosis [174]. Furthermore, Dnmt1 expression in human umbilical cord was positively associated with the degree of methylation of the glucocorticoid receptor promoter [172], and reduced expression of the receptor protein [172]. These findings, in combination with those from adult cohorts, suggest that epigenetic modulation of the glucocorticoid axis in early life may set up longterm patterns of bone gain and loss [159,160]. This area of science is still in its infancy, but offers an exciting insight into possible mechanisms which might underlie the relationships between maternal 25(OH)-vitamin D status, placental calcium transport and offspring bone mass described earlier in this chapter.

SUMMARY It is increasingly clear that the early life environment is a critical factor in the life long health of the skeleton. Life begins at conception, not at delivery, and the supply and metabolism of key factors such as vitamin D and calcium should not be ignored at any age. Although neonates now 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 medications such as 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 to prevent short-term morbidity associated with metabolic bone disease and fractures. Whether early mineral intake or nutrition influences later bone health is not completely clear. Most follow-up studies of preterm infants suggest that there is “catch-up” in bone mass, although in the only study with follow up into early adult life, preterm subjects were significantly shorter with low lumbar spine bone mineral density compared to population reference data, and these deficits were greatest in subjects born SGA with birth weight <1250 g. Despite large variations in early nutrient and mineral intake (and occurrence of metabolic bone disease) during the neonatal period, randomized infant diet in these subjects did not influence peak bone mass or turnover. However, the proportion of (unsupplemented) human milk in the neonatal diet was significantly positively associated with later whole body bone size and mineral content. The higher whole body bone mass associated with human milk intake, despite its very low nutrient content, may instead reflect nonnutritive factors in breast milk. However, the lack of effect of randomized infant diet on peak bone mass suggests that the observed deficits in height and lumbar

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spine bone mass of these former preterm subjects may not be directly related to suboptimal early nutrient or mineral intake. Although further studies are clearly required to investigate later bone health of preterm infants, it is unlikely that contemporary preterm infants are exposed to the extremely low early mineral intakes experienced by some infants in the cited studies, and one might conclude that they are less likely to experience long-term adverse consequences for bone health.

References [1] Ferrari S, Rizzoli R, Bonjour JP. Genetic aspects of osteoporosis. Curr Opin Rheumatol 1999;11:294e300. [2] Harvey N, Cooper C. The developmental origins of osteoporotic fracture. J Br Menopause Soc 2004;10:29. [3] Gluckman PD, Hanson MA, Cooper C, Thornburg KL. Effect of in utero and early-life conditions on adult health and disease. N Engl J Med 2008;359:61e73. [4] Kanis JA, Delmas P, Burckhardt P, Cooper C, Torgerson D. Guidelines for diagnosis and management of osteoporosis. The European Foundation for Osteoporosis and Bone Disease. Osteoporos Int 1997;7:390e406. [5] Sambrook P, Cooper C. Osteoporosis. Lancet 2006;367:2010e8. [6] Incidence of vertebral fracture in Europe: results from the European Prospective Osteoporosis Study (EPOS). J Bone Miner Res 2002;17:716e24. [7] Ferrari S, Rizzoli R, Slosman D, Bonjour JP. Familial resemblance for bone mineral mass is expressed before puberty. J Clin Endocrinol Metab 1998;83:358e61. [8] Wang Q, Alen M, Lyytikainen A, et al. Familial resemblance and diversity in bone mass and strength in the population are established during the first year of postnatal life. J Bone Miner Res 2010;25:1512e20. [9] Wang Q, Cheng S, Alen M, Seeman E. Bone’s structural diversity in adult females is established before puberty. J Clin Endocrinol Metab 2009;94:1555e61. [10] Hernandez CJ, Beaupre GS, Carter DR. A theoretical analysis of the relative influences of peak BMD, age-related bone loss and menopause on the development of osteoporosis. Osteoporos Int 2003;14:843e7. [11] Salle BL, Glorieux FH, Delvin EE. Perinatal vitamin D metabolism. Biol Neonate 1988;54:181e7. [12] Seki K, Furuya K, Makimura N, Mitsui C, Hirata J, Nagata I. Cord blood levels of calcium-regulating hormones and osteocalcin in premature infants. J Perinat Med 1994;22:189e94. [13] Whitsett JA, Ho M, Tsang RC, Norman EJ, Adams KG. Synthesis of 1,25-dihydroxyvitamin D3 by human placenta in vitro. J Clin Endocrinol Metab 1981;53:484e8. [14] Ron M, Levitz M, Chuba J, Dancis J. Transfer of 25hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 across the perfused human placenta. Am J Obstet Gynecol 1984;148:370e4. [15] Koo WWK, Tsang RC. Calcium, magnesium, phosphorus, and vitamin D. In: Tsang RC, Lucas A, Uauy R, Zlotkin S, editors. Nutritional Needs of the Preterm Infant. New York: Williams and Wilkins; 1993. p. 135e55. [16] Kovacs CS, Chafe LL, Fudge NJ, Friel JK, Manley NR. PTH regulates fetal blood calcium and skeletal mineralization independently of PTHrP. Endocrinology 2001;142:4983e93. [17] Lester GE. Cholecalciferol and placental calcium transport. Fed Proc 1986;45:2524e7. [18] Kovacs CS, Woodland ML, Fudge NJ, Friel JK. The vitamin D receptor is not required for fetal mineral homeostasis or for the

PEDIATRIC BONE

674

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

24. METABOLIC BONE DISEASE IN THE NEONATAL PERIOD AND ITS LATER SEQUELAE

regulation of placental calcium transfer in mice. Am J Physiol Endocrinol Metab 2005;289:E133e44. Kovacs CS. Skeletal physiology: fetus and neonate. In: Favus MJ, editor. Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. 5th ed. Washington: ASBMR; 2003. p. 65e71. Care AD, Caple IW, Abbas SK, Pickard DW. The effect of fetal thyroparathyroidectomy on the transport of calcium across the ovine placenta to the fetus. Placenta 1986;7:417e24. Kovacs CS, Lanske B, Hunzelman JL, Guo J, Karaplis AC, Kronenberg HM. Parathyroid hormone-related peptide (PTHrP) regulates fetal-placental calcium transport through a receptor distinct from the PTH/PTHrP receptor. Proc Natl Acad Sci USA 1996;93:15233e8. Kovacs CS, Manley NR, Moseley JM, Martin TJ, Kronenberg HM. Fetal parathyroids are not required to maintain placental calcium transport. J Clin Invest 2001;107:1007e15. Ardawi MS, Nasrat HA, HS BAA. Calcium-regulating hormones and parathyroid hormone-related peptide in normal human pregnancy and postpartum: a longitudinal study. Eur J Endocrinol 1997;137:402e9. Belkacemi L, Bedard I, Simoneau L, Lafond J. Calcium channels, transporters and exchangers in placenta: a review. Cell Calcium 2005;37:1e8. Stauffer TP, Hilfiker H, Carafoli E, Strehler EE. Quantitative analysis of alternative splicing options of human plasma membrane calcium pump genes. J Biol Chem 1993;268:25993e6003. Glazier JD, Atkinson DE, Thornburg KL, et al. Gestational changes in Ca2þ transport across rat placenta and mRNA for calbindin9K and Ca(2þ)-ATPase. Am J Physiol 1992;263:R930e5. Kip SN, Strehler EE. Vitamin D3 upregulates plasma membrane Ca2þ-ATPase expression and potentiates apico-basal Ca2þ flux in MDCK cells. Am J Physiol Renal Physiol 2004;286:F363e9. Martin R, Harvey NC, Crozier SR, et al. Placental calcium transporter (PMCA3) gene expression predicts intrauterine bone mineral accrual. Bone 2007;40:1203e8. Weir EC, Philbrick WM, Amling M, Neff LA, Baron R, Broadus AE. Targeted overexpression of parathyroid hormonerelated peptide in chondrocytes causes chondrodysplasia and delayed endochondral bone formation. Proc Natl Acad Sci USA 1996;93:10240e5. Calvi LM, Schipani E. The PTH/PTHrP receptor in Jansen’s metaphyseal chondrodysplasia. J Endocrinol Invest 2000;23:545e54. Iwamoto M, Jikko A, Murakami H, et al. Changes in parathyroid hormone receptors during chondrocyte cytodifferentiation. J Biol Chem 1994;269:17245e51. Kato Y, Shimazu A, Nakashima K, Suzuki F, Jikko A, Iwamoto M. Effects of parathyroid hormone and calcitonin on alkaline phosphatase activity and matrix calcification in rabbit growth-plate chondrocyte cultures. Endocrinology 1990;127:114e8. Vortkamp A, Lee K, Lanske B, Segre GV, Kronenberg HM, Tabin CJ. Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 1996;273:613e22. Karaplis AC, Luz A, Glowacki J, et al. Lethal skeletal dysplasia from targeted disruption of the parathyroid hormone-related peptide gene. Genes Dev 1994;8:277e89. Lanske B, Karaplis AC, Lee K, et al. PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth. Science 1996;273:663e6.

[36] Lanske B, Amling M, Neff L, Guiducci J, Baron R, Kronenberg HM. Ablation of the PTHrP gene or the PTH/ PTHrP receptor gene leads to distinct abnormalities in bone development. J Clin Invest 1999;104:399e407. [37] Calvi LM, Sims NA, Hunzelman JL, et al. Activated parathyroid hormone/parathyroid hormone-related protein receptor in osteoblastic cells differentially affects cortical and trabecular bone. J Clin Invest 2001;107:277e86. [38] Antoniades L, MacGregor AJ, Andrew T, Spector TD. Association of birth weight with osteoporosis and osteoarthritis in adult twins. Rheumatology (Oxford) 2003;42:791e6. [39] Bagnoli F, Sardelli S, Vispi L, et al. [Serum calcitonin in full-term infants]. Boll Soc Ital Biol Sper 1982;58:556e61. [40] Hillman LS, Hoff N, Walgate J, Haddad JG. Serum calcitonin concentrations in premature infants during the first 12 weeks of life. Calcif Tissue Int 1982;34:470e3. [41] Romagnoli C, Zecca E, Tortorolo G, Diodato A, Fazzini G, Sorcini-Carta M. Plasma thyrocalcitonin and parathyroid hormone concentrations in early neonatal hypocalcaemia. Arch Dis Child 1987;62:580e4. [42] Sann L, David L, Chayvialle JA, Lasne Y, Bethenod M. Effect of early oral calcium supplementation on serum calcium and immunoreactive calcitonin concentration in preterm infants. Arch Dis Child 1980;55:611e5. [43] Cooper LJ, Anast CS. Circulating immunoreactive parathyroid hormone levels in premature infants and the response to calcium therapy. Acta Paediatr Scand 1985;74:669e73. [44] Holtrop ME, King GJ, Cox KA, Reit B. Time-related changes in the ultrastructure of osteoclasts after injection of parathyroid hormone in young rats. Calcif Tissue Int 1979;27:129e35. [45] Suda T, Takahashi N, Udagawa N, Jimi E, Gillespie MT, Martin TJ. Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr Rev 1999;20:345e57. [46] Wright C, David L, Chapuy MC, Delvin E, Putet G, Salle BL. [Late neonatal hypocalcemia. Apropos of 33 cases treated with 1 alpha-hydroxycholecalciferol]. Pediatrie 1989;44:289e95. [47] Anderson HC. Matrix vesicle calcification: review and update. In: Bone and Mineral Research. Amsterdam: Elsevier; 1985. p. 109e50. [48] Anderson HC. Introduction to the second conference on matrix vesicle calcification. Metab Bone Dis Relat Res 1978;1:83e7. [49] Simon DR, Berman I, Howell DS. Relationship of extracellular matrix vesicles to calcification in normal and healing rachitic epiphyseal cartilage. Anat Rec 1973;176:167e79. [50] Widdowson EM. Nutrition. In: Davis JA, Dobbing J, editors. Scientific Foundations of Paediatrics. 2nd ed. London: Heinemann; 1981. p. 41e3. [51] Bishop NJ. Bone mineralisation in preterm infants; effect of early diet. [MD] University of Manchester 1993. [52] Ziegler EE, O’Donnell AM, Nelson SE. Body composition of the reference fetus. Growth 1976;40:329e41. [53] Moyer-Mileur LJ, Brunstetter V, McNaught TP, Gill G, Chan GM. Daily physical activity program increases bone mineralization and growth in preterm very low birth weight infants. Pediatrics 2000;106:1088e92. [54] Kurl S, Heinonen K, Lansimies E. Effects of prematurity, intrauterine growth status, and early dexamethasone treatment on postnatal bone mineralisation. Arch Dis Child Fetal Neonatal Ed 2000;83:F109e11. [55] Weiler HA, Paes B, Shah JK, Atkinson SA. Longitudinal assessment of growth and bone mineral accretion in prematurely born infants treated for chronic lung disease with dexamethasone. Early Hum Dev 1997;47:271e86.

PEDIATRIC BONE

REFERENCES

[56] Brunton JA, Weiler HA, Atkinson SA. Improvement in the accuracy of dual energy x-ray absorptiometry for whole body and regional analysis of body composition: validation using piglets and methodologic considerations in infants. Pediatr Res 1997;41:590e6. [57] Crofton PM, Shrivastava A, Wade JC, et al. Effects of dexamethasone treatment on bone and collagen turnover in preterm infants with chronic lung disease. Pediatr Res 2000;48:155e62. [58] Shrivastava A, Lyon A, McIntosh N. The effect of dexamethasone on growth, mineral balance and bone mineralisation in preterm infants with chronic lung disease. Eur J Pediatr 2000;159:380e4. [59] Ylppo A. Das Wachstum der Fruhgeborenen von der Geburt bis zum Schulalter. Ztschr Kinderh 1919;24:111. [60] Benjamin HR, Gordon HH, Marples E. Calcium and phosphorus requirements of premature infants. Am J Dis Child 1943;65:412e25. [61] Von Sydow G. A study of the development of rickets in premature infants. Acta Paediatr 1946;22:2. [62] Eek S, Gabrielson LH. Prematurity and rickets. Pediatrics 1957;20:63e77. [63] Lewin PK, Reid M, Reilly BJ, Swyer PR, Fraser D. Iatrogenic rickets in low-birth-weight infants. J Pediatr 1971;78:207e10. [64] Tulloch AL. Rickets in the premature. Med J Aust 1974;2(1):137e40. [65] Gross SJ. Bone mineralization in preterm infants fed human milk with and without mineral supplementation. J Pediatr 1987;111:450e8. [66] Chan GM, Mileur LJ. Posthospitalization growth and bone mineral status of normal preterm infants. Feeding with mother’s milk or standard formula. Am J Dis Child 1985;139:896e8. [67] Greer FR, McCormick A. Bone growth with low bone mineral content in very low birth weight premature infants. Pediatr Res 1986;20:925e8. [68] Rowe J, Rowe D, Horak E, et al. Hypophosphatemia and hypercalciuria in small premature infants fed human milk: evidence for inadequate dietary phosphorus. J Pediatr 1984;104:112e7. [69] Fewtrell MS, Cole TJ, Bishop NJ, Lucas A. Neonatal factors predicting childhood height in preterm infants: evidence for a persisting effect of early metabolic bone disease? J Pediatr 2000;137:668e73. [70] Lyon AJ, Hawkes DJ, Doran M, McIntosh N, Chan F. Bone mineralisation in preterm infants measured by dual energy radiographic densitometry. Arch Dis Child 1989;64:919e23. [71] Lucas A, Gore SM, Cole TJ, et al. Multicentre trial on feeding low birthweight infants: effects of diet on early growth. Arch Dis Child 1984;59:722e30. [72] Minton SD, Steichen JJ, Tsang RC. Bone mineral content in term and preterm appropriate-for-gestational-age infants. J Pediatr 1979;95:1037e42. [73] James JR, Congdon PJ, Truscott J, Horsman A, Arthur R. Osteopenia of prematurity. Arch Dis Child 1986;61:871e6. [74] Pohlandt F. Hypothesis: myopia of prematurity is caused by postnatal bone mineral deficiency. Eur J Pediatr 1994;153:234e6. [75] Pohlandt F, Mathers N. Bone mineral content of appropriate and light for gestational age preterm and term newborn infants. Acta Paediatr Scand 1989;78:835e9. [76] Minton SD, Steichen JJ, Tsang RC. Decreased bone mineral content in small-for-gestational-age infants compared with appropriate-for-gestational-age infants: normal serum 25-hydroxyvitamin D and decreasing parathyroid hormone. Pediatrics 1983;71:383e8.

675

[77] Petersen S, Gotfredsen A, Knudsen FU. Total body bone mineral in light-for-gestational-age infants and appropriate-forgestational-age infants. Acta Paediatr Scand 1989;78:347e50. [78] Greer FR, Steichen JJ, Tsang RC. Effects of increased calcium, phosphorus, and vitamin D intake on bone mineralization in very low-birth-weight infants fed formulas with Polycose and medium-chain triglycerides. J Pediatr 1982;100:951e5. [79] Steichen JJ, Gratton TL, Tsang RC. Osteopenia of prematurity: the cause and possible treatment. J Pediatr 1980;96:528e34. [80] Pittard 3rd WB, Geddes KM, Sutherland SE, Miller MC, Hollis BW. Longitudinal changes in the bone mineral content of term and premature infants. Am J Dis Child 1990;144:36e40. [81] Chan GM, Mileur L, Hansen JW. Calcium and phosphorus requirements in bone mineralization of preterm infants. J Pediatr 1988;113:225e9. [82] Venkataraman PS, Blick KE. Effect of mineral supplementation of human milk on bone mineral content and trace element metabolism. J Pediatr 1988;113:220e4. [83] DHSS. The composition of mature human milk. Report on Health and Social Subjects No. 12. London: HMSO; 1977. [84] DHSS. Artificial feeds for the young infant: Report on Health and Social Subjects No. 18. Report of the Committee on Medical Aspects of Food Policy. London: HMSO; 1980; Department of Health and Social Security. Present day practice in infant feeding, 1980. London: HMSO; 1981 (Reports on Health and Social Subjects, No 20.) [85] DHSS. Present day practice in infant feeding, third report. Report on Health and Social Subjects No. 32. London: HMSO; 1988. [86] Macy IG, Kelly HJ, Sloan RE. The composition of milks. Publication 254. Washington DC: National Academy of Science, National Research Council; 1953. [87] Mettler AE. Infant milk powder feeds compared on a common basis. Postgraduate Medical Journal 1976;52(Suppl 8):3e20. [88] Greer FR, McCormick A. Improved bone mineralization and growth in premature infants fed fortified own mother’s milk. J Pediatr 1988;112:961e9. [89] Pettifor JM, Rajah R, Venter A, et al. Bone mineralization and mineral homeostasis in very low-birth-weight infants fed either human milk or fortified human milk. J Pediatr Gastroenterol Nutr 1989;8:217e24. [90] Lapillonne AA, Glorieux FH, Salle BL, et al. Mineral balance and whole body bone mineral content in very low-birth-weight infants. Acta Paediatr Suppl 1994;405:117e22. [91] Wauben IP, Atkinson SA, Grad TL, Shah JK, Paes B. Moderate nutrient supplementation of mother’s milk for preterm infants supports adequate bone mass and short-term growth: a randomized, controlled trial. Am J Clin Nutr 1998;67:465e72. [92] Faerk J, Petersen S, Peitersen B, Michaelsen KF. Diet and bone mineral content at term in premature infants. Pediatr Res 2000;47:148e56. [93] McDevitt H, Tomlinson C, White MP, Ahmed SF. Changes in quantitative ultrasound in infants born at less than 32 weeks’ gestation over the first 2 years of life: influence of clinical and biochemical changes. Calcif Tissue Int 2007;81:263e9. [94] Fewtrell MS, Loh KL, Chomtho S, Kennedy K, Hawdon J, Khakoo A. Quantitative ultrasound (QUS): a useful tool for monitoring bone health in preterm infants? Acta Paediatr 2008;97:1625e30. [95] Lucas A, Brooke OG, Baker BA, Bishop N, Morley R. High alkaline phosphatase activity and growth in preterm neonates. Arch Dis Child 1989;64:902e9. [96] Beyers N, Alheit B, Taljaard JF, Hall JM, Hough SF. High turnover osteopenia in preterm babies. Bone 1994;15:5e13.

PEDIATRIC BONE

676

24. METABOLIC BONE DISEASE IN THE NEONATAL PERIOD AND ITS LATER SEQUELAE

[97] Bhandari V, Fall P, Raisz L, Rowe J. Potential biochemical growth markers in premature infants. Am J Perinatol 1999;16:339e49. [98] Crofton PM, Shrivastava A, Wade JC, et al. Bone and collagen markers in preterm infants: relationship with growth and bone mineral content over the first 10 weeks of life. Pediatr Res 1999;46:581e7. [99] Seibold-Weiger K, Wollmann HA, Ranke MB, Speer CP. Plasma concentrations of the carboxyterminal propeptide of type I procollagen (PICP) in preterm neonates from birth to term. Pediatr Res 2000;48:104e8. [100] Shiff Y, Eliakim A, Shainkin-Kestenbaum R, Arnon S, Lis M, Dolfin T. Measurements of bone turnover markers in premature infants. J Pediatr Endocrinol Metab 2001;14:389e95. [101] Gfatter R, Braun F, Herkner K, Kohlross C, Hackl P. Urinary excretion of pyridinium crosslinks and N-terminal crosslinked peptide in preterm and term infants. Int J Clin Lab Res 1997;27:238e43. [102] Smurthwaite D, Wright NB, Russell S, Emmerson AJ, Mughal MZ. How common are rib fractures in extremely low birth weight preterm infants? Arch Dis Child Fetal Neonatal Ed 2009;94:F138e9. [103] Jones S, Bell MJ. Distal radius fracture in a premature infant with osteopenia caused by handling during intravenous cannulation. Injury 2002;33:265e6. [104] Bishop N, Sprigg A, Dalton A. Unexplained fractures in infancy: looking for fragile bones. Arch Dis Child 2007;92:251e6. [105] Salle BL, Braillon P. Bone mineral content (BMC) of the lumbar spine in preterm infants: A longitudinal study during the first year of life. Pediatr Res 1992;31:294A. [106] Koo WW, Sherman R, Succop P, et al. Sequential bone mineral content in small preterm infants with and without fractures and rickets. J Bone Miner Res 1988;3:193e7. [107] Congdon PJ, Horsman A, Ryan SW, Truscott JG, Durward H. Spontaneous resolution of bone mineral depletion in preterm infants. Arch Dis Child 1990;65:1038e42. [108] Abrams SA, Schanler RJ, Garza C. Bone mineralization in former very low birth weight infants fed either human milk or commercial formula. J Pediatr 1988;112:956e60. [109] Bishop NJ, King FJ, Lucas A. Increased bone mineral content of preterm infants fed with a nutrient enriched formula after discharge from hospital. Arch Dis Child 1993;68:573e8. [110] Brunton JA, Saigal S, Atkinson SA. Growth and body composition in infants with bronchopulmonary dysplasia up to 3 months corrected age: a randomized trial of a high-energy nutrient-enriched formula fed after hospital discharge. J Pediatr 1998;133:340e5. [111] Amir J, Katz K, Grunebaum M, Yosipovich Z, Wielunsky E, Reisner SH. Fractures in premature infants. J Pediatr Orthop 1988;8:41e4. [112] Fewtrell MS, Prentice A, Cole TJ, Lucas A. Effects of growth during infancy and childhood on bone mineralization and turnover in preterm children aged 8-12 years. Acta Paediatr 2000;89:148e53. [113] Fewtrell MS, Williams JE, Singhal A, Murgatroyd PR, Fuller N, Lucas A. Early diet and peak bone mass: 20 year follow-up of a randomized trial of early diet in infants born preterm. Bone 2009;45:142e9. [114] Rubinacci A, Sirtori P, Moro G, Galli L, Minoli I, Tessari L. Is there an impact of birth weight and early life nutrition on bone mineral content in preterm born infants and children? Acta Paediatr 1993;82:711e3.

[115] Hori C, Tsukahara H, Fujii Y, et al. Bone mineral status in preterm-born children: assessment by dual-energy X-ray absorptiometry. Biol Neonate 1995;68:254e8. [116] Helin I, Landin LA, Nilsson BE. Bone mineral content in preterm infants at age 4 to 16. Acta Paediatr Scand 1985;74:264e7. [117] Kurl S, Heinonen K, Lansimies E, Launiala K. Determinants of bone mineral density in prematurely born children aged 6e7 years. Acta Paediatr 1998;87:650e3. [118] Bowden LS, Jones CJ, Ryan SW. Bone mineralisation in expreterm infants aged 8 years. Eur J Pediatr 1999;158:658e61. [119] Backstrom MC, Maki R, Kuusela AL, et al. The long-term effect of early mineral, vitamin D, and breast milk intake on bone mineral status in 9- to 11-year-old children born prematurely. J Pediatr Gastroenterol Nutr 1999;29:575e82. [120] Bishop NJ, Dahlenburg SL, Fewtrell MS, Morley R, Lucas A. Early diet of preterm infants and bone mineralization at age five years. Acta Paediatr 1996;85:230e6. [121] Fewtrell MS, Prentice A, Jones SC, et al. Bone mineralization and turnover in preterm infants at 8e12 years of age: the effect of early diet. J Bone Miner Res 1999;14:810e20. [122] Dahlenburg SL, Bishop NJ, Lucas A. Are preterm infants at risk for subsequent fractures? Arch Dis Child 1989;64:1384e5. [123] Tsang R, Uauy R, Koletzko B, Zlotkin S, editors. Nutrition of the Preterm Infant. Scientific Basis and Practical Guidelines. 2nd ed. Cincinnati: Digital Educational Publishing; 2005. [124] Agostoni C, Buonocore G, Carnielli VP, et al. Enteral nutrient supply for preterm infants: commentary from the European Society of Paediatric Gastroenterology, Hepatology and Nutrition Committee on Nutrition. J Pediatr Gastroenterol Nutr 2010;50:85e91. [125] Klein CJ, editor. Nutrient requirements for preterm infant formulas. J Nutr 2002;132:6SI. [126] Bishop NJ, Morley R, Day JP, Lucas A. Aluminum neurotoxicity in preterm infants receiving intravenous-feeding solutions. N Engl J Med 1997;29(336):1557e61. [127] Fewtrell MS, Bishop NJ, Edmonds CJ, Isaacs EB, Lucas A. Aluminum exposure from parenteral nutrition in preterm infants: bone health at 15-year follow-up. Pediatrics 2009;124:1372e9. [128] Poole RL, Hintz SR, Mackenzie NI, Kerner Jr JA. Aluminum exposure from pediatric parenteral nutrition: meeting the new FDA regulation. J Parenter Enteral Nutr 2008;32:242e6. [129] Poole RL, Schiff L, Hintz SR, Wong A, Mackenzie N, Kerner Jr JA. Aluminum content of parenteral nutrition in neonates: measured versus calculated levels. J Pediatr Gastroenterol Nutr 2010;50:208e11. [130] MHRA. Available from: http://www.mhra.gov.uk/ Safetyinformation/Safetywarningsalertsandrecalls/ Safetywarningsandmessagesformedicines/CON093935 [131] Backstrom MC, Maki R, Kuusela AL, et al. Randomised controlled trial of vitamin D supplementation on bone density and biochemical indices in preterm infants. Arch Dis Child Fetal Neonatal Ed 1999;80:F161e6. [132] Teaema FH, Al Ansari K. Nineteen cases of symptomatic neonatal hypocalcemia secondary to vitamin D deficiency: a 2year study. J Trop Pediatr 2010;56:108e10. [133] Poomthavorn P, Ongphiphadhanakul B, Mahachoklertwattana P. Transient neonatal hypoparathyroidism in two siblings unmasking maternal normocalcemic hyperparathyroidism. Eur J Pediatr 2008;167:431e4. [134] Santi MD, Henry GW, Douglas GL. Magnesium sulfate treatment of preterm labor as a cause of abnormal neonatal bone mineralization. J Pediatr Orthop 1994;14:249e53.

PEDIATRIC BONE

677

REFERENCES

[135] Schanler RJ, Smith Jr LG, Burns PA. Effects of long-term maternal intravenous magnesium sulfate therapy on neonatal calcium metabolism and bone mineral content. Gynecol Obstet Invest 1997;43:236e41. [136] Egbuna OI, Brown EM. Hypercalcaemic and hypocalcaemic conditions due to calcium-sensing receptor mutations. Best Pract Res Clin Rheumatol 2008;22:129e48. [137] Garabedian M. Hypocalcemia and chromosome 22q11 microdeletion. Genet Couns 1999;10:389e94. [138] Delvin EE, Salle BL, Glorieux FH, Adeleine P, David LS. Vitamin D supplementation during pregnancy: effect on neonatal calcium homeostasis. J Pediatr 1986;109:328e34. [139] Maiya S, Sullivan I, Allgrove J, et al. Hypocalcaemia and vitamin D deficiency: an important, but preventable, cause of life-threatening infant heart failure. Heart 2008;94:581e4. [140] Cooper C, Cawley M, Bhalla A, et al. Childhood growth, physical activity, and peak bone mass in women. J Bone Miner Res 1995;10:940e7. [141] Dennison EM, Arden NK, Keen RW, et al. Birthweight, vitamin D receptor genotype and the programming of osteoporosis. Paediatr Perinat Epidemiol 2001;15:211e9. [142] Dennison EM, Syddall HE, Sayer AA, Gilbody HJ, Cooper C. Birth weight and weight at 1 year are independent determinants of bone mass in the seventh decade: the Hertfordshire cohort study. Pediatr Res 2005;57:582e6. [143] Javaid MK, Lekamwasam S, Clark J, et al. Infant growth influences proximal femoral geometry in adulthood. J Bone Miner Res 2006;21:508e12. [144] Oliver H, Jameson KA, Sayer AA, Cooper C, Dennison EM. Growth in early life predicts bone strength in late adulthood: the Hertfordshire Cohort Study. Bone 2007;41:400e5. [145] Cooper C, Eriksson JG, Forsen T, Osmond C, Tuomilehto J, Barker DJ. Maternal height, childhood growth and risk of hip fracture in later life: a longitudinal study. Osteoporos Int 2001;12:623e9. [146] Godfrey K, Walker-Bone K, Robinson S, et al. Neonatal bone mass: influence of parental birthweight, maternal smoking, body composition, and activity during pregnancy. J Bone Miner Res 2001;16:1694e703. [147] Cole ZA, Gale CR, Javaid MK, et al. Maternal dietary patterns during pregnancy and childhood bone mass: a longitudinal study. J Bone Miner Res 2009;24:663e8. [148] Javaid MK, Crozier SR, Harvey NC, et al. Maternal vitamin D status during pregnancy and childhood bone mass at age 9 years: a longitudinal study. Lancet 2006;367:36e43. [149] Harvey NC, Javaid MK, Poole JR, et al. Paternal skeletal size predicts intrauterine bone mineral accrual. J Clin Endocrinol Metab 2008;93:1676e81. [150] Sayers A, Tobias JH. Estimated maternal ultraviolet B exposure levels in pregnancy influence skeletal development of the child. J Clin Endocrinol Metab 2009;94:765e71. [151] Ganpule A, Yajnik CS, Fall CH, et al. Bone mass in Indian children e relationships to maternal nutritional status and diet during pregnancy: the Pune Maternal Nutrition Study. J Clin Endocrinol Metab 2006;91:2994e3001. [152] Javaid MK, Godfrey KM, Taylor P, et al. Umbilical cord leptin predicts neonatal bone mass. Calcif Tissue Int 2005;76:341e7. [153] Javaid MK, Godfrey KM, Taylor P, et al. Umbilical venous IGF-1 concentration, neonatal bone mass, and body composition. J Bone Miner Res 2004;19:56e63. [154] Olney RC, Mougey EB. Expression of the components of the insulin-like growth factor axis across the growth-plate. Mol Cell Endocrinol 1999;156:63e71. [155] Thomas T, Gori F, Khosla S, Jensen MD, Burguera B, Riggs BL. Leptin acts on human marrow stromal cells to enhance

[156]

[157]

[158]

[159]

[160]

[161]

[162] [163]

[164] [165]

[166]

[167] [168]

[169]

[170] [171]

[172]

[173]

[174]

differentiation to osteoblasts and to inhibit differentiation to adipocytes. Endocrinology 1999;140:1630e8. Lepercq J, Challier JC, Guerre-Millo M, Cauzac M, Vidal H. Hauguel-de Mouzon S. Prenatal leptin production: evidence that fetal adipose tissue produces leptin. J Clin Endocrinol Metab 2001;86:2409e13. Masuzaki H, Ogawa Y, Sagawa N, et al. Nonadipose tissue production of leptin: leptin as a novel placenta-derived hormone in humans. Nat Med 1997;3:1029e33. Harvey NC, Mahon PA, Robinson SM, et al. Different indices of fetal growth predict bone size and volumetric density at 4 years of age. J Bone Miner Res 2010;25:920e7. Phillips DI, Barker DJ, Fall CH, et al. Elevated plasma cortisol concentrations: a link between low birth weight and the insulin resistance syndrome? J Clin Endocrinol Metab 1998;83:757e60. Dennison E, Hindmarsh P, Fall C, et al. Profiles of endogenous circulating cortisol and bone mineral density in healthy elderly men. J Clin Endocrinol Metab 1999;84:3058e63. Fall C, Hindmarsh P, Dennison E, Kellingray S, Barker D, Cooper C. Programming of growth hormone secretion and bone mineral density in elderly men: a hypothesis. J Clin Endocrinol Metab 1998;83:135e9. Bateson P, Barker D, Clutton-Brock T, et al. Developmental plasticity and human health. Nature 2004;22(430):419e21. Lee TM. Development of meadow voles is influenced postnatally by maternal photoperiodic history. Am J Physiol 1993;265:R749e55. Bateson P. Fetal experience and good adult design. Int J Epidemiol 2001;30:928e34. Dennison EM, Syddall HE, Rodriguez S, Voropanov A, Day IN, Cooper C. Polymorphism in the growth hormone gene, weight in infancy, and adult bone mass. J Clin Endocrinol Metab 2004;89:4898e903. Jaenisch R, Bird A. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet 2003;33(Suppl.):245e54. Morgan HD, Santos F, Green K, Dean W, Reik W. Epigenetic reprogramming in mammals. Hum Mol Genet 2005;14:R47e58. Gicquel C, El-Osta A, Le Bouc Y. Epigenetic regulation and fetal programming. Best Pract Res Clin Endocrinol Metab 2008;22:1e16. Gluckman PD, Hanson MA, Beedle AS. Non-genomic transgenerational inheritance of disease risk. Bioessays 2007;29:145e54. Bird A. Perceptions of epigenetics. Nature 2007;447:396e8. Lillycrop KA, Phillips ES, Torrens C, Hanson MA, Jackson AA, Burdge GC. Feeding pregnant rats a protein-restricted diet persistently alters the methylation of specific cytosines in the hepatic PPAR alpha promoter of the offspring. Br J Nutr 2008;100:278e82. Lillycrop KA, Slater-Jefferies JL, Hanson MA, Godfrey KM, Jackson AA, Burdge GC. Induction of altered epigenetic regulation of the hepatic glucocorticoid receptor in the offspring of rats fed a protein-restricted diet during pregnancy suggests that reduced DNA methyltransferase-1 expression is involved in impaired DNA methylation and changes in histone modifications. Br J Nutr 2007;97:1064e73. Godfrey KM, Lillycrop KA, Burdge GC, Gluckman PD, Hanson MA. Epigenetic mechanisms and the mismatch concept of the developmental origins of health and disease. Pediatr Res 2007;61:5Re10R. Bird A. DNA methylation patterns and epigenetic memory. Genes Dev 2002;16:6e21.

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Rickets Due to Hereditary Abnormalities of Vitamin D Synthesis or Action Anthony A. Portale, Farzana Perwad, Walter L. Miller Department of Pediatrics, University of California San Francisco, San Francisco, California, USA

INTRODUCTION As one of the principal hormonal regulators of calcium and phosphorus metabolism, 1,25-dihydroxyvitamin D (1,25(OH)2D) is critically important for normal growth and mineralization of bone. The classical actions of 1,25(OH)2D 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.

BIOSYNTHESIS OF VITAMIN D A detailed discussion of the metabolism and action of vitamin D is provided in Chapter 8. Briefly, vitamin D exists as either ergocalciferol (vitamin D2) produced by plants, or cholecalciferol (vitamin D3) produced by animal tissues and by the action of near ultraviolet radiation (290e320 nm) on 7-dehydrocholesterol in human skin. Both forms of vitamin D are biologically inactive pro-hormones that must undergo successive hydroxylations at carbons 25 and 1 before they can bind to and activate the vitamin D receptor (Fig. 25.1). The 25-hydroxylation of vitamin D occurs in the liver, catalyzed by one or more enzymes including the mitochondrial enzyme CYP27A1 and the microsomal enzyme CYP2R1 (see below). The activity of hepatic 25-hydroxylation is not under tight physiologic regulation, and thus circulating concentrations of 25OHD are determined primarily by dietary intake of vitamin D and exposure to sunlight. Although 25OHD 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.

Pediatric Bone, Second Edition DOI: 10.1016/B978-0-12-382040-2.10025-5

The active form of vitamin D, 1,25(OH)2D, is produced by the 1a-hydroxylation of 25OHD by the mitochondrial enzyme, 25-hydroxyvitamin D-1a-hydroxylase (1ahydroxylase, P450c1a) [1] (see Fig. 25.1). The circulating concentration of 1,25(OH)2D primarily reflects its synthesis in the kidney; however, 1a-hydroxylase activity also is found in keratinocytes, macrophages, and osteoblasts [2e4]. The 1a-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, fibroblast growth factor 23, and 1,25(OH)2D. Because of the importance of this enzyme in normal physiology and because synthesis of 1,25(OH)2D is impaired in chronic renal insufficiency, Fanconi syndrome, X-linked hypophosphatemic rickets, autosomal recessive 1a-hydroxylase deficiency, and other disorders, the 1a-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 (24-hydroxylase 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 25OHD to 24,25(OH)2D and that of 1,25(OH)2D to 1,24,25(OH)3D; both reactions initiate the metabolic inactivation of vitamin D via the C24oxidation pathway. The kidney and intestine are major sites of hormonal inactivation of vitamin D by virtue of their abundant 24-hydroxylase activity.

VITAMIN D BIOSYNTHETIC ENZYMES The vitamin D biosynthetic enzymes include both mitochondrial (type I) and microsomal (type II) cytochrome P450 enzymes. These enzymes are hemecontaining, mixed function oxidases that must receive

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Cholecalciferol

OH

OH

1α-OHase kidney

25-OHase liver HO

HO

HO

24-OHase kidney

OH

24-OHase kidney

OH OH

HO

FIGURE 25.1 Biosynthesis of vitamin D3. Near

1,25(OH)2D

25OH D

ultraviolet light (290e320 nm) 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 25OHD, which is the most abundant form of vitamin D in the human circulation, can undergo 1a-hydroxylation in the kidney by P450c1a to yield the active hormonal compound 1,25(OH)2D. Both 25OHD and 1,25(OH)2D also can undergo 24-hydroxylation in the kidney by P450c24 to yield either 24,25(OH)2D or 1,24,25(OH)3D, respectively.

OH

(1),24,25(OH)2(3)D

electrons from NADPH to mediate catalysis. Type I P450 enzymes receive electrons via an electron-transfer chain consisting of two proteins, a flavoprotein termed ferredoxin reductase and an iron-sulfur protein termed ferredoxin; type II enzymes utilize a single di-flavin electron-transfer protein termed P450 oxidoreductase [6]. The heme iron in both types of P450 ultimately receives the electrons and donates them to molecular oxygen as the terminal electron acceptor, mediating catalysis.

Vitamin D 25-Hydroxylase The identity of the hepatic 25-hydroxylase remained elusive for many years, with cell fractionation and biochemical studies providing evidence that both mitochondrial and microsomal enzymes catalyze this reaction. Screening a rat liver cDNA expression library with polyclonal antisera raised against a purified rat liver 25-hydroxylase preparation yielded a cDNA then called P450c25 [7,8]. This enzyme could hydroxylate sterol carbons 26 and 27 to initiate bile acid synthesis, and is often referred to as P450c27 or CYP27A1 [9]. This enzyme is structurally related to the mitochondrial 1a- and 24-hydroxylases, suggesting that CYP27A1 is a major 25-hydroxylase. However, patients with CYP27A1 mutations have cerebrotendinous xanthomatosis without a disorder in calcium metabolism. The identity of the physiologically relevant 25-hydroxylating enzyme(s) remained elusive because very few patients had been described having apparent 25hydroxylase deficiency, and there are no generally agreed-upon criteria for this potential diagnosis. The best available clinical description was that of Casella et al. [10], who measured vitamin D metabolites in

blood and showed decreased intestinal absorption of vitamin D. A case that appeared to be both clinically and hormonally similar was reported by Abdullah et al. [11]. However, the genes for CYP27A1 and another candidate, CYP2D6, lacked mutations in these reported families [12]. Cheng et al. identified microsomal CYP2R1 as a mouse and human vitamin D 25-hydroxylase [13]. They then identified the CYP2R1 mutation L99P in an EBV-transformed cell line established from one of the patients described by Casella, and showed that this mutation dramatically reduced 25-hydroxylase activity [14]. Others confirmed the genetic findings in the same family, but found no CYP2R1 mutations in the family described by Abdullah, suggesting that vitamin D 25-hydroxylase deficiency is indeed rare and underscoring the difficulty in making this diagnosis by classical hormonal approaches rather than by vitamin D absorption studies [15]. Thus, the diagnostic criteria and prevalence of 25-hydroxylase deficiency remain unclear. It seems likely that both CYP2R1 and another enzyme (possibly CYP27A1) are effective 25hydroxylases in vivo, so that symptomatic disease is only seen when a CYP2R1 mutation occurs in the presence of another stressor such as neonatal hypoparathyroidism or nutritional vitamin D deficiency. CYP2R1 expression is ubiquitous and not confined to the liver [16], possibly accounting for the lack of change in vitamin D 25-hydroxylation in patients with liver failure. Although CYP2R1 belongs to a family of hepatic drug-metabolizing enzymes that have broad substrate specificity, CYP2R1 is highly specific for vitamin D 25hydroxylation. The crystal structure of CYP2R1 with vitamin D3 bound in its catalytic site shows a typical microsomal cytochrome P450 structure, but with a more closed, tight conformation and with

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hydrophobic residues lining the substrate-binding pocket, so that the geometry is only suited to binding planar hydrophobic molecules such as sterols [17]. The L99 residue mutated in the family described by Casella is located in the B-helix, near to, but not directly involved in binding vitamin D. A genome-wide association study in z34 000 individuals of European descent revealed that common variants at the CYP2R1 locus are associated with circulating 25OHD concentrations, providing strong evidence that CYP2R1 is the enzyme underlying the crucial first step in vitamin D metabolism [18].

Vitamin D 24-Hydroxylase The 24-hydroxylase (P450c24 or CYP24) was cloned by purifying the protein from rat renal mitochondria, raising a polyclonal antiserum, and screening a rat kidney cDNA expression library [19]. Subsequently, the human cDNA [20] and gene [21] were cloned. Studies with the purified rat kidney enzyme [19] and with cells expressing the human CYP24 cDNA [20] confirmed that this enzyme can catalyze the 24-hydroxylation of both 25OHD and 1,25(OH)2D. The 24-hydroxylase is induced by 1,25(OH)2D, thus promoting its inactivation by 24-hydroxylation as a mechanism to regulate the amount of available active 1,25(OH)2D [22]. Rat P450c24 is the first mitochondrial cytochrome P450 to have its structure determined by crystallography [23]. The structure shows a classic cytochrome P450 fold with an open conformation, more reminiscent of CYP3A4 than of CYP2R1; despite this, P450c24, like other mitochondrial P450 exhibits narrow substrate specificity. The open conformation may facilitate binding of substrate by diffusion from the membrane, followed by closure to exclude solvent and facilitate catalysis [23].

Vitamin D 1a-Hydroxylase It was not until 1997 that four groups of investigators working independently and using different approaches reported the cloning of the human, rat, and mouse vitamin D-1a-hydroxylase cDNAs [24e28], and subsequently the human gene (CYP27B1) [25,29]. Efforts to purify the 1a-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 approached the problem of low renal abundance of P450c1a by using a different tissue system, primary cultures of human keratinocytes [24]. These cells, when grown in serumfree medium in the presence of low concentrations of

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calcium, exhibit substantial 1a-hydroxylase activity [2]. Using keratinocytes as a source of RNA enriched for P450c1a mRNA, degenerate-sequence oligonucleotides were prepared based on the relatively well-conserved sequences of the ferredoxin-binding sites and hemebinding 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 P450c1a. This was used to screen a keratinocyte cDNA library yielding a partial-length, 1.9-kb cDNA, whose complete sequence was obtained by rapid amplification of cDNA ends (50 -RACE) [24]. The human P450c1a cDNA is 2.4 kb in length and encodes a protein of 508 amino acids that has a predicted molecular mass of 56 kDa [24]. P450c1a is one of only seven mitochondrial P450 enzymes encoded by the human genome. The hemebinding region of P450c1a is similar to that of the other mitochondrial P450 enzymes, but its overall sequence identity to the other mitochondrial P450 enzymes is limited [24]. Although the P450c1a cDNA was cloned from human keratinocytes, four lines of evidence demonstrated that the keratinocyte and renal P450c1a enzymes are encoded by the same gene [24]. First, when the keratinocyte P450c1a cDNA was transfected into mouse Leydig MA-10 cells, the transfected cells catalyzed the conversion of 25OHD3 to authentic 1,25(OH)2D3, as determined by high pressure liquid chromatography and confirmed by gas chromatography/mass spectrometry of the 1a-hydroxylated product. Second, the cloned P450c1a had a Km for 25OHD of 2.7  107 M, which closely approximates the concentration of 25OHD found in vivo. Third, reverse transcription/polymerase chain reaction (RT/PCR) was used to show that those sequences that were cloned from keratinocytes also were 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 1ahydroxylase 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 P450c1a and the first proof that VDDR-1 is caused by mutations in vitamin D-1a-hydroxylase gene [24]. Soon after the P450c1a cDNAs were cloned, the human gene was cloned [25,29], localized to chromosome 12 by somatic cell hybrid analysis [29], and mapped to 12q13.1-13.3 by fluorescence in situ hybridization [26,30,31]. The human gene for 1a-hydroxylase, designated CYP27B1, is only 5 kb in length, is single copy, and comprises nine exons and eight introns (Fig. 25.2) [29]. Although it is substantially smaller

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- G102E - gggcg → cttcgg - G73R - 958ΔG - R107H - P112L - 1921ΔG - G125E - 1984ΔC - IVS2+1g →a - W241X

- 212ΔG - Q65H

- T409I - Y413C - R429P - W433X - S323Y - 3398 7bp dup - W328X - 3403 2bp dup - R335P - R453C - L343F - IVS6+1g →a - R453H

- Q135K - T321R - P143L - D164N - E189G - E189K - IVS3+1g →a

- P382S - R389C - R389G - R389H - IVS7+1g →a

- V478G - R492P - P497R - 3922ΔA

1 kb

FIGURE 25.2 Scale diagram of the intron/exon organization of the human P450c1a gene, as reported by Fu et al. [29]. All mutations causing 1a-hydroxylase deficiency reported through 2010 are shown.

than the genes for other mitochondrial P450 enzymes, its intron/exon organization is very similar, especially to that of P450scc [29]. This strongly suggests that although the mitochondrial P450 enzymes retain only 30e40% amino acid sequence identity with each other, they all belong to a single evolutionary lineage. The mouse CYP27B1 also has been cloned [32,33].

RICKETS DUE TO ABNORMALITIES OF VITAMIN D METABOLISM 1a-Hydroxylase Deficiency In 1961, Prader et al. [34] described a new form of rickets that differed from the hypophosphatemic rickets first reported by Albright et al. in 1937 [35], 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) [34], vitamin D dependency because of its responsiveness to vitamin D [36], or vitamin D-dependent rickets type I, this disease is now known to be caused by defective renal conversion of 25OHD to 1,25(OH)2D as a consequence of loss-of-function mutations in the renal 1a-hydroxylase gene, CYP27B1 [24]. Herein, we refer to this form of rickets as vitamin D 1a-hydroxylase deficiency. Clinical Features Patients with 1a-hydroxylase deficiency usually are normal at birth but come to medical attention within

the first 24 months of life, most commonly because of 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 fontanels, or craniotabes (softening of the parieto-occipital area). Muscle traction on the softened rib cage can give rise to thoracic deformity, including pectus carinatum. Dental development often is 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 parathyroid hormone (PTH) concentrations are observed [34,37e43], as is typical of patients with vitamin D deficiency rickets. When severe, hypocalcemia can cause tetany and seizures. Metabolic balance studies in patients with 1a-hydroxylase deficiency reveal malabsorption of calcium and phosphorus and

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reduced urinary calcium excretion [38,39,42,44]. The renal tubular abnormalities of hyperchloremic metabolic acidosis and generalized hyperaminoaciduria are observed in some patients [34,37,39e42], findings also seen in patients with vitamin D deficiency [45]. The hallmarks of 1a-hydroxylase deficiency are greatly reduced serum concentrations of 1,25(OH)2D despite normal concentrations of 25OHD, and the reversal of clinical and laboratory abnormalities by administration of physiologic amounts of 1,25(OH)2D3 [43,46,47]. These findings support the hypothesis of Fraser et al. [46] that the genetic defect results from defective renal conversion of 25OHD to 1,25(OH)2D, and they serve to distinguish patients with 1a-hydroxylase deficiency from those with nutritional vitamin D deficiency in whom serum levels of 25OHD are reduced, or hereditary 1,25(OH)2D-resistant rickets in whom serum levels of 1,25(OH)2D are greatly increased. In some patients with 1a-hydroxylase deficiency, serum concentrations of 1,25(OH)2D are nominally within the normal range [44,48], 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 1a-hydroxylase deficiency, in contrast to the increase induced in control subjects [49]. Serum concentrations of 24,25(OH)2D are normal in patients with 1a-hydroxylase deficiency [44,49,50], indicating that the 24-hydroxylase enzyme is intact. Molecular Genetics of 1a-Hydroxylase Deficiency The hereditary nature of 1a-hydroxylase deficiency was observed by Prader et al. in their initial description of the disease [34], with autosomal recessive inheritance reported soon thereafter [37,40,41]. 1a-Hydroxylase deficiency is rare in most populations, but is particularly common among French Canadians, with an apparent carrier rate of 1/26 in the Charlevoix-Saguenay-Lac Saint Jean area of Quebec [51]. Using linkage analysis in French Canadian families with 1a-hydroxylase deficiency, the disease was mapped to a region in band 14 of the long arm of chromosome 12 (12q14) [52]. Microsatellite haplotype analysis of 32 affected families mapped the gene proximal to D12S305 and distal to D12S305, D12S04 [53]. 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 [53].

683

With the cloning of the 1a-hydroxylase gene, the molecular genetics of 1a-hydroxylase deficiency have now been studied thoroughly by several groups [24,30,31,48,54e59]. Gene localization via human/ rodent somatic-cell hybrids [29] and fluorescent in situ hybridization (FISH) [26,30] reveal that the gene maps to 12q13.3, consistent with the linkage analysis of Labuda et al. [53] and with the autosomal recessive inheritance of the disease. The first mutation in the 1ahydroxylase gene was identified by Fu and colleagues in a Caucasian-American girl with 1a-hydroxylase deficiency who was a compound heterozygote for two deletion/frameshift mutations in exon 2 that predicted premature truncation of the protein (Fig. 25.3A) [24]. Cultured skin keratinocytes from the patient were devoid of 1a-hydroxylase activity (Fig. 25.3B) [24]. Subsequently, four unrelated Japanese patients studied by Kitanaka and colleagues [30] confirmed that mutations in the 1a-hydroxylase gene could cause the clinical syndrome of 1a-hydroxylase deficiency. However, several questions remained. It was not yet known whether all patients with the typical clinical syndrome of 1a-hydroxylase deficiency had the same disease.

FIGURE 25.3 Mutation of CYP27B1 causes 1a-hydroxylase deficiency. (A) Keratinocytes from a healthy person and from a patient with 1a-hydroxylase deficiency were used to prepare mRNA, which was reverse-transcribed, and the CYP27B1 cDNA was PCR-amplified using specific primers. The upper panel displays normal (left) and patient (right) cDNA sequence in the region of codon 211, showing that the G (arrow) in the normal sequence is deleted in the patient. The lower panel displays 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) 1a-hydroxylase activity in keratinocytes from human neonatal foreskin (N), adult skin (Ad), and skin from a patient with 1a-hydroxylase deficiency (Pt). No activity was detected in the patient. The scale is logarithmic and begins at the level of detection of the assay. (Reprinted from [24].)

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25. RICKETS DUE TO HEREDITARY ABNORMALITIES OF VITAMIN D SYNTHESIS OR ACTION

Furthermore, although it was established that 1ahydroxylase deficiency was common in French Canada, it was not known whether such patients had two distinct mutations, as had been suggested by linkage analysis [53]. Finally, there was no information relating enzyme structure to its function. Wang et al. studied the 1a-hydroxylase genes of 19 patients with the clinical syndrome of 1a-hydroxylase deficiency from 17 families representing multiple ethnic groups, including five French Canadian families, three Polish families, four Caucasian American families, one Filipino family, one Chinese family, one Haitian family, one African-American family, and one Hispanic family [48]. 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 1a-hydroxylase deficiency: hypocalcemia, hypophosphatemia, increased serum concentrations of alkaline phosphatase and PTH, normal serum concentrations of 25OHD, 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 CYP27B1 mutations was identified and the mutations were correlated with the microsatellite haplotyping of chromosome 12q13, using the markers D12S90, D12S305, and D12S104. As noted previously, Labuda et al. had examined the microsatellite genetic markers on chromosome 12 and observed that the French Canadian patients with 1a-hydroxylase deficiency carried one of two haplotypes [53]. 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 [53]. Among the five French Canadian families studied by Wang et al. [48], nine of 10 unique alleles carried the 4-7-1 haplotype, and all nine of these carried the identical mutation in codon 88, the deletion of guanosine at position 958 (958delG), 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 958delG

mutation identifies it as the Charlevoix mutation. This mutation deletes the G in the sequence 50 ACGT30 , which is normally recognized by the endonucleases Tai I and Mae II. This feature was used to design a rapid, accurate PCR-based diagnostic test that can detect this mutation in genomic DNA from any source [48]. Yoshida et al. characterized the P450c1a genes of four French Canadian patients [31], and found three to be homozygous for the 958delG mutation and one homozygous for the duplication of a 7-bp sequence in exon 8 [31]. Based on the geographic origins of each patient, Yoshida et al. suggested that mutation 958delG 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. found this 7-bp duplication, upstream from codon 441, on seven separate alleles in six families (Fig. 25.4) [48]. 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 that allele carried the missense mutation P497R, rather than the 7-bp duplication. Smith et al. identified two unrelated patients from the UK who were homozygous for this same 7-bp duplication [54]. 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. identified a total of 14 different mutations in the 19 patients, including seven missense mutations [48]. To determine the effect of these mutations on enzyme activity, each mutant was recreated using sitedirected mutagenesis and expressed in MA-10 cells. None of the missense mutations encoded a protein with 1a-hydroxylase activity significantly above the low endogenous activity of MA-10 cells [48]. Eight additional missense mutations were identified by Kitanaka et al. 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 [30,55]. None of the eight FIGURE 25.4 The 7-bp duplication. (A) The sequence CCCACCC is normally duplicated in exon 8, encoding residues 438 to 442 (Pro-Thr-Pro-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.

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mutants showed any activity, consistent with the phenotype of the patients. Two other missense mutations were identified by Smith et al. in a compound heterozygous patient from the UK [54]; 1a-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 1a-hydroxylase deficiency, but the molecular basis of such variation is unknown. To address this question, Wang et al. analyzed six patients with clinical and radiographic features of rickets; in four patients the laboratory abnormalities were typical of 1a-hydroxylase deficiency, but in two they were unusually mild; i.e. mild hypocalcemia and normal serum 1,25(OH)2D concentrations [56]. 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 þ 1g/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 (CGT to GGT) in exon 7 was totally inactive, but mutant L343F (CTC to TTC) in exon 6 retained 2.3% of wild-type activity, and mutant E189G (GAA to GGA) in exon 3 retained 22% of wild-type activity (Fig. 25.5). 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

100

Relative activity (%)

100 80 60 40 22

20 0

FIGURE 25.5

0

0

2.3

Ve ctor

R389G

L343F

E189G

WT

1a-Hydroxylase activity of the P450c1a mutants. The three novel missense mutations, R389G, L343F and E189G, were recreated in a P450c1a cDNA expression vector and transfected into mouse Leydig MA-10 cells. The mutation R389G had no 1a-hydroxylase 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. Data are expressed as a percentage of the activity of the wild type cDNA. (From Wang et al. [56].)

685

variable phenotype observed in patients with 1ahydroxylase deficiency [56]. In 2007, Kim et al. reported CYP27B1 mutations in 10 patients with 1a-hydroxylase deficiency, and reviewed the 44 other cases then reported [57]. They found four known mutations and four novel mutations; among five Korean patients, nine of 10 alleles carried either a mutation in intron 3 that led to incorrect RNA splicing or a frameshift mutation in exon 8, suggesting founder effects in this population. Alzahrani et al. described an extended Saudi Arabian family in which all six affected members were homozygous for the missense mutation G102E; when expressed in transfected CHO cells, this mutant form of 1a-hydroxylase had about 20% of normal activity [58]. Structure and Function of the 1a-Hydroxylase Although vitamin D 1a-hydroxylase deficiency is rare, to date at least 43 different mutations have been found in over 100 patients since the first description of gene mutations in 1997 (Table 25.1; see Fig. 25.2) [24,56e59]. The mutations observed most frequently are 958delG, commonly found in French Canadian patients [48] due to a founder effect [53], and a 7-bp duplication that arose independently in several populations [48]. All of the frameshift and nonsense (premature translation arrest) mutants eliminate the heme-binding site of the 1a-hydroxylase, resulting in a protein devoid of enzymatic activity. Of the many missense mutations reported, all except three [56,58] were totally inactive when assayed for enzymatic activity in vitro. The tertiary structures of P450c24 [23] and of several bacterial type I P450 enzymes have been determined by x-ray crystallography [61e63]. Comparisons of the structures of these enzymes reveals remarkable conservation of their topology and tertiary structure, despite low amino acid sequence identity [64]. Based on both linear sequence alignments [48] and on comparisons with the crystal structure of P450c24, it is possible to infer the location and function of the amino acids whose replacement mutations disrupt enzyme activity. Mutation Q65H is in a-helix A’, T409I is in strand 3 of b-sheet 1, and R389H is in strand 4 of b-sheet 1. Although distinct from one another in terms of their amino acid numbers, these mutations lie in the clustered b-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 1a-hydroxylase.

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686 TABLE 25.1

25. RICKETS DUE TO HEREDITARY ABNORMALITIES OF VITAMIN D SYNTHESIS OR ACTION

Known Mutations of CYP27B1 in Patients with Vitamin D 1a-Hydroxylase Deficiency

Amino acid change

Nucleotide change*

Exon

Reference

TABLE 25.1 Known Mutations of CYP27B1 in Patients with Vitamin D 1a-Hydroxylase DeficiencydCont’d Amino acid change

Nucleotide change*

Exon

Reference

Insertions

Missense mutations

frameshift after 441H

33983406insCCCACCC

8

[31,48, 54,57]

frameshift after 441H

3403-3406insAC

8

[48]

897-901delGGGCG; 897-902insCTTCGG

2

frameshift after 129A

IVS2 þ 1 G to A (1083G/A)

intron 2

[56]

frameshift after 196E

IVS3 þ 1 G to A (1796G/A)

intron 3

[55,57]

frameshift after 379R

IVS6 þ 1 G to A (2715G/T)

intron 6

[60]

frameshift after 405N

IVS7 þ 1 G to A (G2997G/A)

intron 7

[57]

Q65H

246G>T

1

[48]

G73R

913G>C

2

[59]

G102E

305G>A

2

[58]

R107H

1016G>A

2

[30,55]

P112L

1031C>T

2

[57]

G125E

1070G>A

2

[30]

Splice-site mutations

P143L

1634C>T

3

[55]

D164N

1696G>A

3

[55]

E189G

1772A>G

3

[56]

E189K

1771G>A

3

[48]

T321R

2337C>G

5

[55]

S323Y

2546C>A

6

[54]

R335P

2582G>C

6

[30]

L343F

2605C>T

6

[56]

P382S

2925C>T

7

[30]

R389C

2946C>T

7

[55]

R389G

2946C>G

7

[56]

R389H

2947G>A

7

[48,56,57]

T409I

3299C>T

8

[48,56]

Y413C

3311A>G

8

[59]

R429P

3359G>C

8

[48]

R453C

3430C>T

8

[48]

R453H

3430G>A

8

[59]

V478G

3680T>G

9

[54]

R492P

3902G>C

9

[59]

P497R

3917C>G

9

[48]

W241X

2014G>A

4

[48]

W328X

2561G>A

6

[57]

W433X

3372G>A

8

[55]

frameshift from 55K

212delG

1

[48]

frameshift from 87Y

958delG

2

[31,48,57]

frameshift after 209C

1921delG

4

[24,48]

frameshift after 230V

1984delC

4

[24,48]

frameshift after 498E

3922delA

9

[57]

Q135K

1609delC

3

[59]

Nonsense mutations

Deletions

Deletioneinsertion frameshift after 66V

[56]

* Nucleotide numbers refer to genomic DNA and are numbered from the transcription start site [29]. The reference sequence is available on the NCBI, Entrez, Nucleotide database: http://www.ncbi.nlm.nih.gov/Entrez; accession number AF 027152.

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 17ahydroxylase deficiency [65], and like the R435C mutation in P450arom, which causes complete aromatase deficiency [66]. Mutant P497R lies near strand 3 of b-sheet 3, which participates in defining the top of the substrate-binding pocket; the directional change in the a-carbon backbone that results from insertion of a proline could disrupt substrate binding. Three different missense mutations have been described in codon 389, R389G [56], R389H [48] and R389C [55], all three of which are totally inactive. This would be predicted, because R389 aligns with the highly conserved arginine in the b1e4 helix of type 1 P450 proteins [48]; this arginine coordinates one of the heme propionate side chains [64,67] 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 [56] 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

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catalytically. The mutation L343F could disrupt activity by creating a conformational mutant. Sawada et al. performed structureefunction analysis of missense mutations identified in Japanese patients [68]; their sequence alignments were virtually identical to those of Wang et al. [48]. All mutations were functionally inactive when expressed in vitro [68]. 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 substrateeheme pocket. They further suggested that residues R389 and R453 are involved in heme-propionate binding, and residue D164, 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 [68]. The first missense mutation described that retains partial activity in vitro is mutation E189G, which retained 22% of wild-type activity (see Fig. 25.5) [56]. 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 E189G mutation [56] 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)2D were not reduced. The diagnosis of 1a-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 mg per day of 1,25(OH)2D3. In another patient whose mutation retained 2.3% of wild-type activity [56], serum concentrations of 25OHD and 1,25(OH)2D 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 1a-hydroxylase deficiency may fail to identify patients with partial but significant defects in this enzyme, and hence 1a-hydroxylase deficiency syndromes may be more common than previously appreciated. Treatment In the earliest reports of treatment of 1a-hydroxylase deficiency, administration of large doses of vitamin D2, 50 000 to 200 000 units per day, was associated with reversal of clinical, chemical, and radiographic abnormalities, and improvement in growth rate [34,37e39]. However, the availability of activated forms of vitamin D has now rendered such therapy obsolete.

687

Hypocalcemia and hyperparathyroidism were reversed and rickets was healed by administration of 1e3 mg/day (80e100 ng/kg) of 1aOHD3 [69], a 1a-hydroxylated analog of vitamin D that requires 25-hydroxylation in the liver. The synthetic analog dihydrotachysterol (DHT) is not 1a-hydroxylated, but carries a hydroxyl group in the 3a-position; rotation of the A-ring about the 6e7 carbon bond brings this group into a pseudo-1a-hydroxyl configuration, so that DHT is active in the absence of 1a-hydroxylation. Typical doses are 50 mg/kg/day in infancy or 0.5 to 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 0.25e2.0 mg/day (10e400 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 [43]. The maintenance dosage of 1,25(OH)2D3 typically is lower than that needed to initiate healing of rickets; therapy must be life-long and is predictably successful [48]. 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 the bone healing that accompanies the initial phase of therapy. One generally aims to increase the total serum calcium concentration into the low-normal range (8.5e9 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-hour urinary excretion of calcium should remain less than 4 mg/kg. The long-term results recently reported by Edouard et al. support this approach [59]. These authors reported up to 26-year follow up of a well-characterized cohort of 39 patients in Quebec. Among 21 patients diagnosed by 2.6 years of age, the most common presenting features were rickets, motor delay, inability to walk and short stature. Treatment with calcitriol in infancy and childhood normalized hormonal and chemical values and lumbar spine bone mineral density within 3 months; catch-up growth took about 2 years. Among adult patients, final average height was similar to that of the general population in those patients treated before the pubertal growth spurt but was decreased in those treated after puberty. Nine affected women treated with calcitriol had a total of 19 pregnancies. Pregnancies were uncomplicated; newborn infants were normocalcemic at birth and without intrauterine growth retardation.

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25. RICKETS DUE TO HEREDITARY ABNORMALITIES OF VITAMIN D SYNTHESIS OR ACTION

Whether or not “minor” variations in 1a-hydroxylase activity contribute to phenotypic variation is not established. A recent study reported an association of a single nucleotide polymorphism (SNP) in the promoter of CYP27B1 with an increased risk for bone fractures among 153 men and 596 women above the age of 65 [70]. The SNP was located 1260 bp from the transcriptional start site; when recreated in a 1.3 kb promoter-reporter construct, it had the same or minimally decreased activity. Furthermore, there was no hormonal evidence for the suspected lowered 1a-hydroxylase activity, as there was no correlation between the SNP and PTH levels. Thus, current data do not support a role for 1a-hydroxylase genetics in osteopenia and fracture risk, but little work has been done in this area.

RICKETS DUE TO ABNORMALITIES OF VITAMIN D ACTION Hereditary 1,25-Dihydroxyvitamin D Resistant Rickets In 1978, Brooks et al. described a young woman with hypocalcemia, secondary hyperparathyroidism, osteomalacia, and osteitis fibrosa in whom serum concentrations of 25OHD were normal and those of 1,25(OH)2D were greatly increased [71]. The authors suggested that the disorder resulted from impaired end-organ response to 1,25(OH)2D and proposed that it be called vitamin Ddependent rickets type II (VDDR-II) to distinguish it from VDDR-I. That same year, Marx et al. described a condition with similar clinical and laboratory findings in a brother and sister in whom rickets first became evident between the ages of 5 and 20 months (Fig. 25.6) [72]. The hypocalcemia in these patients responded to oral administration of 1,25(OH)2D in doses of 15e20 mg/day, approximately 20 times that needed to reverse hypocalcemia in patients with 1a-hydroxylase deficiency. These authors suggested that the condition could result from a hereditary abnormality in the receptor for 1,25(OH)2D [72]. We now know 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, pseudovitamin D deficiency type II (PDDR II), calcitriolresistant rickets, hypocalcemic vitamin D-resistant rickets, and hereditary 1,25(OH)2D-resistant rickets (HVDRR). Herein, we refer to the disease as HVDRR, as suggested in a comprehensive review [74]. Clinical and Laboratory Features Most of the clinical, laboratory, and radiographic findings in patients with HVDRR [71e3,75] are similar to

those in patients with 1a-hydroxylase deficiency or nutritional vitamin D deficiency. However, a striking difference is the finding of either sparse body hair or total alopecia in the majority of affected patients [73,77e78]. 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 [78]. Serum concentrations of 1,25(OH)2D are greatly increased in patients with HVDRR; values range from approximately threefold to 30-fold higher than the normal mean value [71e73,75,76,79]. This feature readily distinguishes such patients from those with 1a-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 [75,76,80e82] or undetectable [79]. Patients are resistant to treatment with physiological and even supraphysiological doses of vitamin D. Cellular and Molecular Defect: Vitamin D Receptor The action of 1,25(OH)2D in target tissues depends on its binding to the vitamin D receptor (VDR), a member of the thyroid-retinoid group of receptors, which is a subgroup of the steroid-thyroid-retinoid gene superfamily of nuclear transcription factors [83]. 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 elements (VDRE), on target genes, thereby regulating gene transcription. The transcriptional activity of the liganded VDReRXR heterodimer is influenced by posttranslational modifications and by association with nuclear receptor co-activators. 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. 25.7). Region A/B includes those residues amino terminal to the C region, which is the highly conserved DNA binding domain (DBD) 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 DNA binding domain and region E/F, the ligand-binding domain (LBD). 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 co-activators and activating transcription [84e86]. Ligand binding appears to induce a conformational change of the AF-2 helix that allows recruitment of co-activators of the p160 family of co-activator proteins, including steroid receptor co-activator 1 (SRC-1) [84,87]. Another class of

PEDIATRIC BONE

RICKETS DUE TO ABNORMALITIES OF VITAMIN D ACTION

FIGURE 25.6

Two sisters, 7 (left) and 3 years of age, with HVDRR and alopecia. (Reprinted from [73].)

co-activators, vitamin D receptor-interacting protein (DRIP), binds to the liganded VDR, strongly potentiates transcription, and is essential for ligand-dependent transactivation [88]. The VDR is found in numerous tissues including skin fibroblasts; therefore, skin obtained by biopsy from patients with HVDRR was used to determine the nature of their vitamin D “resistance”. In early studies of cultured skin fibroblasts from affected E -1

patients, two abnormalities were found: absence of high-affinity nuclear binding of [3H]1,25(OH)2D3 [89,90], and failure of 1,25(OH)2D to induce 24hydroxylase activity [90], a well-characterized biomarker for cellular responsiveness to 1,25(OH)2D. Subsequent studies showed that, in some patients, [3H]1,25(OH)2D3 binding was normal. In either case, however, cellular responsiveness to 1,25(OH)2D was greatly impaired [90e92]. Patients in whom AF-2

hVDR 1 domains

24

A/B

89 C DNA binding domain

689

242 D

272

320

404

427 aa

E/F 1,25(OH)2D binding domain

PEDIATRIC BONE

FIGURE 25.7 Schematic illustration of the VDR. The protein is comprised of 427 aa 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.

690

25. RICKETS DUE TO HEREDITARY ABNORMALITIES OF VITAMIN D SYNTHESIS OR ACTION

[3H]1,25(OH)2D3 binding was very low or undetectable were designated as “receptor negative” or “hormone binding negative” [89,92e96]. Fibroblasts from such patients did contain immunoreactive VDR protein, suggesting that abnormal [3H]1,25(OH)2D3 binding was caused by a defect in the LBD of the VDR rather than defective synthesis of VDR protein [97e100]. Patients whose cells had detectable [3H]1,25(OH)2D3 binding were designated “receptor positive” or “hormone binding positive” [91,93,94,101) and, in such patients, DNA-binding affinity of the VDR was defective [81,101e103]. In some receptor positive patients, DNA-binding affinity was normal but the ability of the VDR to localize to the nucleus was impaired [101].

Molecular Genetics of HVDRR Cloning of the avian VDR [104] and human [105] VDR permitted analysis of the molecular genetic basis of HVDRR. The human VDR gene is localized to chromosome 12q13-14 [106], the same region in which the gene encoding the vitamin D 1a-hydroxylase is found. The VDR gene spans 75 kb of DNA and comprises eight coding exons (exons 2e9) and at least three short 50 noncoding exons (exons 1a, 1b, and 1c). Exons 2 and 3 encode the highly conserved DNA-binding domain (DBD) of the receptor, and exons 6 through 9 encode the ligand-binding domain (LBD).

M

DNA-Binding Defects Hughes et al. identified the first mutations in the VDR gene in two families in which the affected children were homozygous for the disorder. Binding of the receptor to 1,25(OH)2D was normal but affinity of the liganded VDR for DNA was decreased [107]. One family carried a missense mutation (R73Q) in exon 3, which encodes the second zinc finger region (Fig. 25.8). (In this chapter, the numbering system of Baker et al. [105] 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 observed in receptor isolated from the patients [107]. The mutant receptor was transcriptionally inactive in vitro as well [108]. At least 13 different mutations in the DBD of the VDR have been identified (Table 25.2; see Fig. 25.8) [80,107,109e113, 115e118,138). In most cases, fibroblasts from affected patients had normal [3H]1,25(OH)2D3binding capacity but reduced DNA-binding affinity. Based on structural similarities with the crystal structures of the related glucocorticoid receptor [139], RXR, and thyroid receptor (TR) [140], one can predict the likely consequences of the mutations in the VDR. Mutations V26M, G33D, H35Q, K45E, G46D, and F47I affect regions of the receptor that contact DNA. Mutation

A

A

A

E

D

S

M

Q F

T

G

S L

10

X

P

30

D P

H F

T A

N

R

A

G

T

G M

D

V

F

G

D R

N

V 20

C

P

R

C

X

Q

G D

G C

I

K

E

H C

G

D

F

R F

R S

M K

F R

50

K

A

L

P F

C T

M

C Q

Z n+ +

N

E

60

A

Q

G

C 80

90

M I

R

D

L K

I

F E K

R R

40

X

R

I

C Z n+ +

K

fs

Q N

T

M

D

D

70

V R C

R

FIGURE 25.8

Model of the DNA binding domain (DBD) of the VDR and location 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 location of the mutations. Mutations are shown as large shaded circles. FS refers to frame shift. The numbers indicate the amino acid number. All mutations in the DBD reported through 2010 are shown. (Modified from [74].)

PEDIATRIC BONE

691

RICKETS DUE TO ABNORMALITIES OF VITAMIN D ACTION

L45E 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 [112]. Mutations R30X in exon 2, and R50X and R73X in exon 3, result in receptors that are truncated in zinc finger region and thus are devoid of both DNA- and ligand-binding functions. Ligand-Binding Defects Ritchie et al. identified the first VDR mutation associated with a ligand-binding negative phenotype in three TABLE 25.2

unrelated patients with HVDRR [130]. The mutation, T295X, resulted in truncation of 132 amino acids of the carboxy terminus of the VDR, thereby deleting a major portion of the LBD and creating the ligand-binding negative phenotype. This mutation has been identified in several additional families [117,131,141]. Twentyseven mutations in the LBD of the VDR have been identified to date (Fig. 25.9; see Table 25.2) [116e118,120e124,126e128,130e137,140e144]. The crystal structure of the LBD of the human VDR has been described, confirming that its overall topology is similar to that of the other nuclear receptors and is composed of 13 alpha helices, H1eH12 and H3n,

Known Mutations in the VDR in Patients with HVDRR

Amino acid change*

Nucleotide change

Exon

Domain

Ligand binding

Alopecia

Reference

V26M

GTG-ATG

2

DBD

þ

þ

[109]

R30X

CGA-TGA

2

DBD



þ

[110,111]

G33D

GCC-GAC

2

DBD

þ

þ

[107]

H35Q

CAC-CAG

2

DBD

þ

þ

[80]

K45E

AAA-GAA

2

DBD

þ

þ

[112]

G46D

GGC-GAC

2

DBD

þ

þ

[113]

F47I

TTC-ATC

2

DBD

þ

þ

[112]

R50Q

CGA-CAA

3

DBD

þ

þ

[114]

R50X

CGA-TGA

3

DBP



þ

[115]

R73Q

CGA-CAA

3

DBD

þ

þ

[107]

R73X

CGA-TGA

3

DBD



þ

[116e118]

R80Q

CGG-CAG

3

DBD

þ

þ

[116]

C84R

3

DBP

E92fs

intron e

[119] 

þ

[120]

P122fs

c.366delC

4

LBD

[121]

141KTYN144 to 141LWAY144

5 bp deletion/8 bp insertion

4

LBD





[122]

Q152X

CAG-TAG

4

LBD



þ

[123]

frameshift

delete splice

intron f

LBD



þ

[124]

C190W

TGT-TGG

5

LBD

?

þ

[123]

create splice, fs

c.702C>G

6

LBD



þ

[116]

K240Rfs

AAG-AGG

6

LBD



þ

[125]

K246del

3 bp deletion

6

LBD

þ

þ

[126]

F251C

TTC-TGC

6

LBD



þ

[105]

Q259E

CAG-GAG

7

LBD

nr

þ

[118]

Q259P

CAG-CCG

7

LBD

þ

þ

[116]

L263R

CTG-CGG

7

LBD



þ

[127]

I268T

ATT-ACT

7

LBD





[128] (Continued)

PEDIATRIC BONE

692 TABLE 25.2

25. RICKETS DUE TO HEREDITARY ABNORMALITIES OF VITAMIN D SYNTHESIS OR ACTION

Known Mutations in the VDR in Patients with HVDRRdCont’d

Amino acid change*

Nucleotide change

Exon

Domain

Ligand binding

Alopecia

Reference

R274L

CGC-CTC

7

LBD

þ



[123]

W286R

TGG-CGG

7

LBD





[129]

Y295X

TAC-TAA

7

LBD



þ

[117,130,131]

H305Q

CAC-CAG

8

LBD

þ



[132]

I314S

ATC-AGC

8

LBD

þ



[133]

Q317X

CAG-TAG

8

LBD



þ

[134]

G319V

GAC-TAC

8

LBD

nr

þ

[118]

E329K

GAG-AAG

8

LBD

nr

þ

[121]

deletion exon 8

intron j

LBD



þ

[135]

V346M

GTG-ATG

9

LBD

nr

þ

[136]

R391C

CGC-TGC

9

LBD

þ

þ

[133]

R391S

CGC-AGC

9

LBD



þ

[127]

Y401X

insertion/duplication

9

LBD





[132]

E420K

CAA-AAA

9

LBD

þ



[137]

LBD



þ

[123]

deletion exons 7e9

* Mutations are numbered according to Baker et al. [91]. Fs: frameshift; nr: not reported. Introns are designated aej, following exons 1a, 1b, 1c, 2 to 9, respectively.

organized in three layers, and a three-stranded b-sheet [145]. 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 Ser275 (loop H5-b), Trp286 (b-1), and Leu233 (H3), with a “lid” formed by H12. The ligand-binding pocket is lined predominately by hydrophobic residues; two of these residues, H305

(loop H6eH7) and H397 (loop H11), are hydrogen bonded to the 25-hydroxyl group of the 1,25(OH)2D ligand [145]. This structure predicts that H305Q, described by Malloy et al. [143], results in defective ligand binding, as observed in fibroblasts from the affected patient. Mutation F251C, is located in the E1 region (amino acids 244e263), which overlaps the Cterminal portion of helix H3, loop 3e4, and the Nβ _turns

E1 H1

H2

C190W

H3

H4

H5

S1-3 H6

AF-2 H7

H8

H9

V346M E329K G319V I314S H305Q W286R R274L

H10

H11

H12

E420K R391C/S

I268T L263R Q259P/E F251C FIGURE 25.9 Model of the ligand binding domain (LBD) of the VDR and location of missense mutations in patients with HVRDR. The helices are depicted as shaded rectangles and the single b-turn (S1) as an open rectangle. The E1 and AF-2 regions are shown above the a-helices. Missense mutations in the LBD reported through 2010 are shown. (Modified from [74].)

PEDIATRIC BONE

RICKETS DUE TO ABNORMALITIES OF VITAMIN D ACTION

terminal portion of helix H4 [144]. 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]1,25(OH)2D3-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 [144]. Mutations G259P (which occurs in H4), R274L (H5), I314S (H7), and R391C (H10) should interfere with either ligand binding or heterodimerization. Mutation E420K, located in helix H12 of the LBD, alters the coactivator binding site, preventing proper interaction between the VDR and co-activators SRC-1 and DRIP205, and thus results in loss of transactivation [137]. Additional Mutations 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 [117,142]. The donor splice site mutation eliminates the 50 -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 [120]. A second splice-site mutation introduces a cryptic 50 -donor splice site in exon 6, causing a 56-bp deletion in exon 6, a shift of the reading frame (L233fs), and premature truncation of 194 amino acids of the VDR, leading to a hormone-binding negative, defective transactivation phenotype [116]. Two other splicing mutations have been reported [124,135], one resulting in skipping of exon 8 and deletion of 39 amino acids in the LBD [135]. A patient with HVDRR in whom exons 7e9 of the VDR gene were deleted has been reported [123]. Hewison et al. [146] 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)2D3 failed to induce 24-hydroxylase activity in these cells. When the patient’s cDNA was expressed in vitro, 1,25(OH)2D3-induced 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. Alopecia Molecular analysis of the VDR from patients with HVDRR and with and without alopecia has provided insight into unique features of the VDR. Alopecia is observed in patients with VDR mutations in the DBD, in regions that inhibit RXR heterodimerization, or with premature stop mutations, whereas patients

693

with VDR mutations in the LBD or co-activator binding regions without defective heterodimerization do not develop alopecia [122,128,137]. VDR null mice develop their first coat of hair normally but develop alopecia by the second week of life [147,148]. When wild type VDR is expressed in keratinocytes of VDR null mice, alopecia is prevented [149]. However, alopecia does not develop in CYP27B1 null mice or in patients with 1a-hydroxylase deficiency. Thus, the intact VDR protein is required for normal hair growth. Studies in VDR null mice helped to establish the critical role of VDR in hair follicle development [150e152]. The VDR, independently of its ligand, heterodimerizes with RXR and binds to co-repressors like hairless, to inhibit transcription of genes involved in the hair follicle cycle and hair growth. Data suggest involvement of Wnt signaling via b-catenin and hedgehog signaling pathways in the regulation of hair follicle cycling by the VDR [153]. Treatment Patients with HVDRR have been treated with large doses of vitamin D2, 25OHD, 1a-OHD, or 1,25(OH)2D, with highly variable results. A few patients responded to 4000e40 000 units/day of vitamin D2 [71,72,154]; 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 [73,75,76,90]. Patients with HVDRR without alopecia are generally more responsive to treatment with metabolites of vitamin D than patients with alopecia [78]. Some patients without alopecia responded to treatment with 25OHD3 in doses ranging from 20 to 200 mg/day and 1,25(OH)2D3 in doses of 17e20 mg/day [72]. 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 [143]. Treatment with 12.5 mg/day of 1,25(OH)2D3 induced reversal of 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 [143]. Thus, patients with mutations in the LBD are more likely to respond to high-dose vitamin D therapy than those with mutations in the DBD of the receptor [133,143]. Although many patients are refractory to treatment [80,93,155,156), some have responded to administration of 1a-OHD3, in doses as high as 90 mg/ day [82,157] or to 1,25(OH)2D3, in doses as high as 20 mg/day [81,82,92,157e159]. For patients who are refractory to treatment, intravenous administration of large amounts of elemental calcium, 400e1400 mg/m2/day, for periods of up to 3.8 years, was associated with resolution of bone pain,

PEDIATRIC BONE

694

25. RICKETS DUE TO HEREDITARY ABNORMALITIES OF VITAMIN D SYNTHESIS OR ACTION

normalization of the serum calcium and phosphorus concentrations, reversal of hyperparathyroidism [80,125,135,155,157], radiographic and histologic healing of skeletal lesions [155], and increased growth velocity [155,156]. After clinical improvement was induced with intravenous calcium infusions, high doses of oral calcium were required to maintain normocalcemia [135,156]. However, such intensive therapy can be complicated by cardiac arrhythmias, hypercalciuria, nephrolithiasis, and sepsis [156]. 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 1a-hydroxylase deficiency with physiologic replacement doses of calcitriol is straightforward and uniformly successful [48], treatment of patients with mutations of the VDR remains daunting.

References [1] Henry HL. The 25-hydroxyvitamin D 1-a-hydroxylase. In: Feldman D, Pike JW, Glorieux FH, editors. Vitamin D. San Diego: Elsevier Academic Press; 2005. p. 69e83. [2] Bikle DD, Nemanic MK, Gee E, Elias P. 1,25-dihydroxyvitamin D3 production by human keratinocytes. J Clin Invest 1986;78:557e66. [3] Adams JS, Sharma OP, Gacad MA, Singer FR. Metabolism of 25-hydroxyvitamin D3 by cultured pulmonary alveolar macrophages in sarcoidosis. J Clin Invest 1983;72:1856e60. [4] Howard GA, Turner RT, Sherrard DJ, Baylink DJ. Human bone cells in culture metabolize 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3. J Biol Chem 1981;256:7738e40. [5] Armbrecht HJ, Okuda K, Wongsurawat N, Nemani RK, Chen ML, Boltz MA. Characterization and regulation of the vitamin D hydroxylases. J Steroid Biochem Mol Biol 1992;43:1073e81. [6] Miller WL. Minireview: regulation of steroidogenesis by electron transfer. Endocrinology 2005;146:2544e50. [7] Usui E, Noshiro M, Okuda K. Molecular cloning of cDNA for vitamin D3 25-hydroxylase from rat liver mitochondria. FEBS Lett 1990;262:135e8. [8] Su P, Rennert H, Shayiq RM, et al. 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 RNA in ovaries. DNA Cell Biol 1990;9:657e67. [9] Cali JJ, Russell DW. Characterization of human sterol 27-hydroxylase. J Biol Chem 1991;266:7774e8. [10] Casella SJ, Reiner BJ, Chen TC, Holick MF, Harrison HE. A possible genetic defect in 25-hydroxylation as a cause of rickets. J Pediatr 1994;124:929e32. [11] Abdullah MA, Salhi HS, Bakry LA, et al. Adolescent rickets in Saudi Arabia: a rich and sunny country. J Pediatr Endocrinol Metab 2002;15:1017e25. [12] Lin CJ, Dardis A, Wijesuriya SD, Abdullah MA, Casella SJ, Miller WL. Lack of mutations in CYP2D6 and CYP27 in patients with apparent deficiency of vitamin D 25-hydroxylase. Mol Gene Metab 2003;80:469e72.

[13] Cheng JB, Motola DL, Mangelsdorf DJ, Russell DW. Deorphanization of cytochrome P450 2R1: a microsomal vitamin D 25-hydroxilase. J Biol Chem 2003;278:38084e93. [14] Cheng JB, Levine MA, Bell NH, Mangelsdorf DJ, Russell DW. Genetic evidence that the human CYP2R1 enzyme is a key vitamin D 25-hydroxylase. Proc Natl Acad Sci USA 2004; 101:7711e5. [15] Dong Q, Miller WL. Vitamin D 25-hydroxylase deficiency. Mol Genet Metab 2004;83:197e8. [16] Bieche I, Narjoz C, Asselah T, et al. Reverse transcriptase-PCR quantification of mRNA levels from cytochrome (CYP)1, CYP2 and CYP3 families in 22 different human tissues. Pharmacogenet Genomics 2007;17:731e42. [17] Strushkevich N, Usanov SA, Plotnikov AN, Jones G, Park HW. Structural analysis of CYP2R1 in complex with vitamin D3. J Mol Biol 2008;380:95e106. [18] Wang TJ, Zhang F, Richards JB, et al. Common genetic determinants of vitamin D insufficiency: a genome-wide association study. Lancet 2010;376:180e8. [19] Ohyama Y, Noshiro M, Okuda K. Cloning and expression of cDNA encoding 25-hydroxyvitamin D3 24-hydroxylase. FEBS Lett 1991;278:195e8. [20] Chen KS, Prahl JM, DeLuca HF. Isolation and expression of human 1,25-dihydroxyvitamin D3 24-hydroxylase cDNA. Proc Natl Acad Sci USA 1993;90:4543e7. [21] Chen KS, DeLuca HF. Cloning of the human 1a,25-dihydroxyvitamin D-3 24-hydroxylase gene promoter and identification of two vitamin D-responsive elements. Biochim Biophys Acta 1995;1263:1e9. [22] Xie Z, Munson SJ, Huang N, et al. The mechanism of 1,25dihydroxyvitamin D3 auto-regulation in keratinocytes. J Biol Chem 2002;277:36987e90. [23] Annalora AJ, Goodin DB, Hong WX, Zhang Q, Johnson EF, Stout CD. Crystal structure of CYP24A1, a mitochondrial cytochrome P450 involved in vitamin D metabolism. J Mol Biol 2010;396:441e51. [24] Fu GK, Lin D, Zhang MYH, Bikle DD, Miller WL, Portale AA. Cloning of human 25-hydroxyvitamin D-1a-hydroxylase and mutations causing vitamin D-dependent rickets type 1. Mol Endocrinol 1997;11:1961e70. [25] Monkawa T, Yoshida T, Wakino S, et al. Molecular cloning of cDNA and genomic DNA for human 25-hydroxyvitamin D3 1ahydroxylase. Biochem Biophys Res Commun 1997;239:527e33. [26] St-Arnaud R, Messerlian S, Moir JM, Omdahl JL, Glorieux FH. The 25-hydroxyvitamin D 1-alpha-hydroxylase gene maps to the pseudovitamin D-deficiency rickets (PDDR) disease locus. J Bone Miner Res 1997;12:1552e9. [27] Shinki T, Shimada H, Wakino S, et al. Cloning and expression of rat 25-hydroxyvitamin D3-1a-hydroxylase cDNA. Proc Natl Acad Sci USA 1997;94:12920e5. [28] Takeyama K, Kitanaka S, Sato T, Kobori M, Yanagisawa J, Kato S. 25-hydroxyvitamin D3 1a-hydroxylase and vitamin D synthesis. Science 1997;277:1827e30. [29] Fu GK, Portale AA, Miller WL. Complete structure of the human gene for the vitamin D 1a-hydroxylase, P450c1a. DNA Cell Biol 1997;16:1499e507. [30] Kitanaka S, Takeyama K, Murayama A, et al. Inactivating mutations in the 25-hydroxyvitamin D3 1a-hydroxylase gene in patients with pseudovitamin D-deficiency rickets. N Engl J Med 1998;338:653e61. [31] Yoshida T, Monkawa T, Tenenhouse HS, et al. Two novel 1a-hydroxylase mutations in French-Canadians with vitamin D dependency rickets type 1. Kidney Int 1998;54:1437e43.

PEDIATRIC BONE

REFERENCES

[32] Kimmel-Jehan C, DeLuca HF. Cloning of the mouse 25-hydroxyvitamin D3-1a-hydroxylase (CYP1a) gene. Biochim Biophys Acta 2000;1475:109e13. [33] Panda DK, Al Kawas S, Seldin MF, Hendy GN, Goltzman D. 25-hydroxyvitamin D 1alpha-hydroxylase: structure of the mouse gene, chromosomal assignment, and developmental expression. J Bone Miner Res 2001;16:46e56. [34] Prader A, Illig R, Heierli E. Eine besondere form des primare vitamin-D-resistenten rachitis mit hypocalcamie und autosomal-dominanten erbgang: die hereditare pseudomangelrachitis. Helv Paediatr Acta 1961;16:452e68. [35] Albright F, Butler AM, Bloomberg E. Rickets resistant to vitamin D therapy. Am J Dis Child 1937;54:529e47. [36] Scriver CR. Vitamin D dependency. Pediatrics 1970;45:361e3. [37] Stoop JW, Schraagen MJ, Tiddens HA. Pseudo vitamin D deficiency rickets. Report of four new cases. Acta Paediatr Scand 1967;56:607e16. [38] Dent CE, Friedman M, Watson L. Hereditary pseudo-vitamin D deficiency rickets ("Hereditare pseudo-mangelrachitis"). J Bone Joint Surg Br 1968;50:708e19. [39] Matsuda I, Sugai M, Ohsawa T. Laboratory findings in a child with pseudo-vitamin D deficiency rickets. Helv Paediatr Acta 1969;24:329e36. [40] Fanconi A, Prader A. Hereditary pseudo-vitamin D deficiency rickets. Helv Paediatr Acta 1969;24:423e47. [41] Arnaud C, Maijer R, Reade T, Scriver CR, Whelan DT. Vitamin D dependency: an inherited postnatal syndrome with secondary hyperparathyroidism. Pediatrics 1970;46:871e80. [42] Hamilton R, Harrison J, Fraser D, Radde I, Morecki R, Paunier L. The small intestine in vitamin D dependent rickets. Pediatrics 1970;45:364e73. [43] Delvin EE, Glorieux FH, Marie PJ, Pettifor JM. Vitamin D dependency: Replacement therapy with calcitriol. J Pediatr 1981;99:26e34. [44] Balsan S. Hereditary pseudo-deficiency rickets or vitamin D-dependency type I. In: Glorieux FH, editor. Rickets. New York: Vevey/Raven Press, Ltd; 1991. p. 155e65. [45] Fraser D, Kooh SW, Scriver CR. Hyperparathyroidism as the cause of hyperaminoaciduria and phosphaturia in human vitamin D deficiency. Pediatr Res 1967;1:425e35. [46] Fraser D, Kooh SW, Kind HP, Holick MF, Tanaka Y, DeLuca HF. Pathogenesis of hereditary vitamin-D-dependent rickets. An inborn error of vitamin D metabolism involving defective conversion of 25-hydroxyvitamin D to 1a,25-dihydroxyvitamin D. N Engl J Med 1973;289:817e22. [47] Scriver CR, Reade TM, DeLuca HF, Hamstra AJ. Serum 1,25dihydroxyvitamin D levels in normal subjects and in patients with hereditary rickets or bone disease. N Engl J Med 1978;299:976e9. [48] Wang JT, Lin CJ, Burridge SM, et al. Genetics of vitamin D 1a-hydroxylase deficiency in 17 families. Am J Hum Genet 1998;63:1694e702. [49] Aarskog D, Aksnes L, Markestad T. Effect of parathyroid hormone on cAMP and 1,25-dihydroxyvitamin D formation and renal handling of phosphate in vitamin D-dependent rickets. Pediatrics 1983;71:59e63. [50] Mandla S, Jones G, Tenenhouse HS. Normal 24-hydroxylation of vitamin D metabolites in patients with vitamin D-dependency rickets type I. Structural implications for the vitamin D hydroxylases. J Clin Endocrinol Metab 1992;74:814e20. [51] De Braekeleer M, Larochelle J. Population genetics of vitamin D-dependent rickets in northeastern Quebec. Ann Hum Genet 1991;55:283e90.

695

[52] Labuda M, Morgan K, Glorieux FH. Mapping autosomal recessive vitamin D dependency type I to chromosomal 12q14 by linkage analysis. Am J Hum Genet 1990;47:28e36. [53] Labuda M, Labuda D, Korab-Laskowska M, et al. Linkage disequilibrium analysis in young populations: pseudo-vitamin D-deficiency rickets and the founder effect in French Canadians. Am J Hum Genet 1996;59:633e43. [54] Smith SJ, Rucka AK, Berry JL, et al. Novel mutations in the 1a-hydroxylase (P450c1) gene in three families with pseudovitamin D-deficiency rickets resulting in loss of functional enzyme activity in blood-derived macrophages. J Bone Miner Res 1999;14:730e9. [55] Kitanaka S, Murayama A, Sakaki T, et al. No enzyme activity of 25-hydroxyvitamin D3 1a-hydroxylase gene product in pseudovitamin D deficiency rickets, including that with mild clinical manifestation. J Clin Endocrinol Metab 1999;84:4111e7. [56] Wang X, Zhang MYH, Miller WL, Portale AA. Novel gene mutations in patients with 1a-hydroxylase deficiency that confer partial enzyme activity in vitro. J Clin Endocrinol Metab 2002;87:2424e30. [57] Kim CJ, Kaplan LE, Perwad F, et al. Vitamin D 1a-hydroxylase gene mutations in patients with 1a-hydroxylase deficiency. J Clin Endocrinol Metab 2007;92:3177e82. [58] Alzahrani AS, Zou M, Baitei EY, et al. A novel G102E mutation of CYP27B1 in a large family with vitamin D-dependent rickets type 1. J Clin Endocrinol Metab 2010;95:4176e83. [59] Edouard T, Alos N, Chabot G, Roughley P, Glorieux FH, Rauch F. Short- and long-term outcome of patients with pseudo-vitamin D deficiency rickets treated with calcitriol. J Clin Endocrinol Metab 2011;96:82e9. [60] Porcu L, Meloni A, Casula L, et al. A novel splicing defect (IVS6 þ 1G>T) in a patient with pseudovitamin D deficiency rickets. J Endocrinol Invest 2002;25:557e60. [61] Poulos TL, Finzel BC, Howard AJ. High-resolution crystal structure of cytochrome P450cam. J Mol Biol 1987;195:687e700. [62] Hasemann CA, Ravichandran KG, Peterson JA, Deisenhofer J. Crystal structure and refinement of cytochrome P450terp at 2.3 A resolution. J Mol Biol 1994:236. [63] Cupp-Vickery JR, Poulos TL. Structure of cytochrome P450eryF involved in erythromycin biosynthesis. Nat Struct Biol 1995;2:144e53. [64] Hasemann CA, Kurumbail RG, Boddupalli SS, Peterson JA, Deisenhofer J. Structure and function of cytochromes P450: a comparative analysis of three crystal structures. Structure 1995;3:41e62. [65] Fardella CE, Hum DW, Homoki J, Miller WL. Point mutation of Arg440 to His in cytochrome P450c17 causes severe 17ahydroxylase deficiency. J Clin Endocrinol Metab 1994;79:160e4. [66] Conte FA, Grumbach MM, Ito Y, Fisher CR, Simpson ER. A syndrome of female pseudohermaphrodism, hypergonadotropic hypogonadism, and multicystic ovaries associated with missense mutations in the gene encoding aromatase (P450arom). J Clin Endocrinol Metab 1994;78:1287e92. [67] Auchus RJ, Miller WL. Molecular modeling of human P450c17 (17a-hydroxylase/17,20-lyase): insights into reaction mechanisms and effects of mutations. Mol Endocrinol 1999;13:1169e82. [68] Sawada N, Sakaki T, Kitanaka S, Kato S, Inouye K. Structurefunction analysis of CYP27B1 and CYP27A1. Studies on mutants from patients with vitamin D-dependent rickets type I (VDDR-I) and cerebrotendinous xanthomatosis (CTX). Eur J Biochem 2001:268. [69] Reade TM, Scriver CR, Glorieux FH, et al. Response to crystalline 1a-hydroxyvitamin D3 in vitamin D dependency. Pediatr Res 1975;9:593e9.

PEDIATRIC BONE

696

25. RICKETS DUE TO HEREDITARY ABNORMALITIES OF VITAMIN D SYNTHESIS OR ACTION

[70] Clifton-Bligh RJ, Nguyen TV, Au A, et al. Contribution of a common variant in the promoter of the 1-a-hydroxylase gene (CYP27B1) to fracture risk in the elderly. Calcif Tissue Int 2011;88:109e16. [71] Brooks MH, Stern PH, Bell NH. Vitamin D-dependent rickets type II. N Engl J Med 1980;302:810. [72] Marx SJ, Spiegel AM, Brown EM, et al. A familial syndrome of decrease in sensitivity to 1,25-dihydroxyvitamin D. J Clin Endocrinol Metab 1978;47:1303e10. [73] Rosen JF, Fleishman AR, Finberg L, Hamstra A, DeLuca HF. Rickets with alopecia: an inborn error of vitamin D metabolism. J Pediatr 1979;94:729e35. [74] Malloy PJ, Pike JW, Feldman D. The vitamin D receptor and the syndrome of hereditary 1,25-dihydroxyvitamin D-resistant rickets. Endocr Rev 1999;20:156e88. [75] Beer S, Tieder M, Kohelet D, et al. Vitamin D resistant rickets with alopecia: A form of end organ resistance to 1,25-dihydroxyvitamin D. Clin Endocrinol 1981;14:395e402. [76] Hochberg Z, Benderli A, Levy J, et al. 1,25-dihydroxyvitamin D resistance, rickets, and alopecia. Am J Med 1984;77:805e11. [77] Hochberg Z, Gilhar A, Haim S, Friedman-Birnbaum R, Levy J, Benderly A. Calcitriol-resistant rickets with alopecia. Arch Dermatol 1985;121:646e7. [78] Marx SJ, Bliziotes MM, Nanes M. Analysis of the relation between alopecia and resistance to 1,25-dihydroxyvitamin D. Clin Endocrinol (Oxf) 1986;25:373e81. [79] Liberman UA, Halabe A, Samuel R, et al. End-organ resistance to 1,25-dihydroxycholecalciferol. Lancet 1980:504e7. [80] Yagi H, Ozono K, Miyake H, Nagashima K, Kuroume T, Pike JW. A new point mutation in the deoxyribonucleic acidbinding domain of the vitamin D receptor in a kindred with hereditary 1,25-dihydroxyvitamin D-resistant rickets. J Clin Endocrinol Metab 1993;76:509e12. [81] Hirst MA, Hochman HI, Feldman D. Vitamin D resistance and alopecia: a kindred with normal 1,25-dihydroxyvitamin D binding, but decreased receptor affinity for deoxyribonucleic acid. J Clin Endocrinol Metab 1985;60:490e5. [82] Takeda E, Kuroda Y, Saijo T, et al. 1a-hydroxyvitamin D3 treatment of three patients with 1,25-dihydroxyvitamin Dreceptor-defect rickets and alopecia. Pediatrics 1987;80:97e101. [83] Haussler MR, Whitfield GK, Haussler CA, et al. The nuclear vitamin D receptor: biological and molecular regulatory properties revealed. J Bone Miner Res 1998;13:325e49. [84] Jurutka PW, Hsieh JC, Remus LS, et al. Mutations in the 1,25dihydroxyvitamin D3 receptor identifying C-terminal amino acids required for transcriptional activation that are functionally dissociated from hormone binding, heterodimeric DNA binding, and interaction with basal transcription factor IIB, in vitro. J Biol Chem 1997;272:14592e9. [85] Masuyama H, Brownfield CM, St-Arnaud R, MacDonald PN. Evidence for ligand-dependent intramolecular folding of the AF-2 domain in vitamin D receptor-activated transcription and coactivator interaction. Mol Endocrinol 1997;11:1507e17. [86] Thompson PD, Remus LS, Hsieh JC, et al. Distinct retinoid X receptor activation function-2 residues mediate transactivation in homodimeric and vitamin D receptor heterodimeric contexts. J Mol Endocrinol 2001;27:211e27. [87] Nakajima S, Hsieh JC, MacDonald PN, et al. The C-terminal region of the vitamin D receptor is essential to form a complex with a receptor auxiliary factor required for high affinity binding to the vitamin D-responsive element. Mol Endocrinol 1994;8:159e72. [88] Rachez C, Lemon BD, Suldan Z, et al. Ligand-dependent transcription activation by nuclear receptors requires the DRIP complex. Nature 1999;398:824e8.

[89] Eil C, Liberman UA, Rosen JF, Marx SJ. A cellular defect in hereditary vitamin-D dependent rickets type II: Defective nuclear uptake of 1,25-dihydroxyvitamin D in cultured skin fibroblasts. N Engl J Med 1981;304:1588e91. [90] Feldman D, Chen T, Cone C, et al. Vitamin D resistant rickets with alopecia: Cultured skin fibroblasts exhibit defective cytoplasmic receptors and unresponsiveness to 1,25(OH)2D3. J Clin Endocrinol Metab 1982;55:1020e2. [91] Griffin JE, Zerwekh JE. Impaired stimulation of 25-hydroxyvitamin D-24-hydroxylase in fibroblasts from a patient with vitamin D-dependent rickets, type II. A form of receptorpositive resistance to 1,25-dihydroxyvitamin D3. J Clin Invest 1983;72:1190e9. [92] Clemens TL, Adams JS, Horiuchi N, et al. Interaction of 1,25dihydroxyvitamin-D3 with keratinocytes and fibroblasts from skin of normal subjects and a subject with vitamin-D-dependent rickets, type II: a model for study of the mode of action of 1,25dihydroxyvitamin D3. J Clin Endocrinol Metab 1983;56:824e30. [93] Balsan S, Garabedian M, Liberman UA, et al. Rickets and alopecia with resistance to 1,25-dihydroxyvitamin D: two different clinical courses with two different cellular defects. J Clin Endocrinol Metab 1983;57:803e11. [94] Liberman UA, Eil C, Marx SJ. Resistance to 1,25-dihydroxyvitamin D. Association with heterogeneous defects in cultured skin fibroblasts. J Clin Invest 1983;71:192e200. [95] Chen TL, Hirst MA, Cone CM, Hochberg T, Tietze HU, Feldman D. 1,25-dihydroxyvitamin D resistance, rickets, and alopecia: Analysis of receptors and bioresponse in cultured fibroblasts from patients and parents. J Clin Endocrinol Metab 1984;59:383e8. [96] Liberman UA, Eil C, Holst P, Rosen JF, Marx SJ. Hereditary resistance to 1,25-dihydroxyvitamin D: Defective function of receptors for 1,25-dihydroxyvitamin D in cells cultured from bone. J Clin Endocrinol Metab 1983;57:958e62. [97] Dokoh S, Haussler MR, Pike JW. Development of a radioligand immunoassay for 1,25-dihydroxycholecalciferol receptors utilizing monoclonal antibody. Biochem J 1984;221:129e36. [98] Pike JW, Donaldson CA, Marion SL, Haussler MR. Development of hybridomas secreting monoclonal antibodies to the chicken intestinal 1 a,25-dihydroxyvitamin D3 receptor. Proc Natl Acad Sci USA 1982;79:7719e23. [99] Pike JW, Dokoh S, Haussler MR, Liberman UA, Marx SJ, Eil C. Vitamin D3-resistant fibroblasts have immunoassayable 1,25dihydroxyvitamin D3 receptors. Science 1984;224:879e81. [100] Pike JW. Monoclonal antibodies to chick intestinal receptors for 1,25-dihydroxyvitamin D3. Interaction and effects of binding on receptor function. J Biol Chem 1984;259:1167e73. [101] Liberman UA, Eil C, Marx SJ. Receptor-positive hereditary resistance to 1,25-dihydroxyvitamin D: chromatography of hormone-receptor complexes on deoxyribonucleic acid-cellulose shows two classes of mutation. J Clin Endocrinol Metab 1986;62:122e6. [102] Gamblin GT, Liberman UA, Eil C, Downs RWJ, DeGrange DA, Marx SJ. Vitamin D-dependent rickets type II. Defective induction of 25-hydroxyvitamin D3-24-hydroxylase by 1,25dihydroxyvitamin D3 in cultured skin fibroblasts. J Clin Invest 1985;75:954e60. [103] Malloy PJ, Hochberg Z, Pike JW, Feldman D. Abnormal binding of vitamin D receptors to deoxyribonucleic acid in a kindred with vitamin D-dependent rickets, type II. J Clin Endocrinol Metab 1989;68:263e9. [104] McDonnell DP, Mangelsdorf DJ, Wesley Pike JW, Haussler MR, O’Malley BW. Molecular cloning of complementary DNA encoding the avian receptor for vitamin D. Science 1987;235:1214e7.

PEDIATRIC BONE

697

REFERENCES

[105] Baker AR, McDonnell DP, Hughes M, et al. Cloning and expression of full-length cDNA encoding human vitamin D receptor. Proc Natl Acad Sci USA 1988;85:3294e8. [106] Szpirer J, Szpirer C, Riviere M, et al. The Sp1 transcription factor gene (SP1) and the 1,25-dihydroxyvitamin D3 receptor gene (VDR) are colocalized on human chromosome arm 12q and rat chromosome 7. Genomics 1991;11:168e73. [107] Hughes MR, Malloy PJ, Kieback DG, et al. Point mutations in the human vitamin D receptor gene associated with hypocalcemic rickets. Science 1988;242:1702e5. [108] Sone T, Scott RA, Hughes MR, et al. Mutant vitamin D receptors which confer hereditary resistance to 1,25-dihydroxyvitamin D3 in humans are transcriptionally inactive in vitro. J Biol Chem 1989;264:20230e4. [109] Malloy PJ, Wang J, Srivastava T, Feldman D. Hereditary 1,25dihydroxyvitamin D-resistant rickets with alopecia resulting from a novel missense mutation in the DNA-binding domain of the vitamin D receptor. Mol Genet Metab 2010;99:72e9. [110] Mechica JB, Leite MO, Mendonca BB, Frazzatto ES, Borelli A, Latronico AC. A novel nonsense mutation in the first zinc finger of the vitamin D receptor causing hereditary 1,25-dihydroxyvitamin D3-resistant rickets. J Clin Endocrinol Metab 1997;82:3892e4. [111] Zhu W, Malloy PJ, Delvin E, Chabot G, Feldman D. Hereditary 1,25-dihydroxyvitamin D-resistant rickets due to an opal mutation causing premature termination of the vitamin D receptor. J Bone Miner Res 1998;13:259e64. [112] Rut AR, Hewison M, Kristjansson K, Luisi B, Hughes MR, O’Riordan JL. Two mutations causing vitamin D resistant rickets: modelling on the basis of steroid hormone receptor DNA-binding domain crystal structures. Clin Endocrinol (Oxf) 1994;41:581e90. [113] Lin NU, Malloy PJ, Sakati N, al-Ashwal A, Feldman D. A novel mutation in the deoxyribonucleic acid-binding domain of the vitamin D receptor causes hereditary 1,25-dihydroxyvitamin Dresistant rickets. J Clin Endocrinol Metab 1996;81:2564e9. [114] Saijo T, Ito M, Takeda E, et al. A unique mutation in the vitamin D receptor gene in three Japanese patients with vitamin Ddependent rickets type II: utility of single-strand conformation polymorphism analysis for heterozygous carrier detection. Am J Hum Genet 1991;49:668e73. [115] Forghani N, Lum C, Krishnan S, et al. Two new unrelated cases of hereditary 1,25-dihydroxyvitamin D-resistant rickets with alopecia resulting from the same novel nonsense mutation in the vitamin D receptor gene. J Pediatr Endocrinol Metab 2010;23:843e50. [116] Cockerill FJ, Hawa NS, Yousaf N, Hewison M, O’Riordan JL, Farrow SM. Mutations in the vitamin D receptor gene in three kindreds associated with hereditary vitamin D resistant rickets. J Clin Endocrinol Metab 1997;82:3156e60. [117] Wiese RJ, Goto H, Prahl JM, et al. Vitamin D-dependency rickets type II: truncated vitamin D receptor in three kindreds. Mol Cell Endocrinol 1993;90:197e201. [118] Macedo LC, Soardi FC, Ananias N, et al. Mutations in the vitamin D receptor gene in four patients with hereditary 1,25dihydroxyvitamin D-resistant rickets. Arq Bras. Endocrinol Metabol 2008;52:1244e51. [119] Asunis I, Marini MG, Porcu L, et al. A novel missense mutation (C84R) in a patient with type II vitamin D-dependent rickets. Exp Clin Endocrinol Diabetes 2010;118:177e9. [120] Hawa NS, Cockerill FJ, Vadher S, et al. Identification of a novel mutation in hereditary vitamin D resistant rickets causing exon skipping. Clin Endocrinol (Oxf) 1996;45:85e92. [121] Miller J, Djabali K, Chen T, et al. Atrichia caused by mutations in the vitamin D receptor gene is a phenocopy of generalized

[122]

[123]

[124]

[125]

[126]

[127]

[128]

[129]

[130]

[131]

[132]

[133]

[134]

[135]

atrichia caused by mutations in the hairless gene. J Invest Dermatol 2001;117:612e7. Malloy PJ, Xu R, Cattani A, Reyes L, Feldman D. A unique insertion/substitution in helix H1 of the vitamin D receptor ligand binding domain in a patient with hereditary 1,25-dihydroxyvitamin D-resistant rickets. J Bone Miner Res 2004;19:1018e24. Thompson E, Kristjansson K, Hughes M. Molecular scanning methods for mutation detection: application to the 1,25-dihydroxyvitamin D receptor. In: Norman AW, Bouillon R, Thomasset M, editors. Vitamin D Receptor Mutations and Hereditary 1,25-dihydroxyvitamin D Resistant Rickets. New York: Walter de Gruyter; 1991. p. 6 (abstract). Katavetin P, Katavetin P, Wacharasindhu S, Shotelersuk V. A girl with a novel splice site mutation in VDR supports the role of a ligand-independent VDR function on hair cycling. Horm Res 2006;66:273e6. Kanakamani J, Tomar N, Kaushal E, Tandon N, Goswami R. Presence of a deletion mutation (c.716delA) in the ligand binding domain of the vitamin D receptor in an Indian patient with vitamin D-dependent rickets type II. Calcif Tissue Int 2010;86:33e41. Zhou Y, Wang J, Malloy PJ, Dolezel Z, Feldman D. Compound heterozygous mutations in the vitamin D receptor in a patient with hereditary 1,25-dihydroxyvitamin D-resistant rickets with alopecia. J Bone Miner Res 2009;24:643e51. Nguyen M, d’Alesio A, Pascussi JM, et al. Vitamin D-resistant rickets and type 1 diabetes in a child with compound heterozygous mutations of the vitamin D receptor (L263R and R391S): dissociated responses of the CYP-24 and rel-B promoters to 1,25-dihydroxyvitamin D3. J Bone Miner Res 2006;21:886e94. Malloy PJ, Xu R, Peng L, Peleg S, al-Ashwal A, Feldman D. Hereditary 1,25-dihydroxyvitamin D resistant rickets due to a mutation causing multiple defects in vitamin D receptor function. Endocrinology 2004;145:5106e14. Nguyen TM, Adiceam P, Kottler ML, et al. Tryptophan missense mutation in the ligand-binding domain of the vitamin D receptor causes severe resistance to 1,25-dihydroxyvitamin D. J Bone Miner Res 2002;17:1728e37. Ritchie HH, Hughes MR, Thompson ET, et al. An ochre mutation in the vitamin D receptor gene causes hereditary 1,25dihydroxyvitamin D3-resistant rickets in three families. Proc Natl Acad Sci USA 1989;86:9783e7. Malloy PJ, Hughes MR, Pike JW, Feldman D. In: Norman AW, Bouillon R, Thomasset M, editors. Vitamin D Receptor Mutations and Hereditary 1,25-dihydroxyvitamin D Resistant Rickets. New York: Walter de Gruyter; 1991. p. 116e24. Malloy PJ, Wang J, Peng L, et al. A unique insertion/duplication in the VDR gene that truncates the VDR causing hereditary 1,25-dihydroxyvitamin D-resistant rickets without alopecia. Arch Biochem Biophys 2007;460:285e92. Whitfield GK, Selznick SH, Haussler CA, et al. 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 1996;10:1617e31. Malloy PJ, Zhu W, Bouillon R, Feldman D. A novel nonsense mutation in the vitamin D receptor causes hereditary 1,25dihydroxyvitamin D-resistant rickets. Mol Genet Metab 2002;77:314e8. Ma NS, Malloy PJ, Pitukcheewanont P, Dreimane D, Geffner ME, Feldman D. Hereditary vitamin D resistant rickets: identification of a novel splice site mutation in the vitamin D

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[136]

[137]

[138]

[139]

[140]

[141]

[142]

[143]

[144]

[145]

[146]

[147]

25. RICKETS DUE TO HEREDITARY ABNORMALITIES OF VITAMIN D SYNTHESIS OR ACTION

receptor gene and successful treatment with oral calcium therapy. Bone 2009;45:743e6. Arita K, Nanda A, Wessagowit V, Akiyama M, Alsaleh QA, McGrath JA. A novel mutation in the VDR gene in hereditary vitamin D-resistant rickets. Br J Dermatol 2008;158:168e71. Malloy PJ, Xu R, Peng L, Clark PA, Feldman D. A novel mutation in helix 12 of the VDR impairs coactivator interaction and causes hereditary 1,25-dihydroxyvitamin D-resistant rickets without alopecia. Mol Endocrinol 2002;16:2538e46. Malloy PJ, Weisman Y, Feldman D. Hereditary 1a,25-dihydroxyvitamin D-resistant rickets resulting from a mutation in the vitamin D receptor deoxyribonucleic acid-binding domain. J Clin Endocrinol Metab 1994;78:313e6. Luisi BF, Xu WX, Otwinowski Z, Freedman LP, Yamamoto KR, Sigler PB. Crystallographic analysis of the interaction of the glucocorticoid receptor with DNA. Nature 1991;352:497e505. Rastinejad F, Perlmann T, Evans RM, Sigler PB. Structural determinants of nuclear receptor assembly on DNA direct repeats. Nature 1995;375:203e11. Malloy PJ, Hochberg Z, Tiosano D, Pike JW, Hughes MR, Feldman D. The molecular basis of hereditary 1,25-dihydroxyvitamin D3 resistant rickets in seven related families. J Clin Invest 1990;86:2071e9. Kristjansson K, Rut AR, Hewison M, O’Riordan JL, Hughes MR. Two mutations in the hormone binding domain of the vitamin D receptor cause tissue resistance to 1,25 dihydroxyvitamin D3. J Clin Invest 1993;92:12e6. Malloy PJ, Eccleshall TR, Gross C, Van Maldergem L, Bouillon R, Feldman D. 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 1997;99:297e304. Malloy PJ, Zhu W, Zhao XY, Pehling GB, Feldman D. A novel inborn error in the ligand-binding domain of the vitamin D receptor causes hereditary vitamin D-resistant rickets. Mol Genet Metab 2001;73:138e48. Tocchini-Valentini G, Rochel N, Wurtz JM, Mitschler A, Moras D. Crystal structures of the vitamin D receptor complexed to superagonist 20-epi ligands. Proc Natl Acad Sci USA 2001;98:5491e6. Hewison M, Rut AR, Kristjansson K, et al. Tissue resistance to 1,25-dihydroxyvitamin D without a mutation of the vitamin D receptor gene. Clin Endocrinol (Oxf) 1993;39:663e70. Li YC, Pirro AE, Amling M, et al. Targeted ablation of the vitamin D receptor: an animal model of vitamin D-dependent

[148]

[149]

[150]

[151]

[152]

[153] [154]

[155]

[156] [157]

[158]

[159]

rickets type II with alopecia. Proc Natl Acad Sci USA 1997;94:9831e5. Yoshizawa T, Handa Y, Uematsu Y, et al. Mice lacking the vitamin D receptor exhibit impaired bone formation, uterine hypoplasia and growth retardation after weaning. Nat Genet 1997;16:391e6. Chen CH, Sakai Y, Demay MB. Targeting expression of the human vitamin D receptor to the keratinocytes of vitamin D receptor null mice prevents alopecia. Endocrinology 2001;142:5386e9. Hsieh JC, Slater SA, Whitfield GK, et al. Analysis of hairless corepressor mutants to characterize molecular cooperation with the vitamin D receptor in promoting the mammalian hair cycle. J Cell Biochem 2010;110:671e86. Cianferotti L, Cox M, Skorija K, Demay MB. Vitamin D receptor is essential for normal keratinocyte stem cell function. Proc Natl Acad Sci USA 2007;104:9428e33. Teichert A, Elalieh H, Bikle D. Disruption of the hedgehog signaling pathway contributes to the hair follicle cycling deficiency in Vdr knockout mice. J Cell Physiol 2010;225:482e9. Bikle DD. Vitamin D and the skin. J Bone Miner Metab 2010;28:117e30. Zerwekh JE, Glass K, Jowsey J, Pak CYC. A unique form of osteomalacia associated with end organ refractoriness to 1,25dihydroxyvitamin D and apparent defective synthesis of 25hydroxyvitamin D. J Clin Endocrinol Metab 1979;49:171e5. Balsan S, Garabedian M, Larchet M, et al. Long-term nocturnal calcium infusions can cure rickets and promote normal mineralization in hereditary resistance to 1,25-dihydroxyvitamin D. J Clin Invest 1986;77:1661e7. Hochberg Z, Tiosano D, Even L. Calcium therapy for calcitriolresistant rickets. J Pediatr 1992;121:803e8. Kudoh T, Kumagai T, Uetsuji N, et al. Vitamin D dependent rickets: decreased sensitivity to 1,25-dihydroxyvitamin D. Eur J Pediatr 1981;137:307e11. Castells S, Greig F, Fusi MA, et al. 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 1986;63:252e6. Kruse K, Feldmann E. Healing of rickets during vitamin D therapy despite defective vitamin D receptors in two siblings with vitamin D-dependent rickets type II. J Pediatr 1995;126:145e8.

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Familial Hypophosphatemia and Related Disorders Ingrid A. Holm 1, Michael J. Econs 2, Thomas O. Carpenter 3 1

Harvard Medical School, Boston, Massachusetts, USA 2 Indiana University School of Medicine, Indianapolis, Indiana, USA 3 Yale University School of Medicine, New Haven, Connecticut, USA

INTRODUCTION The precise physiologic basis of the hypophosphatemic disorders has eluded clinicians and scientists for years. Key discoveries in the past two decades have led to several significant advances in the understanding of these disorders, particularly with the identification of PHEX as the mutated gene in X-linked hypophosphatemia (XLH), and the recognition that a novel member of the fibroblast growth factor (FGF) family, FGF-23, is central to the mediation of the renal abnormalities of phosphate reclamation and altered vitamin D metabolism in these disorders. Nevertheless, a number of the clinical observations in XLH remain inadequately explained, and the elucidation of the relevant pathophysiology will be critical in developing successful therapies to manage the significant clinical complications of these disorders. Indeed, XLH is one of the most common bone diseases seen in children, with available therapies that only partially address the long-term morbidity of the condition. These suboptimal therapeutic approaches require frequent biochemical monitoring, and the panoply of chronic features that complicate the condition persist throughout life. This chapter provides a detailed 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. The biochemistry of FGF-23 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),

Pediatric Bone, Second Edition DOI: 10.1016/B978-0-12-382040-2.10026-7

and tumor-induced osteomalacia (TIO) are compared to those observed in XLH. Current strategies of management and therapeutics are reviewed as well as their long-term complications.

REGULATION OF PHOSPHATE HOMEOSTASIS In healthy adults, 65e75% of ingested phosphate is absorbed in the intestine, independent of the amount of phosphate ingested [1]. Thus, as dietary phosphate content increases, the amount of absorbed phosphate increases. 1,25(OH)2D promotes intestinal phosphate absorption, although the magnitude of the 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 [1]. 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 15e20% in the proximal straight tubule [1]. Phosphate transport in the renal tubules occurs via the type 2 sodiumephosphate (NaPi-II)-dependent cotransporters NaPi-IIa and NaPi-IIc, in the brush border membrane of the luminal surface [2]. Active transport of sodium and phosphate into the cell from the lumen of the tubule via these co-transporters is driven by the downhill sodium gradient into the cell. This gradient is maintained by a Na/K-ATPase pump at the

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basolateral surface of the cell, which pumps Na out of the cell. Phosphate efflux from the cell is poorly understood, but thought to occur passively at the basolateral surface, driven by an anion-exchange pump [1]. There is also a NaePi co-transporter 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 [1]. The abundance of NaPi-IIa and NaPi-IIc at the brush border membrane determines the capacity of the proximal tubule to reabsorb phosphate (see Bastepe [3] and Prie [4] for review). The most physiologically important regulators of phosphate transport in the proximal tubule are parathyroid hormone (PTH) and FGF-23. PTH acutely stimulates the translocation of NaPi-II transporters from the brush border membrane. Membrane sorting of NaPi-IIa requires its interaction with sodiumehydrogen exchanger regulatory factor 1 (NHERF1); inactivation of NHERF1 results in this removal of NaPi-IIa from the brush border [5,6]. In contrast, FGF-23 appears to act by downregulating expression of the genes encoding NaPi-IIa and NaPi-IIc (SLC34A1 and SLC34A3). Other 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)2D stimulate tubular reabsorption of phosphate. PTH-related protein (PTHrP), calcitonin, atrial natriuretic factor (ANF), transforming growth factor-a (TGF-a), TGF-b, and glucocorticoids inhibit these renal transport mechanisms [7,8]. All these hormonal factors directly or indirectly affect the availability or abundance of the high-affinity, low-capacity class of NaPi-II cotransporters [1]. In sum, regulation of total body phosphate homeostasis primarily occurs at the level of the kidney.

CLINICAL DISORDERS OF PHOSPHATE HOMEOSTASIS XLH XLH is characterized by renal phosphate wasting leading to hypophosphatemia and low or normal concentrations of 1,25-dihydroxyvitamin D (1,25(OH)2D), 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 [9]. XLH is the most common inherited form of rickets in the USA and the most common inherited defect in renal

tubular phosphate transport. The incidence has been estimated at 1 in 20 000 in England [10]. There is a great deal of variability in the manifestations of XLH. In the mildest cases, only hypophosphatemia is evident. In more severe disease, 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, as the lack of a family history usually results in a delayed diagnosis. Biochemical findings in the serum of XLH patients that differentiate XLH from other forms 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 PTH levels. Clinical Manifestations of XLH SKELETAL FINDINGS

In children, XLH is characterized by rickets, although this is not an invariant feature of the disease [11]. The skeletal manifestations are most severe in the lower extremities, and bowing (genu varum) or a knock-knee (genu valgum) deformity is common. Radiographic findings are typical of rickets, with flaring, fraying, and cupping of the ends of the metaphyses of the femur and tibia (Fig. 26.1). Although the upper extremities may be affected with similar changes, findings at the distal radius or ulna are usually not as striking as those in the lower extremities. Corrective surgical osteotomy is often required to correct the severe bow deformity. In addition, tibial torsion may complicate the bowing defect, and a rotational osteotomy is occasionally required when correction of the torsion does not reverse with medical therapy. In adults, osteomalacia, bone pain, arthritis, stiffness, and enthesopathy are all frequently encountered and may be quite severe and progressive. 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 [12e16]. These bone density changes often persist into adulthood [13,15], and are not usually affected by standard therapy for the disease [16]. Decreased joint mobility is seen in most adults with XLH [17]. Despite corrective osteotomies as children, many adults will continue to have an abnormal leg axis [17]. 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 [18]. Bone and joint pain, pseudofractures, and enthesopathy (proliferation of bone at sites of attachment of ligaments) are common findings [9,18,19].

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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 (SD) [9,24]. The mean final adult height in females is 150e152.4 cm and in males it is 157.3e158 cm [9,17]. Upper segment length is more normal (
0.91 SD) compared to lower segment length (
2.59 SD) [25]. The disease appears to have a greater impact on height in early childhood; children remain approximately 2 SD below the mean for height after 5 years of age and have a normal pubertal growth spurt [26]. Conventional treatment has a significant positive impact on final adult height [27]. However, some reports refute this finding [28], and the response to therapy can be variable. DENTAL FINDINGS

FIGURE 26.1 Radiograph of 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.

Osteomalacia is the primary bone histomorphometric finding in both adults and children with XLH (Fig. 26.2). Osteoid volume is increased, mineralization lag time is prolonged, and rates of bone formation are decreased [9,20,21]. However, despite the osteomalacia, the trabecular calcified bone volume is normal or increased, suggesting an imbalance between the rates of bone resorption and formation [20]. The formation rate of new bone remodeling units is decreased, but the formation component of the turnover cycle is prolonged [21]. These findings suggest that abnormalities of the osteoblasts contribute to osteomalacia [21]. Recent evidence in Hyp mice indicate that a fragment (ASARM, or acidic serine and aspartate rich MEPE-associated motif) matrix glycoprotein, MEPE, is elevated in Hyp mice, and can serve as a direct inhibitor of mineralization [22]. In adults, the degree of osteomalacia, as measured by the osteoid volume on bone histomorphometry, is positively correlated with the degree of bone pain [23].

Dental abscesses are common in children with XLH and start in early childhood. Twenty-five percent of patients developed abscesses of the primary dentition [29]. Dental problems continue into adulthood [9,17], during which more than 85% of patients have reported dental problems, many of whom required dental clearance [9]. Individuals who develop one abscess go on to develop multiple abscesses, indicating that the development of one abscess predicts future abscesses [29]. The primary tooth defect in XLH is in the dentin, whereas the enamel is relatively normal [30,31]. In normal teeth, calciumephosphate calcospheres form and coalesce, forming the dentin matrix [1]. In XLH, the dentin is undermineralized and characterized by calcospherites that do not coalesce normally and thus are

FIGURE 26.2 Biopsy of adult XLH osteomalacia. Goldner-stained undecalcified sections of iliac crest bone from an adult with XLH (magnification, 360). Note the excess osteoid accumulation with relatively normal abundance of mineralized bone. (See color plate section.)

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separated by large amounts of interglobular dentin [31,32]. 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 [31,33]. Since the enamel barrier is weakened, microorganisms can penetrate [31], possibly through microclefts [31] or infractures [33]. Microorganisms pass through the dentinoenamel junction, invading the dentin [31]. Dental taurodontism is seen in males [33,34]. The globular nature of the dentin and undermineralization does not appear to be impacted by therapy [35]. As a result, the impact of therapy on the dental manifestations of XLH, including the occurrence of abscesses, is not significant [29,36]. Other dental findings include a normal rate of dental development and an increased prevalence of ectopic permanent canine teeth in males with XLH [34]. OTHER MANIFESTATIONS

Ossification of the posterior longitudinal ligament leading to cervical myelopathy [37], ossification of the ligamentum flavum [38], spinal cord compression [39,40], and spinal canal stenosis [41,42] have all been reported in XLH. Chiari I malformation has also been described [43]. Hearing loss is a common finding in XLH [44e47] and may be more common in adults [46]. Cardiovascular problems, including hypertension and left ventricular hypertrophy, have been described in XLH patients, all of whom also had nephrocalcinosis [48]. The relationship to the disease and/or treatment is unclear [48]. Although psychological problems are common in XLH [17], 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. Biochemical Findings The biochemical findings in serum that characterize XLH include low phosphorus, normal calcium, inappropriately normal 1,25(OH)2D, normal 25-hydroxyvitamin D [25(OH)D], elevated alkaline phosphatase, and a normal or slightly elevated PTH [49]. 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, when assessing serum phosphorus levels it is critical to know the normal range at the given age. There have been many instances when hypophosphatemia was missed because a low level was mistakenly considered normal because the clinician was not aware of this fact [50]. Other causes of hypophosphatemia include dietary

phosphate deficiency which is extremely rare as most diets are rich in phosphate. Another common cause of hypophosphatemia, particularly in the hospital setting, is due to extracellulareintracellular shifts of phosphate associated with refeeding the malnourished individual, or in the correction of diabetic ketoacidosis. In order to determine whether hypophosphatemia is secondary to poor intake or renal phosphate wasting, measurement of urinary phosphate excretion is critical. We collect a 2-hour urine sample after at least a 4-hour fast, with a serum sample obtained in the midpoint of the collection [51]. Phosphorus and creatinine are measured in the serum and urine. The tubular reabsorption of phosphate (TRP), which is the fraction of excreted phosphate that is reabsorbed by the kidney, can then be determined as follows [52]: TRP ¼ 1
(urine phosphorus concentration  serum creatinine concentration)/(serum phosphorus concentration  urine creatinine concentration).

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 derived using a nomogram developed by Walton and Bijvoet [53,54] (Fig. 26.3). The normal range for TMP/GFR in adults is 2.5e4.2 mg/dL [53,54]; it is higher in children. The normal range for TMP/GFR in children is approximately the same as the normal range for serum phosphorus [51]. Once urinary phosphate wasting is established using such measures, the possibility of other solute losses in the urine should be considered, as generalized renal tubular dysfunction with accompanying phosphate losses may occur in several rare disorders that can lead to Fanconi syndrome, phosphate deficiency, and rickets. These disorders include cystinosis, Lowe syndrome, tyrosinemia type I, and certain drugs (such as ifosfamide), heavy metals, and other toxins [55]. Wasting of glucose, amino acids, and bicarbonate often occurs in these disorders. XLH is the most common cause of isolated renal phosphate wasting leading to rickets. Coincident inappropriately normal 1,25(OH)2D levels and the lack of significant renal losses of other minerals distinguish XLH from most other forms of phosphate wasting, although mild and intermittent renal glycosuria has been described in XLH. 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

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PTH

Although PTH is usually elevated in XLH as a result of treatment with phosphate, PTH can be mildly elevated at presentation [49]. Occasionally, patients undergoing treatment of XLH subsequently develop autonomous parathyroid hyperplasia requiring parathyroidectomy. Unlike the near universal observation of elevated serum PTH levels in the setting of vitamin D-deficiency rickets, marked hyperparathyroidism is less commonly a prominent feature of XLH.

FIGURE 26.3 Nomogram to calculate the TMP/GFR from the TRP

[TRP ¼ 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].)

Long-Term Sequelae Long-term sequelae of XLH in adults, some of which develop in later life, include osteomalacia, pseudofractures, skeletal pain, dental abscesses, arthritis, spinous ligament calcification, and calcification of tendons and ligaments (enthesopathy). These complications can be severe and very debilitating. Spinal stenosis may occur, potentially resulting in severe pain or other neurologic symptoms. Many older affected individuals have undergone multiple root canal procedures and may have lost most of their teeth. Medical treatment in adulthood has been shown to improve osteomalacia and, in some instances, associated skeletal pain, but it is unclear as to whether treatment impacts any of these other longterm sequelae of XLH. Genetics IDENTIFICATION OF THE GENE DEFECT IN XLH

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 1a-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, and is due to elevated FGF-23 levels (see below).

Efforts to understand the defect in XLH led to the localization of the XLH locus to Xp22.1 by linkage analysis in some families [56e67]. PEX, now termed PHEX (phosphate regulating gene with homologies to endopeptidases, on the X chromosome), was identified as the gene defective in XLH using positional cloning techniques [68]. The name was derived from the homology of PHEX to a family of neutral endopeptidases, including neprilysin (NEP) [69], endothelin converting enzyme-1 (ECE-1) [70], and the Kell antigen [71]. The full-length PHEX cDNA [72,73] consists of a 2247-bp coding region spanning 22 exons [74]. The full-length PHEX is 60% similar to NEP and 57% similar to ECE-1 at the amino acid level [73].

ALKALINE PHOSPHATASE

STUDIES OF PHEX IN FAMILIES WITH XLH

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.

The identification of PHEX made it possible to perform mutational analysis in XLH patients [74e83] and to distinguish individuals with XLH from those who may have ADHR. In these studies, PHEX mutations were identified in 60e95% of familial hypophosphatemic rickets (FHR) patients. Although most of the mutations identified are in the coding region, mutations in the non-coding regions have also been identified [84]. In most studies, PHEX mutations were

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detected in a smaller percentage of sporadic cases (28e93%) compared to familial cases (71e100%) [74,75,80,81]. 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 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 [85]. Although a variety of mutations has been detected, suggesting that some of the variability in XLH may be explained by the variety of PHEX mutations, our phenotypeegenotype analysis of PHEX in XLH has disputed this hypothesis [76]. There is now a database of PHEX mutations, phenotypes, and authors (http:// data.mch.mcgill.ca/phexdb) [86]. XLH IS AN X-LINKED DOMINANT DISORDER

XLH is inherited in an X-linked dominant fashion [87], characterized by lack of male-to-male transmission and a female-to-male prevalence ratio of approximately 2 to 1. Unlike many X-linked dominant disorders, however, the presence of a gene dosage effect is not overtly evident in XLH. One would expect a hemizygous male (who carries only one X chromosome and thus has only one defective gene and no normal 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 dosage effect [1]. In practice, however, it has been very difficult to prove even a minor gene dosage effect in humans [88]. Males and females have similar degrees of hypophosphatemia [89,90] indicating lack of a gene dosage effect on serum phosphate levels. In one study [88], biochemical parameters, including serum phosphate levels and height z scores, were similar between untreated prepubertal girls (heterozygous) and boys (hemizygous). Although there does not seem to be a gene dosage effect in untreated prepubertal children, there is evidence suggesting a gene dosage effect with treatment and in adults. Girls appear to respond better to calcitriol and phosphate therapy compared to boys [91]. In addition, radiographic and bone scan findings are less severe in adult women compared to men [92] 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 [9]. 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 [90], and males are more likely to be affected with dental abscesses [29]. 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) [89]. 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 [93]. This does not rule out the possibility that the X inactivation pattern in the bone, teeth, and/or kidney is not random. Another explanation could be that the PHEX gene escapes X inactivation and has a homolog on the Y chromosome; however, there is no evidence for a Y homolog. FAMILIAL VERSUS SPORADIC CASES

In most studies of individuals with XLH, approximately one-third of cases are sporadic. There are no differences in clinical severity between familial and sporadic cases, although serum phosphate levels are lower in sporadic cases [88]. 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. THE ROLE OF PHEX IN THE PATHOPHYSIOLOGY OF XLH

Although PHEX is known to be an endopeptidase, the role of PHEX in the pathophysiology of XLH is still unclear (see Gattineni [94], Ramon [95], and Strom [96] for review). What is known is that FGF23 levels are elevated in XLH and this elevation in FGF-23 is responsible for the phosphate wasting and lack of elevation in 1,25(OH)2D levels. FGF-23 downregulates SLC34A1 expression thus suppressing NaPiIIa protein levels and leading to phosphate wasting. FGF-23 also decreases levels of 1a-hydroxylase and increases levels of 25-hydroxyvitamin D-24-hydroxylase, resulting in decreased synthesis and increased inactivation of 1,25(OH)2D. FGF-23 is inactivated by cleavage, and it seems logical that FGF-23 would be a substrate for the PHEX, which is an endopeptidase, and that loss of PHEX activity would result in less inactivation of FGF-23 and result in high serum FGF-23 levels. However, the evidence to date suggests that PHEX does not act on FGF-23, and that FGF-23 may be downstream of PHEX. Thus, how mutations in PHEX lead to elevated levels of FGF-23 is not known.

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Treatment Activated vitamin D metabolites and phosphate salts are currently the best available treatments for XLH. Goals of treatment are to facilitate radiographic healing of the growth plate lesions and to maintain optimal growth. Normalization of the serum phosphate concentration is not a goal. Most children are treated until completion of growth, so as to minimize the degree of leg deformities, decrease the number of necessary surgeries, and improve final adult height. The treatment also aims to improve the degree of osteomalacia, although biopsy evidence of this lesion is seldom monitored in the clinical setting. 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, in the absence of informative data, again 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 radiographic evidence of rachitic changes, but the vast majority of patients do go on therapy. We have seen normalappearing adults with XLH who never received therapy. INITIAL DOSES

We generally initiate therapy at dosages of 20e30 ng/ kg/day of calcitriol in two to three divided doses, and 20e40 mg/kg/day of phosphorus in three to five divided doses; a given child may require more or less of each. Calcitriol is available in a liquid formulation, which is convenient to administer to infants and small children. For treatment of the small child, oral sodium phosphate preparations are available over-the-counter; most contain 127 mg of elemental phosphorus per milliliter. The older child may use Neutra-Phos or NeutraPhos K powder (250 mg 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 have used KPhos Neutral (250 mg of elemental phosphorus per tablet) or K-Phos No. 2, a urinary acidifier with decreased sodium content compared to K-Phos Neutral. These tablets may also be crushed and provided with soft foods to assist with administration to small children. In older children, dosages of phosphorus are 1 g per day on average and rarely exceed 2 g per day. Diarrhea

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or persistent abdominal discomfort indicates the need to reassess the dosage schedule and usually reduce the total phosphate dose. 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 [49]. MONITORING

Adjustments in dosing may be necessary in order to achieve the treatment goals stated above. Within 3e4 weeks after the onset of therapy, or after a change in dose is made, serum calcium and PTH levels and urinary calcium and creatinine excretion are assessed. The goal is to maintain normal serum calcium and PTH concentrations and to avoid hypercalciuria. 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 4 months. 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. Thus its value as an indicator of therapeutic response is limited. After 1e2 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 growth plate is optimally responsive to therapy. Thus, the radiographic appearance of the physes is an important gauge of medical therapy and can be an important indicator of the need for dosage adjustments. ADJUSTMENTS IN DOSAGE

The dosage for phosphate and calcitriol can range widely, 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 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

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follow-up laboratory investigation should be performed within 1 month of the change in dose. A recurrent observation is that after correction of the bone disease, perhaps related to optimizing skeletal mineralization, a dose that was appropriate for months to years is suddenly excessive. Thus continual monitoring of this situation is useful from a therapeutic perspective and to avoid toxicity. It is often necessary to increase dosages as the pubertal growth spurt begins e 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. After the growth spurt and achievement of final height, dosages may be lowered to adult maintenance doses. In later childhood, we usually use doses of calcitriol that average 0.75 mg per day, rarely exceeding 1.5 mg per day; 10e50 ng/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 (10e25 ng/kg/day) except for transient increases during puberty. Measurement of PTH is informative to therapeutic decisions. 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 clinical setting 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. An elevated PTH level, without hypercalcemia or hypercalciuria, generally calls for an increase in calcitriol alone. Hypercalciuria or hypercalcemia with a normal PTH indicates the need to decrease the calcitriol dose; if hypercalcemia is severe, temporary cessation of all therapy is recommended. Treatment of adults is somewhat controversial. Adult patients frequently complain of bone pain. Treatment reduces osteomalacia and bone pain, and it is reasonable to treat patients with this complaint. Additionally, it is advisable to treat patients who have non-union 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 asymptomatic patients who do not have pseudofractures. Adults who are treated are prescribed medications based on symptomatology; a dose of 0.75e1.0 mg of calcitriol daily is typical. ADJUNCTIVE THERAPIES HUMAN GROWTH HORMONE Human growth hormone (hGH) has been used as adjunctive therapy in XLH. However, there is no clear evidence that benefits of growth hormone outweigh cost and potential side effects, and thus growth hormone is not recommended as part of therapy. One study [97] noted an increased effect of treatment on truncal height, thereby potentially exaggerating the disproportionate short stature observed in XLH. Others [98] have documented an accelerated increase in skeletal age during treatment with hGH. 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 [99]. Thus, it is difficult to make a case for routine treatment of XLH with hGH. OTHER ADJUNCTIVE THERAPIES It is possible that non-hypercalcemic vitamin D analogs (such as paricalcitol or doxercalciferol) would be of use in the setting of hyperparathyroidism in XLH, but no data specific to this issue in XLH are available. The calcimimetic agent, cinacalcet, has been shown to lower acutely the PTH response to a phosphate load in XLH [100], and may also be useful in this regard, although it is relatively expensive. Thiazide diuretics or amiloride have been suggested to increase renal calcium reabsorption and to enhance mineralization [101]. The long-term effects of this measure have not been reported. 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 [102]. 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 [103]. We have seen that healing of osteotomy in young children may be prolonged. Surgical procedures vary. Fixation with plating and stapling has been performed, and external fixation devices have recently been used [104]. An

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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. Leg-lengthening procedures have been attempted in a few patients with XLH [24]. Newer, less invasive approaches include epiphysiodesis, which induces corrective differential growth of the growth plate [105]. TREATMENT-RELATED COMPLICATIONS

Complications of therapy for XLH include hypervitaminosis D, hyperparathyroidism, and soft-tissue calcification, particularly in kidney (Fig. 26.4). Significant clinical consequences of these complications can usually be avoided with careful monitoring. Treatment is essentially a compromise in this setting: In order effectively to 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 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

FIGURE 26.4 Renal ultrasound of nephrocalcinosis. Diffuse evidence for calcifications is seen.

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about medical therapy in surviving family members. The 1a-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 1a-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. Less frequently, hyperparathyroidism can progress to autonomous tertiary hyperparathyroidism requiring parathyroidectomy. The stimulatory effect of treatment with phosphate salts on PTH secretion in XLH is well described [106,107]. 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. Elevations in circulating PTH occur at night in most individuals with XLH [49]. SOFT-TISSUE CALCIFICATION Soft-tissue calcification, particularly at the level of the renal medullary pyramids (nephrocalcinosis), can be detected by renal ultrasound examination [108]. 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. [109], 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 [108]. 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 [110]. Others have shown that the lesions are composed of calcium phosphate precipitates and can be reproduced in the Hyp mouse by administration of phosphate [111]. It is also possible that increased PTH levels predispose the tissue to calcification. Other related problems in XLH include calcification of the entheses [112], which is not thought to be treatment dependent. In addition, ocular calcifications [113] and myocardial and aortic valve calcifications [114] have been described.

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Autosomal Dominant Hypophosphatemic Rickets ADHR is a rare renal phosphate wasting disorder. Bianchine et al. [115] 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 [116] evaluated a large ADHR kindred with 23 affected individuals who presented with hypophosphatemia for age, normocalcemia, normal renal function, and inappropriately normal calcitriol concentrations. In six of seven patients tested, there was no amino aciduria. In one 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 1 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 ¼ 6) or a history of treatment for rickets or lower extremity deformity as a child and documented hypophosphatemia as an adult (n ¼ 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 women and they presented during the reproductive years. 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  0.39 mg/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 and these women presented during the reproductive years. Preliminary data demonstrate an inverse correlation between serum iron concentrations and intact FGF-23 levels in ADHR patients, highlighting the possibility that iron deficiency may play a role in adult onset of hypophosphatemia in ADHR patients [117]. An additional novel feature of ADHR 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 and recent data indicate that clinical disease can wax and wane in females [118]. ADHR is the only hereditary disorder of renal phosphate wasting in

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which patients may regain the ability to conserve phosphate. The mechanism that underlies the ability of patients to attain normal phosphate homeostasis is being investigated. In summary, ADHR patients present with isolated renal phosphate wasting and inappropriately normal calcitriol concentrations, similar to patients with XLH. In contrast to XLH, ADHR displays variable age of onset, incomplete penetrance, and loss of the phenotype.

Autosomal Recessive Hypophosphatemic Rickets Familial hypophosphatemic rickets occurring with an apparent autosomal recessive mode of inheritance was described in 2006. Five separate families were described in two separate reports [119,120]. Consideration of the observed biochemical and skeletal phenotypes in dentin matrix protein 1 (DMP1) null mice led one group of investigators to employ a candidate gene approach, and sequence analysis of the DMP1 gene in affected family members revealed a deletion in one family and a start codon mutation in another [119]. Other investigators performed a genome-wide linkage analysis [120], which revealed a cluster of potential candidate genes on chromosome 4, encoding members of the SIBLING (small integrin-binding ligand, N-linked glycoproteins) family [121]. DMP1 was identified as the mutated gene in this region, and each family was found to have separate mutations including a deletion leading to a premature stop codon, a splice site acceptor mutation, and the same start codon mutation identified above. Affected individuals demonstrated biochemical parameters in the range typical of those observed in XLH, including hypophosphatemia, reduced %TRP and TMP/GFR, with inappropriately normal (or low) circulating 1,25(OH)2D levels. Moreover, FGF-23 levels tended to be elevated in a similar range as reported in XLH. DMP1 is a product of the osteocyte, and the coincident expression of PHEX and FGF-23 in this cell type has further emphasized a central role of this cell in the maintenance of phosphorus homeostasis. More recently, a curious novel etiology of autosomal recessive hypophosphatemia has been identified, related to homozygous loss-of-function mutations in ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). This enzyme has been thought to be critical in regulating local concentrations of the mineralization inhibitor, pyrophosphate, at matrix vesicles and mineralization sites [122]. Two reports have identified nine affected patients in five families with the disorder, and four loss-of-function mutations in ENPP1 [123,124]. Affected individuals are described to have biochemical features consistent with those seen in XLH, including

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normocalcemia and hypophosphatemia secondary to renal phosphate wasting, with “inappropriately normal” circulating 1,25(OH)2D levels. Circulating FGF-23 levels are reported as convincingly elevated in four of the nine reported patients. Circulating 1,25(OH)2D levels vary in these reports, with most in the low-normal to normal ranges in the setting of hypophosphatemia; one case is reported to have an elevated 1,25(OH)2D value. Paradoxically, loss-of-function mutations in ENPP1 have previously been reported to account for a severe disorder of vascular calcification, generalized arterial calcification of infancy (GACI) [125], and in fact, the same mutation has been reported to be associated with both disorders independently in father and son [126]. Thus the variable effect of loss-offunction of ENPP1 is likely related to other genetic and environmental modifiers, rather than a specific genotype/phenotype effect.

Hereditary Hypophosphatemic Rickets With Hypercalciuria In 1985, Tieder et al. [127] described an unusual variant of hypophosphatemic rickets in a Bedouin tribe in which consanguineous marriage 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)2D levels were elevated, indicating that the renal pathophysiology of this condition is strictly limited to defective epithelial renal phosphate transport. The normal 1a-hydroxylase trophism of low blood phosphate levels is intact, in contrast to XLH, in which the renal defect involves both a disruption of the normal hypophosphatemia-1a hydroxylase axis and renal tubular phosphate handling. This contrast with XLH represents the fact that HHRH is not a disorder of FGF-23-mediated hypophosphatemia. 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 evident from an early age. This finding differs from the typical pattern of skeletal disease in XLH, in

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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 [127], but others have noted decreased numbers of osteoclasts [128,129]. 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. [127], 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 [127]. Renal losses can be significant, however, a short-term balance study in one patient demonstrated negative phosphate balance [128]. 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. [10], 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 1a-hydroxylase axis is fully intact in HHRH. Relation to Idiopathic Hypercalciuria HHRH has interesting implications regarding the pathophysiology of idiopathic hypercalciuria. Tieder et al. [129] 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 correlate 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 [130]. Management and Course HHRH is usually managed by administration of phosphate salts alone. In older children and adults, we have employed doses in the range of 1e2.5 g of elemental phosphorus per day, given in four to five divided doses daily, or 20e50 mg/kg of elemental phosphorus daily. Attention to both the bone disease and hypercalciuria is important in monitoring the clinical response to therapy. 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, in general, patients continue to have low bone density. Thus, therapies directed toward the osteoporotic component of the disorder may be important in the long-term management of affected adults, although there are theoretical concerns about using agents such as bisphosphonate in the setting of osteomalacia. Generous hydration is important, and avoidance of a high sodium intake is recommended. The Genetic Basis of HHRH The identification of mutations in the NaPi-IIc renal phosphate co-transporter (encoded by SLC34A3) came as a surprise, as the most abundant renal tubular phosphate transporter in mammalian kidney is the type IIa sodiumephosphate co-transporter, NaPi-IIa (encoded by SLC34A1, and also a member of the SLC34 family of sodiumephosphate co-transporters). Both transporters localize to the brush border surface of proximal renal tubular cells, and are regulated by PTH and dietary phosphate. Although SLC34A1 was originally considered the prime candidate for the site of mutation in HHRH, multiple attempts to identify mutations or linkage of the disease to this gene were unsuccessful [131,132]. Subsequently, the identification of homozygous loss-of-function mutations in SLC34A3, was identified as causative to the syndrome in multiple pedigrees. It has been suggested that NaPi-IIc may play a relatively greater role in humans than in rodents, where data

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regarding its abundance in comparison to NaPi-IIa have been derived. There appears to be a more widespread tissue distribution of NaPi-IIc than NaPi-IIa. One specific mutation (T137M) in SLC34A3 when examined in in vitro functional studies demonstrates sodium transport alterations; this potential association with sodium wasting, suggests that certain mutations in SLC34A3 may play a role in the formation of urinary tract stones [133]. Most recently, homozygous loss-offunction mutations in NaPi-IIa have been identified to cause a hypophosphatemic rickets syndrome, however, generalized renal tubular dysfunction (Fanconi syndrome) was evident. In contrast to HHRH, circulating 1,25(OH)2D levels were not elevated and hypercalciuria was not evident, which the authors attributed to concomitant vitamin D deficiency [134].

Tumor-Induced 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 [135]. Most reports of TIO identify the characteristic hypophosphatemia and renal phosphate wasting observed in patients with XLH [136e139]. The serum phosphate levels may be lower than those seen in XLH, but there is considerable overlap of serum 1,25(OH)2D 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

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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 [137]. Spindle cells are prominent in many reports. Other frequent pathologic descriptions are ossifying and non-ossifying 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 [140]. The similarity in appearance of many of these tumors has been noted by Weidner and Santa Cruz [141], 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; osteoblastomalike; ossifying fibroma; and non-ossifying fibroma. The latter three groups tend to occur in bone. Most of these tumors are phosphaturic mesenchymal tumors of the mixed connective tissue type [142]. 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 resonance imaging (MRI) has been suggested. Alternatively, scintigraphy using indium-111 pentetreotide or octreotide has been useful in tumor localization [143]. Technetium-99 methylene diphosphonate 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. More recently, the development of co-register techniques employing simultaneous CT and positron emission tomography

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(PET) scans have been particularly useful imaging measures for tumor location in TIO cases. Sites within bones often occur, pelvic tumors are described, and other presentations described include plantar warts and tibial stress fractures [144]. The development of reliable assays for circulating FGF-23 has resulted in confirmation of tumors using a selective venous sampling technique that can survey for informative concentration gradients of this compound. With increasing awareness of this syndrome, together with improved imaging and other diagnostic techniques, the detection of these tumors may certainly increase, 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 [145]. 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 [146]. 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 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 longterm intravenous phosphate in concert with oral calcium and vitamin D [147]. 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 [148]. A TIOassociated tumor was detected by indium-111 octreotide scintigraphy, providing evidence for the presence of somatostatin receptors on the tumor. The patient was then given 50e100 mg 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)2D 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. Long-term monitoring postresection is clearly important. 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-1a-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 [149]. 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 limited amounts of source material from the small excised tumors, and the limited duration of secretion of material from tumor cells in culture. In time, pursuit of this factor resulted in the identification of FGF-23 as the predominant causative factor in TIO [150e152], although other potential phosphatonins have been identified in the process. Overproduction of FGF-23 as a paraneoplastic phenomenon is

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in keeping with the discovery that ADHR, another disorder that manifests the XLH phenotype, results from specific mutations in FGF-23 that retard its degradation [85]. Upon delivery of FGF-23 via transfected cells overexpressing the material or via transgenic overexpression, mice developed a syndrome with characteristic features of TIO [151,153]. Other substances expressed by these tumors include matrix extracellular phosphoglycoprotein (MEPE), a large glycosylated protein, frizzledrelated protein 4 (FRP4), a potential decoy of Wnt signaling, and FGF-7 [154e156]. The role of these substances in the pathogenesis of TIO is not clear.

Related Syndromes Other forms of inherited hypophosphatemic rickets occur. Dent’s disease is an X-linked recessive disorder due to mutations in the voltage-gated chloride-channel gene, CLCN5. Fanconi syndrome, and Lowe’s syndrome are disorders which manifest a variety of clinical features beyond that usually observed in the setting of either isolated hypophosphatemia or the “XLH” phenotype; therefore the diagnosis is usually identified for reasons other than those discussed in this chapter. Several other circumstances may arise in which the XLH or TIO phenotype is evident secondary to unusual lesions or disease processes. These are discussed below.

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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 that some cases involve skeletal anomalies in anatomic proximity to the skin lesions, it has been speculated that this syndrome may manifest regional genetic expression of disease [158]. Increased levels of FGF-23 have been reported in several cases of ENS [159,160], and some cases are due to activating mutations of an FGF receptor, FGFR3 [161]. 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. McCuneeAlbright Syndrome

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 [157]. 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

McCuneeAlbright syndrome (MAS) is a chimeric disorder usually characterized by cafe´-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 Gsa, 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 [162]. The renal tubular phosphate wasting in MAS likely occurs as a result of a circulating material generated by the skeletal lesions. This hypothesis was put forward by Dent and Gertner [163] after noting the correction of hypophosphatemic rickets following surgical excision from bone of fibrous dysplasia

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lesions. More recently, elevated FGF-23 levels have been described in this condition, and correlate with the degree of hypophosphatemia. Furthermore, correlation of biochemical parameters of renal phosphate wasting with the extent of skeletal involvement has been demonstrated, as well as immunohistochemical staining for FGF-23 in bone cells obtained at biopsy of such patients [164]. 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 [136,165,166].

MOLECULES, PATHOPHYSIOLOGY, AND LESSONS FROM ANIMAL MODELS Mouse Models for XLH Murine models of XLH include Hyp [167] and Gy [168] mice; the Hyp mouse is the more extensively characterized, and its manifestations are very similar to those of XLH in humans. Hyp mice are characterized by hypophosphatemia due to phosphate wasting, a slightly decreased serum calcium level, elevated serum alkaline phosphatase activity, slow growth, rickets, and osteomalacia [7]. Additional phenotypic features are found in the Gy mouse, which has a contiguous gene syndrome, include sterility in the male, circling behavior, and reduced viability [168]. Phosphate Wasting The Hyp mouse has a defect in renal phosphate transport [169], confined to the renal brush border membrane [170,171]. Hyp mice demonstrate an z50% decrease in sodium-dependent phosphate transport in the brush border membrane of the proximal tubule [171,172], mostly due to reduced abundance of the high-affinity, low-capacity sodium-dependent phosphate cotransporters NaPi-IIa and NaPi-IIc [173]. This occurs in concert with marked elevations in FGF-23 [174]. In addition, Hyp mice have higher PTH levels compared to normal mice [175e177]. Bone and Teeth As in humans, Hyp mice demonstrate rickets and osteomalacia. Osteoid thickness and volume are increased, and bone mineral content is decreased [178]. Although hypophosphatemia is 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 [179] and, although there is further improvement when 1,25(OH)2D is added, osteomalacia persists [180]. 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 [178,181e183]. These studies revealed that the impaired mineralization of Hyp cells is improved but not corrected when placed in a normal mouse [178], and that prior exposure to a hypophosphatemic environment does not explain the abnormality [181]. 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 [182,183]. Most recently, mineralization has been shown to be impaired in concert with elevated levels of the ASARM peptide fragment of the SIBLING protein, MEPE, in Hyp mice [22]. Like humans with XLH, the Hyp mouse has a defect in dentin formation [184e187] that is not resolved by correcting the hypophosphatemia [188]. Vitamin D Metabolism Regulation of 1,25(OH)2D metabolism is abnormal in the Hyp mouse due to abnormal renal 25-hydroxyvitamin D-1a-hydroxylase (1-OHase) activity (the enzyme converting 25(OH)D to 1,25(OH)2D) and 25-hydroxyvitamin D-24-hydroxylase (24-OHase) activity (the enzyme converting 25(OH)D to 24,25(OH)2D). 24-OHase is the first enzymatic step in the inactivation of 1,25(OH)2D. When normal mice are made hypophosphatemic (by phosphate deprivation), 1-OHase activity [189] and subsequently 1,25(OH)2D levels [190] increase, and there is no change in 24-OHase products [190], activity, or mRNA levels [191]. However, in the Hyp mouse, 1,25(OH)2D levels [190,192] and 1-OHase activity [189] 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 [190]. 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 [189,193] and increase in renal 24-OHase activity, protein, and mRNA levels [190,191,194], leading to even lower 1,25(OH)2D levels [190,192,195]. The Hyp mouse also responds paradoxically to phosphate supplementation with an increase in 1,25(OH)2D levels and decrease in the products of 24OHase, whereas in phosphate-supplemented normal mice 1,25(OH)2D levels and the products of 24-OHase do not change [190].

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In the Hyp mouse, 1,25(OH)2D increases in response to vitamin D and calcium deprivation but blunted compared to normal control mice showing less of an increase in 1-OHase activity and less of a decrease in 24-OHase activity [196e198]. The Hyp mouse also has a blunted 1-OHase response to exogenous PTH [199], but calcitonin stimulates 1-OHase activity to similar levels in Hyp and normal mice [200]. Although vitamin D metabolism is abnormal in the Hyp mouse, the renal response to 1,25(OH)2D supplementation is appropriate. 1,25(OH)2D supplementation leads to an increase in calcium and phosphate, no increase in renal phosphate transport [201], and an increase in renal 24-OHase mRNA and activity [191]. As with expression of the renal phosphate transporters, expression of the vitamin D hydroxylaseencoding genes CYP27B1 (1-OHase) and CY24A1 (24-OHase) is regulated by FGF-23, in vivo and in vitro. In normal mice, expression of CYP27B1 is decreased with application of FGF-23 (as well as phosphate loading). Although the regulation of FGF-23 by phosphate remains intact in Hyp mice, the end effect on CYP27B1 expression in Hyp mice under varying dietary phosphate conditions is less clear [174,202]. The response of bone from Hyp mice to 1,25(OH)2D is abnormal. In vitro, 1,25(OH)2D inhibits bone collagen synthesis in both Hyp mice and normal mice [203]. This 1,25(OH)2D-induced inhibition increases in the presence of a lowered phosphate concentration in normal mice but not in Hyp mice [203]. In addition, Hyp osteoblasts in culture do not respond to physiologic doses of 1,25(OH)2D 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 [204]. 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 [205e209] and is associated with a greater than 50% decrease in levels of intestinal calcium-binding protein (CaBP) [210,211]. 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) [209,212], nor by differences in the basal levels of intestinal VDR mRNA [213], In addition, the Hyp mouse responds normally to 1,25(OH)2D supplementation with increased intestinal absorption of calcium and phosphate and increased duodenal and renal CaBP [211,214]. Instead, the decreased intestinal absorption of calcium and phosphate appears to be due to the defect in 1,25(OH)2D metabolism [7]. The Hyp mouse has decreased nuclear uptake of 1,25(OH)2D in intestinal mucosal cells [215],

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which is corrected after phosphate supplementation [216]. 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)2D and phosphate levels. PHEX/Phex Soon after the identification of PHEX, the mouse Phex was cloned [217]. 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 at the amino acid level [74,217e219]. The Hyp mouse has a 3’ deletion of Phex, including exons 16e22 [73,218,220] and the 3’ untranslated region [220]. The Gy mouse has a deletion of exons 1e3 and upstream sequences [218], including a partial deletion of the spermine synthase gene, which could explain some of the additional phenotypic features of the Gy mouse [221e223]. Phex is a 100- to 105-kDa glycoprotein and a member of the neutral endopeptidase family [224,225]. PHEX/ Phex is expressed primarily in human and mouse fetal and adult bone [73,217,224e227] and in mouse teeth [217,224,226,227]. Expression is two- to 10-fold higher in bone than in other tissues [225]. Phex mRNA expression remained high in brain but decreased to undetectable levels with age in the other tissues [228]. In the femur and calvaria, Phex protein has also been shown to decrease with age [224]. In most [73,74,217] but not all studies [225], no PHEX expression has been detected in adult kidney. Human PHEX expression has also been detected in ovary [73,74] and lung [73,74]. PHEX expression was detected in fetal liver in one study [74] but not in others [73,225], and it has been detected in fetal muscle [73,225]. PHEX is also expressed in the parathyroid glands [229]. Phex appears to be a marker of mature osteoblasts, osteocytes and odontoblasts and is associated with matrix mineralization [226,227]. Although Phex mRNA is not detectable in mouse MC3T3-E1 pre-osteoblasts, mRNA and proteins levels increase with differentiation of the osteoblast and concomitant matrix mineralization [219,227]. Phex expression is downregulated by 1,25(OH) 2 D, which inhibits matrix mineralization [227]. By in situ hybridization, Phex mRNA 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 [226]. The amount of Phex transcript is decreased in adult bone and non-growing teeth [226]. Phex protein expression mirrors that of mRNA and is found in osteoblasts, osteocytes, and odontoblasts but not in osteoblast precursors [224]. Of interest, in one study transplantation of bone marrow from normal to

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Hyp mice led to increased serum phosphorus, increased renal NaePi co-transporter gene expression, and decreased alkaline phosphatase activity [230]. Targeted overexpression of Phex in Hyp osteoblasts will partially, but not completely, correct the mineralization defect or the hypophosphatemia [231,232]. This may be secondary to lack of adequate expression in the osteoblasts, but it could also indicate that expression in other cells is critically important. The PHEX protein is glycosylated and located on the cell surface with the carboxy domain facing extracellularly [225]. Three missense mutations responsible for HYP have been shown to interfere with the membrane targeting of the mutant PHEX protein [233]. PHEX has also been shown to degrade PTH-derived peptides [225,234]. Osteocalcin and phosphate inhibit the degradation of PHEX [234]. After the discovery that FGF-23 concentrations were elevated in XLH and in the Hyp mouse the concept that PHEX might cleave FGF-23 was an enticing hypothesis. However, it was soon found that PHEX does not metabolize FGF-23. Instead, osteoblasts and osteocytes in the Hyp mouse markedly overexpress FGF-23 [235,74]. Overexpression of FGF-23 leads to the biochemical abnormalities seen in the disease. Moreover, overexpression of FGF-23 explains why XLH is an X-linked dominant disorder because the mutant osteoblasts and osteocytes in the female overproduce FGF-23 even if the cells with the normal allele downregulate FGF-23 expression. However, serum phosphate concentrations in affected men, in whom all cells contain the mutant allele, are the same as affected women, who are heterozygous with half their cells containing the mutant allele and half with the normal allele. Preliminary data from our laboratory [236] indicate that PHEX mutations may alter the set point for phosphate and it is possible that the mutant osteoblasts and osteocytes in the female, which constitute only half the cells, are making as much FGF-23 as the male cells (i.e. twice the amount per cell). If such a set point/phosphate sensing defect exists, this would have important implications for therapy because the patient’s bone may produce more FGF-23 in response to any attempt to raise the serum phosphate concentration. Indeed, studies have demonstrated that FGF-23 concentrations rise with high dose phosphate and calcitriol treatment [237,238]. In summary, PHEX is the gene defective in XLH. As a membrane-bound endopeptidase that is primarily expressed in bone and is not expressed in kidney, PHEX is not a direct regulator of phosphate transport. Indeed, there is currently no proof that PHEX has a role in normal phosphate homeostasis, only data demonstrating that PHEX mutations result in elevated FGF-23 concentrations, which results in hypophosphatemia and inappropriately normal calcitriol concentrations.

FGF-23 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 [239]. Further analysis revealed missense mutations in a novel member of the fibroblast growth factor family, FGF-23 [85]. Sequencing of FGF-23 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. FGF-23 is primarily expressed in bone in osteoblasts and osteocytes [240]. Using reverse transcriptase-polymerase chain reaction, FGF-23 RNA was detected in heart, liver, and thyroid/parathyroid [85]. This technique has also amplified products from thymus [241]. In light of the clinical similarity between ADHR and TIO, tissue from tumors that caused TIO was analyzed for FGF-23 expression [150]. 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 Cterminal portion of FGF-23 detected a 32-kDa band on Western blot [150]. These results were subsequently confirmed by others [151,152]. These data implicated FGF-23 as the factor previously called phosphatonin that is responsible for the phosphate wasting in this disorder. Additional important evidence implicating FGF-23 as phosphatonin derived from studies performed by Shimada et al. [152]. These investigators also found that FGF-23 was highly expressed in tumors that cause TIO. They found that intraperitoneal administration of recombinant human FGF-23 resulted in increased renal phosphate excretion and decreased serum phosphorus concentrations. In subsequent experiments, they made Chinese hamster ovary cell lines that stably expressed FGF-23 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. To elucidate the mechanism by which the FGF-23 mutations in arginine 176 and 179 cause ADHR Western blots of cells transfected with wild-type and mutant FGF-23 were probed with a C-terminal FGF-23 antibody. Wild-type FGF-23 demonstrated bands of approximately 32 and 12 kDa [150]. Further analysis indicated that the 12-kDa fragment was a C-terminal cleavage

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product of the wild-type 32-kDa protein [150]. Similar studies done in all three ADHR mutations yielded only the intact, 32-kDa FGF-23 protein [242]. Of note, the mutated arginines form an RXXR motif, which is a recognition sequence for the furin class of proteolytic enzymes [243]. These and other data indicate that the ADHR mutations protect the protein from degradation and thereby elevate circulating FGF-23 concentrations in ADHR patients. In summary, ADHR is caused by missense mutations in arginines 176 or 179 of FGF-23, which protect the protein from degradation. Furthermore, FGF-23 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 FGF-23 has a role in the pathogenesis of these disorders. FGF-23 Receptors and Klotho FGF-23, even in the presence of heparin, has low affinity for the classical FGF receptors. However, recent data demonstrate that the protein Klotho is a critical coreceptor that allows FGF-23 to bind to select FGFRs [244]. Klotho, which was originally thought to be an “aging” gene [245], is expressed primarily in the kidney (distal tubule), and choroid plexus [246]. Mice deficient in Klotho exhibit hyperphosphatemia, increased calcitriol concentrations as well as a variety of other phenotypic features, including premature death (presumably from renal failure). Correcting the phosphate and vitamin D abnormalities essentially normalizes the phenotype [247]. So far, only one human Klotho lossof-function mutation has been identified [248]. This patient exhibited hyperphosphatemia, increase calcitriol concentrations, and soft-tissue calcifications, but no evidence of premature aging. In contrast to the Klothodeficient mouse, PTH concentrations were elevated. In contrast, a genetic translocation disrupting the upstream region of Klotho resulting in Klotho overexpression has been associated with hypophosphatemic rickets due to renal phosphate-wasting phenotype and coincident hyperparathyroidism [249]. FGF-23 binds to the FGFR/Klotho complex and initiates signaling via the MAP kinase pathway [250]. There is general agreement that FGFR-1c is an important receptor for FGF-23 [244,251]. There has been controversy as to whether FGFR-3c and FGFR-4 also bind FGF-23 [244,251]. However, recent data support the roles of FGFR-3c and FGFR-4 as receptors [252]. In any event, there is general agreement that Klotho is a required coreceptor for FGF-23 binding and signaling. Since FGFRs are widely distributed throughout the body and play roles in multiple processes, it is necessary to have a coreceptor system for the endocrine FGFs, like FGF-23, to prevent unwanted receptor activation in tissues

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unrelated to phosphate and vitamin D homeostasis. Figure 26.5 illustrates the central role of FGF-23 in XLH, TIO, and ADHR, as the primary mediatior of these disorders.

Non-FGF Mediated Hypophosphatemia Primary Defects in the Phosphate Transporters and Related Proteins Renal phosphate transport is primarily under the influence of two members of the SLC34 family (type II sodiumephosphate co-transporters), NaPi-IIa and NaPi-IIc. Expression of these transporters is regulated by PTH, FGF-23, and dietary phosphate, all significant regulators of total body phosphate economy. Initial attention was directed toward NaPi-IIa, as it is the most abundant renal Pi transporter. Targeted inactivation of NaPi-IIa in mice generates a biochemical phenotype similar to that seen in HHRH [253], with hypophosphatemia and increased renal excretion of phosphate. A slight but significant hypercalcemia occurs, with PTH levels suppressed to the low range. Circulating 1,25(OH)2D levels, urinary calcium excretion, and serum alkaline phosphatase activity are elevated. The heterozygous mice demonstrated an elevation in 1,25(OH)2D levels intermediate between homozygous mice and wild-type mice but no significant difference in serum calcium, urinary calcium excretion, or alkaline phosphatase activity. The skeletal phenotype of this model is complex, with growth plate expansion consistent with rickets at 2 weeks of age, but resolving by 5 weeks of age [253,254]. Based on the similarities in biochemical phenotype to the human disorder HHRH, it was originally considered that the human disorder would likely result from a primary disruption in renal tubular brush border phosphate transport. However, mutations in NaPi-IIa (SLC34A1) were excluded as causal for this disease [131], and the condition was later found to be a result of loss-of-function mutations in the related SLC34 family member, NaPi-IIc (SLC34A3) [255]. The NaPi-IIc transporter may have a more important role for Pi transport in humans as compared to its relative role in rodents, and appears to have a more widespread tissue distribution. Heterozygous NaPi-IIa loss-of-function mutations in humans have been described, associated with calcium nephrolithiasis and osteopenia, but have not resulted in features of phosphate-wasting rickets. As noted above, homozygous loss-of-function mutations in SLC34A1 have been shown to result in a phosphate-wasting form of rickets with generalized renal tubular dysfunction [134]. Another class of NaePi co-transporters, the type III (SLC20 family) transporters, Pit1 and Pit2, are also expressed in kidney, but their role in this process,

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FIGURE 26.5 FGF-23 is shown as central to the process of renal phosphate conservation. FGF-23 is a normal product of bone, secreted by osteocytes. PHEX, a zinc-binding protein, is found in the membrane of normal osteocytes. In the setting of XLH, loss of function of PHEX results in increased FGF-23 production (heavy arrow). Increased circulating levels of FGF-23 may also result from production in tumors associated with tumor-induced osteomalacia (TIO). In autosomal dominant hypophosphatemic rickets (ADHR), a mutation in the RXXR protease recognition site of FGF-23 results in impaired breakdown of the molecule, and accumulation of increased circulating levels of FGF-23 occur due to the reduced clearance of the intact form, which is necessary for the renal actions. FGF-23 requires an FGF receptor and Klotho for downstream signaling. This initial event in FGF-23 action on the kidney occurs in the distal tubule, whereas the effects of impaired renal tubular phosphate reabsorption and inhibition of 1,25(OH)2D production are associated with proximal tubular functions. It is unclear how the distal tubular interaction of FGF-23, its receptor, and Klotho result in actions at the proximal tubule. Autosomal recessive hypophosphatemic rickets may occur with loss of function of the osteocyte-expressed protein DMP1 (not shown), resulting in increased FGF-23 secretion. Finally, loss of function of NaPi-IIc results in hereditary hypophosphatemic rickets with hypercalciuria (proximal renal tubular cell). See text for detailed explanations of each of these disorders.

although not well established, is not felt to be as dominant as the type II transporters at this time. The third member of this family of sodiumephosphate co-transporters, NaPi-IIb, is predominantly found in the intestine, and has been found to be associated with pulmonary microlithiasis [256,257]. Other related proteins that directly affect NaPi-II function include the PTH receptor (PTH1R) and NHERF1. Gain-of-function mutations in the PTH receptor can result in Jansen’s metaphyseal dysplasia, which can be accompanied by hypercalcemia and hypophosphatemia. Curiously, a recent report describes the coincident finding of elevated circulating FGF-23 levels in this disorder [258]. NHERF1 binds the PTH receptor and NaPi-II transporters, and are thought to modulate cAMP production, and perhaps coordinate the movement of NaPi-II from the apical membrane in response to PTH. Mutations in NHERF1 have been associated with hypophosphatemia and nephrolithiasis but variability exists in the phenotype of individuals identified with such mutations [259].

CURRENT PROBLEMS AND UNRESOLVED QUESTIONS Directions for Future Research The metabolic bone community has made great strides in understanding the pathogenesis of XLH, ADHR, and TIO, however, there are many avenues for future research. Recent data by Farrow et al. [260] demonstrate that FGF-23 acts on the renal distal tubule. However, the renal effects on phosphate and vitamin D homeostasis take place in the proximal tubule. Solving this apparent paradox will be important for further progress in the field. Are there paracrine signals between the distal and proximal tubules? If so, what lies between FGF-23 binding the FGF receptor/Klotho complex in the distal tubule and downregulation of the sodiumephosphate cotransporters (NaPi-IIa, NaPi-IIc), downregulation of 1 hydroxylase and upregulation of 24 hydroxylase in the proximal tubule?

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FGF-23 excess clearly plays a role in the pathogenesis of XLH, and FGF-23 neutralizing antibodies are being tested as a new therapy for the disease, but do PHEX mutations have adverse effects outside of the resulting increase in FGF-23? Is there receptor redundancy for FGF-23 and might strategies aimed at blocking the receptor(s) be a useful therapy for FGF-23 mediated hypophosphatemic disorders? 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? If this is the case, can we pharmacologically manipulate the sensor? If the molecular basis of XLH involves an altered set point for the sensor, how can we change therapy to minimize the rise in FGF-23 concentrations? What is the pathogenesis of the enthesopathy in XLH? If patients do not properly mineralize bone, why do they mineralize their tendons and ligaments? Finally, can we use these new data to design new therapeutic approaches for phosphate wasting disorders that have less toxicity than current regimens? Such approaches could include decreasing circulating FGF23 concentrations with neutralizing antibody or receptor blockade. Indeed, novel approaches to therapy are already under development. Clearly, there is great cause for optimism for patients who suffer from these diseases.

References [1] Tenenhouse HS, Econs MJ. Mendelian hypophosphatemias. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The Metabolic and Molecular Basis of Inherited Disease. New York: McGrawHill; 2001. p. 5039e68. [2] Tenenhouse HS. Phosphate transport: molecular basis, regulation and pathophysiology. J Steroid Biochem Mol Biol 2007;103:572e7. [3] Bastepe M, Ju¨ppner H. Inherited hypophosphatemic disorders in children and the evolving mechanisms of phosphate regulation. Rev Endocr Metab Disord 2008;9:171e80. [4] Prie´ D, Friedlander G. Genetic disorders of renal phosphate transport. N Engl J Med 2010;362:2399e409. [5] Gisler SM, Stagljar I, Traebert M, Bacic D, Biber J, Murer H. Interaction of the type IIa Na/Pi cotransporter with PDZ proteins. J Biol Chem 2001;276:9206e13. [6] Hernando N, Deliot N, Gisler SM, et al. PDZ domain interactions and apical expression of type IIa Na/P(i) cotransporters. Proc Natl Acad Sci USA 2002;99:11957e62. [7] Rasmussen H, Tenenhouse HS. Mendelian hypophosphatemias. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The Metabolic and Molecular Basis of Inherited Disease. New York: McGraw-Hill; 1995. p. 3717e45. [8] Tenenhouse HS. Cellular and molecular mechanisms of renal phosphate transport. J Bone Miner Res 1997;12:159e64. [9] Reid IR, Hardy DC, Murphy WA, Teitelbaum SL, Bergfeld MA, Whyte MP. X-linked hypophosphatemia: a clinical, biochemical, and histopathologic assessment of morbidity in adults. Medicine 1989;68:336e52.

719

[10] Burnett CH, Dent CE, Harper C, Warland BJ. Vitamin D-resistant rickets. Am J Med 1964;36:222e32. [11] Econs MJ, Feussner JR, Samsa GP, et al. X-linked hypophosphatemic rickets without “rickets”. Skeletal Radiol 1991;20:109e14. [12] Block JE, Piel CF, Selvidge R, Genant HK. Familial hypophosphatemic rickets: bone mass measurements in children following therapy with calcitriol and supplemental phosphate. Calcif Tissue Int 1989;44:86e92. [13] Reid IR, Murphy WA, Hardy DC, Teitelbaum SL, Bergfeld MA, Whyte MP. X-linked hypophosphatemia: skeletal mass in adults assessed by histomorphometry, computed tomography, and absorptiometry. Am J Med 1991;90:63e9. [14] Oliveri MB, Cassinelli H, Bergada C, Mautalen CA. Bone mineral density of the spine and radius shaft in children with X-linked hypophosphatemic rickets (XLH). Bone Miner 1991;12:91e100. [15] Rosenthall L. DEXA bone densitometry measurements in adults with X-linked hypophosphatemia. Clin Nucl Med 1993;18:564e6. [16] Shore RM, Langman CB, Poznanski AK. Lumbar and radial bone mineral density in children and adolescents with X-linked hypophosphatemia: evaluation with dual X-ray absorptiometry. Skeletal Radiol 2000;29:90e3. [17] Berndt M, Ehrich JH, Lazovic D, et al. Clinical course of hypophosphatemic rickets in 23 adults. Clin Nephrol 1996;45:33e41. [18] Ferris B, Walker C, Jackson A, Kirwan E. The orthopaedic management of hypophosphataemic rickets. J Pediatr Orthop 1991;11:367e73. [19] Chalmers J. Enthesopathy as the presenting feature of X-linked hypophosphatemia. A case report. Acta Orthop Scand 1993;64:221e3. [20] Marie PJ, Glorieux FH. Bone histomorphometry in asymptomatic adults with hereditary hypophosphatemic vitamin Dresistant osteomalacia. Metab Bone Dis Relat Res 1982;4:249e53. [21] Marie PJ, Glorieux FH. Histomorphometric study of bone remodeling in hypophosphatemic vitamin D-resistant rickets. Metab Bone Dis Relat Res 1981;3:31e8. [22] Rowe PS, Matsumoto N, Jo OD, et al. Correction of the mineralization defect in hyp mice treated with protease inhibitors CA074 and pepstatin. Bone 2006;39:773e86. [23] Sullivan W, Carpenter T, Glorieux F, Travers R, Insogna K. A prospective trial of phosphate and 1,25-dihydroxyvitamin D3 therapy in symptomatic adults with X-linked hypophosphatemic rickets. J Clin Endocrinol Metab 1992;75:879e85. [24] Verge CF, Cowell CT, Howard NJ, Donaghue KC, Silink M. Growth in children with X-linked hypophosphataemic rickets. Acta Paediatr Suppl 1993;388:70e6. [25] Steendijk R, Herweijer TJ. Height, sitting height and leg length in patients with hypophosphataemic rickets. Acta Paediatr Scand 1984;73:181e4. [26] Steendijk R, Hauspie RC. The pattern of growth and growth retardation of patients with hypophosphataemic vitamin Dresistant rickets: a longitudinal study. Eur J Pediatr 1992;151:422e7. [27] Balsan S, Tieder M. Linear growth in patients with hypophosphatemic vitamin D-resistant rickets: influence of treatment regimen and parental height [see Comments]. J Pediatr 1990;116:365e71. [28] Friedman NE, Lobaugh B, Drezner MK. Effects of calcitriol and phosphorus therapy on the growth of patients with X-linked hypophosphatemia. J Clin Endocrinol Metab 1993;76:839e44.

PEDIATRIC BONE

720

26. FAMILIAL HYPOPHOSPHATEMIA AND RELATED DISORDERS

[29] McWhorter AG, Seale NS. Prevalence of dental abscess in a population of children with vitamin D-resistant rickets. Pediatr Dent 1991;13:91e6. [30] Abe K, Ooshima T, Lily TS, Yasufuku Y, Sobue S. Structural deformities of deciduous teeth in patients with hypophosphatemic vitamin D-resistant rickets. Oral Surg Oral Med Oral Pathol 1988;65:191e8. [31] Hillmann G, Geurtsen W. Pathohistology of undecalcified primary teeth in vitamin D-resistant rickets: Review and report of two cases. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1996;82:18e24. [32] Seeto E, Seow WK. Scanning electron microscopic analysis of dentin in vitamin D-resistant rickets e assessment of mineralization and correlation with clinical findings. Pediatr Dent 1991;13:43e8. [33] Goodman JR, Gelbier MJ, Bennett JH, Winter GB. Dental problems associated with hypophosphataemic vitamin D resistant rickets. Int J Paediatr Dent 1998;8:19e28. [34] Seow WK, Needleman HL, Holm IA. Effect of familial hypophosphatemic rickets on dental development: a controlled, longitudinal study. Pediatr Dent 1995;17:346e50. [35] Larmas M, Hietala EL, Simila S, Pajari U. Oral manifestations of familial hypophosphatemic rickets after phosphate supplement therapy: A review of the literature and report of case. J Dent Child 1991;58:328e34. [36] Seow WK. The effect of medical therapy on dentin formation in vitamin D-resistant rickets. Pediatr Dent 1991;13:97e102. [37] Velan GJ, Currier BL, Clarke BL, Yaszemski MJ. Ossification of the posterior longitudinal ligament in vitamin D-resistant rickets: case report and review of the literature. Spine 2001;26:590e3. [38] Matsui H, Katoh Y, Tsuji H. Untreated hypophosphatemic vitamin D-resistant rickets with symptomatic ossification of the ligamenturn flavum. J Spinal Disord 1991;4:110e3. [39] Dunlop DJ, Stirling AJ. Thoracic spinal cord compression caused by hypophosphataemic rickets: a case report and review of the world literature. Eur Spine J 1996;5:272e4. [40] Bradbury PG, Brenton DP, Stern GM. Neurological involvement in X-linked hypophosphataemic rickets. J Nenrol Neurosurg Psychiatr 1987;50:810e2. [41] Ballantyne ES, Findlay GF. Thoracic spinal stenosis in two brothers due to vitamin D-resistant rickets. Eur Spine J 1996;5:125e7. [42] Yamamoto Y, Onofrio BM. Spinal canal stenosis with hypophosphatemic vitamin D-resistant rickets: case report. Neurosurgery 1994;35:512e5. [43] Caldemeyer KS, Boaz JC, Wappner RS, Moran CC, Smith RR, Quets JP. Chiari I malformation: association with hypophosphatemic rickets and MR imaging appearance. Radiology 1995;195:733e8. [44] O’Malley SP, Adams JE, Davies M, Ramsden RT. The petrous temporal bone and deafness in X-linked hypophosphataemic osteomalacia. Clin Radiol 1988;39:528e30. [45] Boneh A, Reade TM, Scriver CR, Rishikof E. Audiometric evidence for two forms of X-linked hypophosphatemia in humans, apparent counterparts of Hyp and Gy mutations in mouse. Am J Med Genet 1987;27:997e1003. [46] Meister M, Johnson A, Popelka GR, Kim GS, Whyte MP. Audiologic findings in young patients with hypophosphatemic bone disease. Ann Otol Rhinol Laryngol 1986;95:415e20. [47] O’Malley S, Ramsden RT, Latif A, Kane R, Davies M. Electrocochleographic changes in the hearing loss associated with Xlinked hypophosphataemic osteomalacia. Acta Otolaryngol 1985;100:13e8.

[48] Nehgme R, Fahey JT, Smith C, Carpenter TO. Cardiovascular abnormalities in patients with X-linked hypophosphatemia. J Clin Endocrinol Metab 1997;82:2450e4. [49] Carpenter TO, Mitnick MA, Ellison A, Smith C, Insogna KL. Nocturnal hyperparathyroidism: a frequent feature of X-linked hypophosphatemia. J Clin Endocrinol Metab 1994;78:1378e83. [50] Carpenter TO, Key LL. Disorders of the metabolism of calcium, phosphorus, and other minerals. In: Ichikawa I, editor. Pediatric Textbook of Fluids and Electrolytes. Baltimore: Williams & Wilkins; 1990. p. 237e68. [51] Carpenter TO. New perspectives on the biology and treatment of X-linked hypophosphatemic rickets. Pediatr Clin North Am 1997;44:43e66. [52] Bijvoet OLM. Kidney function in calcium and phosphate metabolism. In: Avioli LV, Krane SM, editors. Metabolic Bone Disease. New York: Academic Press; 1977. p. 49e69. [53] Walton RJ, Bijvoet OL. A simple slide-rule method for the assessment of renal tubular reaborption of phosphate in man. Clin Chim Acta 1977;81:273e6. [54] Walton RJ, Bijvoet OL. Nomogram for derivation of renal threshold phosphate concentration. Lancet 1975;2:309e10. [55] Chesney RW. Fanconi syndrome and renal tubular acidosis. In: Favus MJ, editor. Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. Philadelphia: Lippincotte Raven; 1999. p. 340e3. [56] Francis F, Rowe PS, Econs MJ, et al. A YAC contig spanning the hypophosphatemic rickets disease gene (HYP) candidate region. Genomics 1994;21:229e37. [57] Rowe PS, Goulding J, Read A, et al. Refining the genetic map for the region flanking the X-linked hypophosphataemic rickets locus (Xp22.1e22.2). Hum Genet 1994;93:291e4. [58] Thakker RV, Farmery MR, Sakati NA, Milner RD. Genetic linkage studies of X-linked hypophosphataemic rickets in a Saudi Arabian family. Clin Endocrinol 1992;37:338e43. [59] Alitalo T, Kruse TA, Ahrens P, Albertsen HM, Eriksson AW, Chapelle ADL. Genetic mapping of 12 marker loci in the Xp22.3ep21.2 region. Hum Genet 1991;86:599e603. [60] Thakker RV, Davies KE, Read AP, et al. Linkage analysis of two cloned DNA sequences, DXS197 and DXS207, in hypophosphatemic rickets families. Genomics 1990;8:189e93. [61] Thakker RV, Read AP, Davies KE, et al. Bridging markers defining the map position of X linked hypophosphataemic rickets. J Med Genet 1987;24:756e60. [62] Machler M, Frey D, Gal A, et al. X-linked dominant hypophosphatemia is closely linked to DNA markers DXS41 and DXS43 at Xp22. Hum Genet 1986;73:271e5. [63] Read AP, Thakker RV, Davies KE, et al. Mapping of human X-linked hypophosphataemic rickets by multilocus linkage analysis. Hum Genet 1986;73:267e70. [64] Econs MJ, Barker DF, Speer MC, Pericak-Vance MA, Fain P, Drezner MK. Multilocus mapping of the human X-linked hypophosphatemic rickets gene locus. J Bone Miner Res 1990;5: S108. [65] Econs MJ, Barker DF, Speer MC, Pericak-Vance MA, Fain PR, Drezner MK. Multilocus mapping of the X-linked hypophosphatemic rickets gene. J Clin Endocrinol Metab 1992;75:201e6. [66] Econs MJ, Fain PR, Norman M, et al. Flanking markers define the X-linked hypophosphatemic rickets gene locus. J Bone Miner Res 1993;8:1149e52. [67] Econs MJ, Rowe PS, Francis F, et al. Fine structure mapping of the human X-linked hypophosphatemic rickets gene locus. J Clin Endocrinol Metab 1994;79:1351e4.

PEDIATRIC BONE

REFERENCES

[68] Consortium HYP. A gene (PEX) with homologies to endopeptidases is mutated in patients with X-linked hypophosphatemic rickets. Nature Genet 1995;11:130e6. [69] D’Adamio L, Shipp MA, Masteller EL, Reinherz EL. Organization of the gene encoding common acute lymphoblast miniexons and separate 5’ untranslated regions. Proc Natl Acad Sci USA 1989;86:7103e7. [70] Xu D, Emoto N, Giaid A, et al. ECE-1: a membrane-bound metalloprotease that catalyzes the proteolytic activation of big endothelin-1. Cell 1994;78:473e85. [71] Lee S, Zambas ED, Marsh WL, Redman CM. Molecular cloning and primary structure of Kell blood group protein. Proc Natl Acad Sci USA 1991;88:6353e7. [72] Beck L, Soumounou Y, Martel J, et al. Pex/PEX tissue distribution and evidence for a deletion in the 3’ region of the Pex gene in X-linked hypophosphatemic mice. J Clin Invest 1997;99:1200e9. [73] Grieff M, Mumm S, Waeltz P, et al. Expression and cloning of the human X-linked hypophosphatemia gene cDNA. Biochem Biophys Res Commun 1997;231:635e9. [74] Francis F, Strom TM, Hennig S, et al. Genomic organization of the human PEX gene mutated in X-linked dominant hypophosphatemic rickets. Genome Res 1997;7:573e85. [75] Holm IA, Huang X, Kunkel LM. Mutational analysis of the PEX gene in patients with X-linked hypophosphatemic rickets. Am J Hum Genet 1997;60:790e7. [76] Holm IA, Nelson AE, Robinson BG, et al. Mutational analysis and genotype-phenotype correlation of the PHEX gene in X-linked hypophosphatemic rickets. J Clin Endocrinol Metab 2001;86:3889e99. [77] Dixon PH, Wooding C, Trump D, Schlessinger D, Whyte MP, Thakker RV. Seven novel mutations in the PEX gene indicate molecular heterogeneity for X-linked hypophosphatemic rickets. J Bone Miner Res 1996;11. S136. [78] Dixon PH, Wooding C, Christie P, et al. Twelve novel PEX mutations in X-linked hypophosphatemic rickets indicate molecular heterogeneity. [Abstract OC12]. J Endocrinol 1997:152. [79] Dixon PH, Christie PT, Wooding C, et al. Mutational analysis of PHEX gene in X-linked hypophosphatemia. J Clin Endocrinol Metab 1998;83:3615e23. [80] Rowe PS, Oudet CL, Francis F, et al. Distribution of mutations in the PEX gene in families with X-linked hypophosphataemic rickets (HYP). Hum Mol Genet 1997;6:539e49. [81] Tyynismaa H, Kaitila I, Nanto-Salonen K, Ala-Houhala M, Alitalo T. Identification of fifteen novel PHEX gene mutations in Finnish patients with hypophosphatemic rickets. Hum Mutat 2000;15:383e4. [82] Filisetti D, Ostermann G, Von Bredow M, et al. Non-random distribution of mutations in the PHEX gene, and under-detected missense mutations at non-conserved residues. Eur J Hum Genet 1999;7:615e9. [83] Sato K, Tajima T, Nakae J, et al. Three novel PHEX gene mutations in Japanese patients with X-linked hypophosphatemic rickets. Pediatr Res 2000;48:536e40. [84] Christie PT, Harding B, Nesbit MA, Whyte MP, Thakker RV. Xlinked hypophosphatemia attributable to pseudoexons of the PHEX gene. J Clin Endocrinol Metab 2001;86:3840e4. [85] Consortium ADHR. Autosomal dominant hypophosphataemic rickets is associated with mutations in FGF-23. Nature Genet 2000;26:345e8. [86] Sabbagh Y, Jones AO, Tenenhouse HS. PHEXdb, a locus-specific database for mutations causing X-linked hypophosphatemia. Hum Mutat 2000;16:1e6.

721

[87] Winters RW, Graham JB, Williams TF, McFalls VW, Burnett CH. A genetic study of familial hypophosphatemia and vitamin Dresistant rickets with a review of the literature. Medicine 1958;37:97. [88] Whyte MP, Schranck FW, Armamento-Villareal R. X-linked hypophosphatemia: a search for gender, race, anticipation, or parent of origin effects on disease expression in children. J Clin Endocrinol Metab 1996;81:4075e80. [89] Shields ED, Scriver CR, Reade T, et al. X-linked hypophosphatemia: the mutant gene is expressed in teeth as well as in kidney. Am J Hum Genet 1990;46:434e42. [90] Scriver CR, Tenenhouse HS. X-linked hypophosphataemia: a homologous phenotype in humans and mice with unusual organ-specific gene dosage. J Inherit Metab Dis 1992;15:610e24. [91] Petersen DJ, Boniface AM, Schranck FW, Rupich RC, Whyte MP. X-linked hypophosphatemic rickets: a study (with literature review) of linear growth response to calcitriol and phosphate therapy. J Bone Miner Res 1992;7:583e97. [92] Hardy DC, Murphy WA, Siegel BA, Reid IR, Whyte MP. Xlinked hypophosphatemia in adults: prevalence of skeletal radiographic and scintigraphic features. Radiology 1989;171:403e14. [93] Orstavi KH, Orstavik RE, Halse J, Knudtzon J. X chromosome inactivation pattern in female carriers of X linked hypophosphataemic rickets. J Med Genet 1996;33:700e3. [94] Gattineni J, Baum M. Regulation of phosphate transport by fibroblast growth factor 23 (FGF-23): implications for disorders of phosphate metabolism. Pediatr Nephrol 2010;25:591e601. [95] Ramon I, Kleynen P, Body JJ, Karmali R. Fibroblast growth factor 23 and its role in phosphate homeostasis. Eur J Endocrinol 2010;162:1e10. [96] Strom TM, Ju¨ppner H. PHEX, FGF-23, DMP1 and beyond. Curr Opin Nephrol Hypertens 2008;17:357e62. [97] Seikaly MG, Brown R, Baum M. The effect of recombinant human growth hormone in children with X-linked hypophosphatemia. Pediatrics 1997;100:879e84. [98] Haffner D, Wuhl E, Blum WF, Schaefer F, Mehls O. Disproportionate growth following long-term growth hormone treatment in short children with X-linked hypophosphataemia. Eur J Pediatr 1995;154:610e3. [99] Patel L, Clayton PE, Brain C, et al. Acute biochemical effects of growth hormone treatment compared with conventional treatment in familial hypophosphataemic rickets. Clin Endocrinol 1996;44:687e96. [100] Alon US, Levy-Olomucki R, Moore WV, Stubbs J, Liu S, Quarles LD. Calcimimetics as an adjuvant treatment for familial hypophosphatemic rickets. Clin J Am Soc Nephrol 2008;3:658e64. [101] Alon U, Chan JC. Effects of hydrochlorothiazide and amiloride in renal hypophosphatemic rickets. Pediatrics 1985;75:754e63. [102] Dietz FR. A primer of osteotomy of the weight bearing long bones in children. Iowa Orthop J 1993;13:136e48. [103] Rubinovitch M, Said SE, Glorieux FH, Cruess RL, Rogala E. Principles and results of corrective lower limb osteotomies for patients with vitamin D-resistant hypophosphatemic rickets. Clin Orthop Rel Res 1988;237:264e70. [104] Kanel JS, Price CT. Unilateral external fixation for corrective osteotomies in patients with hypophosphatemic rickets. J Pediatr Orthop 1995;15:232e5. [105] Novais E, Stevens PM. Hypophosphatemic rickets: the role of hemiepiphysiodesis. J Pediatr Orthop 2006;26:238e44. [106] Bettinelli A, Bianchi ML, Mazzucchi E, Gandolini G, Appiani AC. Acute effects of calcitriol and phosphate salts on mineral metabolism in children with hypophosphatemic rickets. J Pediatr 1991;118:372e6.

PEDIATRIC BONE

722

26. FAMILIAL HYPOPHOSPHATEMIA AND RELATED DISORDERS

[107] Yamaoka K, Tanaka H, Kurose H, et al. Effect of single oral phosphate loading on vitamin D metabolites in normal subjects and in X-linked hypophosphatemic rickets. Bone Miner 1989;7:159e69. [108] Verge CF, Lam A, Simpson JM, Cowell CT, Howard NJ, Silink M. Effects of therapy in X-linked hypophosphatemic rickets. N Engl J Med 1991;325:1843e8. [109] Kooh SW, Binet A, Daneman A. Nephrocalcinosis in X-linked hypophosphataemic rickets: its relationship to treatment, kidney function, and growth. Clin Invest Med 1994;17:123e30. [110] Reusz GS, Latta K, Hoyer PF, Byrd DJ, Ehrich JH, Brodehl J. Evidence suggesting hyperoxaluria as a cause of nephrocalcinosis in phosphate-treated hypophosphataemic rickets. Lancet 1990;335:1240e3. [111] Alon U, Donaldson DL, Hellerstein S, Warady BA, Harris DJ. Metabolic and histologic investigation of the nature of nephrocalcinosis in children with hypophosphatemic rickets and in the Hyp mouse. J Pediatr 1992;120:899e905. [112] Polisson RP, Martinez S, Khoury M, et al. Calcification of entheses associated with X-linked hypophosphatemic osteomalacia. N Engl J Med 1985;313:1e6. [113] Caldemeyer KS, Smith RR, Edwards-Brown MK. Familial hypophosphatemic rickets causing ocular calcification and optic canal narrowing. Am J Neuroradiol 1995;16:1252e4. [114] Moltz KC, Friedman AH, Nehgme RA, Kleinman CS, Carpenter TO. Ectopic cardiac calcification associated with hyperparathyroidism in a boy with hypophosphatemic rickets. Curr Opin Pediatr 2001;13:373e5. [115] Bianchine JW, Stambler AA, Harrison HE. Familial hypophosphatemic rickets showing autosomal dominant inheritance. Birth Defects Orig Artic Ser 1971;7:287e94. [116] Econs MJ, McEnery PT. Autosomal dominant hypophosphatemic rickets/osteomalacia: clinical characterization of a novel renal phosphate-wasting disorder. J Clin Endocrinol Metab 1997;82:674e81. [117] Imel E, Gray A, Econs M. FGF-23 is related to serum iron concentrations in ADHR. J Bone Miner Res(S1). Available at, http://www.asbmr.org/Meetings/AnnualMeeting/ AbstractDetail.aspx?aid¼d5dcac8e-264e-47d3-9291b37ecb57c8e6, 2009;24. Accessed February 10, 2011. [118] Imel EA, Hui SL, Econs MJ. FGF-23 concentrations vary with disease status in autosomal dominant hypophosphatemic rickets. J Bone Miner Res 2007;22:520e6. [119] Feng JQ, Ward LM, Liu S, et al. Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism. Nat Genet 2006;38:1310e5. [120] Lorenz-Depiereux B, Bastepe M, Benet-Page`s A, et al. DMP1 mutations in autosomal recessive hypophosphatemia implicate a bone matrix protein in the regulation of phosphate homeostasis. Nat Genet 2006;38:1248e50. [121] Zhang B, Sun Y, Chen L, Guan C, Guo L, Qin C. Expression and distribution of SIBLING proteins in the predentin/dentin and mandible of hyp mice. Oral Dis 2010;16:453e64. [122] Harmey D, Hessle L, Narisawa S, Johnson KA, Terkeltaub R, Milla´n JL. Concerted regulation of inorganic pyrophosphate and osteopontin by akp2, enpp1, and ank: an integrated model of the pathogenesis of mineralization disorders. Am J Pathol 2004;164:1199e209. [123] Lorenz-Depiereux B, Schnabel D, Tiosano D, Ha¨usler G, Strom TM. Loss-of-function ENPP1 mutations cause both generalized arterial calcification of infancy and autosomalrecessive hypophosphatemic rickets. Am J Hum Genet 2010;86:267e72. [124] Levy-Litan V, Hershkovitz E, Avizov L, et al. Autosomalrecessive hypophosphatemic rickets is associated with an

[125]

[126]

[127]

[128]

[129]

[130]

[131]

[132]

[133]

[134]

[135]

[136]

[137] [138]

[139] [140]

[141]

[142]

inactivation mutation in the ENPP1 gene. Am J Hum Genet 2010;86:273e8. Rutsch F, Ruf N, Vaingankar S, et al. Mutations in ENPP1 are associated with ‘idiopathic’ infantile arterial calcification. Nat Genet 2003;34:379e81. Rutsch F, Bo¨yer P, Nitschke Y, et al. GACI Study Group. Hypophosphatemia, hyperphosphaturia, and bisphosphonate treatment are associated with survival beyond infancy in generalized arterial calcification of infancy. Circ Cardiovasc Genet 2008;1:133e40. Tieder M, Modai D, Samuel R, et al. Hereditary hypophosphatemic rickets with hypercalciuria. N Engl J Med 1985;312:611e7. Chen C, Carpenter T, Steg N, Baron R, Anast C. Hypercalciuric hypophosphatemic rickets, mineral balance, bone histomorphometry, and therapeutic implications of hypercalciuria. Pediatrics 1989;84:276e80. Tieder M, Modai D, Shaked U, et al. “Idiopathic” hypercalciuria and hereditary hypophosphatemic rickets. Two phenotypical expressions of a common genetic defect. N Engl J Med 1987;316:125e9. Pak CYC. Kidney stones. In: Wilson JD, Foster DW, Kronenberg HM, Larsen PR, editors. Williams Textbook of Endocrinology. Philadelphia: Saunders; 1998. p. 1241e57. Jones A, Tzenova J, Frappier D, et al. Hereditary hypophosphatemic rickets with hypercalciuria is not caused by mutations in the Na/Pi cotransporter NPT2 gene. J Am Soc Nephrol 2001;12:507e14. Van den Heuvel L, Op de Koul K, Knots E, Knoers N, Monnens L. Autosomal recessive hypophosphataemic rickets with hypercalciuria is not caused by mutations in the type II renal sodium/phosphate cotransporter gene. Nephrol Dial Transplant 2001;16:48e51. Jaureguiberry G, Carpenter TO, Forman S, Ju¨ppner H, Bergwitz C. A novel missense mutation in SLC34A3 that causes hereditary hypophosphatemic rickets with hypercalciuria in humans identifies threonine 137 as an important determinant of sodium-phosphate cotransport in NaPi-IIc. Am J Physiol Renal Physiol 2008;295:F371e9. Magen D, Berger L, Coady MJ, et al. A loss-of-function mutation in NaPi-IIa and renal Fanconi’s syndrome. N Engl J Med 2010;362:1102e9. Klein GL, Dallas JS, Hawkins HK, Swischuk LE, McCauley RL. Congenital linear sebaceous nevus syndrome. J Bone Miner Res 1998;13:1056e7. Drezner MK. Tumor-induced rickets and osteomalacia. In: Favus MJ, editor. Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. Philadelphia: LippincottRaven; 1999. p. 331e7. Agus ZS. Oncogenic hypophosphatemic osteomalacia. Kidney Int 1983;24:113e23. Eyskens B, Proesmans W, Van Damme B, Lateur L, Bouillon R, Hoogmartens M. Tumour-induced rickets: a case report and review of the literature. Eur J Pediatr 1995;154:462e8. Schapira D, Ben Izhak O, Nachtigal A, et al. Tumor-induced osteomalacia. Sem Arthritis Rheum 1995;25:35e46. Cheng CL, Ma J, Wu PC, Mason RS, Posen S. Osteomalacia secondary to osteosarcoma. A case report. J Bone Joint Surg Am 1989;71:288e92. Weidner N, Santa Cruz D. Phosphaturic mesenchymal tumors. A polymorphous group causing osteomalacia or rickets. Cancer 1987;59:1442e54. Folpe AL, Fanburg-Smith JC, Billings SD, et al. Most osteomalacia-associated mesenchymal tumors are a single

PEDIATRIC BONE

REFERENCES

[143]

[144]

[145]

[146]

[147]

[148] [149]

[150]

[151]

[152]

[153]

[154]

[155]

[156]

[157]

[158]

[159]

[160]

histopathologic entity: an analysis of 32 cases and a comprehensive review of the literature. Am J Surg Pathol 2004;28:1e30. Nguyen BD, Wang EA. Indium-111 pentetreotide scintigraphy of mesenchymal tumor with oncogenic osteomalacia. Clin Nucl Med 1999;24:130e1. Ohashi K, Ohnishi T, Ishikawa T, Tani H, Uesugi K, Takagi M. Oncogenic osteomalacia presenting as bilateral stress fractures of the tibia. Skeletal Radiol 1999;28:46e8. Reyes-Mugica M, Arnsmeier SL, Backeljauw PF, Persing J, Ellis B, Carpenter TO. Phosphaturic mesenchymal tumorinduced rickets. Pediatr Dev Pathol 2000;3:61e9. Shane E, Parisien M, Henderson JE, et al. Tumor-induced osteomalacia: clinical and basic studies. J Bone Miner Res 1997;12:1502e11. Yeung SJ, McCutcheon IE, Schultz P, Gagel RF. Use of long-term intravenous phosphate infusion in the palliative treatment of tumor-induced osteomalacia. J Clin Endocrinol Metab 2000;85:549e55. Seufert J, Ebert K, Muller J, et al. Octreotide therapy for tumorinduced osteomalacia. N Engl J Med 2001;345:1883e8. Aschinberg LC, Solomon LM, Zeis PM, Justice P, Rosenthal IM. Vitamin D-resistant rickets associated with epidermal nevus syndrome: demonstration of a phosphaturic substance in the dermal lesions. J Pediatr 1977;91:56e60. White KE, Jonsson KB, Cam G, et al. The autosomal dominant hypophosphatemic rickets (ADHR) gene is a secreted polypeptide overexpressed by tumors that cause phosphate wasting. J Clin Endocrinol Metab 2001;86:497e500. Bowe AE, Finnegan R, Jan de Beur SM, et al. FGF-23 inhibits renal tubular phosphate transport and is a PHEX substrate. Biochem Biophys Res Commun 2001;284:977e81. Shimada T, Mizutani S, Muto T, et al. Cloning and characterization of FGF-23 as a causative factor of tumor-induced osteomalacia. Proc Natl Acad Sci USA 2001;98:6500e5. Shimada T, Urakawa I, Yamazaki Y, et al. FGF-23 Transgenic mice demonstrate hypophosphatemic rickets with reduced expression of sodium phosphate cotransporter type IIa. Biochem Biophys Res Common 2004;314:409e14. Kumar R. New insights into phosphate homeostasis: fibroblast growth factor 23 and frizzled-related protein-4 are phosphaturic factors derived from tumors associated with osteomalacia. Curr Opin Nephrol Hypertens 2002;11:547e53. Schiavi SC, Moe OW. Phosphatonins: a new class of phosphateregulating proteins. Curr Opin Nephrol Hypertens 2002;11:423e30. Vassiliadis J, Jan de Beur SM, Bowe AE, et al. Frizzled related protein 4 expression is elevated in tumors associated with oncogenic osteomalacia and inhibits phosphate transport in vitro. J Bone Miner Res 2001;16:S225. Olivares JL, Ramos FJ, Carapeto FJ, Bueno M. Epidermal naevus syndrome and hypophosphataemic rickets: description of a patient with central nervous system anomalies and review of the literature. Eur J Pediatr 1999;158:103e7. Heike CL, Cunningham ML, Steiner RD, et al. Skeletal changes in epidermal nevus syndrome: does focal bone disease harbor clues concerning pathogenesis? Am J Med Genet A 2005;139A:67e77. Hoffman WH, Jueppner HW, Deyoung BR, O’dorisio MS, Given KS. Elevated fibroblast growth factor-23 in hypophosphatemic linear nevus sebaceous syndrome. Am J Med Genet 2005;134:233e6. Sethi SK, Hari P, Bagga A. Elevated FGF-23 and parathormone in linear nevus sebaceous syndrome with resistant rickets. Pediatr Nephrol 2010;25:1577e8.

723

[161] Hafner C, van Oers JM, Vogt T, et al. Mosaicism of activating FGFR3 mutations in human skin causes epidermal nevi. J Clin Invest 2006;116:2201e7. [162] Collins MT, Chebli C, Jones J, et al. Renal phosphate wasting in fibrous dysplasia of bone is part of a generalized renal tubular dysfunction similar to that seen in tumor-induced osteomalacia. J Bone Miner Res 2001;16:806e13. [163] Dent CE, Gertner JM. Hypophosphataemic osteomalacia in fibrous dysplasia. Q J Med 1976;45:411e20. [164] Riminucci M, Collins MT, Fedarko NS, et al. FGF-23 in fibrous dysplasia of bone and its relationship to renal phosphate wasting. J Clin Invest 2003;112:683e92. [165] John MR, Wickert H, Zaar K, et al. A case of neuroendocrine oncogenic osteomalacia associated with a PHEX and fibroblast growth factor-23 expressing sinusoidal malignant schwannoma. Bone 2001;29:393e402. [166] Konishi K, Nakamura M, Yamakawa H, et al. Hypophosphatemic osteomalacia in von Recklinghausen neurofibromatosis. Am J Med Sci 1991;301:322e8. [167] Eicher EM, Southard JL, Scriver CR, Glorieux FH. Hypophosphatemia: mouse model for human familial hypophosphatemic (vitamin D-resistant) rickets. Proc Natl Acad Sci USA 1976;73:4667e71. [168] Lyon MF, Scriver CR, Baker LR, Tenenhouse HS, Kronick J, Mandla S. The Gy mutation: another cause of X-linked hypophosphatemia in mouse. Proc Natl Acad Sci USA 1986;83:4899e903. [169] O’Doherty PJ, DeLuca HF, Eicher EM. Lack of effect of vitamin D and its metabolites on intestinal phosphate transport in familial hypophosphatemia of mice. Endocrinology 1977;101:1325e30. [170] Tenenhouse HS, Scriver CR. The defect in transcellular transport of phosphate in the nephron is located in brush-border membranes in X-linked hypophosphatemia (Hyp mouse model). Can J Biochem 1978;56:640e6. [171] Tenenhouse HS, Scriver CR, McInnes RR, Glorieux FH. 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 1978;14:236e44. [172] Tenenhouse HS, Scriver CR, Vizel EJ. Alkaline phosphatase activity does not mediate phosphate transport in the renalcortical brush-border membrane. Biochem J 1980;190:473e6. [173] Tenenhouse HS, Klugerman AH, Neal JL. Effect of phosphonoformic acid, dietary phosphate and the Hyp mutation on kinetically distinct phosphate transport processes in mouse kidney. Biochim Biophys Acta 1989;984:207e13. [174] Perwad F, Azam N, Zhang MY, Yamashita T, Tenenhouse HS, Portale AA. Dietary and serum phosphorus regulate fibroblast growth factor 23 expression and 1,25-dihydroxyvitamin D metabolism in mice. Endocrinology 2005;146:5358e64. [175] Kiebzak GM, Roos BA, Meyer Jr RA. Secondary hyperparathyroidism in X-linked hypophosphatemic mice. Endocrinology 1982;111:650e2. [176] Posillico JT, Lobaugh B, Muhlbaier LH, Drezner MK. Abnormal parathyroid function in the X-linked hypophosphatemic mouse. Calcif Tissue Int 1985;37:418e22. [177] Meyer Jr RA, Meyer MH, Morgan PL. Effects of altered diet on serum levels of 1,25-dihydroxyvitamin D and parathyroid hormone in X-linked hypophosphatemic (Hyp and Gy) mice. Bone 1996;18:23e8. [178] Ecarot-Charrier B, Glorieux FH, Travers R, Desbarats M, Bouchard F, Hinek A. Defective bone formation by transplanted Hyp mouse bone cells into normal mice. Endocrinology 1988;123:768e73.

PEDIATRIC BONE

724

26. FAMILIAL HYPOPHOSPHATEMIA AND RELATED DISORDERS

[179] Marie PJ, Travers R, Glorieux FH. Healing of rickets with phosphate supplementation in the hypophosphatemic male mouse. J Clin Invest 1981;67:911e4. [180] Marie PJ, Travers R, Glorieux FH. Bone response to phosphate and vitamin D metabolites in the hypophosphatemic male mouse. Calcif Tissue Int 1982;34:158e64. [181] Ecarot B, Glorieux FH, Desbarats M, Travers R, Labelle L. Defective bone formation by Hyp mouse bone cells transplanted into normal mice: evidence in favor of an intrinsic osteoblast defect. J Bone Miner Res 1992;7:215e20. [182] Ecarot B, Glorieux FH, Desbarats M, Travers R, Labelle L. Effect of 1,25-dihydroxyvitamin D3 treatment on bone formation by transplanted cells from normal and X-linked hypophosphatemic mice. J Bone Miner Res 1995;10:424e31. [183] Ecarot B, Glorieux FH, Desbarats M, Travers R, Labelle L. Effect of dietary phosphate deprivation and supplementation of recipient mice on bone formation by transplanted cells from normal and X-linked hypophosphatemic mice. J Bone Miner Res 1992;7:523e30. [184] Iorio RJ, Bell WA, Meyer MH, Meyer Jr RA. Radiographic evidence of craniofacial and dental abnormalities in the Xlinked hypophosphatemic mouse. Ann Dent 1979;38:31e7. [185] Iorio RJ, Bell WA, Meyer MH, Meyer Jr RA. Histologic evidence of calcification abnormalities in teeth and alveolar bone of mice with X-linked dominant hypophosphatemia (VDRR). Ann Dent 1979;38:38e44. [186] Sofaer JA, Southam JC. Naturally-occurring exposure of the dental pulp in mice with inherited hypophosphataemia. Arch Oral Biol 1982;27:701e3. [187] Abe K, Ooshima T, Masatomi Y, Sobue S, Moriwaki Y. Microscopic and crystallographic examinations of the teeth of the Xlinked hypophosphatemic mouse. J Dent Res 1989;68:1519e24. [188] Abe K, Masatomi Y, Nakajima Y. The occurrence of interglobular dentin in incisors of hypophosphatemic mice fed a high-calcium and high-phosphate diet. J Dental Res 1992;71:478e83. [189] Yamaoka K, Seino Y, Satomura K, Tanaka Y, Yabuuchi H, Haussier MR. Abnormal relationship between serum phosphate concentration and renal 25-hydroxycholecalciferol-1-alphahydroxylase activity in X-linked hypophosphatemic mice. Miner Electrolyte Metab 1986;12:194e8. [190] Tenenhouse HS, Jones G. Abnormal regulation of renal vitamin D catabolism by dietary phosphate in murine X-linked hypophosphatemic rickets. J Clin Invest 1990;85:1450e5. [191] Roy S, Martel J, Ma S, Tenenhouse HS. Increased renal 25hydroxyvitamin D3-24-hydroxylase messenger ribonucleic acid and immunoreactive protein in phosphate-deprived Hyp mice: a mechanism for accelerated 1,25-dihydroxyvitamin D3 catabolism in X-linked hypophosphatemic rickets. Endocrinology 1994;134:1761e7. [192] Meyer RA, Gray RW, Meyer MH. Abnormal vitamin D metabolism in the X-linked hypophosphatemic mouse. Endocrinology 1980;107:1577e81. [193] Lobaugh B, Drezner MK. Abnormal regulation of renal 25hydroxyvitamin D-1 alpha-hydroxylase activity in the X-linked hypophosphatemic mouse. J Clin Invest 1983;71:400e3. [194] Roy S, Tenenhouse HS. Transcriptional regulation and renal localization of 1,25-dihydroxyvitamin D3-24-hydroxylase gene expression: effects of the Hyp mutation and 1,25-dihydroxyvitamin D3. Endocrinology 1996;137:2938e46. [195] Tenenhouse HS, Martel J. Renal adaptation to phosphate deprivation: lessons from the X-linked Hyp mouse. Pediatr Nephrol 1993;7:312e8.

[196] Carpenter TO, Shiratori T. Renal 25ehydroxyvitamin D-1 alpha-hydroxylase activity and mitochondrial phosphate transport in Hyp mice. Am J Physiol 1990;259:E814e21. [197] Tenenhouse HS. Metabolism of 25-hydroxyvitamin D3 in renal slices from the X-linked hypophosphatemic (Hyp) mouse: abnormal response to fall in serum calcium. Cell Calcium 1984;5:43e55. [198] Tenenhouse HS. Abnormal renal mitochondrial 25-hydroxyvitamin D3-1-hydroxylase activity in the vitamin D and calcium deficient X-linked Hyp mouse. Endocrinology 1983;113:816e8. [199] Nesbitt T, Drezner MK, Lobaugh B. Abnormal parathyroid hormone stimulation of 25-hydroxyvitamin D-1 alpha-hydroxylase activity in the hypophosphatemic mouse. Evidence for a generalized defect of vitamin D metabolism. J Clin Invest 1986;77:181e7. [200] Nesbitt T, Lobaugh B, Drezner MK. Calcitonin stimulation of renal 25-hydroxyvitamin D-1 alpha-hydroxylase activity in hypophosphatemic mice. Evidence that the regulation of calcitriol production is not universally abnormal in X-linked hypophosphatemia. J Clin Invest 1987;79:15e9. [201] Tenenhouse HS, Scriver CR. Effect of 1,25-dihydroxyvitamin D3 on phosphate homeostasis in the X-linked hypophosphatemic (Hyp) mouse. Endocrinology 1981;109:658e60. [202] Perwad F, Zhang MY, Tenenhouse HS, Portale AA. Fibroblast growth factor 23 impairs phosphorus and vitamin D metabolism in vivo and suppresses 25-hydroxyvitamin D-1alphahydroxylase expression in vitro. Am J Physiol Renal Physiol 2007;293:F1577e83. [203] Ecarot-Charrier B, Glorieux FH. Effects of phosphate and 1,25(OH)2D3 on in vitro bone collagen synthesis in the hypophosphatemic mouse. Calcif Tissue Int 1983;35:383e91. [204] Yamamoto T, Ecarot B, Glorieux FH. Abnormal response of osteoblasts from Hyp mice to 1,25-dihydroxyvitamin D3. Bone 1992;13:209e15. [205] Tenenhouse HS, Fast DK, Scriver CR, Koltay M. Intestinal transport of phosphate anion is not impaired in the Hyp (hypophosphatemic) mouse. Biochem Biophys Res Commun 1981;100:537e43. [206] Nakagawa N, Ghishan FK. Sodium-phosphate transport in the kidney and intestine of the hypophosphatemic mouse. Pediatr Nephrol 1993;7:815e8. [207] Brault BA, Meyer MH, Meyer Jr RA. Malabsorption of phosphate by the intestines of young X-linked hypophosphatemic mice. Calcif Tissue Int 1988;43:289e93. [208] Meyer MH, Meyer Jr RA, Iorio RJ. A role for the intestine in the bone disease of juvenile X-linked hypophosphatemic mice: malabsorption of calcium and reduced skeletal mineralization. Endocrinology 1984;115:1464e70. [209] Meyer Jr RA, Meyer MH, Erickson PR, Korkor AB. Reduced absorption of 45calcium from isolated duodenal segments in vivo in juvenile but not adult X-linked hypophosphatemic mice. Calcif Tissue Int 1986;38:95e102. [210] Bruns ME, Meyer Jr RA, Meyer MH. Low levels of intestinal vitamin D-dependent calcium-binding protein in juvenile Xlinked hypophosphatemic mice. Endocrinology 1984;115:1459e63. [211] Bruns ME, Christakos S, Huang YC, Meyer MH, Meyer RA. Vitamin D-dependent calcium binding proteins in the kidney and intestine of the X-linked hypophosphatemic mouse: changes with age and responses to 1,25-dihydroxycholecalciferol. Endocrinology 1987;121:1e6. [212] Seino Y, Sierra RI, Ichikawa M, Avioli LV. I Alpha, 25-dihydroxyvitamin D3 receptor in the X-linked hypophosphatemic mouse. Endocrinology 1982;111:329e31.

PEDIATRIC BONE

REFERENCES

[213] Nakajima S, Yamaoka K, Okada S, Pike JW, Seino Y, Haussier MR. 1,25-Dihydroxyvitamin D3 does not up-regulate vitamin D receptor messenger ribonucleic acid levels in hypophosphatemic mice. Bone Miner 1992;19:201e13. [214] Meyer Jr RA, Meyer MH, Gray RW, Bruns ME. Evidence that low plasma 1,25-dihydroxyvitamin D causes intestinal malabsorption of calcium and phosphate in juvenile X-linked hypophosphatemic mice. J Bone Miner Res 1987;2:67e82. [215] Yamamoto T, Seino Y, Yamaoka K, Yabuuchi H. Decreased nuclear uptake of 1,25-dihydroxyvitamin D3 by duodenal mucosal cells in the X-linked hypophosphatemic mouse. Endocrinology 1985;117:2252e4. [216] Yamamoto T, Seino Y, Tanaka H, et al. Effects of the administration of phosphate on nuclear 1,25-dihydroxyvitamin D3 uptake by duodenal mucosal cells of Hyp mice. Endocrinology 1988;122:576e80. [217] Du L, Desbarats M, Viel J, Glorieux FH, Cawthorn C, Ecarot B. cDNA cloning of the murine Pex gene implicated in X-linked hypophosphatemia and evidence for expression in bone. Genomics 1996;36:22e8. [218] Strom TM, Francis F, Lorenz B, et al. Pex gene deletions in Gy and Hyp mice provide mouse models for X-linked hypophosphatemia. Hum Mol Genet 1997;6:165e71. [219] Guo R, Quarles LD. Cloning and sequencing of human PEX from a bone cDNA library: evidence for its developmental state-specific regulation in osteoblasts. J Bone Miner Res 1997;12:1009e17. [220] Wang L, Du L, Ecarot B. Evidence for Phex haploinsufficiency in murine X-linked hypophosphatemia. Mamm Genome 1999;10:385e9. [221] Grieff M, Whyte MP, Thakker RV, Mazzarella R. Sequence analysis of 139 kb in Xp22.1 containing spermine synthase and the 5’ region of PEX. Genomics 1997;44:227e31. [222] Lorenz B, Francis F, Gempel K, et al. Spermine deficiency in Gy mice caused by deletion of the spermine synthase gene. Hum Mol Genet 1998;7:541e7. [223] Meyer Jr RA, Henley CM, Meyer MH, et al. Partial deletion of both the spermine synthase gene and the Pex gene in the Xlinked hypophosphatemic, gyro (Gy) mouse. Genomics 1998;48:289e95. [224] Ruchon AF, Tenenhouse HS, Marcinkiewicz M, et al. Developmental expression and tissue distribution of Phex protein: effect of the Hyp mutation and relationship to bone markers. J Bone Miner Res 2000;15:1440e50. [225] Lipman ML, Panda D, Bennett HP, et al. Cloning of human PEX cDNA. Expression, subcellular localization, and endopeptidase activity. J Biol Chem 1998;273:13729e37. [226] Ruchon AF, Marcinkiewicz M, Siegfried G, et al. Pex mRNA is localized in developing mouse osteoblasts and odontoblasts. J Histochem Cytochem 1998;46:459e68. [227] Ecarot B, Desbarats M. 1,25-(OH)2D3 down-regulates expression of Phex, a marker of the mature osteoblast. Endocrinology 1999;140:1192e9. [228] Meyer Jr RA, Young CG, Meyer MH, Garges PL, Price DK. Effect of age on the expression of Pex (Phex) in the mouse. Calcif Tissue Int 2000;66:282e7. [229] Blydt-Hansen TD, Tenenhouse HS, Goodyer P. PHEX expression in parathyroid gland and parathyroid hormone dysregulation in X-linked hypophosphatemia. Pediatr Nephrol 1999;13:607e11. [230] Miyamura T, Tanaka H, Inoue M, Ichinose Y, Seino Y. The effects of bone marrow transplantation on X-linked hypophosphatemic mice. J Bone Miner Res 2000;15:1451e8.

725

[231] Liu S, Guo R, Tu Q, Quarles LD. Overexpression of Phex in osteoblasts fails to rescue the Hyp mouse phenotype. J Biol Chem 2002;277:3686e97. [232] Boskey A, Frank A, Fujimoto Y, et al. The PHEX transgene corrects mineralization defects in 9-month-old hypophosphatemic mice. Calcif Tissue Int 2009;84:126e37. [233] Sabbagh Y, Boileau G, DesGroseillers L, Tenenhouse HS. Disease-causing missense mutations in the PHEX gene interfere with membrane targeting of the recombinant protein. Hum Mol Genet 2001;10:1539e46. [234] Boileau G, Tenenhouse HS, Desgroseillers L, Crine P. Characterization of PHEX endopeptidase catalytic activity: identification of parathyroid-hormone-related peptide 107e139 as a substrate and osteocalcin, PPi and phosphate as inhibitors. Biochem J 2001;355:707e13. [235] Liu S, Guo R, Simpson LG, Xiao ZS, Burnham CE, Quarles LD. Regulation of fibroblastic growth factor 23 expression but not degradation by PHEX. J Biol Chem 2003;278:37419e26. [236] Ichikawa S, Austin AM, Gray AK, Econs M. Phex mutations in a murine model of X-linked hypophosphatemia result in impaired phosphate sensing. J Bone Miner Res(S1). Available at: http:// www.asbmr.org/Meetings/AnnualMeeting/AbstractDetail.aspx? aid¼3e23c74e-dd91-461b-b631-4fc47be274a9, 2010;25. Accessed February 10, 2011. [237] Imel EA, DiMeglio LA, Hui SL, Carpenter TO, Econs MJ. Treatment of X-linked hypophosphatemia with calcitriol and phosphate increases circulating fibroblast growth factor 23 concentrations. J Clin Endocrinol Metab 2010;95:1846e50. [238] Carpenter TO, Insogna KL, Zhang JH, et al. Circulating levels of soluble klotho and FGF-23 in X-linked hypophosphatemia: circadian variance, effects of treatment, and relationship to parathyroid status. J Clin Endocrinol Metab 2010;95:E352e7. [239] Econs MJ, McEnery PT, Lennon F, Speer MC. Autosomal dominant hypophosphatemic rickets is linked to chromosome 12p13. J Clin Invest 1997;100:2653e7. [240] Liu S, Zhou J, Tang W, Jiang X, Rowe DW, Quarles LD. Pathogenic role of FGF-23 in Hyp mice. Am J Physiol Endocrinol Metab 2006;29:E38e49. [241] Meyer RA. Elevated mRNA gene expression of fibroblast growth factor 23 (FGF-23) in the thymus of X-linked hypophosphatemic (Hyp) mice. J Bone Miner Res 2001;16(S251). Available at: http:// www.abstractsonline.com/viewer/viewAbstractPrintFriendly. asp?CKey¼{F45961FC-93EF-4C35-86A5-B4796302EA43}&SKey¼ {EB5BC322-4BB8-47A0-92C9-F1C5DE967B90}&MKey¼A3C75A6C -2DD6-4EB3-9C22-D35081DC2990&AKey¼{D0C01D4F-E23B45E2-ACD4-0AF8AC866B8B} Accessed February 10, 2011. [242] White KE, Carn G, Lorenz-Depiereux B, Benet-Pages A, Strom TM, Econs MJ. Autosomal-dominant hypophosphatemic rickets (ADHR) mutations stabilize FGF-23. Kidney Int 2001;60:2079e86. [243] Seidah NG, Chretien M. Proprotein and prohormone convertases: a family of subtilases generating diverse bioactive polypeptides. Brain Res 1999;848:45e62. [244] Kurosu H, Ogawa Y, Miyoshi M, et al. Regulation of fibroblast growth factor-23 signaling by klotho. J Biol Chem 2006;281:6120e3. [245] Kuro-o M, Matsumura Y, Aizawa H, et al. Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature 1997;390:45e51. [246] Kurosu H, Kuro-O M. The Klotho gene family as a regulator of endocrine fibroblast growth factors. Mol Cell Endocrinol 2009;299:72e8. [247] Razzaque MS, Sitara D, Taguchi T, St-Arnaud R, Lanske B. Premature aging-like phenotype in fibroblast growth factor 23

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[248]

[249]

[250]

[251]

[252]

[253]

[254]

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null mice is a vitamin D mediated process. FASEB J 2006;20:720e2. Ichikawa S, Imel EA, Kreiter ML, et al. A homozygous missense mutation in human KLOTHO causes severe tumoral calcinosis. J Clin Invest 2007;117:2684e91. Brownstein CA, Adler F, Nelson-Williams C, et al. A translocation causing increased alpha-klotho level results in hypophosphatemic rickets and hyperparathyroidism. Proc Natl Acad Sci USA 2008;105:3455e60. Farrow EG, Summers LJ, Schiavi SC, McCormick JA, Ellison DH, White KE. Altered renal FGF-23-mediated activity involving MAPK and Wnt: effects of the Hyp mutation. J Endocrinol 2010;207:67e75. Urakawa I, Yamazaki Y, Shimada T, et al. Klotho converts canonical FGF receptor into a specific receptor for FGF-23. Nature 2006;444:770e4. Li H, Martin AC, David V, Quarles LD. Compound deletion of Fgfr3 and Fgfr4 partially rescues the Hyp mouse phenotype. Am J Physiol Endocrinol Metab 2010;Dec 7. [Epub ahead of print.]. Beck L, Karaplis AC. Amizuka N, Hewson AS, Ozawa H, Tenenhouse HS. Targeted inactivation of Npt2 in mice leads to severe renal phosphate wasting, hypercalciuria, and skeletal abnormalities. Proc Natl Acad Sci USA 1998;95:5372e7. Miedlich SU, Zhu ED, Sabbagh Y, Demay MB. The receptordependent actions of 1,25-dihydroxyvitamin D are required for

[255]

[256]

[257]

[258]

[259]

[260]

normal growth plate maturation in NPt2a knockout mice. Endocrinology 2010;151:4607e12. Bergwitz C, Roslin NM, Tieder M, et al. SLC34A3 mutations in patients with hereditary hypophosphatemic rickets with hypercalciuria predict a key role for the sodium-phosphate cotransporter NaPi-IIc in maintaining phosphate homeostasis. Am J Hum Genet 2006;78:179e92. Sabbagh Y, O’Brien SP, Song W, et al. Intestinal npt2b plays a major role in phosphate absorption and homeostasis. J Am Soc Nephrol 2009;20:2348e58. Corut A, Senyigit A, Ugur SA, et al. Mutations in SLC34A2 cause pulmonary alveolar microlithiasis and are possibly associated with testicular microlithiasis. Am J Hum Genet 2006;79:650e6. Brown WW, Ju¨ppner H, Langman CB, et al. Hypophosphatemia with elevations in serum fibroblast growth factor 23 in a child with Jansen’s metaphyseal chondrodysplasia. J Clin Endocrinol Metab 2009;94:17e20. Karim Z, Ge´rard B, Bakouh N, et al. NHERF1 mutations and responsiveness of renal parathyroid hormone. N Engl J Med 2008;359:1128e35. Farrow EG, Davis SI, Summers LJ, White KE. Initial FGF-23mediated signaling occurs in the distal convoluted tubule. J Am Soc Nephrol 2009;20:955e60.

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Hereditary Tubular Disorders of Mineral Handling Daniella Magen, Israel Zelikovic Division of Pediatric Nephrology, Meyer Children’s Hospital, Rambam Health Care Campus, Laboratory of Molecular Medicine, and Laboratory of Developmental Nephrology, Department of Physiology and Biophysics, Rappaport Faculty of Medicine and Research Institute, Technion e Israel Institute of Technology, Haifa, Israel

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Hereditary tubular transport disorders comprise a group of diseases that lead to profound derangements in the homeostasis of electrolytes, minerals or organic solutes in the body, and can be associated with significant morbidity. For decades, the study of inherited tubular transport disorders has focused on the physiological and metabolic alterations leading to impaired solute handling by the tubular epithelial cell. Over the past decade, the breakthrough in molecular biology and molecular genetics has provided the tools to investigate hereditary tubulopathies at the molecular level. As a result, exciting discoveries have been made and the underlying molecular defects in many of these disorders have been defined. The molecular study of hereditary tubulopathies has been important not only in clarifying the genetic basis of these disorders, but also in providing new and important insight into the function of specific transport proteins and into the physiology of renal tubular reclamation of solutes. This chapter summarizes normal renal handling of phosphate, calcium and magnesium as well as renal acidebase handling, reviews the molecular pathophysiology and genetic aspects of hereditary tubular disorders affecting mineral handling, and describes the clinical and laboratory features of these tubulopathies including the associated bone disease. In Tables 27.1, 27.3 and 27.4, the reviewed disorders are thematically grouped and the major findings are summarized.

Pediatric Bone, Second Edition DOI: 10.1016/B978-0-12-382040-2.10027-9

Overview of Renal Phosphate Handling Inorganic phosphate (Pi) is one of the most abundant anions in the human body, and plays a crucial role in skeletal mineralization and cellular metabolism. Whole body Pi balance is maintained by a finely orchestrated and highly controlled equilibrium between intestinal dietary Pi uptake, skeletal Pi deposition and renal Pi excretion. Under normal conditions of dietary Pi intake, parathyroid function and vitamin D status, the net intestinal Pi uptake equals urinary Pi excretion [1,2]. Among the organ systems and tissues involved in Pi homeostasis, the mammalian kidney serves as the major regulator of Pi balance, and is capable of modulating its Pi reabsorptive capacity according to the needs of the organism. Renal Pi handling consists of glomerular filtration, tubular reabsorption and urinary excretion. The rate limiting step in renal Pi handling occurs predominantly in the proximal tubule, where approximately 70e80% of glomerular filtered Pi is reclaimed [3,4]. Pi reabsorption from the lumen across the apical membrane of the proximal tubular cells is transcellular, unidirectional and saturable. Clearance studies in humans and in rodents show that as the filtered load of Pi is progressively increased, there is a proportionate increase in Pi reabsorption until a maximum tubular reabsorptive Pi rate (TmP) is reached. Since TmP measurements vary in the same individual and between individuals according to variation in the glomerular filtration rate (GFR), it is more accurate to estimate the

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overall tubular Pi reabsorptive capacity by calculating the ratio of TmP/GFR (i.e. the maximum tubular reabsorption of Pi per volume of filtrate). The TmP/GFR reflects the quantity of available Pi transporters and their activity in relation to kidney mass, and thus represents the overall renal reabsorptive capacity for Pi. The total renal Pi reabsorptive capacity is the major determinant of extracellular and serum Pi concentration [5]. Pi reabsorption along the proximal tubule is characterized by axial heterogeneity, namely, a gradual decrease in the Pi reabsorptive capacity from early to late proximal tubular segments. Most of the filtered Pi (z70%) is reabsorbed in the proximal convoluted tubule (PCT), with greater reabsorption in the early PCT (S1 segment) as compared to the late PCT (S2 segment), whereas only z10% of the filtered Pi is reabsorbed in the proximal straight tubule (S3 segment) (Fig. 27.1) [6e9]. Proximal tubular Pi transport is mediated by specialized Naþ/Pi-co-transporters in a sodium-dependent mechanism. The driving force for Pi influx into the proximal tubular epithelial cells is generated by the inwardly directed transmembrane electrochemical Naþ gradient, and maintained by the Naþ/Kþ-ATPase pump at the basolateral membrane. The precise mechanism of Pi efflux across the basolateral membrane remains obscure (Fig. 27.2) [3,4]. Three types of Naþ/Pi-co-transporters have been identified in the brush border membrane (BBM) of mammalian proximal tubules: type I, type II and type III, which are encoded by the solute carrier gene families SLC17, SLC34 and SLC20, respectively [10e14]. Of these three Naþ/Pi-co-transporter types, two members of the type II Naþ/Pi-co-transporter family, designated as NaPi-IIa and NaPi-IIc and encoded by SLC34A1 and

FIGURE 27.1 Profile of Pi reabsorption along the mammalian nephron, as derived from micropuncture data. PCT: proximal convoluted tubule; PST: proximal straight tubule; TALH: thick ascending limb of Henle’s loop; DCT: distal convoluted tubule; CCD: cortical collecting duct; IMCD: inner medullary collecting duct. (modified from [5])

FIGURE 27.2 Scheme of renal Pi reabsorption in the proximal tubule. The majority of filtered Pi is reabsorbed in the early proximal tubule (shaded region) involving at least two apical sodium-dependent Pi co-transporters (Na/Pi-co-transporters): NaPi-IIa (SLC34A1) and NaPi-IIc (SLC34A3) expressed in the brush border membrane. Transport is energized by basolateral Naþ/Kþ-ATPases providing a transmembrane electrochemical gradient. The basolateral exit pathway for Pi has not been identified on a molecular level to date. (Derived from: Wagner CA, Hernando N, Forster IC, Biber J, Murer H. Genetic defects in renal phosphate handling. In: Lifton RP, Somlo S, Giebisch GH, Seldin DW, eds. Genetic Disoders of the Kidney. Philadelphia: Elsevier; 2009. p. 718.)

SLC34A3, respectively, play the most critical role in proximal tubular Pi reabsorption [14e17]. By contrast, the type I Naþ/Pi-co-transporter most probably functions as an organic anion transporter [18e20], with no apparent major role in renal Pi handling [21]. The type III Naþ/Pi-co-transporter, PiT-2 (encoded by SLC20A2) has been shown to be expressed in the apical membrane of rat proximal tubular cells, and to be regulated by dietary Pi intake. However, since its level of expression is much lower than that of the type II Naþ/Pi-co-transporters NaPi-IIa and NaPi-IIc, and since it reaches its maximal activity at a pH level of less than 6, the importance of PiT-2 in overall renal Pi reabsorption is not completely established as yet [13,22,23].

The Renal Type II NaD/Pi-co-transporter Family Members NaPi-IIa and NaPi-IIc Based on accumulating data derived from animal models and from human monogenic disorders of renal Pi handling, it is widely accepted that the bulk of the filtered Pi is reabsorbed in the proximal tubule via the

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activity of NaPi-IIa and NaPi-IIc, which are both members of the type II Naþ/Pi-co-transporter family [15,24]. The human SLC34A1 gene encoding NaPi-IIa is located on chromosome 5q35 [25,26], spans 14.4 kb of genomic DNA, contains 13 exons and is translated into a transmembrane protein of 639 amino acids [27]. The human SLC34A3 gene encoding NaPi-IIc resides on chromosome 9q34, is 5.8 kb long, consists of 13 exons and encodes a 599 amino acids protein of eight transmembrane spanning domains [28,29]. Both NaPi-IIa and NaPi-IIc share a high degree of sequence homology [30], are exclusively expressed in the BBM of renal proximal tubular cells [31,32] with highest abundance in the S1 nephron segment [11], preferentially transport divalent Pi ions, show similar Naþ and Pi affinities and pH dependency and are both subject to hormonal (i.e. parathyroid hormone [PTH], fibroblast growth factor 23 [FGF-23]) and nutritional (dietary Pi) control [4,24]. Despite these similarities, the two renal NaPi-II subtypes exhibit several major differences in their developmental stages of expression, proximal tubular distribution, nature of molecular interactions with other proteins and kinetic characteristics [3,24]. From murine experiments and in vitro models, it is well established that while NaPi-IIa is expressed throughout life [33] along the entire proximal tubule (S1eS3 segments), interacts with PDZ domain containing proteins and is electrogenic with a Naþ/Pi coupling ratio of 3:1, NaPi-IIc is most prominently expressed during weaning [17], is confined to the proximal convoluted tubule (S1eS2 segments), does not interact with other proteins through PDZ domain binding and exhibits electroneutrality with a Naþ/Pi coupling ratio of 2:1 [3,24]. Studies in mice demonstrate that NaPi-IIa ablation reduces the overall proximal tubular Naþ/Pi co-transporter activity by 70e80%, which improves over time and adults show no evidence for hypophosphatemia [34]. In contrast, homozygous disruption of NaPi-IIc does not cause significant reduction in overall proximal tubular BBM Naþ/Pi co-transport in affected mice, and is sufficiently compensated for by increasing expression of NaPi-IIa [35]. These findings strongly suggest that, in mice, NaPi-IIa is the predominant mediator of renal Pi reabsorption. However, the importance of NaPi-IIa and NaPi-IIc in human renal Pi handling has only been established in the last decade, following the identification of disease-causing mutations in their encoding genes as underlying hereditary disorders of renal Pi wasting (see below).

Regulation of Renal Phosphate Transport Renal Pi handling is subject to intense regulatory control. The most important regulators of renal Pi reabsorption are dietary Pi intake, PTH and FGF-23 [32,36].

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All these factors exert their regulatory affect primarily by modulating the abundance of Naþ/Pi co-transporters at the apical membrane of the proximal tubular epithelium. Dietary Pi Dietary Pi intake is inversely correlated with renal Pi reabsorption. Increased Pi intake leads to decreased renal Pi reabsorption and increased phosphaturia, whereas dietary Pi restriction causes increased Pi reabsorption and negligible urinary Pi loss. In vivo studies have shown that dietary adaptive changes in Pi reabsorption may occur independently of shifts in extracellular fluid status, calciotropic or phosphaturic hormones, and can be attributed to changes in the net activity of Naþ/Pi co-transporters in proximal tubular BBM [36,37]. Interestingly, similar adaptive responses to changes in extacellular Pi concentration have been demonstrated using in vitro experiments in cultured opossum kidney (OK) cells, suggesting the presence of autonomous cellular regulatory mechanism for proximal tubular Pi handling [38,39]. In experimental animals, acute reduction in Pi intake induces a severalfold increase in NaPi-IIa and NaPi-IIc protein abundance within hours [17,36,40,41]. Acute (hours) Pi deprivation was found to increase mainly the abundance of NaPi-IIa protein, with negligible effect on its mRNA levels. This observation indicates that the adaptive response of NaPi-IIa to acute Pi restriction may be mediated by post-transcriptional modifying factors causing either increased protein translation or enhanced protein stability [40,42]. Chronic (days) Pi restriction, however, was shown to increase both NaPi-IIa mRNA and protein levels, implicating both transcriptional and translational stimulatory effects [40,42e48]. High Pi intake in experimental animals causes abrupt downregulation of NaPiIIa protein expression in the BBM, which is mediated by its endosomal internalization and subsequent lysosomal degradation [39,49]. In contrast to NaPi-IIa, NaPi-IIc does not undergo lysosomal degradation in response to high dietary Pi, but is rather partially and more slowly internalized into the subapical compartment, from where it may be recycled back to the plasma membrane [32,50]. These observations further emphasize the divergent pathways of NaPi-IIa and NaPi-IIc regulatory control. Parathyroid Hormone PTH is a major hormonal regulator of renal Pi handling, the function of which results in decreased proximal tubular Pi reabsorption [51]. PTH exerts its immediate (minutes) phosphaturic effect by reducing the abundance of NaPi-IIa in the BBM of proximal tubular cells via receptor-mediated endocytosis into clathrin coated vesicles, leading to endosomal

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internalization and lysosomal degradation [49,52e56]. The PTH-dependent regulation of NaPi-IIc is less well studied, but accumulating data indicate that the rate of NaPi-IIc disappearance from the proximal tubular BBM in response to PTH is much slower as compared to that of NaPi-IIa [4,32,57]. In addition, NaPi-IIc does not undergo lysosomal degradation in response to PTH-induced internalization, but is rather recycled and reincorporated into the plasma membrane, in a similar way to the above described effect of increased dietary Pi intake [58]. The signaling cascades induced by PTH in proximal tubular cells are complex, and involve the stimulation of two different G-protein coupled PTH receptors, localized at the apical and basolateral cell membranes (Fig. 27.3) [59]. Stimulation of the apical PTH receptor by luminal PTH activates phospholipase C (PLC) and protein kinase C (PKC). In contrast, stimulation of the basolateral PTH receptor by blood PTH activates adenylate cyclase and protein kinase A (PKA). Both PKC and PKA cascades converge into a single ERK1/2 kinase-dependent pathway, subsequently inducing internalization of NaPi-IIa [36,53,56]. The

apical PTH protein receptor is part of a macromolecular signaling complex including also PLC and the PDZdomain-containing scaffold protein NHERF1 (Naþ/Hþ exchanger regulatory factor 1), all of which jointly bind NaPi-IIa and mediate its stabilization at the BBM [60,61]. Of note, NEHRF1 deficiency in mice disrupts this complex, which causes enhanced internalization of NaPi-IIa and decreases renal Pi reabsorption [62,63] (see below). Another indirect path by which PTH also influences renal Pi handling involves its stimulatory effect on the expression and activity of 1a hydroxylase in the kidney, that catalyzes the hydroxylation of inactive 25-(OH)-vitamin D3 to its active metabolite, 1,25-(OH)2-vitamin D3 [64].

FIGURE 27.3 Regulation of NaPi-IIa co-transporter localization and activity in renal proximal tubule cells by parathyroid hormone (PTH). PTH receptors are expressed on the apial and basolateral membranes of proximal tubular cells. Apical PTH receptors couple Gq G-proteins and phospholipase C (PLC), which all form part of an apical protein complex including also the Naþ/phosphate cotransporter NaPi-IIa and the PDZ protein NHERF1. Activation of PLC leads to protein kinase C dependent internalization of NaPi-IIa cotransporters from the apical membrane. In contrast, stimulation of the basolateral PTH receptors activates protein kinase A via Gs G-proteins, adenylate cyclases (AC) and increased intracellular cAMP levels. Protein kinase C (PKC) and protein kinase A (PKA)-dependent cascades converge to an ERK 1/2 kinase dependent pathway. (Derived from: Wagner CA, Hernando N, Forster IC, Biber J, Murer H. Genetic defects in renal phosphate handling. In: Lifton RP, Somlo S, Giebisch GH, Seldin DW, eds. Genetic Disoders of the Kidney. Philadelphia: Elsevier; 2009. p. 719.)

FGF-23 and other Phosphatonins Phosphatonins are a continuously growing group of Pi-regulating substances with phosphaturic activity [70]. The term “phosphatonin” was originally used to describe a previously non-identified circulating phosphaturic factor which was postulated to underlie the renal Pi wasting observed in tumor induced osteomalacia (TIO), autosomal dominant hypophosphatemic rickets (ADHR) and X-linked hypophosphatemic rickets (XLH). Speculation regarding the existence of a phosphatonin followed the observation that renal Pi wasting and mineral deficiency bone disease in all of the above mentioned hypophosphatemic disorders could not be attributed to dysregulation of serum calcium, PTH or

Vitamin D Vitamin D3 is well known for its major effect on intestinal Pi reabsorption. However, its direct effect on proximal tubular Pi handling is less well established. Although previous studies in thyroparathyroidectomized rats showed reduced proximal tubular Pi reabsorption in response to vitamin D3 administration [65], more recent studies report a direct stimulatory effect of vitamin D3 on renal Pi reabsorption [66,67]. Nevertheless, the complex interactions of active vitamin D3 with PTH and serum calcium, and the direct effect of these factors on serum Pi levels, poses great difficulty in data interpretation of these more recent studies. In addition to these conflicting findings, several in vitro experiments have identified a vitamin D3 response element in the promoter region of the NaPi-IIa gene, suggesting a stimulatory effect of vitamin D3 at the transcriptional level [48]. However, in double knockout mice deficient in both the vitamin D3 receptor and the 1a hydroxylase encoding genes, the abundance of NaPi-IIa protein in proximal tubular BBM vesicles was comparable to that of wild-type animals, indicating that NaPi-IIa transcription may not depend on vitamin D3 activity [68,69]. Hence, the precise direct effect of vitamin D3 on renal Pi handling awaits further elucidation.

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1,25-(OH)2-vitamin D3 alone [71,72]. In addition, extraction of tumor tissues from patients with TIO was shown to inhibit Pi transport in vitro [73,74]. Subsequently, a novel member of the fibroblast growth factor family proteins designated as fibroblast growth factor 23 (FGF-23) was identified as the most important factor underlying the phsophaturia in this group of diseases, and was eventually proven to be the first long sought substance with phosphatonin activity [4,70,75]. The current list of identified phosphatonins is composed of proteins with overlapping but distinct functions, and includes, among others, FGF-23, fibroblast growth factor 7 (FGF-7), secreted frizzled related protein 4 (sFRP4) and matrix extracellular phosphoglycoprotein (MEPE). Of these, FGF-23 is the most studied phosphatonin, and is presently considered as one of the key regulators of renal Pi reabsorption. The cardinal role of FGF-23 in Pi homeostasis and its various functions in health and disease have been extensively reviewed elsewhere [75e79]. In brief, the human FGF-23 is a circulating glycoprotein, mainly expressed and secreted by bone osteocytes and osteoblasts [80e82]. The full-length FGF-23 precursor consists of 251 amino acids, and is encoded by the FGF23 gene on chromosome 12p13.3. The precursor protein contains a hydrophobic leader sequence of 24 residues that is cleaved off before secretion into the blood circulation, generating a 227-amino acid protein of approximately

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32 kD [83]. The second post-translational modification of FGF-23 involves O-linked glycosylation by GALNT3 [84,85], which protects the intact protein from proteolytic cleavage by enzymes such as subtilin-like proprotein convertase, that recognize the 176R-177X-178-X-179R motif in FGF-23, just before the cleavage site at 179 R-180Ser [84,86e88]. FGF-23 is subject to complex regulatory control at various levels. Its synthesis is upregulated by dietary Pi [89e91] and by 1,25-(OH)2-vitamin D3 [92], whereas both its synthesis and secretion are downregulated by the Pi regulating protein with homology to endopeptidase on the X chromosome (PHEX), by dentin matrix protein 1 (DMP1) and by ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) [93e95]. FGF-23 exerts its biological functions by binding to various FGF receptors (FGFRs), of which the c-splice variant of FGFR1 (FGFR1c) was shown to be the most sensitive to FGF-23 activation [96,97]. In order for FGFR1c to be activated by FGF-23 it must recruit Klotho, which functions as an FGF-23 co-receptor [96e98]. Hence, FGF-23, FCR1c and Klotho form a signaling complex, which is essential for FGF-23 bioactivity [96]. The overall effect of FGF-23 is to increase renal Pi excretion and to decrease intestinal Pi uptake (Fig. 27.4). In the kidney, FGF-23 decreases proximal tubular Pi reabsorption by decreasing the expression of NaPi-IIa and NaPi-IIc [98], and by reducing NaPi-IIa

FIGURE 27.4 The boneekidney axis. In this model, osteocytes in bone are the principal cells that coordinate the mineralization process with renal handling of phosphate. Osteocytes secrete FGF-23 and sclerostin (SOST), matrix extracellular phosphoglycoprotein (MEPE), and dentin matrix protein 1 (DMP1) and express Phex. FGF-23 targets organs, particularly the kidney, via an FGF receptor:Klotho complex that inhibits sodium-dependent phosphate uptake and 1a-hydroxylase activity by a mechanism that remains to be elucidated. In addition, SOST inhibits osteoblastic function by disrupting Wnt and bone morphogenic protein (BMP) signals that regulate osteoblast-mediated bone formation. Also, the extracellular matrix proteins DMP1 and MEPE respectively positively and negatively regulate the process of extracellular matrix mineralization. Inhibition of bone formation would be associated with decreased phosphate buffering capacity of bone and require increased FGF-23-mediated phosphaturia, whereas accelerated bone formation and increased bone phosphate utilization would be associated with suppressed FGF-23 and renal phosphate retention to meet the need for bone mineralization. (Derived from reference [76].)

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abundance [59,99] using a similar signaling pathway as described above for PTH-induced phosphaturia [100]. In addition, FGF-23 also reduces the production of active vitamin D3 by decreasing the activity of the enzyme 25-(OH)D3-1a-hydroxylase in the proximal tubule, and by enhancing the expression of the vitamin D3 inactivating enzyme, 25-(OH)D3-24-hydroxylase in the same nephron segment [76,95]. These effects, in turn, lead to reduced 1,25-(OH)2-vitamin D3, and result in decreased intestinal Pi uptake. Although FGF-23 exerts its phosphaturic actions by modifying Pi reabsorption in the proximal tubule, and although Klotho is an essential mediator in the FGF23-induced signaling cascade, it has been surprisingly found that renal Klotho expression is confined mainly to the distal tubule [101]. Hence, the exact site and mechanism of FGF-23 action in the kidney is still unresolved, and awaits further delineation. In addition to FGF-23, other phosphatonins, including sFRP-4, MEPE and FGF-7 were also identified and shown to be overexpressed and secreted in tumor tissues from patients with TIO and oncogenic hypophosphatemic osteomalacia. All these factors inhibit renal Pi reabsorption in vitro and in vivo, and play an important role in the pathogenesis of renal Pi wasting associated with these neoplasms [102e106]. However, as opposed to FGF-23, which has unquestionable importance in the regulation of Pi homeostasis under normal conditions, the role of these additional phosphatonins in the control of renal Pi handling and systemic Pi balance during health remains to be established, as they are currently believed to be important mainly in the pathophysiology of tumor-induced renal Pi wasting [107].

Rickets due to Disorders of Renal Phosphate Transport Renal Pi wasting disorders constitute a complex and heterogeneous group of acquired and hereditary diseases, the understanding of which has dramatically improved over the last decade. Much of the progress in delineating the pathophysiology of these disorders is attributed to genetic analysis of families with rare forms of hereditary renal Pi wasting. Such genetic studies, combined with complementary animal and in vitro experimental models, have led to the discovery of novel genes and biological pathways involved in the regulation of Pi homeostasis in health and disease. The heterogeneous group of renal Pi wasting disorders can generally be divided into three main subgroups, based on the underlying mechanism of renal Pi wasting (Table 27.1): 1. renal Pi wasting disorders attributed to primary impairment in the function of specific proximal tubular proteins that are directly involved in renal Pi

transport. In this subgroup, the circulating level of FGF-23 is usually at the low-normal range (with the exception of hypophosphatemic rickets due to Klotho overexpression; see below), which is indicative of FGF-23-independent phosphaturia 2. renal Pi wasting disorders due to disruption of extrarenal proteins that are normally involved in the regulation of renal Pi handling. In this subgroup of diseases FGF-23 serves as the major mediator of renal Pi wasting, and its circulating level is usually elevated 3. renal Pi wasting disorders secondary to a more generalized proximal tubular dysfunction, designated as renal Fanconi’s syndrome. The phosphaturia in renal Fanconi’s syndrome is accompanied by urinary wasting of additional solutes, and is usually secondary to a major insult to the structural and/or functional integrity of proximal tubular cells.

Rickets due to Impairment of Proximal Tubular Proteins Directly Involved in Renal Phosphate Transport Primary Defects of NaPi-IIa (Fanconi renotubular syndrome 2 [FRTS2]) The significance of NaPi-IIa for renal Pi handling in rodents has been extensively studied and is well established. By using in vivo models of genetically engineered mice, it was repeatedly shown that homozygous disruption of Slc34a1, encoding the murine homolog of the human NaPi-IIa, results in severe renal Pi wasting, skeletal abnormalities, hypercalciuria and renal calcifications [33,34,108,109]. Based on these data, NaPi-IIa was a long-sought candidate for various human diseases chracterized by hereditary hypophosphatemic rickets, which were eventually all found to result from mutations in genes encoding proteins other than NaPi-IIa [110,111] (see below). Based on the finding of hypercalciuria and renal calcifications in experimental animals with slc34a1 gene disruption [33,34,108,109], combined with reports in humans on the association between renal Pi wasting and calcium nephrolithiasis [112,113], NaPi-IIa was considered as a candidate gene underlying this clinical constellation [114,115]. The first apparent human disease-causing mutations in NaPi-IIa were reported by Prie´ et al. [125] (nephrolithiasis/osteoporosis, hypophosphatemic, 1[NPHLOP1]) in two out of 20 individuals with renal Pi leak associated with either urolithiasis or bone demineralization. Each of the two identified NaPiIIa sequence variations (A48F in exon 3 and V147M in exon 5) was carried in the heterozygous state, suggesting an autosomal dominant inheritance. Notwithstanding initial functional analysis of these variants suggesting a dominant effect of the mutant gene product,

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TABLE 27.1 Primary disorders of renal Pi wasting OMIM#

Inheritance

Serum Mutant gene (protein) Serum Pi TmP/GFR calcium

Urinary calcium

1,25(OH)2D3

PTH

Fanconi renotobular syndrome 2 (FRTS2) [116,117]

613388

AR

SLC34A1 (NaPi-IIa)

Low

High

High

Normal Low-normal Generalized proximal tubulopathy, renal failure

Hereditary 241530 hypophosphatemic rickets with hypercalciuria (HHRH) [28,29,118]

AR

SLC34A3 (NaPi-IIc)

Normal/ Low Low

Normal/ High High

High

Normal Low-normal Nephrolithiasis, nephrocalcinosis

Nephrolithiasis/ osteoporosis, hypophosphatemia 2 (NPHLOP2) [119]

612287

AD

SLC9A3R1 (NHERF1) Low

Low

Normal

ND

High

Normal Normal

Nephrolithiasis

Hypophosphatemic rickets and hypeparathyroidism [120]

612089

Klotho (KLOTHO) De-novo balanced translocation

Low

Low

Normal

Normal

Low/ inappropriately normal

High

Facial and skeletal abnormalities

X-linked hypophosphatemic rickets (XLH) [121]

307800

X-linked dominant

PHEX (PHEX)

Low

Low

Normal/ Normal high

Low/ inappropriately normal

Normal High

Dental abnormalities

Autosomal dominant hypophosphatemic rickets (ADHR) [122]

193100

AD

FGF23 (FGF-23)

Low

Low

Normal

Normal

Low/ inappropriately normal

Normal High

Dental abnormalities

Autosomal recessive hypophosphatemic rickets 1 (ARHR1) [123,124]

241520

AR

DMP1 (DMP1)

Low

Low

Normal

Normal

Low/ inappropriately normal

Normal Normal-high Dental abnormalities

Autosomal recessive hypophosphatemic rickets 2 (ARHR2) [125,126]

613312

AR

ENPP1 (ENPP1)

Low

Low

Normal

Normal

Low/ inappropriately normal

Normal Normal-high Generalized arterial calcification of infancy in some patients

Low

High

FGF-23

High

Additional features

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Disease

AR: autosomal recessive; ND: not determined; OMIM#: Online Mendelian Inheritance in Man (database at http://www.ncbi.nlm.hih.gov/Omim)

733

734

27. HEREDITARY TUBULAR DISORDERS OF MINERAL HANDLING

subsequent studies [115] effectively ruled out any such effect, and were consistent with the mutations being non-functional polymorphic variants. In a more recent report by Lapointe et al. [127] several heterozygous NaPi-IIa sequence variants were identified in 28 probands from a large cohort of 98 pedigrees with renal Pi leak and hypercalciuria. Detailed functional analysis of the identified NaPi-IIa variants, including one in-frame 21-bp small deletion at the N-terminal intracellular segment, demonstrated either normal or near normal Pi transport, and no correlation of genotype with renal Pi leak or hypercalciuria. It was thus concluded that the identified NaPi-IIa variants are common in the general population, and that at least in their heterozygous state, do not account for renal Pi wasting. The cardinal role of NaPi-IIa in human renal Pi handling was only recently established by the report of Magen et al. describing a homozygous loss-of-function mutation in NaPi-IIa as the cause of autosomal recessive hypophosphatemic rickets associated with proximal tubulopathy and renal failure in an inbred kindred of Israeli Arab origin [116]. In this report, two affected siblings (one male and one female) and 10 of their close relatives were clinically reassessed and genetically studied, two decades after being clinically described by Tieder et al. [117]. During childhood, both patients suffered from vitamin D-refractory hypophosphatemic rickets, associated with hypercalciuria, elevated 1,25-(OH)2-vitamin D3 and normal PTH levels. In addition, the patients were also shown to have generalized proximal tubulopathy (renal Fanconi’s syndrome) without an acidification defect, and their GFR was normal. No biochemical abnormalities were identified in healthy, first-degree relatives of affected individuals, in accordance with the autosomal recessive mode of inheritance. Oral Pi therapy during childhood resulted in normalization of serum Pi and 1,25-(OH)2-vitamin D3 levels, reversal of hypercalciuria and radiologic resolution of rickets, but had no effect on the proximal tubulopathy. Clinical and biochemical reassessment of both patients after two decades showed persistent proximal tubulopathy, renal Pi wasting and osteomalacia, accompanied by mild to moderate reduction in GFR. However, in contrast to the hypercalciuria and hypervitaminosis D reported during childhood, and attributed to compensatory renal overproduction of 1,25-(OH)2-vitamin D3 triggered by the hypophosphatemia, re-evaluation showed normocalciuria and reduced 1,25-(OH)2-vitamin D3, which were related to nutritional vitamin D deficiency at adulthood. Of note, plasma intact and C-terminal levels of FGF-23 in both adult patients were within lownormal range, indicating primary renal Pi wasting that is FGF-23-independent, as underlying their disease. Finally, genetic analysis in the studied family revealed a homozygous in-frame duplication of 21 bp in exon 5

of SLC34A1 causing a duplicated mutation of seven amino acids in the NaPi-IIa protein (p.I154_V160dup) as the causative mutation in affected individuals. Functional studies in Xenopus laevis oocytes and in opossum kidney cells revealed complete loss of function of the mutant NaPi-IIa co-transporter, as a consequence of its failure to localize at the plasma membrane. Taken together, these findings support a major role for NaPiIIa in human renal Pi handling, and establish its importance in the maintenance of systemic Pi balance. The findings by Magen et al. indicate considerable phenotypic similarity between humans and mice with NaPi-IIa disruption, as manifested by renal Pi wasting, hypercalciuria, elevated serum 1,25-(OH)2-vitamin D3 levels, impaired growth, and metabolic bone disease in both affected species. However, there are several as yet unexplained differences between humans and mice, including the absence of classic rickets and the lack of renal Fanconi’s syndrome in homozygous, NaPi-IIa-disrupted mice [34], and the absence of renal calcifications in affected humans [33,117,108]. The causes for these phenotypic differences remain to be delineated, but, as previously suggested [34,128], may imply fundamental differences in regulatory pathways controlling human and murine skeletal and renal responses to calciotropic hormones and to hypophosphatemia. Although the mechanism of renal Fanconi’s syndrome in association with human NaPi-IIa disruption awaits further clarification, it may suggest a deleterious effect of the intracellular accumulated mutant NaPi-IIa on other proximal tubular BBM transporters. A possible explanation for the absence of Fanconi’s syndrome in NaPi-IIa deficient mice may arise from the fact that assessment of proximal tubular function in relation to NaPi-IIa activity has so far been restricted to the NaPiIIa knockout mouse model, where no NaPi-IIa gene product is formed that could potentially disrupt other tubule functions [34]. Data derived from Iwaki’s report on a NaPi-IIa missense mutations in mice do not provide sufficient information regarding the presence of proximal tubulopathy in affected animals [108]. Hence, further investigation is needed to determine whether NaPi-IIa mutations other than complete gene knockout may also cause renal Fanconi’s syndrome in rodents. In addition, it remains to be seen whether the observed Fanconi’s syndrome accompanying NaPi-IIa disruption in human is limited to NaPi-IIa trafficking defects, or is rather a universal manifestation of NaPi-IIa impairment. Most recently, Levtchenko et al. reported on a patient with Sotos syndrome (also known as cerebral gigantism, MIM 117550), in whom significant hypophosphatemia was diagnosed at early infancy, and gradually improved with age. The hypophosphatemia could not be attributed to the Sotos syndrome per se, and was not identified in other patients with Sotos syndrome secondary

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to defects in the NSD1 gene [129]. There was no evidence of nephrocalcinosis or mineral deficiency bone disease in the reported patient, although a potential bone mineralization defect could be masked in this case, given the bone overgrowth and advanced bone age inherent in Sotos syndrome. Biochemical investigation was compatible with renal Pi wasting, based on the findings of reduced TmP/GFR, normal levels of serum calcium and PTH and slightly elevated 1,25-(OH)2-vitamin D3 levels. Renal Fanconi’s syndrome was excluded (personal communication). Genetic analysis revealed a heterozygous microdeletion on chromosome 5q35, which removed both the NSD1 and the nearby SLC34A1 genes. Sequence analysis of the remaining allele excluded a mutation in SLC34A1 as the cause of the hyperphosphaturia (Levtchenko E and Magen D, unpublished data). This finding suggests that haploinsufficiency of NaPi-IIa underlies the patients’s renal Pi wasting, and reinforces the importance of the human NaPi-IIa in renal Pi reclamation. Of note, this report on a heterozygous deletion of NaPi-IIa as a cause for renal Pi wasting is in contrast to the observed normal Pi balance in the heterozygous carriers of the NaPi-IIa defect reported by Magen et al., and warrants further clarification. It is not impossible, however, that the age-related improvement in the hypophosphatemia of the NaPi-IIa haploinsufficient patient could be attributed to the normal function of the second intact NaPi-IIa allele, and that transient, early-infantile hypophosphatemia might have been overlooked in heterozygous carriers of the NaPi-IIa loss-of-function mutation associated with hypophosphatemic rickets and renal Fanconi’s syndrome [116]. Primary Defects of NaPi-IIc (hereditary hypophosphatemic rickets with hypercalciuria [HHRH]) In 1987, Tieder et al. reported a new hereditary autosomal recessive syndrome of hypophosphatemic rickets with hypercalciuria (HHRH) in a highly inbred kindred of Israeli Bedouin origin [118]. The disease is compatible with an autosomal recessive pattern of inheritance, and obligate carriers exhibit only mild hypercalciuria with normal Pi balance [29]. Similar to other genetic Pi wasting disorders such as XLH and ADHR, affected individuals with HHRH were shown by Tieder and others to present at early childhood with rickets, bone pain, short stature, muscle weakness and renal Pi wasting [28,29,118,130e133]. However, as opposed to the former FGF-23-dependent hereditary renal Pi wasting disorders (see below), in which 1,25-(OH)2vitamin D3 levels are inappropriately low in the presence of hypophosphatemia, HHRH is characterized by appropriately increased 1,25-(OH)2-vitamin D3 level which causes absorptive hypercalciuria and suppressed

735

PTH secretion, and by low-normal plasma FGF-23 levels [29]. Moreover, long-term Pi supplementation as the sole therapy in HHRH patients causes reversal of all clinical and biochemical abnormalities except for the renal Pi wasting [134]. Taken together, this clinical constellation suggested that HHRH was a primary disorder of renal Pi handling. Nevertheless, no disease-causing mutations in the major renal Naþ/Pi-co-transporter, NaPi-IIa, could be identified in affected individuals as underlying their disease [110,111]. Rather, subsequent studies employing homozygosity mapping and positional cloning in HHRH families of various ethnicities have mapped the disorder to a linked interval on chromosome 9 [28,29], and resulted in the identification of various homozygous or compound heterozygous mutations in SLC34A3 encoding the less abundant renal Naþ/Pi co-transporter, NaPi-IIc, in affected individuals [28,29,135e139]. The fact that NaPi-IIc disruption in humans profoundly impairs renal Pi homeostatsis and leads to significant clinical disease suggests that this transporter may have a more significant role in human renal Pi handling than recognized so far. Nevertheless, these clinical findings are in sharp contrast with data derived from murine models, in which homozygous disruption of NaPi-IIc does not cause renal Pi wasting or mineral deficiency bone disease [24,35,140]. On the other hand, the finding that Npt2a/ and Npt2c/ double knockout mice are more severly affected than mice with isolated Npt2a/ knockout suggests that NaPiIIc does have a role in renal Pi handling of mice, which is non-redundant of NaPi-IIa [140]. Taken together, this conflicting information, combined with the above mentioned evidence for phenotypic discrepancy between humans and mice with NaPi-IIc disruption, suggests that it is not yet clear whether data derived from experimental models in rodents regarding the function of the renal type II Naþ/Pi co-transporter family members may be applicable to the human kidney in health and disease. Primary Defects of NHERF1 (nephrolithiasis/ osteoporosis, hypophosphatemic, 2 [NPHLOP2]) In 2008, Karim et al. reported three different heterozygous missense mutations in the coding region of SLC9A3R1 encoding the proximal tubular Naþ/Hþ exchanger regulatory factor 1 (NHERF1), in four of 92 unrelated patients with impaired renal Pi reabsorption and either calcium nephrolithiasis or bone mineral deficiency [119,141]. Affected individuals did not exhibit additional features of proximal tubular dysfunction other than phosphaturia, and displayed normal levels of serum calcium, 1,25-(OH)2-vitamin D3 and PTH. FGF-23 level measured in one of these patients was found within normal range. An extended genetic analysis in close

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27. HEREDITARY TUBULAR DISORDERS OF MINERAL HANDLING

relatives of one of the probands revealed co-segregation of the heterozygous NHERF1 mutation with the disease phenotype. Additional biochemical investigation revealed significantly increased urinary cAMP levels in patients with NHERF1 mutations as compared to matched controls with a similar phenotype but without NHERF1 mutations, indicating an increased effect of PTH in the kidneys of NHERF1 mutation carriers. This finding was followed by in vitro experiments aimed at measuring cAMP secretion and Pi uptake in OK cells transfected with either wild-type or mutant variants of NHERF1 under PTH stimulation. In accordance with findings in affected individuals, OK cells transfected with mutant NHERF1 exhibited significantly higher cAMP accumulation in response to PTH stimulation as compared to OK cells transfected with wild-type NHERF1. In addition, the inhibitory effect of PTH on Pi uptake was markedly accentuated in OK cells transfected with mutant as compared to wild-type variants of NHERF1. These findings suggest that NHERF1 mutations potentiate PTH-induced renal cAMP generation and consequent inhibition of proximal tubular renal Pi transport. As indicated above, NHERF1 is a PDZ-domain containing scaffolding protein, which participates in the formation of a subapical multiprotein complex, allowing the coupling of the PTHR1 to its downstream signaling effector, PLC [60]. Accordingly, its role in the apical proximal tubular trafficking and stabilization of NaPi-IIa and NaPi-IIc has been well established using various animal and in vitro experimental models. In mice, targeted disruption of Nherf1 was found to promote the internalization of the NaPi-IIa from the BBM, leading to its routing to lysosomal degradation [62] and resulting in decreased renal Pi reabsorption. In addition, physiologic adaptation to low Pi diet was shown to be defective in both Nherf1deficient mice and in primary proximal tubular cultured cells derived from Nherf1-deficient mice, due to impaired apical trafficking of NaPi-IIa into the BBM [142,143]. Similarly, in vivo experimental evidence for the interaction of NaPi-IIc with NHERF1 in rat kidney cortex [22] suggests that NHERF1 deficiency could also impair the stabilization of NaPi-IIc at the proximal tubular apical membrane, thereby further jeopardizing renal Pi reabsorption. Taken together, these cumulative data suggest that NHERF1 plays a key role in mammalian renal Pi handling, and implicate its involvement in human renal Pi wasting disorders associated with mineral deficiency bone disease. Primary Impairment of Klotho (hypophosphatemic rickets and hyperparathyroidism) In 2008, Brownstein et al. reported a 23-year-old woman with hypophosphatemic rickets and hyperparathyroidism diagnosed at infancy [120]. In addition to

the typical clinical and radiographic features of rachitic bone changes at presentation, this patient also exhibited facial and skeletal abnormalities, consisting of a prominent forehead with frontal bossing, macrocephaly, nasal bone dysplasia and exaggerated midfacial protrusion. Biochemical investigation revealed hypophosphatemia secondary to renal Pi wasting, normocalcmia, hyperparathyroidism, inappropriately normal vitamin D level relative to the degree of hypophosphatemia and normocalciuria. Rickets was unresponsive to high-dose oral vitamin D2 therapy, but was markedly improved with oral Pi and 1,25-(OH)2-vitamin D3 supplements. The patient’s clinical course was complicated by worsening hyperparathyroidism and hypercalcemia secondary to parathyroid hyperplasia, which necessitated repeated partial parathyroidectomy. Although parathyroid gland surgery resulted in normalization of serum calcium and PTH levels, the hypophosphatemia and renal Pi wasting persisted, indicating their unrelatedness to the hyperparathroidism, per se. Direct sequencing of PHEX, FGF23, DMP1 and FGFR1 excluded the existence of disease-causing mutations in these genes as the cause for the renal Pi wasting. Further genetic analysis subsequently revealed a de novo balanced translocation between chromosomes 9 and 13: t(9,13)(q21.13;q13.1), with a break point adjacent to the Klotho gene on chromosome 13q. Since the levels of circulating a-Klotho and of its associated b-glucoronidase enzymatic activity were found to be markedly elevated in this patient, the authors concluded that the identified translocation near the Klotho gene has caused dysregulated overexpression of Klotho, leading to the patient’s novel phenotype. Interestingly, serum FGF-23 levels in this patient were markedly elevated as well, and did not normalize after temporary cessation of calcitriol therapy. The elevated PTH and FGF-23 levels in this patient suggest the in vivo effect of Klotho on these pathways, by mechanisms that still await further delineation. Of note, it has been shown that both mice and humans with Klotho gene deficiency or loss of function exhibit severe hyperphosphatemia, elevated 1,25-(OH)2-vitamin D3 and increased renal Pi reabsorption [144], which is, in part, a mirror image of the phenotype in the patient with Klotho overexpression as described in Brownstein’s report. Taken together, these findings suggest that Klotho overexpression results in renal Pi wasting and hypophosphatemic rickets, with FGF-23, and not PTH, being the mediator of hyperphosphaturia.

Rickets due to Defects in Extrarenal Proteins Involved in Renal Phosphate Transport X-linked Hypophosphatemic Rickets X-linked hypophosphatemic rickets (XLH) is considered as the most common cause of hereditary rickets,

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PHOSPHATE

with a prevalence of 1:20 000. Although the phenotype may be variable, it usually consists of renal Pi wasting leading to short stature, leg deformities, bone pain, dental abscesses and radiographic evidence for rickets and osteomalacia. Serum 1,25-(OH)2-vitamin D3 levels are inappropriately normal to low relative to the degree of hypophosphatemia [113]. XLH, which is inherited in an X-linked dominant pattern, has been mapped by linkage analysis to chromosome Xp22 [145e147]. By using positional cloning in affected individuals, mutations in the PHEX (phosphate regulating gene with homologies to endopeptidase on the X chromosome) gene have been subsequently identified as underlying XLH [113]. PHEX encodes a zinc-dependent metalloproteinase, which is expressed in cell membranes of osteoblasts, osteocytes and odontoblasts, as well as in tumor tissue associated with paraneoplastic renal Pi wasting, but not in the normal kidney [148]. Data derived from cross-transplant experiments in XLH Hyp and Gy mouse models [121,149,150] have shown that the defect in XLH was due to an extrarenal circulating factor with phosphaturic activity [151]. In addition, renal transplantation from a healthy donor to an XLH patient did not correct the renal Pi wasting [152]. The finding of elevated circulating FGF-23 levels in most XLH patients [153,154] eventually led to the conclusion that overexpression of bone FGF-23 in affected individuals underlies the renal Pi wasting of XLH. Notwithstanding this observation, the mechanism whereby mutations in PHEX result in elevated circulating FGF-23 remains unclear. Initial reports on the finding that FGF-23 is a substrate of PHEX, and that PHEX disruption impairs FGF-23 inactivation [148,155,156] could not be replicated in subsequent studies, which have shown that FGF-23 is not directly cleaved by PHEX [157,158]. Thus, the exact role of PHEX in FGF-23 regulation, and its function in the pathogenesis of XLH remain to be established. Therapy of XLH with high pharmacological doses of Pi and 1,25-(OH)2-vitamin D3 supplements was shown to have a beneficial effect on growth and bone disease in some patients [159]. However, this combination therapy has been reported to result in nephrocalcinosis and occasional kidney failure, a complication which mandates close follow up and cautious Pi and vitamin D dose adjustment in affected individuals [160]. Autosomal Dominant Hypophosphatemic Rickets Autosomal dominant hypophosphatemic rickets (ADHR) is a rare cause of hereditary renal Pi wasting. The disease is characterized by variable penetrance, and is associated with a wide range of clinical severity, depending on the age at presentation. While disease onset during childhood usually manifests with hypophosphatemic rickets, bone deformities, short stature and dental abnormalities, presentation after puberty is

737

characterized by hypophosphatemic osteomalacia, weakness, bone pain and fractures, but without short stature or bone deformities [161]. As in XLH, serum 1,25-(OH)2-vitamin D3 is inappropriately normal, and clinical improvement is observed with administration of Pi salts combined with high doses of 1,25-(OH)2vitamin D3 therapy. Using positional cloning, XLH was mapped to chromosome 12p13 [162], and was thereafter shown to be caused by heterozygous “gain-of-function” mutations in the FGF23 gene residing within the linked interval [122]. Although FGF-23 was previously identified and cloned in mouse embryonic tissue based on its homology to other members of the FGF protein family [163], its major role in Pi balance has been appreciated only when pathogenic mutations in its encoding gene have been identified in human disorders of renal Pi handling, including in ADHR, among others. Of note, all FGF-23 disease-causing mutations identified so far in patients with ADHR reside at the 176R-177X-178X-179R motif of the protein sequence, affecting either amino acid residue 179 or 176. All of these mutations cause disruption of the FGF-23 cleavage site, rendering the mutant protein resistant to proteolytic cleavage. Hence, ADHR is caused by increased circulating levels of excessively stable FGF-23 variants, resulting in enhanced FGF-23 signaling and eventually leading to FGF-23 mediated renal Pi wasting [122]. Autosomal Recessive Hypophosphatemic Rickets 1 In 2006, two groups (Lorenz-Depiereux et al. and Feng et al.) simultaneously reported patients with hereditary hypophosphatemic rickets, whose phenotype was similar to XLH and ADHR, and was characterized by hypophosphatemic bone disease, renal Pi wasting, normal PTH, inappropriately normal 1,25-(OH)2vitamin D3 and elevated circulating FGF-23. The inheritance pattern was compatible with autosomal recessive transmission [123], and no mutations in the coding regions of PHEX or FGF23 could be identified in affected individuals [124]. Using homozygosity mapping [123] and candidate gene approach [124], both groups identified homozygous loss-of-function mutations in the DMP1 gene, encoding dentin matrix protein 1, as underlying the disease in all affected individuals. Dentin matrix protein 1 is a member of non-collagenous matrix proteins which are highly expressed in tooth and bone, and are considered as cardinally important for osteoid and dentin mineralization [123]. Data derived from experimental Dmp1-null mice suggest that DMP1 disruption causes defective osteocyte maturation and increased FGF-23 expression, resulting in impaired bone mineralization and in FGF-23 mediated renal Pi wasting. The observation, both in experimental mice and in affected humans,

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27. HEREDITARY TUBULAR DISORDERS OF MINERAL HANDLING

that the phenotype of DMP1 disruption is only partially rescued by high Pi intake, suggested that apart from its indirect effect on bone mineralization through regulation of FGF-23 expression, DMP1 may also have a direct effect on osteoblast differentiation, independent of systemic Pi homeostasis [124]. Autosomal Recessive Hypophosphatemic Rickets 2 In 2010, Lorenz-Depiereux et al. and Levy-Litan et al. reported loss-of-function mutations in ENPP1, encoding the ectonucleotide pyrophosphatase/phosphodiesterase 1, in families with autosomal recessive hypophosphatemic rickets of unexplained etiology [125,126]. The phenotype of affected individuals was highly variable and consisted of renal Pi wasting, mineral deficiency bone disease of varying severity, normal PTH and 1,25-(OH)2-vitamin D3 levels, and elevated intact FGF23 in some patients. ENPP1 is a cell surface enzyme, playing a key role in generation and bone deposition of inorganic pyrophosphate [164,165]. The mechanism of renal Pi wasting and increased FGF-23 production secondary to ENPP1 disruption remains to be elucidated. Interestingly, a previous finding of inactivating mutations in ENPP1 in association with generalized arterial calcification at infancy [166], suggests an as yet unidentified mechanism that balances arterial calcification with bone mineralization [125].

Rickets due to Renal Fanconi’s Syndrome Renal Fanconi’s syndrome is a general disorder of proximal tubular transport, which is named after Guido Fanconi, a Swiss pediatrician, who, together with De Toni and Debre´ described this clinical entity in the 1930s [167e169]. In its complete form, Fanconi’s syndrome is characterized by excessive renal wasting of glucose, Pi, amino acids, uric acid, bicarbonate, lowmolecular-weight (LMW) proteins, albumin, carnitine, electrolytes and water, and results in dehydration, electrolyte imbalance, renal tubular acidosis, hypophosphatemic rickets, growth retardation in children, and osteomalacia in adults. In most cases, decreased activity of 1a-hydroxylase in the proximal tubule leads to low circulating levels of 1,25-(OH)2-vitamin D3. Hypercalciuria with or without nephrocalcinosis may also be observed, either as a primary manifestation of the proximal tubulopathy, or secondary to vitamin D and calcium therapy for rickets [170]. Fanconi’s syndrome comprises a heterogeneous group of disorders which, based on its etiology, can be divided into two main categories (Table 27.2): 1. hereditary proximal tubulopathies. This heterogeneous group of disorders comprises primary renal and systemic inborn errors of metabolism,

TABLE 27.2 Causes of Fanconi syndrome Hereditary Cystinosis Galactosemia Hereditary fructose intolerance Glycogen storage disease type I (von Gierke disease) Tyrosinemia FanconieBickel syndrome Dent disease Lowe syndrome Mitochondriopathies Wilson disease Idiopathic Fanconi syndrome Acquired Nephrotic syndrome Renal transplantation Acute tubulointerstitial nephritis with uveitis (TINU) syndrome Autoimmune interstitial nephritis and membranous nephropathy Sjo¨gren syndrome Myeloma Anorexia nervosa Untreated condition of distal renal tubular acidosis Exogenous substances Drugs Chemical compounds Heavy metals

transmitted by either autosomal dominant, autosomal recessive, X-linked or mitochondrial inheritance 2. Acquired proximal tubulopathies accompanying acquired renal or systemic disorders, or secondary to the toxic effect of exogenous chemicals and drugs. Hereditary Renal Fanconi’s Syndrome NEPHROPATHIC CYSTINOSIS

Infantile nephropathic cystinosis is an autosomal recessive lysosomal storage disorder characterized by excessive intralysosomal accumulation of free cystine, the disulfite of the amino acid cysteine. Lysosomal storage of cystine is systemic, leading to widespread organ involvement, which is most notable in the kidneys, cornea, bone marrow, thyroid, liver and spleen [171]. Cystinosis is comprised of several phenotypic variants (infantile, MIM 219800; juvenile, MIM 219900; ocular, MIM 219750), based on the age at presentation and on clinical severity. The most common and most severe form of the disease is infantile nephropathic cystinosis, which is also the most frequent cause of renal Fanconi’s syndrome in children. This variant of cystinosis is manifested at 6e12 months of age with complete renal Fanconi’s syndrome, failure to thrive, fluids and electrolyte imbalance, renal tubular acidosis and hypophosphatemic rickets. Without specific treatment,

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subsequent deterioration of renal function in affected children results in end-stage renal failure by 10 years of age [172]. Cystinosis is caused by mutations in the CTNS gene on chromosome 17p13 encoding cystinosin [173,174], which is a 367 amino acids lysosomal integral membrane protein with cystine transport activity [175]. To date, nearly 100 different mutations in CTNS have been described in patients with all three variants of cystinosis. The most frequent CTNS mutation is a 57-kb deletion removing a large portion of the 50 region of the gene, which is identified in 76% of cystinosis patients of Northern European origin. Cystinosin functions as an Hþ-driven transporter responsible for cystine export from the lysosome [175]. Impairment of lysosomal cystine exit results in its cellular accumulation, crystallization and tissue damage. However, although excessively studied, the exact pathophysiology of the proximal tubulopathy in nephropathic cystinosis secondary to the defective lysosomal cystine transport is poorly understood. Based on in vitro experiments showing that cystine-loaded proximal tubular cells manifest decreased ATPase activity and consequent reduced Naþ-dependent transport [176,177], it was postulated that mitochondrial oxidative phosphorylation may be impaired in cystinosis proximal tubular cells, leading to reduced proximal tubular reabsorptive capacity and to renal solute wasting. This theory was supported by the observation of swollen mitochondria in renal biopsy specimens from cystinosis patients and from Ctns knockout mice [178e180]. Another potential mechanism for the proximal tubulopathy of nephropathic cystinosis is increased apoptotic cell death, based on the observation that both cultured nephropathic cystinosis fibroblasts and cystin-loaded proximal tubular cells exhibit enhanced apoptosis [181]. Nevertheless, the exact molecular pathways leading to impaired proximal tubular function in cystinosis still await further clarification by in vivo experimental models, especially in view of the surprising finding that Ctns knockout mice do not exhibit the clinical and biochemical features of renal Fanconi’s syndrome, despite accumulation of lysosomal cystine in their proximal tubular cells [178]. The diagnosis of nephropathic cystinosis is usually established on clinical grounds, and is confirmed by the finding of corneal crystals by ocular slit-lamp examination, and by demonstration of elevated cystine levels in peripheral leukocytes using the cystine binding protein assay [171]. The natural course of nephropathic cystinosis has dramatically improved with the introduction of oral cysteamine therapy. Cysteamine is an orally administered cystine depleting agent, which when ingested by cystinosis patients early after presentation and in adequately high doses, can significantly delay

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the progression of systemic organ involvement and of end stage kidney disease [182]. Nevertheless, cysteamine therapy has no effect on the renal Fanconi’s syndrome associated with cystinosis, which should be managed by vigorous replacement of water, electrolytes, bicarbonate and Pi losses, as well as treatment with 1,25(OH)2-vitamin D3. FANCONIeBICKEL SYNDROME

FanconieBickel syndrome is a rare autosomal recessive hepatorenal glycogen storage disease characterized by hepatomegaly, fasting hypoglycemia, galactose intolerance, “doll-like” facial appearance, failure to thrive, short stature, renal Fanconi’s syndrome and severe rickets [183e186]. Although most affected individuals live to adulthood, some patients suffer from neonatal diabetes and galactosemia, and succumb during infancy due to hepatic failure [187]. FanconieBickel syndrome is caused by mutations in the SLC2A2 gene encoding the facilitative glucose transporter, GLUT2. This protein mediates bidirectional glucose transport across the hepatocyte plasma membrane, is involved in glucose uptake by pancreatic islet cells, and is responsible for cellular exit of glucose across the basolateral membranes of the small intestinal and of proximal tubular cells [185,188]. The mechanism of proximal tubulopathy in Fanconie Bickel syndrome is not completely understood, but is thought to result from the failure of glucose exit at the proximal tubular basolateral membrane, leading to intracellular accumulation of glucose and secondarily of glycogen, which is toxic to the proximal tubular cells [189]. As in other renal Fanconi’s syndrome variants, therapy of FanconieBickel syndrome is directed at replacement of renal solute losses by supplements of water, electrolytes and bicarbonate. Rickets is managed with Pi and active vitamin D3 supplements. To prevent hypoglycemia and ketosis, patients should avoid prolonged fasting. Night feeding with uncooked starch has been shown to reduce hypoglycemia and improve growth during infancy and early childhood [190]. DENT’S DISEASE DUE TO DEFECTS IN THE CHLORIDE CHANNEL CLC-5

X-linked hypercalciuric nephrolithiasis is a group of hereditary proximal tubulopathies in which hypercalciuria is a dominant manifestation. Variants of the syndrome include Dent’s disease, X-linked recessive nephrolithiasis, X-linked recessive hypophosphatemic rickets and LMW-proteinuria with hypercalciuria and nephrocalcinosis. All these syndromes are characterized by multiple proximal tubular transport defects of varying severity, hypercalciuria with nephrolithiasis and/or nephrocalcinosis, renal Pi wasting leading to

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rickets or osteomalacia, decreased serum PTH, elevated serum 1,25-(OH)2-vitamin D3, progressive renal insufficiency and male predominance [191,192]. Most carrier females have LMW proteinuria without clinical disease. All four syndromes are inherited in an X-linked recessive pattern, and are caused by mutations in the CLCN5 gene on chromosome Xp11.22, encoding the kidney-specific voltage-gated chloride channel, ClC-5 [193e197]. Currently, “Dent’s disease” is the accepted generic term for all disorders caused by mutations in CLCN5. ClC-5 plays a key role in endosomal acidification, a process that is essential for the megalin/cubulin

receptor-mediated endocytosis [198] (Fig. 27.5). In the proximal tubular cell, the recycling of integral plasma proteins and endocytosis of various glomerular-filtered substances, including LMW proteins, albumin, PTH and vitamin D3, among others, is mediated by megalin, which is a recycling receptor with multiligand binding capacity. Megalin, together with the plasma membrane surface protein cubilin, attaches to their various ligands, generating a receptor-ligand complex which is internalized by endocytosis into endosomal vesicles. Inside the endosome, dissociation of the receptor from its reabsorbed ligand is followed by recycling of the receptor back to the plasma membrane, and direction of the

FIGURE 27.5

The chloride channel ClC-5 is essential for proximal tubular endocytosis by providing an electric shunt necessary for the efficient acidification of vesicles in the endocytic pathway. Adsorptive or receptor mediated endocytosis is responsible for the reabsorption of albumin, PTH, and vitamin D3. The polyspecific receptor megalin binds a variety of ligands including vitamin-binding proteins, carrier and transporter proteins, lipoproteins, hormones, drugs, enzymes, immune-related proteins, and myoglobin. Megalin also acts as a membrane anchor for the peripheral membrane protein cubilin and the two form a scavenger receptor complex. During the process of endocytosis, small plasma membrane invaginations detach from the membrane to form endocytic vesicles. Once albumin, PTH, vitamin D3 and other ligands have been taken up by receptor-mediated endocytosis, their final destination is the lysosome and the common mechanism triggering receptor-ligand dissociation is the drop in pH in different endocytic compartments. Megalin and cubilin recycle back to the plasma membrane and are not directed to the lysosome. (Modified from: Bonnardeaux A, Bichet DG. Inherited disorders of the renal tubule. In: Brenner BM, ed. Brenner and Rector’s The Kidney, 8th edn. Philadelphia: W.B. Saunders Company; 2008. p. 1393.)

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ligand to lysosomal proteolysis. Intra-endosomal receptor-ligand dissociation is dependent on acidification of the endosomal lumen by influx of Hþ and Cl, which is mediated by the vacuolar Hþ-ATPase pump and the ClC-5 chloride channel. Experimental mouse models of Dent’s disease have shown that defective function of ClC-5 causes impaired proximal tubular endosomal acidification. This, in turn, results in impaired receptor-mediated endocytosis of LMW proteins and calciotropic hormones, eventually leading to the biochemical and clinical sequelae of Dent’s disease [199e201]. While rickets and clinical bone disease due to renal Pi wasting only occur in a minority of patients with Dent’s disease, in those patients it is often severe and disabling [192]. One of the explanations for the hypophosphatemic rickets associated with Dent’s disease stems from the excessive accumulation of PTH inside the proximal tubular lumen, as a consequence of its defective endocytosis. Intraluminal persistence of PTH stimulates the proximal tubular apical PTH receptor, which causes enhanced internalization of the Naþ/Pi-co-transporter NaPi-IIa. This PTH-dependent decrease in the BBM abundance of NaPi-IIa causes renal Pi wasting both in ClC-5 knockout mice and in patients with Dent’s disease, and underlies the hypophosphatemic rickets and osteomalacia observed in affected individuals [192,202]. LOWE OCULOCEREBRORENAL SYNDROME

Lowe’s syndrome is a rare X-linked disorder characterized by congenital cataracts or glaucoma, severe mental retardation and partial renal Fanconi’s syndrome [203]. Renal involvement is a major clinical feature of Lowe syndrome, usually occurring during the first year of life with varying degrees of severity. Affected patients exhibit LMW proteinuria, aminoaciduria, hypercalciuria, hyperphosphaturia with hypophosphatemic rickets, variable glycosuria, renal tubular acidosis and progressive renal insufficiency [204]. Lowe’s syndrome was mapped to Xq24-25 [205] by linkage analysis, and was subsequently found to be caused by mutations in the ubiquitously expressed OCRL1 [206]. OCRL1 encodes the enzyme phosphatidyl-inositol 4,5-biphosphate 5-phosphatase (PIP2 5-phosphatase) [207,208], which is localized to the Golgi complex [209], and is involved in signal transduction, actin polymerization and receptor-mediated endosomal trafficking and sorting [210]. In proximal tubular cells, PIP2 5-phosphatase co-localizes with megalin at the BBM, where it is involved in early steps of receptormediated endocytosis. This may explain the phenotypic similarity between Dent’s disease and Lowe’s syndrome, as well as the pathophysiology of renal Pi wasting in both disorders [211].

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To date, various mutations have been described in patients with Lowe’s syndrome, including missense, nonsense, complete gene deletions and mosaicism [212,213]. Most patients with OCRL1 gene disruption exhibit the typical ocular-cerebral-renal triad of classic Lowe’s syndrome. However, OCRL1 mutations can also cause a milder variant of Lowe’s syndrome which is indistinguishable from classic Dent’s disease and is designated as Dent-2 disease (MIM 300555). This variant, which accounts for approximately 15% of patients with a Dent’s phenotype, is characterized by isolated renal manifestations and lacks the ocular and neurologic abnormalities of full-blown Lowe’s syndrome [214]. The cause for this phenotypic heterogeneity among carriers of OCRL1 mutations is unknown. MITOCHONDRIAL CYTOPATHIES

Mitochondrial cytopathies comprise a heterogeneous group of genetic disorders resulting from impaired oxidative phosphorylation secondary to defects of one or more of the enzymatic complexes of the mitochondrial respiratory chain [215]. The most important role of the mitochondria is cellular energy supply by ATP production through the process of oxidative phosphorylation. Oxidative phosphorylation is carried out in the inner mitochondrial membrane by proteins of the respiratory chain, which comprises five multisubunit protein complexes, encoded either by the mitochondrial or by the nuclear genomes [216]. The mitochondrial genome consists of a circular, double-stranded DNA molecule spanning 16.6 kb, and containing 37 genes, encoding two ribosomal RNAs (rRNA), 22 transfer RNAs (tRNA), and 13 polypeptides of the respiratory chain. These 13 polypeptides are contained within subunits I, III, IV and V of the respiratory chain complexes. All other respiratory chain subunit proteins and mitochondrial structural proteins are encoded by the nuclear genome, and are specifically targeted to the mitochondria after translation. Hence, mitochondrial cytopathies can arise from genetic defects in the mitochondrial genome or in nuclear genes encoding mitochondrial targeted proteins. Since mitochondria are almost exclusively transmitted maternally, the classic inheritance pattern of mitochondrial cytopathies is maternal. In contrast, mitochondrial diseases due to defects in nuclear-encoded genes with mitochondrial function are transmitted in an autosomal recessive or autosomal dominant pattern of inheritance [217]. Since mutant and wild-type copies of mitochondrial DNA can coexist in the same cell, and since the ratio of wild-type to mutant copies of mitochondrial DNA is greatly variable between tissues (a concept known as heteroplasmy), hereditary mitochondrial cytopathies are characterized by multiorgan involvement and by a wide range of clinical manifestations of varying

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severity. The organs most susceptible to mitochondrial impairment are those with highest dependence on mitochondrial energy supply, including the brain, muscle, heart, endocrine glands, liver and kidney [215,218]. Renal involvement in mitochondrial cytopathies may result from impairment of various nephron segments, including the glomerulus, the proximal tubule or the tubulo-interstitium. Renal injury due to mitochondrial impairment may manifest alone [219e221], or appear concomitantly with other organ involvement, during any time of the disease course [222]. Given the heavy energy dependence of molecular processes associated with proximal tubular solute handling, it is not surprising that the most frequent presentation of renal involvement due to mitochondrial impairment is renal Fanconi’s syndrome [218]. As in other forms of renal Fanconi’s syndrome, patients with mitochondrial cytopathies exhibit the typical features of dehydration, failure to thrive, multiple solute wasting, hypophosphatemic rickets and renal tubular acidosis. In addition, patients may have extrarenal manifestation [223e226], compatible with the diagnoses of Pearson’s disease (pancytopenia, hepatocellular dysfunction, exocrine pancreatic dysfunction, and proximal tubulopathy) [227,228], KearnseSayre syndrome (progressive ophthalmoplegia, pigmentary retinopathy, ataxia and heart block) [219,229] and Leigh’s disease (subacute necrotizing encephalomyelopathy) [230], among others. There is no effective treatment for renal Fanconi’s syndrome secondary to hereditary mitochondrial disorders, and therapy is mostly symptomatic, aimed at replacing the renal losses by supplements of sodium bicarbonate, potassium, vitamin D3, Pi, carnitine and water, as required [231]. OTHER CAUSES OF HEREDITARY FANCONI’S SYNDROME

Hereditary renal Fanconi’s syndrome may also occur secondary to additional inborn errors of metabolism such as galactosemia (MIM 230400), hereditary fructose intolerance (MIM), glycogen storage disease type I (MIM 229600), tyrosinemia type I (MIM 276700) and Wilson’s disease (MIM 277900). In all these disorders, renal involvement in the form of proximal tubulopathy is part of a multisystem disorder, with predominant extrarenal manifestations [231]. Hereditary idiopathic Fanconi’s syndrome is diagnosed by exclusion of all other known causes of this disorder. Idiopathic Fanconi’s syndrome can be transmitted by various modes of inheritance [232e238], although autosomal dominant transmission is most commonly described. Renal manifestations are similar to those described in other forms of renal Fanconi’s syndrome, and treatment is symptomatic. Many of the

idiopathic forms of renal Fanconi’s syndrome progress to chronic renal failure during adulthood [233,238]. Acquired Renal Fanconi’s Syndrome ACQUIRED FANCONI’S SYNDROME SECONDARY TO SYSTEMIC DISORDERS

Generalized proximal tubulopathy as part of an acquired renal or systemic disorder is usually manifested during adulthood, and rarely affects pediatric patients. Disorders typically associated with acquired renal Fanconi’s syndrome include plasma cell dyscrasias such as multiple myeloma [239], secondary amyloidosis [240] and Sjo¨gren syndrome [241e243]. Rarely, proximal tubulopathy may occur after renal transplantation [244], or secondary to various immune-mediated renal disorders such as acute tubulointerstitial nephritis [245] or focal segmental glomerulosclerosis [246]. Anorexia nervosa can result in transient renal Fanconi’s syndrome associated with profound renal Pi wasting, which is reversible with nutritional recovery [247]. ACQUIRED FANCONI’S SYNDROME SECONDARY TO EXOGENOUS SUBSTANCES

There are numerous drugs, chemical substances and natural herbs with toxic effects on the proximal tubule, leading to renal Fanconi’s syndrome. Many ingested chemicals gain access into proximal tubular cells after undergoing glomerular filtration, followed by proximal tubular reabsorption through the luminal membrane. Other substances accumulate within proximal tubular cells via the basolateral membrane from peritubular capillaries. Outdated tetracyclines, aminoglycosides, salicilates, valproic acid and Chinese herbs are all well-established proximal tubular nephrotoxins, exerting their toxic effect by various cellular mechanisms including reduced intracellular glucose transport, impaired lysosomal function, mitochondrial injury and respiratory chain arrest [248e254]. Nucleoside analog reverse-transcriptase inhibitors (NARTIs) and nucleotide analog reverse-transcriptase inhibitors (NtARTIs) are two subclasses of antiretroviral drugs used for the treatment of human immunodeficiency virus (HIV) and other retroviral infections. Members of both subclasses (including stavudine, lamivudine, adefovir, cidofovir and tenofovir) have been implicated in renal Fanconi’s syndrome, renal Pi wasting and osteomalacia [255e257]. Some NtARTIs gain access into the proximal tubular cells via activated organic anion transporters (OAT) located in the basolateral cell membrane, and are thought to accumulate within the cells due to inhibited efflux via the luminal membrane [258]. The mechanism of proximal damage secondary to NARTIs and NtARTIs involves mitochondrial toxicity due to mitochondrial DNA depletion. In

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some instances, dose adjusting or discontinuing treatment have resulted in resolution of renal Fanconi’s syndrome [255]. Deferasirox is a recently approved oral iron chelator used for the treatment of iron overload in patients with chronic transfusion therapy for diseases such as b-thalassemia or sickle cell anemia. While generally well tolerated, there is an increasing number of reported renal side effects of deferasirox, including proximal tubular dysfunction [259e261]. The mechanism of deferasirox renal injury has been postulated to result from chelation of proximal tubular mitochondrial iron, leading to ATP depletion. Renal Fanconi’s syndrome due to deferasirox can be reversible with appropriate dose adjustment or with temporary suspension of treatment. Several anticancer chemotherapeutic drugs have been associated with proximal tubulopathy and renal Pi wasting. The alkylating agent ifosfamide exerts its nephrotoxic effect by conversion to the toxic metabolite chloroacetylaldehyde (CAA) [262]. CAA impairs proximal tubular function by decreasing gluthatione and ATP levels, and by inhibiting the endosomal Hþ-ATPase leading to impaired endocytosis [263]. Cisplatin is a platinum-containing anticancer drug with significant nephrotoxic side effects. Cisplatin-associated proximal tubulopathy is mediated by reactive oxygen species formation leading to mitochondrial disruption, lysosomal damage and increased apoptotic cell death [264,265]. Prior administration of cisplatin or carboplatin was shown to aggravate the proximal tubular nephrotoxicity of ifosfamide, thus highlighting the additive nephrotoxic effect of these chemotherapeutic drugs. The proximal tubulopathy of ifosfamide and of carboplatin is usually irreversible, and requires prolonged and extended therapy with electrolytes and Pi supplements [266]. Heavy metals such as lead, cadmium, mercury, chromium and platinum are environmental and occupational hazardous chemicals with high toxicity at very low doses. The kidneys are the first target organs of heavy metal toxicity, owing to their ability to reabsorb and accumulate divalent metals. Both acute and chronic intoxication of varying severity has been implicated in heavy metal induced nephropathy [267]. Of the list of toxic heavy metals, cadmium and lead are the most prevalent and most nephrotoxic metals known to humans [268]. Cadmium intoxication causes renal Fanconi’s syndrome after prolonged exposure. The best characterized example of intoxication of a large population by environmental exposure to cadmium is itai-itai-byo or “ouch-ouch” disease, endemic to the Jinzu river basin in Japan, and named after its crippling and painful osteomalacic bone involvement [268]. The major target for proximal tubular cadmium toxicity is the

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mitochondria where it accumulates and inhibits the respiratory chain, resulting in generation of reactive oxygen species and leading to activation of apoptotic pathways and to proximal tubular cell death [198,269]. As opposed to cadmium exposure, which may cause proximal tubulopathy at any age group, lead intoxication produces renal Fanconi’s syndrome predominantly in the pediatric age group [268]. Acute lead nephropathy is characterized by proximal tubular dysfunction and renal Fanconi’s syndrome, associated with ultrastructural changes of mitochondrial structure accompanied by cytosolic and nuclear inclusion bodies. Acute lead nephropathy may be reversible with appropriate chelating therapy [268]. Chronic lead nephropathy, however, is irreversible and leads to chronic renal failure end stage kidney disease [270].

CALCIUM AND MAGNESIUM Renal Handling of Calcium The body homeostasis of calcium is largely regulated at the level of the intestine [271,272], with the renal handling of calcium being far less important than the degree of intestinal calcium absorption. However, without a means of reabsorbing the amounts of calcium appearing in the glomerular ultrafiltrate, massive urinary calcium losses would ensue and hypercalciuria, nephrocalcinosis, and nephrolithiasis would develop. Plasma-ionized calcium and calcium complexed with various anions e a fraction that forms 0.6 of the total plasma concentration of calcium e is freely filterable. The fraction of calcium bound to plasma protein is non-filtered [273]. Ninety-eight percent of the filtered calcium is reabsorbed in the renal tubule: 60e70% in the proximal convoluted tubule, 20% in the thick ascending limb of Henle, 10% in the distal convoluted tubule, and less than 5% in the cortical collecting duct (Fig. 27.6) [272]. The renal handling of Ca2þ has been the subject of intensive investigation over the past decade and several transport pathways along the nephron involved in Ca2þ reabsorption have been elucidated at the cellular and molecular levels [5,271e274]. Hereditary defects in some of these transport pathways have been shown to result in deranged renal Ca2þ handling with either increased or decreased urinary Ca2þ excretion (see later) [272,274]. Proximal Tubule The proximal tubule (PT), which includes the proximal convoluted tubule (PCT; S1 and S2 segments) and the proximal straight tubule (PST; S3 segment), is responsible for the reabsorption of the bulk (60e70%)

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B

A Bowman’s Capsule

100%

Collecting Duct

Distal Convoluted Tubule

Proximal Convoluted Tubule

60-70%

Bowman’s Capsule

Ca2+

100%

Proximal Convoluted Tubule

10%

10-15%

Collecting Duct

Distal Convoluted Tubule

10-15%

Thick Ascending Limb of Loop

Thick Ascending Limb of Loop

60-70%

20% Thin Descending Limb of the Loop of Henle

Thin Descending Limb of the Loop of Henle

Thin Ascending Limb of Loop

Thin Ascending Limb of Loop

3-5%

3-5%

FIGURE 27.6

Mg2+

Segmental calcium (A) and magnesium (B) reabsorption along the nephron.

of the filtered Ca2þ (see Fig. 27.6) [271,275,276]. Microporeture studies have shown that tubular Ca2þ transport in the proximal tubule is primarily passive, paracellular, and is driven by solvent drag produced by high rate of Naþ and water reabsorption across the tight junction of this nephron segment [275,276]. Additional factors promoting this passive, diffusional Ca2þ reabsorption in the PT include a favorable ionized calcium concentration gradient, a high permeability for calcium in this nephron segment and, in the late PCT (S2 segment), a lumen positive transepithelial voltage [271,273]. Claudin-2, a tight junction protein expressed in the proximal nephron is a strong candidate for the paracellular Ca2þ channel in the PT [273]. There also is evidence for some active Ca2þ reabsorption in the proximal tubule, mainly in the PST (S3 segment).

tubule in health and disease [278e281]. The CaSR belongs to the family of G protein- coupled receptors and, although present in several nephron segments, is most abundant in the TAL [278,281,282], where claudin-16 and claudin-19 expression is also maximal (see Fig. 27.7). Activation of the CaSR by high concentrations of extracellular Ca2þ or Mg2þ triggers a series of intracellular signaling events including production of arachidonic acid and inhibition of adenylate cyclase [278]. Both actions result in inhibition of the luminal Naþ-Kþ2Cl co-transporter (NKCC2) and Kþ channel (ROMK) (see Fig. 27.7). This, in turn, leads to reduction in the lumen positive electrical potential in the TAL, resulting in increased urinary Mg2þ and Ca2þ excretion [278,280].

Loop of Henle

The distal convoluted tubule (DCT) and the connecting tubule (CT) which reabsorb up to 10% of the filtered Ca2þ load (see Fig. 27.6) are responsible for the fine tuning of Ca2þ excretion in the kidney. Ca2þ transport in the DCT and CT is transcellular, active and occurs against on electrochemical gradient (Fig. 27.8) [5,273,277]. This transport process involves three steps which include entry of luminal Ca2þ into cell via the apical membrane, transient receptor potential cation channel TRPV5 (also known as ECaC1), transcellular movement mediated by vitamin D-dependent calbindin-D28K, and basolateral extrusion of Ca2þ via the Ca2þ-ATPase (Ca2þ-pump) PMCA1b or the Naþ/Ca2þ exchanger NCX1 (see Fig. 27.8) [5,274,277]. The intracellular free Ca2þ level in most cells is several orders of magnitude

Ca2þ reabsorption in the thin descending and ascending limbs of the loop of Henle is negligible. The thick ascending limb (TAL), however, reabsorbs 20% of the filtered Ca2þ (see Fig. 27.6) [273]. Ca2þ (and Mg2þ) reabsorption in the TAL is paracellular, primarily mediated by the tight junction proteins claudin-16 (paracellin-1) and claudin-19 (see later), and is driven by the lumen positive transepithelial voltage. This voltage is generated by Cl entry into cell via the luminal membrane, bumetanide-sensitive, Naþ-Kþ-2Cl co-transporter (NKCC2) and Kþ exit from cell via the luminal membrane, Kþ channel ROMK (Fig. 27.7) [271,273,277]. Increasing evidence suggests that the calcium/magnesium sensing receptor (CaSR) plays an important role in controlling divalent cation reabsorption in the renal

Distal Convoluted Tubule and the Connecting Tubule

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Transcellular and paracellular transport pathways in the TAL. Cl reabsorption across the luminal membrane occurs via the Na -K -2Cl co-transporter (NKCC2). This co-transporter is driven by the low intracellular Naþ and Cl concentrations generated by the basolateral Naþ Kþ-ATPase and ClC-Kb, respectively. In addition, ROMK enables function of NKCC2 by recycling Kþ back to the lumen. The lumen-positive electrical potential, which is generated by Cl entry into the cell and Kþ exit from the cell, drives paracellular, claudin-16-, and claudin-19- mediated, Ca2þ and Mg2þ transport from lumen to blood. Activation of the basolateral calcium sensing receptor (CaSR) by high concentrations of extracellular Ca2þ or Mg2þ triggers a series of intracellular signaling events, resulting in an inhibition of the luminal ROMK channel and the luminal NKCC2 co-transporter. This, in turn, results in decreased NaCl reabsorption and (secondary to the reduction in the intraluminal positive potential) increased urinary Ca2þ and Mg2þ excretion. Hereditary tubulopathies caused by defects in these transport mechanisms are depicted. AD, autosomal dominant.

FIGURE 27.7 þ

þ



below that of extracellular fluid and the membrane potential inside is negative [273]. Hence, the apical entry step is passive whereas the basolateral exit step is active. Collecting Duct (CD) The collecting duct plays a minor role in Ca2þ reabsorption reclaiming less than 5% of the filtered Ca2þ. In the CD, Ca2þ reabsorption is active and mediated by calbindin-D28k, Ca2þ-ATPase and Naþ/Ca2þ exchange that are expressed in the human CD [273]. Many factors are known to influence renal tubular Ca2þ reabsorption in various nephron segments. Factors increasing net Ca2þ reabsorption include Pi load, alkalosis, the hormones PTH, vitamin D, calcitonin and estrogens and the diuretics thiazides and amiloride. Factors increasing Ca2þ excretion include volume expansion, hypercalcemia, acidosis, the hormones insulin, glucagon and glucocorticoids and the diuretic furosemide [271e273,277,283].

Renal Handling of Magnesium Magnesium is the second most abundant divalent cation in the intracellular fluid. It plays a critical role in numerous cellular and metabolic processes.

Abnormalities in magnesium homeostasis are relatively common in clinical practice and may lead to neuromuscular disturbances, central nervous system manifestations and cardiovascular dysfunction [284e286]. Most of the magnesium in the body (99%) is found intracellularly and only 1% is in the extracellular fluid. Whole body Mg2þ balance is regulated by dietary intake and urinary excretion. In mammals, the kidney is the principal organ responsible for Mg2þ balance [284,286,287]. Approximately 80% of plasma magnesium is not bound to proteins and is filtered through the glomerulus. Normally, more than 95% of the filtered Mg2þ is reabsorbed by the renal tubule (15e20% in the proximal tubule, 70% in the TAL and 5e10% in the distal convoluted tubule), and only about 5% is excreted in the urine (see Fig. 27.6) [286,287]. Intensive research over the past decade has defined several transport processes responsible for tubular Mg2þ reabsorption along the nephron, has clarified many of the molecular mechanisms and signaling pathways involved in these processes, and has identified several kidney-specific genes, mutations in which result in renal Mg2þ wasting and severe hypomagnesemia associated with major morbidity (see later) [273,274,286,288e290].

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Transport mechanisms in the distal convoluted tubule. Naþ and Cl reabsorption occurs via the luminal, thiazide-sensitive NaCl co-transporter (TSC; NCCT), the activity of which is modulated, among other factors, by the action of the intracellular serin/threonine protein kinases WNK4 and WNK1. Naþ exits the cell via the basolateral Naþ-Kþ-ATPase and Cl exit to blood is mediated by the basolateral Cl channel ClC-Kb. Ca2þ enters the cell via the luminal Ca2þ channel TRPV5, is transported transcellularly by vitamin D-dependant calbindinD28K, and exits the cell via the Ca2þ-ATPase, PMCA1b, and the Naþ/Ca2þ exchange, NCX1. Mg2þ enters the cell via the luminal Mg2þ channel, TRPM6, and exits the cell via a putalive basolateral Naþ/Mg2þ exchanger. TRPM6-mediated Mg2þ entry into cell is promoted by the negative intracellular membrane potential which is maintained by the activity of the Naþ-Kþ-ATPase as well as by the action of the luminal Kþ channel Kv1.1 and the basolateral Kþ channel Kir 4.1. The latter channel recycles Kþ entering the cell via the Naþ-Kþ-ATPase back into the interstitium. The g-subunit of Naþ-Kþ-ATPase, FXYD2, which is transcriptionally regulated by the hepatocyte nuclear factor 1b (HNF1b), modulates the activity of Naþ-Kþ-ATPase by affecting its afflinity to Kþ and Naþ. The basolateral EGF receptor (EGFR) activates TRPM6 by shuttling it from intracellular compartments to the apical membrane. Hereditary tubulopathies caused by defects in these transport mechanisms are depicted. AD, autosomal dominant; AR, autosomal recessive.

FIGURE 27.8

Proximal Tubule Only 15e20% of the Mg2þ filtered in the glomerulus is reabsorbed by the proximal tubule compared with 60e70% of filtered Naþ, Ca2þ and water (see Fig. 27.6) [286,287]. This leads to an increasing ratio of [Mg2þ] in the tubular lumen to that in plasma along the length of the PT as water is reabsorbed [291,292]. Mg2þ transport in the PT is generally thought to be paracellular, diffusive, and driven by the rising intraluminal Mg2þ concentration [284,285]. However, an active, transcellular Mg2þ reabsorption cannot be excluded [285,293]. Interestingly, in immature animals, the DCT plays a more important role in Mg2þ reabsorption reclaiming 60e70% of the filtered load [294]. The most likely explanation for this phenomenon may be connected with maturational changes in tight junction permeability. Loop of Henle There is likely no Mg2þ reabsorption in the thin descending and ascending limbs of the loop of Henle. About 70% of Mg2þ filtered in the glomerulus is

reabsorbed in the cortical portion of the TAL (cTAL) (see Fig. 27.6) [287,295]. The medullary TAL (mTAL) does not appear to have any capacity for Mg2þ reclamation [296]. Mg2þ (and Ca2þ) transport in the TAL segment occurs primarily through paracellular conductance [285,286,295] and is mediated by the tight junction proteins claudin-16 [297,298] and claudin-19 [299,300] (see Fig. 27.7). As indicated earlier, the lumen to cell flux of Cl via the Naþ-Kþ-2Cl co-transporter (NKCC2) in the TAL and the exit of Kþ from cell to lumen via the Kþ channel (ROMK) generate a lumen positive electrical potential which, in turn, drives paracellular Mg2þ and Ca2þ transport from lumen to blood (see Fig. 27.7) [273,274,301]. Loop diuretics, such as furosemide and bumetanide, inhibit the NKCC2 transporter and prevent the generation of the transepithelial potential difference thereby reducing Mg2þ reabsorption [302,303]. The CaSR expressed in the basolateral membrane of the TAL (see earlier) [278,281,282] also appears to participate in Mg2þ handling in this nephron segment. Activation of the CaSR by hypercalcemia or

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TABLE 27.3

Hereditary tubular disorders of calcium and magnesium handling Defective gene

Locus

Defective protein

Antenatal Bartter syndrome (type I)

SLC12A1

15q21

Naþ-Kþ-2Cl cotranoporter, NKCC2

Luminal

Antenatal Bartter syndrome (type II)

KCNJ1

11q24

Classic Bartter syndrome (type III)

CLCNKB

Bartter syndrome with deafness (type IV)

Disorder

Localization Mode of of defect inheritance

Magnesium

Calcium

Serum

Urine Serum Urine Additional features OMIM #

Reference

AR

N

N

N

[

Hypokalemia, 241200 hypochloremic metabolic alkalosis, osteopenia

[304,305]

Kþ channel, ROMK Luminal

AR

N

N

N

[

Hypokalemia, hypochloremic metabolic alkalosis

601678

[306]

1p36

Cl channel, CIC-Kb

Basolateral

AR

NY

N[

N

N[

Hypokalemia, hypochloremic metabolic alkalosis

602023

[307]

BSND

1p31

Barttin (b-subunit of Basolateral CIC-Ka/CIC-KB)

AR

N

N

N

N[

Hypokalemia, 602522 hypochloremic metabolic alkalosis, chronic renal failure, senorineural deafness

[308,309]

Autosomal dominant CASR hypocalcemia with Bartter syndrome (type V)

3q21

Ca2þ/Mg2þ sensing Basolateral receptor (CaSR)

AD

Y

[

Y

[

Hypokalemia, hypochloremic metabolic alkalosis

601198

[310,311]

Familial benign CASR hypercalcemia/ neonatal severe primary hyperparathyroidism

3q21

Ca2þ/Mg2þ sensing Basolateral receptor (CaSR)

AD/AR

N

Y

[

Y

-

145980/ 259200

[312,313]

3q28 1p34

Claudin-16 (paracellin1), Claudin-19

AR

Y

[

N

[

Ocular abnormalities (in particular, in CLDN19 defect)

603959 610036

[297,299]

Loop of Henle

CLDN16 CLDN19

Tight junction

CALCIUM AND MAGNESIUM

PEDIATRIC BONE

Familial hypomagnesemiahypercalciurianephrocalcinosis syndrome

(Continued)

747

748

TABLE 27.3 Hereditary tubular disorders of calcium and magnesium handlingdcont’d

Disorder

Defective gene

Locus

Defective protein

Localization Mode of of defect inheritance

Magnesium

Calcium

Serum

Urine Serum Urine Additional features OMIM #

Reference

[314e316]

Distal convoluted tubule SLC12A3 CLCNKB

16q13 1p36

Luminal Naþ-Cle cotransporter, TSC Basolateral (NCCT), Clechannel, CIC-Kb

AR

Y

[

N

YY

Hypokalemia, hypochloremic metabolic alkalosis

Hypomagnesemia with secondary hypocalcemia

TRPM6

9q22

Mg2þ channel, TRPM6

Luminal

AR

YY

[

Y

N

Mental retardation, 602014 epilepsy

[317,318]

12p13

Kþ channel, Kv1.1

Luminal

AD

YY

N

N

N

Episodic ataxia

160120

[319]

Autosomal dominant KCNA1 hypomagnesemia

263800

Isolated autosomal recessive hypomagnesemia

EGF

4q25

Pro EGF, epidermal Basolateral growth factor

AR

YY

N

N

N

Mental retardation, 611718 epilepsy

[320]

Isolated dominant hypomagnesemia

FXYD2

11q23

g subunit of the Naþ-Kþ-ATPase

Basolateral

AD

YY

[[

N

YY

-

154020

[321]

Maturity onset HNF1B diabetes of the young, type 5

17q12

HNF1B, hepatocye nuclear factor 1B

Nuclear/ Basolateral

AD

Y

[

N

Y

Cystic kidneys, diabetes mellitus

137920

[322]

1q23

Kþ channel, Kir4.1

Basolateral

AR

Y

[

N

YY

Epilepsy, ataxia, sensorineural deafness

612780

[323,324]

EAST, SESAME syndrome

KCNJ10

AR: autosomal recessive; AD: autosomal dominant; OMIM#: Online Mendelian Inheritance in Man (database at http://www.ncbi.nlm.hih.gov/Omim)

27. HEREDITARY TUBULAR DISORDERS OF MINERAL HANDLING

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Gitelman syndrome

CALCIUM AND MAGNESIUM

hypermagnesemia inhibits NKCC2 and ROMK activity, reduces lumen positive voltage and hence, inhibits Mg2þ and Ca2þ reabsorption in the TAL which results in urinary loss of these divalent cations (see Fig. 27.7) [278,280]. Distal Convoluted Tubule Ten percent of the filtered Mg2þ is reclaimed in the DCT (see Fig. 27.6). This amount is significant because it represents 70e80% of the Mg2þ delivered from the loop of Henle. Unlike the TAL, reabsorption of Mg2þ in the DCT is an active, transcellular process which occurs against lumen-to-interstitium electrical and concentration gradients [325]. In 2002, Konrad and coworkers [317] identified the TRPM6 gene, as the mutated gene in hereditary hypomagnesemia with secondary hypocalcemia (see later). The TRPM6 gene encodes the Mg2þ permeable ion channel, transient receptor potential melastatin 6 (TRPM6), which is a member of the transient receptor potential (TRP) family of ion channels and is exclusively expressed in the luminal membrane of the DCT and small intestine (see Fig. 27.8) [317,326]. TRPM6 appears to constitute the apical Mg2þ entry channel in transcellular Mg2þ reabsorption in the DCT [301,326,327]. TRPM6mediated uptake of Mg2þ across the apical membrane is dictated largely by the negative intracellular membrane potential present in the DCT cell [328,329,309]. The nature of the basolateral Mg2þ transport process is obscure. It has been suggested that this active transport step is mediated by an Naþ/Mg2þ exchanger, the molecular identity of which remains to be elucidated [273]. Several novel DCT-based magnesiotropic proteins have been recently identified as a result of molecular genetic studies in patients with rare hypomagnesemic disorders [274,209,301] (see later). These include: 1. the epidermal growth factor (EGF): EGF is the first reported magnesiotropic hormone which directly regulates the activity of TRPM6 [301,330]. EGF binds to its basolateral membrane-bound receptor (EGFR) which, in turn, activates TRPM6 by shuttling it from intracellular compartments to the apical membrane (see Fig. 27.8) [320,331]. A mutation in the gene encoding EGF leads to incorrect processing of proEGF to the DCT basolateral membrane and results in isolated autosomal recessive hypomagnesemia (IRH) [331] 2. the shaker-related voltage-gated Kþ channel (Kvl.l): as stated earlier, Mg2þ uptake from the lumen into the DCT cell via TRPM6 is primarily driven by the negative intracellular potential. This negative potential appears to be maintained by an apical Kþ efflux via the Kv1.1 Kþ channel, which is encoded by the gene KCNA1. Kv1.1 co-localizes with TRPM6

749

along the luminal membrane of the DCT, and is energized by the action of the basolateral Naþ-KþATPase (see Fig. 27.8) [319,332,333]. A genetic defect in Kv1.1 leads to TRPM6 malfunction and results in autosomal dominant hypomagnesemia [319,332,333] 3. ATP-sensitive inward rectifier Kþ channel 10 (Kir4.1): the Kþ channel Kir4.1 which is encoded by the gene KCNJ10, is expressed in the brain, inner ear and kidney [323,324]. In the kidney, it is expressed in the basolateral membrane of the DCT cell where it recycles Kþ ions entering the cell, via the Naþ-KþATPase, back into the interstitial space. This action of Kir4.1 contributes to the inside-negative electrical potential, which is essential for TRPM6-mediated Mg2 entry into cell (see Fig. 27.8) [301,324]. A genetic defect in Kir4.1 leads to EAST [323] or SeSAME [317] syndrome which, among other features, includes renal Mg2þ wasting and hypomagnesemia 4. the Naþ-Kþ-ATPase subunit, FXYD2 and HNF1B: The g-subunit of the basolateral Naþ-Kþ-ATPase, also termed FXYD2, is a single transmembrane protein which is encoded by the gene FXYD2 and is localized to the DCT (see Fig. 27.8) [334e336]. The FXYD2 subunit affects the kinetics of the Naþ-Kþ-ATPase altering the affinity for Naþ and Kþ [337,338] and, hence, plays an important role in generating the inside negative electrical potential in the DCT cell which, in turn, is the driving force for apical Mg2þ entry into cell. A genetic defect in FXYD2 results in isolated dominant hypomagnesemia [334,335]. Recently, additional support has been provided for the role of FXYD2 in renal Mg2þ transport. Hypatocyte nuclear factor 1B (HNF1B), a transcription factor implicated in maturity onset diabetes of the young (MODY) type 5 has been shown to regulate the FXYD2 gene (see Fig. 27.8) [301,322]. The FXYD2 gene contains several putative HNF1Bbinding sites [322], and HNF1B stimulates FXYD2 protein expression. Not surprisingly a genetic defect in HNF1B has been shown to result in a phenotype of renal Mg2þ wasting and hypomagnesemia [322] similar to FXYD2 defect-induced isolated dominant hypomagnesemia. The DCT seems to play a final role in determining urinary Mg2þ concentration since there is no evidence for significant magnesium reabsorption in the collecting duct. Many factors are known to modulate Mg2þ reabsorption in various nephron segments. These factors include volume expansion, Mg2þ restriction/load, Ca2þ restriction/load, acidebase changes, and potassium depletion, hormones such as 1,25 (OH)2 Vit D, insulin, aldosterone and parathyroid hormone and, finally, drugs such as

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27. HEREDITARY TUBULAR DISORDERS OF MINERAL HANDLING

cyclosporine A, cisplatin, furosemide and thiazides [284,286,325,339]. Some of these factors influence transcellular, TRPM6-mediated Mg2þ transport in the DCT [340,341] or paracellular, claudin-16-mediated transport of this cation in the TAL [342,343].

Tubular Transport Defects Affecting Ca2D and Mg2D Handling Proximal Tubule DENT’S DISEASE AND LOWE SYNDROME

Dent’s disease and Lowe syndrome, hereditary disorders of endosomal trafficking caused by mutations in the genes CLCN5 and OCRL1, respectively, which lead to Fanconi’s syndrome, are described earlier. Both disorders can be associated with hypercalciuria and nephrocalcinosis but no abnormalities in renal Mg2þ handling [274]. It remains unclear, however, why mutations in these two genes produce hypercalciuria. The elevated 1,25(OH)2 vitamin D level in patients with Dent’s disease may increase intestinal calcium absorption and contribute to the hypercalciuria [274]. Also, it has been suggested that defective proximal tubular endocytosis of calciotropic hormones could lead to impaired Ca2þ homeostasis and hypercalciuria [202,344,345]. Alternatively, since ClC5 is also expressed in the TAL, a major site of Ca2þ reabsorption, it is possible that impaired ClC5 function in this nephron segment contributes to the hypercalciuria. There are few reports on non-acidotic proximal tubulopathy and hypercalciuria inherited in a non-X-linked fashion [170,346]. The genetic defect in these syndromes, most likely a mutation in a gene other than CLCN5, has not yet been elucidated. Loop of Henle BARTTER SYNDROME

Bartter syndrome is a group of closely related hereditary tubulopathies. All variants of the syndrome share several clinical characteristics including renal salt wasting, hypokalemic metabolic alkalosis, hyperreninemic hyperaldosteronism with normal blood pressure, and hyperplasia of the juxtaglomerular apparatus [347e349]. All forms of the syndrome are transmitted as autosomal recessive traits. Generally, Bartter syndrome results from defective transepithelial transport of Cl in TAL or the DCT [347,349]. Transepithelial Cl transport in the TAL is a complex process that involves coordinated interplay between the luminal, bumetanide-sensitive, Naþ-Kþ2Cl co-transporter (NKCC2), the luminal, Kþ channel (ROMK), the basolateral Cl channel (CIC-Kb), as well as other co-transporters and channels (see Fig. 27.7) [273,274,301,347]. Chloride is reabsorbed across the

luminal membrane of the TAL cell by the activity of NKCC2. This co-transporter is driven by the low intracellular Naþ and Cl concentration generated by the Naþ-Kþ-ATPase and CIC-Kb, respectively. In addition, as indicated earlier, ROMK enables functioning of NKCC2 by recycling Kþ back to the renal tubular lumen. Chloride transport in the DCT occurs primarily via the luminal, thiazide-sensitive NaCl co-transporter (TSC) (see Fig. 27.8) [273,274]. Cl exit to blood in the DCT is mediated via basolateral Cl channels. The genetic variants of Bartter syndrome that have been identified include (Table 27.3; see Figs 27.7 and 27.8) [304,306,307,314,347,348]: 1. Bartter syndrome type I is caused by mutations in the NKCC2 gene, SLC12A1. This gene belongs to the family of electroneutral chloride-coupled co-transporter genes [350] and resides on chromosome 15q15-21. This genetic variant leads to antenatal Bartter syndrome which is the most severe form of the disease. It is characterized by polyhydramnios, premature birth, life-threatening episodes of salt and water loss in the neonatal period, hypokalemic alkalosis and failure to thrive, as well as osteopenia, hypercalciuria and early onset nephrocalcinosis [304,305,347] 2. Bartter syndrome type II is caused by mutations in the ROMK gene (KCNJ1), that is located on chromosome 11q24-25 [347]. ROMK mutations lead to the clinical phenotype of antenatal Bartter syndrome [306] 3. Bartter syndrome type III is caused by mutations in the ClC-Kb gene, CLCNKB [307]. This gene, which is located on chromosome 1p36, belongs to the family of genes encoding voltage-gated Cl channels to which CLCN5 (Dent’s disease gene) also belongs [347,349]. Patients with CLCNKB mutation usually have classic Barrter syndrome which occurs in infancy or early childhood. It is characterized by marked salt wasting and hypokalemia leading to polyuria, polydipsia, volume contraction, muscle weakness and growth retardation. Hypercalciuria and nephrocalcinosis may occur [347,349] 4. Bartter syndrome type IV is caused by mutations in the barttin gene, BSND. Barttin serves as a b-subunit for ClC-Ka and ClC-Kb chloride channels (see Fig. 27.7) [308,309]. Hereditary defect in barttin leads to antenatal Bartter syndrome associated with sensorineural deafness and renal failure. Barttin colocalizes with the subunit of the Cl channel in basolateral membranes of the renal tubule and inner car epithelium [309] 5. Bartter syndrome type V is caused by gain-offunction mutations in the gene CASR, encoding the calcium-sensing receptor (CaSR) (see Fig. 27.7). This

PEDIATRIC BONE

CALCIUM AND MAGNESIUM

genetic defect leads to autosomal dominant hypocalcemia with Bartter syndrome [310,311]. As indicated earlier, activation of the basolateral CaSR inhibits the luminal NKCC2 co-transporter and the ROMK channel which, in turn, results in decreased NaCl reabsorption and increased urinary Ca2þ and Mg2þ excretion 6. Gitelman syndrome is caused by mutations in the gene SLC12A3 encoding the thiazide-sensitive NaCl cotransporter (NCCT) operating in the DCT (see later). Recent data, however, have suggested that the genotypeephenotype correlation is not so clear-cut and that phenotypic overlap may occur. It has been shown that mutations in CLCNKB can also cause phenotypes that overlap with either antenatal or Gitelman syndrome [315,316]. The loss-of-function mutations in the genes of TAL cell transporters in Bartter syndrome lead to the derangements in tubular handling of minerals observed in this syndrome [273,274,301,347]. Normally, the lumen-to-cell flux of Cl via the NKCC2 co-transporter in the TAL and the exit of Kþ from cell to lumen generate lumen-positive electrical potential which, in turn, drives paracellular Ca2þ and Mg2þ transport from lumen to blood (see earlier) (see Fig. 27.7). Impaired function of NKCC2 or ROMK in antenatal Bartter syndrome results in reduction of intraluminal positive voltage, which leads to hypercalciuria and nephrocalcinosis. However, normal serum Ca2þ levels are maintained. Possible mechanisms responsible for the maintenance of normocalcemia include 1,25(OH)2VitD-induced increase in intestinal Ca2þ absorption and PTH-induced retrieval of Ca2þ from bone [273,274]. The transport defect in the TAL should have resulted in inhibition of Mg2þ reabsorption and hypomagnesemia. The absence of hypomagnesemia in patients with antenatal Bartter syndrome has been explained by compensatory stimulation of Mg2þ reabsorption in the distal convoluted tubule induced by the high level of aldosterone, which is a characteristic of the syndrome [347]. FAMILIAL HYPOMAGNESEMIA WITH HYPERCLACIURIA AND NEPHROCALCINOSIS (FHHNC)

Familial hypomagesemia with hypercalciuria and nephrocalcinosis syndrome (FHHNC, MichelliseCastrillo syndrome), is a rare autosomal recessive disorder [351,352]. The disease is characterized by marked renal Mg2þ wasting which leads to severe hypomagnesemia [301,352]. Patients also have hypercalciuria resulting in nephrocalcinosis and renal failure. In addition, ocular abnormalities, such as myopia and horizontal nystagmus, may occur [301].

751

In 1999, Simon et al. [297] first reported that FHHNC was caused by mutations in the gene CLDN16 (also known as PCLN-1). The gene encodes a protein, claudin-16 (paracellin-1), which is located in the paracellular tight junctions of the TAL (see Fig. 27.7) and is a member of the claudin family of tight junction proteins [353e355]. Claudin-16, which appears to regulate the paracellular transport of Mg2þ in the TAL, is the first reported tight junction protein involved in ion resorption. Mutations in the gene CLDN16 gene render the claudin-16 protein either non-functional [297,356] or, when occurring in the PDZ domain, cause mislocalization of the protein to lysosomes [357]. The disease mutations found in claudin-16 result in an increase in the permeability of the tight junction to anions thereby dissipating the transepithelial voltage gradient and selectively impeding Mg2þ (and Ca2þ) resorption [358]. Despite the concomitant impairment in Ca2þ reabsorption, these patients maintain normal serum Ca2þ, most likely by using alternative routes of renal and intestinal Ca2þ reclamation. More recently, FHHNC associated with severe ocular abnormalities has been shown to be caused also by mutations in CLDN19, the gene encoding the tight junction protein claudin-19 [299]. Claudin-19 co-localizes with claudin-16 in the TAL, the two proteins interact in conferring a tight junction with cation selectivity and, hence, control divalent ion reabsorption [359,360]. AUTOSOMAL DOMINANT HYPOCALCEMIC HYPERCALCIURIA (ADHH)

Normally, extracellular Ca2þ regulates PTH activity by interacting with the CaSR located on the surface of parathyroid cells [361,362]. Autosomal dominant hypercalemic hypercalciuria (ADHH) is caused by heterozygous activating mutations of the CaSR gene which cause the receptor to be hyperresponsive to extracellular calcium therby decreasing PTH production [361e363]. Patients with ADHH have mild hypocalcemia, low PTH levels and occasionally hypomagnesemia [310,311]. In addition, the activation of renal CaSR inhibits Ca2þ and Mg2þ reabsorption in the TAL leading to hypercalciuria and hypermagnesiuria (see Fig. 27.7). In some cases, due to the effect of CaSR on TAL transporters, ADHH is associated with Bartter-like syndrome (Bartter syndrome type V; see earlier). FAMILIAL BENIGN HYPERCALCEMIA AND NEONATAL SEVERE PRIMARY HYPERPARATHYROIDISM

Patients with familial benign hypercalcemia (FHH) or neonatal severe primary hyperparathyroidism (NSHPT) have inactivating mutations in the CaSR gene that are heterzygous and homozygous, respectively [361,364]. These mutations shift the set point of Ca2þ in this condition blunting the normal ability to

PEDIATRIC BONE

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27. HEREDITARY TUBULAR DISORDERS OF MINERAL HANDLING

reduce PTH secretion in response to elevated Ca2þ and Mg2þ levels and directly increasing renal Ca2þ and Mg2þ reabsorption (see Table 27.3 and Fig. 27.7) [312,313]. Whereas FHH is a mild disease inherited in an autosomal dominant fashion, NSHPT is a severe, life-threatening condition caused by homozygous CaSR mutations [364]. The genetic defect leads in both diseases to elevated or “inappropriately normal” PTH levels, hypercalcemia, hypermagnesemia and an inappropriate reduction in urinary Ca2þ and Mg2þ excretion. Renal hyperabsorption of divalent cations is partly caused by increased circulating PTH levels, and partly by reduced functionality of the CaSR in the TAL [365]. The persistence of hypocalciuria even after parathyroidectomy in FHH patients confirms the role of CaR in regulating renal calcium handling [366]. Distal Convoluted Tubule GITELMAN SYNDROME

Gitelman syndrome, which is a variant of Bartter syndrome, is characterized by a mild clinical presentation in older children or adults [347,348]. Patients may be asymptomatic and present with transient muscle weakness, abdominal pain, symptoms of neuromuscular irritability or unexplained hypokalemia. Hypocalciuria and hypomagnesemia are typical [347,348]. The disease is usually caused by mutations in the gene SLC12A3 encoding the DCT apical membrane thiazidesensitive NaCl co-transporter (NCCT) (see Fig. 27.8) [314]. Hence, the Gitelman phenotype is mimicked by chronic thiazide treatment, a potent NCC blocker. However, mutations in the gene CLCNKB encoding the basolateral membrane Cl channel ClC-Kb have also been reported to result in Gitelman syndrome phenotype [315,316]. The exact mechanism underlying the hypocalciuria and hypomagnesemia in Gitelman syndrome remain to be elucidated. It has been hypothesized that the loss-of-function mutation in NCCT causes hypocalciuria by the same mechanism as thiazides (see Fig. 27.8) [367,368]. According to this hypothesis, impaired Naþ reabsorption across the luminal membrane of the DCT cell, coupled with continued exit of intracellular Cl through basolateral ClC-Kb channels, causes the cell to hyperpolarize. This, in turn, stimulates entry of Ca2þ into the cell via the luminal voltage-activated Ca2þ channels, TRPV5 [277,367]. In addition, the lowering of intracellular Naþ concentration facilitates Ca2þ exit via the basolateral Naþ/Ca2þ exchanger, NCX1, and the Ca2þATPase, PMCA1b. Other studies have suggested that the hypocalciuria in Gitelman syndrome or in chronic thiazide administration is secondary to volume contraction which increases proximal tubular Na2þ

reabsorption and, hence, facilitates hyperabsorption of Ca2þ in the proximal tubule [274,369]. The reasons for renal Mg2þ wasting and hypomagnesemia typical of Gitelman syndrome are unknown [368]. Slc12a3 deficient mice have decreased expression of TRPM6 in the DCT [369] but the mechanism responsible for this decrease is unknown. HYPOMAGNESEMIA WITH SECONDARY HYPOCALCEMIA

Hypomagnesemia with secondary hypocalcemia (HSH) is a rare autosomal recessive disorder that is characterized by very low serum Mg2þ and Ca2þ levels and manifests in early infancy with seizures, tetany and muscle spasms (see Table 27.3) [301,317,318]. Affected patients have evidence of both defective intestinal absorption and impaired renal reabsorption of Mg2þ [317,326]. The hypocalcemia, which is secondary to Mg2þ deficiency-induced parathyroid failure, is resistant to Ca2þ or vitamin D therapy [274]. The hypomagnesemia and the hypocalcemia respond to large doses of oral or parenteral Mg2þ [318,370]. The disorder is caused by a mutation in the gene TRPM6, which is localized on chromosome 9q22, and encodes the transient receptor potential Mg2þ channel, TRPM6 that is exclusively expressed in the apical membrane of the DCT (see earlier) (see Fig. 27.8) and the small intestine [317,318]. Although the hypomagnesemia of HSH appears to be primarily the result of deficient TRPM6mediated intestinal Mg2þ absorption, there is also evidence for renal Mg2þ leak [317,318]. ISOLATED DOMINANT HYPOMAGNESEMIA

Isolated dominant hypomagnesemia (IDH) is a rare disorder which is characterized by renal Mg2þ wasting, mild hypomagnesemic symptoms, as well as hypocalciuria (see Table 27.3) [301,321]. Linkage analysis has mapped the syndrome to chromosome 11q23 [301]. Subsequent molecular analysis [321] has identified the gene FXYD2 encoding the Naþ-Kþ-ATPase g-subunit, also termed FXYD2, as the mutated gene in this disorder (see Fig. 27.8). The FXYD2 protein is localized on the basolateral membrane of the DCT (see Fig. 27.8) [334,335]. Isolated renal Mg2þ wasting is the first human disease in which a mutation in a gene encoding a NaþKþ-ATPase subunit has been implicated. As indicated earlier, FXYD2 proteins are a family of single transmembrane proteins known to modulate Naþ/Kþ-ATPase function [337,338]. The FXYD2 protein changes the Naþ/Kþ-ATPase kinetics by reducing the affinity for Naþ while increasing that for Kþ. In IDH, the dominant negative mutant FXYD2 protein binds to wild-type FXYD2 proteins and retains them inside the cell [321,371]. It has been hypothesized that this retention results in reduced outward basolateral Naþ transport

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753

ACIDeBASE

in the polarized DCT cell which, in turn, reduces intracellular voltage thereby reducing inward electric driving force for Mg2þ entry via TRPM6 [274]. Recently, mutations in the gene encoding the hepatocyte nuclear factor 1B (HNF1B) implicated in maturityonset diabetes of the young (MODY) type 5 have been shown to result in hypomagnesemia and renal Mg2þ wasting as well as hypocalciuria (see Table 27.3) [301,323]. The HNF1B gene encodes a transcription factor which regulates the FXYD2 gene (see Fig. 27.8) [301]. Defective transcription of FXYD2 appears to promote renal Mg2þ wasting. ISOLATED AUTOSOMAL RECESSIVE HYPOMAGNESEMIA

Isolated autosomal recessive hypomagnesemia (IRH) was first reported in two siblings who showed renal Mg2þ wasting, normal urinary Ca2þ excretion, as well as epileptic seizures and psychomotor deficit (see Table 27.3) [372]. The molecular defect in these patients was identified as a mutation in the epidermal growth factor (EGF) gene causing a defect in the routing of proEGF to the basolateral membrane of the DCT cell (see Fig. 27.8) [330,331]. As indicated earlier, the binding of EGF protein to the EGF receptor located in the basolateral membrane is essential for the function of the apical membrane-bound Mg2þ channel, TRPM6 [320,331]. The EGF mutation results in disruption of this pathway, dysfunction of TRPM6 and renal Mg2þ wasting. EGF is the first reported autocrine/paracrine magnesiotropic hormone [274]. AUTOSOMAL DOMINANT HYPOMAGNESEMIA

Autosomal dominant hypomagnesemia is a rare disease that was recently reported in a large Brazilian family [319]. The patients displayed recurrent muscle cramps, tetany, tremor, muscle weakness, cerebellar atrophy and hypomagnesemia (see Table 27.3). A mutation was found in the gene KCNA1 encoding the voltagegated Kþ channel (Kv1.1) [319]. As described earlier, this channel co-localizes with TRPM6 in the apical membrane of DCT cells (see Fig. 27.8) and normally generates the inside negative voltage essential for Mg2þ entry into cell. A heterozygous Kv1.1 N255D mutation results in a non-functional channel with a dominant negative effect on the wild-type Kv1.1 channel, an effect that is in line with the autosomal dominant inheritance pattern of this disorder [301]. EAST OR SESAME SYNDROME

Recently, two groups reported a new syndrome of hereditary hypomagnesemia. The syndrome includes epilepsy, ataxia, sensorineural deafness and a Gitelman-like tubulopathy with normotensive hypokalemic metabolic alkalosis and hypomagnesemia (see

Table 27.3) [323,324]. The syndrome is caused by a defect in the gene KCNJ10 encoding the basolateral Kþ channel Kir4.1 (see Fig. 27.8). Kir4.1 malfunction leads to reduced basolateral Kþ cycling which, in turns impedes electrogenic Naþ-Kþ-ATPase transport [301]. This results in depolarization of the DCT apical membrane and inhibition of NCCT-driven NaCl reabsorption and TRPM6-driven Mg2þ reabsorption explaining the Gitelman-like phenotype. MITOCHONDRIAL HYPOMAGNESEMIA

In 2004, a large Caucasian family was identified with hypomagnesemia, hypercholesterolemia and hypertension [373]. A pattern of maternal transmission suggested a mitochondrial disease and, indeed, mutation analysis discovered a thymine-to-cytidine conversion near the anticodon of the isoleucine tRNA [373]. Family members with hypomagnesemia had evidence of renal Mg2þ wasting. The authors have speculated about an impaired energy metabolism of DCT cells as the consequence of the mitochondrial defect which could impede transcellular Mg2þ transport [374].

ACIDeBASE Renal Acid-base Handling The kidney plays a vital role in acidebase homeostasis. Acid generated in the body, mostly from dietary protein, must be excreted by the kidneys to preserve acidebase balance. To accomplish this, the kidneys reabsorb filtered bicarbonate primarily in the proximal tubule and excrete acid as titratable acid and ammonium in the cortical collecting duct (CCD) [375e377]. Proximal Tubule Most (80e90%) of the filtered load of HCO3  is reabsorbed in the proximal tubule [375,376]. Several membrane transport proteins participate in acidebase handling in the proximal tubule (Fig. 27.9) [376,377]. Hþ and HCO3  are formed in the proximal tubular cell from CO2 and H2O as a result of the action of intracellular carbonic anhydrase II (CAII). Hþ efflux from cell to lumen occurs via the apical Naþ/Hþ exchanger (NHE3), and to a small extent via the apical Hþ-ATPase pump. HCO3  exit to blood is mediated by the basolateral electrogenic Naþ-HCO3  co-transporter (NBC1) which functions with a probable stoichiometry of 3HCO3  to 1Naþ [378]. The secreted Hþ combines in the tubular lumen with filtered HCO3  to form carbonic acid which, in the presence of the brush border membrane-bound carbonic anhydrase IV (CA IV), dissociates to carbon dioxide and water (see Fig. 27.9). The

PEDIATRIC BONE

754

27. HEREDITARY TUBULAR DISORDERS OF MINERAL HANDLING

LUMEN

BLOOD

CELL

NaHCO3

HCO3-

Na +

NHE3 H+

Na +

H+

NBC1

FIGURE 27.9 Transport mechanisms participating in acidebase handling in a proximal tubular cell. Hþ and HCO3  are formed in the cell as a result of carbonic anhydrase II (CAII) action. Hþ exits the cell via the apical Naþ/Hþ exchanger (NHE3) and Hþ-ATPase pump. HCO3  exit occurs via the basolateral Naþ/ HCO3  co-transporter (NBC1). Hereditary renal tubular acidosis syndromes caused by defects in NBC1 and CAII, respectively, are depicted.

HCO3H2CO3

CA IV H 2O

ATP

H+

H2CO3

ADP

CA II CO2

Isolated Proximal RTA

H2O + CO2

Mixed Proximal and Distal RTA (Type 3)

CO2 diffuses rapidly across the luminal membrane into the cell [375]. Factors affecting proximal acidification include plasma and luminal HCO3  concentration, luminal flow rate, extracellular fluid volume status and modulators of Na/Hþ exchange such as PTH and angiotensin II [375,377]. Distal Tubule Acidebase handling in the distal tubule occurs primarily in the CCD. There are two classes of cells in the CCD, which can be distinguished by morphologic and functional criteria: the principal cell and the intercalated cell (Fig. 27.10) [379]. The principal cells are involved in sodium, potassium, and water transport, and the intercalated cells, which make up a third of the cells in the CCD, are responsible for acidebase transport, i.e. proton and bicarbonate secretion and reabsorption, in this nephron segment [379,380]. Hþ and HCO3  are formed in the CCD cell as a result of the action of intracellular carbonic anhydrase (see Fig. 27.10). The CCD is capable of secreting Hþ or HCO3  depending on the acidebase status of the body [375,380]. Two functionally distinct subtypes of intercalated cells have been identified in the CCD. a- (or type A) intercalated cells secrete protons to the lumen and reabsorb HCO3  to the blood (see Fig. 27.10) [381]. These cells harbor the Hþ-ATPase (proton pump) and the Hþ/ Kþ-ATPase in the luminal membrane and the kidney splice variant of anion exchanger 1 (AE1), a Cl/HCO3  exchanger, in the basolateral membrane. b- (or type B) intercalated cells operate in the reverse orientation: they reabsorb protons to the blood and secrete HCO3  (in exchange for Cl) across the apical membrane into the lumen [380,381]. In b-intercalated cells, Hþ-ATPase is

present on the basolateral membrane, whereas the Cl/ HCO3  exchanger pendrin is localized on the apical membrane of these cells [382]. A third type of intercalated cells, the so-called non-anon-b-cell, is present in the CCD. These cells harbor both Hþ-ATPase and pendrin in the apical membrane, and no AE1 in the basolateral membrane [383]. The exact function of these cells remains unknown. Secretion of Hþ in the a-intercalated cells results in: 1. reclamation of 10e20% of the filtered HCO3  that escaped absorption in the proximal tubule and 2. titration of the major urinary buffers, HPO4 2 and NH3 to form H2PO4  (titratable acid) and ammonium ion (NH4), respectively (see Fig. 27.10). NH3 is synthesized primarily in the proximal tubule and reaches the distal tubular lumen by a series of specialized transport processes [375,376]. Hþ secretion into the tubular lumen is mediated by two mechanisms: 1. Hþ-ATPase located at the luminal membrane of the a-intercalated cells 2. the lumen negative electrical potential difference created by electrogenic, epithelial Naþ channel (ENaC)-mediated Naþ reabsorption in the principal cells [376,377] (see Fig. 27.10). These cells are also responsible for Kþ secretion. Factors affecting distal tubular acidification include the integrity of the Hþ-ATPase pump, intraluminal and intracellular pH, distal Naþ delivery and transepithelial potential difference, permeability of the luminal membrane to protons and, finally, aldosterone which stimulates both Hþ-ATPase activity in the a-intercalated cells and Naþ reabsorption in the principal cells thereby

PEDIATRIC BONE

TABLE 27.4

Hereditary tubular disorders of acidebase handling Defective gene

Locus

Defective protein

Cell/nephron segment involved

Localization of defect

OMIM #

Reference

Autosomal dominant distal RTA

SLC4A1

17q21-22

AE1

a-intercalated/ CCD

Basolateral

Mild metabolic acidosis, hypokalemia, hypercalciuria, hypocitraturia, nephrolithiasis, nephrocalcinosis, rickets/ osteomalacia

179800

[384,385]

Autosomal recessive distal RTA

SLC4A1

17q21-22

AE1

a-intercalated/ CCD

Basolateral

Metabolic acidosis, hemolytic anemia (Southeast Asia only)

602722

[386]

Autosomal recessive distal RTA with deafness

ATP6V1B1

2p13

B1 subunit of Hþ-ATPase

a-intercalated/ CCD

Luminal

Early metabolic acidosis, nephrocalcinosis, vomiting, dehydration, growth retardation. rickets, bilateral sensorineural hearing loss

267300

[387]

Autosomal recessive distal RTA without or with late onset deafness

ATP6VO14

7q33

a4 subunit of Hþ-ATPase

a-intercalated/ CCD

Luminal

Early metabolic acidosis, nephrocalcinosis, vomiting, dehydration, growth retardation, rickets, late onset sensorineural hearing loss or normal hearing

602722

[388,389]

Isolated autosomal recessive proximal RTA

SLC4A4

4q21

Naþ-HCO 3 cotransporter, NBCl

Proximal tubule

Basolateral

Metabolic acidosis, hypokalemia, ocular abnormalities, growth retardation, intellectual deficit, basal ganglia calcification

604278

[390,391]

Mixed proximal and distal RTA (type 3)

CA2

8q22

Carbonic anhydrase II

Proximal tubule and a-intercalated/CCD

Cytoplasm

Metabolic acidosis, hypokalemia, osteopetrosis, blindness, deafness, early nephrocalcinosis

259730

[392,393]

Autosomal dominant pseudohypoaldosteronism type 1

MR

4q31

Mineralocorticoid receptor

Principal/CCD

Cytoplasm

Mild hyponatremia, hyperkalemia, metabolic acidosis

600983

[394]

Autosomal recessive pseudohypoaldosteronism type 1

SCNN1A SCNN1B SCNN1C

12p13,1 16p13 16p12

Naþ channel ENaC (a-, b-, gsubunits)

Principal/CCD

Luminal

Neonatal salt wasting, severe dehydration, hyperkalemic metabolic acidosis, hyponatremia, respiratory disease

264350

[395,396]

Pseudohypoaldosteronism type 2 (Gordon syndrome)

WNK4 WNK1

17q21 12p13

WNK4 WNK1

Distal convoluted tubule

Luminal

Thiazide-sensitive hypertension, hyperkalemia, hyperchloremic metabolic acidosis

601844 605232

[397,398]

Disorder

Clinical features

Distal RTA (type 1)

ACIDeBASE

PEDIATRIC BONE

Proximal RTA (type 2)

RTA type 4

755

CCD: cortical collecting duct; OMIM#: Online Mendelian Inheritance in Man (database at http://www.ncbi.nlm.hih.gov/Omim)

756

27. HEREDITARY TUBULAR DISORDERS OF MINERAL HANDLING LUMEN

BLOOD

CELL

A

-

ENaC

Na+

Na+ ATP

AR Pseudohypoaldosteronism Type I (RTA Type 4)

K+

8 8 8 8 K+ Mineralocorticoid Receptor

ROMK AD Pseudohypoaldosteronism Type I (RTA Type 4)

Aldosterone AE1

B

Cl-

ATP

H+ ADP

HCO3-

AR Distal RTA +/– Hearing Loss

AD/AR Distal RTA

H2CO3 HPO42-

H+

NH3

K+

ATP

ClCA II

H+ H2PO4-

Mixed Proximal and Distal RTA (Type 3)

H2O + CO2

NH4+

Titratable acid

C

Pendrin

FIGURE 27.10

Transport mechanisms in principal cell (A), a-intercalated cell (B) and b-intercalated cell (C) of the cortical collecting duct. (A) Aldosteronemineralocorticoid receptor complex interacts with hormone-responsive elements of DNA in the nucleus of the principal cell. This results in production of specific proteins, which stimulate ENaC-mediated Naþ entry (and ROMK-mediated Kþ exit) at the luminal membrane and Naþ-Kþ-ATPase at the basolateral membrane. (B) Hþ and HCO3  are formed in the a-intercalated cell as a result of intracellular carbonic anhydrase (CAII) action. Hþ is secreted into the lumen via Hþ-ATPase and Hþ-Kþ-ATPase and binds to the major urinary buffers, HPO4 2 and NH3 to form titratable acid and ammonium ion. (C) Hþ formed in the b-intercalated cell exits the cell to the interstitium via the basolateral Hþ-ATPase. HCO3  is secreted into the lumen in exchange for Cl via the activity of pendrin present in the apical membrane. Depicted are hereditary renal tubular acidosis syndromes caused by defects in these transport mechanisms. AD, autosomal dominant; AR, autosomal recessive.

ATP

H+

HCO3-

ADP

ClH2CO3

Cl-

CA II H2O + CO2

increasing the electrical gradient [375,376]. Aldosterone and Kþ depletion affect distal Hþ secretion also by enhancing ammonium production.

various degrees of abnormalities in renal handing of minerals and, in some instances, metabolic bone disease.

Renal Tubular Acidosis

Proximal renal tubular acidosis (pRTA) is characterized by hyperchloremic metabolic acidosis due to impaired capacity of the proximal tubule to reabsorb HCO3  [400,402]. pRTA may occur either as a manifestation of a generalized proximal tubular dysfunction (Fanconi syndrom, see earlier) or as an isolated entity. Inheritance of isolated pRTA is autosomal recessive and occurs consistently in association with ocular abnormalities including glaucoma, band keratopathy, and cataracts (Table 27.4; see Fig. 27.10) [390,403,404]. Additional manifastations include short stature, calcification of basal ganglia and mental retardation (see later) [386,403]. The most prominent clinical feature of pRTA is failure to thrive. Other manifestations, which are related to untreated hypokalemia, include polyuria, polydipsia, dehydration, vomiting, anorexia, constipation, and muscle weakness. The hypokalemia occurs because

Renal tubular acidosis (RTA) is a diverse group of tubular transport disorders characterized by an impairment of urinary acidification and hyperchloremic metabolic acidosis with a normal plasma anion gap [366,376,386,399e401]. RTA syndromes involve defects in the reabsorption of bicarbonate or the excretion of hydrogen ions or both. Glomerular filtration rate remains relatively normal. The four main types of RTA include: 1. 2. 3. 4.

distal RTA (type 1) proximal RTA (type 2) combined proximal and distal RTA (type 3) hyperkalemic RTA (type 4) [386,399e401].

This review will describe hereditary RTA syndromes. All types of hereditary RTAs can be associated with

Proximal Renal Tubular Acidosis

PEDIATRIC BONE

ACIDeBASE

increased delivery of Naþ to the distal nephron results in enhanced secretion of Kþ in the principal cell of the CCD (see Fig. 27.10) and mild volume depletion secondary to Naþ loss results in secondary hyperaldosteronism that increases Kþ secretion [376,400]. Hypercalciuria, nephrocalcinosis, and nephrolithiasis typically are not observed. Metabolic bone disease usually occurs in patients with Fanconi syndrome, and is attributed to hypophosphatemia and impaired vitamin D metabolism, but may also be induced by the chronic acidosis (see later) in isolated pRTA [386,403]. The genes encoding the transporters operating in the proximal tubular cell (see Fig. 27.9) have been logical candidate genes for involvement in inherited pRTA. Several studies have focused on the Naþ/Hþ exchanger, NHE3, the major acid secreting pump in the proximal tubule (see Fig. 27.9). Targeted NHE3 deletion in mice has been shown to result in proximal tubular dysfunction [405]. As yet, no human mutations of NHE3 have been identified. The Naþ/ HCO3  co-transporter, NBCl (see Fig. 27.9), encoded by the SLC4A4 gene, which is located on chromosome 4q21, has been implicated in autosomal recessive proximal RTA [389,390]. NBCl belongs to the bicarbonate transporter SLC4 superfamily, to which the AE1 Cl/ HCO3  exchanger also belongs [406,407]. The two known NBCl isoforms include the pancreatic isoform, which has 1079 residues, and the shorter, kidney isoform, which has 994 residues. Igarashi et al. [390] identified two homozygous missense mutations in kidney NBCl in two individuals with autosomal recessive pRTA and ocular abnormalities (see Table 27.4). Functional anaylsis in transfected cells revealed 50% reduction in mutated NBCl activity. Both patients had cataracts, glaucoma and band keratopathy. A number of other mutations have subsequently been described [400,408,409]. In addition to reduced functional activity, defects of intracellular trafficking have been demonstrated for some of these mutations [410e412]. Since NBCl transcripts have been found in human corneal epithelium [404], it is possible that defective corneal NBCl function in these patients results in impaired HCO3  transport which, in turn, leads to abnormal calcium carbonate deposition in cornea and band keratopathy [403,404]. Autosomal dominant pRTA with short stature and decreased bone density was described in a single Costa Rican kindred [413]. The responsible gene, however, is unknown. Distal Renal Tubular Acidosis Primary distal renal tubular acidosis (dRTA) is characterized by hyperchloremic metabolic acidosis due to failure of hydrogen ion secretion in the distal nephron [386,399,400,414]. Patients with primary dRTA usually

757

present in early infancy with polyuria, vomiting, dehydration, failure to thrive, hypokalemia, and urine pH above 6.0 despite systemic metabolic acidosis. As in pRTA, the urinary Kþ loss in dRTA is due to extracellular fluid volume contraction and secondary hyperaldosteronism [376,400]. Hypercalciuria and hypocitraturia may occur, in which case nephrocalcinosis usually develops. Distal RTA is accompanied by rickets or osteomalacia which are primarily the result of chronic acidosis-induced mobilization of skeletal calcium and inhibition of renal conversion of 25(OH)vitD to 1,25(OH)2vitD [415]. Acidosis-induced renal phosphate wasting leading to hypophosphatemia may also contribute to the bone disease. The hypercalciuria is the result of Ca2þ release from bone [416] as well as inhibition of distal tubular Ca2þ reabsorption by chronic metabolic acidosis, first demonstrated by micropuncture experiments [417]. These findings are in line with more recents studies [274,418] showing decreased renal expression of TRPV5 during experimental acidosis and demonstrating that renal Ca2þ excretion is refractory to NH4Cl-induced metabolic acidosis in TRPV5-deficient mice. Patients with dRTA have normal serum Mg2þ levels but my present with hypermagnesiuria [419]. Chronic metabolic acidosis has been shown to decrease renal abundance of TRPM6 [418]. Alkali therapy of patients with dRTA leads to correction of all biochemical abnormalities [376,400]. Distal RTA in children is most commonly a primary entity. Primary dRTA is inherited as either an autosomal dominant or autosomal recessive trait (see Table 27.4 and Fig. 27.10) [366,399]. Patients with the autosomal dominant form usually have a mild disease, whereas those with autosomal recessive dRTA may be severely affected in infancy with growth retardation and early nephrocalcinosis leading to renal failure [366,385]. Autosomal recessive dRTA is subdivided into two variants: with or without sensorineural hearing loss (see Table 27.4) [386,399,420,421]. Molecular studies exploring hereditary dRTA have focused on the transporters operating in the a-intercalated cell, which were obvious candidates for involvement in the disease. AUTOSOMAL DOMINANT dRTA

The AE1 gene, SLC4A1, located on chromosome 17q21-22, and a member of the anion exchanger SLC4 gene family, has been implicated in autosomal dominant dRTA (see Table 27.4) [386,399,422]. The AE1 gene product, also termed band 3, consists of 12e14 transmembrane domains and functions as an anion exchanger in erythroid cells and in the basolateral membrane of a-intercalated cells (see Fig. 27.10). Erythrocyte AE1 (eAE1) is 65 amino acids longer at its NH2 terminus than the kidney isoform (kAE1) [422]. Several mutations in this N-terminal region of band 3 have

PEDIATRIC BONE

758

27. HEREDITARY TUBULAR DISORDERS OF MINERAL HANDLING

been identified as the cause of hereditary spherocytosis and Southeast Asian ovalocytosis and normal acidebase handling [386,423]. However, various missense and deletion mutations of AE1 have been found in the COOH terminus and other regions of AE1 in several families with autosomal dominant dRTA (see Fig. 27.10) [384,424]. It has been demonstrated that AE1 mutations associated with autosomal dominant dRTA have normal function when expressed in Xenopus oocytes but cause abnormalities in trafficking and targeting of AE1 to the renal basolateral membrane [385,386,424]. It is noteworthy that AE1 mutations causing autosomal recessive dRTA in association with hemolytic anemia have been demonstrated in Southeast Asian kindreds [386]. AUTOSOMAL RECESSIVE dRTA

The ATP6V1B1 gene, localized on chromosome 12q13, and encoding the B1-subunit of the apical proton pump expressed in a-intercalated cells has been implicated in autosomal recessive dRTA with early onset sensorineural deafness (see Table 27.4 and Fig. 27.10) [387,425,426]. The B1-containing Hþ-ATPase is a member of the vacuolar (V)-ATPase family that has a complex structure of at least 10 subunits [426e428]. An intracellular domain of Hþ-ATPase which, among other subunits, contains three B subunits, catalyzes ATP hydrolysis providing energy for active Hþ transport across the membrane spanning Vo domain [386,427,428]. Various mutations causing autosomal recessive dRTA with sensorineural deafness have been identified within the ATP6V1B1 gene [386,399,425]. Consistent with the finding of hearing loss, expression of ATP6V1B1 in cochlea and endolymphatic sac has been demonstrated [386,387]. This suggested that mutations in inner ear Hþ-ATPase likely affect auditory function by altering the normally acidic endolymphatic pH [386,387]. Several homozygous mutations in the ATP6VOA4 gene, located on chromosome 7q33-34, and encoding the a4 accessory subunit of Hþ-ATPase, were found to cause dRTA without or with later onset deafness (see Table 27.4) [388,389]. Mixed Proximal and Distal Renal Tubular Acidosis (Type 3) This variant of RTA shares the features of both proximal (reduced HCO3  reabsorption) and distal (impaired urine acidification) RTA (see Figs 27.9 and 27.10). It is caused by autosomal recessive mutations of the CAII gene located on chromosome 8q22 and expressed in kidney, bone and brain [376,392,393]. Since CAII is present in the cytosol of both proximal and distal tubules, these mutations lead to this mixed syndrome. Since the expression of CAII is affected also in bone

and brain, additional manifestations include osteopetrosis, cerebral calcifications, and mental retardation [394,395]. The osteopetrosis, which is secondary to osteoclast dysfunction, is a condition of increased bone density but also increased bone fragility leading to increased fracture risk [386]. Excess bone growth leads to conductive deafness and can also cause blindness through compression of the optic nerve. Hyperkalemic Renal Tubular Acidosis (Type 4) Hyperkalemic RTA is characterized by normal ability to acidify the urine during acidosis but reduced urinary concentration of ammonium and hence of net acid [376,386,400]. The primary pathogenic mechanism in RTA type 4 is aldosterone deficiency or resistance that results in impairment of Hþ and Kþ secretion in the principal cells of the collecting tubule (see Fig. 27.10). The ensuing hyperkalemia leads to an impairment of ammonium production. Decreased ENaC-mediated Naþ reabsorption by principal cells also contributes to impaired electrogenic secretion of Hþ by intercalated cells. Two hereditary aldosterone resistance states, pseudohypoaldosteron (PHA) type I and type II, occurring primarily in children, lead to RTA type 4 (see Table 27.4). PSEUDOHYPOALDOSTERONISM TYPE I

Pseudohypoaldosteronism type I (PHAI) is a rare inherited disorder characterized by renal salt wasting and end-organ unresponsiveness to mineralocorticoids [366,376,429]. The manifestations of the disease include hyponatremia, hyperkalemia, hyperchloremic metabolic acidosis and elevated plasma aldosterone and plasma renin activity. The disorder is divided into two forms of inheritance with distinct pathphysiological and clinical features. The autosomal dominant form is a relatively mild disease, which remits with age, is restricted to the kidney, and is caused by heterozygous loss-offunction mutations in the mineralocorticoid receptor (MR) gene (see Fig. 27.10) [394]. The autosomal recessive form presents with severe Naþ transport defects in all aldosterone target tissues including the kidney, colon, and salivary and sweat glands, as well as in lungs [366,395,430]. Autosomal recessive PHAI is characterized by neonatal salt wasting with dehydration, hypotension, life-threatening hyperkalemia, RTA type 4 and failure to thrive [395,429,430]. Sweat test is usually positive. The manifestations of the disease do not respond to mineralocorticoids but improve with salt supplementation. Neonatal respiratory distress syndrome and respiratory tract infections in affected children are common [431]. Autosomal recessive PHAI is caused by homozygous or compound heterozygous loss-of-function mutations, which have been described in each of the three subunits a, b, and g of the amiloride-sensitive, epithelial

PEDIATRIC BONE

REFERENCES

Naþ channel (ENaC) of the principal cell apical membrane (see Fig. 27.10) [366,395,396,432]. PSEUDOHYPOALDOSTERONISM TYPE II

Pseudohypoaldosteronism type II (PHAII), or Gordon syndrome, is an autosomal dominant disorder characterized by hypertension, hyperkalemia, hyperchloremic metabolic acidosis and low plasma aldosterone levels [432e434]. The underlying pathogenic mechanism is chloride-dependent Naþ retention and the disease is highly responsive to low dose thiazide treatment [433,434]. PHAII is caused by mutations in the genes encoding the WNKI and WNK4 serinethreonine protein kinases which are particular subtypes of these kinases lacking a lysine residue at the active site (WNK ¼ with no K [lysine]) (see Fig. 27.8) [397,398]. WNK1 and WNK4 serve as both intracellular [Cl] sensors and ion transport regulators [398,435] with an inhibitory activity on the thiazide-sensitive NaCl cotransporter (NCCT) of the DCT (see Fig. 27.8). Both genetic types of PHAII increase surface expression and activity of the NCCT in the DCT [433,436]. Consequent decreased Naþ delivery to the cortical collecting duct results in reduced Naþ reabsorption which, in turn, results in decreased electrogenic secretion of Hþ and Kþ. No changes in serum Mg2þ levels have been observed in patients with PHAII. In addition to the hypertensive phenotype, hypercalciuria, nephrolithiasis and decreased bone mineral density have been reported in patients with PHAII [437e439]. The mechanisms that have been proposed as responsible for these abnormalities in mineral handling include: volume expansion which reduces proximal tubule Na2þ reabsorption thereby decreasing electrochemical driving force for Ca2þ reabsorption in this nephron segment [274,440] and possible reduced expression of the TRPV5 Ca2þ channel at the CCD cell membrane [274,441].

CONCLUSION In the past decade, remarkable progress has been made in our understanding of the molecular pathogenesis of hereditary tubulopathies affecting mineral balance. Molecular genetics and molecular biology studies have led to the identification of numerous tubular disease-causing mutations, have provided important insight into the defective molecular mechanisms underlying various tubulopathies leading to deranged mineral homeostasis and bone disease, and have greatly increased our understanding of the physiology of renal tubular transport of minerals. Nevertheless, numerous issues remain unsettled and warrant additional research. Future studies are needed to

759

identify additional genetic defects responsible for various tubular transport disorders, better to define genotypeephenotype correlation in these disorders, and to explore how a specific mutation in a specific transporter gene leads to a specific aberration in renal mineral transport. Functional and expression studies using Xenopus oocytes and transfected cells are needed to shed more light on the molecular mechanisms and functional defects underlying the impaired transport of minerals in various tubulopathies. Future studies utilizing the powerful models of transgenic and knockout mice and analyzing their phenotype will undoubtedly provide very important insight into the molecular pathogenesis of these tubular transport disorders. Finally, the identification of the molecular defects in inherited tubulopathies may provide a basis for future design of targeted therapeutic interventions and, possibly, strategies for gene therapy of these complex disorders.

Acknowledgment We are indebted to Mrs Ora Bider for her expert secretarial assistance.

References [1] Amanzadeh J, Reilly RF. Hypophosphatemia: an evidencebased approach to its clinical consequences and management. Nat Clin Pract Neph 2006;2:136e48. [2] Hilfiker H, Hattenhauer O, Traebert M, Forster I, Murer H, Biber J. Characterization of a murine type II sodium-phosphate cotransporter expressed in mammalian small intestine. Proc Natl Acad Sci USA 1998;95:14564e9. [3] Biber J, Hernando N, Forster I, Murer H. Regulation of phosphate transport in proximal tubules. Pflu¨gers Arch 2009;458:39e52. [4] Tenenhouse HS. Phosphate transport: molecular basis, regulation and pathophysiology. J Steroid Biochem Mol Biol 2007;103:572e7. [5] Portale AA, Perwad F. Calcium and Phosphorus. In: Avner ED, Harmon WE, Niaudet P, et al., editors. Pediatric Nephrology. 6th edn. Berlin: Springer-Verlag; 2009. p. 247e8. [6] Baumann K, de Rouffignac C, Roinel N, Rumrich G, Ullrich K. Renal phosphate transport: inhomogeneity of local proximal transport rates and sodium dependence. Pflu¨gers Arch. 1975;356:287e98. [7] Ullrich K, Rumrich G, Klo¨ss S. Phosphate transport in the proximal convolution of the rat kidney. I. Tubular heterogeneity, effect of parathyroid hormone in acute and chronic parathyroidectomized animals and effect of phosphate diet. Pflu¨gers Arch 1977;372:269e74. [8] McKeown J, Brazy P, Dennis V. Intrarenal heterogeneity for fluid, phosphate, and glucose absorption in the rabbit. Am J Physiol 1979;237:F312e8. [9] Lang F, Greger R, Marchand G. Stationary microperfusion study of phosphate reabsorption in proximal and distal nephron segments. Pflu¨gers Arch 1977;368:45e8. [10] Werner A, Moore ML, Mantei N, Biber J, Semenza G, Murer H. Cloning and expression of cDNA for a Na/Pi cotransport system of kidney cortex. Proc Natl Acad Sci USA 1991;88:9608e12.

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760

27. HEREDITARY TUBULAR DISORDERS OF MINERAL HANDLING

[11] Custer M, Lo¨tscher M, Biber J, Murer H, Kaissling B. Expression of Na-P(i) cotransport in rat kidney: localization by RT-PCR and immunohistochemistry. Am J Physiol 1994;266:F767e74. [12] Custer M, Meier F, Greger R, et al. Localization of NaPi-1, a NaPi cotransporter, in rabbit kidney proximal tubules. I. mRNA localization by reverse transcription/polymerase chain reaction. Pflu¨gers Arch 1993;424:203e9. [13] Villa-Bellosta R, Ravera S, Sorribas V, et al. The Naþ-Pi cotransporter PiT-2 (SLC20A2) is expressed in the apical membrane of rat renal proximal tubules and regulated by dietary Pi. Am J Physiol Renal Physiol 2009;296:F691e9. [14] Villa-Bellosta R, Sorribas V. Compensatory regulation of the sodium/phosphate cotransporters NaPi-IIc (SCL34A3) and Pit2 (SLC20A2) during Pi deprivation and acidosis. Pflu¨gers Arch 2010;459:499e508. [15] Murer H, Forster I, Biber J. The sodium phosphate cotransporter family SLC34. Pflu¨gers Arch. 2004;447:763e7. [16] Tenenhouse HS, Roy SP, Martel JE, Gauthier C. Differential expression, abundance, and regulation of Naþ-phosphate cotransporter genes in murine kidney. Am J Physiol Renal Physiol 1998;275:F527e54. [17] Segawa H, Kaneko I, Takahashi A, et al. Growth-related renal type II Na/Pi cotransporter. J Biol Chem 2002;277:19665e72. [18] Jutabha P, Kanai Y, Hosoyamada M, et al. Identification of a novel voltage-driven organic anion transporter present at apical membrane of renal proximal tubule. J Biol Chem 2003;278:27930e8. [19] Anzai N, Jutabha P, Kanai Y, Endou H. Integrated physiology of proximal tubular organic anion transport. Curr Opin Nephrol Hypertens 2005;4:472e9. [20] Busch AE, Schuster A, Waldegger S, et al. Expression of a renal type I sodium/phosphate transporter (NaPi-1) induces a conductance in Xenopus oocytes permeable for organic and inorganic anions. Proc Natl Acad Sci USA 1996;93:5347e51. [21] Reimer R, Edwards RH. Organic anion transport is the primary function of the SLC17/type I phosphate transporter family. Pflu¨gers Arch 2004;447:629e35. [22] Villa-Bellosta R, Barac-Nieto M, Breusegem SY, Barry NP, Levi M, Sorribas V. Interactions of the growth-related, type IIc renal sodium/phosphate cotransporter with PDZ proteins. Kidney Int 2008;73:456e64. [23] Moe OW. PiT-2 Coming out of the pits. Am J Physiol Renal Physiol 2009;296:F689e90. [24] Segawa H, Aranami F, Kaneko I, Tomoe Y, Miyamoto K. The roles of Na/Pi-II transporters in phosphate metabolism. Bone 2009;45:S2e7. [25] Kos CH, Tihy F, Econs MJ, Murer H, Lemieux N, Tenenhouse HS. Localization of a renal sodium-phosphate cotransporter gene to human chromosome 5q35. Genomics 1994;19:176e7. [26] Hartmann CM, Hewson AS, Kos CH, et al. Structure of murine and human renal type II Naþ-phosphate cotransporter genes (Npt2 and NPT2). Proc Natl Acad Sci USA 1996;93:7409e14. [27] Magagnin S, Werner A, Markovich D, et al. Expression cloning of human and rat renal cortex Na/Pi cotransport. Proc Natl Acad Sci USA 1993;90:5979e83. [28] Bergwitz C, Roslin NM, Tieder M, et al. SLC34A3 Mutations in patients with hereditary hypophosphatemic rickets with hypercalciuria predict a key role for the sodium-phosphate cotransporter NaPi-IIc in naintaining phosphate homeostasis. Am J Hum Genet 2006;78:179e92. [29] Lorenz-Depiereux B, Benet-Pages A, Eckstein G, et al. Hereditary hypophosphatemic rickets with hypercalciuria is caused by mutations in the sodium-phosphate cotransporter gene SLC34A3. Am J Hum Genet 2006;78:193e201.

[30] Forster I, Ko¨hler K, Biber J, Murer H. Forging the link between structure and function of electrogenic cotransporters: the renal type IIa Naþ/Pi cotransporter as a case study. Prog Biophys Mol Biol 2002;80:69e108. [31] Tenenhouse HS. Regulation of phosphorus homeostasis by the type IIa Na/Phosphate cotransporter. Annu Rev Nutr 2005;25:197e214. [32] Miyamoto K, Ito M, Tatsumi S, Kuwahata M, Segawa H. New aspect of renal phosphate reabsorption: The type IIc sodiumdependent phosphate transporter. Am J Nephrol 2007;27:503e15. [33] Chau H, El-Maadawy S, McKee MD, Tenenhouse HS. Renal calcification in mice homozygous for the disrupted type IIa Na/ Pi cotransporter gene Npt2. J Bone Miner Res 2003;18:644e57. [34] Beck L, Karaplis AC, Amizuka N, Hewson AS, Ozawa H, Tenenhouse HS. Targeted inactivation of Npt2 in mice leads to severe renal phosphate wasting, hypercalciuria, and skeletal abnormalities. Proc Natl Acad Sci USA 1998;95:5372e7. [35] Segawa H, Onitsuka A, Kuwahata M, et al. Type IIc sodiumdependent phosphate transporter regulates calcium metabolism. J Am Soc Nephrol 2009;20:104e13. [36] Murer H, Hernando N, Forster I, Biber J. Proximal tubular phosphate reabsorption: Molecular mechanisms. Physiol Rev 2000;80:1373e409. [37] Murer H, Hernando N, Forster L, J. B. Molecular mechanisms in proximal tubular and small intestinal phosphate reabsorption (plenary lecture). Mol Membr Biol 2001;18:3e11. [38] Biber J, Forgo J, Murer H. Modulation of Naþ-Pi cotransport in opossum kidney cells by extracellular phosphate. Am J Physiol 1988;255(2):C155e61. [39] Pfister MF, Hilfiker H, Forgo J, Lederer E, Biber J, Murer H. Cellular mechanisms involved in the acute adaptation of OK cell Na/Pi-cotransport to high- or low-Pi medium. Pflu¨gers Arch 1998;435:713e9. [40] Levi M, Lo¨tscher M, Sorribas V, et al. Cellular mechanisms of acute and chronic adaptation of rat renal P(i) transporter to alterations in dietary P(i). Am J Physiol 1994;267:F900e8. [41] Ohkido I, Segawa H, Yanagida R, Nakamura M, Miyamoto K. Cloning, gene structure and dietary regulation of the type-IIc Na/Pi cotransporter in the mouse kidney. Pflu¨gers Arch 2003;446:106e15. [42] Lo¨tscher M, Kaissling B, Biber J, Murer H, Levi M. Role of microtubules in the rapid regulation of renal phosphate transport in response to acute alterations in dietary phosphate content. J Clin Invest 1997;99:1302e12. [43] Werner A, Kempson SA, Biber J, Murer H. Increase of Na/Picotransport encoding mRNA in response to low Pi diet in rat kidney cortex. J Biol Chem 1994;269:6637e9. [44] Tenenhouse H, Martel J, Biber J, H. M. Effect of P(i) restriction on renal Na(þ)-P(i) cotransporter mRNA and immunoreactive protein in X-linked Hyp mice. Am J Physiol 1995;268:F1062e9. [45] Verri T, Markovich D, Perego C, et al. Cloning of a rabbit renal Na-Pi cotransporter, which is regulated by dietary phosphate. Am J Physiol 1995;268:F626e33. [46] Beck L, Meyer R, Meyer M, Biber J, Murer H, Tenenhouse H. Renal expression of Naþ-phosphate cotransporter mRNA and protein: Effect of the Gy mutation and low phosphate diet. Pflu¨gers Arch 1996;431:936e41. [47] Katai K, Segawa H, Haga H, et al. Acute regulation by dietary phosphate of the sodium-dependent phosphate transporter (NaP(i)-2) in rat kidney. J Biochem 1997;121:50e5. [48] Takahashi F, Morita K, Katai K, et al. Effects of dietary Pi on the renal Naþ-dependent Pi transporter NaPi-2 in thyroparathyroidectomized rats. Biochem J 1998;333:175e81.

PEDIATRIC BONE

REFERENCES

[49] Keusch I, Traebert M, Lotscher M, Kaissling B, Murer H, Biber J. Parathyroid hormone and dietary phosphate provoke a lysosomal routing of the proximal tubular Na/Pi-cotransporter type II. Kidney Int 1998;54:1224e32. [50] Segawa H, Yamanaka S, Ito M, et al. Internalization of renal type IIc Na-Pi cotransporter in response to a high-phosphate diet. Am J Physiol Renal Physiol 2005;288:F587e96. [51] Mizgala CL, Quamme GA. Renal handling of phosphate. Physiol Rev 1985;65:431e66. [52] Pfister MF, Lederer E, Forgo J, et al. Parathyroid hormonedependent degradation of type II Naþ/Pi cotransporters. J Biol Chem 1997;272:20125e30. [53] Bacic D, Schulz N, Biber J, Kaissling B, Murer H, Wagner C. Involvement of the MAPK-kinase pathway in the PTH-mediated regulation of the proximal tubule type IIa Naþ/Pi cotransporter in mouse kidney. Pflu¨gers Arch 2003;446:52e60. [54] Bacic D, LeHir M, Biber J, Kaissling B, Murer H, Wagner CA. The renal Naþ/phosphate cotransporter NaPi-IIa is internalized via the receptor-mediated endocytic route in response to parathyroid hormone. Kidney Int 2006;69:495e503. [55] Bachmann S, Schlichting U, Geist B, et al. Kidney-specific inactivation of the megalin gene impairs trafficking of renal inorganic sodium phosphate cotransporter (NaPi-IIa). J Am Soc Nephrol 2004;15:892e900. [56] Traebert M, Roth J, Biber J, Murer H, Kaissling B. Internalization of proximal tubular type II Na-Pi cotransporter by PTH: immunogold electron microscopy. Am J Physiol Renal Physiol 2000;278:F148e54. [57] Virkki LV, Biber J, Murer H, Forster IC. Phosphate transporters: a tale of two solute carrier families. Am J Physiol Renal Physiol 2007;293:F643e54. [58] Picard N, Capuano P, Stange G, et al. Acute parathyroid hormone differentially regulates renal brush border membrane phosphate cotransporters. Pflu¨gers Arch 2010;460:677e87. [59] Gensure RC, Gardella TJ, Ju¨ppner H. Parathyroid hormone and parathyroid hormone-related peptide, and their receptors. Biochem Biophys Res Commun 2005;328:666e78. [60] Mahon MJ, Cole JA, Lederer ED, Segre GV. Naþ/Hþ exchangerregulatory factor 1 mediates inhibition of phosphate transport by parathyroid hormone and second messengers by acting at multiple sites in opossum kidney cells. Mol Endocrinol 2003;17:2355e64. [61] Weinman EJ, Cunningham R, Wade JB, Shenolikar S. The role of NHERF-1 in the regulation of renal proximal tubule sodiumhydrogen exchanger 3 and sodium-dependent phosphate cotransporter 2a. J Physiol 2005;567:27e32. [62] Shenolikar S, Voltz JW, Minkoff CM, Wade JB, Weinman EJ. Targeted disruption of the mouse NHERF-1 gene promotes internalization of proximal tubule sodium-phosphate cotransporter type IIa and renal phosphate wasting. Proc Natl Acad Sci USA 2002;99:11470e5. [63] Capuano P, Bacic D, Roos M, et al. Defective coupling of apical PTH receptors to phospholipase C prevents internalization of the Naþ-phosphate cotransporter NaPi-IIa in Nherf1-deficient mice. Am J Physiol Cell Physiol 2007;292:C927e34. [64] Kong XF, Zhu XH, Pei YL, Jackson DM, Holick MF. Molecular cloning, characterization, and promoter analysis of the human 25-hydroxyvitamin D3-1alpha-hydroxylase gene. Proc Natl Acad Sci USA 1999;96:6988e93. [65] Mu¨hlbauer R, Bonjour J. Tubular handling of Pi: localization of effects of 1,25(OH)2D3 and dietary Pi in TPTX rats. Am J Physiol 1981;241:F123e8. [66] Kurnik B, Hruska K. Effects of 1,25-dihydroxycholecalciferol on phosphate transport in vitamin D-deprived rats. Am J Physiol 1984;247:F177e84.

761

[67] Kurnik BRC, Hruska KA. Mechanism of stimulation of renal phosphate transport by 1,25-dihydroxycholecalciferol. Biochim Biophys Acta 1985;817:42e50. [68] Capuano P, Radanovic T, Wagner CA, et al. Intestinal and renal adaptation to a low-Pi diet of type II NaPi cotransporters in vitamin D receptor- and 1aOHase-deficient mice. Am J Physiol Cell Physiol 2005;288:C429e34. [69] Segawa H, Kaneko I, Yamanaka S, et al. Intestinal Na-Pi cotransporter adaptation to dietary Pi content in vitamin D receptor null mice. Am J Physiol Renal Physiol 2004;287:F39e47. [70] Schiavi SC, Moe OW. Phosphatonins: a new class of phosphateregulating proteins. Curr Opin Nephrol Hypertens 2002;11:423e30. [71] Gattineni J, Baum M. Regulation of phosphate transport by fibroblast growth factor 23 (FGF23): implications for disorders of phosphate metabolism. Pediatr Nephrol 2010;25:591e601. [72] Kumar R. Tumor-induced osteomalacia and the regulation of phosphate homeostasis. Bone 2000;27:333e8. [73] Cai Q, Hodgson SF, Kao PC, et al. Brief report: inhibition of renal phosphate transport by a tumor product in a patient with oncogenic osteomalacia. N Engl J Med 1994;330:1645e9. [74] Jonsson KB, Mannstadt M, Miyauchi A, et al. Extracts from tumors causing oncogenic osteomalacia inhibit phosphate uptake in opossum kidney cells. J Endocrinol 2001;169:613e20. [75] Marsell R, Jonsson KB. The phosphate regulating hormone fibroblast growth factor-23. Acta Physiol 2010;200:97e106. [76] Liu S, Quarles LD. How fibroblast growth factor 23 works. J Am Soc Nephrol 2007;18:1637e47. [77] Saito T, Fukumoto S. Fibroblast growth factor 23 (FGF23) and disorders of phosphate metabolism. Int J Pediatr Endocrinol 2009. doi:10.1155/2009/496514. [78] Bergwitz C, Ju¨ppner H. Regulation of phosphate homeostasis by PTH, vitamin D, and FGF23. Ann Rev Med 2010;61:91e104. [79] Ju¨ppner H, Wolf M, Salusky IB. FGF-23: More than a regulator of renal phosphate handling? J Bone Miner Res 2010;25:2091e7. [80] Sitara D, Razzaque MS, Hesse M, et al. Homozygous ablation of fibroblast growth factor-23 results in hyperphosphatemia and impaired skeletogenesis, and reverses hypophosphatemia in Phex-deficient mice. Matrix Biol 2004;23:421e32. [81] Liu S, Zhou J, Tang W, Jiang X, Rowe DW, Quarles LD. Pathogenic role of Fgf23 in Hyp mice. Am J Physiol Endocrinol Metab 2006;291:E38e49. [82] Pereira RC, Ju¨ppner H, Azucena-Serrano CE, Yadin O, Salusky IB, Wesseling-Perry K. Patterns of FGF-23, DMP1, and MEPE expression in patients with chronic kidney disease. Bone 2009;45:1161e8. [83] Shimada T, Mizutani S, Muto T, et al. Cloning and characterization of FGF23 as a causative factor of tumor-induced osteomalacia. Proc Natl Acad Sci USA 2001;98:6500e5. [84] Benet-Page`s A, Orlik P, Strom TM, Lorenz-Depiereux B. An FGF23 missense mutation causes familial tumoral calcinosis with hyperphosphatemia. Hum Mol Genet 2005;14:385e90. [85] Topaz O, Shurman DL, Bergman R, et al. Mutations in GALNT3, encoding a protein involved in O-linked glycosylation, cause familial tumoral calcinosis. Nat Genet 2004;36:579e81. [86] Larsson T, Davis SI, Garringer HJ, et al. Fibroblast growth factor-23 mutants causing familial tumoral calcinosis are differentially processed. Endocrinology 2005;146:3883e91. [87] Araya K, Fukumoto S, Backenroth R, et al. A novel mutation in fibroblast growth factor 23 gene as a cause of tumoral calcinosis. J Clin Endocrinol Metab 2005;90:5523e7. [88] Bergwitz C, Banerjee S, Abu-Zahra H, et al. Defective O-glycosylation due to a novel homozygous S129P mutation is

PEDIATRIC BONE

762

[89]

[90]

[91]

[92]

[93]

[94] [95] [96]

[97]

[98]

[99]

[100]

[101]

[102]

[103]

[104]

[105]

[106]

27. HEREDITARY TUBULAR DISORDERS OF MINERAL HANDLING

associated with lack of fibroblast growth factor 23 secretion and tumoral calcinosis. J Clin Endocrinol Metab 2009;94:4267e74. Perwad F, Azam N, Zhang MYH, Yamashita T, Tenenhouse HS, Portale AA. Dietary and serum phosphorus regulate fibroblast growth factor 23 expression and 1,25-dihydroxyvitamin D metabolism in mice. Endocrinology 2005;146:5358e64. Ferrari SL, Bonjour J-P, Rizzoli R. Fibroblast growth factor-23 relationship to dietary phosphate and renal phosphate handling in healthy young men. J Clin Endocrinol Metab 2005;90:1519e24. Burnett S-AM, Gunawardene SC, Bringhurst FR, Ju¨ppner H, Lee H, Finkelstein JS. Regulation of C-terminal and intact FGF23 by dietary phosphate in men and women. J Bone Miner Res 2006;21:1187e96. Kolek OI, Hines ER, Jones MD, et al. 1alpha,25-Dihydroxyvitamin D3 upregulates FGF23 gene expression in bone: the final link in a renal-gastrointestinal-skeletal axis that controls phosphate transport. Am J Physiol Gastrointest Liver Physiol 2005;289:G1036e42. White KE, Larsson TE, Econs MJ. The roles of specific genes implicated as circulating factors involved in normal and disordered phosphate homeostasis: frizzled related protein-4, matrix extracellular phosphoglycoprotein, and fibroblast growth factor 23. Endocr Rev. 2006;27:221e41. Quarles LD. Endocrine functions of bone in mineral metabolism regulation. J Clin Invest 2008;118:3820e8. Strom TM, Ju¨ppner H. PHEX, FGF23, DMP1 and beyond. Curr Opin Nephrol Hypertens 2008;17:357e62. Kurosu H, Ogawa Y, Miyoshi M, et al. Regulation of fibroblast growth factor-23 signaling by Klotho. J Biol Chem 2006;281:6120e3. Urakawa I, Yamazaki Y, Shimada T, et al. Klotho converts canonical FGF receptor into a specific receptor for FGF23. Nature 2006;444:770e4. Gattineni J, Bates C, Twombley K, et al. FGF23 decreases renal NaPi-2a and NaPi-2c expression and induces hypophosphatemia in vivo predominantly via FGF receptor 1. Am J Physiol Renal Physiol 2009;297:F282e91. Baum M, Schiavi S, Dwarakanath V, Quigley R. Effect of fibroblast growth factor-23 on phosphate transport in proximal tubules. Kidney Int 2005;68:1148e53. Yamashita T, Konishi M, Miyake A, Inui K-i, Itoh N. Fibroblast growth factor (FGF)-23 inhibits renal phosphate reabsorption by activation of the mitogen-activated protein kinase pathway. J Biol Chem 2002;277:28265e70. Farrow EG, Davis SI, Summers LJ, White KE. Initial FGF23mediated signaling occurs in the distal convoluted tubule. J Am Soc Nephrol 2009;20:955e60. De Beur SMJ, Finnegan RB, Vassiliadis J, et al. Tumors associated with oncogenic osteomalacia express genes important in bone and mineral metabolism. J Bone Miner Res 2002;17:1102e10. Rowe PSN, de Zoysa PA, Dong R, et al. MEPE, a new gene expressed in bone marrow and tumors causing osteomalacia. Genomics 2000;67:54e68. Berndt T, Craig TA, Bowe AE, et al. Secreted frizzled-related protein 4 is a potent tumor-derived phosphaturic agent. J Clin Invest 2003;112:785e94. Carpenter TO, Ellis BK, Insogna KL, Philbrick WM, Sterpka J, Shimkets R. Fibroblast growth factor 7: an inhibitor of phosphate transport derived from oncogenic osteomalacia-causing tumors. J Clin Endocrinol Metab 2005;90:1012e20. Schiavi SC, Kumar R. The phosphatonin pathway: New insights in phosphate homeostasis. Kidney Int 2004;65:1e14.

[107] Berndt T, Kumar R. Novel mechanisms in the regulation of phosphorus homeostasis. Physiology 2009;24:17e25. [108] Iwaki T, Sandoval-Cooper MJ, Tenenhouse HS, Castellino FJ. A missense mutation in the sodium phosphate co-transporter Slc34a1 impairs phosphate homeostasis. J Am Soc Nephrol 2008;19:1753e62. [109] Khan SR, Glenton PA. Calcium oxalate crystal deposition in kidneys of hypercalciuric mice with disrupted type IIa sodiumphosphate cotransporter. Am J Physiol Renal Physiol 2008;294:F1109e15. [110] Jones AO, Tzenova J, Frappier D, et al. Hereditary hypophosphatemic rickets with hypercalciuria is not caused by mutations in the Na/Pi cotransporter NPT2 gene. J Am Soc Nephrol 2001;12:507e14. [111] van den Heuvel L, Op de Koul K, Knots E, Knoers N, Monnens L. Autosomal recessive hypophosphataemic rickets with hypercalciuria is not caused by mutations in the type II renal sodium/phosphate cotransporter gene. Nephrol Dial Transplant 2001;16:48e51. [112] Shen F, Ivey J, Sherrard D, Nielsen R, Haussler M, Dj B. Further evidence supporting the phosphate leak hypothesis of idiopathic hypercalciuria. Adv Exp Med Biol 1978;103: 217e23. [113] Prie´, Dominique, Ravery V, Boccon-Gibod L, Friedlander G. Frequency of renal phosphate leak among patients with calcium nephrolithiasis. Kidney Int 2001;60:272e6. [114] Prie´ D, Huart V, Bakouh N, et al. Nephrolithiasis and osteoporosis associated with hypophosphatemia caused by mutations in the type 2a sodium-phosphate cotransporter. N Engl J Med 2002;347:983e91. [115] Virkki LV, Forster IC, Hernando N, Biber J, Murer H. Functional characterization of two naturally occurring mutations in the human sodium-phosphate cotransporter type IIa. J Bone Miner Res 2003;18:2135e41. [116] Magen D, Berger L, Coady MJ, et al. A loss-of-function mutation in NaPi-IIa and renal Fanconi’s syndrome. N Engl J Med 2010;362:1102e9. [117] Tieder M, Arie R, Modai D, Samuel R, Weissgarten J, Liberman UA. Elevated serum 1,25-dihydroxyvitamin D concentrations in siblings with primary Fanconi’s syndrome. N Engl J Med 1988;319:845e9. [118] Tieder M, Modai D, Shared U, et al. Idiopathic hypercalciuria and hereditary hypophosphatemic rickets. N Engl J Med 1987;316:125e9. [119] Karim Z, Ge´rard B, Bakouh N, et al. NHERF1 mutations and responsiveness of renal parathyroid hormone. N Engl J Med 2008;359:1128e35. [120] Brownstein CA, Adler F, Nelson-Williams C, et al. A translocation causing increased alpha-Klotho level results in hypophosphatemic rickets and hyperparathyroidism. Proc Natl Acad Sci USA 2008;105:3455e60. [121] Consortium TH. A gene (PEX) with homologies to endopeptidases is mutated in patients with X-linked hypophosphatemic rickets. Nat Genet 1995;11:130e6. [122] White KE, Evans WE, O’Riordan JLH, et al. Autosomal dominant hypophosphataemic rickets is associated with mutations in FGF23. Nat Genet 2000;26:345e8. [123] Lorenz-Depiereux B, Bastepe M, Benet-Pages A, et al. DMP1 mutations in autosomal recessive hypophosphatemia implicate a bone matrix protein in the regulation of phosphate homeostasis. Nat Genet 2006;38:1248e50. [124] Feng JQ, Ward LM, Liu S, et al. Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism. Nat Genet 2006;38:1310e5.

PEDIATRIC BONE

REFERENCES

[125] Lorenz-Depiereux B, Schnabel D, Tiosano D, Ha¨usler G, Strom TM. Loss-of-function ENPP1 mutations cause both generalized arterial calcification of infancy and autosomalrecessive hypophosphatemic rickets. Am J Hum Genet 2010;86:267e72. [126] Levy-Litan V, Hershkovitz E, Avizov L, et al. Autosomalrecessive hypophosphatemic rickets is associated with an inactivation mutation in the ENPP1 gene. Am J Hum Genet 2010;86:273e8. [127] Lapointe JY, Tessier J, Paquette Y, et al. NPT2a gene variation in calcium nephrolithiasis with renal phosphate leak. Kidney Int 2006;69:2261e7. [128] Clemens TL, Tang H, Maeda S, et al. Analysis of osteocalcin expression in transgenic mice reveals a species difference in vitamin D regulation of mouse and human osteocalcin genes. J Bone Miner Res 1997;12:1570e6. [129] Levtchenko E, Schoeber J, Jaeken J. Genetic disorders of renal phosphate transport. N Engl J Med 2010;363:1774e5. [130] Nishiyama S. A single case of hypophosphatemic rickets with hypercalciuria. J Pediatr Gastroenterol Nutr 1986;5:829. [131] Chen C, Carpenter T, Steg N, Baron R, Anast C. Hypercalciuric hypophosphatemic rickets, mineral balance, bone histomorphometry, and therapeutic implications of hypercalciuria. Pediatrics 1989;84:276e80. [132] Gazit DAN, Tieder M, Liberman UA, Passi-Even LEA, Bab IA. Osteomalacia in hereditary hypophosphatemic rickets with hypercalciuria: a correlative clinical-histomorphometric study. J Clin Endocrinol Metab 1991;72:229e35. [133] Tieder M, Arie R, Bab I, Maor J, Liberman U. A new kindred with hereditary hypophosphatemic rickets with hypercalciuria: implications for correct diagnosis and treatment. Nephron 1992;62:176e81. [134] Tieder M, Modai D, Samuel R, et al. Hereditary hypophosphatemic rickets with hypercalciuria. N Engl J Med 1985;312:611e7. [135] Ichikawa S, Sorenson AH, Imel EA, Friedman NE, Gertner JM, Econs MJ. Intronic deletions in the SLC34A3 gene cause hereditary hypophosphatemic rickets with hypercalciuria. J Clin Endocrinol Metab 2006;91:4022e7. [136] Tencza AL, Ichikawa S, Dang A, et al. Hypophosphatemic rickets with hypercalciuria due to mutation in SLC34A3/type IIc sodium-phosphate cotransporter: presentation as hypercalciuria and nephrolithiasis. J Clin Endocrinol Metab 2009;94:4433e8. [137] Kremke B, Bergwitz C, Ahrens W, et al. Hypophosphatemic rickets with hypercalciuria due to mutation in SLC34A3/NaPiIIc can be masked by vitamin D deficiency and can be associated with renal calcifications. Exp Clin Endocrinol Diabetes 2009;117:49e56. [138] Jaureguiberry G, Carpenter TO, Forman S, Ju¨ppner H, Bergwitz C. A novel missense mutation in SLC34A3 that causes hereditary hypophosphatemic rickets with hypercalciuria in humans identifies threonine 137 as an important determinant of sodium-phosphate cotransport in NaPi-IIc. Am J Physiol Renal Physiol 2008;295:F371e9. [139] Page K, Bergwitz C, Jaureguiberry G, Harinarayan C, Insogna K. A patient with hypophosphatemia, a femoral fracture, and recurrent kidney stones: report of a novel mutation in SLC34A3. Endocr Pract 2008;14:869e74. [140] Segawa H, Onitsuka A, Furutani J, et al. Npt2a and Npt2c in mice play distinct and synergistic roles in inorganic phosphate metabolism and skeletal development. Am J Physiol Renal Physiol 2009;297:F671e8. [141] Prie´ D, Friedlander G. Genetic disorders of renal phosphate transport. N Engl J Med 2010;362:2399e409.

763

[142] Cunningham R, Steplock D, Wang F, et al. Defective parathyroid hormone regulation of NHE3 activity and phosphate adaptation in cultured NHERF-1/ renal proximal tubule cells. J Biol Chem 2004;279:37815e21. [143] Weinman EJ, Boddeti A, Cunningham R, et al. NHERF-1 is required for renal adaptation to a low-phosphate diet. Am J Physiol Renal Physiol 2003;285:F1225e32. [144] Kuro-o M, Matsumura Y, Aizawa H, et al. Mutation of the mouse Klotho gene leads to a syndrome resembling ageing. Nature 1997;390:45e51. [145] Read AP, Thakker RV, Davies KE, et al. Mapping of human Xlinked hypophosphataemic rickets by multilocus linkage analysis. Hum Genet 1986;73:267e70. [146] Ma¨chler M, Frey D, Gal A, et al. X-linked dominant hypophosphatemia is closely linked to DNA markers DXS41 and DXS43 at Xp22. Hum Genet 1986;73:271e5. [147] Thakker RV, Read AP, Davies KE, et al. Bridging markers defining the map position of X linked hypophosphataemic rickets. J Med Genet 1987;24:756e60. [148] Guo R, Quarles LD. Cloning and sequencing of human PEX from a bone cDNA library: evidence for its developmental stage-specific regulation in osteoblasts. J Bone Miner Res 1997;12:1009e17. [149] Eicher EM, Southard JL, Scriver CR, Glorieux FH. Hypophosphatemia: mouse model for human familial hypophosphatemic (vitamin D-resistant) rickets. Proc Natl Acad Sci USA 1976;73:4667e71. [150] Strom TM, Francis F, Lorenz B, et al. Pex gene deletions in Gy and Hyp mice provide mouse models for X-linked hypophosphatemia. Hum Mol Genet 1997;6:165e71. [151] Nesbitt T, Coffman TM, Griffiths R, Drezner MK. Crosstransplantation of kidneys in normal and Hyp mice. Evidence that the Hyp mouse phenotype is unrelated to an intrinsic renal defect. J Clin Invest 1992;89:1453e9. [152] Morgan JM, Hawley WL, Chenoweth AI, Retan WJ, Diethelm AG. Renal transplantation in hypophosphatemia with vitamin D-resistant rickets. Arch Int Med 1974;134:549e52. [153] Yamazaki Y, Okazaki R, Shibata M, et al. Increased circulatory level of biologically active full-length FGF-23 in patients with hypophosphatemic rickets/osteomalacia. J Clin Endocrinol Metab 2002;87:4957e60. [154] Jonsson KB, Zahradnik R, Larsson T, et al. Fibroblast growth factor 23 in oncogenic osteomalacia and X-linked hypophosphatemia. N Engl J Med 2003;348:1656e63. [155] Bowe AE, Finnegan R, Jan de Beur SM, et al. FGF-23 Inhibits renal tubular phosphate transport and is a PHEX substrate. Biochem Biophys Res Commun 2001;284:977e81. [156] Campos M, Couture C, Hirata I, et al. Human recombinant endopeptidase PHEX has a strict S1’ specificity for acidic residues and cleaves peptides derived from fibroblast growth factor-23 and matrix extracellular phosphoglycoprotein. Biochem J 2003;373:271e9. [157] Benet-Page`s A, Lorenz-Depiereux B, Zischka H, White KE, Econs MJ, Strom TM. FGF23 is processed by proprotein convertases but not by PHEX. Bone 2004;35:455e62. [158] Liu S, Guo R, Simpson LG, Xiao Z-S, Burnham CE, Quarles LD. Regulation of fibroblastic growth factor 23 expression but not degradation by PHEX. J Biol Chem 2003;278:37419e26. [159] Berndt M, Ehrich J, Lazovic D, et al. Clinical course of hypophosphatemic rickets in 23 adults. Clin Nephrol 1996;45:33e41. [160] Taylor A, Sherman NH, Norman ME. Nephrocalcinosis in X-linked hypophosphatemia: effect of treatment versus disease. Pediatr Nephrol 1995;9:173e5. [161] Econs MJ, McEnery PT. Autosomal dominant hypophosphatemic rickets/osteomalacia: clinical characterization of

PEDIATRIC BONE

764

[162]

[163]

[164]

[165]

[166]

[167] [168]

[169]

[170]

[171] [172]

[173]

[174]

[175]

[176]

[177]

[178]

[179]

[180]

[181]

27. HEREDITARY TUBULAR DISORDERS OF MINERAL HANDLING

a novel renal phosphate-wasting disorder. J Clin Endocrinol Metab 1997;82:674e81. Econs MJ, McEnery PT, Lennon F, Speer MC. Autosomal dominant hypophosphatemic rickets is linked to chromosome 12p13. J Clin Invest 1997;100:2653e7. Yamashita T, Yoshioka M, Itoh N. Identification of a novel fibroblast growth factor, FGF-23, preferentially expressed in the ventrolateral thalamic nucleus of the brain. Biochem Biophys Res Commun 2000;277:494e8. Terkeltaub R. Physiologic and pathologic functions of the NPP nucleotide pyrophosphatase/phosphodiesterase family focusing on NPP1 in calcification. Purinergic Signal 2006;2:371e7. Terkeltaub RA. Inorganic pyrophosphate generation and disposition in pathophysiology. Am J Physiol Cell Physiol 2001;281:C1e11. Rutsch F, Ruf N, Vaingankar S, et al. Mutations in ENPP1 are associated with ’idiopathic’ infantile arterial calcification. Nat Genet 2003;34:379e81. De Toni G. Remarks on the relation between renal rickets (renal dwarfism) and renal diabetes. Acta Paediatr 1933;16:479e84. Debre´ R, Marie J, Cleret F, Messimy R. Rachitisme tardif coexistant avec une ne´phrite chronique et une glycosurie. Arch Me´d Enf Paris 1934;37:597e606. Fanconi G. Der fru¨hinfantile nephrotisch-glykosurische Zwergwuchs mit Hypophosphata¨mischer Rachitis. Jahrbuch Kinderheilk 1936;147:299e338. Magen D, Adler L, Mandel H, Efrati E, Zelikovic I. Autosomal recessive renal proximal tubulopathy and hypercalciuria: a new syndrome. Am J Kidney Dis 2004;43:600e6. Gahl WA, Thoene JG, Schneider JA. Cystinosis. N Engl J Med 2002;347:111e21. Nesterova G, Gahl W. Nephropathic cystinosis: late complications of a multisystemic disease. Pediatr Nephrol 2008;23:863e78. McDowell GA, Gahl WA, Stephenson LA, et al. Linkage of the gene for cystinosis to markers on the short arm of chromosome 17. Nat Genet 1995;10:246e8. Town M, Jean G, Cherqui S, et al. A novel gene encoding an integral membrane protein is mutated in nephropathic cystinosis. Nat Genet 1998;18:319e24. Kalatzis V, Cherqui S, Antignac C, Gasnier B. Cystinosin, the protein defective in cystinosis, is a Hþ-driven lysosomal cystine transporter. EMBO J 2001;20:5940e9. Coor C, Salmon RF, Quigley R, Marver D, Baum M. Role of adenosine triphosphate (ATP) and NaK ATPase in the inhibition of proximal tubule transport with intracellular cystine loading. J Clin Invest 1991;87:955e61. Cetinkaya I, Schlatter E, Hirsch JR, Herter P, Harms E, Kleta R. Inhibition of Na(þ)-dependent transporters in cystine-loaded human renal cells: electrophysiological studies on the Fanconi syndrome of cystinosis. J Am Soc Nephrol 2002;13:2085e93. Cherqui S, Sevin C, Hamard G, et al. Intralysosomal cystine accumulation in mice lacking cystinosin, the protein defective in cystinosis. Mol Cell Biol 2002;22:7622e32. Jackson J, Smith F, Litman N, Yuile C, Latta H. The Fanconi syndrome with cystinossis. Electron microscopy of renal biopsy specimens from five patients. Am J Med 1962;33:893e910. Spear G, RJ S, Tousimis A, Taylor C. Cystinosis. An ultrastructural and electron-probe study of the kidney with unusual findings. Arch Pathol 1971;91:206e21. Park M, Helip-Wooley A, Thoene J. Lysosomal cystine storage augments apoptosis in cultured human fibroblasts and renal tubular epithelial cells. J Am Soc Nephrol 2002;13:2878e87.

[182] Kleta R, Gahl WA. Pharmacological treatment of nephropathic cystinosis with cysteamine. Expert Opin Pharmacother 2004;5:2255e62. [183] Fanconi G, Bickel H. Die chronische Aminoacidurie (Aminosaeurediabetes oder nephrotisch-glukosurischer Zwergwuchs) bei der Glykogenose und der Cystinkrankheit. Helv Paediatr Acta 1949;4:359e96. [184] Manz F, Bickel H, Brodehl J, et al. FanconieBickel syndrome. Pediatr Nephrol 1987;1:509e18. [185] Santer R, Schneppenheim R, Dombrowski A, Gotze H, Steinmann B, Schaub J. Mutations in GLUT2, the gene for the liver-type glucose transporter, in patients with FanconieBickel syndrome. Nat Genet 1997;17:324e6. [186] Santer R, Schneppenheim R, Suter D, Schaub J, Steinmann B. FanconieBickel syndrome e the original patient and his natural history, historical steps leading to the primary defect, and a review of the literature. Eur J Pediatr 1998;157:783e97. [187] Yoo H, Shin Y, Seo E, Kim G. Identification of a novel mutation in the GLUT2 gene in a patient with FanconieBickel syndrome presenting with neonatal diabetes mellitus and galactosaemia. Eur J Pediatr 2002;161:351e3. [188] Bell GI, Burant CF, Takeda J, Gould GW. Structure and function of mammalian facilitative sugar transporters. J Biol Chem 1993;268:19161e4. [189] Berry G, Baker L, Kaplan F, Witzleben C. Diabetes-like renal glomerular disease in FanconieBickel syndrome. Pediatr Nephrol 1995;9:287e91. [190] Lee P, Van’t Hoff W, Leonard J. Catch-up growth in FanconieBickel syndrome with uncooked cornstarch. J Inherit Metab Dis 1995;18:153e6. [191] Dent C, Friedman M. Hypercalcuric rickets associated with renal tubular damage. Arch Dis Child 1964;39:240e9. [192] Wrong OM, Norden AGW, Feest TG. Dent’s disease; a familial proximal renal tubular syndrome with low-molecular-weight proteinuria, hypercalciuria, nephrocalcinosis, metabolic bone disease, progressive renal failure and a marked male predominance. Q J Med 1994;87:473e93. [193] Lloyd SE, Pearce SHS, Fisher SE, et al. A common molecular basis for three inherited kidney stone diseases. Nature 1996;379:445e9. [194] Akuta N, Lloyd S, Igarashi T, et al. Mutations of CLCN5 in Japanese children with idiopathic low molecular weight proteinuria, hypercalciuria and nephrocalcinosis. Kidney Int 1997;52:911e6. [195] Igarashi T, Inatomi J, Ohara T, Kuwahara T, Shimadzu M, Thakker RV. Clinical and genetic studies of CLCN5 mutations in Japanese families with Dent’s disease. Kidney Int 2000;58:520e7. [196] Scheinman SJ. X-linked hypercalciuric nephrolithiasis: Clinical syndromes and chloride channel mutations. Kidney Int 1998;53:3e17. [197] Thakker RV. Pathogenesis of Dent’s disease and related syndromes of X-linked nephrolithiasis. Kidney Int 2000;57:767e73. [198] Jentsch TJ, Poe¨t M, Fuhrmann JC, Zdebik AA. Physiological functions of CLC Cl- channels gleaned from human genetic disease and mouse models. Annu Rev Physiol 2004;67:779e807. [199] Gu¨nther W, Piwon N, Jentsch T. The ClC-5 chloride channel knock-out mouse e an animal model for Dent’s disease. Pflu¨gers Arch 2003;445:456e62. [200] Wang SS, Devuyst O, Courtoy PJ, et al. Mice lacking renal chloride channel, CLC-5, are a model for Dent’s disease, a nephrolithiasis disorder associated with defective receptormediated endocytosis. Hum Mol Genet 2000;9:2937e45.

PEDIATRIC BONE

REFERENCES

[201] Moulin P, Igarashi T, Van Der Smissen P, et al. Altered polarity and expression of Hþ-ATPase without ultrastructural changes in kidneys of Dent’s disease patients. Kidney Int 2003;63:1285e95. [202] Piwon N, Gunther W, Schwake M, Bosl MR, Jentsch TJ. ClC-5 Clechannel disruption impairs endocytosis in a mouse model for Dent’s disease. Nature 2000;408:369e73. [203] Lowe C, Terrey M, MacLachlen E. Organic-aciduria, decreased renal ammonia production, hydrophthalmos, and mental retardation; a clinical entity. Am J Dis Child 1952;83:164e84. [204] Charnas LR, Bernardini I, Rader D, Hoeg J, Gahl WA. Clinical and laboratory findings in the oculocerebrorenal syndrome of Lowe, with special reference to growth and renal function. N Engl J Med 1991;324:1318e25. [205] Silver DN, Lewis RA, Nussbaum RL. Mapping the Lowe oculocerebrorenal syndrome to Xq24-q26 by use of restriction fragment length polymorphisms. J Clin Invest 1987;79:282e5. [206] Attree O, Olivos IM, Okabe I, et al. The Lowe’s oculocerebrorenal syndrome gene encodes a protein highly homologous to inositol polyphosphate-5-phosphatase. Nature 1992;358:239e42. [207] Suchy SF, Olivos-Glander IM, Nussbaum RL. Lowe syndrome, a deficiency of a phosphatidyl-inositol 4,5-bisphosphate 5-phosphatase in the Golgi apparatus. Hum Mol Genet 1995;4:2245e50. [208] Zhang X, Jefferson AB, Auethavekiat V, Majerus PW. The protein deficient in Lowe syndrome is a phosphatidylinositol4,5-bisphosphate 5-phosphatase. Proc Natl Acad Sci USA 1995;92:4853e6. [209] Olivos-Glander IM, Janne PA, Nussbaum RL. The oculocerebrorenal syndrome gene product is a 105-kD protein localized to the Golgi complex. Am J Hum Genet 1995;57:817e23. [210] Suchy SF, Nussbaum RL. The deficiemcy of PIP2 5-phosphatase in Lowe syndrome affects actin polymerisation. Am J Hum Genet 2002;71:1420e7. [211] Bo¨kenkamp A, Ludwig M. Disorders of the renal proximal tubule. Nephron Physiol 2011;118:1e6. [212] Loi M. Lowe syndrome. Orphanet J Rare Dis 2006;1:16. [213] Monnier N, Satre V, Lerouge E, Berthoin F, Lunardi J. OCRL1 mutation analysis in French Lowe syndrome patients: implications for molecular diagnosis strategy and genetic counseling. Hum Mutat 2000;16:157e65. [214] Bo¨kenkamp A, Bo¨kenkamp D, Cheong HI, et al. Dent-2 disease: a mild variant of Lowe syndrome. J Pediatr 2009;155:94e9. [215] Niaudet P, Ro¨tig A. Renal involvement in mitochondrial cytopathies. Pediatr Nephrol 1996;10:368e73. [216] Hatefi Y. The mitochondrial electron transport and oxidative phosphorylation system. Annu Rev Biochem 1985;54:1015e69. [217] Clayton D. Structure and function of the mitochondrial genome. J Inherit Metab Dis 1992;15:439e47. [218] Niaudet P. Mitochondrial disorders and the kidney. Arch Dis Child 1998;78:387e90. [219] Eviatar L, Shanske S, Gauthier B, et al. KearnseSayre syndrome presenting as renal tubular acidosis. Neurology 1990;40:1761. [220] Kuwertz-Bro¨king E, Koch HG, Marquardt T, et al. Renal Fanconi syndrome: first sign of partial respiratory chain complex IV deficiency. Pediatr Nephrol 2000;14:495e8. [221] Mochizuki H, Joh K, Kawame H, et al. Mitochondrial encephalomyopathies preceded by de-TonieDebre´eFanconi syndrome or focal segmental glomerulosclerosis. Clin Nephrol 1996;46:347e52. [222] Ueda Y, Ando A, Nagata T, et al. A boy with mitochondrial disease: asymptomatic proteinuria without neuromyopathy. Pediatr Nephrol 2004;19:107e10.

765

[223] Gilbert R, Emms M. Pearson’s syndrome presenting with Fanconi syndrome. Ultrastruct Pathol 1996;20:473e5. [224] Goto Y, Itami N, Kajii N, Tochimaru H, Endo M, Horai S. Renal tubular involvement mimicking Bartter syndrome in a patient with KearnseSayre syndrome. J Pediatr 1990;116:904e10. [225] Matsutani H, Mizusawa Y, Shimoda M, et al. Partial deficiency of cytochrome c oxidase with isolated proximal renal tubular acidosis and hypercalciuria. Child Nephrol Urol 1992;12:221e4. [226] Zaffanello M, Zamboni G. Therapeutic approach in a case of Pearson’s syndrome. Minerva Pediatr 2005;57:143e6. [227] Majander A, Suomalainen ANU, Vettenranta KIM, et al. Congenital hypoplastic anemia, diabetes, and severe renal tubular dysfunction associated with a mitochondrial DNA deletion. Pediatr Res 1991;30:327e30. [228] Niaudet P, Heidet L, Munnich A, et al. Deletion of the mitochondrial DNA in a case of de TonieDebre´eFanconi syndrome and Pearson syndrome. Pediatr Nephrol 1994;8:164e8. [229] Mori K, Narahara K, Ninomiya S, Goto Y-i, Nonaka I. Renal and skin involvement in a patient with complete KearnseSayre syndrome. Am J Med Genet 1991;38:583e7. [230] Ogier H, Lombes A, Scholte HR, et al. de TonieFanconieDebre´ syndrome with Leigh syndrome revealing severe muscle cytochrome c oxidase deficiency. J Pediatr 1988;112:734e9. [231] Igarashi T. Fanconi syndrome. In: Avner ED, Harmon WE, Niaudet P, et al., editors. Pediatric Nephrology. 6th edn. Berlin: Springer-Verlag; 2009. p. 1039e67. [232] Ben-Ishay D, Dreyfuss F, Ullmann T. Fanconi syndrome with hypouricemia in an adult: family study. Am J Med 1961;31:793e800. [233] Friedman A, Trygstad C, Chesney R. Autosomal dominant Fanconi syndrome with early renal failure. Am J Med Genet 1978;2:225e32. [234] Patrick A, Cameron J, Ogg CA. Family with a dominant form of idiopathic Fanconi syndrome leading to renal failure in adult life. Clin Nephrol 1981;16:289e92. [235] Tolaymat A, Sakarcan A, Neiberger R. Idiopathic Fanconi syndrome in a family. Part I. Clinical aspects. J Am Soc Nephrol 1992;2:1310e7. [236] Wen S, Friedman A, Oberley T. Two case studies from a family with primary Fanconi syndrome. Am J Kidney Dis 1989;13:240e6. [237] Neimann N, Pierson M, Marchal C, Rauber G, Grignon G. Glomerulo-tubular nephropathy with the de Toni-Debre´-Fanconi syndrome. Arch Fr Pediatr 1968;25:43e69. [238] Wornell P, Crocker J, Wade A, Dixon J, Acott P. An Acadian variant of Fanconi syndrome. Pediatr Nephrol 2007;22:1711e5. [239] Batuman V. Proximal tubular injury in myeloma. Contrib Nephrol 2007;153:87e104. [240] Rikitake O, Sakemit A, Yoshikawa Y, Nagano Y, Watanabe T. Adult Fanconi syndrome in primary amyloidosis with lambda light-chain proteinuria. Jpn J Med 1989;28:523e6. [241] Ren H, Wang W-M, Chen X-N, et al. Renal involvement and followup of 130 patients with primary Sjo¨gren’s syndrome. J Rheumatol 2008;35:278e84. [242] Wang C-C, Shiang J-C, Huang W-T, Lin S-H. Hypokalemic paralysis as primary presentation of Fanconi syndrome associated with Sjo¨gren syndrome. J Clin Rheumatol 2010;16:178e80. [243] Yang Y-S, Peng C-H, Sia S-K, Huang C-N. Acquired hypophosphatemia osteomalacia associated with Fanconi’s syndrome in Sjo¨gren’s syndrome. Rheumatol Int 2007;27:593e7. [244] Friedman A, Chesney R. Fanconi’s syndrome in renal transplantation. Am J Nephrol 1981;1:45e7. [245] Dobrin R, Vernier R, Fish A. Acute eosinophilic interstitial nephritis and renal failure with bone marrow-lymph node

PEDIATRIC BONE

766

[246]

[247]

[248]

[249]

[250] [251]

[252]

[253]

[254]

[255]

[256]

[257]

[258]

[259]

[260]

[261]

[262]

[263]

[264]

[265]

27. HEREDITARY TUBULAR DISORDERS OF MINERAL HANDLING

granulomas and anterior uveitis. A new syndrome. Am J Med 1975;59:325e33. McVicar M, Exeni R, Susin M. Nephrotic syndrome and multiple tubular defects in children: an early sign of focal segmental glomerulosclerosis. J Pediatr Gastroenterol Nutr 1980;97:918e22. Alexandridis G, Liamis G, Elisaf M. Reversible tubular dysfunction that mimicked Fanconi’s syndrome in a patient with anorexia nervosa. Int J Eat Disord 2001;30:227e30. Cleveland W, Adams W, Mann J, Nyhan W. Acquired Fanconi syndrome following degraded tetracycline. J Pediatr Gastroenterol Nutr 1965;66:333e42. Gainza F, Minguela J, Lampreabe I. Aminoglycoside-associated Fanconi’s syndrome: an underrecognized entity. Nephron 1997;77:205e11. Ghiculescu RA, Kubler PA. Aminoglycoside-associated Fanconi syndrome. Am J Kidney Dis 2006;48:e89e93. Watanabe T, Yoshikawa H, Yamazaki S, Abe Y, Abe T. Secondary renal Fanconi syndrome caused by valproate therapy. Pediatr Nephrol 2005;20:814e7. Hong YT, Fu LS, Chung LH, Hung SC, Huang YT, Chi CS. Fanconi’s syndrome, interstitial fibrosis and renal failure by aristolochic acid in Chinese herbs. Pediatr Nephrol 2006;21:577e9. Takamoto K, Kawada M, Usui T, Ishizuka M, Ikeda D. Aminoglycoside antibiotics reduce glucose reabsorption in kidney through down-regulation of SGLT1. Biochem Biophys Res Commun 2003;308:866e71. Zietse R, Zoutendijk R, Hoorn EJ. Fluid, electrolyte and acidbase disorders associated with antibiotic therapy. Nat Rev Nephrol 2009;5:193e202. Nelson M, Azwa A, Sokwala A, Harania RS, Stebbing J. Fanconi syndrome and lactic acidosis associated with stavudine and lamivudine therapy. AIDS 2008;22:1374e6. Quinn KJ, Emerson CR, Dinsmore WW, Donnelly CM. Incidence of proximal renal tubular dysfunction in patients on tenofovir disoproxil fumarate. Int J STD AIDS 2010;21:150e1. Girgis C, Wong T, Ngu M, et al. Hypophosphataemic osteomalacia in patients on adefovir dipivoxil. J Clin Gastroenterol 2010. doi: 10.1097/MCG.0b013e3181e12ed3. Ho ES, Lin DC, Mendel DB, Cihlar T. Cytotoxicity of antiviral nucleotides adefovir and cidofovir is induced by the expression of human renal organic anion transporter 1. J Am Soc Nephrol 2000;11:383e93. Baum M. Renal Fanconi syndrome secondary to deferasirox: where there is smoke there is fire. J Pediatr Hematol Oncol 2010;32:525e6. Rheault MN, Bechtel H, Neglia JP, Kashtan CE. Reversible Fanconi syndrome in a pediatric patient on deferasirox. Pediatr Blood Cancer 2011;56:674e6. Grange´ S, Bertrand DM, Guerrot D, Eas F, Godin M. Acute renal failure and Fanconi syndrome due to deferasirox. Nephrol Dial Transplant 2010;25:2376e8. Zamlauski-Tucker MJ, Morris ME, Springate JE. Ifosfamide metabolite chloroacetaldehyde causes Fanconi syndrome in the perfused rat kidney. Toxicol Appl Pharmacol 1994;129:170e5. Yaseen Z, Michoudet C, Baverel G, Dubourg L. Mechanisms of the ifosfamide-induced inhibition of endocytosis in the rat proximal kidney tubule. Arch Toxicol 2008;82:607e14. Pourahmad J, Hosseini M-J, Eskandari MR, Shekarabi SM, Daraei B. Mitochondrial/lysosomal toxic cross-talk plays a key role in cisplatin nephrotoxicity. Xenobiotica 2010;40:763e71. Clark JS, Faisal A, Baliga R, Nagamine Y, Arany I. Cisplatin induces apoptosis through the ERK-p66shc pathway in renal proximal tubule cells. Cancer Lett 2010;297:165e70.

[266] Pratt CB, Meyer WH, Jenkins JJ, et al. Ifosfamide, Fanconi’s syndrome, and rickets. J Clin Oncol 1991;9:1495e9. [267] Barbier O, Jacquillet G, Tauc M, Cougnon M, Poujeol P. Effect of heavy metals on, and handling by, the kidney. Nephron Physiol 2005;99:105e10. [268] Gonick H. Nephrotoxicity of cadmium & lead. Indian J Med Res. 2008;128:335e52. [269] Johri N, Jacquillet G, Unwin R. Heavy metal poisoning: the effects of cadmium on the kidney. Biometals 2010;23:783e92. [270] Nolan CV, Shaikh ZA. Lead nephrotoxicity and associated disorders: biochemical mechanisms. Toxicology 1992;73:127e46. [271] Hoenderop JG, Nilius B, Bindels RJ. Calcium absorption across epithelia. Physiol Rev 2005;85:373e422. [272] Suzuki Y, Landowski CP, Hediger MA. Mechanisms and regulation of epithelial Ca2þ absorption in health and disease. Annu Rev Physiol 2008;70:257e71. [273] Mount DB, Yu ASL. Transport of inorganic solutes: sodium, chloride, potassium, magnesium, calcium, and phosphate. In: Brenner BM, editor. The Kidney. 8th edn. Philadelphia: Saunders Elsevier; 2008. p. 156e213. [274] Dimke H, Hoenderop JG, Bindels RJ. Hereditary tubular transport disorders: implications for renal handling of Ca2þ and Mg2þ. Clin Sci (Lond) 2009;118:1e18. [275] Edwards BR, Baer PG, Sutton RA, et al. Micropuncture study of diuretic effects on sodium and calcium reabsorption in the dog nephron. J Clin Invest 1973;52:2418e27. [276] Friedman PA. Calcium transport in the kidney. Curr Opin Nephrol Hypertens 1999;8:589e95. [277] Van de Graaf SFJ, Bindels RJM, Hoenderop JGJ. Physiology of epithelial Ca2þ and Mg2þ transport. Rev Physiol Biochem Pharmacol 2007;158:77e160. [278] Brown EM, MacLeod RJ. Extracellular calcium sensing and extracellular calcium signaling. Physiol Rev 2001;81:237e9. [279] Houillier P, Paillard M. Calcium-sensing receptor and renal cation handling. Nephrol Dial Transplant 2003;18:2467e70. [280] Ward DT, Riccardi D. Renal physiology of the extracellular calcium-sensing receptor. Pflu¨gers Arch 2002;445:169e76. [281] Riccardi D, Brown EM. Physiology and pathophysiology of the calcium-sensing receptor in the kidney. Am J Physiol Renal Physiol 2010;298:F485e99. [282] Huang H, Miller RT. Novel Ca receptor signaling pathways for control of renal ion transport. Curr Opin Nephrol Hypertens 2010;19:106e12. [283] Friedman PA. Renal calcium metabolism. In: Alpern RJ, Hebert SC, editors. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 4th edn. Philadelphia: Saunders Elsevier; 2008. p. 1851e90. [284] Kelepouris E, Agus ZS. Hypomagnesemia: renal Mg2þ handling. Sem Nephrol 1998;18:58e73. [285] Quamme GA. Renal magnesium handling: new insights in understanding old problems. Kidney Int 1997;52:1180e95. [286] Quamme GA, Schlingmann KP, Konard M. Mechanisms and disorders of magnesium metabolism. In: Alpern RJ, Hebert SC, editors. Seldin and Giebisch’s The Kidney: Physiology and Pathophysiology. 4th edn. Philadelphia: Saunders Elsevier; 2008. p. 1747e67. [287] Quamme GA, Rouffignac CD. Epithelial magnesium transport and regulation by the kidney. Front Biosci 2000;5:694e711. [288] Asch W, Lifton RP. Molecular genetics of magnesium homeostasis. In: Lifton RP, Somlo S, Giebisch GH, et al., editors. Genetic Diseases of the Kidney. Philadelphia: Saunders Elsevier; 2009. p. 249e61. [289] Naderi ASA, Reilly Jr RF. Hereditary etiologies of hypomagnesemia. Nat Clin Pract Nephrol 2008;4:80e9.

PEDIATRIC BONE

767

REFERENCES

[290] Knoers NVAM. Inherited forms of renal hypomagnesemia: an update. Pediatr Nephrol 2009;24:697e705. [291] Quamme GA, Smith CM. Magnesium transport in the proximal straight tubule of the rabbit. Am J Physiol 1984;246:F544e50. [292] Wong NL, Whiting SJ, Mizgala CL, et al. Electrolyte handling by the superficial nephron of the rabbit. Am J Physiol 1986;250:F590e5. [293] Monnens L, Starremans P, Bindels R. Great strides in the understanding of renal magnesium and calcium reabsorption. Nephrol Dial Transplant 2000;15:568e71. [294] Lelievre-Pegorier M, Merlet-Benichou C, Roinel N, et al. Developmental pattern of water and electrolyte transport in rat superficial nephrons. Am J Physiol 1983;245:F15e21. [295] Mandon B, Siga E, Chabardes D, et al. Insulin stimulates Naþ, Cl, Ca2þ, and Mg2þ transports in TAL of mouse nephron: cross-potentiation with AVP. Am J Physiol 1993;265:F361e9. [296] Wittner M, Desfleurs E, Pajaud S, et al. Calcium and magnesium: low passive permeability and tubular secretion in the mouse medullary thick ascending limb of Henle’s loop (mTAL). J Membr Biol 1996;153:27e35. [297] Simon DB, Lu Y, Chaote KA, et al. Paracellin-1, a renal tight junction protein required for paracellular Mg2þ resorption. Science 1999;285:103e6. [298] Weber S, Schlingmann KP, Peters M, et al. Primary gene structure and expression studies of rodent paracellin-1. J Am Soc Nephrol 2001;12:2664e7. [299] Konrad M, Schaller A, Seelow DJ, et al. Mutations in the tightjunction gene claudin-19 (CLDN19) are associated with renal magnesium wasting, renal failure and severe ocular involvement. Am J Hum Genet 2006;79:949e57. [300] Haisch L, Almeida JR, Abreu da Silva PR, et al. The role of tight junctions in paracellular ion transport in the renal tubule: lessons learned from a rare inherited tubular disorder. Am J Kidney Dis 2011;57:320e30. [301] Cristobal PS, Dimke H, Hoenderop JGJ, et al. Novel molecular pathways in renal Mg2þ transport: a guided tour along the nephron. Curr Opin Nephrol Hypertens 2010;19:456e62. [302] Wittner M, Mandon B, Roinel N, et al. Hormonal stimulation of Ca2þ and Mg2þ transport in the cortical thick ascending limb of Henle’s loop of the mouse: evidence for a change in the paracellular pathway permeability. Pflu¨gers Arch 1993;423:387e96. [303] Quamme GA. Effect of furosemide on calcium and magnesium transport in the rat nephron. Am J Physiol 1981;241:F340e7. [304] Simon DB, Karet FE, Hamdan JM, et al. Bartter’s syndrome, hypokalemic alkalosis with hypercalciuria, is caused by mutations in the Na-K-2Cl contransporter NKCC2. Nat Genet 1996;13:183e8. [305] Seyberth HW, Rascher W, Schweer H, et al. Congenital hypokalemia with hypercalciuria in pre-term infants: a hyperprostaglandinuric tubular syndrome different from Bartter syndrome. J Pediatr 1985;107:694e701. [306] Simon DB, Karet FE, Rodriguez-Soriano J, et al. Genetic heterogeneity of Bartter’s syndrome revealed by mutations in the Kþ channel, ROMK. Nat Genet 1996;14:152e6. [307] Simon DB, Bindra RS, Mansfield TA, et al. Mutations in the chloride channel gene, CLCNKB, cause Bartter’s syndrome type III. Nat Genet 1997;17:171e8. [308] Birkenhager R, Otto E, Schurmann MJ, et al. Mutation of BSND causes Bartter syndrome with sensorineural deafness and kidney failure. Nat Genet 2001;29:310e4. [309] Estevez R, Boettger T, Stein V, et al. Barttin is a Cl channel betasubunit crucial for renal Cl reabsorption and inner ear Kþ secretion. Nature 2001;414:558e61. [310] Vargas-Poussou R, Huang C, Hulin P, et al. Functional characterization of a calcium-sensing receptor mutation in severe

[311]

[312]

[313]

[314]

[315]

[316]

[317]

[318]

[319]

[320]

[321]

[322]

[323]

[324]

[325] [326]

[327]

[328]

[329]

autosomal dominant hypocalcemia with a Bartter-like syndrome. J Am Soc Nephron 2002;13:2259e66. Watanabe S, Fukumoto S, Chang H, et al. Association between activation mutations of calcium-sensing receptor and Bartter’s syndrome. Lancet 2002;360:692e4. Auwerx J, Demedts M, Bouillon R. Altered parathyroid set point to calcium in familial hypocalciuric hypercalcaemia. Acta Endocrinol 1984;106:215e8. Attie MF, Gill Jr JR, Stock JL, et al. Urinary calcium excretion in familial hypocalciuric hypercalcemia. Persistence of relative hypocalciuria after induction of hypoparathyroidism. J Clin Invest 1983;72:667e76. Simon DB, Nelson-Williams C, Bia MJ, et al. Gitelman’s variant of Bartter’s syndrome, inherited hypokalemic alkalosis, is caused by mutations in the thiazide-sensitive Na-Cl cotransporter. Nat Genet 1996;12:24e30. Jeck N, Konrad M, Peters M, et al. Mutations in the chloride channel gene, CLCNKB, leading to a mixed Bartter-Gitelman phenotype. Pediatr Res 2000;48:754e8. Zelikovic I, Szargel R, Hawash A, et al. A novel mutation in the chloride channel gene, CLCNKB, as a cause of Gitelman and Bartter syndromes. Kidney Int 2003;63:24e32. Schlingmann KP, Weber S, Peters M, et al. Hypomagnesemia with secondary hypocalcemia is caused by mutations in TRPM6, a new member of the TRPM gene family. Nat Genet 2002;31:166e70. Walder RY, Landau D, Meyer P, et al. Mutation of TRPM6 causes familial hypomagnesemia with secondary hypocalcemia. Nat Genet 2002;31:171e4. Glaudemans B, van der Wijst J, Scola RH, et al. A missense mutation in the Kv1.1 voltage-gated potassium channelencoding gene KCNA1 is linked to human autosomal dominant hypomagnesemia. J Clin Invest 2009;119:936e42. Groenestege WM, Thebault S, van der Wijst J, et al. Impaired basolateral sorting of pro-EGF causes isolated recessive renal hypomagnesemia. J Clin Invest 2007;117:2260e7. Meij IC, Koendering JB, van Bokhoven H, et al. Dominant isolated renal magnesium loss is caused by misrouting of the NaþKþ-ATPase gamma-subunit. Nat Genet 2000;26:265e6. Adalat S, Woolf AS, Johnstone KA, et al. HNF1B mutations associate with hypomagnesemia and renal magnesium wasting. J Am Soc Nephrol 2009;20:1123e31. Bockenhauer D, Feather S, Stanescu HC, et al. Epilepsy, ataxia, sensorineural deafness, tubulopathy, and KCNJ10 mutations. N Engl J Med 2009;360:1960e70. Scholl UI, Choi M, Liu T, et al. Seizures, sensorineural deafness, ataxia, mental retardation, and electrolyte imbalance (SeSAME syndrome) caused by mutations in KCNJ10. Proc Natl Acad Sci USA 2009;106:5842e7. Dai LJ, Ritchie G, Kerstan D, et al. Magnesium transport in the renal convoluted tubule. Physiol Rev 2001;81:51e84. Voets T, Nilius B, Hoefs S, et al. TRPM6 forms the Mg2þ influx channel involved in intestinal and renal Mg2þ absorption. J Biol Chem 2004;279:19e25. Glaudemans B, Knoers NVAM, Hoenderop JGJ, et al. New molecular players facilitation Mg2þ reabsorption in the distal convoluted tubule. Kidney Int 2010;77:17e22. Cohen B, Giebisch G, Hansen LL, et al. Relationship between peritubular membrane potential and net fluid reabsorption in the distal renal tubule of Amphiuma. J Physiol 1984;348:115e34. Hansen LL, Schilling AR, Wiederholt M. Effect of calcium, furosemide and chlorothiazide on net volume reabsorption and basolateral membrane potential of the distal tubule. Pflu¨gers Arch 1981;389:121e6.

PEDIATRIC BONE

768

27. HEREDITARY TUBULAR DISORDERS OF MINERAL HANDLING

[330] Muallem S, Moe OW. When EGF is offside, magnesium is wasted. J Clin Invest 2007;117:2086e9. [331] Thebault S, Alexander RT, Tiel Groenestege WM, et al. EGF increases TRPM6 activity and surface expression. J Am Soc Nephrol 2009;20:78e85. [332] Ellison DH. The voltage-gated Kþ channel subunit Kv1.1 links kidney and brain. J Clin Invest 2009;119:763e6. [333] Van der Wijst J, Glaudemans B, Venselaar H, et al. Functional analysis of the Kv1.1 N255D mutation associated with autosomal dominant hypomagnesemia. J Biol Chem 2010;285:171e8. [334] Sweadner KJ, Wetzel RK, Arystarkhova E. Genomic organization of the human FXYD2 gene encoding the gamma subunit of the Naþ-Kþ-ATPase. Biochem Biophys Res Commun 2000;279:196e201. [335] Sweadner KJ, Rael E. The FXYD gene family of small ion transport regulators or channels: cDNA sequence, protein signature sequence, and expression. Genomics 2000;68:41e56. [336] Wetzel RK, Sweadner KJ. Immunocytochemical localization of Naþ-Kþ-ATPase alpha- and gamma-subunits in rat kidney. Am J Physiol Renal Physiol 2001;281:F531e45. [337] Arystarkhova E, Wetzel RK, Asinovski NK, et al. The gamma subunit modulates Naþ and Kþ affinity of the renal Naþ-KþATPase. J Biol Chem 1999;274:33183e5. [338] Beguin P, Wang X, Firsov D, et al. The gamma subunit is a specific component of the Naþ-Kþ-ATPase and modulates its transport function. EMBO J 1997;16:4250e60. [339] Rouffignac CD, Quamme GA. Renal magnesium handling and its hormonal control. Physiol Rev 1994;74:305e22. [340] Cao G, Hoenderop JGJ, Bindels RJM. Insight into the molecular regulation of the epithelial magnesium channel TRPM6. Curr Opin Nephrol Hypertens 2008;17:373e8. [341] Hoenderop JGJ, Bindels RJM. Calciotropic and magnesiotropic TRP channels. Physiology 2008;23:32e40. [342] Efrati E, Arsentiev J, Zelikovic I. The human paracellin-1 gene (hPCLN-1): renal epithelial-specific expression and regulation. Am J Physiol Renal Physiol 2005;288:F272e83. [343] Efrati E, Hirsch A, Kladnitsky O, et al. Transcriptional regulation of the claudin-16 gene by Mg2þ availability. Cell Physiol Biochem 2010;25:705e14. [344] Leheste JR, Melsen F, Wellner M, et al. Hypocalcemia and osteopathy in mice with kidney-specific megalin gene defect. FASEB J 2003;17:247e9. [345] Nykjaer A, Dragun D, Walther D, et al. An endocytic pathway essential for renal uptake and activation of the steroid 25-(OH) vitamin D3. Cell 1999;96:507e15. [346] Vezzoli G, Zerbi S, Baragetti I, et al. Nonacidotic proximal tubulopathy transmitted as autosomal dominant trait. Am J Kid Dis 1997;29:490e5. [347] Zelikovic I. Hypokalemic salt-losing tubulopathies: an evolving story. Nephrol Dial Transpl 2003;18:1696e700. [348] Knoers NVAM. Gitelman syndrome. Adv Chronic Kidney Dis 2006;13:148e54. [349] Seyberth HW. An improved terminology and classification of Bartter-like syndromes. Nat Clin Pract Nephrol 2008;4:560e7. [350] Gamba G. Electroneutral chloride coupled cotransporters. Curr Opin Nephrol Hypertens 2000;9:535e40. [351] Michelis MF, Drash AL, Linarelli LG, et al. Decreased bicarbonate threshold and renal magnesium wasting in a sibship with distal renal tubular acidosis. Evaluation of the pathophysiological role of parathyroid hormone. Metab Clin Exp 1972;21:905e20. [352] Praga M, Vara J, Gonzalez-Parra E, et al. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis. Kidney Int 1995;47:1419e25.

[353] Morita K, Furuse M, Fujimoto K, et al. Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands. Proc Natl Acad Sci USA 1999;96:511e6. [354] Anderson JM. Molecular structure of tight junctions and their role in epithelial transport. News Physiol Sci. 2001;16:126e30. [355] Yu Alan SL. Claudins and epithelial paracellular transport: the end of the beginning. Curr Opin Nephrol Hypertens 2003;12:503e9. [356] Weber S, Schneider L, Peters M, et al. Novel paracellin-1 mutations in 25 families with familial hypomagnesemia with hypercalciuria and nephrocalcinosis. J Am Soc Nephrol 2001;12:1872e81. [357] Muller D, Kausalya PJ, Claverie-Martin F, et al. A novel claudin16 mutation associated with childhood hypercalciuria abolishes binding to ZO-1 and results in lysosomal mistargeting. Am J Hum Genet 2003;73:1293e301. [358] Hou J, Paul DL, Goodenough DA. Paracellin-1 and the modulation of ion selectivity of tight junctions. J Cell Sci 2005;118:5109e18. [359] Hou J, Renigunta A, Konrad M, et al. Claudin-16 and claudin-19 interact and form a cation-selective tight junction complex. J Clin Invest 2008;118:619e28. [360] Hou J, Renigunta A, Gomes AS, et al. Claudin-16 and claudin19 interaction is required for their assembly into tight junctions and for renal reabsorption of magnesium. Proc Natl Acad Sci USA 2009;106:15350e5. [361] Brown EM, Hebert SC. Inherited diseases of the calciumsensing receptor: impact on parathyroid and renal function. In: Lifton RP, Somlo S, Giebisch GH, et al., editors. Genetic Diseases of the Kidney. Philadelphia: Saunders Elsevier; 2009. p. 263e78. [362] Devuyst O, Konrad M, Jeunemaitre X, et al. 38 tubular disorders of electrolyte regulation. In: Avner ED, Harmon WE, Niaudet P, et al., editors. Pediatric Nephrology. 6th edn. Berlin: SpringerVerlag; 2009. p. 929e77. [363] Pollak MR, Brown EM, Estep HL, et al. Autosomal dominant hypocalcaemia caused by a Ca (2þ)-sensing receptor gene mutation. Nat Genet 1994;8:303e7. [364] Egbuna OI, Brown EM. Hypercalcaemic and hypocalcaemic conditions due to calcium-sensing receptor mutations. Best Pract Res Clin Rheumatol 2008;22:129e48. [365] Hebert SC. Extracellular calcium-sensing receptor: implications for calcium and magnesium handling in the kidney. Kidney 1996;50:2129e39. [366] Pollak MR, Seldman CE, Brown EM. Three inherited disorders of calcium sensing. Medicine (Baltimore) 1996;75:115e23. [367] Friedman PA. Codependence of renal calcium and sodium transport. Annu Rev Physiol 1998;60:179e97. [368] Zelikovic I. Molecular pathophysiology of tubular transport disorders. Pediatr Nephrol 2001;16:919e35. [369] Nijenhuis T, Vallon V, Van der Kemp AW, et al. enhanced passive Ca2þ reabsorption and reduced Mg2þ channel abundance explains thiazide-induced hypocalciuria and hypomagnesemia. J Clin Invest 2005;115:1651e8. [370] Shalev H, Phillip M, Galil A, et al. Clinical presentation and outcome in primary familial hypomagnesaemia. Arch Dis Child 1998;78:127e30. [371] Cairo ER, Friedrich T, Swarts HG, et al. Impaired routing of wild type FXYD2 after oligomerisation with FXYD2-G41R might explain the dominant nature of renal hypomagnesemia. Biochim Biophys Acta 2008;1778:398e404. [372] Geven WB, Monnens LA, Willems JL, et al. Isolated autosomal recessive renal magnesium loss in two sisters. Clin Genet 1987;32:398e402.

PEDIATRIC BONE

REFERENCES

[373] Wilson FH, Hariri A, Farhi A, et al. A cluster of metabolic defects caused by mutation in a mitochondrial tRNA. Science 2004;306:1190e4. [374] Reilly RF, Ellison DH. Mammalian distal tubule: physiology, pathophysiology, and molecular anatomy. Physiol Rev 2000;80:277e313. [375] Hamm LL, Alpern RJ, Preisig PA. Cellular mechanisms of renal tubular acidification. In: Alpern RJ, Hebert SC, editors. Seldin and Giebisch’s The Kidney, Physiology and Pathophysiology. 4th edn. Philadelphia: Saunders Elsevier; 2008. p. 1539e85. [376] Soriano JR. Renal tubular acidosis: the clinical entity. J Am Soc Nephrol 2002;13:2160e70. [377] Ingulli E, Mistry K, Mak RHK. Acidebase homeostasis. In: Avner ED, Harmon WE, Niaudet P, et al., editors. Pediatric Nephrology. 6th edn. Berlin: Springer-Verlag; 2009. p. 205e30. [378] Romero MF, Hediger MA, Boulpaep EL, et al. Expression cloning and characterization of a renal electrogenic Naþ/ HCO3contransporter. Nature 1997;387:409e13. [379] Madsen KM, Nielsen S, Tisher CC. Anatomy of the kidney. In: Brenner BM, editor. The Kidney. 8th edn. Philadelphia: Saunders Elsevier; 2008. p. 25e90. [380] Schwartz GJ. Plasticity of intercalated cell polarity: effect of metabolic acidosis. Nephron 2001;87:304e13. [381] Wagner CA. Renal acid-base transport: old and new players. Nephron Physiol 2006;103:1e6. [382] Frische S, Kwon TH, Frokiaer J, et al. Regulated expression of pendrin in rat kidney in response to chronic NH4Cl or NaHCO3 loading. Am J Physiol Renal Physiol 2003;284:F584e93. [383] Wagner CA, Geiber JP. Acidebase transport in the collecting duct. J Nephron 2002;15:S112e27. [384] Bruce LJ, Cope DL, Jones GK, et al. Familial distal renal tubular acidosis is associated with mutations in the red cell anion exchanger (Band 3 AE1) gene. J Clin Invest 1997;100:1693e707. [385] Rungroj N, Devonald MA, Cuthbert AW, et al. A novel missense mutation in AE1 causing autosomal dominant distal renal tubular acidosis retains normal transport function but is mistargeted in polarized epithelial cells. J Biol Chem 2004;279:13833e8. [386] Fry AC, Karet FE. Inherited renal acidoses. Physiology 2007;22:202e11. [387] Karet FE, Finberg KE, Nelson RD, et al. Mutations in the gene encoding B1 subunit of Hþ-ATPase cause renal tubular acidosis with sensorineural deafness. Nat Genet 1999;21:84e90. [388] Smith AN, Skaug J, Choat KA, et al. Mutation in ATP6N1B, encoding a new kidney vacuolar proton pump 116-kD subunit, cause recessive distal renal tubular acidosis with preserved hearing. Nat Genet. 2000;26:71e5. [389] Stover EH, Borthwick KJ, Bavalia C, et al. Novel ATP6V1B1 and ATP6V0A4 mutations in autosomal recessive distal renal tubular acidosis with new evidence for hearing loss. J Med Genet 2002;39:796e803. [390] Igarashi T, Inatomi J, Sekine T, et al. Mutations in SLC4A4 cause permanent isolated proximal renal tubular acidosis with ocular abnormalities. Nat Genet 1999;23:264e6. [391] Horita S, Yamada H, Inatomi J, et al. Functional analysis of NBC1 mutants associated with proximal renal tubular acidosis and ocular abnormalities. J Am Soc Nephrol 2005;16:2270e8. [392] Sly WS, Hewett-Emmett D, Whyte MP, et al. 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 1983;80:2752e6. [393] Roth DE, Venta PJ, Tashian RE, et al. Molecular basis of human carbonic anhydrase H deficiency. Proc Natl Acad Sci USA 1992;89:1804e8.

769

[394] Geller DS, Rodriguez-Soriano J, Vallo A, et al. Mutations in the mineralocorticoid receptor gene cause autosomal dominant pseudohypoaldosteronism Type I. Nat Genet 1998;19:279e81. [395] Rossier BC. Cum grano salis: the epithelial sodium channel and the control of blood pressure. J Am Soc Nephrol 1997;8:980e92. [396] Chang SS, Grunder S, Hanukoglu A, et al. Mutations in subunits of the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, pseudohypoaldosteronism type 1. Nat Genet 1996;12:248e53. [397] Wilson FH, Disse-Nicodeme S, Choate KA, et al. Human hypertension caused by mutations in WNK kinases. Science 2001;293:1107e12. [398] Peng JB, Warnock DG. WNK4-mediated regulation of renal ion transport proteins. Am J Physiol Renal Physiol 2007;293:F961e73. [399] Alper SL. Familial renal tubular acidosis. J Nephrol 2010;23:S57e76. [400] Krapf R, Seldin DW, Alpern RJ. Clinical syndromes of metabolic acidosis. In: Alpern RJ, Hebert SC, editors. Seldin and Giebisch’s The Kidney, Physiology and Pathophysiology. 4th edn. Philadelphia: Saunders Elsevier; 2008. p. 1667e720. [401] Pereira PCB, Miranda DM, Oliveira EA, et al. Molecular pathophysiology of renal tubular acidosis. Curr Genomics 2009;10:51e9. [402] Rodriguez-Soriano J. New insights into the pathogenesis of renal tubular acidosis e from functional to molecular studies. Pediatr Nephrol 2000;14:1121e36. [403] Igarashi T, Sekine T, Inatomi J, et al. Unraveling the molecular pathogenesis of isolated proximal renal tubular acidosis. J Am Soc Nephrol 2002;13:2171e7. [404] Usui T, Hara M, Satoh H, et al. Molecular basis of ocular abnormalities associated with proximal renal tubular acidosis. J Clin Invest 2001;108:107e15. [405] Schultheis PJ, Clarke LL, Meneton P, et al. Renal and intestinal absorptive defects in mice lacking the NHE3 Naþ/Hþ exchanger. Nat Genet 1998;19:282e5. [406] Soleimani M, Burnham CE. Physiologic and molecular aspects of the Naþ:HCO3  cotransporter in health and disease processes. Kidney 2000;57:371e84. [407] Romero MF, Boron WF. Electrogenic Naþ/HCO3cotransporters: cloning and physiology. Annu Rev Physiol 1999;61:699e723. [408] Dinour D, Chang MH, Satoh J, et al. A novel missense mutation in the sodium bicarbonate cotransporter (NBCe1/SLC4A4) causes proximal tubular acidosis and glaucoma through ion transport defects. J Biol Chem 2004;279:52238e46. [409] Igarashi T, Inatomi J, Sekine T, et al. Novel nonsense mutation in the Naþ/HCO3  cotransporter gene (SLC4A4) in a patient with permanent isolated proximal renal tubular acidosis and bilateral glaucoma. J Am Soc Nephrol 2001;12:713e8. [410] Li HC, Szigligeti P, Worrell RT, et al. Missense mutations in Naþ:HCO3  cotransporter NBC1 show abnormal trafficking in polarized kidney cells: a basis of proximal renal tubular acidosis. Am J Physiol Renal Physiol 2005;289:F61e71. [411] Toye AM, Parker MD, Daly CM, et al. The human NBCe1-A mutant R881C, associated with proximal renal tubular acidosis, retains function but is mistargeted in polarized renal epithelia. Am J Physiol Cell Physiol 2006;291:C788e801. [412] Suzuki M, Vaisbich MH, Yamada H, et al. Functional anaylsis of a novel missense NBC1 mutation and of other mutations causing proximal renal tubular acidosis. Pflu¨gers Arch Eur J Physiol 2008;455:583e93. [413] Lemann Jr J, Adams ND, Wilz DR, et al. Acid and mineral balances and bone in familial proximal renal tubular acidosis. Kidney Int 2000;58:1267e77.

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[414] Laing CM, Toye AM, Capasso G, et al. Renal tubular acidosis: developments in our understanding of the molecular basis. Int J Biochem Cell Biol 2005;37:1151e61. [415] Dubose TDJ, Alpern RJ. Renal tubular acidosis. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The Metabolic and Molecular Bases of Inherited Disease. New York: McGraw-Hill; 1995. p. 3655e89. [416] Lemann JRJ, Bushinsky DA, Hamm LL. Bone buffering of acid and base in humans. Am J Physiol Renal Physiol 2003;285:F811e32. [417] Sutton RA, Wong NL, Dirks JH. Effects of metabolic acidosis and alkalosis on sodium and calcium transport in the dog kidney. Kidney Int 1979;15:520e33. [418] Nijenhuis T, Renkema KY, Hoenderop JG, et al. Acid-base status determines the renal expression of Ca2þ and Mg2þ transport proteins. J Am Soc Nephrol 2006;17:617e26. [419] Ariceta G, Vallo A, Rodriguez-Soriano J. Acidosis increases magnesiuria in children with distal renal tubular acidosis. Pediatr Nephrol 2004;19:1367e70. [420] Donckerwolcke RA, Biervliet JP van, Koorevaar G, et al. The syndrome of renal tubular acidosis with nerve deafness. Acta Paediatr Scand 1976;65:100e4. [421] Brown MT, Cunningham MJ, Ingelfinger JR, et al. Progressive sensorineural hearing loss in association with distal renal tubular acidosis. Arch Otolaryngol Head Neck Surg 1993;119:458e60. [422] Alper SL. Molecular physiology of SLC4 anion exchangers. Exp Physiol 2006;91:153e61. [423] Gallagher PG. Red cell membrane disorders. Am Soc Hematol Educ Program 2005:13e8. [424] Shayakul C, Alper SL. Inherited renal tubular acidosis. Curr Opin Nephrol Hypertens 2000;9:541e6. [425] Blake-Palmer KG, Karet FE. Cellular physiology of the renal Hþ ATPase. Curr Opin Nephrol Hypertens 2009:433e8. [426] Wagner CA, Finberg KE, Breton S, et al. Renal vacuolar-ATPase. Physiol Rev 2004;84:1263e314. [427] Jefferies KC, Cipriano DJ, Forgac M. Function, structure and regulation of the vacuolar (Hþ)-ATPases. Arch Biochem Biophys 2008;476:33e42. [428] Toei M, Saum R, Forgac M. Regulation and isoform function of the V-ATPase. Biochemistry 2010;49:4715e23.

[429] Scheinman SJ, Guay-Woodford LM, Thakker RV, et al. Genetic disorders of renal electrolyte transport. N Engl J Med 1999;340:1177e87. [430] Bonny O, Hummler E. Dysfunction of epithelial sodium transport: from human to mouse. Kidney Int 2000;57:1313e8. [431] Kerem E, Bistritzer T, Hanukoglu A, et al. Pulmonary epithelial sodium channel dysfunction and excess airway liquid in pseudohypoaldosteronism. N Engl J Med 1999;341:156e62. [432] Bonny O, Rossier BC. Disturbances of Na/K balance: pseudohypoaldosteronism revisited. J Am Soc Nephrol 2002;13:2399e414. [433] Kahle KT, Wilson FH, Lifton RP. The syndrome of hypertension and hyperkalemia (pseudohypoaldosteronism type II): WNK kinases regulate the balance between renal salt reabsorption and potassium secretion. Philadelphia: Saunders Elsevier; 2009. p. 313e29. [434] Proctor G, Linas S. Type 2 pseudohypoaldosteronism: new insights into renal potassium, sodium, and chloride handling. Am J Kidney Dis 2006;48:674e93. [435] Kahle KT, Ring AM, Lifton RP. Molecular physiology of the WNK kinases. Annu Rev Physiol 2008;70. 11.1-27. [436] Cai H, Cebotaru V, Wang YH, et al. WNK4 kinase regulates surface expression of the human sodium chloride cotransporter in mammalian cells. Kidney Int 2006;69:2162e70. [437] Rodriguez-Soriano J, Vallo A, Dominguez MJ. "Chloride-shunt" syndrome: an overlooked cause of renal hypercalciuria. Pediatr Nephrol 1989;3:113e21. [438] Achard JM, Warnock DG, Disse-Nicodeme S, et al. Familial hyperkalemic hypertension: phenotypic analysis in a larger family with the WNK1 deletion mutation. Am J Med 2003;114:495e8. [439] Mayan H, Vered I, Mouallem M, et al. Pseudohypoaldosteronism type II: marked sensitivity to thiazides, hypercalciuria, normomagnesemia, and low bone mineral density. J Clin Endocrinol Metab 2002;87:3248e54. [440] Lau K, Eby BK. Tubular mechanism for the spontaneous hypercalciuria in laboratory rat. J Clin Invest 1982;70:835e44. [441] Jiang Y, Ferguson WB, Peng JB. WNK4 enhances TRPV5mediated calcium transport: potential role in hypercalciuria of familial hyperkalemic hypertension caused by gene mutation of WNK4. Am J Physiol Renal Physiol 2007:F545e54.

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Hypophosphatasia Michael P. Whyte Center for Metabolic Bone Disease and Molecular Research, Shriners Hospital for Children, St Louis, Missouri; and Division of Bone and Mineral Diseases, Washington University School of Medicine at Barnes-Jewish Hospital, St Louis, Missouri, USA

INTRODUCTION Hypophosphatasia (HPP: OMIM #146300, #241500, #241510) is the rare, heritable form of rickets or osteomalacia that features low serum alkaline phosphatase (ALP) activity (hypophosphatasemia) [1]. Hypophosphatasemia in HPP is explained by a generalized reduction of activity of the “tissue-non-specific” isoenzyme of ALP (TNSALP) caused by loss-of-function mutation(s) within the TNSALP gene [2]. The tissue-specific ALP isoenzyme genes are intact. In HPP, extracellular accumulation of the TNSALP substrate inorganic pyrophosphate (PPi), an inhibitor of mineralization, explains the skeletal disease [3]. Characterization of this inbornerror-of-metabolism verified a role for ALP in bone formation [4]. This chapter begins with a description of ALP in humans, and then provides an overview of HPP.

BIOCHEMISTRY AND MOLECULAR BIOLOGY OF ALKALINE PHOSPHATASE Alkaline phosphatase (ALP) (orthophosphoricmonoester phosphohydrolase, alkaline optimum, EC 3.1.3.1) is found in all animals [5]. In humans, ALPs are encoded by four genes [1,6e8]. Three genes express tissue-specific ALP isoenzymes; i.e. “intestinal”, “placental”, and “germ-cell (placental-like)” ALP. The fourth gene encodes an ALP isoenzyme that is ubiquitous, but especially abundant in liver, bone, and kidney [6,7]. Accordingly, this “liver/bone/kidney” ALP can be designated “tissue-non-specific” ALP (TNSALP) [1e4,6e8]. Perhaps there is also a “fetal intestinal” ALP isoenzyme [1]. The distinctive physicochemical properties of the TNSALPs purified from liver, bone, and kidney are lost following digestion with

Pediatric Bone, Second Edition DOI: 10.1016/B978-0-12-382040-2.10028-0

glycosidases [9]. Thus, TNSALP is a family of “secondary” isoenzymes (isoforms) which differ only by post-translational modifications involving carbohydrates [10]. The TNSALP gene (OMIM 171760) is located near the end of the short arm of chromosome 1 (1p36.1-34) [11]. The genes for intestinal, placental, and germ-cell ALP, and perhaps fetal intestinal ALP (OMIM 171740, 171750, 171800, 171810), are near the tip of the long arm of chromosome 2 (2q34-37) [12]. The human gene mapping symbol for the TNSALP locus is ALPL [13] (“ALP-liver”), but the physiological role of ALP in the liver is not known. Therefore, “TNSALP” seems to be a better designation for the enzyme and its gene (TNSALP). Each ALP gene has been characterized [14e16]. TNSALP is more than 50 kb and has 12 exons. Eleven exons are translated to the 507 amino acid nascent enzyme [7,16]. The promoter region is within 610 nucleotides 5’ to the major transcription start site [17]. TATA and Sp1 sequences seem important for promoter function. Basal expression appears to reflect “housekeeping” promoter activity, whereas higher levels in various tissues may be mediated by a post-transcriptional mechanism [17]. The 5’ untranslated region differs for the bone and liver TNSALP isoforms [18]. The tissue-specific ALP genes are smaller than TNSALP, primarily due to shorter introns. Amino acid profiles deduced from cDNAs suggest 87% positional identity between placental and intestinal ALP, but 50e60% identity between these ALPs and TNSALP [6,7]. Nevertheless, the active site, encoded by six exons, reflects base sequences conserved in ALPs throughout nature [19]. In humans, TNSALP seems to be the ancestral gene from which the tissue-specific ALPs arose by gene duplication [6].

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ALPs are Zn2þ-metalloenzymes [5,6]. Catalytic activity depends on a multimeric configuration of identical subunits with molecular mass ranging from 40 to 75 kDa [7]. Each monomer has one active site, and contains two Zn2þ atoms that stabilize the tertiary structure [20]. TNSALP in symmetric dimeric form has a/b topology for each subunit, with a ten-stranded b-sheet at its center [21]. The cDNA sequence of TNSALP predicts five potential N-linked glycosylation sites [16], a modification necessary for catalytic activity [18]. O-glycosylation involves the bone, but not the liver, isoform of TNSALP [18]. The ALPs are generally considered homodimeric in the circulation [5,7]. In tissues, ALPs may function as plasma membrane attached homotetramers [22]. The ALPs have broad substrate specificity and pH optima that depend on the type and concentration of phosphocompound undergoing degradation [5]. Hydrolytic activity cleaves phosphoesters and PPi [23]. Catalysis involves phosphorylationedephosphorylation of a serine residue, where dissociation of covalently linked phosphate seems to be the rate-limiting step. Inorganic phosphate (Pi) is a potent competitive inhibitor of ALP [5,20], and may also stabilize the enzyme [24]. ALP activity requires Mg2þ as a co-factor [5]. Human ALP gene sequences indicate that the nascent polypeptides have a short signal sequence of 17 to 21 amino acid residues [6,7], and a hydrophobic domain at their carboxy-terminus [14e16]. However, ALPs become tethered to the surface of plasma membranes bound to the polar head group of a phosphatidylinositol-glycan moiety, and can be liberated by phosphatidylinositol-specific phospholipase [22,25]. The precise interaction with phosphatidylinositol may differ among the individual ALP isoenzymes [25]. Although lipid-free ALP is the moiety found in plasma, how ALPs are released from cell surfaces is poorly understood [26]. Mabry syndrome (OMIM #239300), which features hyperphosphatasemia and mental retardation, involves a defect in the gene that encodes an enzyme needed to bind ALPs to plasma membranes (see below) [27]. Clearance of circulating ALPs is presumed to occur, as for many other glycoproteins, in the liver [28]. In healthy adults, most of the ALP in serum represents approximately equal amounts of TNSALP from bone and liver (hepatobiliary) tissue [29]. However, in infants and children, and especially during the growth spurt of adolescence, blood is enriched with the bone isoform of TNSALP [5]. Some people (with B and O blood types and positive secretory status) increase their serum intestinal ALP levels after a fatty meal [5,30], but this value usually represents only a few percent of the total ALP [31]. Normally, placental ALP circulates only in women during the latter stages of pregnancy, when

expression of ALP in the placenta is controlled by the fetal genome [5]. With various malignancies, however, placental ALP or placental-like ALP may appear in serum [6,7].

PHYSIOLOGY OF SKELETAL FORMATION AND ALKALINE PHOSPHATASE FUNCTION Skeletal development is complex and involves bone growth, modeling (shaping), and remodeling (formation and resorption, or “turnover”, of osseous tissue) (see Chapter 4). Growth of bones in the extremities and at many axial sites occurs by endochondral bone formation until after puberty. In healthy physes (growth plates), there is orderly proliferation, hypertrophy, and then degeneration of chondrocytes, with subsequent mineralization of the resulting cartilage matrix (primary spongiosa). Elsewhere, osteoblasts synthesize bone matrix (osteoid) which then mineralizes. Hypertrophic chondrocytes and osteoblasts are rich in the bone isoform of TNSALP. Electron microscopy in the 1960s revealed that the earliest site of mineral deposition during endochondral bone formation occurs within extracellular, membranebound structures called matrix vesicles (MVs) [32]. MVs were presumed to be buds of the chondrocyte plasma membrane, and were subsequently found in cortical and membranous bone and in fracture callus [32]. MVs are rich in enzymes (including TNSALP, pyrophosphatase, and ATPase), and may contain polysaccharides, phospholipids, and glycolipids [32]. Hydroxyapatite (HA) crystals grow within and eventually rupture the membrane of MVs. Then, extravesicular crystal enlargement must continue for HA deposition into osteoid [32]. Accordingly, skeletal mineralization can be described as primary or phase 1 occurring in MVs, and secondary or phase 2 featuring HA crystal enlargement in skeletal matrix [33]. Generalized impairment of skeletal matrix mineralization in infants or children compromises endochondral bone formation and leads to rickets [34]. In adults, whose growth plates are fused, there is osteomalacia. The principal feature that distinguishes rickets from osteomalacia is the disruption of the growth plates and impaired calcification which can lead to short stature and skeletal deformity. Nearly all forms of rickets or osteomalacia feature reduced extracellular levels of ionized calcium (Ca2þ) and/or Pi [34]. HPP is an interesting and instructive exception (see below). In 1923, Robert Robison, PhD, discovered that calcifying cartilage and bone from young rats and rabbits was rich in phosphatase activity and suggested that the associated enzyme conditioned skeletal mineralization

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by hydrolyzing some unidentified phosphate ester to increase the local concentration of Pi [35]. One year later, Robison and Soames found that the catalytic reaction had a distinctly alkaline pH optimum [36]. However, Robison never used the term “alkaline phosphatase”. Instead, he referred to the enzyme as “bone phosphatase” [37]. Ironically, nearly a century after its discovery, the methods used to assay ALP activity still fail to reflect the enzyme’s physiological function [3e5,38]. In both clinical and research laboratories, ALP activity is measured with high concentrations (mM) of artificial, colormetric substrates (e.g. p-nitrophenylphosphate) at non-physiological, alkaline pH (e.g. 9.2e10.5) [5]. The assays for serum ALP were developed primarily to detect and follow the clinical course of skeletal and hepatobiliary disorders [5]. However, the pH optima of ALPs are less alkaline for natural substrates at lower concentrations, although the hydrolytic rates are reduced [3,5,38]. Soon after its discovery, ALP was found to be abundant also in tissues that do not mineralize (e.g. intestine, placenta) [5]. Therefore, a role for ALP in calcification was challenged. The physiological functions postulated for ALP came to include hydrolysis of phosphate esters to supply the non-phosphate moiety, synthesis of phosphate esters with ALP functioning as a transferase, and regulation of a variety of cellular processes in which ALP acted as a phosphoprotein phosphatase [5]. In fact, a considerable variety of hypotheses was proposed for how TNSALP might function in skeletal mineralization [3,4]. It was suggested that the Pi donor could be nucleoside phosphate liberated by degenerating cells [39]. An important alternative hypothesis, however, was consistent with Robison’s recognition that a second factor regulated skeletal mineralization [37]. It was postulated that ALP hydrolyzes an inhibitor of mineralization [3,5]. The subsequent discoveries that PPi impairs the growth of HA crystals [40], ALP can function as a PPi-ase [41,42], and that endogenous levels of PPi are increased in HPP [43] offered a plausible candidate molecule for this inhibitor and a biochemical explanation for the principal clinical feature of HPP [44] (see below). Nevertheless, it has also been suggested that ALP might act in mineralization as a plasma membrane transport protein for Pi, an extracellular Ca2þ-binder that promotes calcium phosphate formation and orients HA crystal deposition into osteoid [45], a Ca2þ/Mg2þATPase, or a phosphoprotein phosphatase that conditions skeletal matrix for ossification [46].

HYPOPHOSPHATASIA In my opinion, it was the characterization and early clinical investigation of HPP that provided proof for

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Robison’s work, and the greatest insight into the physiological role of ALP in humans [3,4,38]. With identification beginning in 1988 of loss-of-function mutation(s) in the TNSALP gene causing this disorder (see below), unequivocal evidence was obtained that Robison was correct and that it was TNSALP that acted critically during skeletal mineralization. Nevertheless, the seemingly undisturbed function in HPP of other organs/ tissues that are normally ALP-rich posed challenging questions for whether TNSALP has biological significance elsewhere in the body [3,7,8,38].

History In 1948, John C. Rathbun, M.D., a Canadian pediatrician, coined the term “hypophosphatasia” when he published the case report of an infant boy who acquired and then died from severe rickets, weight loss, and seizures, yet whose ALP activity in serum, bone, and other tissues obtained at autopsy was paradoxically subnormal [47]. This was the discovery of HPP. Several historical reviews refer to patient descriptions that probably represent earlier encounters with this disorder [48,49]. In 1953, premature loss of deciduous teeth was noted to be another major clinical feature [50]. The discoveries of elevated endogenous levels of three phosphocompounds in HPP clarified the metabolic basis for this inborn-error-of-metabolism and the physiological role of TNSALP [2e4]. In 1955, increased concentrations of phosphoethanolamine (PEA) in urine [51,52] provided a useful biochemical marker for HPP. In 1965 and 1971, high levels of PPI were noted in the urine [53] and in the blood [43], respectively, of patients with HPP and suggested a mechanism for the associated defective mineralization of hard tissues. In 1985, elevated plasma levels of pyridoxal 5’-phosphate (PLP), the major circulating form of vitamin B6, were discovered in HPP and indicated that TNSALP [54] functions as an ectoenzyme (see below). In 1988, the structure of the TNSALP gene was characterized [16] and loss-of-function mutation of the candidate TNSALP gene was documented that same year in HPP [55]. About 350 HPP patients have been described in the medical literature, and the features and range of the severity of the clinical disease are now well documented (see below).

Clinical Features HPP occurs worldwide. It is especially prevalent for inbred Mennonite families in Manitoba, Canada, where z1 in 2500 newborns manifests severe disease, and z1 in 25 individuals is a carrier [56]. The incidence of severe forms of HPP in Toronto, Canada, was estimated in 1957

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to be 1 per 100 000 live births [49]. In 2011, the prevalences of severe and moderate HPP were estimated in Europe, based upon TNSALP mutation data, to be 1/ 300 000 and 1/6370, respectively [57]. Inexplicably, HPP seems to be particularly rare in individuals of black ancestry [58]. Despite the presence in healthy people of relatively high levels of TNSALP not only in bone, but also in liver, kidney, and adrenal tissue (and at least some TNSALP throughout the body), HPP directly disrupts just hard tissues (the skeleton and dentition). However, its expression ranges remarkably with the clinical consequences spanning death in utero to problems with dentition yet no skeletal symptoms in adult life [49,59e62]. Although HPP generally “breeds true” within sibships, significantly different severity can occur even in this setting [60,61,63,64]. Additionally, some individuals who demonstrate characteristic biochemical abnormalities of HPP and harbor a defective TNSALP allele may never become symptomatic and can be considered “carriers” [60,61]. The nosology of HPP for prognostication, recurrence risk estimates, etc. remains a clinical one. Several classification schemes have been proposed that attempt to deal with the disorder’s remarkably variable expression [49,59]. Seven clinical forms, detailed below, represent a useful description. The age at which bone lesions become apparent distinguishes the perinatal, infantile, childhood, and adult forms of HPP [49,62]. Patients who experience only dental manifestations have odontoHPP. An especially rare HPP variant designated pseudoHPP resembles infantile HPP except that serum ALP activity is not subnormal in the clinical laboratory (see below). Importantly, a “benign prenatal” form of HPP is now recognized that features skeletal deformity noted in utero or at birth, but soon after is associated with significant, spontaneous improvement [65e68]. The prognosis for the various forms of HPP typically reflects the severity of the skeletal disease which, in turn, generally corresponds with the age at presentation. Usually, the earlier a patient becomes symptomatic from HPP, the more severe the disorder [3,38,49]. Although the above nosology is useful, there is considerable variability within each clinical form of HPP, and no clear-cut separations between them. Perhaps it is HPP that spans the greatest range of all disease severity. Perinatal Hypophosphatasia This is the most severe form of HPP, and is almost always lethal. It manifests in utero and can cause stillbirth. The pregnancy may be complicated by polyhydramnios. At delivery, there is caput membraneceum and limbs are shortened and deformed from profound skeletal hypomineralization. In some cases, the bones

at birth appear to be completely unmineralized. Unusual, osteochondral (“Bowdler”) spurs may protrude through the skin from the mid-portion of the forearms and legs [69,70]. Some affected neonates live a few days, but suffer increasing respiratory compromise from rachitic defects in the chest and hypoplastic lungs [71]. Clinical findings also often include a high-pitched cry, irritability, periodic apnea with cyanosis and bradycardia, unexplained fever, myelophthisic anemia (perhaps from marrow space crowding by excess osteoid), intracranial hemorrhage, and seizures [59,62.]. Very rarely is survival possible and prolonged [72]. Radiographs of the skeleton show pathognomonic findings [69,70]. Perinatal HPP can be distinguished from even the most severe cases of osteogenesis imperfecta and other forms of congenital dwarfism. Nevertheless, the features are diverse, with considerable patient-to-patient variability [70]. If some skeletal mineralization is present, severe rachitic changes can be apparent. The findings also include irregular extensions of radiolucency into metaphyses together with poorly ossified epiphyses. Individual membranous bones of the cranium may show calcification only centrally, giving the illusion that the cranial sutures are widely separated (Fig. 28.1). However, these sutures can be functionally “closed” [69]. The teeth look poorly formed [70]. Fractures are often present. Other unusual features of perinatal HPP can include parts of (or entire) vertebrae that appear missing, and bony spurs that protrude laterally from the midshaft of the ulnas and fibulas [73]. Infantile Hypophosphatasia This form of HPP presents postnatally, but before 6 months of age [49]. Development may seem normal until the onset of poor feeding, inadequate weight gain, failure-to-thrive, hypotonia, and clinical signs of rickets. The cranial sutures feel wide, but this is explained by the ossification defect in the skull that can instead cause “functional” craniosynostosis. Exceptional patients present with vitamin B6-responsive epilepsy before skeletal disease is clinically apparent [74]. There may be bulging of the anterior fontanel, raised intracranial pressure with papilledema, proptosis, mild hypertelorism, and brachycephaly. Blue sclerae have been reported [75]. A flail chest can occur from rachitic deformity of the thorax and rib fractures that predispose to pneumonia. Hypercalcemia and hypercalciuria are common, and can cause recurrent vomiting and lead to nephrocalcinosis with renal compromise [49,76,77]. Weakness and delayed motor milestones are important complications. True (bony) premature fusion of the cranial sutures may occur if the patient survives infancy [69,78].

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(A)

(B)

FIGURE 28.2 FIGURE 28.1

Perinatal hypophosphatasia. (A) Lateral radiograph of the skull of this newborn shows extreme hypomineralization. (B) Marked rachitic changes are seen in the lower extremities.

The radiographic features of infantile HPP are also pathognomonic and resemble those of the perinatal form, but are somewhat less severe (Fig. 28.2) [69]. Sometimes there may be what appears to be abrupt transition from normal-appearing diaphyses to poorly calcified metaphyses. This finding is interesting because it parallels a seemingly acquired clinical presentation, and suggests a sudden pathological change [49]. Sequential radiographic studies may

Infantile hypophosphatasia.

then disclose not only persistence of defective skeletal mineralization (rickets), but gradual and generalized demineralization of osseous tissue as well [77]. Consequently, there are fractures and progressive deformity. Skeletal scintigraphy in infantile HPP can suggest functional closure of cranial sutures, because these structures show decreased radioisotope uptake although they appear “widened” on conventional radiographs [79]. Functional craniosynostosis can occur despite widely “open” fontanels that are an illusion from hypomineralized areas of calvarium. Vitamin B6-responsive seizures is a grave prognostic sign [74].

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Childhood Hypophosphatasia This form of HPP is also highly variable in severity [49,71,80], but diagnosed after 6 months of age. Premature loss of deciduous teeth (i.e. earlier than age 5 years) occurs without tooth root resorption and because of aplasia, hypoplasia, or dysplasia of dental cementum [81,82]. Cementum may also be destroyed by periodontal infection [83]. Teeth “slide” out from their sockets painlessly and without bleeding. The lower and then upper incisors are typically lost first, but occasionally the entire dentition is exfoliated. Dental radiography may show enlarged pulp chambers and root canals (“shell teeth”). Alveolar bone attrition, especially in the anterior mandible, can result from lack of mechanical stimulation because the defective cementum prevents the periodontal ligament from properly connecting the teeth to the jaw [84]. However, the prognosis for the permanent dentition seems better [85], but poorly-characterized dental manifestations appear to lead often to wearing dentures by early adult life. In childhood HPP, rickets often causes short stature and is associated with delayed walking [49,76]. Rachitic deformities include beading of the costochondral junctions, either bowed legs or knock-knees, enlargement of the wrists, knees, and ankles from flared metaphyses, and occasionally a brachycephalic skull [78]. Patients may complain of skeletal pain and stiffness, as well as isolated episodes of joint discomfort and swelling. Rarely, a painful syndrome occurs that mimics chronic recurrent multifocal osteomyelitis [86,87]. Typically, patients have muscle weakness (especially the thighs) consistent with a non-progressive myopathy that is associated with a waddling gait [49,76,88]. However, children with HPP, unlike some with the infantile form, are not troubled by vitamin B6-responsive seizures. Radiographs of the major long bones usually reveal characteristic focal bony defects that project from the growth plates into the metaphyses. These are often described as “tongues” of radiolucency. This feature, if present, distinguishes HPP from other forms of rickets and metaphyseal dysplasias [3,4,69]. There can also be physeal widening, irregularity of the provisional zone of calcification, and metaphyseal flaring with areas of radiolucency adjacent to areas of osteosclerosis (Fig. 28.3). Secondary centers of ossification (epiphyses) may be well preserved. Premature bony fusion of cranial sutures may cause raised intracranial pressure, proptosis, and cerebral damage. The skull can then have a “beaten-copper” appearance (Fig. 28.4). Adult Hypophosphatasia This form of HPP usually presents during middle age [60,61]. Not infrequently, however, patients mention

FIGURE 28.3 Childhood hypophosphatasia. Anterioposterior radiograph of the right knee of this 6-year-old boy shows a characteristic “tongue” of radiolucency (arrow) projecting from the physis into the metaphysis where there is paradoxical osteosclerosis. The head of the fibula is particularly involved.

that they had a history of rickets or premature loss of deciduous teeth. Following good health in early adult life, osteomalacia manifests with pain in the feet caused by recurrent, poorly-healing, metatarsal stress fractures, and then discomfort in the thighs or hips due to subtrochanteric femoral pseudofractures [89e92]. Early loss or extraction of the adult dentition is not uncommon [60,61,93]. Calcium pyrophosphate dihydrate (CPPD) deposition troubles some patients causing PPi arthropathy [61], including attacks of pseudogout. These rheumatologic complications reflect increased endogenous levels of PPi (see below) [61,94]. There may also be predisposition to primary hyperparathyroidism (personal observation). Assay of serum ALP activity often reveals symptomatic or asymptomatic family members [60,61]. In some kindreds featuring hypophosphatasemia, periarticular

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FIGURE 28.4 Childhood hypophosphatasia. Lateral radiograph of the skull of this 5-year-old girl shows pansuture closure causing a “beaten copper” appearance. A craniectomy defect is present (arrow).

calcium phosphate deposition presents as “calcific periarthritis” and with ossification of ligaments (syndesmophytes) resembling spinal hyperostosis (Forrestier disease) [95,96]. It is uncertain if this condition represents a form of HPP. In adult HPP, radiographs often show pseudofractures (Looser zones) [89,90], a hallmark of osteomalacia (Fig. 28.5). Inexplicably, these defects typically occur in the femurs at the lateral cortex proximally, rather than medially in the femoral neck as in other types of osteomalacia [89]. In this regard, the femoral pseudofractures of adult HPP resemble the subtrochanteric femoral “stress” fractures sometimes reported with prolonged bisphosphonate therapy [90]. In HPP, radiographs may also reveal osteopenia, chondrocalcinosis, features of PPi arthropathy, and calcific periarthritis [61,95,96]. Odontohypophosphatasia This most mild form of HPP is diagnosed when the only clinical abnormality is the dental disease of HPP. There is no radiographic and/or bone biopsy evidence of HPP skeletal disease. OdontoHPP may explain some cases of “early-onset” periodontitis, although hereditary leukocyte abnormalities and other disorders usually account for this condition [97]. Pseudohypophosphatasia This form of HPP is particularly interesting but especially rare, having been documented convincingly in two infants [98,99]. Here, the clinical, radiographic,

FIGURE 28.5 Adult hypophosphatasia. Anteroposterior radiograph of the proximal right femur of this 51-year-old woman shows a subtrochanteric “stress” fracture in the lateral diaphyseal cortex (arrow). Actually, this is a pseudofracture (Looser zone, milkman fracture) characteristic of the osteomalacia of HPP.

and biochemical findings are those of infantile HPP, except serum ALP activity is consistently normal or increased in the clinical laboratory. The enzymatic defect seems to involve a mutant TNSALP that has diminished catalytic activity endogenously, but retains or has enhanced catalytic activity under the non-physiological conditions of routine ALP assay procedures. Consequently, PEA, PPi, and PLP accumulate extracellularly in these patients (see below) [100,101]. Some reports of pseudoHPP are unconvincing [102e104], and probably describe patients with HPP who had transient normalization of serum ALP activity during fracture, illness, etc., or, more likely, represented misinterpretation of reference ranges for serum ALP activity and/or overemphasis on the significance of a slightly elevated urine PEA level (see below).

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TABLE 28.1 Causes of Hypophosphatasemia

Benign Prenatal Hypophosphatasia Beginning in the 1990s, reports emerged which emphasized HPP patients who manifested bowing deformity in utero suggestive of perinatal HPP, yet who had non-lethal clinical courses with spontaneous improvement after birth [65,66]. This presentation for autosomal dominant HPP became referred to as “bent but not broken” [66], or benign prenatal HPP [68]. In 2008, it became apparent that autosomal recessive HPP could also present this way [67]. Recently, a detailed assessment of experience with 17 such patients, together with a comprehensive review of the HPP literature, revealed that skeletal deformity of these patients can improve later during the pregnancy. Patient outcome, when skeletal deformity is first detected in utero by ultrasound, cannot be predicted with certainty [68]. Both autosomal dominant and autosomal recessive inheritance can cause this not uncommon clinical phenotype of HPP [68].

Laboratory Findings Biochemical ALP ACTIVITY

HPP can be diagnosed with confidence when the clinical history, physical findings, and radiographic changes are consistent with this diagnosis and occur together with serum ALP activity that is clearly and consistently subnormal for the patient’s age. In general, the more severe the manifestations of HPP the lower the serum ALP activity compared to reference values appropriate for age [2]. Even patients with odontoHPP are hypophosphatasemic. In the perinatal and infantile forms of HPP, low serum ALP activity is detectable in serum from umbilical cord blood [76,105]. Notably, in types of rickets or osteomalacia other than HPP, serum ALP activity is typically increased [34]. Accordingly, the hypophosphatasemia of HPP is especially striking. Nevertheless, several diagnostic pitfalls should be avoided (Table 28.1). Blood for ALP assay must be collected properly [106]. Chelation of Mg2þ or Zn2þ by ethylenediamine tetra-acetic acid (EDTA) will destroy ALP activity [5]. Levels of serum ALP activity must be interpreted knowing that reference ranges differ significantly depending on age and gender [5]. Healthy infants and children have considerably higher ALP levels compared to adults (due to an abundance of the bone isoform of TNSALP). Also, serum ALP activity is especially high during the growth spurt of adolescence, which occurs earlier in girls than in boys [5]. Although the problem is improving, reference ranges cited by some clinical laboratories still report values for adults exclusively. Consequently, some infants or children with HPP are mistakenly considered

• Hypophosphatasia

• Clofibrate therapy

• Familial benign?

• Starvation

• Pernicious or profound anemia

• Zn2þ or Mg2þ deficiency

• Hypothyroidism • Vitamin C deficiency • Osteogenesis imperfecta, type II • Wilson’s disease • Vitamin D intoxication • Inappropriate reference range • Multiple myeloma

• Cushing’s syndrome • Milkealkali syndrome • Celiac disease • Massive transfusion • Cleidocranial dysplasia • Cardiac bypass surgery • Improperly collected blood (e.g., EDTA, oxalate) • Radioactive heavy metals

to have normal ALP activity, or perhaps pseudoHPP, because the higher pediatric reference ranges are not provided. Also, hypophosphatasemia may occur in hypothyroidism, starvation/severe malnutrition, scurvy, profound anemia, multiple myeloma, celiac disease, Wilson disease, hypomagnesemia, or Zn2þ deficiency, and with exposure to certain drugs (glucocorticoids, chemotherapy, clofibrate, vitamin D toxicity, or milkealkali syndrome), as well as with massive transfusion of blood or plasma, or radioactive heavy metal poisoning [106,107]. However, these clinical situations should be apparent. Rarely, newborns with severe osteogenesis imperfecta can have low serum ALP activity [108], as do some patients with RUNX2 deactivation causing cleidocranial dysplasia [109,110]. To assess such situations, assay of plasma PLP (“vitamin B6”) can be important [111]. Elevated PLP levels are expected only for HPP where both bone and liver TNSALP are reduced. Finally, a few case reports of HPP describe transient increases in serum ALP activity (probably the bone isoform of TNSALP) after orthopedic surgery or fracture [60]. In theory, at least, conditions that increase circulating levels of any type of ALP (e.g. pregnancy, liver disease) could mask the biochemical diagnosis of HPP [76,112]. Accordingly, if a puzzling patient is encountered, documentation that serum ALP activity is low on more than one occasion during clinical stability seems advisable. Quantitation of the levels of serum ALP isoenzymes, or the TNSALP isoforms, may also be helpful in exceptional circumstances, e.g. pregnancy, certain malignancies. Now, however, mutational analysis of the TNSALP gene is available from research and fee-for-service laboratories. In our experience, all patients with HPP have carried one or two defective TNSALP alleles. The diagnosis of

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HPP is straightforward without TNSALP analysis, but this procedure can be important for other reasons, such as understanding recurrence risks, and perhaps prenatal diagnosis [68]. MINERALS

The block of mineral entry into the HPP skeleton caused by extracellular PPi accumulation leads to unique disturbances of calcium and Pi homeostasis [2,3]. In contrast to most types of rickets or osteomalacia [34], serum calcium or Pi levels are not low in HPP. In fact, hypercalcemia occurs frequently in the infantile form of the disease [48,59,62]. In childhood HPP, patients can have hypercalciuria, but only rarely hypercalcemia. Serum levels of the bioactive forms of vitamin D (25-hydroxyvitamin D and 1,25-dihydroxyvitamin D) and parathyroid hormone (PTH) are typically unremarkable [113,114]. However, serum PTH levels are often low in HPP patients with hypercalcemia, and sometimes low when there is only hypercalciuria. The low circulating levels of PTH have been considered from an abnormality in the Ca2þ-PTH feedback system [115], but seem to be explained by the disruption in mineral homeostasis. Years ago, several HPP patients reportedly had elevated serum PTH levels, but renal compromise from hypercalcemia with retention of immunoreactive PTH fragments may have been the explanation in the severe cases. Patients with the childhood and adult forms of HPP have serum Pi levels that are above the mean value for age-matched controls, and approximately 50% of these subjects are distinctly hyperphosphatemic. Enhanced renal reclamation of Pi (increased tubular maximum for Pi/glomerular filtration rate; i.e. TmP/GFR) explains this finding [92,116]. The observation is only sometimes explained by suppressed circulating levels of PTH. Instead, it is possible that TNSALP plays a direct role in renal excretion of Pi. Of interest, patients with generalized arterial calcification of infancy, who have low extracellular levels of PPi, can become hypophosphatemic [117]. Especially rare patients have been reported with HPP who are hypophosphatemic from renal Pi wasting [118,119]. ROUTINE STUDIES

Other standard laboratory tests, including serum parameters of liver or muscle function (e.g. bilirubin, aspartate aminotransferase, lactate dehydrogenase, creatine kinase, aldolase), are typically unremarkable in HPP. Serum acid phosphatase activity is generally normal [120], but tartrate-resistant acid phosphatase from osteoclasts was elevated for more than a decade in one affected woman [121]. Bone turnover markers have not been detailed in published reports. Increased levels of proline in blood and urine have been

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described in a few patients, but the significance is not known [122]. PHOSPHOETHANOLAMINE

Elevated levels of PEA in blood or urine, typically assessed by “inborn error laboratories” providing quantitative amino acid chromatography, support a diagnosis of HPP [123], but are not pathognomonic. Phosphoethanolaminuria can occur in other disorders, including several metabolic bone diseases [124]. Ideally, a 24hour urine collection is assayed and the PEA level is “normalized” to creatinine content. Importantly, PEA excretion in urine is conditioned by patient age, follows a circadian rhythm, depends on the diet, and has been reported to be normal in several mildly affected individuals with HPP [59,125]. The following age-adjusted reference ranges, as micromoles of PEA per gram of urine creatinine, have been published (less than 15 years, 83 to 222; 15 to 30 years, 42 to 146; 31 to 41 years, 38 to 155; and over 45 years, 48 to 93) [124]. PYRIDOXAL 5’-PHOSPHATE

Compared to serum or urine levels of PEA, an increased plasma level of PLP seems to be a more sensitive and specific TNSALP substrate marker for diagnosing HPP [54,100,126]. Commercial assays are available, typically ordered as “vitamin B6”. Generally, the more severely affected the HPP patient is, the greater is the elevation in the plasma PLP level [2]. Even patients with odontoHPP manifest this biochemical finding [54]. Nevertheless, overlap of the levels does occur from one clinical form of HPP to the next. To exclude false positive values, vitamin supplements must not be taken for one week before testing [126]. Conversely, assay of plasma PLP levels after oral challenge with pyridoxine hydrochloride for several days distinguishes HPP patients especially well, and has proven helpful for identifying Canadian Mennonite carriers of severe HPP [127]. INORGANIC PYROPHOSPHATE

Assay of PPi is a research technique. Urine levels of PPi are increased in most HPP patients [111], but occasionally values are unremarkable in mildly affected individuals [44]. Quantitation of urine PPi has been reported to be a sensitive means for HPP carrier detection [128].

Radiological Findings Radiographic study of the skeleton is diagnostic in perinatal and infantile HPP (see Figures 28.1 and 28.2), and in the childhood form when there is significant skeletal disease (see Figures 28.3 and 28.4). The findings in adult HPP are not diagnostic. Bone scanning retains its utility for revealing fractures, and may help to detect

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craniosynostosis [79]. Magnetic resonance imaging is necessary for identifying the unusual occurrence of painful bone marrow edema in HPP that can resemble chronic recurrent multifocal osteomyelitis [86] or malignancy [87]. Dual energy x-ray absorptiometry (DXA) of HPP [129] may be difficult to interpret because of the patient’s short stature, deformity, and the heterogeneous skeletal changes of bone mineralization [130].

Histopathological Findings Histological abnormalities are observed primarily in the hard tissues. In severe cases of HPP, hypoplastic lungs have been reported [71], and extramedullary hematopoiesis is occasionally noted in the liver [33,80]. Biopsy of muscle is usually unremarkable. Skeleton In patients with HPP, except those with odontoHPP [60], non-decalcified sections of bone following in vivo tetracycline labeling show evidence of defective skeletal mineralization [33,34,80]. Impaired skeletal mineralization is confirmed when fluorescence microscopy fails to show characteristic fluorescent bands on bone surfaces where calcification should be occurring at “mineralization fronts”. Unmineralized skeletal matrix (osteoid) accumulates because it does not calcify properly. However, features of secondary hyperparathyroidism, such as peritrabecular fibrosis, which occur as a consequence of hypocalcemia in most other types of rickets or osteomalacia, are typically absent. In the physes (growth plates) of HPP patients, rachitic changes [33,34,80] can include disruption of the normal columnar arrangement of chondrocytes, widening of the zone of provisional calcification, and failure of areas near degenerating cartilage cells to calcify. However, the sources of the bone isoform of TNSALP (chondrocytes and osteoblasts, as well as their MVs) are present, although they have reduced TNSALP activity [33,34]. Cranial “sutures” that appear widened on radiographs are not fibrous tissue, but are largely an illusion due to hypomineralization of the cranial bones [49]. Woven bone, a finding that can reflect either bone repair or defective skeletal formation, may be observed [80]. The severity of the mineralization defect in HPP generally reflects the clinical outcome [80]. In lethal HPP, even the bony structures of the middle ear can be poorly ossified [131]. Some questionable cases of pseudoHPP lack this critical information from tetracycline administration [132]. Unless histochemical or biochemical studies of ALP activity are performed, however, the changes in the skeleton of HPP at the level of the microscope cannot be distinguished from other forms of rickets or

osteomalacia [80]. The numbers and morphology of osteoblasts and osteoclasts, as well as the appearance of unmineralized osteoid, vary from patient to patient. ALP activity in bone tissue does correlate inversely with the degree of osteoid accumulation [80]. Electron microscopy of perinatal HPP bone obtained at autopsy has revealed normal distribution of proteoglycan granules, collagen fibers, and MVs [33,80]. The MVs are deficient in ALP activity, yet contain HA crystals [34]. Accordingly, the initial stage (phase 1) of skeletal mineralization seems intact. However, in the osteoid of HPP bone, only isolated or tiny groups of HA crystals (calcospherites), frequently not associated with MVs, have been observed [33,34,70]. This finding indicates that the extravesicular propagation of the HA crystal growth (phase 2 of skeletal mineralization) is compromised in HPP (see below). Dentition Premature loss of deciduous teeth occurs in several disorders (including toxicities, metabolic errors, and malignancies) [83]. In HPP, this complication is caused by lack of cementum covering tooth roots, despite the presence of cells that look like cementoblasts [82,84,133]. The cementum may be afibrillar [134]. Desiccated teeth exfoliated years earlier may still be useful for microscopic examination [135]. The magnitude of this defect varies from tooth to tooth, but generally reflects the severity of the skeletal disease [136]. Incisors are most vulnerable. Also in HPP, big pulp chambers suggest retarded dentinogenesis. Dentin tubules may be enlarged although reduced in number. The excessive width of predentin, increased amounts of interglobular dentin, and impaired calcification of cementum seem analogous to the osteoidosis observed within bone. Conflicting reports discuss whether enamel is directly affected [83,133]. The histopathological changes of HPP found in the permanent teeth seem similar, but are more mild, compared to the deciduous teeth [85,134].

Biochemical and Genetic Defect TNSALP Deficiency Early on, necropsy studies of perinatal and infantile HPP revealed the nature of the enzymatic defect, and thereby alluded to its etiology. Profound deficiency of ALP activity was documented in liver, bone, and kidney of these patients, yet ALP activity was not diminished in intestine or in placenta (fetal trophoblast) [137,138]. These observations matched results emerging from amino acid sequence analysis of ALPs purified from healthy human tissues [3,7], and indicated that there is

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in HPP selective deficiency of the catalytic activity of all of the secondary isoforms within the TNSALP isoenzyme family. Investigation of the biochemical hallmark of HPP, hypophosphatasemia, supported the autopsy studies. There was deficient activity of both the bone and the liver isoforms of TNSALP in serum [29]. Additionally, there did not seem to be enhanced clearance of circulating TNSALP [60,139]. The bone isoform of TNSALP from the plasma of patients with Paget’s bone disease, and the ALP extracted from healthy placentas, had unremarkable circulating half-lives when given intravenously to infants with life-threatening HPP during early attempts at enzymereplacement therapy (see below) [77]. Furthermore, coincubation experiments with mixtures of serum, as well as cell co-culture and heterokaryon studies using dermal fibroblasts from severely affected HPP patients, excluded a pathogenetic inhibitor or absence of an activator of TNSALP in HPP [49,60,94,140]. Instead, the hypophosphatasemia of HPP appeared to result from failure of especially liver and bone to contribute adequate TNSALP activity into the circulation. Leukocyte ALP activity, first noted to be absent in an adult with HPP [141], is reported to be a mixture of TNSALP and the placental ALP isoenzyme and, therefore, can also be low in any clinical form of HPP except perhaps pseudoHPP [80]. During pregnancy in HPP, low levels of leukocyte ALP activity may correct due to increased expression of the placental isoenzyme [142]. Preliminary observations using a polyclonal antibody to the liver isoform of TNSALP suggested normal amounts of TNSALP protein in HPP tissue specimens [143,144]. However, a monoclonal antibody-based immunoassay that measured dimeric TNSALP demonstrated low levels of bone and liver TNSALP isoforms in the serum of patients with all clinical forms of HPP except pseudoHPP [145]. Accordingly, disruption of TNSALP immunoreactivity seemed to follow its release from cell surfaces [145]. In infants with HPP, some ALP activity is detectable by sensitive methods in liver, bone, and kidney tissue, and in skin fibroblasts in culture [146,147]. The ALP in patient fibroblasts has different physicochemical properties compared to healthy cells [146]. In one patient with infantile HPP, catalytic inhibition and isoelectric focusing studies suggested this was intestinal ALP [138], perhaps reflecting a compensatory expression. Indeed, studies of small bowel mucosa from a family with a clinically mild childhood/adult form of HPP [148], and autopsy tissues from severely affected patients [147], reported increased intestinal ALP. Subsequently, however, fibroblasts in culture from severely affected patients had low ALP activity with physicochemical properties that were TNSALP-like, although the physicochemical [146] and immunological

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properties differed from patient to patient [149]. Accordingly, the precise effects on circulating as well as tissue ALP of the many loss-of-function TNSALP gene mutations throughout the TNSALP molecule now documented in HPP (see below) require further investigation. Autopsy studies of children or adults with HPP have not been reported. However, these non-lethal forms of HPP would also feature globally diminished TNSALP activity within tissues. TNSALP isoform activity can be deficient in serum [29], circulating granulocytes [80], bone [80], and cultivated skin fibroblasts [150]. Immunoreactivity of the TNSALP isoforms in serum is also reduced [145]. Inheritance The first evidence that HPP is a heritable disorder came when affected siblings were reported in 1950 [151]. Early on, family studies of severe disease in infants or children indicated autosomal recessive inheritance. The parents of such patients often had low or low-normal levels of serum ALP activity, and PEA was detectable in their urine [48,49]. Furthermore, consanguinity was reported in some kindreds. We now know from TNSALP mutation analysis that the perinatal and infantile forms of HPP do indeed reflect autosomal recessive inheritance (see below). However, inheritance for the milder forms of HPP is more complex. In some early reports, childhood and adult HPP, as well as odontoHPP, were regarded as autosomal recessive conditions [152e154] because vertical transmission of clinically apparent disease seemed unusual [60,61]. However, multigenerational occurrences of clinical and biochemical abnormalities of HPP were then increasingly recognized indicating that mild disease could be transmitted as an autosomal dominant trait [60,61,85,155e157]. Rarely, family studies showed mildly affected individuals who had severe disease in their offspring [60,157,158]. Identification of carriers for HPP could necessitate quantitation of several biochemical markers including urine PPi [159]. Pyridoxine loading, followed by assay of plasma PLP levels, can be especially helpful in heterozygote detection, as reported in the Mennonite population in Canada [127]. Now, from TNSALP mutation analysis, it is clear that the more mild childhood, adult, and odonto forms of HPP can be inherited either as autosomal recessive or autosomal dominant traits. Gene Defects Morquio syndrome together with HPP has occurred in a Canadian Hutterite kindred, but this appeared a coincidence of two autosomal recessive conditions [160]. Phenylketonuria was described in one infant with hypophosphatasemia, phosphoethanolaminuria,

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and generalized skeletal demineralization [161]. I know of patients who have both mild HPP and osteogenesis imperfecta documented by mutation analyses. In 1984, a preliminary report from skin fibroblast heterokaryon studies of 10 unrelated families with perinatal or infantile HPP described absence of complementation. This indicated a defect involving one gene locus causing HPP [140]. In 1987, genetic linkage of the RH blood group in six inbred Mennonite kindreds from Manitoba, Canada, indicated the “candidate” TNSALP gene caused severe HPP in this population [162]. In 1988, characterization of the gene encoding the TNSALP isoenzyme [16] provided the basis for fully understanding the etiology of HPP [16,163]. That same year, a missense mutation in TNSALP was discovered in an infant boy with perinatal HPP born to second cousins in Nova Scotia [55]. Site-directed mutagenesis and transfection analysis confirmed that the homozygous, missense defect diminished TNSALP catalytic activity. For this patient, information from the threedimensional structure of Escherichia coli ALP [20] suggested compromise of the spatial relationship of metal ligands to an important arginine residue at the catalytic pocket [53]. Later, this mutation was shown to also impair the transport of the enzyme leading to its intracellular aggregation [164]. In 1992, four additional unrelated patients with perinatal or infantile HPP demonstrated a different missense mutation in each of the eight TNSALP alleles examined [163]. Here, two siblings with typical childhood HPP and one unrelated woman with classic adult HPP were compound heterozygotes for the identical TNSALP missense mutations, showing that these clinical forms of HPP can in fact be the same disorder and transmitted as an autosomal recessive trait [163]. Each of these TNSALP missense mutations altered an amino acid residue shared in mammalian TNSALPs [19]. The three-dimensional structure of Escherichia coli ALP [19,20] suggested disruption of metal ligand binding [19]. In 1993, homozygosity for a different TNSALP missense mutation accounted for the severe HPP prevalent in Canadian Mennonites, presumably explained by a founder and inbreeding [56]. However, homozygosity for a TNSALP defect only rarely explains HPP in other populations. Now, >240 different TNSALP defects have been reported worldwide in HPP patients, including missense, nonsense, and donor splice site mutations, and frame shift deletions [55,56,72,163e173]. Of interest, z80% of TNSALP mutations are missense [174]. Accordingly, molecular diagnosis of a new case of HPP typically requires mutation analysis of the splice sites and coding exons of the entire TNSALP gene.

Transfection studies indicate that some TNSALP gene mutations inactivate the enzyme, and others lead to its intracellular accumulation [164,165,175]. Other defects may diminish expression of the mutated allele or mRNA stability [72]. Some TNSALP mutations exert dominant-negative effects, and thereby explain autosomal dominant transmission of HPP. One such missense defect accounts for relatively many instances of autosomal dominant HPP in the USA [176]. All forms of HPP, including pseudoHPP (unpublished), originate from loss-of-function TNSALP mutations. I have not encountered a bona fide case of HPP without at least one defective TNSALP allele. Even odontoHPP can, however, be inherited as an autosomal recessive trait [175]. Epigenetic Effects Some occurrences of HPP suggested a component of TNSALP biosynthesis dysregulation. A boy with infantile HPP showed remarkable, transient remineralization of his skeleton after treatment with prednisone and a PTH fragment, and then a series of i.v. infusions of pooled normal plasma [177]. The 4-month correction of his hypophosphatasemia appeared to be explained by skeletal synthesis of the bone isoform of TNSALP [177]. The observation could not be attributed to the infused ALP, which had a circulating half-life of just several days [77,178. Yet, he was later shown to be homozygous for a missense mutation of TNSALP, and the explanation for his welldocumented, transient improvement remains a mystery [179]. Regulation of TNSALP biosynthesis may condition the severity of HPP in other ways. Patients with childhood HPP usually have higher absolute levels of ALP activity than adult-onset cases. Nevertheless, the degree of hypophosphatasemia (relative to serum ALP level appropriate for age) is similar in affected children and adults. Perhaps, this helps to explain the “overlap” in defining these two clinical forms of HPP. Possibly, physiological decreases in skeletal ALP levels that normally occur after adolescence eventually cause adult HPP. Sometimes there is considerable difference in the severity of HPP among siblings who share the same TNSALP mutation(s) causing their HPP. Accordingly, other genes likely condition HPP expressivity. We wonder if dietary mineral intake also plays a role because excessive calcium could suppress PTH stimulation of TNSALP biosynthesis by osteoblasts, and Pi acts as a competitive inhibitor for TNSALP. Investigation of benign prenatal HPP has shown that mechanical factors (“fetal packing”) as well as the maternal genetic environment can affect HPP pregnancies [68].

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Treatment Prognosis Prior to experimental, bone-targeted, enzyme-replacement therapy for HPP [180] (see below), perinatal HPP was almost always rapidly fatal. Only rarely was there prolonged survival [72]. Infantile HPP has an unpredictable outcome when first diagnosed. In some patients, progressive skeletal deterioration occurs [77]. In others, there is significant spontaneous improvement [181]. Sequential clinical assessments and radiographic studies are critical for prognostication of infantile HPP. Although the probabilities are not known, perhaps 50% of these babies die from respiratory compromise and pneumonia that follows worsening skeletal disease of the chest [49]. In others, there may be significant improvement, particularly after infancy, maybe because growth rates decrease. Indeed, a preliminary report from Canada suggested that the adult stature of survivors of infantile HPP can be normal, but I am aware of many significant exceptions in Canada and in the USA. Childhood HPP may also seem to improve spontaneously in young adult life [49], but recurrence of symptoms later is possible, if not likely [49,60,182]. Adult HPP causes chronic orthopedic problems after the onset of skeletal symptomatology [49,60,89,182]. Worsening osteomalacia, leading to pain and fractures, can occur at menopause in affected women, and does not appear to be prevented by estrogen replacement therapy (personal observation). Supportive Severely affected infants and young children with HPP should be followed carefully to detect neurological complications, such as increased intracranial pressure, from either “functional” or “true” craniosynostosis [78]. Functional craniosynostosis (that may require craniotomy) can occur despite the radiographic illusion of widely open fontanels [69]. In other circumstances, there is skull deformity but without significant neurological sequelae. Vitamin B6-responsive seizures occur only in severe HPP (perinatal or infantile forms), and represent a grave prognostic sign, probably because TNSALP deficiency is especially profound. Fractures in children with HPP do mend, although delayed healing seems likely and has occurred after femoral osteotomy with casting [183]. In adult patients, pseudofractures may remain unchanged for years, but will not mend unless they first progress to completion, or are treated with intramedullary fixation [60]. Use of intramedullary rods or nails, rather than load-sparing plates, etc., seems best for prophylactic or acute surgical management of femoral fractures and pseudofractures [89]. For recurrent metatarsal stress fractures, anklefoot orthoses may be useful.

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Expert dental care is especially important for children with HPP. Severely compromised dentition can impair nutrition, and preservation of teeth in position or use of complete or partial dentures may be necessary [81,82]. One study indicates that proliferation of bacteria on the tooth surface, perhaps related to deficiency of TNSALP activity in leukocytes, may contribute to loss of dentition [134]. Symptoms from CPPD or calcium phosphate crystal deposition may respond to non-steroidal anti-inflammatory medication [95]. One report [184], and our own experience, suggests that naproxen is useful for the pains of children with HPP. Medical There is no established medical therapy for HPP, although a variety of treatments have been attempted [49,60,177,178,180]. Traditional regimens for rickets and osteomalacia (vitamin D and mineral supplements) should be avoided, unless deficiencies are documented, because circulating levels of calcium, Pi, and the vitamin D metabolites are not low. In infantile HPP, excessive vitamin D could provoke or exacerbate the hypercalcemia and hypercalciuria often encountered in severe HPP. However, restriction of vitamin D intake or sunshine exposure should be guarded against because superimposed vitamin D-deficiency rickets has occurred in HPP [114]. Hypercalcemia in infantile HPP can be improved by lowering dietary calcium intake and/or with hydration, diuretics, or glucocorticoid therapy [69,77]. Progressive skeletal demineralization may follow, but is probably due to the HPP per se, because serum levels of calcium and Pi are not low [77,178,182,185]. Synthetic salmon calcitonin [186] or aminobisphosphonate [187] treatment has been tried to control the hypercalcemia, by hopefully blocking mineral loss, but has not been very useful. Because extracellular accumulation of PPi is the key pathogenetic factor in HPP (see below), reduction in endogenous PPi levels might enable skeletal mineralization to proceed normally [3,44]. In 1968, oral Pi supplementation to promote renal PPi excretion reportedly met with some radiographic success [191]. However, in subsequent studies, plasma PPi levels were found to be essentially unchanged. In fact, increased urinary PPi levels after Pi is administered orally may merely reflect enhanced renal synthesis of PPi [3,44]. Any efficacy has not been confirmed [53,137]. It is also concerning that Pi is an inhibitor of ALP activity [5]. In theory, agents that could stimulate TNSALP biosynthesis or enhance the residual activity, especially in bone, might be helpful for HPP. Administration of cortisone to a few patients with severe disease was reportedly

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followed by periods of normalization of serum ALP activity and radiographic improvement [49,178,188], but this has not been a consistent finding [49]. Brief treatments with Zn2þ or Mg2þ have been unsuccessful [60,177]. However, an active fragment of PTH (1e34) or the intact (1e84) molecule to stimulate skeletal ALP biosynthesis has been reported to have success in some patients with adult HPP who had pain and pseudofractures or bony non-unions [92,189,190,192]. Enzyme replacement therapy for the infantile form of HPP has been attempted by i.v. infusion of several types of soluble human ALPs. Generally, the earliest attempts were disappointing [77,177,178,193,194]. Plasma rich in the bone isoform of TNSALP obtained from patients with Paget’s bone disease was given to one affected infant and seemed to accompany slight radiographic improvement [77]. However, subsequent trials for four infants showed no significant clinical or radiographic benefit, and their disease too proved fatal [178]. Weekly i.v. infusions of fresh plasma were followed by clinical and some radiographic improvement in one patient [195]. Also, i.v. infusions of plasma pooled from several normal individuals were followed by transient correction of hypophosphatasemia and marked temporary clinical, radiographic, and histological improvement in one severely affected boy (see above) [177]. A subsequent trial of pooled plasma infusions in a different patient did not reproduce this response [147]. A brief report in 1989 suggested that i.v. administration of ALP purified from liver improved the histological appearance of bone and decreased urinary PEA levels in one patient [196]. Placental ALP, shown to be catalytically active toward PEA, PPi, and PLP in a study of pregnant women who were carriers for HPP [197], caused hyperphosphatasemia when given i.v., but led to only modest decrements of plasma PLP and urinary PEA concentrations and no change in urinary PPi levels, and showed no clinical or radiographic improvement for lethal disease [193]. These cumulative observations may reflect the fact that the amount of ALP in the body is much greater than levels achieved in the circulation by these treatment attempts. Alternatively, perhaps ALP needs to be present on cell surfaces, particularly in the skeleton, or in bone matrix, to act therapeutically [193]. In this regard, it is notable that the extreme skeletal disease of perinatal HPP occurs in utero in an environment that does not seem protective. Hence, in two unrelated girls with worsening infantile HPP [198,199], marrow cell and bone cell transplantation seemed beneficial although the degree of engraftment of donor cells was low. Preliminary findings in the TNSALP gene knockout mouse supported this therapeutic approach [200]. Recently, in TNSALP knockout mice, TNSALP carried by lentiviral gene therapy has had success [201]. Allogeneic mesenchymal

stem cell transplantation has had some benefit in one patient with perinatal HPP [202]. Currently, experimental, bone-targeted, TNSALP replacement therapy is showing excellent promise for HPP [180]. ENB-0040 is a recombinant fusion protein consisting of TNSALP, the Fc fragment of immunoglobin G, and a deca-aspartate motif for bone targeting [203,204]. Marked improvement in the radiographic abnormalities of newborns, infants, and children with HPP has been documented within several weeks or a few months of commencing therapy, and has been accompanied by significant pulmonary and motor improvements [180,205,206]. The static weakness of HPP seems to respond especially quickly to this treatment [205,206]. Circulating levels of PLP and PPi diminish [180,205,206]. Significant titers of anti-ENB0040 antibodies have not been observed. At the time of this writing, there is preliminary experience of more than one year’s duration of therapy in patients with perinatal, infantile, and childhood HPP. ENB-0040 is emerging as a safe and effective treatment for HPP [180,205,206]. Prenatal Diagnosis Measurement of a-fetoprotein in amniotic fluid can help to differentiate anencephaly from severe HPP. Assay of ALP activity in amniotic fluid is not useful [207]. At 14e18 weeks’ gestation, most of it derives from intestinal ALP excreted from the fetus [208]. Assaying cord blood ALP in utero is untested. Several reports considered the diagnosis of perinatal HPP in utero by radiologic techniques as indicating a lethal outcome for the fetus [96]. Additionally, during the first trimester, chorionic villus samples were studied utilizing a monoclonal antibody-based assay specific for TNSALP [209,210]. A precisely timed and carefully prepared specimen is required [209,211], and this procedure is no longer used. Restriction fragment length polymorphism analysis, using a chorionic villus sample, was successfull for a Canadian Mennonite [212] and for a Japanese family [213]. During the second trimester, perinatal HPP has been reported from, ultrasonography (with attention to the limbs as well as to the skull) [214], radiographic study of the fetus, and assay of ALP activity in amniotic fluid cells by an experienced laboratory [215]. An ultrasound study, however, was judged to be normal at 16e19 weeks of gestation in three cases of perinatal HPP in which radiographic study near term showed absence of a fetal skeleton [216,217]. Combined use of radiological techniques, including serial ultrasonography, has until recently seemed best. TNSALP mutation analysis has been used to evaluate pregnancies considered at risk for lethal disease [165,218,219]. Now, TNSALP mutation analysis is

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available in several commercial laboratories. Although not necessary to make the postnatal diagnosis of HPP, the information is critical for understanding the inheritance pattern of HPP, and if requested for prenatal assessment typically when there has been a previously affected sibling with severe disease. However, characterization of the “benign prenatal” form of HPP [65,66] has raised important issues concerning the viability of pregnancies where single or even double TNSALP allelic defects are present in a fetus and the unpredictability of the clinical outcome [68]. In these fetuses, bowing has corrected spontaneously late in the pregnancy or postnatally, with the clinical phenotype otherwise ranging from infantile to odontoHPP [68].

Mouse Model for Hypophosphatasia In 1995 and 1997, two independent research laboratories reported use of homologous recombination to inactivate the equivalent of the TNSALP gene in mice [220,221]. Although there were some minor phenotypic differences, perhaps explained by the background strains [222], the mice developed skeletal disease that resembled a form of rickets [221]. Their skeletons were unremarkable at birth, suggesting in utero protection or redundant or back-up mechanisms for skeletal development [222]. A striking feature was seizures, apparently due to low extracellular and tissue levels of pyridoxal (PL) from diminished hydrolysis of PLP leading to decreased gamma-aminobutyric acid in the brain [220]. Parenteral administration of vitamin B6 could briefly prolong their lives [221,222]. Subsequent investigations showed that these mice were an excellent model for infantile HPP with postnatal development of defective skeletal mineralization, endogenous accumulation of PEA, PPi, and PLP, and a disturbance in epiphyseal and physeal chondrocyte development [222]. In 2007, an N-ethyl-N-nitrosourea-based murine model of mild HPP was reported that exhibited late-onset skeletal disease featuring defective endochondral ossification and bone mineralization leading to arthropathies of the knees and shoulders [223].

PHYSIOLOGICAL ROLE OF TISSUE-NONSPECIFIC ALKALINE PHOSPHATASE EXPLORED IN HYPOPHOSPHATASIA Discovery in 1988 that loss-of-function mutation within the TNSALP gene can cause HPP [55] proved, after 65 years, that Robert Robison was correct [35e37]. ALP does act critically in mineralization of the human skeleton. The process of phase 2 mineralization

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of cartilage and bone seems to be the primary target of the skeletal disturbance in HPP. HA crystals are found appropriately in MVs [34], but are deficient nearby in skeletal matrix [33]. The defects in teeth seem to be analogous to those in the skeleton [3,38,84]. Although liberation of Pi from an unidentified phosphocompound(s) was a key component of Robison’s hypothesis, he also recognized a second, unknown factor that controlled skeletal mineralization [37]. This has proven to be extracellular PPi, a potent inhibitor of HA crystal growth [3,4]. Although the liver, kidneys, and adrenals are normally rich in TNSALP activity [5], these organs do not seem compromised in HPP. Accordingly, TNSALP in humans may have little physiological importance except for mineralization of hard tissues. However, two Japanese siblings with infantile HPP died with sudden unexplained liver failure as teenagers [170]. It has also been suggested that TNSALP deficiency might diminish biosynthesis of the phospholipid surfactant and cause atelectasis in HPP [76], but the pulmonary problems of severely affected patients likely reflect weakness, thoracic deformity, and rib fractures. Several studies have suggested that TNSALP functions in cell growth and differentiation, however, TNSALP-deficient infantile HPP fibroblasts proliferate normally in culture [224]. Two-dimensional gel electrophoresis has shown that these cells have unremarkable profiles of plasma membrane-associated phosphoproteins [225], hence TNSALP is not a phosphoprotein phosphatase. Several roles for TNSALP in calcification have been proposed that could be deranged in HPP. ALPs have been investigated for domains that predict binding to other proteins (including types I, II, and X collagen), which might help to orient TNSALP in skeletal matrix for mineral deposition. As reviewed below, the discovery that PEA, PLP, and PPi accumulate endogenously, and are inferred therefore to be natural substrates for TNSALP, has been important for understanding the physiological role of TNSALP.

Phosphoethanolamine The discovery in 1955 that urinary PEA levels are increased in HPP provided a useful biochemical marker for this inborn error of metabolism and the first evidence for a natural substrate for TNSALP [51,52,123]. Detailed studies of renal handling of PEA in healthy subjects showed that this phosphocompound is excreted when plasma levels are scarcely detectable; i.e. essentially no renal threshold exists for PEA [123]. Although its metabolic origin is unclear, PEA is thought not to be a derivative of phosphatidylethanolamine; i.e. not from plasma membrane phospholipid breakdown. PEA is now

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known to be part of the phosphatidylinositol-glycan linkage apparatus. Accordingly, extracellular PEA could come from degradation of these anchors of cell-surface proteins. In one family with adult HPP [29], urinary levels of PEA correlated inversely with the activity of the liver (but not the bone) isoform of TNSALP in serum. The major source of circulating PEA could also be the liver [226], which otherwise metabolizes PEA to ammonia, acetaldehyde, and Pi in a reaction catalyzed by O-phosphorylethanolamine phosphorlyase. This enzyme requires PLP as a co-factor, and it had been proposed that pseudoHPP might result from its deficiency [226].

Pyridoxal 50 -Phosphate In 1985, discovery that plasma levels of PLP increase in HPP importantly clarified the physiological role of TNSALP [54]. The dietary forms of vitamin B6 (pyridoxine, pyridoxal, pyridoxamine and their phosphorylated derivatives) are absorbed and then converted to PLP in the liver [227]. PLP is the major co-factor form of vitamin B6. PLP is secreted from the liver into the circulation primarily coupled to albumin [227]. A minor fraction of PLP in plasma is bound to various enzymes. Only a small amount of PLP circulates freely. However, like many phosphorylated compounds, PLP cannot traverse plasma membranes and must first be dephosphorylated to pyridoxal (PL). After PL crosses a plasma membrane, it is rephosphorylated to PLP, or converted to pyridoxamine 5’-phosphate, which acts intracellularly as a co-factor for many enzymatic reactions. Ultimately, vitamin B6 is degraded to pyridoxic acid, primarily in the liver, and is then excreted in urine [227]. Increases in plasma levels of PLP in HPP indicated that TNSALP acts importantly in the extracellular dephosphorylation of PLP [54,126]. In fact, when serum levels of the bone or the liver TNSALP isoforms are increased by other skeletal or hepatic diseases, plasma PLP levels are decreased [126,228]. Increased plasma PLP in HPP seems to result from diminished hydrolysis of PLP. Clinical observations show that patients with HPP typically do not have symptoms of vitamin B6 deficiency or toxicity, and indicate that plasma membranebound TNSALP functions as an ectoenzyme [54,126]. Dermatitis, stomatitis, peripheral neuritis, depression, or anemia (hallmarks of vitamin B6 deficiency) [227] are not complications. Similarly, peripheral neuropathy, a manifestation of vitamin B6 toxicity [227], is not a feature of HPP. Biochemical studies indicate that intracellular levels of vitamin B6 are normal in HPP. Urinary concentrations of 4-pyridoxic acid are unremarkable [54,147]. Children with HPP respond normally during a conventional L-tryptophan loading test for vitamin

B6 deficiency (Whyte MP and Coburn SP: unpublished observation). Levels of PLP and total vitamin B6 in homogenates of cultured TNSALP-deficient fibroblasts are normal [228]. Finally, analysis of tissues obtained at autopsy from three perinatal cases, in which plasma PLP concentrations were elevated 50 to 900 times, revealed unremarkable levels of PLP, PL, and total forms of vitamin B6 [147]. Although vitamin B6 deficiency can cause kidney stones and epilepsy, nephrocalcinosis in infants with HPP is likely due to hypercalciuria. Nevertheless, the possibility of altered oxalate metabolism (a consequence of vitamin B6 deficiency) has not been explored [227]. Of interest, PEA was found to be epileptogenic when given intravenously to one infant patient during a study of PEA metabolism, but the epilepsy of severe HPP is partially vitamin B6 responsive [229]. In all but the most severe cases of HPP, normal (or somewhat elevated) plasma PL levels are observed [54]. In most HPP patients, sufficient extracellular dephosphorylation of PLP to PL by some mechanism seems to account for their normal vitamin B6 status. In two patients with perinatal HPP and epilepsy, who had plasma PL levels below assay sensitivity, administration of vitamin B6 did not correct the seizure disorder [147] (personal observation). Perhaps plasma PLP levels can increase, but not PL. Because TNSALP seems to dephosphorylate PLP to PL extracellularly, PL in the circulation could be low in such HPP patients. Seizures that can be controlled by vitamin B6 supplementation are sometimes a major feature of infantile HPP [220e222,230,231] and characterize the TNSALPknockout mouse model of HPP [220]. In 1985, clinical and biochemical observations concerning vitamin B6 metabolism in HPP indicated an ectoenzyme role for TNSALP [54]. Subsequent characterization of TNSALP as a plasma membrane-bound glycoprotein, covalently linked to the polar head group of phosphatidylinositol, provided verification [232]. Studies using cultivated dermal fibroblasts from patients with infantile HPP [195] and human osteosarcoma cells showed that TNSALP is indeed attached to plasma membranes with ectotopography and dephosphorylates PLP and PEA at physiological concentrations and at physiological pH [233,234].

Inorganic Pyrophosphate The discovery in 1965 that PPi levels are increased in the urine [53] in HPP patients presented a mechanism for the disordered HA crystal deposition and the defective skeletal mineralization [43,44]. At high concentrations, PPi adsorbs to amorphous calcium phosphate and prevents the transformation to HA crystals [43].

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Furthermore, adsorption of PPi to HA crystals impairs their growth and dissolution [40,44]. Hence, PPi accumulation surrounding MVs could be expressed clinically as rickets or osteomalacia. Of interest, at low concentrations, PPi enhances precipitation of calcium and Pi to form amorphous calcium phosphate [44]. Perhaps the calcific periarthritis of HPP reflects this action of PPi [95]. CPPD deposition leading to chondrocalcinosis, pseudogout, and pyrophosphate arthropathy [61] likely results from failure of TNSALP to hydrolyze PPi and to destroy CPPD crystals [23]. ALP has been shown to dissolve CPPD crystals in vitro [23]. This pyrophosphatase activity seems to be unrelated to its capacity to hydrolyze phosphoesters. Studies using TNSALP-deficient fibroblasts from perinatal and infantile HPP patients demonstrated that these cells generate PPi at normal rates from extracellular ATP [235]. They have normal levels of nucleoside triphosphate pyrophosphatase (a.k.a. NTP-PPi-ase, ENPP1, or PC1) activity. Accordingly, NTP-PPi-ase seems to differ from TNSALP. Furthermore, clearance studies of 32PPi administered to adults with HPP indicated that the endogenous accumulation of PPi results from defective degradation rather than increased PPi biosynthesis [44].

Tissue-Non-Specific Alkaline Phosphatase in Serum A variety of evidence suggests that circulating ALP is physiologically inactive [3]. Infants with HPP who received i.v. infusions of plasma from patients with Paget bone disease [182], or were given purified placental ALP [193] (causing normal or even elevated levels of ALP activity in serum, respectively) demonstrated essentially no clinical or radiographic improvement. Furthermore, such therapy using these soluble ALPs failed to reduce substantially urinary PEA or PPi levels or plasma PLP concentrations [77,193]. Accordingly, it was possible that deficiency of TNSALP activity within the skeleton explained the rickets or osteomalacia of HPP [236]. In 1955, Fraser and Yendt reported that rachitic rat cartilage would calcify in serum from an infant with HPP, but slices of the patient’s costochondral junction would not mineralize in synthetic calcifying medium or in pooled serum from healthy children [237]. TNSALP bound to cell or MV surfaces seemed to be the physiologically active form of the enzyme.

Overview for Tissue-Non-Specific Alkaline Phosphatase Function Observations from HPP can be formulated into an overview of how TNSALP functions in humans.

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Clinical and biochemical investigations of vitamin B6 metabolism in HPP, confirmed by a variety of tissue and cell culture studies, revealed that TNSALP is an ectoenzyme. We now know that ALPs are bound to all surfaces by the phosphatidylinositol-glycan- (GPI) anchoring system [27]. Increased endogenous levels of PEA, PPi, and PLP indicate that TNSALP is active toward phosphocompound substrates with variable chemical structure. Extracellular accumulation of substrates reflects deficient ecto-TNSALP activity. The source of extracellular PEA is unclear, but could be the GPI moiety that anchors ectoproteins to cell surfaces. Accumulation of membrane-impermeable PLP in plasma, but not in tissues, explains the absence of vitamin B6 toxicity. However, profound deficiency of TNSALP activity blocks conversion of PLP to PL sufficiently to cause vitamin B6-responsive seizures in severely affected HPP patients. Generation of extracellular PPi, perhaps from ATP and by the action of NTPPPi-ase, occurs normally in HPP. PPi accumulation reflects decreased PPi degradation [238e240]. In HPP, calcium phosphate crystal deposition causes calcific periarthritis, and CPPD precipitation results in chondrocalcinosis and/or PPi arthropathy. Calcific periarthritis may reflect the effect of PPi at low concentrations to stimulate calcium phosphate precipitation. Chondrocalcinosis and PPi arthropathy occur from PPi accumulation and failure of TNSALP to hydrolyze CPPD crystals. Rickets and osteomalacia develop in HPP due to the extracellular accumulation of PPi at sites of mineralization near MVs. High concentrations of PPi inhibit HA crystal growth surrounding MVs. TNSALP appears to be physiologically active bound in tissues, but not in the soluble forms found in the circulation. The presence of ALP activity together with fibrillar collagen dictates which tissues will mineralize [241]. Because at least three phosphocompounds (PEA, PPi, PLP) accumulate in extracellular fluid at nanomolar or micromolar concentrations in HPP, TNSALP acts at substrate concentrations that are much lower than those used in routine clinical assays for ALP activity. It is clear that TNSALP functions at physiological pH. Accordingly, the term “alkaline phosphatase” (never used by Robison) is misleading.

Acknowledgments This work was supported by Shriners Hospitals for Children, the Clark and Mildred Cox Inherited Metabolic Bone Disease Research Fund, the Hypophosphatasia Research Fund, and the Barnes-Jewish Hospital Foundation. I am grateful to Sharon McKenzie and Vivienne McKenzie for helping to create the chapter.

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References [1] Online Mendelian Inheritance in Man OMIM (TM). McKusickNathans Institute of Genetic Medicine, John Hopkins University (Baltimore, MD) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, MD), http://www.ncbi.nlm.nih.gov/omim/; July 1, 2011. [2] Whyte MP. Hypophosphatasia. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The Metabolic and Molecular Bases of Inherited Disease. 8th ed. New York: McGraw-Hill; 2001. p. 5313e29. [3] Whyte MP. Hypophosphatasia: Nature’s window on alkaline phosphatase function in humans. In: Bilezikian JP, Raisz LG, Martin TJ, editors. Principles of Bone Biology. 3rd ed. San Diego: Academic Press; 2008. p. 1573e98. [4] Whyte MP. Physiological role of alkaline phosphatase explored in hypophosphatasia. Ann NY Acad Sci 2010;1192:190e200. [5] McComb RB, Bowers GN, Posen S. Alkaline Phosphatase. New York: Plenum Press; 1979. [6] Harris H. The human alkaline phosphatases: what we know and what we don’t know. Clinica Chimica Acta 1990;186:133e50. [7] Henthorn PS. Alkaline phosphatase. In: Bilezikian J, Raisz L, Rodan G, editors. Principles of Bone Biology. San Diego: Academic Press; 1996. p. 197e206. [8] Millan JL. Mammalian Alkaline Phosphatases: from Biology to Applications in Medicine and Biotechnology. Weinheim: WileyVCH; 2006. [9] Moss DW, Whitaker KB. Modification of alkaline phosphatases by treatment with glycosidases. Enzyme 1985;34:212e6. [10] Harris H. The Principles of Human Biochemical Genetics. 3rd ed. Amsterdam: Elsevier/North-Holland Biomedical Press; 1980. [11] Smith M, Weiss MJ, Griffin CA, et al. Regional assignment of the gene for human liver/bone/kidney alkaline phosphatase to chromosome 1p36.1ep34. Genomics 1988;2:139e43. [12] Griffin CA, Smith M, Henthorn PS, et al. Human placental and intestinal alkaline phosphatase genes map to 2q34eq37. Am J Hum Genet 1987;41:1025e34. [13] Human Gene Mapping. Cytogenet Cell Genet 1986;40:1. [14] Berger J, Garattini E, Hua JC, Udenfriend S. Cloning and sequencing of human intestinal alkaline phosphatase cDNA. Proc Natl Acad Sci USA 1987;84:695e8. [15] Henthorn PS, Raducha M, Edwards YH, et al. Nucleotide and amino acid sequences of human intestinal alkaline phosphatase: close homology to placental alkaline phosphatase. Proc Natl Acad Sci USA 1987;84:1234e8. [16] Weiss MJ, Ray K, Henthorn PS, Lamb B, Kadesch T, Harris H. Structure of the human liver/bone/kidney alkaline phosphatase gene. J Biol Chem 1988;263:12002e10. [17] Kiledjian M, Kadesch T. Analysis of the human liver/bone/ kidney alkaline phosphatase promoter in vivo and in vitro. Nucleic Acids Res 1990;18:957e61. [18] Nosjean O, Koyama I, Goseki M, Roux B, Komoda T. Human tissue non-specific alkaline phosphatases: sugar-moietyinduced enzymic and antigenic modulations and genetic aspects. Biochem J 1997;321:297e303. [19] Henthorn PS, Whyte MP. Missense mutations of the tissuenonspecific alkaline phosphatase gene in hypophosphatasia. Clin Chem 1992;38:2501e5. [20] Kim EE, Wyckoff HW. Reaction mechanism of alkaline phosphatase based on crystal structures. Two-metal ion catalysis. J Molec Biol 1991;218:449e64. [21] Hoylaerts MF, Millan JL. Site-directed mutagenesis and epitope-mapped monoclonal antibodies define a catalytically

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35] [36]

[37] [38] [39]

important conformational difference between human placental and germ cell alkaline phosphatase. Eur J Biochem 1991;202:605e16. Hawrylak K, Stinson RA. Tetrameric alkaline phosphatase from human liver is converted to dimers by phosphatidylinositol phospholipase C. FEBS Lett 1987;212:289e91. Xu Y, Cruz TF, Pritzker KP. Alkaline phosphatase dissolves calcium pyrophosphate dihydrate crystals. J Rheumatol 1991;18:1606e10. Farley JR. Phosphate regulates the stability of skeletal alkaline phosphatase activity in human osteosarcoma (SaOS-2) cells without equivalent effects on the level of skeletal alkaline phosphatase immunoreactive protein. Calcif Tissue Int 1995;57:371e8. Seetharam B, Tiruppathi C, Alpers DH. Hydrophobic interactions of brush border alkaline phosphatases: the role of phosphatidyl inositol. Arch Biochem Biophys 1987;253:189e98. Anh DJ, Dimai HP, Hall SL, Farley JR. Skeletal alkaline phosphatase activity is primarily released from human osteoblasts in an insoluble form, and the net release is inhibited by calcium and skeletal growth factors. Calcif Tissue Int 1998;62:332e40. Krawitz PM, Schweiger MR, Ro¨delsperger C, et al. Identity-bydescent filtering of exome sequence data identifies PIGV mutations in hyperphosphatasia mental retardation syndrome. Nat Genet 2010;42:827e9. Young GP, Rose IS, Cropper S, Seetharam S, Alpers DH. Hepatic clearance of rat plasma intestinal alkaline phosphatase. Am J Physiol 1984;247:G419e26. Millan JL, Whyte MP, Avioli LV, Fishman WH. Hypophosphatasia (adult form): quantitation of serum alkaline phosphatase isoenzyme activity in a large kindred. Clin Chem 1980;26:840e5. Langman MJ, Leuthold E, Robson EB, Harris J, Luffman JE, Harris H. Influence of diet on the “intestinal” component of serum alkaline phosphatase in people of different ABO blood groups and secretor status. Nature 1966;212:41e3. Mulivor RA, Boccelli D, Harris H. Quantitative analysis of alkaline phosphatases in serum and amniotic fluid: comparison of biochemical and immunologic assays. J Lab Clin Med 1985;105:342e8. Anderson HC, Hsu HH, Morris DC, Fedde KN, Whyte MP. Matrix vesicles in osteomalacic hypophosphatasia bone contain apatite-like mineral crystals. Am J Pathol 1997;151:1555e61. Ornoy A, Adomian GE, Rimoin DL. Histologic and ultrastructural studies on the mineralization process in hypophosphatasia. Am J Med Genet 1985;22:743e58. Whyte MP. Rickets and osteomalacia (acquired and heritable forms). In: Wass JAH, Shalet SM, editors. The Oxford Textbook of Endocrinology and Diabetes. New York: Oxford University Press; 2002. p. 697e715. Robison R. The possible significance of hexosephosphoric esters in ossification. J Biol Chem 1923;17:286e93. Robison R, Soames KM. The possible significance of hexosephosphoric esters in ossification: Part II. The phosphoric esterase of ossifying cartilage. Biochem J 1924;18:740e54. Robison R. The Significance of Phosphoteric Esters in Metabolism. New York: New York University Press; 1932. Whyte MP. Hypophosphatasia and the role of alkaline phosphatase in skeletal mineralization. Endocr Rev 1994;15:439e61. Majeska RJ, Wuthier RE. Studies on matrix vesicles isolated from chick epiphyseal cartilage. Association of pyrophosphatase and ATPase activities with alkaline phosphatase. Biochim Biophys Acta 1975;391:51e60.

PEDIATRIC BONE

REFERENCES

[40] Fleisch H, Russell RG, Straumann F. Effect of pyrophosphate on hydroxyapatite and its implications in calcium homeostasis. Nature 1966;212:901e3. [41] Moss DW, Eaton RH, Smith JK, Whitby LG. Association of inorganic-pyrophosphatase activity with human alkalinephosphatase preparations. Biochem J 1967;102:53e7. [42] Leone FA, Rezende LA, Ciancaglini P, Pizauro JM. Allosteric modulation of pyrophosphatase activity of rat osseous plate alkaline phosphatase by magnesium ions. Int J Biochem Cell Biol 1998;30:89e97. [43] Russell RG, Bisaz S, Donath A, Morgan DB, Fleisch H. Inorganic pyrophosphate in plasma in normal persons and in patients with hypophosphatasia, osteogenesis imperfecta, and other disorders of bone. J Clin Invest 1971;50:961e9. [44] Caswell AM, Whyte MP, Russell RG. Hypophosphatasia and the extracellular metabolism of inorganic pyrophosphate: clinical and laboratory aspects. Crit Rev Clin Lab Sci 1991;28:175e232. [45] de Bernard B, Bianco P, Bonucci E, et al. Biochemical and immunohistochemical evidence that in cartilage an alkaline phosphatase is a Ca2þ-binding glycoprotein. J Cell Biol 1986;103:1615e23. [46] Lau KH, Farley JR, Baylink DJ. Phosphotyrosyl-specific protein phosphatase activity of a bovine skeletal acid phosphatase isoenzyme. Comparison with the phosphotyrosyl protein phosphatase activity of skeletal alkaline phosphatase. J Biol Chem 1985;260:4653e60. [47] Rathbun JC. Hypophosphatasia: a new developmental anomaly. Am J Dis Child 1948;75:822e31. [48] Currarino G, Neuhauser EB, Reyersbach GC, Sobel EH. Hypophosphatasia. Am J Roentgenol Radium Ther Nucl Med 1957;78:392e419. [49] Fraser D. Hypophosphatasia. Am J Med 1957;22:730e46. [50] Sobel EH, Clark Jr LC, Fox RP, Robinow M. Rickets, deficiency of alkaline phosphatase activity and premature loss of teeth in childhood. Pediatrics 1953;11:309e22. [51] Fraser D, Yendt ER, Christie FH. Metabolic abnormalities in hypophosphatasia. Lancet 1955;268:286. [52] McCance RA, Morrison AB, Dent CE. The excretion of phosphoethanolamine and hypophosphatasia. Lancet 1955;268:131. [53] Russell RG. Excretion of inorganic pyrophosphate in hypophosphatasia. Lancet 1965;10:461e4. [54] Whyte MP, Mahuren JD, Vrabel LA, Coburn SP. Markedly increased circulating pyridoxal-5’-phosphate levels in hypophosphatasia. Alkaline phosphatase acts in vitamin B6 metabolism. J Clin Invest 1985;76:752e6. [55] Weiss MJ, Cole DE, Ray K, et al. A missense mutation in the human liver/bone/kidney alkaline phosphatase gene causing a lethal form of hypophosphatasia. Proc Natl Acad Sci USA 1988;85:7666e9. [56] Greenberg CR, Taylor CL, Haworth JC, et al. A homoallelic Gly317/Asp mutation in ALPL causes the perinatal (lethal) form of hypophosphatasia in Canadian mennonites. Genomics 1993;17:215e7. [57] Mornet E, Yvard A, Taillandier A, Fauvert D, Simon-Bouy B. A molecular-based estimation of the prevalence of hypophosphatasia in the European population. Ann Hum Genet 2011. E-Pub. [58] Whyte MP, Essmyer K, Geimer M, Mumm S. Homozygosity for TNSALP mutation 1348C>T (Arg 433Cys) causes infantile hypophosphatasia manifesting transient disease correction and variably lethal outcome in a kindred of black ancestry. J Pediatr 2006;148:753e8.

789

[59] Taillard F, Desbois JC, Delepine N, Gretillat F, Allaneau C, Herrault A. L’hypophosphatasie affection polymorphe de fre´quence peut-eˆtre sous estime´e. Infantile 1984;91:559e76. [60] Whyte MP, Teitelbaum SL, Murphy WA, Bergfeld MA, Avioli LV. Adult hypophosphatasia. Clinical, laboratory, and genetic investigation of a large kindred with review of the literature. Medicine (Baltimore) 1979;58:329e47. [61] Whyte MP, Murphy WA, Fallon MD. Adult hypophosphatasia with chondrocalcinosis and arthropathy. Variable penetrance of hypophosphatasemia in a large Oklahoma kindred. Am J Med 1982;72:631e41. [62] Terheggen HG, Schildberg C, Schurer W, Van Sande M, Butzler O. Congenital hypophosphatasia. Report on two cases with special reference to phosphoethanolamine excretion. Mono Hum Genet 1972;6:188. [63] Moore CA, Ward JC, Rivas ML, Magill HL, Whyte MP. Infantile hypophosphatasia: autosomal recessive transmission to two related sibships. Am J Med Genet 1990;36:15e22. [64] Macfarlane JD, Kroon HM, van der Harten JJ. Phenotypically dissimilar hypophosphatasia in two sibships. Am J Med Genet 1992;42:117e21. [65] Moore CA, Curry CJ, Henthorn PS, et al. Mild autosomal dominant hypophosphatasia: in utero presentation in two families. Am J Med Genet 1999;86:410e5. [66] Pauli RM, Modaff P, Sipes SL, Whyte MP. Mild hypophosphatasia mimicking severe osteogenesis imperfecta in utero: bent but not broken. Am J Med Genet 1999;86:434e8. [67] Stevenson DA, Carey JC, Coburn SP, et al. Autosomal recessive hypophosphatasia manifesting in utero with long bone deformity but showing spontaneous postnatal improvement. J Clin Endocrinol Metab 2008;93:3443e8. [68] Wenkert D, McAlister WH, Coburn SP et al. Hypophosphatasia: skeletal presentation in utero of non-lethal disease (seventeen new cases and literature review) 2011; J Bone Miner Res (in press). [69] Kozlowski K, Sutcliffe J, Barylak A, et al. Hypophosphatasia. Review of 24 cases. Pediatr Radiol 1976;5:103e17. [70] Shohat M, Rimoin DL, Gruber HE, Lachman RS. Perinatal lethal hypophosphatasia; clinical, radiologic and morphologic findings. Pediatr Radiol 1991;21:421e7. [71] Silver MM, Vilos GA, Milne KJ. Pulmonary hypoplasia in neonatal hypophosphatasia. Pediatr Pathol 1988;8:483e93. [72] Ozono K, Yamagata M, Michigami T, et al. Identification of novel missense mutations (Phe310Leu and Gly439Arg) in a neonatal case of hypophosphatasia. J Clin Endocrinol Metab 1996;81:4458e61. [73] Whyte MP. “Spur-limbed” dwarfism identified as hypophosphatasia [letter]. Dysmorphol Clin Genet 1988;2:126e7. [74] Baumgartner-Sigl S, Haberlandt E, Mumm S, et al. Pyridoxineresponsive seizures as the first symptom of infantile hypophosphatasia caused by two novel missense mutations (c.677T>C, p.M226T; c.1112C>T, p.T371I) of the tissue-nonspecific alkaline phosphatase gene. Bone 2007;40:1655e61. [75] Brenner RL, Smith JL, Cleveland WW, Bejar RL, Lockhart Jr WS. Eye signs of hypophosphatasia. Arch Ophthalmol 1969;81:614e7. [76] Teree TM, Klein LR. Hypophosphatasia: clinical and metabolic studies. J Pediatr 1968;72:41e50. [77] Whyte MP, Valdes Jr R, Ryan LM, McAlister WH. Infantile hypophosphatasia: enzyme replacement therapy by intravenous infusion of alkaline phosphatase-rich plasma from patients with Paget bone disease. J Pediatr 1982;101:379e86. [78] Collmann H, Mornet E, Gattenlohner S, Beck C, Girschick H. Neurosurgical aspects of childhood hypophosphatasia. Childs Nerv Syst 2009;25:217e23.

PEDIATRIC BONE

790

28. HYPOPHOSPHATASIA

[79] Sty JR, Boedecker RA, Babbitt DP. Skull scintigraphy in infantile hypophosphatasia. J Nucl Med 1979;20:305e6. [80] Fallon MD, Teitelbaum SL, Weinstein RS, Goldfischer S, Brown DM, Whyte MP. Hypophosphatasia: clinicopathologic comparison of the infantile, childhood, and adult forms. Medicine (Baltimore) 1984;63:12e24. [81] Kjellman M, Oldfelt V, Nordenram A, Olow-Nordenram M. Five cases of hypophosphatasia with dental findings. Int J Oral Surg 1973;2:152e8. [82] Lundgren T, Westphal O, Bolme P, Modeer T, Noren JG. Retrospective study of children with hypophosphatasia with reference to dental changes. Scand J Dent Res 1991;99:357e64. [83] Chapple IL. Hypophosphatasia: dental aspects and mode of inheritance. J Clin Periodontol 1993;20:615e22. [84] Bixler D. Heritable disorders affecting cementum and the periodontal structure. In: Stewart RE, Prescott GH, editors. Oral Facial Genetics. Saint Louis: Mosby; 1976. p. 262. [85] Lepe X, Rothwell BR, Banich S, Page RC. Absence of adult dental anomalies in familial hypophosphatasia. J Periodontal Res 1997;32:375e80. [86] Whyte MP, Wenkert D, McAlister WH, et al. Chronic recurrent multifocal osteomyelitis mimicked in childhood hypophosphatasia. J Bone Miner Res 2009;24:1493e505. [87] Girschick HJ, Mornet E, Beer M, Warmuth-Metz M, Schneider P. Chronic multifocal non-bacterial osteomyelitis in hypophosphatasia mimicking malignancy. BMC Pediatr 2007;7:3. [88] Seshia SS, Derbyshire G, Haworth JC, Hoogstraten J. Myopathy with hypophosphatasia. Arc Dis Child 1990;65:130e1. [89] Coe JD, Murphy WA, Whyte MP. Management of femoral fractures and pseudofractures in adult hypophosphatasia. J Bone Joint Surg (Am) 1986;68:981e90. [90] Whyte MP. Atypical femoral fractures, bisphosphonates, and adult hypophosphatasia. J Bone Miner Res 2009;24:1132e4. [91] Khandwala HM, Mumm S, Whyte MP. Low serum alkaline phosphatase activity and pathologic fracture: case report and brief review of hypophosphatasia diagnosed in adulthood. Endocr Pract 2006;12:676e81. [92] Whyte MP, Mumm S, Deal C. Adult hypophosphatasia treated with teriparatide. J Clin Endocrinol Metab 2007;92:1203e8. [93] Wendling D, Cassou M, Guidet M. Hypophosphatasia in adults. Apropos of 2 cases. Rev Rhum Mal Osteoartic 1985;52:43e50. [94] O’Duffy JD. Hypophosphatasia associated with calcium pyrophosphate dihydrate deposits in cartilage. Report of a case. Arthritis Rheum 1970;13:381e8. [95] Chuck AJ, Pattrick MG, Hamilton E, Wilson R, Doherty M. Crystal deposition in hypophosphatasia: a reappraisal. Ann Rheum Dis 1989;48:571e6. [96] Lassere MN, Jones JG. Recurrent calcific periarthritis, erosive osteoarthritis and hypophosphatasia: a family study. J Rheumatol 1990;17:1244e8. [97] Page RC, Baab DA. A new look at the etiology and pathogenesis of early-onset periodontitis. Cementopathia revisited. J Periodontol 1985;56:748e51. [98] Scriver CR, Cameron D. Pseudohypophosphatasia. N Engl J Med 1969;281:604e6. [99] Moore CA, Wappner RS, Coburn SP, Mulivor RA, Fedde KN. Pseudohypophosphatasia: clinical, radiographic, and biochemical characterization of a second case (abstract). Am J Human Genet 1990;47. A-68. [100] Cole DE, Salisbury SR, Stinson RA, Coburn SP, Ryan LM, Whyte MP. Increased serum pyridoxal-5’-phosphate in pseudohypophosphatasia. N Engl J Med 1986;314:992e3. [101] Fedde KN, Cole DE, Whyte MP. Pseudohypophosphatasia: aberrant localization and substrate specificity of alkaline

[102]

[103]

[104]

[105]

[106]

[107]

[108]

[109]

[110]

[111]

[112]

[113] [114]

[115]

[116]

[117]

[118]

[119]

phosphatase in cultured skin fibroblasts. Am J Human Genet 1990;47:776e83. Heaton BW, McClendon JL. Childhood pseudohypophosphatasia. Clinical and laboratory study of two cases. Tex Dent J 1986;103:4e8. Mehes K, Klujber L, Lassu G, Kajtar P. Hypophosphatasia: screening and family investigations in an endogamous Hungarian village. Clin Genet 1972;3:60e6. Rubecz I, Mehes K, Klujber L, Bozzay L, Weisenbach J, Fenyvesi J. Hypophosphatasia: screening and family investigation. Clin Genet 1974;6:155e9. Kleinman G, Uri M, Hull S, Keene C. Perinatal ultrasound casebook. Antenatal findings in congenital hypophosphatasia. J Perinatol 1991;11:282e4. Weinstein RS, Whyte MP. Heterogeneity of adult hypophosphatasia. Report of severe and mild cases. Arch Intern Med 1981;141:727e31. Macfarlane JD, Souverijn JH, Breedveld FC. Clinical significance of a low serum alkaline phosphatase. Netherlands J Med 1992;40:9e14. Royce PM, Blumberg A, Zurbrugg RP, Zimmermann A, Colombo JP, Steinmann B. Lethal osteogenesis imperfecta: abnormal collagen metabolism and biochemical characteristics of hypophosphatasia. Euro J Ped 1988;147:626e31. Wyckoff MH, El-Turk C, Laptook A, et al. Neonatal lethal osteochondrodysplasia with low serum levels of alkaline phosphatase and osteocalcin. J Clin Endocrinol Metab 2005;90:1233e40. El-Gharbawy AH, Peeden Jr JN, Lachman RS, Graham Jr JM, Moore SR, Rimoin DL. Severe cleidocranial dysplasia and hypophosphatasia in a child with microdeletion of the Cterminal region of RUNX2. Am J Med Genet A 2010;152A:169e74. Chines A, Coburn S, Mahuren D, Landt M, Ryan L, Whyte MP. Hypophosphatasia (HPP): diagnostic sensitivity of assay for alkaline phosphatase (ALP), phosphoethanolamine (PEA), inorganic pyrophosphate (PPi) and pyridoxal-5’-phosphate (PLP). J Bone Miner Res 1994;9:Se425. Pillans PI, Berman P, Saunders SJ. Cholestatic jaundice with a normal serum alkaline phosphatase level: another case of hypophosphatasia in an adult. Gastroenterology 1983;84:175e7. Whyte MP, Seino Y. Circulating vitamin D metabolite levels in hypophosphatasia. J Clin Endocrinol Metab 1982;55:178e80. Opshaug O, Maurseth K, Howlid H, Aksnes L, Aarskog D. Vitamin D metabolism in hypophosphatasia. Acta Paediatr Scand 1982;71:517e21. Taillard F, Desbois JC, Gueris J, et al. Inorganic pyrophosphates and parathormone in hypophosphatasia. Study of a family. Biomed Pharmacother 1985;39:236e41. Whyte MP, Rettinger SD. Hyperphosphatemia due to enhanced renal reclamation of phosphate in hypophosphatasia. (abstract). J Bone Miner Res 1987:S2. Rutsch F, Bo¨yer P, Nitschke Y, et al. Hypophosphatemia, hyperphosphaturia, and bisphosphonate treatment are associated with survival beyond infancy in generalized arterial calcification of infancy. Circ Cardiovasc Genet 2008;1:133e40. Nusynowitz ML. Low serum alkaline phosphatase level, hypophosphataemia, and aching extremities (letter). J Am Med Assoc 1979;242:2800e1. Juan D, Lambert PW, Vitamin D. Metabolism and phosphorus absorption studies in a case of coexistent vitamin D resistant rickets and hypophosphatasia. In: Cohn DV, Talmage RV, Matthews JL, editors. Hormonal Control of Calcium Metabolism. Amsterdam: Excerpta Medica; 1981. p. 465.

PEDIATRIC BONE

REFERENCES

[120] Rettinger SD, Whyte MP. Normal circulating acid phosphatase activity in hypophosphatasia. J Inherit Metab Dis 1985;8:161e2. [121] Iqbal SJ. Persistently raised serum acid phosphatase activity in a patient with hypophosphatasia: electrophoretic and molecular weight characterisation as type 5. Clin Chim Acta 1998;271: 213e20. [122] De Vries HR, Duran M, De Bree PK, Wadman SK. A patient with hypophosphatasia and hyperprolinaemia. Neth J Med 1978;21:28e34. [123] Rasmussen K. Phosphorylethanolamine and hypophosphatasia. Dan Med Bull 1968;15:1e112. [124] Licata AA, Radfar N, Bartter FC, Bou E. The urinary excretion of phosphoethanolamine in diseases other than hypophosphatasia. Am J Med 1978;64:133e8. [125] Tecza S, Prandota J, Morawska Z, Rudzka M, PankowPrandota L. Hypophosphatasia with normal urinary phosphoethanolamine in a 22-month-old girl. Pediatr Pol 1980;55:791e6. [126] Coburn SP, Whyte MP, Leklem JE, Reynolds RD. Role of phosphatases in the regulation of vitamin B-6 metabolism in hypophosphatasia and other disorders. Clinical and physiological applications of vitamin B-6. Proceedings of the Third International Conference on Vitamin B-6. New York: AR Liss; 1988. p. 65e93. [127] Chodirker BN, Coburn SP, Seargeant LE, Whyte MP, Greenberg CR. Increased plasma pyridoxal-5’-phosphate levels before and after pyridoxine loading in carriers of perinatal/ infantile hypophosphatasia. J Inherit Metab Dis 1990;13:891e6. [128] Macfarlane JD, Poorthuis BJ, Mulivor RA, Caswell AM. Raised urinary excretion of inorganic pyrophosphate in asymptomatic members of a hypophosphatasia kindred. Clin Chim Acta 1991;202:141e8. [129] Girschick HJ, Haubitz I, Hiort O, Schneider P. Long-term follow-up of bone mineral density in childhood hypophosphatasia. Joint Bone Spine 2007;74:263e9. [130] Zhang F, Whyte MP, Wenkert D. Improving dual-energy x-ray absorptiometry (DXA) interpretation: A simple equation for height correction for pre-teenage children. 2011; (Submitted for publication.) [131] Nomura Y, Mori Hypophosphatasia W. Histopathology of human temporal bones. J Laryngol Otol 1968;82:1129e36. [132] Manicourt D, Orloff S, Taverne-Verbanck J. Osteomalacia in hyperphosphoethanolaminuria without hypophosphatasia. Ann Endocrinol (Paris) 1979;40:167e8. [133] Beumer 3rd J, Trowbridge HO, Silverman Jr S, Eisenberg E. Childhood hypophosphatasia and the premature loss of teeth. A clinical and laboratory study of seven cases. Oral Surg Oral Med Oral Pathol 1973;35:631e40. [134] el-Labban NG, Lee KW, Rule D. Permanent teeth in hypophosphatasia: light and electron microscopic study. J Oral Pathol Med 1991;20:352e60. [135] Ramer M, Basta R, Fisher K. Childhood hypophosphatasia. A case report. NY State Dent J 1997;63:36e9. [136] Whyte MP, Mumm S, McAlister WH, et al. Hypophosphatasia: natural history in childhood delineated from 25 years experience with 174 pediatric patients 2011 (In Manuscript). [137] Vanneuville FJ, Leroy JG. Hypophosphatasia: biochemical diagnosis in post-mortem organs, plasma and diploid skin fibroblasts [proceedings]. Arch Int Physiol Biochim 1979;87:854e5. [138] Mueller HD, Stinson RA, Mohyuddin F, Milne JK. Isoenzymes of alkaline phosphatase in infantile hypophosphatasia. J Lab Clin Med 1983;102:24e30.

791

[139] Gorodischer R, Davidson RG, Mosovich LL, Yaffe SJ. Hypophosphatasia: a developmental anomaly of alkaline phosphatase? Pediatr Res 1976;10:650e6. [140] Whyte MP, Vrabel L. Infantile Hypophosphatasia: complementation analysis with skin fibroblast heterokaryons suggests a defect(s) at a single gene locus (abstract). Am J Hum Genet. 1984;26:S209. [141] Beisel WR, Austen KF, Rosen H, Herndon Jr EG. Metabolic observations in adult hypophosphatasia. Am J Med 1960;29:369e76. [142] Iqbal SJ. Increase in leukocyte alkaline phosphatase in a patient with hypophosphatasia during pregnancy. J Inherit Metabolic Dis 1998;21:83e4. [143] Fallon MD, Whyte MP, Weiss MJ, Harris H. Molecular biology of hypophosphatasia: a point mutation or small deletion in the bone/liver/kidney alkaline phosphatase gene results in an intact but functionally inactive enzyme (abstract). J Bone Miner Res 1989;4:S304. [144] Goseki M, Oida S, Takagi Y, Okuyama T, Watanabe J, Sasaki S. Immunological study on hypophosphatasia. Clin Chim Acta 1990;190:263e8. [145] Whyte MP, Walkenhorst DA, Fedde KN, Henthorn PS, Hill CS. Hypophosphatasia: Levels of bone alkaline phosphatase immunoreactivity in serum reflect disease severity. J Clin Endocrinol Metab 1996;81:2142e8. [146] Whyte MP, Rettinger SD, Vrabel LA. Infantile hypophosphatasia: enzymatic defect explored with alkaline phosphatase-deficient skin fibroblasts in culture. Calcif Tissue Int 1987;40:244e52. [147] Whyte MP, Mahuren JD, Fedde KN, Cole FS, McCabe ER, Coburn SP. Perinatal hypophosphatasia: Tissue levels of vitamin B6 are unremarkable despite markedly increased circulating concentrations of pyridoxal-5’-phosphate. Evidence for an ectoenzyme role for tissue-nonspecific alkaline phosphatase. J Clin Invest 1988;81:1234e9. [148] Danovitch SH, Baer PN, Laster L. Intestinal alkaline phosphatase activity in familial hypophosphatasia. N Engl J Med 1968;278:1253e60. [149] Fedde KN, Michell MP, Henthorn PS, Whyte MP. Aberrant properties of alkaline phosphatase in patient fibroblasts correlate with clinical expressivity in severe forms of hypophosphatasia. J Clin Endocrinol Metab 1996;81:2587e94. [150] Whyte MP, Vrabel LA, Schwartz TD. Alkaline phosphatase deficiency in cultured skin fibroblasts from patients with hypophosphatasia: comparison of the infantile, childhood, and adult forms. J Clin Endocrinol Metab 1983;57:831e7. [151] Schneider RW, Corcoran AC. Familial nephrogenic osteopathy due to excessive tubular reabsorption of inorganic phosphate; a new syndrome and a novel mode of relief. J Lab Clin Med 1950;36:985e6. [152] Pimstone B, Eisenberg E, Silverman S. Hypophosphatasia: Genetic and dental studies. Ann Intern Med 1966;65:722e9. [153] Harris H, Robson EB. A genetical study of ethanolamine phosphate excretion in hypophosphatasia. Ann Hum Genet 1959;23:421e41. [154] Barrett AM, Fairweather DV, McCance RA, Morrison AB. Genetic, clinical, biochemical, and pathological features of hypophosphatasia; based on the study of a family. Q J Med 1956;25:523e37. [155] Eberle F, Hartenfels S, Pralle H, Kabisch A. Adult hypophosphatasia without apparent skeletal disease: “odontohypophosphatasia” in four heterozygote members of a family. Klin Wochenschr 1984;62:371e6. [156] Silverman JL. Apparent dominant inheritance of hypophosphatasia. Arch Intern Med 1962;110:191e8.

PEDIATRIC BONE

792

28. HYPOPHOSPHATASIA

[157] Eastman JR, Bixler D. Clinical, laboratory, and genetic investigations of hypophosphatasia: support for autosomal dominant inheritance with homozygous lethality. J Craniofac Genet Dev Biol 1983;3:213e34. [158] Eastman J, Bixler D. Lethal and mild hypophosphatasia in halfsibs. J Craniofac Genet Dev Biol 1982;2:35e44. [159] Sorensen SA, Flodgaard H, Sorensen E. Serum alkaline phosphatase, serum pyrophosphatase, phosphorylethanolamine and inorganic pyrophosphate in plasma and urine. A genetic and clinical study of hypophosphatasia. Mono Hum Genet 1978;10:66e9. [160] Lowry RB, Snyder FF, Wesenberg RL, et al. Morquio syndrome (MPS IVA) and hypophosphatasia in a Hutterite kindred. Am J Med Genet 1985;22:463e75. [161] Blaskovics ME, Shaw KN. Hypophosphatasia with phenylketonuria. Z Kinderheilkd 1974;117:265e73. [162] Chodirker BN, Evans JA, Lewis M, et al. Infantile hypophosphatasiaelinkage with the RH locus. Genomics 1987;1:280e2. [163] Henthorn PS, Raducha M, Fedde KN, Lafferty MA, Whyte MP. Different missense mutations at the tissue-nonspecific alkaline phosphatase gene locus in autosomal recessively inherited forms of mild and severe hypophosphatasia. Pro Natl Acad Sci USA 1992;89:9924e8. [164] Shibata H, Fukushi M, Igarashi A, et al. Defective intracellular transport of tissue-nonspecific alkaline phosphatase with an Ala162/Thr mutation associated with lethal hypophosphatasia. J Biochem (Tokyo) 1998;123:968e77. [165] Mornet E, Taillandier A, Peyramaure S, et al. Identification of fifteen novel mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene in European patients with severe hypophosphatasia. Eur J Hum Genet 1998;6:308e14. [166] Henthorn P, Ferrero A, Fedde K, Coburn SP, Whyte MP. Hypophosphatasia mutation D361V exhibits dominant effects both in vivo and in vitro (abstract). Am J Hum Genet 1996;59. A-199. [167] Whyte MP. Hypophosphatasia. In: Econs MJ, editor. The Genetics of Osteoporosis and Metabolic Bone Disease. Totowa: Humana Press; 2000. p. 335e6. [168] Fukushi M, Amizuka N, Hoshi K, et al. Intracellular retention and degradation of tissue-nonspecific alkaline phosphatase with a Gly317/Asp substitution associated with lethal hypophosphatasia. Biochem Biophys Res Commun 1998;246:613e8. [169] Goseki-Sone M, Orimo H, Iimura T, et al. Hypophosphatasia: identification of five novel missense mutations (G507A, G705A, A748G, T1155C, G1320A) in the tissue-nonspecific alkaline phosphatase gene among Japanese patients. Hum Mutat 1998;(Suppl. 1):S263e7. [170] Sugimoto N, Iwamoto S, Hoshino Y, Kajii E. A novel missense mutation of the tissue-nonspecific alkaline phosphatase gene detected in a patient with hypophosphatasia. J Hum Genet 1998;43:160e4. [171] Orimo H, Hayashi Z, Watanabe A, Hirayama T, Hirayama T, Shimada T. Novel missense and frameshift mutations in the tissue-nonspecific alkaline phosphatase gene in a Japanese patient with hypophosphatasia. Hum Mol Genet 1994;3:1683e4. [172] Orimo H, Goseki-Sone M, Sato S, Shimada T. Detection of deletion 1154-1156 hypophosphatasia mutation using TNSALP exon amplification. Genomics 1997;42:364e6. [173] Mumm S, Jones J, Henthorn PS, Eddy MC, Whyte MP. Mutational analysis of the bone alkaline phosphatase gene in hypophosphatasia (abstract). Bone 1998;23. S281. [174] Mornet E. Tissue nonspecific alkaline phosphatase gene mutations database Yvelines (France). SESEP Laboratory at the University of Versailles-Saint Quentin; July 4, 2007. ed; 2004.

[175] Goseki-Sone M, Orimo H, Iimura T, et al. Expression of the mutant (1735T-DEL) tissue-nonspecific alkaline phosphatase gene from hypophosphatasia patients. J Bone Miner Res 1998;13:1827e34. [176] Mumm S, Wenkert D, Zhang X, Geimer M, Zerega J, Whyte MP. Hypophosphatasia: The c.1133A>T, p.D378V transversion is the most common American TNSALP mutation (abstract). J Bone Miner Res 2006;21:S115. [177] Whyte MP, Magill HL, Fallon MD, Herrod HG. Infantile hypophosphatasia: normalization of circulating bone alkaline phosphatase activity followed by skeletal remineralization. Evidence for an intact structural gene for tissue nonspecific alkaline phosphatase. J Pediatr 1986;108:82e8. [178] Whyte MP, McAlister WH, Patton LS, et al. Enzyme replacement therapy for infantile hypophosphatasia attempted by intravenous infusions of alkaline phosphatase-rich Paget plasma: results in three additional patients. J Pediatr 1984;105:926e33. [179] Whyte MP, Essmyer K, Geimer M, Mumm S. Homozygosity for TNSALP mutation 1348c>T (Arg433Cys) causes infantile hypophosphatasia manifesting transient disease correction and variably lethal outcome in a kindred of black ancestry. J Pediatr 2006;148:753e8. [180] Whyte MP, Greenberg CR, Salman NJ et al. Enzyme replacement for life-threatening hypophosphatasia. 2010; (Submitted for publication). [181] Caswell AM, Whyte MP, Russell RG. Hypophosphatasia: pediatric forms. J Pediatr Endocrinol 1989;3:73e92. [182] Weinstein RS, Whyte MP. Fifty-year follow-up of hypophosphatasia. Arch Int Med 1981;141:1720e1. [183] Jacobson DP, McClain EJ. Hypophosphatasia in monozygotic twins. A case report. J Bone Joint Surg Am 1967;49:377e80. [184] Girschick HJ, Schneider P, Haubitz I, et al. Effective NSAID treatment indicates that hyperprostaglandinism is affecting the clinical severity of childhood hypophosphatasia. Orphanet J Rare Dis 2006;1:24. [185] Scaglione PR, Lucey JF. Further observations on hypophosphatasia. Am J Dis Child 1956;92:493e5. [186] Barcia JP, Strife CF, Langman CB. Infantile hypophosphatasia: treatment options to control hypercalcemia, hypercalciuria, and chronic bone demineralization. J Pediatr 1997;130:825e8. [187] Deeb AA, Bruce SN, Morris AA, Cheetham TD. Infantile hypophosphatasia: disappointing results of treatment. Acta Paediatr 2000;89:730e3. [188] Fraser D, Laidlaw JC. Treatment of hypophosphatasia with cortisone. Lancet 1956;1:553. [189] Camacho PM, Painter S, Kadanoff R. Treatment of adult hypophosphatasia with teriparatide. Endocr Pract 2008;14:204e8. [190] Schalin-Jantti C, Mornet E, Lamminen A, Valimaki MJ. Parathyroid hormone treatment improves pain and fracture healing in adult hypophosphatasia. J Clin Endocrinol Metab 2010;95:5174e9. [191] Bongiovanni AM, Album MM, Root AW, Hope JW, Marino J, Spencer DM. Studies in hypophosphatasia and response to high phosphate intake. Am J Med Sci 1968;255:163e70. [192] Gagnon C, Sims NA, Mumm S, et al. Lack of sustained response to teriparatide in a patient with adult hypophosphatasia. J Clin Endocrinol Metab 2010;95:1007e12. [193] Whyte MP, Habib D, Coburn SP, et al. Failure of hyperphosphatasemia by intravenous infusion of purified placental alkaline phosphatase (ALP) to correct severe hypophosphatasia: evidence against a role for circulating ALP in skeletal mineralization. J Bone Miner Res 1992;7:S155.

PEDIATRIC BONE

793

REFERENCES

[194] Macpherson RI, Kroeker M, Houston CS. Hypophosphatasia. J Can Assoc Radiol 1972;23:16e26. [195] Albeggiani A, Cataldo F. Infantile hypophosphatasia diagnosed at 4 months and surviving at 2 years. Helv Paediatr Acta 1982;37:49e58. [196] Weninger M, Stinson RA, Plenk Jr H, Bock P, Pollak A. Biochemical and morphological effects of human hepatic alkaline phosphatase in a neonate with hypophosphatasia. Acta Paediatr Scand Suppl 1989;360:154e60. [197] Whyte MP, Landt M, Ryan LM, et al. Alkaline phosphatase: placental and tissue-nonspecific isoenzymes hydrolyze phosphoethanolamine, inorganic pyrophosphate, and pyridoxal 5’phosphate. Substrate accumulation in carriers of hypophosphatasia corrects during pregnancy. J Clin Invest 1995;95:1440e5. [198] Whyte MP, Kurtzberg J, McAlister WH, et al. Marrow cell transplantation for infantile hypophosphatasia. J Bone Miner Res 2003;18:624e36. [199] Cahill RA, Wenkert D, Perlman SA, et al. Infantile hypophosphatasia: transplantation therapy trial using bone fragments and cultured osteoblasts. J Clin Endocrinol Metab 2007;92:2923e30. [200] Fedde KN, Blair L, Terzic F, et al. Amelioration of the skeletal disease in hypophosphatasia by bone marrow transplantation using the alkaline phosphatase-knockout mouse model (abstract). Am J Hum Genet 1996;59:A15. [201] Yamamoto S, Orimo H, Matsumoto T, et al. Prolonged survival and phenotypic correction of Akp2(/) hypophosphatasia mice by lentiviral gene therapy. J Bone Miner Res 2011;26:135e42. [202] Tadokoro M, Kanai R, Taketani T, Uchio Y, Yamaguchi S, Ohgushi H. New bone formation by allogeneic mesenchymal stem cell transplantation in a patient with perinatal hypophosphatasia. J Pediatr 2009;154:924e30. [203] Millan JL, Narisawa S, Lemire I, et al. Enzyme replacement therapy for murine hypophosphatasia. J Bone Miner Res 2008;23:777e87. [204] McKee M, Nakano Y, Masica DL, et al. Enzyme replacement therapy prevents dental defects in a model of hypophosphatasia. J Dent Res 2011;90:470e6. [205] Whyte MP, Greenberg CR, Wenkert D, et al. Hypophosphatasia (HPP): enzyme replacement therapy (EzRT) for children using bone-targeted, tissue nonspecific alkaline phosphatase. Annual Endocrine Society Meeting 2010 (abstract). [206] Whyte MP, Greenberg CR, et al. Hypophosphatasia: Enzyme replacement therapy for children using bone-targeted, tissue-nonspecific alkaline phosphatase. J Bone Miner Res 2010;25. S5. [207] Rudd NL, Miskin M, Hoar DI, Benzie R, Doran TA. Prenatal diagnosis of hypophosphatasia. N Engl J Med 1976;295:146e8. [208] Mulivor RA, Mennuti M, Zackai EH, Harris H. Prenatal diagnosis of hypophosphatasia; genetic, biochemical, and clinical studies. Am J Hum Genet 1978;30:271e82. [209] Warren RC, McKenzie CF, Rodeck CH, Moscoso G, Brock DJ, Barron L. First trimester diagnosis of hypophosphatasia with a monoclonal antibody to the liver/bone/kidney isoenzyme of alkaline phosphatase. Lancet 1985;2:856e8. [210] Brock DJ, Barron L. First-trimester prenatal diagnosis of hypophosphatasia: experience with 16 cases. Prenatal Diagn 1991;11:387e91. [211] Muller F, Oury JF, Bussiere P, Lewin F, Boue J. First-trimester diagnosis of hypophosphatasia. Importance of gestational age and purity of CV samples. Prenat Diagn 1991;11:725e30. [212] Greenberg CR, Evans JA, McKendry-Smith S, et al. Infantile hypophosphatasia: localization within chromosome region

[213]

[214]

[215] [216]

[217] [218]

[219]

[220]

[221]

[222]

[223]

[224]

[225]

[226]

[227]

[228]

[229]

1p36.1-34 and prenatal diagnosis using linked DNA markers. Am J Hum Genet 1990;46:286e92. Kishi F, Matsuura S, Murano I, Akita A, Kajii T. Prenatal diagnosis of infantile hypophosphatasia. Prenat Diagn 1991;11:305e9. van Dongen PW, Hamel BC, Nijhuis JG, de Boer CN. Prenatal follow-up of hypophosphatasia by ultrasound: case report. Eur J Obstet Gynecol Reprod Biol 1990;34:283e8. Kousseff BG, Mulivor RA. Prenatal diagnosis of hypophosphatasia. Obstet Gynecol 1981;57(Suppl-12S). Garber AP, Sillence DO, Lachman RS, et al. Discordance between ultrasound and radiographic/biochemical findings in the prenatal diagnosis of congenital lethal hypophosphatasia. The National Foundation March of Dimes Birth Defects Conference; June 24e27. Chicago: Ilinois; 1979. p. 61. Hausser C, Habib R, Poitras P. Hypophosphatasia: complete absence of the fetal skeleton. Union Med Can 1984;113:978e9. Orimo H, Nakajima E, Hayashi Z, et al. First-trimester prenatal molecular diagnosis of infantile hypophosphatasia in a Japanese family. Prenat Diagn 1996;16:559e63. Henthorn PS, Whyte MP. Infantile hypophosphatasia: successful prenatal assessment by testing for tissue-non-specific alkaline phosphatase isoenzyme gene mutations. Prenatal Diagn 1995;15:1001e6. Waymire KG, Mahuren JD, Jaje JM, Guilarte TR, Coburn SP, MacGregor GR. Mice lacking tissue non-specific alkaline phosphatase die from seizures due to defective metabolism of vitamin B-6. Nat Genet 1995;11:45e51. Narisawa S, Frohlander N, Millan JL. Inactivation of two mouse alkaline phosphatase genes and establishment of a model of infantile hypophosphatasia. Dev Dynamics 1997;208:432e46. Fedde KN, Blair L, Silverstein J, et al. Alkaline phosphatase knock-out mice recapitulate the metabolic and skeletal defects of infantile hypophosphatasia. J Bone Miner Res 1999;14:2015e26. Hough TA, Polewski M, Johnson K, et al. Novel mouse model of autosomal semidominant adult hypophosphatasia has a splice site mutation in the tissue nonspecific alkaline phosphatase gene Akp2. J Bone Miner Res 2007;22:1397e407. Whyte MP, Vrabel LA. Infantile hypophosphatasia fibroblasts proliferate normally in culture: evidence against a role for alkaline phosphatase (tissue nonspecific isoenzyme) in the regulation of cell growth and differentiation. Calcif Tissue Int 1987;40:1e7. Fedde KN, Michel MP, Whyte MP. Evidence against a role for alkaline phosphatase in the dephosphorylation of plasma membrane proteins: hypophosphatasia fibroblast study. J Cell Biochem 1993;53:43e50. Gron IH. Mammalian O-phosphorylethanolamine phosphorlyase activity and its inhibition. Scand J Clin Lab Invest 1978;38:107e12. Dolphin D, Poulson R, Avramovic O. Vitamin B6 Pyridoxal Phosphate: Chemical, Biochemical, and Medical Aspects. New York: Wiley; 1986. Whyte MP, Mahuren JD, Scott MJ, Coburn SP. Hypophosphatasia: Pyridoxal-5’-phosphate levels are markedly increased in hypophosphatasemic plasma but normal in alkaline phosphatase-deficient fibroblasts (evidence for an ectoenzyme role for alkaline phosphatase in vitamin B6 metabolism) (abstract). J Bone Miner Res 1986;1:S92. Takahashi T, Iwantanti A, Mizuno S et al. The relationship between phosphoethanolamine level in serum and intractable seizure on hypohosphatasia infantile form. Endocrine control of bone and calcium metabolism. Proceedings of the Eighth International Conference on Calcium Regulating Hormones,

PEDIATRIC BONE

794

[230]

[231]

[232] [233]

[234]

[235]

28. HYPOPHOSPHATASIA

Kobe-Kyoto-Niigata-Osaka-Tokyo, Japan, October 16e24, 1983. Amsterdam: Excerpta Medica; 1984. p. 93e4. Posen S, Whyte MP, Coburn SP, et al. Infantile hypophosphatasia with fatal status epilepticus (abstract). J Bone Miner Res 1997;12:S528. Balasubramaniam S, Bowling F, Carpenter K, et al. Perinatal hypophosphatasia presenting as neonatal epileptic encephalopathy with abnormal neurotransmitter metabolism secondary to reduced co-factor pyridoxal-5’-phosphate availability. J Inherit Metab Dis 2010. E-pub DOI: 10.1007/s10545-009-9012-7. Low MG, Saltiel AR. Structural and functional roles of glycosylphosphatidylinositol in membranes. Science 1988;239:268e75. Fedde KN, Lane CC, Whyte MP. Alkaline phosphatase is an ectoenzyme that acts on micromolar concentrations of natural substrates at physiologic pH in human osteosarcoma (SAOS-2) cells. Arch Biochem Biophysics 1988;264:400e9. Fedde KN, Whyte MP. Alkaline phosphatase (tissue-nonspecific isoenzyme) is a phosphoethanolamine and pyridoxal- 5’-phosphate ectophosphatase: normal and hypophosphatasia fibroblast study. A J Hum Genet 1990;47:767e75. Caswell AM, Whyte MP, Russell RG. Normal activity of nucleoside triphosphate pyrophosphatase in alkaline phosphatase-deficient fibroblasts from patients with infantile hypophosphatasia. J Clin Endocrinol Metab 1986;63:1237e41.

[236] Whyte MP, McAlister WH, Patton LS, et al. Enzyme replacement therapy for infantile hypophosphatasia attempted by intravenous infusions of alkaline phosphatase-rich Paget plasma: results in three additional patients. J Pediatr 1984;105:926e33. [237] Fraser D, Yendt ER, Christie FH. Metabolic abnormalities in hypophosphatasia. Lancet 1955;268:286. [238] Yadav MC, Simao AM, Narisawa S, et al. Loss of skeletal mineralization by the simultaneous ablation of PHOSPHO1 and alkaline phosphatase function: a unified model of the mechanisms of initiation of skeletal calcification. J Bone Miner Res 2011;26:286e97. [239] Harmey D, Hessle L, Narisawa S, Johnson KA, Terkeltaub R, Millan JL. Concerted regulation of inorganic pyrophosphate and osteopontin by akp2, enpp1, and ank: an integrated model of the pathogenesis of mineralization disorders. Am J Pathol 2004;164:1199e209. [240] Orimo H. The mechanism of mineralization and the role of alkaline phosphatase in health and disease. J Nippon Med Sch 2010;77:4e12. [241] Murshed M, Harmey D, Millan JL, McKee MD, Karsenty G. Unique coexpression in osteoblasts of broadly expressed genes accounts for the spatial restriction of ECM mineralization to bone. Genes Dev 2005;19:1093e104.

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Chronic Kidney Disease Mineral and Bone Disorder Katherine Wesseling Perry, Isidro B. Salusky Department of Pediatrics, David Geffen School of Medicine at UCLA, Los Angeles, California, USA

INTRODUCTION The kidney plays a major role in bone and mineral homeostasis by regulating calcium, phosphorus, parathyroid hormone (PTH), fibroblast growth factor-23 (FGF-23) and calcitriol (1,25 dihydroxyvitamin D, 1,25(OH)2D) metabolism. Disordered regulation of mineral metabolism occurs early in the course of chronic kidney disease (CKD) and results in alterations in bone modeling and remodeling. A growing body of evidence demonstrates that cardiovascular calcifications accompany CKD, that cardiovascular disease is the leading cause of mortality in patients with CKD, and that therapies designed to treat the skeletal consequences of CKD affect the progression of vascular pathology. This has led to a reclassification of the mineral, skeletal, and vascular complications associated with progressive kidney disease, resulting in alterations that are now termed “CKD Mineral and Bone Disorder” (“CKD-MBD”) [1]. The CKD-MBD is defined as a systemic disorder of mineral and bone metabolism due to CKD that is manifested by either one or a combination of the following: 1. abnormalities of calcium, phosphorus, PTH, or vitamin D metabolism 2. abnormalities in bone histology, linear growth, or strength, or 3. vascular or other soft tissue calcification. “Renal osteodystrophy” is the specific term used to describe only the bone pathology that occurs as a complication of CKD and is therefore one aspect of the CKDMBD. Traditionally, such lesions have been defined according to alterations in bone turnover, ranging from high bone turnover (secondary hyperparathyroidism, osteitis fibrosa) to lesions of low bone turnover (adynamic bone disease and osteomalacia). However, alterations in skeletal mineralization and volume are

Pediatric Bone, Second Edition DOI: 10.1016/B978-0-12-382040-2.10029-2

also common in patients in CKD [1] and may contribute to such outcomes as fractures and skeletal deformities which persist despite normalization of bone turnover [2]. This chapter summarizes the major aspects of the pathogenesis, clinical manifestations, histologic features, and therapeutic interventions currently used in the management of CKD-MBD. The clinical and histologic features of bone diseases after successful kidney transplantation are also described.

PATHOGENESIS OF CKD-MBD Pathogenesis of Disordered Mineral Metabolism in CKD-MBD Through signaling mechanisms between bone, kidney, and parathyroid glands, alterations in kidney function lead to changes in serum biochemical values and progressive skeletal disease. Early in the course of CKD, at a time when serum calcium and phosphorus levels are still within the normal range, changes in circulating bone and mineral hormones are evident (Fig. 29.1). In CKD stages 2e3, levels of FGF-23 rise in order to enhance urinary phosphate excretion. These rising FGF-23 levels suppress the renal 1a-hydroxylase resulting in reduced 1,25(OH)2D values and as a result, increase PTH levels, which help reduce blood phosphate levels. In advanced CKD, however, rising PTH levels cause a release of calcium and phosphorus from bone and, when compensatory mechanisms fail, severely impaired glomerular filtration rate (GFR) results in phosphate retention which itself directly suppresses 1a-hydroxylase activity [3]. At this stage, hypocalcemia (from decreased intestinal calcium absorption mediated by declining calcitriol levels), hyperphosphatemia, and low circulating 1,25(OH)2D values all combine to

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FIGURE 29.1 Serum parathyroid hormone (PTH), fibroblast growth factor 23 (FGF-23) and phosphate levels across the spectrum of chronic kidney disease (CKD) stages. *P<0.05 CKD 1e2 vs CKD 3, **P<0.001 CKD 1e2 vs CKD 4, ***P<0.001 CKD 1e2 vs CKD 5. Boxes and bars represent the interquartile range and the median value, respectively; whiskers represent the distance to the smallest and largest unbooked sample value. (Reprint by permission from Van Husen et al. Kidney Int. 2010;78:200e6.)

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stimulate PTH secretion, thus acting as additional factors in the development of secondary hyperparathyroidism [4]. As early as stage 2 CKD (GFR between 60 and 90 mL/ 1.73 m2/min), circulating levels of FGF-23 begin to rise in adults and children [5e7]. FGF-23 levels increase due to chronically elevated phosphorus levels [3,8] and these levels rise as CKD progresses, likely due to a combination of decreased renal excretion [9] and increased osteocyte FGF-23 production [10] (Fig. 29.2). Early increases in FGF-23 may be the first sign of altered osteocyte function in CKD [5,7,10] as well as the initial hormonal change in the development of secondary hyperparathyroidism. The effects of increasing FGF-23 levels on mineral ion metabolism are multiple, and include the induction of renal phosphate excretion by reducing expression levels of the renal sodium-dependent phosphate co-transporters, NaPi2a and NaPi2c, the suppression of renal 1a-hydroxylase activity [11], and the stimulation of the 24-hydroxylase enhancing the metabolism of the biologically active 1,25(OH)2D [11]. As CKD progresses, serum values of FGF-23 increase, becoming markedly elevated in individuals with end-stage kidney disease [12]. Simultaneously, 1,25(OH)2D levels decline; indeed, values are inversely

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related to levels of circulating FGF-23 [5]. Recent evidence suggests that FGF-23 may also regulate PTH secretion; in vitro and in vivo experiments indicate that FGF-23, by activating MAPK pathways in the parathyroid gland, also directly suppress PTH release, independent of the action of FGF-23 on vitamin D metabolism [13,14]. In CKD, reduced circulating 1,25(OH)2D contributes to secondary hyperparathyroidism and parathyroid gland hyperplasia in a number of ways: through decreased intestinal calcium absorption, decreased vitamin D receptor (VDR) expression and function, and thus lack of PTH gene suppression, and reduced calcium sensing receptor (CaSR) expression. Indeed, 1,25(OH)2D has been shown to suppress PTH gene transcription, both in vitro (bovine parathyroid cell culture) and in vivo (intact rats). In conjunction with the VDR, 1,25(OH)2D binds to negative vitamin D response elements in the parathyroid glands which inhibit pre-proPTH gene transcription [15,16]. In a positive feedback loop, 1,25(OH)2D itself normally increases VDR gene expression in the parathyroid glands, further suppressing PTH gene transcription. 1,25(OH)2D3 also increases the expression of the CaSR, the expression of which is reduced in hyperplastic parathyroid tissues obtained from patients with

FIGURE 29.2 (A) and (B): Staining of FGF-23 in cancellous bone under 50
magnification and (B) and (D): staining of DMP1 in cancellous bone under 200
magnification. Arrows indicate osteoctyes with positive staining. Trabeculae and bone marrow are labeled TB and BM respectively. (A) and (C): subject with normal renal function. (B) and (D): subjects with stage 2 CKD. (Reprint by permission from Pereira et al. Bone. 2009;45:1161-8.). (See color plate section.)

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secondary hyperparathyroidism (HPT) [17]. 1,25(OH)2 vitamin D deficiency in animals is associated with decreased expression of CaSR mRNA in parathyroid tissue, while 1,25(OH)2D3 therapy increases CaSR mRNA levels in a dose-dependent manner [18]. 25(OH)vitamin D (25(OH)D) deficiency is prevalent in patients with CKD due to multiple factors: many are chronically ill with little outdoor (sunlight) exposure; CKD dietary restrictions, particularly of dairy products, curtail the intake of vitamin D rich food and lead to decreased dietary calcium intake [19]; and patients with CKD (particularly those with darker skin pigment) display decreased skin synthesis of vitamin D3 in response to sunlight compared with individuals with normal kidney function [20,21]. Proteinuric diseases further exacerbate D deficiency in the CKD population, as 25(OH)D in combination with vitamin D binding protein is lost in the urine [22,23]. Recent data suggest that increased catabolism of 25(OH) vitamin D through the 24-hydroxylase may also play an important role in the development and maintenance of 25(OH)vitamin D deficiency [24]. Low levels of 25(OH)vitamin D also contribute to the development of secondary HPT e both directly and through limiting substrate for the formation of 1,25(OH)2D. Apart from its conversion to 1,25(OH)2D3, 25(OH)D may have its own effect on tissues. Indeed, supplementation with ergocalciferol has been shown to decrease serum PTH levels in patients with CKD [25,26]. Recent evidence demonstrates that the 1a-hydroxylase is present in the parathyroid glands; thus, 25(OH)D is converted not only in the kidney, but also inside the gland to 1,25(OH)2 D3, suppressing PTH [27]. Furthermore, 25(OH)D administration suppresses PTH synthesis even when parathyroid gland 1a-hydroxylase is inhibited, indicating that 25(OH)D may contribute to PTH suppression, independent of its conversion to 1,25(OH)2D3 (27). Thus, low levels of the precursor 25(OH)D may exacerbate secondary hyperparathyroidism in the context of CKD. Phosphorus retention and hyperphosphatemia are also important factors in the pathogenesis of secondary hyperparathyroidism, but only in late stages of CKD. The development of secondary hyperparathyroidism is prevented in experimental animals with chronic kidney disease when dietary phosphorus intake is lowered in proportion to the GFR [28]. Dietary phosphate restriction can also reduce previously elevated serum PTH levels in patients with moderate renal failure [3,29]. Phosphorus retention and hyperphosphatemia indirectly promote the secretion of PTH in several ways. Hyperphosphatemia lowers blood ionized calcium levels as free calcium ions complex with excess inorganic phosphate; the ensuing hypocalcemia stimulates PTH release. Phosphorus also enhances the secretion

of FGF-23, thereby impairing renal 1a-hydroxylase activity, which diminishes the conversion of 25(OH)D to 1,25(OH)2D3 [3]. Finally, phosphorus can directly enhance PTH synthesis by decreasing cytosolic phospholipase A2 (normally increased by CaSR activation), leading to a decrease in arachidonic acid production with a subsequent increase in PTH secretion [30]. Hypophosphatemia also decreases PTH mRNA transcript stability in vitro [31], suggesting that phosphorus itself affects serum PTH levels, probably by increasing the stability of the PTH mRNA transcript. Finally, alterations in parathyroid gland CaSR expression also occur in secondary hyperparathyroidism and may, in turn, contribute to parathyroid gland hyperplasia. The CaSR is a seven transmembrane G proteincoupled receptor with a large extracellular N-terminus, which binds acidic amino acids and divalent cations [32]. Low extracellular calcium levels result in decreased calcium binding to the receptor, a conformational relaxation of the receptor and a resultant increase in PTH secretion [33], while activation of the receptor by high levels of serum calcium decrease PTH secretion [34,35]. The expression of the CaSR is reduced by 30e70% as judged by immunohistochemical methods in hyperplastic parathyroid tissue obtained from human subjects with renal failure [17,36]. CaSR gene transcription is regulated by vitamin D through two distinct vitamin D response elements in the gene’s promoter region [37]; thus, alterations in vitamin D metabolism in renal failure could account for changes in calcium sensing by the parathyroid glands and vitamin D may act upstream of the CaSR in preventing parathyroid cell hyperplasia [38]. Decreased expression and activity of CaSR has been linked to decreased responsiveness in PTH secretion due to altered calcium levels [39]. This decreased expression of the CaSR results in an insensitivity to serum calcium levels with subsequent uncontrolled secretion of PTH. Increased stimulation of the CaSR by calcimimetics has been shown to decrease PTH cell proliferation, implicating the CaSR as a regulator of cell proliferation, as well as PTH secretion [40].

RENAL OSTEODYSTROPHY Abnormalities in Bone Turnover, Mineralization, and Volume Bone histomorphometry is the gold standard for the diagnosis of different types of bone diseases associated with CKD and provides both a method for understanding the pathophysiology of renal bone disease and a guide to its proper management. As recommended by the Kidney Disease Improving Global Outcomes (KDIGO) workgroup, three areas of bone histology are

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examined: bone turnover, mineralization and volume, all of which may be altered in patients with chronic kidney disease [1,41]. Turnover Traditionally, renal osteodystrophy has been classified primarily by alterations in bone turnover. Since PTH activates the PTH/PTHrP receptor on osteocytes and osteoblasts, increasing cellular activity of both osteoblasts and, indirectly, of osteoclasts [42,43], excessive levels of circulating PTH result in increased bone turnover [44]. Serum PTH levels are inversely correlated with GFR, and the majority of patients with GFR less than 50 ml/min have increased serum PTH levels and high turnover bone disease [45e47]. The presence of CKD, however, markedly attenuates the effect of PTH on bone [48,49]. Indeed, serum levels of PTH that are two to nine times the upper limit of the normal range are associated with normal bone turnover in patients treated with maintenance dialysis, while similar PTH values in patients with mild to moderate kidney disease are associated with high turnover osteodystrophy [41,46,50,51]. Although the precise mechanisms are poorly understood, uremia has been associated with this “skeletal resistance” to the actions of PTH (Fig. 29.3). Uremic animals and humans display decreased PTH/PTHrP receptor mRNA expression in bone and growth plate [52,53]. Hyperphosphatemia and alterations in vitamin D metabolism, among other factors, have been implicated in these changes and calcitriol administration has been shown partially to restore the calcemic response to PTH in both experimental animals and in patients with moderate CKD [54]. Desensitization or downregulation of the PTH/PTHrP

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receptor (also referred to as PTHR1), mediated, at least in part by elevated circulating PTH(7e84) levels, may also contribute. Indeed, renal failure in rodents is associated with decreased expression of these receptors on osteoblasts [52,55e57], while PTH(7e84) has been shown to reduce PTH/PTHrP receptor expression in bone- and kidney-derived cell lines through mechanisms involving receptor internalization [58]. Moreover, PTH(7e84) has been shown to blunt the calcemic response to PTH(1e84) and PTH(1e34) in parathyroidectomized rats with normal kidney function [59e61] and to inhibit in vitro osteoclast activity and bone resorption through mechanisms that do not involve the PTH/PTHrP receptor [62]. Recent data furthermore suggest that persistent elevation in PTH(7e84) values in dialysis patients with high bone turnover bone disease are accompanied by a reduced calcemic response to PTH(1e34) suggesting that the PTH(7e84) fragment may also modulate the skeletal response to PTH in humans [63]. The bone in secondary hyperparathyroidism exhibits a marked increase in turnover with increased numbers of osteoblasts and osteoclasts and variable degrees of peritrabecular fibrosis. Activation of osteoclasts is mediated through PTH [64e67]; the result is increased resorption of both mineral and matrix along the trabecular surface and within the haversian canals of cortical bone [68]. One characteristic of high bone turnover is increased quantities of woven osteoid, exhibiting haphazard arrangement of collagen fibers in contrast to the usual lamellar pattern of osteoid in normal bone. Woven osteoid can become mineralized in patients with advanced kidney disease in the absence of vitamin D; however, the calcium may be deposited

FIGURE 29.3 Prediction tree for patients with both normal bone turnover and mineralization. Numbers in the circles and the boxes define the number of the patients with both normal bone formation rate and normal osteoid thickness over total number of patients in the cohort (excluding those with adynamic bone). The P-value under each circle represents the significance of the split based on Chi-square test.

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as amorphous calcium phosphate rather than hydroxyapatite [69]. At the other end of the spectrum of bone turnover, adynamic bone disease is characterized by normal osteoid volume, an absence of fibrosis, and a reduced bone formation rate, as indicated by a reduced or absent double tetracycline label on bone histomorphometry [70,71]. A paucity of osteoblasts and osteoclasts is observed [44]. The histologic features of adynamic renal osteodystrophy, in the absence of aluminum deposition in bone, cannot be distinguished from the histologic features of corticosteroid-induced osteoporosis or either age-related or postmenopausal osteoporosis. It is not therefore possible to determine whether osteoporosis accounts for decreases in osteoblastic activity and bone formation in patients with adynamic renal osteodystrophy unless the amount of trabecular bone is reduced. Decreases in bone mass and histologic evidence of trabecular bone loss are not integral features of the adynamic lesion of renal osteodystrophy when other causes of osteoporosis can be excluded. Adynamic bone is associated with low PTH levels, low alkaline phosphatase levels, high serum calcium levels, and a propensity for increased vascular calcification [72,73]. In the 1970s and 1980s, aluminum intoxication was largely responsible for the development of adynamic bone and osteomalacia in patients with chronic kidney disease. Two distinct patterns of aluminum intoxication were identified: 1. from the aluminum content of water used to prepare dialysate solution [74e76] and 2. intestinal aluminum absorption after the ingestion of large doses of aluminum hydroxide [77e83]. The neurologic syndrome of “dialysis encephalopathy” and a bone disease manifested by fractures, pain, persistent hypercalcemia, and osteomalacia were the main clinical features. Although the prevalence of aluminum bone disease in developed countries is now very low, the prevalence of adynamic renal osteodystrophy not associated with aluminum intoxication has increased substantially over the past few years in adult patients receiving regular dialysis and is highly prevalent in adult patients treated with dialysis [84]. Currently, adynamic renal osteodystrophy is more commonly associated with disorders such as age-related or postmenopausal osteoporosis, steroid-induced osteoporosis, hypoparathyroidism (idiopathic or surgically induced), diabetes mellitus, and to overtreatment with calcium and vitamin D therapy [85]. It remains relatively uncommon in the pediatric population [86]. Because PTH is the major determinant of bone formation and skeletal remodeling in renal failure, oversuppression of PTH secretion can also result in adynamic renal osteodystrophy. Approximately 40% of those

treated with hemodialysis and more than half of adult patients undergoing peritoneal dialysis have serum PTH levels that are only minimally elevated or fall within the normal range; such values are typically associated with normal or reduced rates of bone formation and turnover [71]. Prolonged treatment with calciumcontaining phosphate-binding medications and the use of high dialysate calcium concentration also contribute to low bone turnover [87]. Calcitriol may also directly suppress osteoblastic activity when given intermittently in large doses to patients receiving regular dialysis [88]. The long-term consequences of adynamic renal osteodystrophy not attributable to aluminum toxicity remain to be determined, but concerns have been raised about increases in the risk of skeletal fracture and delayed fracture healing because of low rates of bone remodeling [88]. The development of soft tissue and vascular calcifications has also been associated with adynamic bone disease in cross-sectional studies [73]. In prepubertal children, adynamic renal osteodystrophy has been associated with a reduction in linear growth [85]. Mineralization Although renal osteodystrophy has traditionally been defined by lesions in bone turnover, alterations in skeletal mineralization are also prevalent in CKD [51]. Increases in unmineralized bone (osteoid) in conjunction with delayed rates of mineral deposition are common [51,68]. Defective mineralization that is associated with low to normal bone turnover is termed “osteomalacia” [1]. The histomorphometric characteristics of osteomalacia include: the presence of wide osteoid seams, an increased number of osteoid lamellae, an increase in the trabecular surface covered with osteoid, and a diminished rate of mineralization or bone formation, as assessed by double tetracycline labeling. Fibrosis is often absent [44]. In long-term dialysis patients, osteomalacia that is refractory to vitamin D therapy is most commonly a result of aluminum intoxication [89]. Although the mechanisms of skeletal mineralization are incompletely understood, factors such as 25(OH)D deficiency and altered FGF-23 metabolism have been implicated in their pathogenesis. In the general population, nutritional 25(OH)D deficiency results in osteomalacia and a similar phenotype may occur in patients with CKD. FGF-23 may also play a role; both overexpression [90e92] and ablation of FGF-23 ]93,94] in mice with normal renal function are associated with abnormal mineralization of osteoid, although by different mechanisms. The phosphaturic effect of increased FGF-23 may cause rickets and osteomalacia through an insufficiency of mineral substrate. The mechanisms leading to impaired mineralization in FGF-23-null animals, which have severe hyperphosphatemia and normal or elevated serum calcium levels, remain uncertain;

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however, osteomalacia in these animals suggests that FGF-23 may play a direct role in skeletal mineral deposition. While the ramifications of defective mineralization remain to be established, increased fracture rates and bone deformities are prevalent in patients with CKD despite adequate control of bone turnover [2]. These complications may be due, in part, to alterations in bone mineralization. Treatment with anticonvulsant therapy may also contribute to the development of osteomalacia in patients with kidney disease. Long-term ingestion of phenytoin and/or phenobarbital is associated with a high incidence of osteomalacia in non-uremic patients [95]. The etiology of these findings may in part be due to alterations in vitamin D metabolism [96]. Osteitis fibrosa cystica, a finding of secondary hyperparathyroidism, can coexist with defective mineralization in some patients; this pattern is called a “mixed” lesion [97]. Patients with mixed lesion often display high serum PTH and alkaline phosphatase levels along with lower serum calcium levels [86]. The mixed lesion is common in the pediatric dialysis population [86] but relatively rare in adults treated with maintenance dialysis [98]. However, defective skeletal mineralization in combination with high-turnover bone disease may be observed in patients who are developing aluminum toxicity or in patients with low-turnover aluminumrelated bone disease during deferoxamine (DFO) therapy [99] (see below). In these cases, mixed lesion represents a transitional stage between high-turnover and low-turnover bone disease. Volume Since PTH is anabolic at the level of trabecular bone, high levels of serum PTH are typically associated with increases in bone volume, trabecular volume, and trabecular width [51,84,100,101]. However, bone volume may also be low (termed “osteoporosis”), particularly in individuals with underlying age-related bone loss or in those treated with corticosteroids. Low bone volume is rare in the pediatric CKD population [86]. Osteoporosis in the general population is associated with an increased risk of hip fractures and mortality [102]. Thus, bone volume is considered a critically important parameter of bone histology in the adult population; however, the impact of osteoporosis on morbidity and mortality in these patients remains to be defined.

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Bone Pain Bone pain is a common manifestation of severe bone disease in patients with advanced kidney disease. It is usually insidious in appearance and is often aggravated by weight bearing or a change in posture. Physical findings are often absent. Pain is most common in the lower part of the back, hips, and legs, but may occur in the peripheral skeleton. Occasionally, sudden appearance of pain around the knee, ankle, or heel can suggest acute arthritis; such pain is not usually relieved by massage or local heat. Bone pain is more common and often more marked in patients with aluminum-related bone disease than in those with osteitis fibrosa cystica, but marked variability is seen from one patient to another [103]. In long-term dialysis patients, carpal tunnel syndrome and chronic arthralgias often occur in association with the deposition of b2-microglobulin amyloid in articular and periarticular structures [104]. The arthralgias are usually bilateral and most commonly affect the shoulders, knees, wrists, and small joints of the hand; symptoms are typically worse with inactivity and at night [105,106]. Muscle Weakness Proximal myopathy can be marked in patients with advanced kidney disease. Symptoms appear slowly. Patients may note difficulty climbing stairs or rising from a low chair, or they may have difficulty raising their arms to comb their hair. This proximal muscle weakness resembles that found in 25(OH)vitamin D deficiency and in primary hyperparathyroidism. Plasma levels of muscle enzymes are usually normal and electromyographic changes are non-specific. The pathogenesis of this myopathy is not clear, and several different mechanisms have been implicated, including: secondary hyperparathyroidism, phosphate depletion [107], abnormal vitamin D metabolism and aluminum intoxication [108]. Improvements in gait and posture has been reported in children with moderate renal failure after treatment with 1,25(OH)2D, and muscle weakness improves rapidly in affected adult patients with end-stage kidney disease after treatment with 1,25(OH)2D [109]. Improvement in muscular strength has also been observed after treatment with 25(OH)D, after subtotal parathyroidectomy, after successful renal transplantation, and after chelation therapy with deferoxamine for aluminum intoxication. Skeletal Deformities

Clinical Manifestations The symptoms and signs of renal osteodystrophy are usually non-specific, and laboratory and radiographic abnormalities generally predate clinical manifestations. Some specific symptoms and syndromes do occur, however.

Bone deformities are common in uremic children because their bones undergo growth, modeling, and remodeling. In adult patients, skeletal deformities also arise from abnormal remodeling or recurrent fractures [110]. In children, bone deformities of the femur and wrists arise from slipped epiphyses [111]. This problem

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is most common during the preadolescent period and is most frequent in patients with long-standing congenital kidney disease. In adults with kidney disease, particularly those with aluminum-related bone disease, skeletal deformities may be characterized by lumbar scoliosis, kyphosis, and deformities of the thoracic cage [110]. Growth Retardation Growth retardation is the hallmark of CKD in children. Protein and calorie malnutrition, metabolic acidosis, end-organ growth hormone resistance, and renal bone disease are most commonly implicated in growth failure [112]. Despite correction of acidosis and anemia, normalization of serum calcium and phosphorus levels, and vitamin D sterol therapy replacement, the majority of children with CKD continue to grow poorly. Growth failure worsens as renal function declines; the average height of children with even mild CKD (GFR 50e70 mL/minute/1.73 m2) is 1 standard deviation (SDS) below the average for healthy children. Moderate CKD (GFR 25e49 mL/minute/1.73 m2) is associated with a height SDS of 1.5 and, at the time of initiation of dialysis, the mean height SDS is 1.8. Boys, younger patients, and those with prior renal transplants are at greatest risk for growth failure [113]. Acidosis has been linked to delayed linear growth in patients with renal tubular acidosis and normal renal function, and correction of metabolic acidosis often leads to acceleration in growth velocity [114]. Acidotic rats have been found to have decreased growth hormone (GH) secretion, serum insulin-like growth factor 1 (IGF-1), and hepatic IGF-1 mRNA expression. Moreover, metabolic acidosis has been shown to inhibit the effects of GH in rats with normal and decreased renal function [115e117]. Growth plate mRNA levels of GH receptor, IGF-1 receptor, and IGF-1 expression are downregulated, while IGF-binding proteins are upregulated [118]. In adults treated with maintenance dialysis, correction of acidosis has been shown to decrease the progression of secondary hyperparathyroidism and improve skeletal mineralization [119]. Calcitriol deficiency has also been thought to contribute to growth retardation and bone disease in children with CKD. Secondary hyperparathyroidism remains prevalent in children with advanced renal disease, and osteitis fibrosa continues to be the most common skeletal lesion of renal osteodystrophy in those undergoing regular dialysis despite treatment with daily doses of oral calcitriol [84,120]. Secondary hyperparathyroidism contributes to growth retardation, although optimal target values for PTH in children in all stages of CKD remain controversial. In children with moderate CKD, some data indicate that normal growth velocity is achieved when PTH levels are maintained within the normal range [121] while others have

demonstrated a linear correlation between growth and PTH levels in the same patient population e those with the highest PTH values displaying the highest growth velocity [122]. Treatment of secondary hyperparathyroidism with large, intermittent doses of calcitriol and calcium-based phosphate binders has been shown significantly to reduce bone formation and suppress osteoblastic activity in both adults and children [54,123]. However, adynamic bone disease may develop and linear bone growth decrease in dialysis patients, despite serum PTH levels in the KDOQI recommended range [85,124], during intermittent vitamin D sterol therapy. Maintaining serum PTH levels between 300 and 500 pg/ml reduces the frequency of these complications [101]. The mechanisms by which calcitriol inhibits epiphyseal growth plate cartilage remain poorly understood. However, it is well known that calcitriol exerts dose-dependent inhibitory effects on cell proliferation of chondrocytes and osteoblasts in vitro. In addition, vitamin D sterols increase expressions of a number of IGF binding proteins (IGFBPs), IGFBP-2, -3, -4, -5, which sequester IGF-1 and may exert IGF-1independent antiproliferative effects through their own receptors [125e129]. GH resistance also contributes to impaired linear growth in renal failure. In CKD, poor growth develops despite normal or increased serum GH levels [130,131]. Uremia has been associated with diminished hepatic GH receptor and IGF-1 mRNA expression, defects in post-receptor GH-mediated signal transduction [132,133], reductions in serum GH-binding protein levels [134], along with increased synthesis and reduced clearance of IGF binding proteins [108,134,135]. Improved growth velocity during recombinant human GH (rhGH) therapy has been ascribed to increased bioavailability of IGF-1 to target tissues. Children who are treated with maintenance dialysis respond less well to rhGH therapy than children with less severe CKD, but the mechanisms for the differences in response to GH therapy remain to be determined. Extraskeletal Calcification Extraskeletal calcification has been associated with uremia for many years [136] and is included in the definition of the CKD-MBD [1]. Vascular calcifications are associated with increased mortality. These lesions have their origins in CKD prior to dialysis and begin in childhood [137e139]. The mortality rate in adults and children with CKD is markedly higher than that in the general population, and cardiovascular disease is the leading cause of death in this patient population [138,140]. In contrast to the calcified atherosclerotic plaques that develop in the vascular intima of aging individuals with normal kidney function, uremia facilitates calcification of the tunica media. This form of

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calcification is associated with decreased distensibility of blood vessels, causing a rigid “lead pipe” pathology which is associated with increased risk of congestive heart failure [1]. Electron beam computed tomography (EBCT) is used in the assessment of vascular calcifications in the adult population, and measurements in young adults who were treated with maintenance dialysis as children demonstrated that a significant proportion of this population has evidence of vascular calcification [138]. Carotid ultrasound measurement of intimal-medial thickness (IMT) has been validated for the assessment of cardiovascular pathology in children e increased thickness being associated with worsening disease [139,141]. Osteoblasts and vascular smooth muscle cells have a common mesenchymal origin and vascular tissues in the uremic milieu express osteoblast differentiation factors [142,143]. Core binding factor-1 (Cbfa1; also known as Runx1) is thought to trigger mesenchymal cell to osteoblast transformation and arteries obtained from patients with end-stage renal disease show increased levels of this protein [142]. Upregulation of the sodium-dependent phosphate transporter PIT-1 likely also contributes to increased calcification [144], and upregulation of pro-mineralization factors such as osteopontin, bone sialoprotein, osteonectin, alkaline phosphatase, type I collagen, and bone morphogenic protein-2 (BMP-2) is potentiated by the uremic milieu [145e148]. By contrast, expression of calcification inhibitors, such as fetuin A, matrix gla protein, and Klotho is suppressed [149e152]. Hypercalcemia, hyperphosphatemia, elevated levels of the calciume phosphorus product, and high doses of vitamin D sterols [137,138] have all been implicated in the pathogenesis of cardiovascular calcification. However, as many as 40% of adult patients with stage 3 CKD e patients with normal circulating calcium and phosphorus concentrations e have evidence of vascular calcification [153], suggesting that other factors in the uremic milieu contribute to this process. Recently, increased circulating levels of FGF-23 levels [154] have also been implicated in the development of vascular calcification and high circulating levels of this protein are associated with increased mortality in adult dialysis patients [155]. Klotho, the co-factor necessary for mediating the actions of FGF-23 on phosphate homeostasis, has also been implicated in vascular calcification. Animals lacking the Klotho gene display elevated levels of phosphorus, 1,25(OH)2D and, consequently, serum calcium, along with vascular calcification and features of premature aging. CKD is associated with low circulating levels of Klotho [152,156]. As the pathophysiology of cardiovascular disease in CKD is multifactorial, treatment strategies are also

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multifaceted and vary according to stage of CKD. Therapies that are effective in early CKD may not be effective in later stages e lipid lowering agents decrease mortality in adults with CKD [157] and in those with stable renal allografts [158] but have not been shown to benefit patients treated with dialysis [159]. By contrast, normalization of mineral metabolism (by avoiding hypercalcemia and hyperphosphatemia, limiting calcium intake, and avoiding adynamic bone) is effective at slowing the progression of cardiovascular calcification in patients treated with maintenance dialysis [124,138,160,161]. Although high levels of FGF-23 have been associated with vascular calcification, it is unknown whether early control of FGF-23 levels, as with the use of phosphate binders in early CKD [162], might lead to improvements in cardiovascular health. Moreover, treatment with vitamin D sterols has been associated with improved survival in dialysis patients [163e165], despite increasing circulating FGF-23 levels [166]. Thus, at different stages of CKD, the relative importance of individual risk factors and the value of different biomarkers may vary. Calciphylaxis This unique syndrome, which is characterized by ischemic necrosis of the skin, subcutaneous fat, and muscles, can develop in patients with advanced renal failure not yet treated by dialysis, in those treated with regular dialysis, and in patients with well-functioning kidney transplants [167]. The pathogenesis of this syndrome is uncertain. Extensive medial calcifications of medium-sized arteries commonly exist in patients with renal disease without causing gangrene or ulcerations, and it is not clear that vascular calcifications per se are the cause of the ischemic necrosis. Two distinct types of the syndrome are recognized: proximal calciphylaxis, which affects the thighs, abdomen, and chest wall, and acral calciphylaxis, which involves sites distal to the knees and elbows, such as the toes, fingers, and ankles [168]. The former has a terrible prognosis, with death occurring in more than 80e90% of affected patients. This syndrome is often accompanied by morbid obesity and hypoalbuminemia. Many patients have severe secondary hyperparathyroidism, and the majority have a history of severe and uncontrolled hyperphosphatemia [168]. Some patients may have a thrombotic diathesis or defective regulation of coagulation [169]. The appearance of this syndrome in renal transplant recipients receiving glucocorticoids suggests that steroids may also play a role. Patients with calciphylaxis frequently die of secondary infection. A significant number of patients improve after parathyroidectomy, and a few have healed after substantial reductions in serum phosphorus levels. Parathyroidectomy and aggressive control of serum phosphate levels

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are therefore indicated in those with evidence of severe secondary hyperparathyroidism. However, while ischemic lesions and medial vascular calcifications are common in uremic patients with diabetes, such lesions rarely improve after parathyroidectomy. Thus, parathyroid surgery should be reserved for those diabetic patients with calciphylaxis who have clear evidence of severe secondary hyperparathyroidism. Calcimimetics [170] as well as sodium-thiosulfate (a calcium chelating agent) and pamidronate [171] have both been used effectively in some individuals. Hyperbaric oxygen therapy has also been advocated [171].

Diagnostic Evaluations Phosphorus Despite declining renal mass, in early stages of CKD, serum phosphorus levels are typically within the normal range, probably due to increased FGF-23 production by bone, which may accelerate the progression towards end-stage renal disease (ESRD) and may contribute to vascular disease [10,155,172,173]. By the initiation of dialysis, serum phosphorus levels are elevated in approximately 35e40% of patients [174] and hyperphosphatemia is highly prevalent in patients treated with maintenance dialysis. Both hemodialysis and continuous ambulatory peritoneal dialysis (CAPD) remove phosphate; however, 90e95% of patients treated with maintenance dialysis require some adjuvant measures to control serum phosphate concentrations. Since hyperphosphatemia is associated with worsened secondary hyperparathyroidism and vascular calcification and has also been found to be an independent risk factor for mortality in patients treated with maintenance dialysis [103,161], current guidelines suggest that measures, including dietary phosphate restriction and the use of phosphate-binding medications, should be initiated in pre-dialysis CKD and in dialysis patients when serum levels rise above the normal range. Doses of phosphate binders should be titrated to maintain serum phosphorus levels within age-appropriate levels and calcium phosphorus product lower than 55 mg2/ dL [175,176]. Calcium Hypocalcemia develops with advanced stages of CKD and often resolves during treatment with calcium-containing phosphate binders, vitamin D, and with initiation of dialysis. The development of hypercalcemia in patients undergoing regular dialysis warrants prompt and thorough investigation. Conditions associated with hypercalcemia include: marked hyperplasia of the parathyroid glands as a result of severe secondary hyperparathyroidism, aluminum-related bone disease, therapy with calcitriol or other vitamin D sterols,

administration of large doses of calcium carbonate or other calcium-containing compounds, immobilization, malignancy, and granulomatous disorders, such as sarcoidosis or tuberculosis in which extrarenal production of 1,25-dihydroxyvitamin D occurs [70,177,178]. Basal serum calcium levels are also higher in patients with adynamic bone than in subjects with other lesions of renal osteodystrophy, and episodes of hypercalcemia are common [51]. Because skeletal calcium uptake is limited in adynamic lesions, calcium entering the extracellular fluid from dialysate or after intestinal absorption cannot adequately be buffered in bone, and serum calcium levels rise [179]. Current guidelines recommend restricting the dose of calcium-based binders and/or the dose of 1,25(OH)2vitamin D or its analogs in the presence of persistent or recurrent hypercalcemia, the presence of vascular calcifications, the presence of adynamic bone disease, and/or persistently low PTH levels [41]. Magnesium The net intestinal absorption of magnesium is generally normal or only very slightly reduced in patients with renal failure [175], yet serum magnesium levels often increase with advanced kidney disease due to reduced renal excretion. During hemodialysis, serum magnesium levels are generally increased if the dialysate magnesium concentration is maintained at 1.75 mEq/L; however, magnesium levels remain within the upper range of normal with dialysate magnesium concentrations of 0.5 mEq/L. The use of magnesiumcontaining laxatives or antacids should be avoided as they may cause an abrupt increase in serum magnesium levels in patients with kidney disease [180]. Serum magnesium levels should be measured frequently and regularly if magnesium-containing medications are used. Rarely, hypomagnesemia can develop in uremic patients with severe malabsorption or diarrhea [175]. Alkaline Phosphatase Serum alkaline phosphatase values are fair markers of the severity of secondary hyperparathyroidism in patients with renal failure. Osteoblasts normally express large amounts of the bone isoenzyme of alkaline phosphatase, and serum levels are usually elevated when osteoblastic activity and bone formation rates are increased. High levels generally reflect the extent of histologic change in patients with high-turnover lesions of renal osteodystrophy, and values frequently correlate with serum PTH levels [181]. Serum total alkaline phosphatase measurements are also useful for monitoring the skeletal response to treatment with vitamin D sterols in patients with osteitis fibrosa; values that decrease over several months usually indicate histologic improvement [85]. Bone-specific alkaline phosphatase

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activity may be useful in predicting the histologic lesion of renal osteodystrophy, but whether these values are superior to total alkaline phosphatase levels remains to be demonstrated. Serum alkaline phosphatase levels may increase early in the course of treating aluminumrelated bone disease with the chelating agent deferoxamine. Levels may also increase during therapy with recombinant human growth hormone in pediatric patients [182]. Assays for bone-specific alkaline phosphatase and measurements of serum osteocalcin levels provide additional information about the level of osteoblastic activity in patients with CKD [183]. Osteocalcin levels are generally elevated in renal failure, but values may help distinguish between patients with high-turnover or low-turnover skeletal lesions [183,184]. If these assays are not available, measurement of the heat-stabile and heat-labile fractions of alkaline phosphatase helps separate skeletal from hepatic causes of elevated total alkaline phosphatase levels. At present, there is no evidence of advantage of any of these new markers of bone turnover when compared to serum alkaline phosphatase levels [41]. Parathyroid Hormone Although the actions of different fragments of the PTH molecule are still uncharacterized, in vitro and in vivo experimental data indicate that one or more aminoterminally truncated PTH(1e84) fragments antagonize the calcemic actions of PTH(1e84) and diminishes bone cell activity, and may, therefore, modulate bone metabolism. Indeed, synthetic PTH(7e84), which appears to be similar to naturally occurring circulating aminoterminally truncated PTH fragments [185], inhibits the formation of TRAP-positive bone resorbing cells in vitro [62] and inhibits bone formation in vivo [61]. In addition, Slatopolsky et al. and Nguyen-Yamamoto et al. demonstrated that PTH(7e84), with or without a mixture of other carboxyl-terminal PTH-fragments, inhibits the calcemic effect of PTH(1e34) in vivo, indicating that these actions are not mediated through the PTH/PTHrP receptor, but instead via a receptor that interacts only with carboxyl-terminal portions of PTH [59,60]. In humans, Wesseling-Perry et al. demonstrated that dialysis patients with hyperparathyroid bone disease due to increased levels of PTH(1e84) who also have increased circulating levels of PTH(7e84) and are resistant to the calcemic actions of PTH(1e34) [63]. Thus, a growing body of evidence suggests that at least some of the different carboxyl-terminal PTH-fragments have biological activity and circulating amino-terminally truncated PTH fragments may play a role in the skeletal resistance to the full-length PTH molecule. Over the last few years, a series of observations have highlighted important shortcomings of the first

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generation immunometric assays (IMA) for measuring PTH (1st PTH-IMA). Studies by D’Amour and colleagues [186e188] demonstrated that 1st PTH-IMAs detect not only the intact hormone, but also additional PTH fragments truncated at the amino-terminus. Indeed, most of the detection antibodies, which are usually directed against epitopes within the aminoterminus of the hormone, detect not only PTH(1e84), but also one or several amino-truncated fragments of the PTH molecule, some of which co-elute from reverse-phase high-performance liquid chromatography (HPLC) column with synthetic PTH(7e84) [187]. By contrast, second generation immunometric PTH assays (2nd PTH-IMAs) use detection antibodies which interact with epitopes comprising the first four aminoterminal amino acids of human PTH and thus recognizes only PTH(1e84) and possibly PTH fragments that are truncated at the carboxyl-terminus or modified within the 15e20 region [189], but not PTH(7e84) [33,60,189]. PTH levels determined by 1st PTH-IMA overestimate the concentration of PTH(1e84) by 40e50% in both healthy individuals with normal renal function and those with varying degrees of CKD [186,187]. Unlike 1st PTH-IMAs, the 2nd PTH-IMAs do not detect these large amino-terminally truncated PTH fragments [33,60,189e191]. Faugiere et al. suggested that 2nd PTH-IMA and, mainly, the ratio between PTH(1e84)/amino-truncated PTH fragment (calculated from the differences in PTH levels determined between 1st and 2nd PTH-IMAs) could be a better predictor of bone turnover than 1st PTH-IMA [190]; however, these findings were not confirmed by subsequent investigations [100,191]. Current data do not yet support the claim that 2nd PTH-IMAs provide an advantage over 1st PTH-IMAs for the diagnosis of the different subtypes of renal bone diseases [190]. Furthermore, the variation in PTH assays between manufacturers makes interpretation of their values, and their relationships to bone turnover, difficult to assess. Thus, current guidelines suggest that PTH values, measured with the same assay, be maintained within a broad range e between two and nine times the range for normal individuals e in patients treated with maintenance dialysis [41]. Aluminum Aluminum toxicity occurs in dialysis patients or CKD patients with GFR less than 30 mL/min/1.73 m2 because aluminum that is absorbed from the gut, from the dialysate, or from parenteral infusions is inadequately excreted by the diseased kidney. Accumulation occurs in various tissues, including bone, liver, brain, and parathyroid glands, and can produce toxicity such as dialysis encephalopathy, osteomalacia, and microcytic anemia. The gold standard for the diagnosis of aluminum bone disease is a bone biopsy demonstrating increased

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aluminum staining of the bone surface (greater than 15e25%) with histologic evidence of adynamic bone or osteomalacia. The presence of aluminum deposits in the bone and liver do not correlate with plasma levels [192]; however, plasma aluminum levels are useful in monitoring patients who are undergoing chronic dialysis therapy and receiving aluminum-containing phosphate-binding agents for prolonged periods. Deferoxamine (DFO) should be administered to symptomatic patients with aluminum levels between 60 and 200 mg/L or a positive DFO test. The DFO infusion test is performed by infusing 5 mg/kg of DFO during the last hour of the dialysis session. Serum aluminum is measured before DFO infusion and 2 days later, before the next dialysis session. In order to prevent DFOinduced neurotoxicity, DFO should not be administered if serum aluminum concentrations are greater than 200 mg/L [100,193]. Radiography RADIOGRAPHIC FEATURES OF OSTEITIS FIBROSA CYSTICA

The most consistent radiographic feature of secondary hyperparathyroidism is the presence of subperiosteal erosions [194,195]. The degree of subperiosteal erosion can correlate with serum PTH and alkaline phosphatase levels, but radiographs can be normal in patients with moderate to severe histologic features of osteitis fibrosa cystica on bone biopsy [196]. In pediatric patients, metaphyseal changes (i.e. growth zone lesions that are termed “rickets-like lesions”) are common [194]. The radiographic changes arising from secondary hyperparathyroidism may be difficult to differentiate from true rachitic abnormalities. However, the radiographic features of slipped epiphyses resulting from osteitis fibrosa are present in uremic children but absent in rickets resulting from vitamin D deficiency [111]. These findings are best detected by hand radiographs and several techniques, including the use of fine-grain films and magnification with a hand lens, are used to enhance sensitivity [197]. Subperiosteal erosions also occur in the distal ends of clavicles, on the surface of the ischium and pubis, at the sacroiliac joints, and at the junction of the metaphysis and diaphysis of long bones [195,196]. Subperiosteal erosions can also be found in patients with aluminum-related bone disease [198]. This finding represents the residual of earlier hyperparathyroidism with osteitis fibrosa cystica which, due to aluminum toxicity, is unable to remineralize during treatment with either vitamin D sterols or parathyroidectomy [198]. Radiographic abnormalities of the skull in secondary hyperparathyroidism can include: (1) a diffuse “ground-

glass” appearance, (2) a generalized mottled or granular appearance, (3) focal radiolucencies, and (4) focal sclerosis. RADIOGRAPHIC FEATURES OF OSTEOMALACIA

The radiographic features of osteomalacia are both less specific and less common than those of secondary hyperparathyroidism. Typical rachitic lesions, with widening of the epiphyseal growth plate and other deformities, can occur in children with open epiphyses [194]. Looser’s zones or pseudofractures, the only pathognomonic radiographic features of osteomalacia in adult patients, are rare in renal patients with osteomalacia; they occur as straight, wide bands of radiolucency that are perpendicular to the long axis of the bone. Fractures, particularly of the ribs, vertebral bodies, and hips, are more common in patients with osteomalacia than in patients with osteitis fibrosa cystica or mixed osteodystrophy [199]. Decreased bone density is another common radiographic feature present in patients with advanced kidney disease and may arise from secondary hyperparathyroidism, osteomalacia, or osteoporosis [200]. Paradoxically, localized osteosclerosis is quite common in patients with kidney disease and is more frequent in patients with osteitis fibrosa cystica. BONE BIOPSY

Although not routinely performed in the clinical setting, a bone biopsy should be considered in all patients with CKD who have fractures with minimal trauma (pathological fractures), suspected aluminum bone disease, or persistent hypercalcemia despite serum PTH levels between 400 and 600 pg/mL [124]. For bone labeling, a 2-day course of tetracycline is administered at 15 mg/kg/day (divided in twice or thrice daily doses). Phosphate binders should be held during labeling since they may interfere with gut absorption of tetracycline. Fourteen days later, the 2-day course is repeated. For children younger than 8 years, tetracycline dosage is usually kept below 10 mg/kg/day to avoid toxicity. Histochemical staining procedures demonstrate the deposition of abnormal components within bone such as iron, aluminum, and oxalate [84,201].

TREATMENT OF CKD-MBD In order to minimize complications on the skeleton and to prevent extraskeletal calcifications, particular attention must be made to the alterations of bone and mineral metabolism in CKD. The specific aims of the management of CKD-MBD are: 1. to maintain blood levels of serum calcium and phosphorus near normal limits

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2. to prevent hyperplasia of the parathyroid glands and to maintain serum PTH at levels appropriate for stage of CKD 3. to avoid the development of extraskeletal calcifications 4. to prevent or reverse the accumulation of toxic substances such as aluminum and b2-microglobulin.

Dietary Manipulation of Calcium and Phosphorus As active vitamin D levels fall during the progression of renal disease, calcium absorption in the gut and kidney diminishes and hypocalcemia often develops in late stages of CKD. Patients with untreated CKD commonly ingest as little as 400e700 mg of elemental calcium per day in their diet. Calcium rich foods such as dairy products, unfortunately, are also high in phosphorus. Thus, increasing dietary consumption of calcium in order to meet daily needs is accompanied by an excessive intake of phosphorus, which cannot be excreted in the face of renal failure. As a result, calcium supplementation in the form of calcium-containing salts is often required. The total amount of calcium supplementation, however, must be monitored carefully, since overaggressive supplementation (as is common when calcium-containing salts are used as the sole means of binding phosphorus) may lead to hypercalcemia and vascular calcification. The development of hyperphosphatemia occurs in the vast majority of patients with advanced renal insufficiency. Hyperphosphatemia and an elevated calciumephosphorus ion product have been reported as independent risk factors for vascular calcification and mortality in adult dialysis patients [160,161,202]. Thus, treatment goals include maintaining serum phosphorus levels within normal limits for age and avoiding a calciumephosphorus ion product above 55 mg2/dL. The average phosphorus intake of both adults and children in the US population is approximately 1500e2000 mg/ day, and 60e70% of the dietary intake is absorbed. In the early stages of renal failure, dietary phosphate restriction is sufficient in preventing hyperphosphatemia. However, strict adherence to dietary phosphate restriction is often difficult because low phosphate diets are unpalatable, especially to older children and adults, and because phosphorus intake is directly linked with protein intake with 10e12 mg of phosphorus accompanying each gram of protein. Adequate protein intake is necessary for growth in children and for maintenance of lean body mass in adults. Current dietary recommendations suggest that adults with CKD ingest between 0.8 and 1 g/kg/day of protein and that children, depending on age, ingest anywhere from 1 to 2.5 g/kg/day [124,176]. This translates to

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a minimum phosphate ingestion of 800 mg per day in an 80 kg person. Patients treated with dialysis also require dietary phosphorus restriction in addition to phosphate-binder therapy (see below), since standard prescription peritoneal dialysis and hemodialysis remove insufficient amounts of phosphate (300e400 mg/day for peritoneal dialysis and 800 mg/treatment for hemodialysis) to maintain normal serum phosphorus levels. The use of daily slow continuous hemodialysis in some centers has been associated with excellent control of serum phosphorus levels, often allowing phosphate-binding agents to be discontinued [203,204]. Indeed, some patients have developed hypophosphatemia and have required the addition of phosphorus to the dialysate solution in order to prevent the long-term consequences of hypophosphatemia [203,204].

Phosphate-Binding Agents Phosphate-binding agents reduce intestinal phosphate absorption by forming poorly soluble complexes with phosphorus in the intestinal tract. Aluminum-containing phosphate binders were frequently used in the past, but long-term treatment led to bone disease, encephalopathy, and anemia [205]. The use of aluminum-containing phosphate binders should, therefore, be restricted to the treatment of severe hyperphosphatemia (>7 mg/dL) associated with hypercalcemia or an elevated calciumephosphorus ion 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 given only for a limited period of approximately 4e6 weeks [206]. Plasma aluminum levels should be monitored regularly. Concomitant intake of citrate-containing compounds should be avoided since citrate increases intestinal aluminum absorption [207] and increases the risk of acute aluminum intoxication [208,209]. Constipation is a common side effect and can be relieved by stool softeners. In order to avoid aluminum-related bone disease and encephalopathy, the use of aluminum-free phosphate binders has been advocated. Among these, calciumcontaining salts are used worldwide for the control of hyperphosphatemia and 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 commonly used compound and studies in adults and children have shown its efficacy in controlling serum phosphorus levels [70,210,211]. The recommended dose is proportional to the phosphorus content of the meal and adjusted to achieve acceptable

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serum levels of calcium and phosphorus. Large doses of calcium carbonate may lead to hypercalcemia, particularly in patients treated with vitamin D or those with adynamic bone [212,213]. Hypercalcemia is usually reversible with reductions in the dose of oral calcium salts, dose of vitamin D sterol, and dialysate calcium concentrations. To avoid the development and progression of cardiovascular calcifications, it is currently recommended that calcium-based binders be avoided in patients with persistent/recurrent hypercalcemia, those with vascular calcifications, those with persistently low PTH values, and those with adynamic bone [41]. Comparison studies between calcium carbonate and calcium acetate have demonstrated that an equivalent dose of calcium acetate binds twice as much phosphorus, but the relative incidence of hypercalcemia varies among studies [214e216]. Calcium citrate is also an effective phosphate-binding agent but should be used with caution in patients with renal failure due to enhanced intestinal aluminum absorption when given in combination with aluminum-containing phosphate binders [217]. Calcium ketoglutarate is less calcemic than calcium carbonate and has anabolic benefits, but gastrointestinal side effects and high cost of therapy often limit its use [218]. To limit the vascular calcification risks associated with the use of calcium salts and the bone and neurologic toxicity associated with aluminum hydroxide, alternative phosphate binders have been developed. Sevelamer hydrochloride (RenaGelÒ), a calcium- and aluminumfree hydrogel of cross-linked poly-allylamine, has been shown to lower serum phosphorus, the calciumephosphorus ion product, and PTH without inducing hypercalcemia in adult patients treated with hemodialysis [219e221]. Sevelamer also halts the progression of vascular calcification while such lesions increase during calcium-containing binder therapy in adult patients with pre-dialysis CKD and those on dialysis [222,223]. In addition to its effects on serum phosphorus levels, sevelamer has been shown to decrease concentrations of total serum cholesterol and low-density lipoprotein cholesterol, but to increase high-density lipoprotein levels [221]. These effects may offer additional benefits in reducing cardiovascular complications in patients with end-stage renal disease. Acidosis may occur in patients treated with sevelamer, thus, a new form of sevelamer, sevelamer carbonate, has recently been introduced. This new compound is as effective a phosphate binder as sevelamer hydrochloride with less potential to induce acidosis [224]. 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 those treated with dialysis [109,225]. Large doses,

however, result in diarrhea, limiting the use of this compound as a single agent. Iron compounds, such as stabilized polynuclear iron hydroxide and ferric polymaltose complex, are effective phosphate binders in short-term studies in adults with CKD [226,227]. Lanthanum also accumulates in the bone of dialysis patients where its presence persists despite discontinuation for as long as 2 years [228]. Further long-term studies are therefore needed to confirm the absence of toxicity before this agent is recommended for widespread use. Traditionally, phosphate binders have been indicated for the treatment of hyperphosphatemia, however, recently Oliviera et al. demonstrated that treatment of normo-phosphatemic patients with CKD stages 3 and 4 with 6 weeks of sevelamer or one year, respectively, was able safely to reduce serum FGF-23 and PTH levels while increasing 1,25(OH)2D values [162,229]. Interestingly, neither calcium carbonate [162] nor lanthanum carbonate [230] was able to reproduce this effect. While further studies are needed to assess the long-term efficacy of sevelamer in reducing FGF-23 values, these preliminary results may represent a paradigm shift in the prevention and control of secondary hyperparathyroidism e preventing the rise in FGF-23 and subsequent decline in 1,25(OH)2D and rise in PTH. It is important to note that, currently, active vitamin D sterol therapy is indicated in early stages of CKD when PTH levels rise [41]. This form of therapy is associated with increased values of FGF-23 [231] which, in turn, suppresses renal 1a-hydroxylase and increase CYP24 activity, thereby lowering native calcitriol production and exacerbating the underlying pathophysiology of secondary hyperparathyroidism.

Vitamin D Therapy Despite dietary phosphate restriction, the intake of phosphate-binding agents, the use of an appropriate level of calcium in dialysate solution, and an adequate intake of calcium, progressive osteitis fibrosa cystica due to hyperparathyroidism develops in a significant number of uremic patients. Treatment with vitamin D is aimed at controlling serum PTH levels and the resultant high turnover bone disease. Current evidence indicates that three main issues exist in vitamin D therapy. First, treatment of 25(OH)vitamin D deficiency, a common finding in patients with renal disease, may in itself reverse hyperparathyroidism. Secondly, treatment with active vitamin D sterols, by inhibiting the formation of prepro-PTH and by activating the CaSR, is useful in pharmacologically reducing PTH levels and has been associated with improved survival in the dialysis patients. Interestingly, however, this form of therapy also increases circulating FGF-23 levels [166]

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which, in turn, are associated with increased mortality in dialysis patients [155], left ventricular hypertrophy in CKD patients [232], and progressive renal disease [172]. Assessment and Treatment of 25(OH)Vitamin D Deficiency Measurement of 25(OH)D levels and treatment of vitamin D deficiency is an important part of the management of hyperparathyroidism in patients with chronic kidney disease. The current classification system stratifies vitamin D deficiency into three categories [176]: 1. severe deficiency, defined as a serum level less than 5 ng/mL 2. mild deficiency, equivalent to serum concentrations of 5e15 ng/mL 3. vitamin D insufficiency with levels between 16 and 30 ng/mL. Thus, ergocalciferol treatment should be initiated in patients with CKD when 25-hydroxyvitamin D levels fall below 30 ng/mL. Severe deficiency (<5 ng/mL) should be treated with 50 000 IU orally, once a week, for 12 weeks, then 50 000 IU orally once a month for a total of 6 months. Alternatively, 500 000 IU may be given as a single intramuscular dose. Serum 25-hydroxyvitamin D levels in the range of 5 to 15 ng/mL (so called “mild deficiency”) should be treated with 50 000 IU of ergocaciferol orally once a week for 4 weeks followed by 50 000 IU orally once a month for a total of 6 months. Vitamin D insufficiency (serum levels between 16 and 30 ng/mL) should be treated with 50 000 IU of ergocalciferol orally once a month for 6 months. In vitamin D-deficient patients, serum 25(OH)D levels should be rechecked after completion of the 6 month course of therapy [176]. Treatment with Active Vitamin D Sterols As mentioned above, active vitamin D sterols act through a variety of pathways to decrease PTH production e by increasing calcium absorption in the gut and kidney, by binding to the CaSR, by increasing skeletal sensitivity to PTH and by altering prepro PTH transcription. Vitamin D sterols may also suppress PTH indirectly, through increasing osteocytic FGF-23 production [166] which, in turn, suppresses parathyroid gland PTH expression [14]. Calcitriol (RocaltrolÒ) has been widely used for many years to control secondary hyperparathyroidism in both adults and children. The efficacy of daily oral doses of calcitriol for the treatment of patients with symptomatic renal osteodystrophy has been documented in several clinical trials [233,234]. Bone pain diminishes, muscle strength and gait and posture improve, and osteitis fibrosa frequently resolves either partially or completely [89]. When measured by

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reliable assays, serum PTH levels decrease in patients who respond favorably to treatment. Doses of oral calcitriol in most clinical trials have ranged from 0.25 to 1.5 mg/day. In patients with chronic kidney disease, initial doses are determined by target PTH levels and specific stage of kidney disease [178]. In dialysis patients, 1,25(OH)2D3 given thrice weekly either by i.v. injection or by oral pulse therapy is effective in reducing serum PTH levels [123,235]. Dosage regimens range from 0.5e1.0 mg to 3.5e4.0 mg three times weekly or 2.0e5.0 mg twice weekly. Low doses should be used initially, and dosage adjustments should be based on frequent measurements of serum calcium, phosphorus and PTH levels. Oral 1a-hydroxyvitamin D3 (alfacalcidol) undergoes 25-hydroxylation in the liver to form calcitriol [236,237] and this agent is widely used in Europe, Japan, and Canada. Calcitriol and 1a-hydroxyvitamin D3 are similarly effective for the treatment of secondary hyperparathyroidism in patients with chronic kidney disease. Although calcitriol and alfacalcidol are effective in decreasing PTH levels and preventing osteitis fibrosis cystica, treatment with these sterols in combination with calcium-based binders often results in hypercalcemia and hyperphosphatemia, which limit their use and contribute to the development of soft tissue calcification. Thus, new vitamin D analogs have been developed to prevent or minimize the intestinal calcium and phosphorus absorption, while suppressing PTH levels as effectively as calcitriol. Three of these new vitamin D analogs are already on the market for use in patients with chronic kidney disease: 22-oxacalcitrol in Japan and 19-nor-1a1,25-dihydroxyvitamin D2 (paricalcitol) and 1a-hydroxyvitamin D2 (doxercalciferol) in the USA. 19-nor-1a,25(OH)2D2 (paricalcitol), is effective in controlling serum PTH levels in adult patients with stages 3 and 4 CKD [238], as well as in dialysis patients. The long-term consequences of therapy with paricalcitol in conjunction with the use of calcium-containing binders on vascular calcification and cardiovascular complications remain to be determined. However, in a large cohort of patients undergoing hemodialysis, higher survival rates were observed in dialyzed patients treated with paricalcitol when compared to those receiving calcitriol [239]. Another new vitamin analog, 1a-(OH)-vitamin D2 (1a-D2, doxercalciferol), is equipotent to 1a-(OH)vitamin D3 in intestinal calcium absorption and bone calcium mobilization in rats [240]. Doxercalciferol also effectively controls secondary hyperparathyroidism in adult patients with stable chronic kidney disease [241]. A comparative trial of calcitriol and doxercalciferol in the control of secondary hyperparathyroidism

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29. CHRONIC KIDNEY DISEASE MINERAL AND BONE DISORDER

in pediatric patients revealed no differences in the two vitamin D sterols in the control of secondary hyperparathyroidism or the development of hypercalcemia; however, osteoclastogenesis was suppressed to a greater degree during doxercalciferol therapy, although the mechanisms mediating in this specific effect remain to be defined [231]. Active vitamin D therapy has been associated with protective effects on both the heart and the kidney. Active vitamin D sterols ameliorate cardiac hypertrophy in animals [242], and calcitriol therapy improves cardiac systolic function in hemodialysis patients [243]. Administration of active vitamin D sterols also reduces proteinuria, fibrosis, and podocyte hypertrophy in subtotally nephrectomized rats [244] while paricalcitol treatment decreases proteinuria in CKD patients [245]. These effects may be mediated by suppression of the renineangiotensin system (RAS); indeed, in vitro studies have demonstrated that calcitriol, paricalcitol, and doxercalciferol all suppress the RAS to a similar degree [246]. However, all activated vitamin D analogs may also increase FGF-23 secretion. Although the consequences of these increased levels in dialyzed patients remain to be completely determined, current evidence suggests that excessive circulating FGF-23 is associated with increased mortality rates in incident and prevalent dialysis patients [154,155]. Thus, there is a paradoxical phenomenon between the survival advantage associated with therapy with active vitamin D sterols and the risk for greater mortality correlated to more elevated serum FGF-23 levels in dialysis patients. A growing body of evidence suggests additional health benefits of 25(OH)vitamin D therapy, primarily due to its immune-regulatory role. Current observations also suggest a role for 25(OH)vitamin D in improving survival in patients treated with maintenance dialysis [247]. Moreover, improved monocyte function has been observed in hemodialysis patients receiving ergocalciferol therapy [248].

Calicimimetics Cinacalcet, an allosteric activator of the calciumsensing receptor, is now available for the treatment of secondary hyperparathyroidism. This small organic molecule reduces serum PTH levels and has also been shown to decrease the calciumephosphorus ion product in adult patients treated with maintenance dialysis, regardless of the specific phosphate-binding agent [40,249]. Experiments in uremic rats have demonstrated that calcimimetics are able to halt the progression of parathyroid cell hyperplasia [40]; the antiproliferative effect of this agent shows promise for use of the molecule as a “medical parathyroidectomy”. These agents

may play a role in reversing the process of vascular calcification [250] and FGF-23 levels decline during calcimimetic therapy [251]; however, the long-term effect of these consequences on cardiovascular morbidity and mortality remain to be determined. Calcimimetics may also have a direct effect on bone biology; indeed, studies in animals suggest that these agents may increase osteoclast number and activity [252], thus increasing bone erosion. Due to the presence of the calcium-sensing receptor on the growth plate, these agents are not approved and should be used with caution in growing children.

Parathyroidectomy In many cases when parathyroid surgery is needed and undertaken, the parathyroid adenoma has become monoclonal and growth autonomous [253,254]. Patients with severe hyperparathyroidism are often unresponsive to vitamin D therapy and Cinacalcet, developing hypercalcemia and hyperphosphatemia without reduction in PTH values or parathyroid gland size [253]. Clinical features that indicate the need for parathyroidectomy are as follows: the presence of hyperplasia and/ or hypertrophy of the parathyroid glands (as documented by the presence of biochemical and radiographic features and, if necessary, the findings of osteitis fibrosa cystica on bone biopsy), elevated serum PTH levels unresponsive to vitamin D sterol therapy, persistent hypercalcemia, pruritus unresponsive to dialysis or other medical treatment, progressive extraskeletal calcification, severe skeletal pain or fractures, or calciphylaxis. Aluminum-related bone disease must first be ruled out in patients receiving low dose calcitriol with persistent hypercalcemia [255]. Other causes of hypercalcemia, such as sarcoidosis, malignancyrelated hypercalcemia, intake of calcium supplements, and the presence of adynamic/aplastic bone lesions not related to aluminum, should also be considered [68,256]. When a decision to perform parathyroid surgery has been made, it is essential to avoid a marked postoperative fall in serum calcium levels caused by the “hungry bone” syndrome. Because of the severity of the bone disease, this fall can be much more marked and prolonged than after parathyroidectomy for primary hyperparathyroidism. Renal patients should receive either daily oral calcitriol (0.5e1.0 mg) or some sort of intravenous active vitamin D sterol for 2e6 days before parathyroid surgery and during the postoperative period to stimulate intestinal calcium absorption and to maximize the effectiveness of oral calcium salts. By 24e36 hours after surgery, marked hypocalcemia with serum calcium levels below 7e8 mg/dL may develop. This condition can be associated with serious symptoms,

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including seizures resulting in fractures and tendon avulsion. For reasons which are still unclear, these seizures most often occur during the last 1e2 hours of a hemodialysis procedure or immediately thereafter. To reduce the risk of convulsions, an infusion containing calcium gluconate should be started in the operating room, upon removal of the parathyroid glands. Calcium gluconate should be initiated at a rate of 100 mg of calcium ion per hour. Serum calcium should be measured every 4e6 hours and the calcium gluconate infusion rate increased if the serum calcium level continues to fall. The infusion rate may exceed 200 mg/h. Enteral calcium carbonate is initiated once the patient is able to tolerate oral intake, and doses as high as 1.0 g (elemental calcium) given four to six times daily, along with vitamin D sterol in excess of 1.0e2.0 mg/day (for calcitriol e doses of other agents vary according to their potency) is often needed for patients with marked hypocalcemia. The intravenous calcium drip is weaned as soon as the oral intake of calcium salts is able to maintain normal serum calcium levels. The duration of intravenous calcium requirements varies greatly between patients e most patients require i.v. therapy for 2e3 days, but severe hypocalcemia may persist for several weeks or months, necessitating permanent central catheter access for daily home infusions of 800e1000 mg of calcium ion. Serum phosphorus levels may fall to subnormal levels postoperatively; phosphate treatment will markedly aggravate the hypocalcemia, and patients should not be treated with phosphate unless serum phosphorus falls below 2.0 mg/dL [257e260]. Hyperplastic parathyroid glands may also be infiltrated with ethanol or calcitriol to cause sclerosis of the parathyroid tissue. This technique has been used at some centers [261,262] with variable efficacy in reducing hyperplastic tissue. This technique is currently used by only a few centers worldwide.

Growth Hormone Therapy Recombinant growth hormone (rhGH) should be considered in children with growth failure that does not respond to optimization of nutrition, correction of acidosis, and control of renal osteodystrophy. Serum phosphorus and PTH levels should be well controlled prior to the initiation of rhGH in children with CKD. Serum phosphorus levels should be less than 1.5 times the upper limit for age and 1st PTH-IMA levels less than 1.5 times the upper target values for the CKD stage prior to rhGH therapy [124]. Growth hormone therapy will increase serum PTH levels during the initial first months of therapy; therefore, serum PTH levels should be monitored monthly. GH therapy should be temporarily discontinued if PTH

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levels exceed three times the upper target value for the CKD stage [124].

BONE DISEASE AFTER SUCCESSFUL KIDNEY TRANSPLANTATION Successful kidney transplantation corrects many of the abnormalities associated with renal osteodystrophy, but disorders of bone and mineral metabolism remain a major problem in such patients. Several factors have been implicated in the development of bone disease after organ transplantation, including persistent secondary hyperparathyroidism, prolonged immobilization, graft function and, most importantly, use of the different immunosuppressive agents. Hypercalcemia is not uncommon after renal transplantation. During the first several months it can be quite severe, and patients with severe secondary hyperparathyroidism before renal transplantation are at greatest risk. More often, hypercalcemia may be less severe, with serum calcium levels between 10.5 and 12.0 mg/ dL, and usually resolves within the first 12 months [263]. Parathyroidectomy should be considered when serum calcium levels are persistently above 12.5 mg/ dL for more than 1 year after transplantation [264].The calcimimetic agents may be useful in preventing the need for parathyroidectomy in these patients [265]. Hypophosphatemia may occur early in the posttransplant period, mainly in patients with severe secondary hyperparathyroidism, although persistent post-transplant elevation of serum FGF-23 levels may also contribute [266,267].The clinical manifestations are quite variable; some patients complain of malaise, fatigue, and muscle weakness [268,269]. Phosphorus supplementation is required when values are persistently below 2.0 mg/dL, primarily in pediatric patients. Significant bone loss has been shown to occur as early as 3e6 months after kidney transplantation [270,271]. Several factors are implicated in the development of bone disease after transplantation, including persistent alterations in mineral metabolism, prolonged immobilization, and the use of immunosuppressive agents required to maintain graft function. Osteonecrosis, or avascular necrosis, is by far the most debilitating skeletal complication associated with organ transplantation. In approximately 15% of patients, osteonecrosis will develop within 3 years of renal transplantation [272,273]. The occurrence of osteonecrosis in inpatients after cardiac, hepatic, and bone marrow transplantation suggests that glucocorticoids play a critical role in the pathogenesis of this disorder [274,275]. In both adult and pediatric kidney recipients, bone histologic changes associated with secondary hyperparathyroidism have been shown to resolve within

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29. CHRONIC KIDNEY DISEASE MINERAL AND BONE DISORDER

6 months after kidney transplantation [271]. However, some patients have persistently elevated rates of bone turnover, while others develop adynamic lesions, despite moderately elevated serum PTH levels [276]. Bone biopsy data from pediatric kidney recipients indicate that 67% of patients with stable graft function have features of normal bone formation, while 10% have adynamic bone lesion, and 23% have bone lesions characteristic of secondary hyperparathyroidism [277]. Bone resorption is typically increased [278], leading to a net loss of bone mass over time. Serum PTH levels are unable to discriminate between adynamic, normal, and increased bone turnover in the pediatric transplant population [277]. The use of maintenance corticosteroids have been implicated in these alterations; steroids decrease intestinal calcium absorption, enhance urinary calcium excretion, inhibit osteoblastic activity, decrease bone formation, and increase osteoclastic activity and bone resorption [279e282]. Likewise, cyclosporine has been reported to increase both bone formation and bone resorption and reduce cancellous bone volume in the rat [283,284]. By contrast, azathioprine has been shown to have minimal impact on skeletal remodeling [285]. The role of other immunosuppressive agents, such as mycophenolate mofetil, as potential modifiers of bone formation and bone resorption has not been evaluated. While bone turnover may return to normal, defective skeletal mineralization is present in the majority of pediatric transplant recipients [277]. Osteoid volume is increased while mineral apposition rate is markedly reduced [277]. Although the factors responsible for the persistent increase in osteoid formation remain to be fully explained, corticosteroid use may contribute, as may persistent imbalances in PTH, vitamin D, and FGF-23 metabolism [267]. After successful kidney transplantation with standard immunosuppressive regimens (daily corticosteroids, calcineurin inhibitor, and antimetabolite), growth may be accelerated by an improvement in kidney function, but catch-up growth may not be observed even in children who undergo transplantation very early in life [113]. Moreover, height deceleration occurs in approximately 75% of patients who undergo transplantation before the age of 15 years [286]. The etiology of persistent growth retardation is not completely understood, but immunosuppressive agents, persistent secondary hyperparathyroidism, altered vitamin D and FGF-23 metabolism, and the persistence of defective skeletal mineralization may all contribute. Children receiving steroid-free immunosuppressive regimens, those treated with alternate day steroids and those with better height SDS at the time of transplant attain the greatest final adult height [113,286e288]. Recombinant human growth hormone (rhGH) has

been used in children with significant height deficit after kidney transplantation. A substantial increase in linear growth occurs within the first year of rhGH therapy, but the magnitude of growth response may decline thereafter [289]. Cardiovascular disease continues to be the leading cause of death after renal transplantation. In the posttransplant period, the presence of hypertension is strongly linked to increased IMT and poor vessel distensibility in children [290]. Alterations in bone and mineral metabolism also may contribute, as impaired kidney function persists in the majority of patients in the posttransplant period [113,176]. Indeed, EBCT data indicate that vascular calcifications do not regress post-transplantation and contribute to the burden of cardiovascular disease in this population [291].

References [1] Moe S, Drueke T, Cunningham J, et al. Definition, evaluation, and classification of renal osteodystrophy: a position statement from Kidney Disease: Improving Global Outcomes (KDIGO). Kidney Int 2006;69:1945e53. [2] Groothoff JW, Offringa M, Van Eck-Smit BL, et al. Severe bone disease and low bone mineral density after juvenile renal failure. Kidney Int 2003;63:266e75. [3] Portale AA, Booth BE, Halloran BP, Morris Jr RC. Effect of dietary phosphorus on circulating concentrations of 1,25-dihydroxyvitamin D and immunoreactive parathyroid hormone in children with moderate renal insufficiency. J Clin Invest 1984; 73:1580e9. [4] Levin A, Bakris GL, Molitch M, et al. Prevalence of abnormal serum vitamin D, PTH, calcium, and phosphorus in patients with chronic kidney disease: results of the study to evaluate early kidney disease. Kidney Int 2007;71:31e8. [5] Gutierrez O, Isakova T, Rhee E, et al. Fibroblast growth factor-23 mitigates hyperphosphatemia but accentuates calcitriol deficiency in chronic kidney disease. J Am Soc Nephrol 2005;16: 2205e15. [6] Bacchetta J, Dubourg L, Harambat J, et al. The influence of glomerular filtration rate and age on fibroblast growth factor 23 serum levels in pediatric chronic kidney disease. J Clin Endocrinol Metab 2010;95:1741e8. [7] van HM, Fischer AK, Lehnhardt A, et al. Fibroblast growth factor 23 and bone metabolism in children with chronic kidney disease. Kidney Int 2010;78:200e6. [8] Antoniucci DM, Yamashita T, Portale AA. Dietary phosphorus regulates serum fibroblast growth factor-23 concentrations in healthy men. J Clin Endocrinol Metab 2006;91: 3144e9. [9] Wesseling-Perry K, Pereira RC, Wang H, et al. Relationship between plasma FGF-23 concentration and bone mineralization in children with renal failure on peritoneal dialysis. J Clin Endocrinol Metab 2009;94:511e7. [10] Pereira RC, Juppner H, Azucena-Serrano CE, Yadin O, Salusky IB, Wesseling-Perry K. Patterns of FGF-23, DMP1, and MEPE expression in patients with chronic kidney disease. Bone 2009;45:1161e8. [11] Shimada T, Hasegawa H, Yamazaki Y, et al. FGF-23 is a potent regulator of vitamin D metabolism and phosphate homeostasis. J Bone Miner Res 2004;19:429e35.

PEDIATRIC BONE

REFERENCES

[12] Larsson T, Nisbeth U, Ljunggren O, Juppner H, Jonsson KB. Circulating concentration of FGF-23 increases as renal function declines in patients with chronic kidney disease, but does not change in response to variation in phosphate intake in healthy volunteers. Kidney Int 2003;64:2272e9. [13] Krajisnik T, Bjorklund P, Marsell R, et al. Fibroblast growth factor-23 regulates parathyroid hormone and 1alpha-hydroxylase expression in cultured bovine parathyroid cells. J Endocrinol 2007;195:125e31. [14] Ben-Dov IZ, Galitzer H, Lavi-Moshayoff V, et al. The parathyroid is a target organ for FGF23 in rats. J Clin Invest 2007;117:4003e8. [15] Silver J, Russell J, Sherwood LM. Regulation by vitamin D metabolites of messenger ribonucleic acid for preproparathyroid hormone in isolated bovine parathyroid cells. Proc Natl Acad Sci USA 1985;82:4270e3. [16] Silver J, Naveh-Many T, Mayer H, Schmelzer HJ, Popovtzer MM. Regulation by vitamin D metabolites of parathyroid hormone gene transcription in vivo in the rat. J Clin Invest 1986;78: 1296e301. [17] Kifor O, Moore Jr FD, Wang P, et al. Reduced immunostaining for the extracellular Ca2þ-sensing receptor in primary and uremic secondary hyperparathyroidism. J Clin Endocrinol Metab 1996;81:1598e606. [18] Brown AJ, Zhong M, Finch J, et al. Rat calcium-sensing receptor is regulated by vitamin D but not by calcium. Am J Physiol 1996;270:F454e60. [19] Coburn JW, Koppel MH, Brickman AS, Massry SG. Study of intestinal absorption of calcium in patients with renal failure. Kidney Int 1973;3:264e72. [20] Holick MF. Vitamin D and the kidney. Kidney Int 1987;32:912e29. [21] Clemens TL, Adams JS, Henderson SL, Holick MF. Increased skin pigment reduces the capacity of skin to synthesise vitamin D3. Lancet 1982;1:74e6. [22] Koenig KG, Lindberg JS, Zerwekh JE, Padalino PK, Cushner HM, Copley JB. Free and total 1,25-dihydroxyvitamin D levels in subjects with renal disease. Kidney Int 1992;41:161e5. [23] Saha H. Calcium and vitamin D homeostasis in patients with heavy proteinuria. Clin Nephrol 1994;41:290e6. [24] Helvig CF, Cuerrier D, Hosfield CM, et al. Dysregulation of renal vitamin D metabolism in the uremic rat. Kidney Int 2010;78:463e72. [25] Zisman AL, Hristova M, Ho LT, Sprague SM. Impact of ergocalciferol treatment of vitamin D deficiency on serum parathyroid hormone concentrations in chronic kidney disease. Am J Nephrol 2007;27:36e43. [26] Chandra P, Binongo JN, Ziegler TR, et al. Cholecalciferol (vitamin D3) therapy and vitamin D insufficiency in patients with chronic kidney disease: a randomized controlled pilot study. Endocr Pract 2008;14:10e7. [27] Ritter CS, Armbrecht HJ, Slatopolsky E, Brown AJ. 25-Hydroxyvitamin D(3) suppresses PTH synthesis and secretion by bovine parathyroid cells. Kidney Int 2006;70:654e9. [28] Slatopolsky E, Caglar S, Pennell JP, et al. On the pathogenesis of hyperparathyroidism in chronic experimental renal insufficiency in the dog. J Clin Invest 1971;50:492e9. [29] Llach F, Massry SG. On the mechanism of secondary hyperparathyroidism in moderate renal insufficiency. J Clin Endocrinol Metab 1985;61:601e6. [30] Almaden Y, Canalejo A, Ballesteros E, Anon G, Canadillas S, Rodriguez M. Regulation of arachidonic acid production by intracellular calcium in parathyroid cells: effect of extracellular phosphate. J Am Soc Nephrol 2002;13:693e8.

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[31] Silver J, Kilav R, Sela-Brown A, Naveh-Many T. Molecular mechanisms of secondary hyperparathyroidism. Pediatr Nephrol 2000;14:626e8. [32] Brown EM, Gamba G, Riccardi D, et al. Cloning and characterization of an extracellular Ca(2þ)-sensing receptor from bovine parathyroid. Nature 1993;366:575e80. [33] John MR, Goodman WG, Gao P, Cantor TL, Salusky IB, Juppner H. A novel immunoradiometric assay detects fulllength human PTH but not amino-terminally truncated fragments: implications for PTH measurements in renal failure. J Clin Endocrinol Metab 1999;84:4287e90. [34] Freichel M, Zink-Lorenz A, Holloschi A, Hafner M, Flockerzi V, Raue F. Expression of a calcium-sensing receptor in a human medullary thyroid carcinoma cell line and its contribution to calcitonin secretion. Endocrinology 1996;137:3842e8. [35] Kirkwood JR, Ozonoff MB, Steinbach HL. Epiphyseal displacement after metaphyseal fracture in renal osteodystrophy. Am J Roentgenol Radium Ther Nucl Med 1972;115: 547e54. [36] Martin-Salvago M, Villar-Rodriguez JL, Palma-Alvarez A, Beato-Moreno A, Galera-Davidson H. Decreased expression of calcium receptor in parathyroid tissue in patients with hyperparathyroidism secondary to chronic renal failure. Endocr Pathol 2003;14:61e70. [37] Canaff L, Hendy GN. Human calcium-sensing receptor gene. Vitamin D response elements in promoters P1 and P2 confer transcriptional responsiveness to 1,25-dihydroxyvitamin D. J Biol Chem 2002;277:30337e50. [38] Li YC, Pirro AE, Amling M, et al. Targeted ablation of the vitamin D receptor: an animal model of vitamin D-dependent rickets type II with alopecia. Proc Natl Acad Sci USA 1997;94:9831e5. [39] Brown AJ, Zhong M, Ritter C, Brown EM, Slatopolsky E. Loss of calcium responsiveness in cultured bovine parathyroid cells is associated with decreased calcium receptor expression. Biochem Biophys Res Commun 1995;212:861e7. [40] Wada M, Furuya Y, Sakiyama J, et al. 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 1997;100:2977e83. [41] KDIGO Work Group. KDIGO Clinical Practice Guideline for the Diagnosis, Evaluation, Prevention, and Treatment of Chronic Kidney Disease-Mineral and Bone Disorder (CKD-MBD). Kidney Int 2009;76(S113):s1es130. [42] Atkins D, Peacock M. A comparison of the effects of the calcitonins, steroid hormones and thyroid hormones on the response of bone to parathyroid hormone in tissue culture. J Endocrinol 1975;64:573e83. [43] Lee K, Deeds JD, Bond AT, Juppner H, bou-Samra AB, Segre GV. In situ localization of PTH/PTHrP receptor mRNA in the bone of fetal and young rats. Bone 1993;14:341e5. [44] Sherrard DJ. Renal osteodystrophy. Semin Nephrol 1986;6:56e67. [45] Malluche HH, Ritz E, Lange HP, Schoeppe W. Changes of bone histology during maintenance hemodialysis at various levels of dialyzate Ca concentration. Clin Nephrol 1976;6:440e7. [46] Hamdy NA, Kanis JA, Beneton MN, et al. Effect of alfacalcidol on natural course of renal bone disease in mild to moderate renal failure. Br Med J 1995;310:358e63. [47] Norman ME, Mazur AT, Borden S, et al. Early diagnosis of juvenile renal osteodystrophy. J Pediatr 1980;97:226e32. [48] Massry SG, Friedler RM, Coburn JW. Excretion of phosphate and calcium. Physiology of their renal handling and relation to clinical medicine. Arch Intern Med 1973;131:828e59.

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814

29. CHRONIC KIDNEY DISEASE MINERAL AND BONE DISORDER

[49] Galceran T, Martin KJ, Morrissey JJ, Slatopolsky E. Role of 1,25-dihydroxyvitamin D on the skeletal resistance to parathyroid hormone. Kidney Int 1987;32:801e7. [50] Mathias R, Salusky I, Harman W, et al. Renal bone disease in pediatric and young adult patients on hemodialysis in a children’s hospital. J Am Soc Nephrol 1993;3:1938e46. [51] Salusky IB, Ramirez JA, Oppenheim W, Gales B, Segre GV, Goodman WG. Biochemical markers of renal osteodystrophy in pediatric patients undergoing CAPD/CCPD. Kidney Int 1994;45:253e8. [52] Urena P, Ferreira A, Morieux C, Drueke T, de Vernejoul MC. PTH/PTHrP receptor mRNA is down-regulated in epiphyseal cartilage growth plate of uraemic rats. Nephrol Dial Transplant 1996;11:2008e16. [53] Sanchez CP, Salusky IB, Kuizon BD, Abdella P, Juppner H, Goodman WG. Growth of long bones in renal failure: roles of hyperparathyroidism, growth hormone and calcitriol. Kidney Int 1998;54:1879e87. [54] Massry SG, Stein R, Garty J, et al. Skeletal resistance to the calcemic action of parathyroid hormone in uremia: role of 1,25 (OH)2 D3. Kidney Int 1976;9:467e74. [55] Urena P, Mannstadt M, Hruby M, et al. Parathyroidectomy does not prevent the renal PTH/PTHrP receptor down-regulation in uremic rats. Kidney Int 1995;47:1797e805. [56] Iwasaki-Ishizuka Y, Yamato H, Nii-Kono T, Kurokawa K, Fukagawa M. Downregulation of parathyroid hormone receptor gene expression and osteoblastic dysfunction associated with skeletal resistance to parathyroid hormone in a rat model of renal failure with low turnover bone. Nephrol Dial Transplant 2005;20:1904e11. [57] Kuwahara M, Inoshita S, Nakano Y, Terada Y, Takano Y, Sasaki S. Expression of bone type 1 PTH receptor in rats with chronic renal failure. Clin Exp Nephrol 2007;11:34e40. [58] Sneddon WB, Syme CA, Bisello A, et al. Activation-independent parathyroid hormone receptor internalization is regulated by NHERF1 (EBP50). J Biol Chem 2003;278:43787e96. [59] Nguyen-Yamamoto L, Rousseau L, Brossard JH, Lepage R, D’Amour P. Synthetic carboxyl-terminal fragments of parathyroid hormone (PTH) decrease ionized calcium concentration in rats by acting on a receptor different from the PTH/PTHrelated peptide receptor. Endocrinology 2001;142:1386e92. [60] Slatopolsky E, Finch J, Clay P, et al. A novel mechanism for skeletal resistance in uremia. Kidney Int 2000;58:753e61. [61] Langub MC, Monier-Faugere MC, Wang G, Williams JP, Koszewski NJ, Malluche HH. Administration of PTH-(7e84) antagonizes the effects of PTH-(1e84) on bone in rats with moderate renal failure. Endocrinology 2003;144:1135e8. [62] Divieti P, John MR, Juppner H, Bringhurst FR. Human PTH-(7-84) inhibits bone resorption in vitro via actions independent of the type 1 PTH/PTHrP receptor. Endocrinology 2002;143:171e6. [63] Wesseling-Perry K, Harkins GC, Wang H, et al. The calcemic response to continous PTH(1e34) infusion in end-stage kidney disease varies according to bone turnover: a potential role for PTH(7e84). J Clin Endocrinol Metab 2010. [64] Parfitt AM. The actions of parathyroid hormone on bone: relation to bone remodeling and turnover, calcium homeostasis, and metabolic bone disease. Part IV of IV parts: The state of the bones in uremic hyperaparathyroidism e the mechanisms of skeletal resistance to PTH in renal failure and pseudohypoparathyroidism and the role of PTH in osteoporosis, osteopetrosis, and osteofluorosis. Metabolism 1976;25:1157e88. [65] Parfitt AM. The actions of parathyroid hormone on bone: relation to bone remodeling and turnover, calcium homeostasis, and metabolic bone disease. Part I of IV parts: mechanisms of

[66]

[67]

[68] [69]

[70]

[71]

[72]

[73]

[74]

[75]

[76]

[77]

[78]

[79]

[80] [81]

[82]

calcium transfer between blood and bone and their cellular basis: morphological and kinetic approaches to bone turnover. Metabolism 1976;25:809e44. Parfitt AM. The actions of parathyroid hormone on bone: relation to bone remodeling and turnover, calcium homeostasis, and metabolic bone disease. Part III of IV parts; PTH and osteoblasts, the relationship between bone turnover and bone loss, and the state of the bones in primary hyperparathyroidism. Metabolism 1976;25:1033e69. Parfitt AM. The actions of parathyroid hormone on bone: relation to bone remodeling and turnover, calcium homeostasis, and metabolic bone diseases. II. PTH and bone cells: bone turnover and plasma calcium regulation. Metabolism 1976;25:909e55. Malluche H, Faugere MC. Renal bone disease 1990: an unmet challenge for the nephrologist. Kidney Int 1990;38:193e211. el-Husseini AA, el-Agroudy AE, El-Sayed M, Sobh MA, Ghoneim MA. A prospective randomized study for the treatment of bone loss with vitamin D during kidney transplantation in children and adolescents. Am J Transplant 2004;4:2052e7. Salusky IB, Coburn JW, Foley J, Nelson P, Fine RN. Effects of oral calcium carbonate on control of serum phosphorus and changes in plasma aluminum levels after discontinuation of aluminum-containing gels in children receiving dialysis. J Pediatr 1986;108:767e70. Goodman WG, Ramirez JA, Belin TR, et al. Development of adynamic bone in patients with secondary hyperparathyroidism after intermittent calcitriol therapy. Kidney Int 1994;46: 1160e6. Ott SM, Maloney NA, Klein GL, et al. Aluminum is associated with low bone formation in patients receiving chronic parenteral nutrition. Ann Intern Med 1983;98:910e4. London GM, Marty C, Marchais SJ, Guerin AP, Metivier F, de Vernejoul MC. Arterial calcifications and bone histomorphometry in end-stage renal disease. J Am Soc Nephrol 2004;15:1943e51. Ward MK, Feest TG, Ellis HA, Parkinson IS, Kerr DN. Osteomalacic dialysis osteodystrophy: Evidence for a water-borne aetiological agent, probably aluminium. Lancet 1978;1:841e5. Parkinson IS, Ward MK, Feest TG, Fawcett RW, Kerr DN. Fracturing dialysis osteodystrophy and dialysis encephalopathy. An epidemiological survey. Lancet 1979;1:406e9. Pierides AM, Edwards Jr WG, Cullum Jr UX, McCall JT, Ellis HA. Hemodialysis encephalopathy with osteomalacic fractures and muscle weakness. Kidney Int 1980;18:115e24. Nathan E, Pedersen SE. Dialysis encephalopathy in a non-dialysed uraemic boy treated with aluminium hydroxide orally. Acta Paediatr Scand 1980;69:793e6. Felsenfeld AJ, Gutman RA, Llach F, Harrelson JM. Osteomalacia in chronic renal failure: a syndrome previously reported only with maintenance dialysis. Am J Nephrol 1982;2:147e54. Griswold WR, Reznik V, Mendoza SA, Trauner D, Alfrey AC. Accumulation of aluminum in a nondialyzed uremic child receiving aluminum hydroxide. Pediatrics 1983;71:56e8. Kaye M. Oral aluminum toxicity in a non-dialyzed patient with renal failure. Clin Nephrol 1983;20:208e11. Andreoli SP, Bergstein JM, Sherrard DJ. Aluminum intoxication from aluminum-containing phosphate binders in children with azotemia not undergoing dialysis. N Engl J Med 1984;310: 1079e84. Sedman AB, Miller NL, Warady BA, Lum GM, Alfrey AC. Aluminum loading in children with chronic renal failure. Kidney Int 1984;26:201e4.

PEDIATRIC BONE

REFERENCES

[83] Kaehny WD, Hegg AP, Alfrey AC. Gastrointestinal absorption of aluminum from aluminum-containing antacids. N Engl J Med 1977;296:1389e90. [84] Salusky IB, Coburn JW, Brill J, et al. Bone disease in pediatric patients undergoing dialysis with CAPD or CCPD. Kidney Int 1988;33:975e82. [85] Kuizon BD, Goodman WG, Juppner H, et al. Diminished linear growth during intermittent calcitriol therapy in children undergoing CCPD. Kidney Int 1998;53:205e11. [86] Bakkaloglu SA, Wesseling-Perry K, Pereira RC, et al. Value of the new bone classification system in pediatric renal osteodystrophy. Clin J Am Soc Nephrol 2010;5:1860e6. [87] Cohen-Solal ME, Sebert JL, Boudailliez B, et al. Non-aluminic adynamic bone disease in non-dialyzed uremic patients: a new type of osteopathy due to overtreatment? Bone 1992;13:1e5. [88] Atsumi K, Kushida K, Yamazaki K, Shimizu S, Ohmura A, Inoue T. Risk factors for vertebral fractures in renal osteodystrophy. Am J Kidney Dis 1999;33:287e93. [89] Ott SM, Maloney NA, Coburn JW, Alfrey AC, Sherrard DJ. The prevalence of bone aluminum deposition in renal osteodystrophy and its relation to the response to calcitriol therapy. N Engl J Med 1982;307:709e13. [90] De Beur SM, Finnegan RB, Vassiliadis J, et al. Tumors associated with oncogenic osteomalacia express genes important in bone and mineral metabolism. J Bone Miner Res 2002;17:1102e10. [91] Jonsson KB, Zahradnik R, Larsson T, et al. Fibroblast growth factor 23 in oncogenic osteomalacia and X-linked hypophosphatemia. N Engl J Med 2003;348:1656e63. [92] Nelson AE, Bligh RC, Mirams M, et al. Clinical case seminar: Fibroblast growth factor 23: a new clinical marker for oncogenic osteomalacia. J Clin Endocrinol Metab 2003;88:4088e94. [93] Shimada T, Kakitani M, Yamazaki Y, et al. Targeted ablation of Fgf23 demonstrates an essential physiological role of FGF23 in phosphate and vitamin D metabolism. J Clin Invest 2004;113: 561e8. [94] Sitara D, Razzaque MS, Hesse M, et al. Homozygous ablation of fibroblast growth factor-23 results in hyperphosphatemia and impaired skeletogenesis, and reverses hypophosphatemia in Phex-deficient mice. Matrix Biol 2004;23:421e32. [95] Genuth SM, Klein L, Rabinovich S, King KC. Osteomalacia accompanying chronic anticonvulsant therapy. J Clin Endocrinol Metab 1972;35:378e86. [96] Pierides AM, Ellis HA, Ward M, et al. Barbiturate and anticonvulsant treatment in relation to osteomalacia with haemodialysis and renal transplantation. Br Med J 1976;1:190e3. [97] Wang M, Hercz G, Sherrard DJ, Maloney NA, Segre GV, Pei Y. Relationship between intact 1-84 parathyroid hormone and bone histomorphometric parameters in dialysis patients without aluminum toxicity. Am J Kidney Dis 1995;26:836e44. [98] Ferreira A, Frazao JM, Monier-Faugere MC, et al. Effects of sevelamer hydrochloride and calcium carbonate on renal osteodystrophy in hemodialysis patients. J Am Soc Nephrol 2008;19:405e12. [99] NIDDK. USRDS 1994 Annual Report. 2008. [100] Salusky IB, Goodman WG, Kuizon BD, et al. Similar predictive value of bone turnover using first- and second-generation immunometric PTH assays in pediatric patients treated with peritoneal dialysis. Kidney Int 2003;63:1801e8. [101] Salusky IB, Goodman WG, Sahney S, et al. Sevelamer controls parathyroid hormone-induced bone disease as efficiently as calcium carbonate without increasing serum calcium levels during therapy with active vitamin D sterols. J Am Soc Nephrol 2005;16:2501e8.

815

[102] Ho AY, Kung AW. Determinants of peak bone mineral density and bone area in young women. J Bone Miner Metab 2005;23:470e5. [103] Bouillon R, Van CS, Carmeliet G. Intestinal calcium absorption: Molecular vitamin D mediated mechanisms. J Cell Biochem 2003;88:332e9. [104] Noel LH, Zingraff J, Bardin T, Atienza C, Kuntz D, Drueke T. Tissue distribution of dialysis amyloidosis. Clin Nephrol 1987;27:175e8. [105] Kleinman KS, Coburn JW. Amyloid syndromes associated with hemodialysis. Kidney Int 1989;35:567e75. [106] Hampl H, Lobeck H, Bartel-Schwarze S, Stein H, Eulitz M, Linke RP. Clinical, morphologic, biochemical, and immunohistochemical aspects of dialysis-associated amyloidosis. ASAIO Trans 1987;33:250e9. [107] Baker LR, Ackrill P, Cattell WR, Stamp TC, Watson L. Iatrogenic osteomalacia and myopathy due to phosphate depletion. Br Med J 1974;3:150e2. [108] Powell DR. Effects of renal failure on the growth hormoneinsulin-like growth factor axis. J Pediatr 1997;131:S13e6. [109] Zanello SB, Collins ED, Marinissen MJ, Norman AW, Boland RL. Vitamin D receptor expression in chicken muscle tissue and cultured myoblasts. Horm Metab Res 1997;29:231e6. [110] Wright RS, Hunt SM. A radioimmunoassay for 17 alpha 20 betadihydroxy-4-pregnen-3-one: its use in measuring changes in serum levels at ovulation in atlantic salmon (Salmo salar), coho salmon (Oncorhynchus kisutch), and rainbow trout (Salmo gairdneri). Gen Comp Endocrinol 1982;47:475e82. [111] Mehls O, Ritz E, Krempien B, et al. Slipped epiphyses in renal osteodystrophy. Arch Dis Child 1975;50:545e54. [112] Tonshoff B, Schaefer F, Mehls O. Disturbance of growth hormoneeinsulin-like growth factor axis in uraemia. Implications for recombinant human growth hormone treatment. Pediatr Nephrol 1990;4:654e62. [113] North American Pediatric Renal Transplant Cooperative Study (NAPRTCS) 2006 Annual Report. 2006. [114] Nash MA, Torrado AD, Greifer I, Spitzer A, Edelmann Jr CM. Renal tubular acidosis in infants and children. Clinical course, response to treatment, and prognosis. J Pediatr 1972;80:738e48. [115] Challa A, Chan W, Krieg Jr RJ, et al. Effect of metabolic acidosis on the expression of insulin-like growth factor and growth hormone receptor. Kidney Int 1993;44:1224e7. [116] Maniar S, Kleinknecht C, Zhou X, Motel V, Yvert JP, Dechaux M. Growth hormone action is blunted by acidosis in experimental uremia or acid load. Clin Nephrol 1996;46:72e6. [117] Challa A, Krieg Jr RJ, Thabet MA, Veldhuis JD, Chan JC. Metabolic acidosis inhibits growth hormone secretion in rats: mechanism of growth retardation. Am J Physiol 1993;265: E547e53. [118] Green J, Maor G. Effect of metabolic acidosis on the growth hormone/IGF-I endocrine axis in skeletal growth centers. Kidney Int 2000;57:2258e67. [119] Lefebvre A, de Vernejoul MC, Gueris J, Goldfarb B, Graulet AM, Morieux C. Optimal correction of acidosis changes progression of dialysis osteodystrophy. Kidney Int 1989;36:1112e8. [120] Goodman WG, Salusky IB. Evolution of secondary hyperparathyroidism during oral calcitriol therapy in pediatric renal osteodystrophy. Contrib Nephrol 1991;90:189e95. [121] Waller SC, Ridout D, Cantor T, Rees L. Parathyroid hormone and growth in children with chronic renal failure. Kidney Int 2005;67:2338e45. [122] Schmitt CP, Ardissino G, Testa S, Claris-Appiani A, Mehls O. Growth in children with chronic renal failure on intermittent versus daily calcitriol. Pediatr Nephrol 2003;18:440e4.

PEDIATRIC BONE

816

29. CHRONIC KIDNEY DISEASE MINERAL AND BONE DISORDER

[123] Martin KJ, Ballal HS, Domoto DT, Blalock S, Weindel M. Pulse oral calcitriol for the treatment of hyperparathyroidism in patients on continuous ambulatory peritoneal dialysis: preliminary observations. Am J Kidney Dis 1992;19:540e5. [124] National Kidney Foundation. K/DOQI clinical practice guidelines for bone metabolism and disease in children with chronic kidney disease. Am J Kidney Dis 2005;46. S1e121. [125] Nickerson T, Huynh H. Vitamin D analogue EB1089-induced prostate regression is associated with increased gene expression of insulin-like growth factor binding proteins. J Endocrinol 1999;160:223e9. [126] Miyakoshi N, Richman C, Kasukawa Y, Linkhart TA, Baylink DJ, Mohan S. Evidence that IGF-binding protein-5 functions as a growth factor. J Clin Invest 2001;107:73e81. [127] Richman C, Baylink DJ, Lang K, Dony C, Mohan S. Recombinant human insulin-like growth factor-binding protein-5 stimulates bone formation parameters in vitro and in vivo. Endocrinology 1999;140:4699e705. [128] Longobardi L, Torello M, Buckway C, et al. A novel insulin-like growth factor (IGF)-independent role for IGF binding protein-3 in mesenchymal chondroprogenitor cell apoptosis. Endocrinology 2003;144:1695e702. [129] Collard TJ, Guy M, Butt AJ, et al. Transcriptional upregulation of the insulin-like growth factor binding protein IGFBP-3 by sodium butyrate increases IGF-independent apoptosis in human colonic adenoma-derived epithelial cells. Carcinogenesis 2003;24:393e401. [130] Samaan NA, Freeman RM. Growth hormone levels in severe renal failure. Metabolism 1970;19:102e13. [131] Tonshoff B, Veldhuis JD, Heinrich U, Mehls O. Deconvolution analysis of spontaneous nocturnal growth hormone secretion in prepubertal children with preterminal chronic renal failure and with end-stage renal disease. Pediatr Res 1995;37:86e93. [132] Tonshoff B, Sammet A, Sanden I, Mehls O, Waldherr R, Scharer K. Outcome and prognostic determinants in the hemolytic uremic syndrome of children. Nephron 1994;68: 63e70. [133] Schaefer F, Chen Y, Tsao T, Nouri P, Rabkin R. Impaired JAKSTAT signal transduction contributes to growth hormone resistance in chronic uremia. J Clin Invest 2001;108:467e75. [134] Tonshoff B, Cronin MJ, Reichert M, et al. Reduced concentration of serum growth hormone (GH)-binding protein in children with chronic renal failure: correlation with GH insensitivity. The European Study Group for Nutritional Treatment of Chronic Renal Failure in Childhood. The German Study Group for Growth Hormone Treatment in Chronic Renal Failure. J Clin Endocrinol Metab 1997;82:1007e13. [135] Powell DR, Liu F, Baker BK, et al. Modulation of growth factors by growth hormone in children with chronic renal failure. The Southwest Pediatric Nephrology Study Group. Kidney Int 1997;51:1970e9. [136] Conger JD, Hammond WS, Alfrey AC, Contiguglia SR, Stanford RE, Huffer WE. Pulmonary calcification in chronic dialysis patients. Clinical and pathologic studies. Ann Intern Med 1975;83:330e6. [137] Milliner DS, Zinsmeister AR, Lieberman E, Landing B. Soft tissue calcification in pediatric patients with end-stage renal disease. Kidney Int 1990;38:931e6. [138] Goodman WG, Goldin J, Kuizon BD, et al. Coronary-artery calcification in young adults with end-stage renal disease who are undergoing dialysis. N Engl J Med 2000;342:1478e83. [139] Oh J, Wunsch R, Turzer M, et al. Advanced coronary and carotid arteriopathy in young adults with childhood-onset chronic renal failure. Circulation 2002;106:100e5.

[140] Chavers BM, Li S, Collins AJ, Herzog CA. Cardiovascular disease in pediatric chronic dialysis patients. Kidney Int 2002; 62:648e53. [141] Mitsnefes MM, Kimball TR, Kartal J, et al. Cardiac and vascular adaptation in pediatric patients with chronic kidney disease: role of calcium-phosphorus metabolism. J Am Soc Nephrol 2005;16:2796e803. [142] Moe SM, Duan D, Doehle BP, O’Neill KD, Chen NX. Uremia induces the osteoblast differentiation factor Cbfa1 in human blood vessels. Kidney Int 2003;63:1003e11. [143] Shroff RC, McNair R, Skepper JN, et al. Chronic mineral dysregulation promotes vascular smooth muscle cell adaptation and extracellular matrix calcification. J Am Soc Nephrol 2010;21:103e12. [144] Jono S, McKee MD, Murry CE, et al. Phosphate regulation of vascular smooth muscle cell calcification. Circ Res 2000;87: E10e7. [145] Ahmed S, O’Neill KD, Hood AF, Evan AP, Moe SM. Calciphylaxis is associated with hyperphosphatemia and increased osteopontin expression by vascular smooth muscle cells. Am J Kidney Dis 2001;37:1267e76. [146] Bostrom K. Insights into the mechanism of vascular calcification. Am J Cardiol 2001;88:20Ee2E. [147] Moe SM, O’Neill KD, Duan D, et al. Medial artery calcification in ESRD patients is associated with deposition of bone matrix proteins. Kidney Int 2002;61:638e47. [148] Chen NX, O’Neill KD, Duan D, Moe SM. Phosphorus and uremic serum up-regulate osteopontin expression in vascular smooth muscle cells. Kidney Int 2002;62:1724e31. [149] Schafer C, Heiss A, Schwarz A, et al. The serum protein alpha 2-Heremans-Schmid glycoprotein/fetuin-A is a systemically acting inhibitor of ectopic calcification. J Clin Invest 2003;112: 357e66. [150] Schinke T, Amendt C, Trindl A, Poschke O, Muller-Esterl W, Jahnen-Dechent W. The serum protein alpha2-HS glycoprotein/ fetuin inhibits apatite formation in vitro and in mineralizing calvaria cells. A possible role in mineralization and calcium homeostasis. J Biol Chem 1996;271:20789e96. [151] Sweatt A, Sane DC, Hutson SM, Wallin R. Matrix Gla protein (MGP) and bone morphogenetic protein-2 in aortic calcified lesions of aging rats. J Thromb Haemost 2003;1:178e85. [152] Koh N, Fujimori T, Nishiguchi S, et al. Severely reduced production of klotho in human chronic renal failure kidney. Biochem Biophys Res Commun 2001;280:1015e20. [153] Russo D, Palmiero G, De Blasio AP, Balletta MM, Andreucci VE. Coronary artery calcification in patients with CRF not undergoing dialysis. Am J Kidney Dis 2004;44:1024e30. [154] Jean G, Bresson E, Terrat JC, et al. Peripheral vascular calcification in long-haemodialysis patients: associated factors and survival consequences. Nephrol Dial Transplant 2009;24: 948e55. [155] Gutierrez OM, Mannstadt M, Isakova T, et al. Fibroblast growth factor 23 and mortality among patients undergoing hemodialysis. N Engl J Med 2008;359:584e92. [156] Miyamoto K, Ito M, Segawa H, Kuwahata M. Molecular targets of hyperphosphataemia in chronic renal failure. Nephrol Dial Transplant 2003;18(Suppl. 3):iii79e80. [157] Tonelli M, Keech A, Shepherd J, et al. Effect of pravastatin in people with diabetes and chronic kidney disease. J Am Soc Nephrol 2005;16:3748e54. [158] Holdaas H, Fellstrom B, Cole E, et al. Long-term cardiac outcomes in renal transplant recipients receiving fluvastatin: the ALERT extension study. Am J Transplant 2005;5:2929e36.

PEDIATRIC BONE

REFERENCES

[159] Wanner C, Krane V, Marz W, et al. Atorvastatin in patients with type 2 diabetes mellitus undergoing hemodialysis. N Engl J Med 2005;353:238e48. [160] Block GA, Spiegel DM, Ehrlich J, et al. Effects of sevelamer and calcium on coronary artery calcification in patients new to hemodialysis. Kidney Int 2005;68:1815e24. [161] Block GA, Raggi P, Bellasi A, Kooienga L, Spiegel DM. Mortality effect of coronary calcification and phosphate binder choice in incident hemodialysis patients. Kidney Int 2007;71:438e41. [162] Oliveira RB, Cancela AL, Graciolli FG, et al. Early control of PTH and FGF23 in normophosphatemic CKD patients: a new target in CKD-MBD therapy? Clin J Am Soc Nephrol 2009. [163] Teng M, Wolf M, Ofsthun MN, et al. Activated injectable vitamin D and hemodialysis survival: a historical cohort study. J Am Soc Nephrol 2005;16:1115e25. [164] Teng M, Wolf M, Lowrie E, Ofsthun N, Lazarus JM, Thadhani R. Survival of patients undergoing hemodialysis with paricalcitol or calcitriol therapy. N Engl J Med 2003;349:446e56. [165] Tentori F, Hunt WC, Stidley CA, et al. Mortality risk among hemodialysis patients receiving different vitamin D analogs. Kidney Int 2006;70:1858e65. [166] Saito H, Maeda A, Ohtomo S, et al. Circulating FGF-23 is regulated by 1alpha,25-dihydroxyvitamin D3 and phosphorus in vivo. J Biol Chem. 2005;280:2543e9. [167] Gipstein RM, Coburn JW, Adams DA, et al. Calciphylaxis in man. A syndrome of tissue necrosis and vascular calcification in 11 patients with chronic renal failure. Arch Intern Med 1976;136:1273e80. [168] Bleyer AJ, Choi M, Igwemezie B, de la TE, White WL. A case control study of proximal calciphylaxis. Am J Kidney Dis 1998; 32:376e83. [169] Goldsmith DJ. Calciphylaxis, thrombotic diathesis and defects in coagulation regulation. Nephrol Dial Transplant 1997;12:1082e3. [170] Mohammed IA, Sekar V, Bubtana AJ, Mitra S, Hutchison AJ. Proximal calciphylaxis treated with calcimimetic ’Cinacalcet’. Nephrol Dial Transplant 2008;23:387e9. [171] Rogers NM, Teubner DJ, Coates PT. Calcific uremic arteriolopathy: advances in pathogenesis and treatment. Semin Dial 2007;20:150e7. [172] Fliser D, Kollerits B, Neyer U, et al. Fibroblast growth factor 23 (FGF23) predicts progression of chronic kidney disease: the mild to moderate kidney disease (MMKD) study. J Am Soc Nephrol 2007;18:2600e8. [173] Seiler S, Reichart B, Roth D, Seibert E, Fliser D, Heine GH. FGF23 and future cardiovascular events in patients with chronic kidney disease before initiation of dialysis treatment. Nephrol Dial Transplant 2010. [174], Levin A, Bakris GL, Molitch M, et al. Prevalence of abnormal serum vitamin D, PTH, calcium, and phosphorus in patients with chronic kidney disease: results of the study to evaluate early kidney disease. Kidney Int 2007;71:31e8. [175] Alfrey AC, Miller NL, Butkus D. Evaluation of body magnesium stores. J Lab Clin Med 1974;84:153e62. [176] National Kidney Foundation. K/DOQI clinical practice guidelines for bone metabolism and disease in chronic kidney disease. Am J Kidney Dis 2003;42:S1e201. [177] Brown EM, Wilson RE, Eastman RC, Pallotta J, Marynick SP. Abnormal regulation of parathyroid hormone release by calcium in secondary hyperparathyroidism due to chronic renal failure. J Clin Endocrinol Metab 1982;54:172e9. [178] Salusky IB, Fine RN, Kangarloo H, et al. “High-dose” calcitriol for control of renal osteodystrophy in children on CAPD. Kidney Int 1987;32:89e95.

817

[179] Kurz P, Monier-Faugere MC, Bognar B, et al. Evidence for abnormal calcium homeostasis in patients with adynamic bone disease. Kidney Int 1994;46:855e61. [180] Guillot AP, Hood VL, Runge CF, Gennari FJ. The use of magnesium-containing phosphate binders in patients with endstage renal disease on maintenance hemodialysis. Nephron 1982;30:114e7. [181] Sherrard DJ, Hercz G, Pei Y, et al. The spectrum of bone disease in end-stage renal failureean evolving disorder. Kidney Int 1993;43:436e42. [182] van Renen MJ, Hogg RJ, Sweeney AL, Henning PH, Penfold JL, Jureidini KF. Accelerated growth in short children with chronic renal failure treated with both strict dietary therapy and recombinant growth hormone. Pediatr Nephrol 1992;6:451e8. [183] Charhon SA, Delmas PD, Malaval L, et al. Serum bone Glaprotein in renal osteodystrophy: comparison with bone histomorphometry. J Clin Endocrinol Metab 1986;63:892e7. [184] Epstein S, Traberg H, Raja R, Poser J. Serum and dialysate osteocalcin levels in hemodialysis and peritoneal dialysis patients and after renal transplantation. J Clin Endocrinol Metab 1985;60:1253e6. [185] D’Amour P, Brossard JH, Rousseau L, et al. Structure of non-(1-84) PTH fragments secreted by parathyroid glands in primary and secondary hyperparathyroidism. Kidney Int 2005; 68:998e1007. [186] Brossard JH, Cloutier M, Roy L, Lepage R, Gascon-Barre M, D’Amour P. Accumulation of a non-(1-84) molecular form of parathyroid hormone (PTH) detected by intact PTH assay in renal failure: importance in the interpretation of PTH values. J Clin Endocrinol Metab 1996;81:3923e9. [187] Lepage R, Roy L, Brossard JH, et al. A non-(1-84) circulating parathyroid hormone (PTH) fragment interferes significantly with intact PTH commercial assay measurements in uremic samples. Clin Chem. 1998;44:805e9. [188] Brossard JH, Yamamoto LN, D’Amour P. Parathyroid hormone metabolites in renal failure: bioactivity and clinical implications. Semin Dial 2002;15:196e201. [189] Gao P, Scheibel S, D’Amour P, et al. Development of a novel immunoradiometric assay exclusively for biologically active whole parathyroid hormone 1-84: implications for improvement of accurate assessment of parathyroid function. J Bone Miner Res 2001;16:605e14. [190] Monier-Faugere MC, Geng Z, Mawad H, et al. Improved assessment of bone turnover by the PTH-(1-84)/large C-PTH fragments ratio in ESRD patients. Kidney Int 2001;60:1460e8. [191] Coen G, Bonucci E, Ballanti P, et al. PTH 1-84 and PTH “7-84” in the noninvasive diagnosis of renal bone disease. Am J Kidney Dis 2002;40:348e54. [192] Alfrey AC. Aluminum metabolism. Kidney Int Suppl 1986;18:S8e11. [193] Andress DL, Endres DB, Maloney NA, Kopp JB, Coburn JW, Sherrard DJ. Comparison of parathyroid hormone assays with bone histomorphometry in renal osteodystrophy. J Clin Endocrinol Metab 1986;63:1163e9. [194] Wright RS, Mehls O, Ritz E, et al. Musculoskeletal manifestation of chronic renal failure, dialysis and transplantation. In: Bacon P, Hadler N, editors. Renal Manifestations in Rheumatic Disease. London: Butterworth Publishers; 1982. p. 352. [195] Dent CE, Hodson CJ. Radiological changes associated with certain metabolic bone diseases. Br J Radiol 1954;27:605e18. [196] Parfitt AM, Kleerekoper M, Cruz C. Reduced phosphate reabsorption unrelated to parathyroid hormone after renal transplantation: implications for the pathogenesis of hyperparathyroidism in chronic renal failure. Miner Electrolyte Metab 1986;12:356e62.

PEDIATRIC BONE

818

29. CHRONIC KIDNEY DISEASE MINERAL AND BONE DISORDER

[197] Meema HE, Rabinovich S, Meema S, Lloyd GJ, Oreopoulos DG. Improved radiological diagnosis of azotemic osteodystrophy. Radiology 1972;102:1e10. [198] Shimada H, Nakamura M, Marumo F. Influence of aluminium on the effect of 1 alpha (OH)D3 on renal osteodystrophy. Nephron 1983;35:163e70. [199] Simpson W, Ellis HA, Kerr DN, McElroy M, McNay RA, Ppeart KN. Bone disease in long-term haemodialysis: the association of radiological with histological abnormalities. Br J Radiol 1976;49:105e10. [200] Parfitt AM, Massry SG, Winfield AC. Osteopenia and fractures occurring during maintenance hemodialysis. A new form of renal osteodystrophy. Clin Orthop Relat Res 1972;87:287e302. [201] Mallette LE, Patten BM, Engel WK. Neuromuscular disease in secondary hyperparathyroidism. Ann Intern Med 1975;82: 474e83. [202] Block GA, Hulbert-Shearon TE, Levin NW, Port FK. Association of serum phosphorus and calcium x phosphate product with mortality risk in chronic hemodialysis patients: a national study. Am J Kidney Dis 1998;31:607e17. [203] Mucsi I, Hercz G, Uldall R, Ouwendyk M, Francoeur R, Pierratos A. Control of serum phosphate without any phosphate binders in patients treated with nocturnal hemodialysis. Kidney Int 1998;53:1399e404. [204] Fischbach M, Terzic J, Menouer S, et al. Intensified and daily hemodialysis in children might improve statural growth. Pediatr Nephrol 2006;21:1746e52. [205] Goodman WG. Aluminum and renal osteodystrophy. In: Bushinsky DA, editor. Renal Osteodystrophy Lippincott-Raven Philadelphia; 1998. p. 317. [206] Salusky IB, Foley J, Nelson P, Goodman WG. Aluminum accumulation during treatment with aluminum hydroxide and dialysis in children and young adults with chronic renal disease. N Engl J Med 1991;324:527e31. [207] Coburn JW, Mischel MG, Goodman WG, Salusky IB. Calcium citrate markedly enhances aluminum absorption from aluminum hydroxide. Am J Kidney Dis 1991;17:708e11. [208] Bakir AA, Hryhorczuk DO, Berman E, Dunea G. Acute fatal hyperaluminemic encephalopathy in undialyzed and recently dialyzed uremic patients. ASAIO Trans 1986;32:171e6. [209] Portale AA, Booth BE, Tsai HC, Morris Jr RC. Reduced plasma concentration of 1,25-dihydroxyvitamin D in children with moderate renal insufficiency. Kidney Int 1982;21:627e32. [210] Alon U, Davidai G, Bentur L, Berant M, Better OS. Oral calcium carbonate as phosphate-binder in infants and children with chronic renal failure. Miner Electrolyte Metab 1986;12:320e5. [211] Andreoli SP, Dunson JW, Bergstein JM. Calcium carbonate is an effective phosphorus binder in children with chronic renal failure. Am J Kidney Dis 1987;9:206e10. [212] Salusky IB, Kuizon BD, Belin TR, et al. Intermittent calcitriol therapy in secondary hyperparathyroidism: a comparison between oral and intraperitoneal administration. Kidney Int 1998;54:907e14. [213] Salusky IB, Goodman WG. Adynamic renal osteodystrophy: is there a problem? J Am Soc Nephrol 2001;12:1978e85. [214] Pflanz S, Henderson IS, McElduff N, Jones MC. Calcium acetate versus calcium carbonate as phosphate-binding agents in chronic haemodialysis. Nephrol Dial Transplant 1994;9:1121e4. [215] Caravaca F, Santos I, Cubero JJ, et al. Calcium acetate versus calcium carbonate as phosphate binders in hemodialysis patients. Nephron 1992;60:423e7. [216] Wallot M, Bonzel KE, Winter A, Georger B, Lettgen B, Bald M. Calcium acetate versus calcium carbonate as oral phosphate binder in pediatric and adolescent hemodialysis patients. Pediatr Nephrol 1996;10:625e30.

[217] Cushner HM, Copley JB, Lindberg JS, Foulks CJ. Calcium citrate, a nonaluminum-containing phosphate-binding agent for treatment of CRF. Kidney Int 1988;33:95e9. [218] Birck R, Zimmermann E, Wassmer S, Nowack R, van der Woude FJ. Calcium ketoglutarate versus calcium acetate for treatment of hyperphosphataemia in patients on maintenance haemodialysis: a cross-over study. Nephrol Dial Transplant 1999;14:1475e9. [219] Slatopolsky EA, Burke SK, Dillon MA. RenaGel, a nonabsorbed calcium- and aluminum-free phosphate binder, lowers serum phosphorus and parathyroid hormone. The RenaGel Study Group. Kidney Int 1999;55:299e307. [220] Bleyer AJ, Burke SK, Dillon M, et al. A comparison of the calcium-free phosphate binder sevelamer hydrochloride with calcium acetate in the treatment of hyperphosphatemia in hemodialysis patients. Am J Kidney Dis 1999;33:694e701. [221] Chertow GM, Burke SK, Dillon MA, Slatopolsky E. Long-term effects of sevelamer hydrochloride on the calcium x phosphate product and lipid profile of haemodialysis patients. Nephrol Dial Transplant 1999;14:2907e14. [222] Chertow GM, Burke SK, Raggi P. Sevelamer attenuates the progression of coronary and aortic calcification in hemodialysis patients. Kidney Int 2002;62:245e52. [223] Russo D, Battaglia Y, Buonanno E. Phosphorus and coronary calcification in predialysis patients. Kidney Int 2010;78:818. [224] Delmez J, Block G, Robertson J, et al. A randomized, doubleblind, crossover design study of sevelamer hydrochloride and sevelamer carbonate in patients on hemodialysis. Clin Nephrol 2007;68:386e91. [225] O’Donovan R, Baldwin D, Hammer M, Moniz C, Parsons V. Substitution of aluminium salts by magnesium salts in control of dialysis hyperphosphataemia. Lancet 1986;1:880e2. [226] Hergesell O, Ritz E. Phosphate binders on iron basis: a new perspective? Kidney Int Suppl 1999;73:S42e5. [227] Hergesell O, Ritz E. Stabilized polynuclear iron hydroxide is an efficient oral phosphate binder in uraemic patients. Nephrol Dial Transplant 1999;14:863e7. [228] Spasovski GB, Sikole A, Gelev S, et al. Evolution of bone and plasma concentration of lanthanum in dialysis patients before, during 1 year of treatment with lanthanum carbonate and after 2 years of follow-up. Nephrol Dial Transplant 2006;21:2217e24. [229] Cancela AL, Oliveira RB, Graciolli FG, et al. Fibroblast growth factor 23 in hemodialysis patients: effects of phosphate binder, calcitriol and calcium concentration in the dialysate. Nephron Clin Pract 2010;117:c74e82. [230] Isakova T, Gutierrez OM, Smith K, et al. Pilot study of dietary phosphorus restriction and phosphorus binders to target fibroblast growth factor 23 in patients with chronic kidney disease. Nephrol Dial Transplant 2010. [231] Wesseling-Perry K, Pereira RC, Sahney S, et al. Calcitriol and doxercalciferol are equivalent in controlling bone turnover, suppressing parathyroid hormone, and increasing fibroblast growth factor-23 in secondary hyperparathyroidism. Kidney Int 2010. [232] Gutierrez OM, Januzzi JL, Isakova T, et al. Fibroblast growth factor 23 and left ventricular hypertrophy in chronic kidney disease. Circulation 2009;119:2545e52. [233] Baker LR, Muir JW, Sharman VL, et al. Controlled trial of calcitriol in hemodialysis patients. Clin Nephrol 1986;26:185e91. [234] Berl T, Berns AS, Hufer WE, et al. 1,25 dihydroxycholecalciferol effects in chronic dialysis. A double-blind controlled study. Ann Intern Med 1978;88:774e80. [235] Fukagawa M, Okazaki R, Takano K, et al. Regression of parathyroid hyperplasia by calcitriol-pulse therapy in patients on long-term dialysis. N Engl J Med 1990;323:421e2.

PEDIATRIC BONE

REFERENCES

[236] Pierides AM, Ellis HA, Simpson W, Dewar JH, Ward MK, Kerr DN. Variable response to long-term 1alpha-hydroxycholecalciferol in haemodialysis osteodystrophy. Lancet 1976;1:1092e5. [237] Kanis JA, Henderson RG, Heynen G, et al. Renal osteodystrophy in nondialysed adolescents. Long-term treatment with 1alpha-hydroxycholecalciferol. Arch Dis Child 1977;52:473e81. [238] Coyne D, Acharya M, Qiu P, et al. Paricalcitol capsule for the treatment of secondary hyperparathyroidism in stages 3 and 4 CKD. Am J Kidney Dis 2006;47:263e76. [239] Dobrez DG, Mathes A, Amdahl M, Marx SE, Melnick JZ, Sprague SM. Paricalcitol-treated patients experience improved hospitalization outcomes compared with calcitriol-treated patients in real-world clinical settings. Nephrol Dial Transplant 2004;19:1174e81. [240] Sjoden G, Smith C, Lindgren U, DeLuca HF. 1 alpha-Hydroxyvitamin D2 is less toxic than 1 alpha-hydroxyvitamin D3 in the rat. Proc Soc Exp Biol Med 1985;178:432e6. [241] Andress DL, Norris KC, Coburn JW, Slatopolsky EA, Sherrard DJ. Intravenous calcitriol in the treatment of refractory osteitis fibrosa of chronic renal failure. N Engl J Med 1989;321:274e9. [242] Bodyak N, Ayus JC, Achinger S, et al. Activated vitamin D attenuates left ventricular abnormalities induced by dietary sodium in Dahl salt-sensitive animals. Proc Natl Acad Sci USA 2007;104:16810e5. [243] Singh NP, Sahni V, Garg D, Nair M. Effect of pharmacological suppression of secondary hyperparathyroidism on cardiovascular hemodynamics in predialysis CKD patients: A preliminary observation. Hemodial Int 2007;11:417e23. [244] Schwarz U, Amann K, Orth SR, Simonaviciene A, Wessels S, Ritz E. Effect of 1,25 (OH)2 vitamin D3 on glomerulosclerosis in subtotally nephrectomized rats. Kidney Int 1998;53:1696e705. [245] Agarwal R, Acharya M, Tian J, et al. Antiproteinuric effect of oral paricalcitol in chronic kidney disease. Kidney Int 2005;68:2823e8. [246] Nakane M, Ma J, Ruan X, Kroeger PE, Wu-Wong R. Mechanistic analysis of VDR-mediated renin suppression. Nephron Physiol 2007;107:35e44. [247] Wolf M, Shah A, Gutierrez O, et al. Vitamin D levels and early mortality among incident hemodialysis patients. Kidney Int 2007;72:1004e13. [248] Stubbs JR, Idiculla A, Slusser J, Menard R, Quarles LD. Cholecalciferol supplementation alters calcitriol-responsive monocyte proteins and decreases inflammatory cytokines in ESRD. J Am Soc Nephrol 2010;21:353e61. [249] Wada M, Nagano N. Control of parathyroid cell growth by calcimimetics. Nephrol Dial Transplant 2003;18(Suppl. 3):iii13e7. [250] Lopez I, Aguilera-Tejero E, Mendoza FJ, et al. Calcimimetic R568 decreases extraosseous calcifications in uremic rats treated with calcitriol. J Am Soc Nephrol 2006;17:795e804. [251] Wetmore JB, Liu S, Krebill R, Menard R, Quarles LD. Effects of cinacalcet and concurrent low-dose vitamin D on FGF23 levels in ESRD. Clin J Am Soc Nephrol 2010;5:110e6. [252] Nguyen-Yamamoto L, Bolivar I, Strugnell SA, Goltzman D. Comparison of active vitamin D compounds and a calcimimetic in mineral homeostasis. J Am Soc Nephrol 2010. [253] Quarles LD, Yohay DA, Carroll BA, et al. Prospective trial of pulse oral versus intravenous calcitriol treatment of hyperparathyroidism in ESRD. Kidney Int 1994;45:1710e21. [254] Arnold A, Brown MF, Urena P, Gaz RD, Sarfati E, Drueke TB. Monoclonality of parathyroid tumors in chronic renal failure and in primary parathyroid hyperplasia. J Clin Invest 1995;95: 2047e53.

819

[255] Froment DP, Molitoris BA, Buddington B, Miller N, Alfrey AC. Site and mechanism of enhanced gastrointestinal absorption of aluminum by citrate. Kidney Int 1989;36:978e84. [256] Jorna FH, Tobe TJ, Huisman RM, de Jong PE, Plukker JT, Stegeman CA. Early identification of risk factors for refractory secondary hyperparathyroidism in patients with long-term renal replacement therapy. Nephrol Dial Transplant 2004;19:1168e73. [257] Andress DL, Ott SM, Maloney NA, Sherrard DJ. Effect of parathyroidectomy on bone aluminum accumulation in chronic renal failure. N Engl J Med 1985;312:468e73. [258] de Vernejoul MC, Marchais S, London G, Morieux C, Bielakoff J, Miravet L. Increased bone aluminum deposition after subtotal parathyroidectomy in dialyzed patients. Kidney Int 1985;27: 785e91. [259] Kurokawa K, Akizawa T, Suzuki M, Akiba T, Ogata E, Slatopolsky E. Effect of 22-oxacalcitriol on hyperparathyroidism of dialysis patients: results of a preliminary study. Nephrol Dial Transplant 1996;11(Suppl. 3):121e4. [260] Charytan C, Coburn JW, Chonchol M, et al. Cinacalcet hydrochloride is an effective treatment for secondary hyperparathyroidism in patients with CKD not receiving dialysis. Am J Kidney Dis 2005;46:58e67. [261] de Barros Gueiros JE, Chammas MC, Gerhard R, et al. Percutaneous ethanol (PEIT) and calcitrol (PCIT) injection therapy are ineffective in treating severe secondary hyperparathyroidism. Nephrol Dial Transplant 2004;19:657e63. [262] Fukagawa M, Kitaoka M, Tominaga Y, et al. Guidelines for percutaneous ethanol injection therapy of the parathyroid glands in chronic dialysis patients. Nephrol Dial Transplant 2003;18(Suppl. 3):iii31e3. [263] Diethelm AG, Edwards RP, Whelchel JD. The natural history and surgical treatment of hypercalcemia before and after renal transplantation. Surg Gynecol Obstet 1982;154:481e90. [264] D’Alessandro AM, Melzer JS, Pirsch JD, et al. Tertiary hyperparathyroidism after renal transplantation: operative indications. Surgery 1989;106:1049e55. [265] Bergua C, Torregrosa JV, Fuster D, Gutierrez-Dalmau A, Oppenheimer F, Campistol JM. Effect of cinacalcet on hypercalcemia and bone mineral density in renal transplanted patients with secondary hyperparathyroidism. Transplantation 2008;86:413e7. [266] Bhan I, Shah A, Holmes J, et al. Post-transplant hypophosphatemia: tertiary ’hyper-phosphatoninism’? Kidney Int 2006;70:1486e94. [267] Evenepoel P, Naesens M, Claes K, Kuypers D, Vanrenterghem Y. Tertiary ’hyperphosphatoninism’ accentuates hypophosphatemia and suppresses calcitriol levels in renal transplant recipients. Am J Transplant 2007;7:1193e200. [268] Bonomini V, Feletti C, Di FA, Buscaroli A. Bone remodelling after renal transplantation (RT). Adv Exp Med Biol 1984;178:207e16. [269] Nielsen HE, Melsen F, Christensen MS. Aseptic necrosis of bone following renal transplantation. Clinical and biochemical aspects and bone morphometry. Acta Med Scand 1977;202:27e32. [270] Grotz WH, Mundinger FA, Gugel B, Exner VM, Kirste G, Schollmeyer PJ. Bone mineral density after kidney transplantation. A cross-sectional study in 190 graft recipients up to 20 years after transplantation. Transplantation 1995;59:982e6. [271] Julian BA, Laskow DA, Dubovsky J, Dubovsky EV, Curtis JJ, Quarles LD. Rapid loss of vertebral mineral density after renal transplantation. N Engl J Med 1991;325:544e50. [272] Slatopolsky E, Martin K. Glucocorticodids and renal transplant osteonecrosis. Adv Exp Med Biol 1984;171:353e9.

PEDIATRIC BONE

820

29. CHRONIC KIDNEY DISEASE MINERAL AND BONE DISORDER

[273] Parfrey PS, Farge D, Parfrey NA, Hanley JA, Guttman RD. The decreased incidence of aseptic necrosis in renal transplant recipientsea case control study. Transplantation 1986;41:182e7. [274] Isono SS, Woolson ST, Schurman DJ. Total joint arthroplasty for steroid-induced osteonecrosis in cardiac transplant patients. Clin Orthop Relat Res 1987;217:201e8. [275] Enright H, Haake R, Weisdorf D. Avascular necrosis of bone: a common serious complication of allogeneic bone marrow transplantation. Am J Med 1990;89:733e8. [276] Velasquez-Forero F, Mondragon A, Herrero B, Pena JC. Adynamic bone lesion in renal transplant recipients with normal renal function. Nephrol Dial Transplant 1996;11(Suppl. 3): 58e64. [277] Sanchez CP, Salusky IB, Kuizon BD, et al. Bone disease in children and adolescents undergoing successful renal transplantation. Kidney Int 1998;53:1358e64. [278] Lindberg JS, Culleton B, Wong G, et al. Cinacalcet HCl, an oral calcimimetic agent for the treatment of secondary hyperparathyroidism in hemodialysis and peritoneal dialysis: a randomized, double-blind, multicenter study. J Am Soc Nephrol 2005;16:800e7. [279] Allen DB, Goldberg BD. Stimulation of collagen synthesis and linear growth by growth hormone in glucocorticoid-treated children. Pediatrics 1992;89:416e21. [280] Root AW, Bongiovanni AM, Eberlein WR. Studies of the secretion and metabolic effects of human growth hormone in children with glucocorticoid-induced growth retardation. J Pediatr 1969;75:826e32. [281] Ortoft G, Oxlund H. Qualitative alterations of cortical bone in female rats after long-term administration of growth hormone and glucocorticoid. Bone 1996;18:581e90. [282] Wehrenberg WB, Bergman PJ, Stagg L, Ndon J, Giustina A. Glucocorticoid inhibition of growth in rats: partial reversal with somatostatin antibodies. Endocrinology 1990;127:2705e8.

[283] Aubia J, Serrano S, Marinoso L, et al. Osteodystrophy of diabetics in chronic dialysis: a histomorphometric study. Calcif Tissue Int 1988;42:297e301. [284] Movsowitz C, Epstein S, Fallon M, Ismail F, Thomas S. Cyclosporin-A in vivo produces severe osteopenia in the rat: effect of dose and duration of administration. Endocrinology 1988;123:2571e7. [285] Bryer HP, Isserow JA, Armstrong EC, et al. Azathioprine alone is bone sparing and does not alter cyclosporin A-induced osteopenia in the rat. J Bone Miner Res 1995;10:132e8. [286] Hokken-Koelega AC, van Zaal MA, van BW, et al. Final height and its predictive factors after renal transplantation in childhood. Pediatr Res 1994;36:323e8. [287] Sarwal MM, Yorgin PD, Alexander S, et al. Promising early outcomes with a novel, complete steroid avoidance immunosuppression protocol in pediatric renal transplantation. Transplantation 2001;72:13e21. [288] Sarwal MM, Vidhun JR, Alexander SR, Satterwhite T, Millan M, Salvatierra Jr O. Continued superior outcomes with modification and lengthened follow-up of a steroid-avoidance pilot with extended daclizumab induction in pediatric renal transplantation. Transplantation 2003;76:1331e9. [289] Fine RN, Yadin O, Nelson PA, et al. Recombinant human growth hormone treatment of children following renal transplantation. Pediatr Nephrol 1991;5:147e51. [290] Mitsnefes MM, Kimball TR, Witt SA, Glascock BJ, Khoury PR, Daniels SR. Abnormal carotid artery structure and function in children and adolescents with successful renal transplantation. Circulation 2004;110:97e101. [291] Ishitani MB, Milliner DS, Kim DY, et al. Early subclinical coronary artery calcification in young adults who were pediatric kidney transplant recipients. Am J Transplant 2005;5: 1689e93.

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Extraskeletal Bone Formation Eileen M. Shore 1, Frederick S. Kaplan 2 1

Departments of Orthopedic Surgery, Genetics and the Center for Research in FOP and Related Disorders, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA 2 Departments of Orthopedic Surgery, Medicine and the Center for Research in FOP and Related Disorders, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA

INTRODUCTION During embryonic development, induction of bone formation is spatially and temporally regulated to form the skeletal elements. After birth, new bone formation is normally limited to bone regeneration during fracture repair, a process that is also precisely regulated [1]. Induction of extraskeletal bone formation, also known as heterotopic ossification (HO), is a pathological condition in which bone forms in tissues outside of the normal skeleton [2,3]. Heterotopic ossification forms through endochondral or intramembranous processes and produces qualitatively normal bone tissue. It is the induction of the bone formation process that is abnormally regulated in these conditions. Extensive heterotopic ossification that begins during childhood occurs in two clinically distinct genetic diseases: fibrodysplasia ossificans progressiva (FOP) and progressive osseous heteroplasia (POH) [4e6]. In each of these rare disorders, bone formation is progressive and continues throughout life. Both FOP and POH are caused by single gene mutations, although the mutation in each affects different cell signaling pathways. These conditions provide important and unique perspectives to understanding the cellular and molecular regulatory mechanisms of bone formation and will contribute to understanding the causes of more common forms of heterotopic ossification. Extraskeletal bone formation also can occur in cases other than these rare genetic diseases (Table 30.1); such conditions are referred to as non-hereditary heterotopic ossification (NHHO). In adults, heterotopic ossification is a frequent complication of a number of common conditions associated with severe tissue trauma, such as spinal cord and head injuries, hip replacement surgery, severe burns, and high impact

Pediatric Bone, Second Edition DOI: 10.1016/B978-0-12-382040-2.10030-9

war wounds, as well as in age-associated conditions such as atherosclerosis and pressure ulcers [2,3,7e10]. In children, bone formation outside of the skeleton is extremely rare, but is occasionally associated with trauma or in extraskeletal osteosarcoma (ESOS), a rare malignant mesenchymal cell neoplasm. Since the genetic forms of heterotopic ossification have more significant relevance to pediatric care, these conditions will be the main focus of this chapter.

NON-HEREDITARY FORMS OF PEDIATRIC HETEROTOPIC OSSIFICATION In addition to the rare genetic forms of HO that are discussed in detail in following sections, non-familial heterotopic ossification (also known as non-hereditary heterotopic ossification, NHHO) occurs in association with various forms of trauma [2,3,11], however, there are few reports in the pediatric population. Little is known about the etiology of these forms of extraskeletal bone formation. In children, heterotopic ossification has formed in response to severe full-thickness burns [12e14], muscle or joint trauma [15e18], and neurologic injuries, most commonly head trauma and spinal cord injury [19,20]. Heterotopic ossification in children has also been associated with hemophilia [21,22] and hypoxic brain damage [20]. Extraskeletal osteosarcoma (ESOS) is an aggressive malignant tumor of mesenchymal cells in which malignant osteoblasts produce osteoid and bone in extraskeletal soft tissues [23]. ESOS comprise only about 1% of soft-tissue sarcomas and 4% of osteosarcomas; fewer than 300 cases of this rare tumor have been reported in

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TABLE 30.1

Heterotopic Ossification (HO) in Children

A

B

Cause

Non-hereditary HO Severe muscle or joint trauma Burns Head and spinal cord injury

FIGURE 30.1

Hemophilia

Characteristic clinical features of classic FOP. (A) Extensive heterotopic bone formation typical of FOP is seen by three-dimensional reconstructed computed tomography (CT) scan of the back of a 12-year-old child. (B) Anteroposterior radiograph of the feet of a 3-year-old child shows symmetrical great toe malformations. (Reprinted from Shore et al. Nat Genet. 2006;38:525-7.)

Hypoxic brain damage Pseudomalignant heterotopic ossification Extraskeletal osteosarcoma (ESOS)

Genetic/inherited HO Fibrodysplasia ossificans progressiva (FOP) Progressive osseous heteroplasia (POH) Albright’s hereditary osteodystrophy (AHO)a a

Albright hereditary osteodystrophy (AHO) describes a variable constellation of clinical features that includes subcutaneous ossification. AHO features occur in pseudopseudohypoparathyroidism (PPHP) and are frequently associated with pseudohypoparathyroidism (PHP) type 1a and type 1c.

adults. ESOS is exceedingly rare in the pediatric population with only 13 cases under the age of 21 reported [24,25]. Pseudomalignant heterotopic ossification, a benign isolated condition often diagnosed as an extraosseous sarcoma based on radiographic and histologic findings, most commonly affects the limbs of young adults, however, occasional cases have been reported in children [26]. In cases of non-hereditary heterotopic ossification, the bone formation typically results from a single inductive event, such as a traumatic injury or tumor. In such cases, the heterotopic bone has been generally observed to reach an end-point of mature bone formation and the resulting bone can often be effectively removed surgically from the soft tissues.

FIBRODYSPLASIA OSSIFICANS PROGRESSIVA (FOP) Clinical Features of FOP Clinical Diagnosis of FOP The main characteristic clinical feature of FOP (FOP; MIM 135100) is the formation of extraskeletal, or heterotopic, bone within connective tissues (Fig. 30.1). However, in addition, specific skeletal malformations that occur during embryonic development frequently

are noted in patients [27]. Individuals with FOP appear normal at birth except for the characteristic malformations of the great toes which are present in all classically affected individuals (Fig. 30.1) [27,28]. Since the identification of the mutated gene that causes FOP (see below), genetic diagnostics can be used to confirm a clinical diagnosis of FOP. However, the clinical features of FOP are unique and distinct, and a diagnosis can be made through clinical evaluation alone, even prior to radiographic evidence of heterotopic ossification, through associating the rapid appearance of soft tissue swelling with great toe malformations [29]. Progressive Heterotopic Ossification in FOP During the first few years of life, children with FOP develop painful and highly inflammatory soft tissue swellings (known as flare-ups). These swellings appear suddenly and expand rapidly. A flare-up is the first clinical indication of cellular events that promote the degradation of soft connective tissues (including aponeuroses, fascia, ligaments, tendons, and skeletal muscles) leading to their replacement by ribbons, sheets, and plates of extraskeletal bone through a process of endochondral ossification [30e35]. The median age at onset of ossification is 5 years, although it has been observed in some cases during the first year of life and may occur in the scalp as a result of birth trauma [30] (FSK, personal observations). Most patients are affected with restricted heterotopic ossification by the age of seven, with severe restriction of upper limb mobility by 15 years [30]. Minor trauma such as intramuscular immunizations, mandibular blocks for dental work, muscle fatigue, blunt muscle trauma from bumps, bruises, falls, or influenza-like illnesses can trigger painful episodes leading to progressive HO [36e40]. The trauma induced by surgical attempts operatively to remove heterotopic bone also commonly leads to new bone formation [27,41e43].

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FIBRODYSPLASIA OSSIFICANS PROGRESSIVA (FOP)

In the absence of trauma or other interventions, HO in FOP is observed to form in a characteristic anatomic and temporal progression that follows the patterns of normal embryonic skeletal formation. Early heterotopic ossification most commonly occurs in the neck, spine, and shoulder girdle [30]. Bone formation typically is seen first in the dorsal, axial, cranial, and proximal regions of the body and later in the ventral, appendicular, caudal, and distal regions [27,30,35,41e43]. Heterotopic bone is typically asymmetrically distributed. A risk assessment of heterotopic bone formation by age and anatomic site [35] indicated that the risk of heterotopic ossification remains constant with age for the neck, spine, shoulders, elbows and ankles, while risk for regions such as the jaw, wrists, hips, and knees increases with age. In addition, the neck, spine, and shoulders show a 10e15% risk of annualized involvement (indicating that it is likely that these areas will be involved by the age of 10), while wrists, ankles, and jaws have a much lower risk of involvement (2e5%) and may remain unimpaired for many years. Although skeletal muscle is the tissue most often affected by heterotopic ossification, extraskeletal bone also forms in other connective tissues such as aponeuroses, fascia, ligaments, and tendons. Several skeletal muscles including the diaphragm, tongue, and extraocular muscles are spared from FOP. Cardiac muscle and smooth muscle are not affected by heterotopic ossification in FOP patients [27,41,43]. Early stages of FOP lesions that form in the axial regions are often different from those in the appendicular regions [44]. Axial lesions often appear very suddenly, more rapidly than almost any neoplasm and have often been misdiagnosed as tumors. Axial lesions commonly occur as large bulbous lesions on the neck and back. By contrast, pre-osseous swelling in the limbs is often diffuse and can be mistaken for acute thrombophlebitis, a complication that may occur in patients with advanced stage FOP due to generalized immobility and associated venous stasis [44]. The qualitative differences in swelling in the axial and the appendicular regions of the body may reflect regional differences in the anatomy of the subaponeurotic spaces as well as differences in the anatomy of the fascial compartments. The heterotopic bone formation in FOP is episodic, in some cases with extended times (weeks, months) with no new disease activity. The rate of bone formation is highly variable among patients, however, the disability of the bone formation is cumulative and eventually causes permanent immobility. Most patients are confined to a wheelchair by the third decade of life, requiring lifelong assistance in performing activities of daily living [11,27,41e43,45]. Severe weight loss often follows ankylosis of the jaw, and pneumonia and right-sided heart failure are complications of rigid

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fixation of the chest wall [46]. The severe disability of FOP results in low reproductive fitness, and genetic transmission occurs rarely, with fewer than 10 families with inheritance of FOP known worldwide [47]. The median age of survival is approximately 45 years [48], and death often results from complications of thoracic insufficiency syndrome [46]. Skeletal Anomalies in FOP In addition to heterotopic bone formation, patients with FOP also exhibit perturbations of normal skeletal formation during development. Malformations of great toes are the most consistent and characteristic feature, although malformations of fingers, vertebrae, and other skeletal elements are variably observed (Table 30.2) [27,49]. Malformation of the great toes (hallux deformity) is a diagnostic feature of a standard clinical presentation of FOP (see Fig. 30.1). The proximal phalanx of the first toe is abnormally shaped, typically more broad and square, and fused with the terminal first phalanx [50]. An abnormal first metatarsal is usually observed with the severity of dysplasia ranging from distortion of the normal contours of the distal tip to a cylindrical and shorter skeletal element that lacks distal metaphyseal or epiphyseal shape. Fusions of the middle and terminal phalanges of the second through fifth toes are also common. In general, these dysmorphologies are bilaterally symmetrical. Skeletal dysmorphologies affecting the hands are less frequent and marked compared to the foot abnormalities [50]. The most common malformations are short first metacarpals, small middle phalanx of the fifth digit, and radial angulations of the distal interphalangeal joint of the finger (clinodactyly). In each patient, the extent of hand abnormalities generally correlates with the degree of first metatarsal dysmorphology. Like the toe malformations, effects on the hands are usually bilateral. Although it is common that patients with foot abnormalities have no dysmorphology of the hands, no patients with affected hands in the absence of toe abnormalities are observed. Early developmental anomalies are also frequently observed in the cervical spine (greater than 80% prevalence) [49,51]. During infancy and early childhood, stiffness of the neck and impaired range of motion occur in most patients, frequently prior to the appearance of HO at that site. Characteristic anomalies of the cervical spine in FOP patients are large posterior elements, tall narrow vertebral bodies, and fusion of the facet joints between C2 and C7, findings that are similar to those seen in mice with homozygous deletions of the gene encoding Noggin, a secreted bone morphogenetic protein (BMP) antagonist [51]. Of note, these characteristic findings in FOP patients are distinct from those in patients with

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824 TABLE 30.2

30. EXTRASKELETAL BONE FORMATION

Clinical Features of Classic FOP, FOP-Plus, and FOP Variant Patientsa Classic FOP

FOP-plus

FOP variants

Number of cases evaluated

n>100

n¼8

n ¼ 12

ACVR1 mutation: codon change

All R206H

Most R206H

In GS or PK domainsc; no R206H

Gender

M, F

M, F

M, F

1e10

<1e14

<1e2 or 8e26

Normal

Normal

Normal

Characteristic malformations of great toe (hallux valgus, malformed 1st metatarsal, and/or monophalangism)

100%

100%

0%

Progressive heterotopic ossification in characteristic anatomic patterns

100%

100%

z100%

Conductive hearing impairment

>50%

>50%

<50%

Cervical spine malformations

>80%

>80%

<80%

Proximal medial tibial osteochondromas

>90%

>90%

<50%

Short broad femoral necks

>70%

>70%

<70%

Thumb malformations (short 1st metacarpal,  monophalangism)

z50%

z50%

z50%

Severe variable reduction deficits of digits

0%

0%

z50%

Absent finger/toe nails in digits with severe reduction deficits

0%

0%

z50%

Normal or minimal changes in great toes

0%

0%

z50%

Intra-articular synovial osteochondromatosis of hips and DJD of hips

0%

0%

z25%

Sparse, thin scalp hair (more prominent in 2nd decade)

0%

n¼1

z50%

Mild cognitive impairment

0%

n¼1

z50%

Severe growth retardation

0%

n¼2

0%

Cataracts

0%

n¼1

0%

Retinal detachment

0%

n¼1

0%

Childhood glaucoma

0%

n¼2

0%

Craniopharyngioma

0%

n¼1

0%

Persistence of primary teeth in adulthood

0%

n¼1

0%

Anatomic abnormalities of cerebellum

0%

n¼0

n¼2

Diffuse cerebral dysfunction with seizures

0%

n¼1

0%

Polyostotic fibrous dysplasia

0%

n¼1

0%

Primary amenorrhea

0%

n¼1

n¼1

Aplastic anemia

0%

n¼1

0%

Hypospadias

0%

n¼0

n¼1

Cerebral cavernous malformations

0%

n¼0

n¼1

Age of HO onset (years) High resolution karyotype

b

Classic/defining FOP features

Common variable FOP features

Atypical FOP clinical features

a

This table is modified from the detailed information presented in Kaplan FS, Xu M, Seemann P et al. Classic and atypical FOP phenotypes are caused by mutations in the BMP type I receptor ACVR1.Hum Mutat. 2009;30:379-90. b When examined. c GS: glycineeserine domain, PK: protein kinase domain.

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KlippeleFeil syndrome, a disorder characterized by fusions of cervical vertebrae and associated with a short neck, low posterior hairline, and decreased cervical spine range of motion [51]. Other skeletal anomalies often associated with FOP include short broad femoral necks (greater than 70% prevalence) and proximal medial tibial osteochondromas (greater than 90% prevalence). The features are also found in patients with multiple hereditary exostoses (MHE). Although the genes associated with multiple hereditary exostoses are not mutated in patients who have FOP, these shared clinical effects may indicate effects on the same cellular pathways [11,27,41,43,45]. Although osteochondromas (benign osteochondral neoplasms or orthotopic lesions of skeletal remodeling) likely occur on other skeletal elements in FOP patients, those on the tibia are most easily surveyed through available x-rays and physical exams. Proximal tibial osteochondromas on FOP are usually located at the pes anserinus. These osteochondromas are usually asymptomatic, frequently bilateral, and most often pedunculated [52]. Some patients with FOP have developmental anomalies of the temporomandibular joints (TMJs), although a comprehensive study of the anatomy of this joint has not yet been undertaken. Spontaneous or post-traumatic extra-articular ankylosis of the TMJs is common and leads to severe disability with resultant difficulties in eating and poor oral hygiene [11,27,41,43,45]. Although a rigorous anthropometric and photogrammetric approach to craniofacial features of FOP is not available, there is strong anecdotal evidence of a facial phenotype of FOP that is characterized by mandibular hypoplasia, a maxillary overbite, and possibly other more subtle features involving a wide range of aponeuroses, fasciae, muscles, bones, joints, and growth plates of the craniofacial region [27]. Many of these noted abnormalities are secondary to FOP flare-ups involving the craniofacial structures, while others may result from growth disturbances involving the bones of the head and face. Additional Complications of FOP Although ankylosis of the temporomandibular joint is common in later stages of FOP, submandibular heterotopic ossification is relatively uncommon [37]. Submandibular swelling can be a life-threatening complication, especially when associated with massive anterior neck swelling and difficulty in swallowing [37]. Efforts to reduce swelling, including treatment with glucocorticoids and respiratory support, are recommended [37,53]. Hearing impairment occurs in about half of FOP patients. The onset is usually in childhood or

825

adolescence with slow progression during the lifetime. Hearing loss is most often conductive, possibly due to middle ear ossification, however, in some patients, the hearing impairment is neurologic [54]. Patients with FOP develop thoracic insufficiency syndrome (TIS). This life-threatening complication of cardiopulmonary function can cause pneumonia and right-sided heart failure and is associated with costovertebral malformations and orthotopic ankylosis of the costovertebral joints, ossification of intercostal muscles, paravertebral muscles and aponeuroses, and progressive spinal deformity including kyphoscoliosis or thoracic lordosis. Prophylactic measures to maximize pulmonary function and minimize respiratory compromise help to reduce morbidity and mortality from TIS in patients with FOP [46,48,55,56]. Assiduous attention should be directed toward the prevention and therapy of intercurrent chest infections. Such measures include prophylactic pneumococcal pneumonia and influenza vaccinations (subcutaneous injection), chest physiotherapy, and antibiotic treatment at early stages of chest infection. Upper abdominal surgery interferes with diaphragmatic respiration and should be avoided if possible. Sleep studies to assess sleep apnea and positive pressure assisted breathing devices such as bipap masks without the use of supplemental oxygen may be helpful [46]. Patients with FOP who have advanced TIS and who use unmonitored oxygen have a high risk of death caused by sudden correction of oxygen tension in the presence of chronic carbon dioxide retention that suppresses respiratory drive. Patients who have FOP and severe TIS should not use supplemental oxygen in an unmonitored setting [46,48]. Patients with FOP have numerous oral and dental health-care issues [57,58]. Most eventually develop heterotopic ossification of the chewing muscles with resultant ankylosis of the temporomandibular joints, and preventive and restorative dental care, as well as endodontic, periodontic, and orthodontic care present therapeutic challenges to the dental practitioner, the anesthesiologist, and to the FOP patient throughout his or her life. Assiduous attention should be paid to prevention and to minimization of risks when restorative care is necessary.

Radiographic Features of FOP Heterotopic ossification in soft tissues and joint malformations are characteristically noted by radiographic evaluation of FOP. Skeletal malformations (great toes, thumbs, cervical spine, and proximal femurs) along with the presence of proximal medial tibial osteochondromas are also observed and

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30. EXTRASKELETAL BONE FORMATION

can assist in the diagnosis of FOP hygiene [11,27,41,43,45]. Radiographic and bone scan findings support that heterotopic bone undergoes normal modeling and remodeling [59,60]. The incidence of fractures is not increased in patients with FOP, and fracture healing of heterotopic bone is characteristically accelerated [61]. Bone scans are abnormal before HO can be detected by conventional radiographs. Computerized tomography and magnetic resonance imaging of early lesions have been described, but contribute no additional information that is necessary for diagnosis, however, can provide a three-dimensional view of disease progression [27,42,62].

Laboratory Findings in FOP Routine evaluations of bone mineral metabolism biochemistry are usually normal in FOP patients, although increased serum alkaline phosphatase activity and erythrocyte sedimentation rate (ESR) have been observed during disease activity [27,42,43]. Elevated urinary basic fibroblast growth factor levels have also been correlated with disease flare-ups during the preosseous angiogenic phase of fibroproliferative lesions [63]. Nephrolithiasis is occasionally observed in older patients with FOP and may be correlated with increased immobilization and dehydration in the setting of generalized increased bone remodeling and mineral turnover [27]. Recently, increased abundance of circulating osteogenic progenitor cells has also been associated with active FOP lesion formation [64].

Histopathology of FOP Lesions Since tissue trauma can stimulate episodes of FOP lesion formation, biopsies are never obtained after FOP has been diagnosed. However, until relatively recently, early stage FOP was frequently misdiagnosed, often mistaken for tumor formation, and tissue biopsies were obtained and examined. The histological stages of FOP lesions have been well described [31e34,65], and involve an initial phase of tissue destruction and turnover, followed by a tissue formation and replacement phase (Fig. 30.2). Initial stages of early FOP lesions are indicated by the presence of mononuclear cells, monocytes, macrophages, mast cells, and B and T lymphocytes. The presence of mononuclear inflammatory cells within affected skeletal muscle and other connective tissues precedes widespread destruction of the connective tissue integrity [31]. Following this inflammatory tissue turnover phase, the tissue formation phase begins with an intense fibroproliferative stage that is associated with robust angiogenesis and neovascularity [31,32]. At

this stage, the appearance of FOP lesions is indistinguishable from aggressive juvenile fibromatosis. As the lesion matures through a normal process of endochondral ossification, the fibroproliferative tissue transitions into an avascular cartilage stage followed by revascularization and osteogenesis. The heterotopic bone that forms is normal, histologically mature lamellar bone tissue that may contain marrow elements [31e34]. Biopsies of actively forming FOP lesions exhibit multiple histological stages of lesion progression, indicating that different regions of the lesion are induced or mature at different rates. Although heterotopic bone formation in FOP is analogous to endochondral bone formation during embryogenesis and postnatal fracture healing, key differences are the absence of inflammation during embryonic skeletal development and the limited presence of lymphocytic inflammatory cells in early fracture healing [34,66]. Recent cell lineage tracing studies investigated the origins of the progenitor cells that participate in the various stages of BMP-induced heterotopic ossification in mouse models [67]. Vascular smooth muscle cells did not contribute to any stage of heterotopic ossification. Despite the osteogenic response of skeletal myoblasts to BMPs in vitro, skeletal muscle precursors contributed minimally to heterotopic ossification (<5%). However, cells that were marked with the vascular endothelial marker Tie2/Tek contribute to all stages of BMP-induced heterotopic ossification, comprising 40e50% of lesional cells at the fibroproliferative, chondrogenic, and osteogenic stages of developing heterotopic endochondral anlagen. These findings indicate that at least one additional progenitor population (Tie2-negative) can participate in ectopic bone formation.

Circulating Stem Cells and Bone Marrow Transplantation in FOP Contributions of circulating cells of bone marrow origin to the induction of heterotopic ossification have also been suggested. Studies have shown that hematopoietic stem cells and bone marrow mesenchymal stem cells can function as osteoblast precursors [68e73]. Osteoprogenitor cells also have been identified in the peripheral blood of normal individuals [74] and in patients who have FOP [64]. Participation of bone marrow-derived cells in BMPinduced heterotopic ossification was investigated through bone marrow transplantation studies in mice [66] and determined that bone marrow cells contributed to the early inflammatory stage of BMP-induced bone formation, but were not present in the fibroproliferative, chondrogenic or osteogenic stages of the FOP-like

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FIBRODYSPLASIA OSSIFICANS PROGRESSIVA (FOP)

827

FIGURE 30.2 Histological stages of heterotopic ossification in FOP. Histological analysis of biopsies obtained from FOP patients (obtained prior to diagnosis for FOP) revealed a tissue degradation phase (1AeC) that includes perivascular lymphocyte infiltration (1A), immune cell infiltration (1B), and connective tissue degradation (1C) that is followed by a tissue formation phase that includes fibroproliferation (2A), early cartilage (2B), and endochondral bone formation (2C) stages. (Adapted from Glaser et al. J Bone Joint Surg. 2003;85A:2332-42.) (See color plate section.)

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30. EXTRASKELETAL BONE FORMATION

lesions, except as contributing to repopulate marrow cavities in formed heterotopic bone. Replacement of bone marrow stem cells through bone marrow transplantation has been suggested as a possible cure for FOP. Clinical observations of an FOP patient who had bone marrow transplantation for the treatment of intercurrent aplastic anemia [66] revealed that while replacement with normal donor bone marrow cured the aplastic anemia in the patient, the normal bone marrow was not sufficient to prevent heterotopic ossification and progression of FOP. However, during the 14 years following his bone marrow transplant that the patient was pharmacologically treated to suppress immune system activity, the acute immunoablation and chronic immunosuppression was correlated with the absence of heterotopic ossification. Progression of heterotopic ossification resumed in the patients following discontinuation of the immunosuppressive drugs [66]. This case demonstrates that bone marrow transplantation did not cure FOP in this patient, most likely because the cells from the bone marrow are not the source of cells that form heterotopic bone. The case additionally demonstrated that even normal bone marrowderived cells are capable of supporting heterotopic ossification in a genetically susceptible individual [66]. These findings are of immense research interest and vital clinical importance, and they exemplify powerfully how much can be learned by careful observation in an individual patient. They also illustrate the importance of the immune system in triggering FOP flare-ups. At present, however, the general use of potent immunosuppressive medications is not advocated in the routine management of FOP, and would likely be extremely dangerous and possibly life-threatening if it were applied broadly to the FOP community. At the present time (and until further studies are performed in appropriate animal models), the international clinical consortium recommends against the use of chronic immunosuppressive medications in the management of FOP.

Immune system and FOP Several lines of evidence suggest involvement of the inflammatory component of the immune system in FOP. The presence of macrophages, lymphocytes and mast cells in early FOP lesions, macrophage and lymphocyte-associated death of skeletal muscle, flareups following viral infections, the intermittent timing of flare-ups, and the suppression of early flare-ups in response to corticosteroids support involvement of the innate immune system in the pathogenesis of FOP lesions [38,66,75]. In addition, studies in mouse models of FOP support the role of the innate immune system in triggering heterotopic ossification [67,76,77].

Mast cells have been identified at every histological stage, and at higher frequency than found in normal skeletal muscle and non-lesional FOP muscle. During the fibroproliferative stage of the lesion formation, mast cells are found at a density that is higher than that found in any other inflammatory myopathy [78].

Atypical FOP Phenotypes Among patients with FOP-type progressive heterotopic ossification, occasional cases that are associated with clinical features unusual for FOP have also been identified [49]. (See also section on FOP ACVR1 variant mutations.) These atypical FOP patients have been clinically categorized into two groups (see Table 30.2). Patients who are described as “FOP-plus” have one or more features that are uncommon in FOP patients, along with the classic defining FOP features of progressive endochondral heterotopic ossification and great toe malformations. Patients described as “FOP variants” present with significant deviation from the standard clinical presentation of one or both of the two classic defining features of FOP. Table 30.2 illustrates the range of clinical features that are present in a set of atypical FOP patients.

Misdiagnosis of FOP FOP has frequently been misdiagnosed, as clinicians often fail to link the finding of rapidly developing soft tissue swellings on the head, neck, and upper back of young children with malformed great toes [79]. When such associations are not made, FOP has been misdiagnosed as aggressive juvenile fibromatosis (extra-abdominal desmoid tumors), lymphedema, or soft tissue sarcomas that have resulted in unnecessary and harmful diagnostic biopsies that stimulate disease progression and exacerbate the condition [79]. Such biopsies have high risk at any anatomic site, but can be particularly harmful and life-threatening when performed in the neck or back where asymmetric HO can lead to rapidly progressive spinal deformity and exacerbation of thoracic insufficiency syndrome, or in the jaw which can cause ankylosis of the temporomandibular joints.

DNA Diagnostic Testing for FOP Diagnosis of patients with classic FOP can be made based on clinical evaluation alone, by associating the great toe malformations with rapid appearance of soft tissue lesions [27,29,60]. Clinical diagnosis of FOP can be confirmed by DNA diagnostic testing to determine the DNA sequence of the ACVR1 gene (see below). DNA sequencing can also be used to evaluate suspected cases of atypical FOP (described above).

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FIBRODYSPLASIA OSSIFICANS PROGRESSIVA (FOP)

Epidemiologic, Genetic and Environmental Factors in FOP FOP is a rare condition with a worldwide prevalence of approximately one in two million. Phenotypic variability is observed [80,81]. There is no evidence of ethnic, racial, gender, or geographic predisposition [42,43]. Since reproductive fitness in FOP is low, most cases are the result of spontaneous new mutations in individuals. Maternal mosaicism may occur [82] and a paternal age effect has been reported [83]. Fewer than 10 families with inheritance of FOP have been reported [47]; in these cases, the pattern of inheritance is autosomal dominant, indicating that one mutant gene copy is sufficient to cause FOP. Inheritance can be from mothers or fathers [84]. In addition to a genetic cause of FOP (see section on Genetics of FOP), environmental factors also influence the phenotype of this condition. A study of three pairs of monozygotic twins with FOP [85] demonstrated that within each pair, the congenital toe malformations were identical. However, postnatal heterotopic ossification varied depending on life history and environmental influences such as tissue trauma and viral infections. This study supported that genetic factors direct the disease phenotype during prenatal development while environmental factors have a major influence on postnatal progression of heterotopic ossification [85].

Genetics of FOP ACVR1 R206H Mutations in FOP Most cases of FOP arise as a result of a spontaneous new mutation within an individual. Reproductive fitness is low, however, in the few cases observed, genetic transmission is autosomal dominant and can be inherited from either mothers or fathers [28,47,84]. Families with a classic clinical presentation of FOP were used for genetic linkage analysis and positional cloning to identify ACVR1 as the mutated gene in FOP [28]. ACVR1/ALK2 (activin A type I receptor/activinlike kinase 2) is a type I receptor for BMPs. All individuals (sporadic and familial cases) with classic clinical features of FOP (progressive heterotopic ossification and great toe malformation; see Fig. 30.1) contain the identical heterozygous single nucleotide substitution (c.617G/A) that changes amino acid 206 from arginine to histidine (R206H). Codon 206 is highly conserved and occurs within the glycineeserine (GS) region of the cytoplasmic domain of ACVR1/ALK2. The ACVR1/ALK2 R206H mutation is fully penetrant; all persons examined who carry this mutation have FOP. Although mutations in the BMP antagonist Noggin have been suggested [86,87], such mutations could not be verified [88e91]. DNA sequencing of

829

patient genomic DNAs supports that there is no locus heterogeneity in FOP; all patients with FOP type heterotopic ossification thus far examined have a mutation in the ACVR1 gene. FOP ACVR1 Variant Mutations Patients with atypical clinical presentations and FOPtype progressive heterotopic ossification have also been identified (described in earlier section on Atypical FOP Phenotypes; see Table 30.2). Mutational analysis of atypical FOP patients by DNA sequencing of the ACVR1 gene has been conducted [49,92e97] and shown that, like patients with a classic clinical presentation of FOP, all atypical patients examined have heterozygous ACVR1 missense mutations in conserved amino acids. The c.617G/A; R206H mutation that occurs in all cases of classic FOP also has been found in most cases of atypical FOP. However, novel ACVR1 mutations occur in each of the FOP variants and in two cases of FOP-plus. All mutations identified in classic, FOP-plus, and variant FOP patients are single nucleotide substitutions that cause missense mutations, with the exception of a single case with a 3-nucleotide deletion that replaces two amino acids with a single codon [49]. No frameshift or nonsense mutations were identified, supporting that in each case a mutant protein with altered function is produced. All of the identified mutations occur in either the glycineeserine (GS) activation domain or the protein kinase domains, regions of the ACVR1/ALK2 receptor that are important in conferring downstream signal transduction. Each of the mutated amino acids in FOP variant and FOP-plus is highly conserved evolutionarily and also invariant among all three human type I BMP receptors. Protein structure homology modeling predicts that each of the amino acid substitutions activates the ACVR1/ALK2 protein and enhances receptor signaling [49,92]. None of the identified ACVR1 mutations have been found to be present in unaffected individuals. Although the rate of progression and the severity of heterotopic ossification vary greatly among individuals with classic and atypical FOP forms, progressive postnatal heterotopic ossification is the common feature shared by all classic FOP, FOP-plus, and FOP variant patients. Among the identified ACVR1 mutations in these patients, there is limited correlation between the severity of heterotopic ossification and specific mutations, supporting that all of the identified ACVR1 missense mutations influence the promiscuous postnatal induction of cartilage and bone cell differentiation in similar ways on a cellular level. However, given that the identified mutations occur in more than one functional domain of the ACVR1/ALK2 protein, some variation in specific molecular mechanisms that are altered by the different

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30. EXTRASKELETAL BONE FORMATION

mutations likely occur. Among patients with classic FOP, FOP-plus, and FOP variants, a wide range of variability in the malformations of the great toe is observed, and genotypeephenotype correlations may explain at least some of this variation. However, given the rarity of FOP-plus and variant FOP patients and that some of the ACVR1 mutations have been identified in a single patient, such correlations are difficult to determine through currently available clinical information. Consequences of ACVR1 Mutation on BMP Signaling BMP SIGNALING AND ACVR1/ALK2

BMPs mediate signal transduction through heterotetramer receptor complexes of two type I and two type II serineethreonine kinase receptors that reside on the cell surface (Fig. 30.3). In response to ligand binding to

I

the extracellular domain, type II receptors phosphorylate the glycineeserine (GS) domain of type I receptors [98e103]. In addition to ACVR1/ALK2, the BMP type I receptor mutated in FOP, BMP signal transduction is mediated through the BMPR1A/ALK3 and BMPRIB/ ALK6 type I receptors. The signaling specificity by these BMP receptors is likely established through stage and tissue specific expression of the receptors and through mechanisms such as receptoreprotein interactions, including type I receptor heterodimer combinations [98,101,104e108]. Activated type I receptors induce downstream signaling through BMP pathway-specific Smad proteins and through MAP kinase pathways [98,105,108,109]. BMPs are members of the transforming growth factor b (TGF-b) family of signaling proteins. The TGF-b/BMP family regulates a wide range of cellular activities including differentiation, proliferation, apoptosis,

II

FIGURE 30.3 Schematic of the BMP signaling pathway. Type I and type II BMP receptors span the cell membrane and bind extracellular BMP ligands. Ligand binding to type I/type II receptor complexes activates signaling through type II receptor phosphorylation of the type I receptor at the GS domain. The activated type I receptor phosphorylates cytoplasmic signal transduction proteins such as R-Smads (that bind to the co-Smad, Smad4) and MAPKs, which in turn, directly or indirectly regulate transcription of target genes in the nucleus. (Adapted from a figure by Julia Haupt; Charite´-Universita¨t Medizin Berlin and the Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Berlin, Germany.)

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FIBRODYSPLASIA OSSIFICANS PROGRESSIVA (FOP)

migration, positional information, and stem cell renewal [110e116]. BMP signaling has important functions in embryonic development and skeletal formation. Distinct from other members of the family, many members of the BMP subgroup can induce the complete process of endochondral bone formation [117] and BMPs and their receptors are expressed in many adult tissues including skeletal muscles and chondrocytes. BMPs act as morphogens, regulating receptor activation in a dosedependent manner that is established, in part, by availability of BMP antagonists that prevent BMP binding to receptors [118]. BMP signaling is a highly regulated process that is dependent on negative and positive regulatory feedback [101]. ACVR1 is expressed in chondrocytes and osteoblasts and a constitutive active form of ACVR1 (caALK2) has been shown in animal models to enhance chondrogenesis, expand cartilage elements, promote joint fusions, and induce heterotopic ossification [77,119]. These features are similar to clinical findings in FOP patients and occur in response to dysregulation of the BMP signaling pathway [120e123]. Effects of FOP ACVR1 Mutation on Cell Functions All of the ACVR1 mutations identified in classic and atypical FOP patients occur in highly conserved amino acids, indicating their functional importance [49]. Protein structure homology modeling of the resulting ACVR1/ALK2 proteins predicts that each of these mutant receptors is likely to activate the ACVR1/ ALK2 protein and enhance receptor signaling [49,92,96,124]. A series of studies demonstrated that signal transduction through the BMP pathway is altered in cells from FOP patients [120e123], with increased phosphorylation of BMP pathway signaling mediators (BMP-specific Smads and p38MAPK) and increased expression of BMP transcriptional targets in the absence of exogenous BMP ligand. Subsequent in vitro and in vivo analyses demonstrated that this BMP-independent signaling activation can be specifically induced by the mutant ACVR1 R206H receptor which activates BMP signaling without requiring BMP to initiate the signaling cascade and stimulates an additional increased pathway activation in response to BMP [125e128]. Codon 206 is within the glycineeserine (GS) activation domain, adjacent to the protein kinase domain of ACVR1. Protein homology modeling of the ACVR1 receptor predicts that the protein conformation of the ACVR1 R206H mutant is altered and could lead to changes in the ability of the receptor to interact with proteins that bind the receptor GS domain [28,124]. The GS domain of all type I TGFb/BMP superfamily receptors is a critical site for binding and activation of the pathway-specific Smad signaling proteins and is

831

a specific binding site for FKBP1A (also known as FKBP12), a highly conserved inhibitory protein that prevents leaky activation of type I receptors in the absence of ligand [99,129,130]. Investigations support that the ACVR1 R206H protein has reduced binding to FKBP1A in the absence of BMP [126,127], indicating that an impaired FKBP1A-ACVR1 interaction contributes to BMP-independent BMP pathway signaling. The ACVR1 R206H mutation does not appear fully to prevent FKBP1A interaction suggesting that FKBP1Amutant ACVR1 interactions are less stable and/or of shorter duration, allowing the mutant GS domain to interact with FKBP1A in a manner that allows activation of Smads and downstream signaling in the presence of aberrant FKBP1A binding [126]. In vivo, disruption of the BMP pathway through ACVR1/ALK2 has severe viability and morphological consequences, as illustrated by knockout mouse models [131,132] and by overactivation or by mutants of Alk8, the functional ACVR1 homolog in zebrafish [106,133e135]. Therefore, it was unexpected that the mutation that causes FOP overactivates the BMP pathway yet allows human embryonic development to occur relatively unimpaired, with only mild skeletal effects. One explanation could be that the ACVR1 mutations in FOP patients are only moderately activating. Comparison of FOP ACVR1 R206H to a constitutively active form of ACVR1 (caALK2; Q207D) in micromass chondrogenesis assays, demonstrated that the ACVR1 R206H receptor has a milder stimulation of cell differentiation compared to caALK2 [126]. The effects of the FOP ACVR1 mutant receptor during embryonic development may therefore induce only relatively minor effects that are compatible with life. In postnatal connective tissues, increased BMP signaling from the mutant receptor may be only moderately “on” under basal in vivo conditions allowing for the quiescent periods that are observed in patients between active episodes of heterotopic bone formation, but priming the cells to respond to changes in the local tissue environment by forming extraskeletal bone. A transgenic mouse model expressing constitutively active ACVR1 (caALK2; Q207D) is embryonic lethal, however, a mouse with conditional activation of the BMP type I receptor ACVR1/ALK2 has provided a model for investigating activated ACVR1/ALK2 gene function in a cell-specific manner [136]. Although caALK2 does not cause FOP, this mutation can induce heterotopic ossification through adenovirus-mediated Cre recombinase induction of floxed caALK2 [77]. Initial characterization of an ACVR1 R206H knockin mouse model demonstrates that this mutation can induce both the embryonic skeletal malformations and postnatal formation of heterotopic bone that are characteristic of FOP patients [137].

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Treatment Options/Strategies for Heterotopic Ossification in FOP Although the heterotopic bone in FOP often forms as discrete, skeletal-like elements that would appear to be good candidates for surgical resection, surgery is always avoided in these patients since the surgical trauma to tissues presents a high risk of inducing additional bone formation [41,53,56,79]. Currently, there are no effective medical treatment options to prevent or regress the formation of heterotopic bone. Guidelines for symptomatic management of FOP are available through the IFOPA website (www.ifopa.org). For FOP, glucocorticoids appear effective in managing new flare-ups affecting major joints of the appendicular skeleton, especially when used at early stages of onset [53,138]. Non-steroidal anti-inflammatory agents, cyclooxygenase-2 inhibitors, mast cell stabilizers, and leukotrine inhibitors are reported by patients to manage chronic pain and ongoing disease progression. The discovery of the FOP gene immediately identifies ACVR1 (ALK2) as a specific and ideal druggable target for FOP. The identification of the recurrent point mutation that causes FOP in all classically affected individuals provides a specific target for drug development and intervention [139,140]. Plausible therapeutic approaches to inhibiting basally overactive and conditionally hyperresponsive BMP signaling in FOP include soluble BMP antagonists, inhibitory RNA technology, monoclonal antibodies directed against ACVR1(ALK2) and, most plausibly, orally available small molecule selective signal transduction inhibitors of ACVR1/ ALK2 receptor activity (see Fig. 30.3).

Distinct from FOP, POH is not associated with inflammation, predictable regional patterns of heterotopic ossification, or FOP-like great toe malformations. Heterotopic bone in POH forms in an asymmetric, mosaic distribution and shows a predominance of intramembranous bone formation (Fig. 30.4) [4,141e143]. POH is among a number of related genetic disorders, including Albright hereditary osteodystrophy (AHO), pseudohypoparathyroidism (PHP), and osteoma cutis (OC), that have the common feature of superficial ossification and that are associated with inactivating mutations of the GNAS gene [144e146]. However, in these other GNAS-associated disorders, heterotopic ossification typically remains limited to subcutaneous tissues. Clinically, POH is defined by cutaneous ossification that usually begins during childhood and progresses

PROGRESSIVE OSSEOUS HETEROPLASIA (POH) Clinical Progressive osseous heteroplasia (POH) was first described as a distinct disorder in 1994 [141]. Similarly to FOP, patients with POH (MIM 166350) develop extensive bone formation within soft connective tissues, and the bone formation occurs episodically and is cumulative over time. However, POH can be distinguished from FOP by several clinical criteria [4,5,47,141]. Unlike FOP patients, POH patients characteristically develop ossification within the dermis, often in association with adipose tissue [4,141]. POH heterotopic ossification progresses from the more superficial cutaneous and subcutaneous tissues to the underlying deep connective tissues, with bone forming within skeletal muscle and in some cases fusing with skeletal bone.

FIGURE 30.4 Heterotopic ossification in POH. (A) Radiographic appearance of heterotopic ossification. Lateral serial x-rays of the lower leg of a child show progression of heterotopic ossification from the ages of 18 months to 30 months to 8 years (amputation specimen). Extensive ossification of the soft tissues of the superficial and deep posterior compartments of the leg, disuse osteopenia, and anterior bowing of the tibia (due to fusion of heterotopic bone with skeletal bone) can be seen. (Reprinted from Kaplan et al. J Bone Joint Surg Am. 1994;76:425-36.) (B) Enhanced computed tomographic image of the thighs of a 10-year-old boy. The right (R) side is normal while the tissues of the left side are atrophied; extensive ossification of the skin with progression of bone formation through the quadriceps muscles and fusion to the femur is present. (Reprinted from Shore et al. N Engl J Med. 2002;346:99-106.)

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PROGRESSIVE OSSEOUS HETEROPLASIA (POH)

from subcutaneous tissues to involve subcutaneous and deep connective tissues in the absence of multiple features of AHO or hormone resistance [141,143]. These GNAS-associated disorders are discussed in detail in the chapter on Parathyroid disorders (see Chapter 21), and will be only briefly described here. AHO describes a variable set of features, in addition to superficial ossification, that includes short adult stature, obesity, a round face, brachydactyly, and neurobehavioral problems (including mental retardation). PHP, or end-organ resistance to parathyroid hormone (PTH), is classified as types 1a, 1b, and 1c [144e147]. The clinical features of PHP1a and PHP1c are the same and can include AHO features, defective responses to PTH, and multiple hormone resistance. PHP1a is distinguished from PHP1c by identified inactivating GNAS mutations and/or reduced activity of Gsa, the major protein product encoded by the GNAS locus; the causative mutations in PHP1c patients remain undetermined. Patients with PHP1b also have hormone resistance, which is generally limited to PTH target tissues, but these patients show no AHO features or reduced Gsa activity. PHP1b is associated with a defect in GNAS imprinting [144,148e151]. Pseudopseudohypoparathyroidism (PPHP) refers to patients with AHO features who have normal target-organ responses to PTH. Osteoma cutis describes heterotopic ossification that is limited to superficial tissues in the absence of hormone resistance or AHO features. In a recent study, the clinical features and GNAS mutations were examined in patients with heterotopic ossification of the skin and subcutaneous tissues [143]. Although all of these disorders of superficial ossification are associated with heterozygous inactivating GNAS mutations, these related disorders can be distinguished solely by clinical criteria. GNAS-based disorders of HO can be grouped into those with heterotopic bone that remains limited to cutaneous tissues and those whose cutaneous lesions progress into deeper tissues (Table 30.3). The non-progressive forms include osteoma cutis, AHO/PPHP, and PHP1a/ c, while the progressive forms are POH and the POHlike syndromes. A small number of patients reveal that POH occasionally presents with additional features previously thought to occur exclusively in other GNAS-associated disorders of HO. Patients with such POH-like syndromes share the progressive ossification characteristic of POH with hormone resistance (POH/PHP1a/ 1c) or multiple AHO features (POH/AHO) (see Table 30.3). Eddy et al. [152] reported two cases in which patients exhibited POH-like HO along with characteristics of AHO (short stature, round face, and brachydactyly) and reduced levels of Gsa protein with or without a heterozygous GNAS mutation. Another

TABLE 30.3

Clinical Features of POH and other GNASInactivation Disordersa

Diagnosis

Superficial (dermal) HO

HO in deep connective tissues

>2 AHOb features

PTH resistance

POH

þ

þ





POH/ AHO

þ

þ

þ



POH/ PHP1a/1c

þ

þ

þ

þ

Osteoma cutis

þ







AHO

þ



þ



PHP1a/1c

þ



þ

þ

a

This table is modified from Adegbite NS, Xu M, Kaplan FS, Shore EM, Pignolo RJ. Clinical features, GNAS mutational analysis, and diagnostic criteria for progressive osseous heteroplasia (POH) and POH-like syndromes. Amer J Med Genet. 2008;146A(14):1788-96. All (þ) or no () patients within the diagnostic category displayed the indicated characteristic. b AHO features include: short stature, obesity, round face, brachydactaly, and neurobehavioral abnormalities.

patient with POH was described with severe plate-like osteoma cutis and also possessed a mutation in the GNAS gene [153]. These cases support that POH is part of a clinical spectrum of HO disorders that are caused by inactivating GNAS mutations [143]. Although some POH patients possess occasional AHO features, POH patients are never obese [143]. Mouse models [154] with heterozygous disruption of GNAS exon 2 that are paternally (þ/p) or maternally inherited (m/þ) have contrasting metabolic phenotypes. The (þ/p) mice are very lean, hypermetabolic, and hyperactive; conversely, (m/þ) mice are obese, hypometabolic, and hypoactive. Since all reported familial POH cases are paternally inherited [142,155], these data suggest that adipose tissues and metabolic activity are regulated through the parental allele expression of GNAS in both mouse and human. Furthermore, obesity is associated with maternal transmission of GNAS mutations and PHP1a [156].

Tissue/Histology e Heterotopic Ossification in POH Bone formation in POH originates spontaneously in the dermis with an initial clinical presentation that is indistinguishable from the subcutaneous ossification that is one of the developmental effects collectively described as Albright hereditary osteodystrophy. In POH, heterotopic bone formation appears usually to form through an intramembranous process, however

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30. EXTRASKELETAL BONE FORMATION

about the function of its protein product. In addition, the GNAS locus encodes non-protein-coding antisense transcripts that have been implicated in transcriptional regulation of other transcripts within the locus. Fewer than 60 clinically-confirmed cases of POH are known. This rarity appears to be due, at least in part, to the incomplete penetrance of inactivating GNAS mutations [143] and the variable range of clinical phenotypes caused by these mutations.

Cellular Functions FIGURE 30.5 Histopathology of a POH lesion. Initial bone-forming lesions in POH are typically observed as irregular deposits of bone within the dermal tissue are often observed in proximity to subcutaneous adipose tissue. Hematoxylin-eosin staining. (See color plate section.)

evidence of ectopic cartilage has been occasionally observed [141,143]. As the heterotopic ossification progresses from cutaneous (Fig. 30.5) to connective tissues, the appearance of the bone is diffuse and weblike; discrete skeletal-like elements do not form [4,141,142].

Genetics/Gene Mutation Most cases of POH, PHP1a, and AHO are associated with heterozygous inactivating mutations of the GNAS gene, which is regulated by genomic imprinting [142,150,157e177]. Maternally-inherited mutations in GNAS lead to PHP1a, whereas paternally-inherited mutations are associated with POH. AHO is more frequently associated with maternally-inherited mutations; AHO caused by a paternally-inherited mutation has been referred to as PPHP. GNAS is a transcriptionally complex locus that includes multiple promoters and alternate splicing [145,146,178,179] (see Chapter 21, Fig. 21.4). Genomic imprinting mechanisms, which differentially methylate the maternally- and paternally-inherited gene copies, contribute to promoter selection and transcriptional regulation within the locus. The most abundant protein product of this gene is Gsa, a ubiquitously expressed heterotrimeric G-protein a-subunit that activates adenylyl cyclases and cyclic AMP (see Chapter 21, Fig. 21.4) The Gsa transcript is biallelically-expressed in most but not all tissues. The GNAS locus also encodes XLas, a variant form of Gsa that appears to function similarly to Gsa but with a more restricted expression pattern at lower expression levels. Transcription of XLas is restricted to the paternally-inherited GNAS allele. The Nesp55 transcript is limited to expression from the maternally-inherited allele; little is known

In addition to subcutaneous ectopic ossification, AHO features including short stature, round face, short metacarpals and metatarsals (brachydactyly), and dental hypoplasia, demonstrate that GNAS inactivation affects bone and skeletal formation [145,146,178]. The association of AHO features with both PHP1a and POH suggests that these features are caused by haploinsufficiency of either the maternally- or paternally-inherited GNAS allele during early skeletal development. Further support has come from mouse models of GNAS haploinsufficiency [145,179]. Mouse models [145,179] that carry a maternallyinherited GNAS null allele show hormone resistance and increased adiposity, features of AHO and PHP1a. By contrast, a paternally-inherited GNAS null allele results in a normal hormone response and lean body mass, similar to POH patients. GNAS transcript-specific knockout models demonstrated that the lean body mass associated with paternal GNAS allele inactivation is associated with the loss of the paternally-expressed GNASXL (XLas) transcript [180]. GNAS mouse models have also identified roles for Gsa in osteoblast and chondrocyte differentiation [145,179]. Growth plates of long bones that are chimeric for Gsa deficiency revealed that Gsa null or haploinsufficient chondrocytes accelerate differentiation to hypertrophic chondrocytes resulting in shorter growth plates and bones [181,182]. Osteoblast-specific Gsanull models showed reduced long bone size, reduced trabecular bone and thicker cortical bone with an overall reduced bone turnover rate [183]. This phenotype suggests that Gsa haploinsufficiency has different roles during the growth and maintenance of bone at the growth plate compared to the initiation of ectopic skeletal formation. The GNAS gene is widely expressed in cells and tissues, therefore its altered expression is expected to effect many tissues and organs. Such effects are observed in patients and mouse models and include, in addition to effects on skeletal development and subcutaneous ossification, roles in energy metabolism, accumulation of adipose tissue, renal function, cognitive abnormalities, and bone marrow hematopoiesis [145,179,184].

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Treatment Options/Strategies for Heterotopic Ossification in POH In cases of POH, the infiltrating nature of the bone within soft tissues results in bone that cannot easily be removed without extensive damage to the involved soft tissues. There are no effective medical treatment options for POH, and current care is mainly palliative.

CONCLUSIONS In cases of non-hereditary heterotopic ossification, the bone formation typically results from a single inductive event, such as a traumatic injury or tumor. In such cases, the heterotopic bone has been generally observed to reach an end-point of mature bone formation and, in adults, the resulting bone can often be effectively removed surgically from the soft tissues. However, although non-hereditary heterotopic ossification in adults may be treated through surgical intervention, currently there are no effective treatment options for children. For both children and adults with FOP or POH, care is mainly palliative. In these inherited forms of heterotopic ossification, the underlying genetic mutations induce a series of bone-forming episodes throughout the lifespan of the individual. In cases of POH, the infiltrating nature of the bone within soft tissues results in bone that cannot be removed without extensive damage to the involved soft tissues. For FOP patients, surgery is always avoided since the surgical trauma to tissues presents a high risk of inducing additional bone formation. Hereditary and non-hereditary heterotopic ossification is rare in children. However, the formation of bone within soft tissues can cause severe disability and chronic pain. Effective strategies to prevent heterotopic ossification or to intervene in its progression have not been identified and clinical management of this condition is difficult. The identification of the genetic causes of FOP and POH provides important insights into the molecular and cellular events that promote extraskeletal bone formation. While both disorders are autosomal dominantly-inherited diseases of extensive and progressive heterotopic ossification, these conditions can be distinguished clinically and are caused by gene mutations in different cellular pathways (Table 30.4). Ongoing investigations into the cellular mechanisms that regulate chondrogenesis and osteogenesis that are perturbed in these disorders will eventually lead to treatments for these conditions and for non-hereditary heterotopic ossification, in children and in adults, which are likely caused by alterations in the same cellular processes and pathways that are altered in FOP and POH.

TABLE 30.4

Comparison of FOP and POH FOP

POH

Progressive anatomic site involvement with age

þ

þ

Progressive involvement of superficial (skin) to deeper tissues



þ

Induced by trauma

þ



Endochondral HO

þ

a

Intramembranous HO



þ

Pre-osseous inflammation

þ



Toe malformation

þ



Autosomal dominant inheritance

þ

þ

Most cases are sporadic (new mutations in family)

þ

þ

Mutated gene

ACVR1

GNAS

Heterotopic ossifcation:

a

Occasional evidence of endochondral ossification has been observed in POH biopsies, however, the main mechanism of HO in POH is intramembranous.

Acknowledgments We thank the members of our research laboratory and our many collaborators for their contributions. We also thank the NIH/NIAMS-supported Penn Center for Musculoskeletal Disorders (AR050950). This work was supported in part by the Center for Research in FOP and Related Disorders, the International FOP Association (IFOPA), the Ian Cali Endowment, the Weldon Family Endowment, the Isaac and Rose Nassau Professorship of Orthopaedic Molecular Medicine, the Rita Allen Foundation, and by grants from the National Institutes of Health (R01-AR41916 and R01-AR046831).

References [1] Al-Aql ZS, Alagl AS, Graves DT, Gerstenfeld LC, Einhorn TA. Molecular mechanisms controlling bone formation during fracture healing and distraction osteogenesis. J Dent Res 2008;87:107e18. [2] McCarthy EF, Sundaram M. Heterotopic ossification: a review. Skeletal Radiol 2005;34:609e19. [3] Pignolo RJ, Foley KL. Nonhereditary heterotopic ossification. Clin Rev Bone Miner Metab 2005;3:261e6. [4] Kaplan FS, Shore EM. Progressive osseous heteroplasia. J Bone Miner Res 2000;15:2084e94. [5] Shore EM, Kaplan FS. Insights from a rare genetic disorder of extra-skeletal bone formation, fibrodysplasia ossificans progressiva (FOP). Bone 2008;43:427e33.

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[6] Shore EM, Kaplan FS. Inherited human diseases of heterotopic bone formation. Nat Rev Rheumatol 2010;6:518e27. [7] Forsberg JA, Pepek JM, Wagner S, et al. Heterotopic ossification in high-energy wartime extremity injuries: prevalence and risk factors. J Bone Joint Surg (Am) 2009;91:1084e91. [8] Neal B, Gray H, MacMahon S, Dunn L. Incidence of heterotopic bone formation after major hip surgery. Aust NZ J Surg 2002;72:808e21. [9] Potter BK, Burns TC, Lacap AP, Granville RR, Gajewski D. Heterotopic ossification in the residual limbs of traumatic and combat-related amputees. J Am Acad Orthoped Surg 2006;14: S191e7. [10] van Kuijk AA, Geurts ACH, van Kuppevelt HJM. Neurogenic heterotopic ossification in spinal cord injury. Spinal Cord 2002; 40:313e26. [11] Kaplan FS, Glaser DL, Hebela N, Shore EM. Heterotopic ossification. J Am Acad Orthoped Surg 2004;12:116e25. [12] Davoodi P, Fernandez JM, Seung-Jun O. Postburn sequelae in the pediatric patient: Clinical presentations and treatment options. J Craniofac Surg 2008;19:1047e52. [13] Gaur A, Sinclair M, Caruso E, Peretti G, Zaleske D. Heterotopic ossification around the elbow following burns in children: Results after excision. J Bone Joint Surg (Am) 2003;85A: 1538e43. [14] Koch BM, Wu CM, Randolph J, Eng GD. Heterotopic ossification in children with burns e 2 case reports. Arch Phys Med Rehab 1992;73:1104e6. [15] Hedin H, Hjorth K, Rehnberg L, Larsson S. External fixation of displaced femoral shaft fractures in children: A consecutive study of 98 fractures. J Orthoped Trauma 2003;17: 250e6. [16] Jarvis J, Davidson D, Letts M. Management of subtrochanteric fractures in skeletally immature adolescents. J Trauma-Injury Infect Crit Care 2006;60:613e9. [17] Sawyer JR, Kapoor M, Gonzales MH, Warner WC, Canale ST, Beaty JH. Heterotopic ossification of the hip after non-accidental injury in a child: case report. J Pediatr Orthoped 2009;29:865e7. [18] Zagaja GP, Cromie WJ. Heterotopic bone formation in association with pelvic fracture and urethral disruption. J Urol 1999;161:1950e3. [19] Garland DE, Shimoyama ST, Lugo C, Barras D, Gilgoff I. Spinal cord insults and heterotopic ossification in the pediatric population. Clin Orthop Rel Res 1989;245:303e10. [20] Kluger G, Kochs A, Holthausen H. Heterotopic ossification in childhood and adolescence. J Child Neurol 2000;15:406e13. [21] Massey GV, Kuhn JG, Nogi J, et al. The spectrum of myositis ossiticans in haemophilia. Haemophilia 2004;10:189e93. [22] Mortazavi SMJ, Asadollahi S, Motamedi M. Operative treatment of anterior heterotopic bone formation of the elbow in a patient with severe haemophilia A. Haemophilia 2006;12: 444e7. [23] Gebhardt MC, Springfield D, Neff JR. Sarcomas of bone. In: Abeloff MD, Armitage JO, Niederhuber JE, Kastan MB, McKenna WG, editors. Abeloff’s Clinical Oncology. 4th ed. Philadelphia: Churchill Livingstone Elsevier; 2008. [24] Beall DP, Ly J, Bell JP, et al. Pediatric extraskeletal osteosarcoma. Pediatr Radiol 2008;38:579e82. [25] Siraj F, Jain D, Chopra P. Extraskeletal osteosarcoma of abdominal wall in a child e a rare case report with review of literature. Ann Diagn Pathol 2010. [26] Kaplan FS, Gannon FH, Hahn GV, Wollner N, Prauner R. Pseudomalignant heterotopic ossification e differential diagnosis and report of two cases. Clin Orthop Rel Res 1998;346:134e40.

[27] Kaplan FS, Glaser DL, Shore EM, et al. The phenotype of fibrodysplasia ossificans progressiva. Clin Rev Bone Miner Metab 2005;3:183e8. [28] Shore EM, Xu MQ, Feldman GJ, et al. A recurrent mutation in the BMP type I receptor ACVR1 causes inherited and sporadic fibrodysplasia ossificans progressiva. Nat Genet 2006;38:525e7. [29] Kaplan FS, Xu MQ, Glaser DL, et al. Early diagnosis of fibrodysplasia ossificans progressiva. Pediatrics 2008;121: E1295e300. [30] Cohen RB, Hahn GV, Tabas JA, et al. The natural history of heterotopic ossification in patients who have fibrodysplasia ossificans progressiva. A study of forty-four patients. J Bone Joint Surg (Am) 1993;75:215e9. [31] Gannon FH, Valentine BA, Shore EM, Zasloff MA, Kaplan FS. Acute lymphocytic infiltration in an extremely early lesion of fibrodysplasia ossificans progressiva. Clin Orthop Rel Res 1998;346:19e25. [32] Glaser DL, Economides AN, Wang LL, et al. In vivo somatic cell gene transfer of an engineered noggin mutein prevents BMP4induced heterotopic ossification. J Bone Joint Surg (Am) 2003;85A:2332e42. [33] Kaplan FS, Tabas JA, Gannon FH, Finkel G, Hahn GV, Zasloff MA. The histopathology of fibrodysplasia ossificans progressiva. An endochondral process. J Bone Joint Surg (Am) 1993;75:220e30. [34] Pignolo RJ, Suda RK, Kaplan FS. The fibrodysplasia ossificans progressiva lesion. Clin Rev Bone Miner Metab 2005;3:195e200. [35] Rocke DM, Zasloff M, Peeper J, Cohen RB, Kaplan FS. Age- and joint-specific risk of initial heterotopic ossification in patients who have fibrodysplasia ossificans progressiva. Clin Orthop Rel Res 1994;301:243e8. [36] Glaser DL, Rocke DM, Kaplan FS. Catastrophic falls in patients who have fibrodysplasia ossificans progressiva. Clin Orthop Rel Res 1998;346:110e6. [37] Janoff HB, Zasloff MA, Kaplan FS. Submandibular swelling in patients with fibrodysplasia ossificans progressiva. Otolaryngol Head Neck Surg 1996;114:599e604. [38] Lanchoney TF, Cohen RB, Rocke DM, Zasloff MA, Kaplan FS. Permanent heterotopic ossification at the injection site after diphtheria-tetanus-pertussis immunizations in children who have fibrodysplasia ossificans progressiva. J Pediatr 1995;126: 762e4. [39] Luchetti W, Cohen RB, Hahn GV, et al. Severe restriction in jaw movement after routine injection of local anesthetic in patients who have fibrodysplasia ossificans progressiva. Oral Surg Oral Med Oral Path Oral Radiol 1996;81:21e5. [40] Scarlett RF, Rocke DM, Kantanie S, Patel JB, Shore EM, Kaplan FS. Influenza-like viral illnesses and flare-ups of fibrodysplasia ossificans progressiva. Clin Orthop Rel Res 2004;423:275e9. [41] Connor JM, Evans DA. Fibrodysplasia ossificans progressiva. The clinical features and natural history of 34 patients. J Bone Joint Surg (Br) 1982;64:76e83. [42] Kaplan FS, Shore EM. Fibrodysplasia (myositis) ossificans progressiva. In: Rosen CJ, editor. Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. 7th ed. Washington, DC: American Society for Bone and Mineral Research; 2008. p. 442e6. [43] Kaplan FS, Shore EM, Connor JM. Fibrodysplasia ossificans progressiva. In: Royce P, Steinmann B, editors. Connective Tissue and its Heritable Disorders. New York: Wiley-Liss; 2002. p. 827e40. [44] Moriatis JM, Gannon FH, Shore EM, Bilker W, Zasloff MA, Kaplan FS. Limb swelling in patients who have fibrodysplasia ossificans progressiva. Clin Orthop Rel Res 1997;336:247e53.

PEDIATRIC BONE

REFERENCES

[45] Smith R. Fibrodysplasia (myositis) ossificans progressiva. Clinical lessons from a rare disease. Clin Orthop Rel Res 1998;346:7e14. [46] Kaplan FS, Glaser DL. Thoracic insufficiency syndrome in patients wiith fibrodysplasia ossificans progressiva. Clin Rev Bone Miner Metab 2005;3:213e6. [47] Shore EM, Feldman GJ, Xu M, Kaplan FS. The genetics of fibrodysplasia ossificans progressiva. Clin Rev Bone Miner Metab 2005;3:201e4. [48] Kaplan FS, Zasloff MA, Kitterman JA, Shore EM, Hong CC, Rocke DM. Early mortality and cardiorespiratory failure in patients with fibrodysplasia ossificans progressiva. J Bone Joint Surg (Am) 2010;92:686e91. [49] Kaplan FS, Xu M, Seemann P, et al. Classic and atypical fibrodysplasia ossificans progressiva (FOP) phenotypes are caused by mutations in the bone morphogenetic protein (BMP) type I receptor ACVR1. Hum Mutat 2009;30:379e90. [50] Schroeder HW, Zasloff M. The hand and foot malformations in fibrodysplasia ossificans progressiva. Johns Hopkins Med J 1980;147:73e8. [51] Schaffer AA, Kaplan FS, Tracy MR, et al. Developmental anomalies of the cervical spine in patients with fibrodysplasia ossificans progressiva are distinctly different from those in patients with Klippel-Feil syndrome e clues from the BMP signaling pathway. Spine 2005;30:1379e85. [52] Deirmengian GK, Hebela NM, O’Connell M, Glaser DL, Shore EM, Kaplan FS. Proximal tibial osteochondromas in patients with fibrodysplasia ossificans progressiva. J Bone Joint Surg (Am) 2008;90:366e74. [53] Kaplan FS, Le Merrer M, Glaser DL, et al. Fibrodysplasia ossificans progressiva. Best Pract Res Clin Rheum 2008;22:191e205. [54] Levy CE, Lash AT, Janoff HB, Kaplan FS. Conductive hearing loss in individuals with fibrodysplasia ossificans progressiva. Am J Audiol 1999;8:29e33. [55] Kussmaul WG, Esmail AN, Sagar Y, Ross J, Gregory S, Kaplan FS. Pulmonary and cardiac function in advanced fibrodysplasia ossificans progressiva. Clin Orthop Rel Res 1998; 346:104e9. [56] Shah PB, Zasloff MA, Drummond D, Kaplan FS. Spinal deformity in patients who have fibrodysplasia ossificans progressiva. J Bone Joint Surg (Am) 1994;76:1442e50. [57] Nussbaum BL, Ohara I, Kaplan FS. Fibrodysplasia ossificans progressiva: Report of a case with guidelines for pediatric dental and anesthetic management. ASDC J Dent Child 1996;63:448. [58] Nussbaum BL, Grunwald Z, Kaplan FS. Oral and dental health care and anesthesia for persons with fibrodysplasia ossificans progressiva. Clin Rev Bone Miner Metab 2005;3:239e42. [59] Kaplan FS, Strear CM, Zasloff MA. Radiographic and scintigraphic features of modeling and remodeling in the heterotopic skeleton of patients who have fibrodysplasia ossificans progressiva. Clin Orthop Rel Res 1994;304:238e47. [60] Mahboubi S, Glaser DL, Shore EM, Kaplan FS. Fibrodysplasia ossificans progressiva. Pediatr Radiol 2001;31:307e14. [61] Einhorn TA, Kaplan FS. Traumatic fractures of heterotopic bone in patients who have fibrodysplasia ossificans progressiva. A report of 2 cases. Clin Orthop Rel Res 1994;308:173e7. [62] Reinig JW, Hill SC, Fang M, Marini J, Zasloff MA. Fibrodysplasia ossificans progressiva e CT appearance. Radiology 1986;159:153e7. [63] Kaplan F, Sawyer J, Connors S, et al. Urinary basic fibroblast growth factor. A biochemical marker for preosseous fibroproliferative lesions in patients with fibrodysplasia ossificans progressiva. Clin Orthop Rel Res 1998;346:59e65.

837

[64] Suda RK, Billings PC, Egan KP, et al. Circulating osteogenic precursor cells in heterotopic bone formation. Stem Cells 2009;27:2209e19. [65] Hegyi L, Gannon FH, Glaser DL, Shore EM, Kaplan FS, Shanahan CM. Stromal cells of fibrodysplasia ossificans progressiva lesions express smooth muscle lineage markers and the osteogenic transcription factor Runx2/Cbfa-1: clues to a vascular origin of heterotopic ossification? J Pathol 2003;201: 141e8. [66] Kaplan FS, Glaser DL, Shore EM, et al. Hematopoietic stem-cell contribution to ectopic skeletogenesis. J Bone Joint Surg (Am) 2007;89A:347e57. [67] Lounev VY, Ramachandran R, Wosczyna MN, et al. Identification of progenitor cells that contribute to heterotopic skeletogenesis. J Bone Joint Surg (Am) 2009;1(91):652e63. [68] Bianco P, Riminucci M, Gronthos S, Robey PG. Bone marrow stromal stem cells: Nature, biology, and potential applications. Stem Cells 2001;19:180e92. [69] Dominici M, Pritchard C, Garlits JE, Hofmann TJ, Persons DA, Horwitz EM. Hematopoietic cells and osteoblasts are derived from a common marrow progenitor after bone marrow transplantation. Proc Natl Acad Sci USA 2004;101:11761e6. [70] Gronthos S, Zannettino ACW, Hay SJ, et al. Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow. J Cell Sci 2003;116:1827e35. [71] Modder UI, Khosla S. Skeletal stem/osteoprogenitor cells: current concepts, alternate hypotheses, and relationship to the bone remodeling compartment. J Cell Biochem 2008;103: 393e400. [72] Olmsted-Davis EA, Gugala Z, Camargo F, et al. Primitive adult hematopoietic stem cells can function as osteoblast precursors. Proc Natl Acad Sci USA 2003;100:15877e82. [73] Prockop DJ. Marrow stromal cells as steam cells for nonhematopoietic tissues. Science 1997;276:71e4. [74] Eghbali-Fatourechi GZ, Lamsam J, Fraser D, Nagel D, Riggs BL, Khosla S. Circulating osteoblast-lineage cells in humans. N Eng J Med 2005;352:1959e66. [75] Kaplan FS, Shore EM, Gupta R, et al. Immunological features of fibrodysplasia ossificans progessiva and the dysregulated BMP4 pathway. Clin Rev Bone Miner Metab 2005;3:189e93. [76] Kan LX, Liu YJ, McGuire TL, et al. Dysregulation of local stem/ progenitor cells as a common cellular mechanism for heterotopic ossification. Stem Cells 2009;27:150e6. [77] Yu PB, Deng DY, Lai CS, et al. BMP type I receptor inhibition reduces heterotopic ossification. Nat Med 2008;14:1363e9. [78] Gannon FH, Glaser D, Caron R, Thompson LDR, Shore EM, Kaplan FS. Mast cell involvement in fibrodysplasia ossificans progressiva. Hum Pathol 2001;32:842e8. [79] Kitterman JA, Kantanie S, Rocke DM, Kaplan FS. Iatrogenic harm caused by diagnostic errors in fibrodysplasia ossificans progressiva. Pediatrics 2005;116:654e61. [80] Janoff HB, Tabas JA, Shore EM, et al. Mild expression of fibrodysplasia ossificans progressiva e a report of 3 cases. J Rheumatol 1995;22:976e8. [81] Virdi AS, Shore EM, Oreffo ROC, et al. Phenotypic and molecular heterogeneity in fibrodysplasia ossificans progressiva. Calcif Tissue Int 1999;65:250e5. [82] Janoff HB, Muenke M, Johnson LO, et al. Fibrodysplasia ossificans progressiva in two half-sisters: evidence for maternal mosaicism. Am J Med Genet 1996;61:320e4. [83] Rogers JG, Chase GA. Paternal age effect in fibrodysplasia ossificans progressiva. J Med Genet 1979;16:147e8. [84] Kaplan FS, McCluskey W, Hahn G, Tabas JA, Muenke M, Zasloff MA. Genetic transmission of fibrodysplasia ossificans

PEDIATRIC BONE

838

[85]

[86]

[87]

[88]

[89] [90]

[91] [92]

[93]

[94]

[95]

[96]

[97]

[98]

[99]

[100]

[101]

[102] [103]

30. EXTRASKELETAL BONE FORMATION

progressiva. Report of a family. J Bone Joint Surg (Am) 1993;75:1214e20. Hebela N, Shore EM, Kaplan FS. Three pairs of monozygotic twins with fibrodysplasia ossificans progressiva. Clin Rev Bone Miner Metab 2005;3:205e8. Lucotte G, Semonin O, Lutz P. A de novo heterozygous deletion of 42 base-pairs in the noggin gene of a fibrodysplasia ossificans progressiva patient. Clin Genet 1999;56:469e70. Lucotte G, Houzet A, Hubans C, Lagarde JP, Lenoir G. Mutations of the noggin and of the activin a type I receptor genes in a series of twenty-seven French fibrodysplasia ossificans progressiva (FOP) patients. Genet Counsel 2009;20:53e62. Kaplan FS, Xu M, Feldman G, et al. Response to "Mutations of the NOGGIN and of the activin A type I receptor genes in fibrodysplasia ossificans progressiva (FOP)" by Lucotte, et al. Genet Counsel 2008;19:357e9. Seemann P, Mundlos S. The tale of FOP, noggin and myristolation: no data, no proof! Genet Counsel 2008;19:353e5. Xu MQ, Feldman G, Le Merrer M, et al. Linkage exclusion and mutational analysis of the noggin gene in patients with fibrodysplasia ossificans progressiva (FOP). Clin Genet 2000;58:291e8. Xu MQ, Shore EM, Kaplan FS. Reported noggin mutations are PCR errors. Am J Med Genet 2002;109(2):161. Bocciardi R, Bordo D, Di Duca M, Di Rocco M, Ravazzolo R. Mutational analysis of the ACVR1 gene in Italian patients affected with fibrodysplasia ossificans progressiva: confirmations and advancements. Eur J Hum Genet 2009;17:311e8. Carvalho DR, Navarro MMM, Martins B, et al. Mutational screening of ACVR1 gene in Brazilian fibrodysplasia ossificans progressiva patients. Clin Genet 2010;77:171e6. Fukuda T, Kanomata K, Nojima J, et al. A unique mutation of ALK2, G356D, found in a patient with fibrodysplasia ossificans progressiva is a moderately activated BMP type I receptor. Biochem Biophys Res Commun 2008;19(377):905e9. Furuya H, Ikezoe K, Wang LX, et al. A unique case of fibrodysplasia ossificans progressiva with an ACVR1 mutation, G356D, other than the common mutation (R206H). Amer J Med Genet Pt A 2008;146A(4):459e63. 5. Petrie KA, Lee WH, Bullock AN, et al. Novel mutations in ACVR1 result in atypical features in two fibrodysplasia ossificans progressiva patients. Plos One 2009;4(3). Ratbi I, Borcciadi R, Regragui A, Ravazzolo R, Sefiani A. Rarely occurring mutation of ACVR1 gene in Moroccan patient with fibrodysplasia ossificans progressiva. Clin Rheumatol 2010;29: 119e21. Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature 2003;425:577e84. Huse M, Muir TW, Xu L, Chen YG, Kuriyan J, Massague J. The TGF beta receptor activation process: an inhibitor- to substratebinding switch. Mol Cell 2001;8:671e82. Krause C, de Gorter DJJ, Karperien M, ten Dijke P. Signal transduction cascades controlling osteoblast differentiiation. In: Rosen CJ, editor. Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. 7th ed. Washington, DC: American Society of Bone and Mineral Research; 2008. p. 10e6. Schmierer B, Hill CS. TGFbeta-SMAD signal transduction: molecular specificity and functional flexibility. Nat Rev Mol Cell Biol 2007;8:970e82. Shi Y, Massague J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 2003;13(113):685e700. ten Dijke P, Hill CS. New insights into TGF-beta-Smad signalling. Trends Biochem Sci 2004;29:265e73.

[104] Chen YG, Hata A, Lo RS, et al. Determinants of specificity in TGF-beta signal transduction. Genes Dev 1998;12:2144e52. [105] Guo X, Wang X- F. Signaling cross-talk between TGF-beta/BMP and other pathways. Cell Res 2009;19:71e88. [106] Little SC, Mullins MC. BMP heterodimers assemble hetero-type I receptor complexes that pattern the DV axis. Nat Cell Biol 2009;11:637e43. [107] Massague J. How cells read TGF-beta signals. Nat Rev Mol Cell Biol 2000;1:169e78. [108] Moustakas A, Heldin C- H. Non-Smad TGF-beta signals. J Cell Sci 2005;118:3573e84. [109] Nohe A, Hassel S, Ehrlich M, et al. The mode of bone morphogenetic protein (BMP) receptor oligomerization determines different BMP-2 signaling pathways. J Biol Chem 2002;277:5330e8. [110] Derynck R, Akhurst RJ. Differentiation plasticity regulated by TGF-beta family proteins in development and disease. Nat Cell Biol 2007;9:1000e4. [111] Eivers E, Fuentealba LC, De Robertis EM. Integrating positional information at the level of Smad1/5/8. Curr Opin Genet Devel 2008;18:304e10. [112] Heisenberg C-P, Solnica-Krezel L. Back and forth between cell fate specification and movement during vertebrate gastrulation. Curr Opin Genet Devel 2008;18:311e6. [113] Hogan BLM. Bone morphogenetic proteins: multifunctional regulators of vertebrate development. Genes Devel 1996;10:1580e94. [114] Watabe T, Miyazono K. Roles of TGF-beta family signaling in stem cell renewal and differentiation. Cell Res 2009;19:103e15. [115] Wu MY, Hill CS. Tgf-beta superfamily signaling in embryonic development and homeostasis. Dev Cell 2009;16:329e43. [116] Xu J, Lamouille S, Derynck R. TGF-beta-induced epithelial to mesenchymal transition. Cell Res 2009;19:156e72. [117] Urist MR. Bone: formation by autoinduction. Science 1965;150:893e9. [118] Gazzerro E, Canalis E. Bone morphogenetic proteins and their antagonists. Rev Endocr Metab Disord 2006;7:51e65. [119] Zhang D, Schwarz EM, Rosier RN, Zuscik MJ, Puzas JE, O’Keefe RJ. ALK2 functions as a BMP type I receptor and induces Indian hedgehog in chondrocytes during skeletal development. J Bone Miner Res 2003;18:1593e604. [120] Billings PC, Fiori JL, Bentwood JL, et al. Dysregulated BMP signaling and enhanced osteogenic differentiation of connective tissue progenitor cells from patients with fibrodysplasia ossificans progressiva (FOP). J Bone Miner Res 2008;23:305e13. [121] Fiori JL, Billings PC. Serrano de la Pena LS, Kaplan FS, Shore EM. Dysregulation of the BMP-p38 MAPK signaling pathway in cells from patients with fibrodysplasia ossificans progressiva (FOP). J Bone Miner Res 2006;21:902e9. [122] Serrano de la Pena LS, Billings PC, Fiori JL, Ahn J, Kaplan FS, Shore EM. Fibrodysplasia ossificans progressiva (FOP), a disorder of ectopic osteogenesis, misregulates cell surface expression and trafficking of BMPRIA. J Bone Miner Res 2005;20:1168e76. [123] Shafritz AB, Shore EM, Gannon FH, et al. Overexpression of an osteogenic morphogen in fibrodysplasia ossificans progressiva. N Engl J Med 1996;335:555e61. [124] Groppe JC, Shore EM, Kaplan FS. Functional Modeling of the ACVR1 (R206H) mutation in FOP. Clin Orthop Rel Res 2007;462:87e92. [125] Fukuda T, Kohda M, Kanomata K, et al. Constitutively activated ALK2 and increased SMAD1/5 cooperatively induce bone morphogenetic protein signaling in fibrodysplasia ossificans progressiva. J Biol Chem 2009;284:7149e56.

PEDIATRIC BONE

REFERENCES

[126] Shen Q, Little SC, Xu M, et al. The fibrodysplasia ossificans progressiva R206H ACVR1 mutation activates BMP-independent chondrogenesis and zebrafish embryo ventralization. J Clin Invest 2009;119:3462e72. [127] Song GA, Kim HJ, Woo KM, et al. Molecular consequences of the ACVR1(R206H) mutation of fibrodysplasia ossificans progressiva. J Biol Chem 2010;285:22542e53. [128] van Dinther M, Visser N, de Gorter DJJ, et al. ALK2 R206H mutation linked to fibrodysplasia ossificans progressiva confers constitutive activity to the BMP type I receptor and sensitizes mesenchymal cells to BMP-induced osteoblast differentiation and bone formation. J Bone Miner Res 2010;25:1208e15. [129] Huse M, Chen YG, Massague J, Kuriyan J. Crystal structure of the cytoplasmic domain of the type I TGF beta receptor in complex with FKBP12. Cell 1999;96:425e36. [130] Yao D, Dore Jr JJ, Leof EB. FKBP12 is a negative regulator of transforming growth factor-beta receptor internalization. J Biol Chem 2000;275:13149e54. [131] Gu Z, Reynolds EM, Song J, et al. The type I serine/threonine kinase receptor ActRIA (ALK2) is required for gastrulation of the mouse embryo. Development 1999;126:2551e61. [132] Mishina Y, Crombie R, Bradley A, Behringer RR. Multiple roles for activin-like kinase-2 signaling during mouse embryogenesis. Dev Biol 1999;213:314e26. [133] Korchynskyi O, ten Dijke P. Identification and functional characterization of distinct critically important bone morphogenetic protein-specific response elements in the Id1 promoter. J Biol Chem 2002;277:4883e91. [134] Mintzer KA, Lee MA, Runke G, Trout J, Whitman M, Mullins MC. Lost-a-fin encodes a type I BMP receptor, Alk8, acting maternally and zygotically in dorsoventral pattern formation. Development 2001;128:859e69. [135] Payne TL, Postlethwait JH, Yelick PC. Functional characterization and genetic mapping of alk8. Mech Dev. 2001;100:275e89. [136] Fukuda T, Scott G, Komatsu Y, et al. Generation of a mouse with conditionally activated signaling through the BMP receptor, ALK2. Genesis 2006;44:159e67. [137] Chakkalakal SA, Zhang D, Culbert A, et al. The ACVR1 R206H mutation recapitulates the clinical phenotype of FOP in a knockin mouse model. J Bone Miner Res 2010;25(Suppl 1). S4. [138] Glaser DL, Kaplan FS. Treatment considerations for the management of fibrodysplasia ossificans progressiva. Clin Rev Bone Miner Metab 2005;3:243e50. [139] Fishman MC, Porter JA. Pharmaceuticals e a new grammar for drug discovery. Nature 2005;437:491e3. [140] Kaplan FS, Glaser DL, Pignolo RJ, Shore EM. A new era for fibrodysplasia ossificans progressiva: a druggable target for the second skeleton. Expert Opin Biol Ther 2007;7:705e12. [141] Kaplan FS, Craver R, MacEwen GD, et al. Progressive osseous heteroplasia: a distinct developmental disorder of heterotopic ossification. Two new case reports and follow-up of three previously reported cases. J Bone Joint Surg (Am) 1994;76:425e36. [142] Shore EM, Ahn J, de Beur SJ, et al. Paternally inherited inactivating mutations of the GNAS1 gene in progressive osseous heteroplasia. N Eng J Med 2002;346:99e106. [143] Adegbite NS, Xu M, Kaplan FS, Shore EM, Pignolo RJ. Diagnostic and mutational spectrum of progressive osseous heteroplasia (POH) and other forms of GNAS-based heterotopic ossification. Am J Med Genet 2008;146A:1788e96. [144] Bastepe M, Juppner H. GNAS locus and pseudohypoparathyroidism. Horm Res 2005;63:65e74. [145] Plagge A, Kelsey G, Germain-Lee EL. Physiological functions of the imprinted Gnas locus and its protein variants Galpha(s) and XLalpha(s) in human and mouse. J Endocrinol 2008;196: 193e214.

839

[146] Weinstein LS, Chen M, Xie T, Liu J. Genetic diseases associated with heterotrimeric G proteins. Trends Pharmacol Sci 2006;27:260e6. [147] Levine MA. Pseudohypoparathyroidism. In: Bilezikian JP, Raisz LG, Rodan GA, editors. Principles of Bone Biology. New York: Academic Press; 1996. p. 853e76. [148] Bastepe M, Lane AH, Juppner H. Paternal uniparental isodisomy of chromosome 20q e and the resulting changes in GNAS1 methylation e as a plausible cause of pseudohypoparathyroidism. Am J Hum Genet 2001;68:1283e9. [149] Bastepe M, Frohlich LF, Linglart A, et al. Deletion of the NESP55 differentially methylated region causes loss of maternal GNAS imprints and pseudohypoparathyroidism type Ib. Nat Genet 2005;37:25e7. [150] Jan de Beur SM, O’Connell JR, et al. The pseudohypoparathyroidism type 1b locus is linked to a region including GNAS1 at 20q13.3. J Bone Miner Res 2003;18:424e33. [151] Juppner H, Schipani E, Bastepe M, et al. The gene responsible for pseudohypoparathyroidism type Ib is paternally imprinted and maps in four unrelated kindreds to chromosome 20q13.3. Proc Natl Acad Sci USA 1998;95:11798e803. [152] Eddy MC, De Beur SMJ, Yandow SM, et al. Deficiency of the alpha-subunit of the stimulatory G protein and severe extraskeletal ossification. J Bone Miner Res 2000;15:2074e83. [153] Yeh GL, Mathur S, Wivel A, et al. GNAS1 mutation and Cbfa1 misexpression in a child with severe congenital platelike osteoma cutis. J Bone Miner Res 2000;15:2063e73. [154] Weinstein LS, Yu S, Warner DR, Liu J. Endocrine manifestations of stimulatory G protein alpha-subunit mutations and the role of genomic imprinting. Endocrine Rev 2001;22:675e705. [155] Lebrun M, Richard N, Abeguile G, et al. Progressive osseous heteroplasia: a model for the imprinting effects of GNAS inactivating mutations in humans. J Clin Endocrinol Metab 2010;95:3028e38. [156] Long DN, McGuire S, Levine MA, Weinstein LS, GermainLee EL. Body mass index differences in pseudohypoparathyroidism type 1a versus pseudopseudohypoparathyroidism may implicate paternal imprinting of Galpha(s) in the development of human obesity. J Clin Endocrinol Metab 2007;92:1073e9. [157] Ahmed SF, Dixon PH, Bonthron DT, et al. GNAS1 mutational analysis in pseudohypoparathyroidism. Clin Endocrinol (Oxf) 1998;49:525e31. [158] Ahrens W, Hiort O, Staedt P, Kirschner T, Marschke C, Kruse K. Analysis of the GNAS1 gene in Albright’s hereditary osteodystrophy. J Clin Endocrinol Metab 2001;86:4630e4. [159] Aldred MA, Trembath RC. Activating and inactivating mutations in the human GNAS1 gene. Hum Mutat 2000;16:183e9. [160] Farfel Z, Iiri T, Shapira H, Roitman A, Mouallem M, Bourne HR. Pseudohypoparathyroidism, a novel mutation in the betagamma-contact region of Gsalpha impairs receptor stimulation. J Biol Chem 1996;271:19653e5. [161] Fischer JA, Egert F, Werder E, Born W. An inherited mutation associated with functional deficiency of the alpha-subunit of the guanine nucleotide-binding protein Gs in pseudo- and pseudopseudohypoparathyroidism. J Clin Endocrinol Metab 1998; 83:935e8. [162] Linglart A, Carel JC, Garabedian M, Le T, Mallet E, Kottler ML. GNAS1 lesions in pseudohypoparathyroidism Ia and Ic: genotype phenotype relationship and evidence of the maternal transmission of the hormonal resistance. J Clin Endocrinol Metab 2002;87:189e97. [163] Luttikhuis ME, Wilson LC, Leonard JV, Trembath RC. Characterization of a de novo 43-bp deletion of the Gs alpha gene (GNAS1) in Albright hereditary osteodystrophy. Genomics 1994;21:455e7.

PEDIATRIC BONE

840

30. EXTRASKELETAL BONE FORMATION

[164] Miric A, Vechio JD, Levine MA. Heterogeneous mutations in the gene encoding the alpha-subunit of the stimulatory G protein of adenylyl cyclase in Albright hereditary osteodystrophy. J Clin Endocrinol Metab 1993;76:1560e8. [165] Nakamoto JM, Zimmerman D, Jones EA, et al. Concurrent hormone resistance (pseudohypoparathyroidism type Ia) and hormone independence (testotoxicosis) caused by a unique mutation in the G alpha s gene. Biochem Mol Med 1996;58: 18e24. [166] Nakamoto JM, Sandstrom AT, Brickman AS, Christenson RA, Van Dop C. Pseudohypoparathyroidism type Ia from maternal but not paternal transmission of a Gsalpha gene mutation. Am J Med Genet 1998;77:261e7. [167] Patten JL, Johns DR, Valle D, et al. Mutation in the gene encoding the stimulatory G protein of adenylate cyclase in Albright’s hereditary osteodystrophy. N Engl J Med 1990;322:1412e9. [168] Schwindinger WF, Miric A, Zimmerman D, Levine MA. A novel Gs alpha mutant in a patient with Albright hereditary osteodystrophy uncouples cell surface receptors from adenylyl cyclase. J Biol Chem 1994;269:25387e91. [169] Shapira H, Mouallem M, Shapiro MS, Weisman Y, Farfel Z. Pseudohypoparathyroidism type Ia: two new heterozygous frameshift mutations in exons 5 and 10 of the Gs alpha gene. Hum Genet 1996;97:73e5. [170] Walden U, Weissortel R, Corria Z, et al. Stimulatory guanine nucleotide binding protein subunit 1 mutation in two siblings with pseudohypoparathyroidism type 1a and mother with pseudopseudohypoparathyroidism. Eur J Pediatr 1999;158: 200e3. [171] Warner DR, Gejman PV, Collins RM, Weinstein LS. A novel mutation adjacent to the switch III domain of G(S alpha) in a patient with pseudohypoparathyroidism. Mol Endocrinol 1997;11:1718e27. [172] Warner DR, Weng G, Yu S, Matalon R, Weinstein LS. A novel mutation in the switch 3 region of Gsalpha in a patient with Albright hereditary osteodystrophy impairs GDP binding and receptor activation. J Biol Chem 1998;273:23976e83. [173] Weinstein LS, Gejman PV, Friedman E, et al. Mutations of the Gs alpha-subunit gene in Albright hereditary osteodystrophy detected by denaturing gradient gel electrophoresis. Proc Natl Acad Sci USA 1990;87:8287e90.

[174] Weinstein LS, Gejman PV, de Mazancourt P, American N, Spiegel AM. A heterozygous 4-bp deletion mutation in the Gs alpha gene (GNAS1) in a patient with Albright hereditary osteodystrophy. Genomics 1992;13:1319e21. [175] Yokoyama M, Takeda K, Iyota K, Okabayashi T, Hashimoto KA. 4-base pair deletion mutation of Gs alpha gene in a Japanese patient with pseudohypoparathyroidism. J Endocrinol Invest 1996;19:236e41. [176] Yu S, Yu D, Hainline BE, et al. A deletion hot-spot in exon 7 of the Gs alpha gene (GNAS1) in patients with Albright hereditary osteodystrophy. Hum Mol Genet 1995;4:2001e2. [177] Yu D, Yu S, Schuster V, Kruse K, Clericuzio CL, Weinstein LS. Identification of two novel deletion mutations within the Gs alpha gene (GNAS1) in Albright hereditary osteodystrophy. J Clin Endocrinol Metab 1999;84:3254e9. [178] Rubin MR, Levine MA. Hypoparathyroidism and pseudohypoparathyroidism. In: Rosen CJ, editor. Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. 7th ed. Washington, DC: American Society for Bone and Mineral Research; 2008. p. 354e61. [179] Weinstein LS, Xie T, Zhang Q-H, Chen M. Studies of the regulation and function of the Gs alpha gene Gnas using gene targeting technology. Pharmacol Therapeut 2007;115:271e91. [180] Plagge A, Gordon E, Dean W, et al. The imprinted signaling protein XL alpha s is required for postnatal adaptation to feeding. Nat Genet 2004;36:818e26. [181] Bastepe M, Raas-Rothschild A, Silver J, et al. A form of Jansen’s metaphyseal chondrodysplasia with limited metabolic and skeletal abnormalities is caused by a novel activating parathyroid hormone (PTH)/PTH-related peptide receptor mutation. J Clin Endocrinol Metab 2004;89:3595e600. [182] Sakamoto A, Chen M, Kobayashi T, Kronenberg HM, Weinstein LS. Chondrocyte-specific knockout of the G protein G(s)alpha leads to epiphyseal and growth plate abnormalities and ectopic chondrocyte formation. J Bone Miner Res 2005;20:663e71. [183] Sakamoto A, Chen M, Nakamura T, Xie T, Karsenty G, Weinstein LS. Deficiency of the G-protein alpha-subunit G(s) alpha in osteoblasts leads to differential effects on trabecular and cortical bone. J Biol Chem 2005;280:21369e75. [184] Wu JY, Scadden DT, Kronenberg HM. Role of the osteoblast lineage in the bone marrow hematopoietic niches. J Bone Miner Res 2009;24:759e64.

PEDIATRIC BONE

Color Plates

FIGURE 1.2 Osteoclast differentiation and activity. The activation of osteoclast precursors to fuse and form active osteoclasts is shown in the schematic image (upper panel). Arising from a hematopoietic precursor that is activated by M-CSF and RANKL, the pre-osteoclast is formed and fuses with others to produce multinucleated TRAP positive cells, which finally form a sealing zone to attach to the bone matrix to form the bone resorption cavity underneath a ruffled border. Active, TRAP positive stained osteoclasts (red) can be seen on the histological image taken from the distal region of a mouse femur. Methyl green was used as counterstaining to label all cell nuclei.

FIGURE 1.3 The osteoblast lineage. The upper panel represents the differentiation of a pre-osteoblast into an osteocyte. While the preosteoblast descended from the mesenchymal progenitor cell is mainly but not entirely expressing type I collagen, the mature osteoblast also expresses a variety of non-collagenous proteins for ECM synthesis, including bone sialoprotein and osteocalcin, a bone specific marker. During the differentiation into mature osteocytes, several proteins are upregulated such as E11/gp38, a marker for embedding and early osteocytes and sclerostin a marker for late, mature osteocytes. The histological image from the distal region of a mouse femur (stained with hematoxylin and eosin) shows an active cuboidal osteoblast on the bone surface (purple arrow), an early osteocyte (blue arrow) and a mature, stellar-shaped osteocyte deeply embedded in the bone matrix (green arrow).

FIGURE 17.16 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 fibro-osseous 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 20 .

FIGURE 17.17 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.

FIGURE 22.1

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.

FIGURE 22.9 Cafe´ 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.

FIGURE 22.10 (A) Mutations at codon 201 demonstrated by sequencing of the relevant PCR-amplifled 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].

FIGURE 22.12 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).

FIGURE 22.13 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.

FIGURE 22.14 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.

FIGURE 22.15 Images from a 24-year-old patient with polyostotic FD and hypophosphatemia, 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.

FIGURE 22.17 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). Undecalcifled methyl-metacrylate embedding; Goldner’s stain, hac, hydroxyapatite carrier.

FIGURE 26.2 Biopsy of adult XLH osteomalacia. Goldner-stained undecalcified sections of iliac crest bone from an adult with XLH (magnification, 360). Note the excess osteoid accumulation with relatively normal abundance of mineralized bone.

FIGURE 29.2 (A) and (B): Staining of FGF-23 in cancellous bone under 50 magnification and (B) and (D): staining of DMP1 in cancellous bone under 200 magnification. Arrows indicate osteoctyes with positive staining. Trabeculae and bone marrow are labeled TB and BM respectively. (A) and (C): subject with normal renal function. (B) and (D): subjects with stage 2 CKD. (Reprint by permission from Pereira et al. Bone. 2009;45:1161-8.)

FIGURE 30.2 Histological stages of heterotopic ossification in FOP. Histological analysis of biopsies obtained from FOP patients (obtained prior to diagnosis for FOP) revealed a tissue degradation phase (1AeC) that includes perivascular lymphocyte infiltration (1A), immune cell infiltration (1B), and connective tissue degradation (1C) that is followed by a tissue formation phase that includes fibroproliferation (2A), early cartilage (2B), and endochondral bone formation (2C) stages. (Adapted from Glaser et al. J Bone Joint Surg. 2003;85A:2332-42.)

FIGURE 30.5

Histopathology of a POH lesion. Initial bone-forming lesions in POH are typically observed as irregular deposits of bone within the dermal tissue are often observed in proximity to subcutaneous adipose tissue. Hematoxylin-eosin staining.

Index

A

Abuse, see Child abuse Acidebase balance, see Renal tubular acidosis Acute lymphoblastic leukemia (ALL) bone histomorphometry findings, 398 osteoporosis association with treatment, 457e458 ACVR1, fibrodysplasia ossificans progressiva mutations bone morphogenetic protein signaling effects, 830e831 cell function effects, 831 DNA testing, 828e829 R206H mutation, 829e830 variant disease mutations, 829 ADHH, see Autosomal dominant hypocalcemic hypercalciuria ADHR, see Autosomal dominant hypophosphatemic rickets AH, see Autoimmune acquired hypoparathyroidism AHO, see Albright’s hereditary osteodystrophy Albright’s hereditary osteodystrophy (AHO) differential diagnosis, 293 extraskeletal ossification, 833e834 radiographic findings, 293 Alkaline phosphatase, see also Bone-specific alkaline phosphatase; Tissuenon-specific alkaline phosphatase chronic kidney disease bone and mineral disorder evaluation, 804e805 skeletal formation function, 772e773 types and structures, 771e772 X-linked hypophosphatemia activity, 703 ALL, see Acute lymphoblastic leukemia ALPL, see Tissue-non-specific alkaline phosphatase Aluminum, chronic kidney disease bone and mineral disorder evaluation, 805e806 AN, see Anorexia nervosa Androgens fetal levels and functions, 253 peak bone mass effects, 197e198 postnatal bone growth regulation, 72e73 ANKH, craniometaphyseal dysplasia mutations, 549 Anorexia nervosa (AN), osteoporosis association, 469e472 APECED, see Autoimmune polyendocrinopathy candidiasisectodermal dystrophy

Aromatase, inhibitor effects on bone turnover markers, 374e375 Asphyxiating thoracic dysplasia (ATD), radiographic findings, 430 ATD, see Asphyxiating thoracic dysplasia Atf4 knockout mouse, 213 osteoblast differentiation role, 47 Atlas technique, bone age assessment, 348e349 Autoimmune acquired hypoparathyroidism (AH), 566 Autoimmune polyendocrinopathy candidiasis-ectodermal dystrophy (APECED), hypoparathyroidism, 561 Autosomal dominant hypocalcemic hypercalciuria (ADHH), 751 Autosomal dominant hypomagnesemia, 753 Autosomal dominant hypophosphatemic rickets (ADHR) features, 708e709 mouse model, 716e717 renal phosphate handling defects, 737 Autosomal recessive hypophosphatemic rickets features, 709 renal phosphate handling defects, 737e738

B

BALP, see Bone-specific alkaline phosphatase Bartter syndrome hypoparathyroidism, 566 renal calcium handling defects, 748, 751 BDE, see Brachydactyly type E BFR/BS, see Bone formation rate per bone surface Biglycan, 19 Biopsy, see Histomorphometry, bone Bisphosphonates fibrous dysplasia management, 614 osteogenesis imperfecta management, 524e527 osteoporosis treatment in children, 488e489 Blomstrand dysplasia, features and gene mutations, 541, 570e571 BMD, see Bone mineral density BMPs, see Bone morphogenetic proteins BN, see Bulimia nervosa Bone age, see also Maturation assessment Atlas technique, 348e349

841

BonXpert, 353 comparison of methods, 354 Fels method, 284e285, 353 GreulichePyle method, 284e285, 349 overview, 284e285 Oxford method, 349e350 reliability, 353e354 RocheeWainereThissen technique, 352 TannereWhitehouse method, 284e285, 350e352 radiographic assessment, 284e285 sexual dimorphism in maturation, 344e345, 347 Bone biopsy, see Histomorphometry, bone Bone formation rate per bone surface (BFR/BS), histomorphometry, 389 Bone histomorphometry, see Histomorphometry, bone Bone mineral density (BMD), see also Peak bone mass assessment, see also Dual energy X-ray energy absorptiometry; Quantitative computed tomography; Quantitative ultrasonography children, 311e312 indications, 309e310 overview, 190 radiation dose, 312 radiogrammetry, 312e313 radiographic absorptiometry, 313 classification in children, 311e312 infant early growth and later development, 671 osteoporosis scores in children, 440e441 preterm infants, 661e662, 664, 666e667, 669 structural development, 190e191, 193 Bone morphogenetic proteins (BMPs) fetal bone development role chondrocyte differentiation, 44e45 condensation, 43e44 epithelialemesenchymal interaction, 42 fibrodysplasia ossificans progressiva signaling, 830e831 osteoblast differentiation and activity role in growth plate ossification, 64e65 osteoblast differentiation role, 48 tooth bud development role, 83e84 Bone scan, see Radionuclide scanning Bone sialoprotein (BSP), 24 Bone-specific alkaline phosphatase (BALP), bone formation marker, 364e365 Bone turnover biochemical considerations, 364 lactation, 233e234 markers

842

Bone turnover (Continued) adult clinical practice, 372 aromatase inhibitor effects, 374e375 bone disease finding interpretation and problems, 370e372 chronic disease effects, 374 EhlerseDanlos syndrome type VI effects, 376 formation markers bone-specific alkaline phosphatase, 364e365 osteocalcin, 365 procollagen type I C-terminal propeptide, 365e366 procollagen type I N-terminal propeptide, 365e366 growth prediction, 372e373 normal findings infancy and childhood, 370 perinatal period, 368, 370 postnatal period, 370 puberty, 370 osteogenesis imperfecta, 373e374 pediatric reference data biological variation, 367 clinical considerations, 367e368 growth spurts, 368 reference curve generation, 368e369 sex differences, 368 prospects for study, 376 resorption markers C-terminal cross-linked telopeptide, 362 C-terminal telopeptide of type I collagen, 366 cathepsin K, 367 deoxypyridinoline, 366 Dickkopf-1, 367 N-terminal cross-linked telopeptide, 362 osteoprotegerin, 367 pyridinoline, 366 RANKL, 367 sclerostin, 367 tartrate-resistant acid phosphatase, 366e367 rickets effects, 375e376 table, 362 overview in children, 361e364 pregnancy, 225e226 preterm infants, 664e667, 669 renal osteodystrophy, 799e800 Bone volume per tissue volume (BV/TV), histomorphometry, 387 BonXpert, bone age assessment, 353 Brachydactyly type E (BDE), hyperparathyroidism, 578 Breast milk, see Lactation Bruck syndrome (BS) osteogenesis imperfecta, 517 osteoporosis, 444 BS, see Bruck syndrome BSP, see Bone sialoprotein

INDEX

Bulimia nervosa (BN), osteoporosis association, 469e472 Burn injury, bone histomorphometry findings, 398 BV/TV, see Bone volume per tissue volume

C Cadmium, renal Fanconi’s syndrome induction, 743 Caffey disease, 542 Calbindin-D9K, placental expression, 257 Calciphylaxis, chronic kidney disease bone and mineral disorder, 803e804 Calcitonin (CT) fetal levels and functions, 252 lactation changes, 227 placental expression, 258 pregnancy changes, 227 Calcium bone histomorphometry in deficiency, 393 chronic kidney disease bone and mineral disorder evaluation, 804 deficiency, see Rickets fetus integrated calcium homeostasis blood calcium regulation, 267 placental calcium transfer, 267 skeletal mineralization, 267 metabolism, 247e248 lactation dietary intake, 237e238 metabolism, 232e233 maternal nutrition effects on offspring bone, 671 osteogenesis imperfecta management, 526 parathyroid hormone expression and secretion regulation by calcium, 115e117 homeostasis bone actions, 119e121 kidney actions, 118e119 peak bone mass effects dietary recommendations, 210 geneeenvironment interactions, 209 individual differences of bone response, 208e209 interventional studies, 204e210 observational studies, 204 physical activity interactions, 206e207 pubertal maturation influences on supplementation effects, 205e206 skeletal site responsiveness, 205 placental transport fetal regulation parathyroid hormone, 262e263 parathyroid hormone-related peptide, 260e262 vitamin D, 263 gene expression, 257e258 maternal regulation, 260 measurement, 258e260 overview, 256e257 placental structure, 257

pregnancy dietary intake, 229e230 maternal fluxes from mother to offspring, 223e224 metabolism, 224 preterm infant intake recommendations, 669e670 protein intake effects on bone metabolism, 210e211 renal handling collecting duct, 745 connecting tubule, 744 defects autosomal dominant hypocalcemic hypercalciuria, 751 Bartter syndrome, 748, 751 Dent’s disease, 748 familial benign hypercalcemia, 751e752 familial hypomagnesemia with hypercalciuria and nephrocalcinosis, 751 Gitelman syndrome, 752 Lowe’s syndrome, 748 overview of diseases, 749e750 distal convoluted tubule, 744 loop of Henle, 744 proximal tubule, 743e744 vitamin D and homeostasis 1a,25-dihydroxyvitamin D actions, 170e178 vitamin D metabolites, 178e179 Calcium-sensing receptor (CaSR) hyperparathyroidism defects, 577 hypoparathyroidism defects, 566 knockout mouse and fetal effects, 255e256 placental expression, 258 Campomelia, differential diagnosis, 429 CamuratieEngelmann disease, see Progressive diaphyseal dysplasia Carbonic anhydrase II, osteopetrosis mutations, 544 Cartilage, see also Chondrocyte; Growth plate fibrous dysplasia pathology, 607 histology in skeletal dysplasia diagnosis, 431e433 vascularization, 60e61 Cartilage-hair hypoplasia (CHH) genetics, 434 radiographic findings, 427 Cartilage oligomeric matrix protein, see Thrombospondins CaSR, see Calcium-sensing receptor b-Catenin, see Wnt Cathepsin K bone resorption marker, 367 pycnodysostosis mutation, 547 CCD, see Cleidocranial dysplasia Celiac disease, osteoporosis association, 462e462, 464e466

INDEX

Cementum composition, 89e90 types and structural differences, 89 Cerebral palsy (CP), osteoporosis association, 451e454 CF, see Cystic fibrosis Cherubism, dental agenesis, 96 CHH, see Cartilage-hair hypoplasia Child abuse dating of fractures, 305 diaphyseal fracture, 303 differentiation from bone fragility conditions, 490e491, 517e518 imaging modalities, 302 metaphyseal fracture, 303 post-mortem imaging, 303 rib fracture, 304e305 skeletal survey, 302e303 skull fracture, 303 soft tissue injury, 303 subperiosteal new bone formation, 305 Chondrocyte fetal bone development differentiation, 44e45 proliferation and maturation, 45e47 growth plate control of proliferation and differentiation, 57e59 Chondrocyte, phosphate function, 143e144 Chondrodysplasia punctata, differential diagnosis, 429e430 Chronic kidney disease bone and mineral disorder (CKD-MBD) clinical manifestations bone pain, 801 calciphylaxis, 803e804 extraskeletal calcification, 802e803 growth retardation, 802 muscle weakness, 801 skeletal deformities, 801e802 diagnostic evaluation alkaline phosphatase, 804e805 aluminum, 805e806 calcium, 804 magnesium, 804 parathyroid hormone, 805 phosphate, 804 kidney transplantation effects on bone, 811e812 osteoporosis association, 576 pathogenesis, 795e798 radiographic features, 806 renal osteodystrophy bone histomorphometry findings, 398, 806 bone turnover, 799e800 bone volume, 801 mineralization abnormalities, 800e801 treatment calcimimetics, 810 goals, 806e807 growth hormone therapy, 811 mineral nutrition, 807 parathyroidecomy, 810e811

phosphate-binding agents, 807e808 vitamin D therapy, 808e810 Cinacalet, chronic kidney disease bone and mineral disorder management, 810 CKD-MBD, see Chronic kidney disease bone and mineral disorder CLC-5, Dent’s disease defects, 740e741, 748 CLCN7, osteopetrosis mutations, 543e545 Cleft palate, dental agenesis, 96e97 Cleidocranial dysplasia (CCD), hyperdontia, 98 CMD, see Craniometaphyseal dysplasia CoffineLowry syndrome, dietary protein in management, 213 ColeeCarpenter syndrome, 517 Collagen osteogenesis imperfecta mutations, 513e517, 521 type I Caffey disease mutations, 542 fibril assembly, 12, 15e16 genes, 11 processing, 11e14 structure, 11, 14e15 type V genes, 17 processing, 16e17 structure, 16e17 Computed tomography (CT) bone imaging overview, 278e279 quantitative, see Quantitative computed tomography Constitutional delay of puberty, osteoporosis association, 478, 480 CP, see Cerebral palsy Craniometaphyseal dysplasia (CMD), features and gene mutations, 548e549 Craniotubular hyperostoses, features and gene mutations, 552 Crohn’s disease, see Inflammatory bowel disease CRTAP, osteogenesis imperfecta mutations, 290, 516 CT, see Calcitonin; Computed tomography C-terminal cross-linked telopeptide (CTX), 362, 638 C-terminal telopeptide of type I collagen (ICTP), bone resorption marker, 366 CTX, see C-terminal cross-linked telopeptide CYP24A1, see 24-Hydroxylase CYP27B1, see 1a-Hydroxylase CYP2R1, see 25-Hydroxylase Cystic fibrosis (CF), osteoporosis association, 466e469

D

DBP, see Vitamin D binding protein Decorin, 19 Deferasirox, renal Fanconi’s syndrome induction, 743

843

Dent’s disease, renal Fanconi’s syndrome, 739e741, 748 Dental development, see Dental occlusion development; Periodontal ligament; Tooth bud; Tooth eruption Dental occlusion development, see also Tooth eruption eruption throughout life, 95 pattern of eruption, 94 speed of eruption, 94 timing of eruption, 93e94 Dentin cell and tissue organization cellular compartment, 88 dentin compartment, 88e89 predentin compartment, 88 circumpulpal dentin, 87e88 composition, 87 defects, 101 extracellular matrix composition, 89 outer peripheral dentin, 86e87 Dentin matrix acidic phosphoprotein-1 (DMP1), 24e25, 70, 709 Dentinogenesis imperfecta (DGI), 101e102, 520 Deoxypyridinoline (DPD), bone resorption marker, 366 Desmosterolosis, 541e542 DGI, see Dentinogenesis imperfecta DGS, see DiGeorge syndrome Diabetes, osteoporosis association, 473e475 Dickkopf-1 (DKK-1), bone resorption marker, 367 DiGeorge syndrome (DGS), hypoparathyroidism, 561 Digital X-ray radiogrammetry (DXR), bone imaging overview, 283 DKK-1, see Dickkopf-1 DLX3, trichodento-osseous dysplasia mutations, 550 DMD, see Duchenne muscular dystrophy DMP1, see Dentin matrix acidic phosphoprotein-1 DNA methylation, see Epigenetics Down syndrome, dental agenesis, 97 DPD, see Deoxypyridinoline Dual energy X-ray energy absorptiometry (DXA), see also Bone mineral density bone edge detection, 317e318 bone size and confounding effect, 318e319 fracture discrimination, 322e323 instrumentation, 316 limitations in infants and children, 315e316 overview, 283, 313e315 osteoporosis diagnosis, 439e440 peak bone mass, 189e190 precision by scan site and subject age, 315 reference data, 319e322 scan acquisition, 316e317

844

INDEX

Duchenne muscular dystrophy (DMD), osteoporosis association, 454e455 DXA, see Dual energy X-ray energy absorptiometry DXR, see Digital X-ray radiogrammetry

E East syndrome, 753 Eating disorders, osteoporosis association, 469e473 Ectodermal dysplasia, dental agenesis, 96 EDS, see EhlerseDanlos syndrome EhlerseDanlos syndrome (EDS) bone turnover marker effects of type VI disease, 376 osteoporosis, 446e447 Eiken syndrome, hyperparathyroidism, 578 Elastin, Williams syndrome defects, 578 Enamel composition, 86 defects, 100e101 formation, 85e86 structure, 85 Endochondral ossification fetal bone development, 40e41 postnatal bone growth, 55e57 ENS, see Epidermal nevus syndrome Epidermal nevus syndrome (ENS), clinical features, 713 Epigenetics hypophosphatasia, 782 maternal effects on offspring bone, 672e673 vitamin D receptor effects, 170 Eroded surface per bone surface (ES/BS), histomorphometry, 390 ES/BS, see Eroded surface per bone surface Estrogen fetal levels and functions, 253 peak bone mass effects, 197e198 postnatal bone growth regulation, 72e73 Exercise, peak bone mass effects calcium intake interactions, 206e207 fracture prevention in later life, 200e201 geneeenvironment interactions, 202 intense exercise negative effects, 202e203 protein intake interactions, 212e213 public health programs, 201 skeletal site specificity, 201 Extraskeletal calcification chronic kidney disease bone and mineral disorder, 802e803 hereditary forms, see Albright’s hereditary osteodystrophy; Fibrodysplasia ossificans progressiva; Progressive osseous heteroplasia non-hereditary heterotopic ossification, 821e822 prospects for study, 835

F

FAM20C, Raine dysplasia mutations, 542 Familial benign hypercalcemia (FHH), 751e752

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC), 751 Fanconi renotubular syndrome 2 (FRTS2), 733e735 FanconieBickel syndrome, 739 FBN1, Marfan syndrome mutations, 447 FD, see Fibrous dysplasia FDH, see Focal dermal hypoplasia Fels method, bone age assessment, 284e285, 353 Fetal bone development, see also Pregnancy adaptive goals, 247 calcium sensing receptor, 255e256 chondrocyte differentiation, 44e45 proliferation and maturation, 45e47 endochondral ossification, 40e41, 264e265 histomorphometry studies, 392 hormone levels and functions androgens, 253 calcitonin, 252 estrogen, 253 fibroblast growth factor-23, 252 parathyroid hormone, 249e250 parathyroid hormone-related peptide, 252e253 vitamin D, 250e252 integrated calcium homeostasis blood calcium regulation, 267 placental calcium transfer, 267 skeletal mineralization, 267 intramembranous ossification, 39e40, 264 kidney function, 264 maternal parathyroid hormone disturbances and fetal response hyperparathyroidism, 265e266 hypoparathyroidism, 266 mineral metabolism calcium, 247e248 magnesium, 248e249 phosphate, 248 twin studies of placental supply versus genetic inheritance, 656e657 molecular regulation condensation, 42e44 epithelialemesenchymal interaction, 41e42 osteoblast differentiation, 47e48 overview, 655e656 parathyroid gland development, 254e255 placental calcium transport fetal regulation parathyroid hormone, 262e263 parathyroid hormone-related peptide, 260e262 vitamin D, 263 gene expression, 257e258 maternal regulation, 260 measurement, 258e260 overview, 256e257 placental structure, 257 placental magnesium transport, 263e264

placental phosphate transport, 263e264 thymus development, 254 timing and sequence, 41 FGFs, see Fibroblast growth factors FHH, see Familial benign hypercalcemia FHHNC, see Familial hypomagnesemia with hypercalciuria and nephrocalcinosis Fibroblast growth factors (FGFs) chondrocyte proliferation and maturation role, 47, 58e59 FGF-23 autosomal dominant hypophosphatemic rickets mutations, 716e717 fetal levels and functions, 252 osteocyte function, 5e6 phosphate homeostasis role, 146, 149e151 receptor interactions, 717 renal phosphate handling regulation, 731e733 rickets findings, 641 secretion regulation by phosphate, 153 osteoblast differentiation and activity role in growth plate ossification, 48, 65 Fibrodysplasia ossificans progressiva (FOP) ACVR1 mutations bone morphogenetic protein signaling effects, 830e831 cell function effects, 831 DNA testing, 828e829 R206H mutation, 829e830 variant disease mutations, 829 bone marrow transplantation, 826, 828 complications, 825 diagnosis and misdiagnosis, 822, 828 epidemiologic factors, 829 histopathology, 826e827 immune system effects, 828 laboratory findings, 826 progressive heterotopic ossification, 822e823 progressive osseous heteroplasia comparison, 835 radiographic features, 825e826 skeletal anomalies, 823, 825 treatment, 832 types and clinical features, 824, 828 Fibromodulin, 19e20 Fibrous dysplasia (FD) bone histomorphometry findings, 397 bone pathology cartilage, 607 deposition and internal structure, 603 lesion growth, 607 mineralization, 603e604 modeling, 602e603 remodeling, 604e606 tumors, 608 vascularity, 606e607 extraskeletal lesions, 592e596 fibro-osseous lesion differential diagnosis, 607

INDEX

GNAS mutations, 597e599 hypophosphatemia, 713e714 management bisphosphonates, 614 mutation analysis, 613 overview, 612e613 prospects, 614e615 surgery, 613e614 overview, 589 pathogenesis, 609e612 phenotypic variation determinants allelic expression, 601e602 mutated clone site and time of origin, 600e601 somatic mosaicism, 600 survival and adaptation, 601 radiological findings, 297e298, 591e592 skeletal lesions, 590e591 FKBP10, osteogenesis imperfecta mutations, 290 Focal dermal hypoplasia (FDH), 101 FOP, see Fibrodysplasia ossificans progressiva Formation period (FP), histomorphometry, 390 FP, see Formation period Fracture child abuse differentiation from bone fragility conditions, 490e491, 517e518 metaphyseal fracture, 303 exercise in childhood and prevention in later life, 200e201 osteoporotic fracture, see Osteoporosis preterm infant risks birth to hospital discharge, 666 hospital discharge to two years, 667 two years onwards, 669 FRTS2, see Fanconi renotubular syndrome 2

G

GATA3, hypoparathyroidism, deafness, and renal anomalies syndrome defects, 564e565 GCMB, see Glial cells missing B GFD, see Gluten-free diet GH, see Growth hormone Ghosal syndrome, features and gene mutations, 553 Gitelman syndrome, 752 GJA1, oculodental-osseous dysplasia mutations, 550 Gla proteins, see Osteocalcin Glial cells missing B (GCMB), hypoparathyroidism, 562e564 Glucocorticoids Duchenne muscular dystrophy management, 454e455 osteoporosis induction by therapy, 460, 463e464, 483e485 postnatal bone growth regulation, 74 Gluten-free diet (GFD), bone effects in celiac disease, 465e466

GNAS expression regulation, 597 fibrous dysplasia expression, 609 functional consequences of mutation, 599, 609 mutations, 597e599 functions, 834 progressive osseous heteroplasia mutations, 834 pseudohypoparathyroidism mutations, 566e570 stem cell expression, 597 structure, 596 transcript processing, 596 GreulichePyle method, bone age assessment, 284e285, 349 Growth hormone (GH) deficiency and osteoporosis association, 476e478 peak bone mass effects, 198 postnatal bone growth regulation, 71e72 therapy chronic kidney disease bone and mineral disorder, 811 osteogenesis imperfecta, 527e528 X-linked hypophosphatemia, 706 Growth plate chondrocyte proliferation and differentiation, 57e59 vascularization, 60e61

H Heat shock protein-47 (HSP47), collagen type I processing role, 13 Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) clinical physiology, 710 idiopathic hypercalciuria relationship, 710 kidney stones, 710 management and course, 710 mixed skeletal phenotype, 709e710 SLC34A3 mutations, 710e711 sodium/phosphate co-transporter defects, 735 Hertwig’s root sheath, 84 Heterotropic ossification, see Extraskeletal calcification HHRH, see Hereditary hypophosphatemic rickets with hypercalciuria HIF-1a, see Hypoxia-inducible factor-1a Histomorphometry, bone biopsy sample processing, 386 technique, 384e386 disease studies calcium deficiency, 393 fibrous dysplasia, 397 hypophosphatemic rickets, 393e394 idiopathic juvenile osteoporosis, 396e397 osteogenesis imperfecta

845

bisphosphonate treatment findings, 398e399 type I, 394e396 type III, 394e395 type IV, 394e395 type V, 395e396 type VI, 395e396 type VII, 396 osteopetrosis, 397 secondary bone disorders acute lymphoblastic leukemia, 398 burn injury, 398 inflammatory bowel disease, 398 renal osteodystrophy, 398, 806 b-thalassemia, 398 vitamin D deficiency, 393 fetal bone development studies, 392 indications, 399 microscopy, 386 nomenclature, 386e387 parameters dynamic formation parameters, 389e390 reproducibility, 390 static formation parameters, 388e389 static resorption parameters, 390 structural parameters, 387e388 principles, 384e384 reference data for pediatric iliac bone histomorphometry, 390e392 HIV, see Human immunodeficiency virus Homocystinuria, osteoporosis, 448 HRPT2, mutation and hyperparathyroidism-jaw tumor syndrome, 574e575 HSH, see Hypomagnesemia with secondary hypocalcemia HSP47, see Heat shock protein-47 Human immunodeficiency virus (HIV), renal Fanconi’s syndrome induction by treatment, 742e743 1a-Hydroxylase deficiency clinical features, 682e683 laboratory findings, 683 molecular genetics, 683e685 overview, 682 treatment, 687e688 knockout mouse, 174e175 secretion regulation by phosphate, 153 structure and function, 685e687 Toll-like receptor activation, 172 vitamin D metabolism, 164e167, 681e682 24-Hydroxylase knockout mouse, 173e174 vitamin D metabolism, 166e167, 681 25-Hydroxylase knockout mouse, 172 vitamin D metabolism, 163e164, 681 Hyperdontia, 97e98 Hyperparathyroidism brachydactyly type E, 578 calcium-sensing receptor defects, 577 chronic renal failure association, 576

846

Hyperparathyroidism (Continued) Eiken syndrome, 578 familial isolated hyperparathyroidism, 574, 576 gene defects, 560 Jansen’s disease, 577e578 maternal disturbance and fetal response, 265e266 Ollier’s disease, 578 tumors and gene mutations HRPT2 and hyperparathyroidism-jaw tumor syndrome, 574e575 LRP5, 575 MEN1, 573e574 MEN2, 574 PRAD1, 572e573 Rb, 576 RIZ1, 576 Wnt pathway, 575e576 Williams syndrome, 578e579 Hyperphosphatasia differential diagnosis, 294 radiographic findings, 293e294 Hyperprolactinemia, osteoporosis association, 482e483 Hyperthyroidism, osteoporosis association, 475e476 Hypodontia, 98e99 Hypomagnesemia with secondary hypocalcemia (HSH), 752 Hypoparathyroidism, see also Pseudohypoparathyroidism DiGeorge syndrome, 561e562 familial syndromes, 565 gene defects, 559e560 glial cells missing B, 562e564 hypoparathyroidism, deafness, and renal anomalies syndrome, 564e565 maternal disturbances and fetal response, 266 maternal disturbances and fetal response, 266 mitochondrial disorders, 561 pluriglandular autoimmune hypoparathyroidism, 561 X-linked recessive hypoparathyroidism, 559, 561 Hypophosphatasia clinical features adult hypophosphatasia, 776e777 benign prenatal hypophosphatasia, 778 childhood hypophosphatasia, 776 epidemiology, 773 infantile hypophosphatasia, 774e775 odonto hypophosphatasia, 777 perinatal hypophosphatasia, 774 pseudohypophosphatasia, 777 diagnosis alkaline phosphatase activity, 778e779 calcium and phosphate levels, 779 histopathological findings dentition, 780 skeleton, 780

INDEX

phosphoethanolamine levels, 779 pyridoxal phosphate levels, 779 pyrophosphate levels, 779 radiographic findings, 293, 779e780 differential diagnosis, 293 epigenetics, 782 history of study, 773 inheritance, 781 mouse model, 785 prenatal diagnosis, 784e785 prognosis, 783 tissue-non-specific alkaline phosphatase deficiency, 780e781 function, 785, 787 gene defects, 781e782 treatment, 783e784 Hypophosphatemia, see also Tumor-induced osteomalcia; X-linked hypophosphatemia autosomal dominant hypophosphatemic rickets features, 708e709 mouse model, 716e717 autosomal recessive hypophosphatemic rickets, 709 bone histomorphometry in hypophosphatemic rickets, 393e394 differential diagnosis, 293 hereditary hypophosphatemic rickets with hypercalciuria clinical physiology, 710 idiopathic hypercalciuria relationship, 710 kidney stones, 710 management and course, 710 mixed skeletal phenotype, 709e710 SLC34A3 mutations, 710e711, 717 phosphate transporter defects, 717e718 prospects for study, 718e719 radiographic findings, 292 Hypothyroidism, osteoporosis association, 476 Hypoxia-inducible factor-1a (HIF-1a), chondrogenesis role, 60

I

IBD, see Inflammatory bowel disease ICTP, see C-terminal telopeptide of type I collagen IDH, see Isolated dominant hypomagnesemia Idiopathic juvenile osteoporosis, see Osteoporosis IGFs, see Insulin-like growth factors Ihh, see Indian Hedgehog Immobilization, osteoporosis association, 455e457 Incontinentia pigmenti, dental agenesis, 96 Indian Hedgehog (Ihh), osteoblast differentiation and activity role in growth plate ossification, 64e65 Infants, see Neonatal hypocalcemia; Preterm infants

Inflammatory bowel disease (IBD) bone histomorphometry findings, 398 osteoporosis association, 461e464 Insulin-like growth factors (IGFs) chondrocyte proliferation and maturation role, 46e47 IGF-1 peak bone mass effects, 198 postnatal bone growth regulation, 71e72 umbilical venous cord blood levels and bone development, 671e672 Intramembranous ossification fetal bone development, 39e40 postnatal bone growth, 55e57 Ionizing radiation dose and regulations, 285 effective dose by imaging modality, 312 IRH, see Isolated autosomal recessive hypomagnesemia Isolated autosomal recessive hypomagnesemia (IRH), 753 Isolated dominant hypomagnesemia (IDH), 752e753

J Jansen’s disease. hyperparathyroidism, 577e578 JDMS, see Juvenile dermatomyositis JIA, see Juvenile idiopathic arthritis Juvenile dermatomyositis (JDMS), osteoporosis association, 458, 461 Juvenile idiopathic arthritis (JIA), osteoporosis association, 459 Juvenile Paget’s disease, features and gene mutations, 550

K KearnseSayre syndrome (KSS), hypoparathyroidism, 561 KenneyeCaffey syndrome, hypoparathyroidism, 565 Kidney transplantation, effects on bone, 811e812 KirkeRichardson syndrome, hypoparathyroidism, 565 Klotho, 717, 736 KSS, see KearnseSayre syndrome

L Lactation bone impact in later life, 236e237 bone metabolism, 231e232 bone turnover, 233e234 breast milk bone growth modulators, 237 mineral composition, 237 dietary effects calcium, 237e238 general nutrition, 239 magnesium, 238e239 phosphate, 238e239 vitamin D, 239 zinc, 238e239

847

INDEX

hormone levels calcitonin, 234 parathyroid hormone, 234e235 parathyroid hormone-related peptide, 234e235 vitamin D, 234 mineral metabolism calcium, 232e233 magnesium, 233 phosphate, 233 zinc, 233 osteoporosis, 236 skeletal changes, 235e236 LEMD3, osteopoikilosis mutations, 547 LenzeMajewski hyperostotic dysplasia, features and gene mutations, 552e553 LEPRE1, osteogenesis imperfecta mutations, 290 Leptin, umbilical venous cord blood levels and bone development, 671e672 LIM-kinase, Williams syndrome defects, 578 Low-density lipoprotein receptor-related protein-5 (LRP5) autosomal dominant craniotubular hyperostoses mutations, 552 gene polymorphisms and peak bone mass effects, 196e197, 202 osteoporosisepseudoglioma syndrome mutations, 444, 446, 516 parathyroid tumor mutations, 575 Lowe’s syndrome, renal Fanconi’s syndrome, 741, 748 LRP5, see Low-density lipoprotein receptorrelated protein-5

M Macrodontia, 99 Magnesium chronic kidney disease bone and mineral disorder evaluation, 804 fetal metabolism, 248e249 lactation dietary intake, 238e239 metabolism, 233 placental transport, 263e264 pregnancy dietary intake, 230 maternal fluxes from mother to offspring, 223e224 metabolism, 225 renal handling defects autosomal dominant hypocalcemic hypercalciuria, 751 autosomal dominant hypomagnesemia, 753 East syndrome, 753 familial hypomagnesemia with hypercalciuria and nephrocalcinosis, 751 Gitelman syndrome, 752

hypomagnesemia with secondary hypocalcemia, 752 isolated autosomal recessive hypomagnesemia, 753 isolated dominant hypomagnesemia, 752e753 mitochondrial hypomagnesemia, 753 overview of diseases, 749e750 distal convoluted tubule, 747e748 loop of Henle, 746e747 overview, 745 proximal tubule, 746 Magnetic resonance imaging (MRI), bone imaging overview, 282e283 MAPK, see Mitogen-activated protein kinase MAR, see Mineral apposition rate Marfan syndrome (MFS), osteoporosis, 447e448 Matrix, extracellular phosphoglycoprotein (MEP), 26, 70 Maturation, see also Bone age; Puberty indicators, 345e346 initial considerations in assessment, 343e345 sexual dimorphism, 344e345, 347 size in assessment, 347 time concept, 345 variation, 346e347 McCuneeAlbright syndrome, see Fibrous dysplasia Mechanostat model, muscleebone interaction in bone growth and development, 441e443 Megalin, knockout mouse, 172e173 MELAS syndrome, hypoparathyroidism association, 561 Melorheostosis, features and gene mutations, 557e548 MEN1, parathyroid tumor mutations, 573e574 MEN2, parathyroid tumor mutations, 574 Menarche, age at, 357e358 MEP, see Matrix, extracellular phosphoglycoprotein Methotrexate, osteoporosis induction, 457e459, 486 MFS, see Marfan syndrome Microdontia, 98e99 Mineral apposition rate (MAR), histomorphometry, 389 Mineralization, bone, 26e28 Mineralization lag time (Mlt), histomorphometry, 389 Mineralizing surface per bone surface (MS/ BS), histomorphometry, 389 Mineralizing surface per osteoid surface (MS/OS), histomorphometry, 389 Mitogen-activated protein kinase (MAPK), phosphate activation, 151e152 Mlt, see Mineralization lag time MRI, see Magnetic resonance imaging

MS/BS, see Mineralizing surface per bone surface MS/OS, see Mineralizing surface per osteoid surface

N N-cadherin, condensation role, 42e43 NCAM, see Neural cell adhesion molecule Neonatal hypocalcemia, features, 670 Nephrolithiasis/osteoporosis, hypophosphatemic-2 (NPHLOP2), 735e736 Nephropathic cystinosis, renal Fanconi’s syndrome, 738e739 Neural cell adhesion molecule (NCAM), condensation role, 42e43 NF-kB, see Nuclear factor-kB NHERF1 hypophosphatemia mutations, 718 nephrolithiasis/osteoporosis, hypophosphatemic-2 defects, 735e736 Non-accidental injury, see Child abuse Notch chondrogenesis suppression, 45 osteoblast differentiation suppression, 48 NPHLOP2, see Nephrolithiasis/ osteoporosis, hypophosphatemic-2 NPTm than, see Sodium/phosphate cotransporter N-terminal cross-linked telopeptide (NTX) bone resorption marker, 362 rickets findings, 638 NTX, see N-terminal cross-linked telopeptide Nuclear factor-kB (NF-kB), fetal bone development role, 253

O

Ob.S/BS, see Osteoblast surface per bone surface Oc, see Osteocalcin Oc.S/BS, see Osteoclast surface per bone surface Oculodental-osseous dysplasia (ODOD), features and gene mutations, 549e550 ODOD, see Oculodental-osseous dysplasia OI, see Osteogenesis imperfecta Ollier’s disease, hyperparathyroidism, 578 OPG, see Osteoprotegerin OPPG, see Osteoporosisepseudoglioma syndrome OS/BS, see Osteoid surface per bone surface OSCS, see Osteopathia striata with cranial sclerosis Osteitis fibrosa cystica, radiographic features, 806 Osteoblast differentiation, 3e5 fetal bone development and differentiation, 47e48

848

Osteoblast (Continued) growth plate ossification differentiation and activity bone morphogenetic proteins, 64e65 fibroblast growth factors, 65 Indiam Hedgehog, 64e65 Osterix, 63e64 Runx2, 63e64 Wnt, 62e63 morphology, 4 osteogenesis imperfecta function, 521, 523 phosphate function, 143e144 vitamin D receptors, 171 Osteoblast surface per bone surface (Ob.S/BS), histomorphometry, 388e389 Osteocalcin (OC) bone formation marker, 365 function, 23 gene, 23 osteoblast expression, 4 processing, 22e23 structure, 23 Osteoclast abundance, 1 bone modeling and remodeling role, 65e66 bone resorption, 68e69 differentiation, 1e3, 67e68 functional overview, 2 markers, 2 phosphate function, 143 Osteoclast surface per bone surface (Oc.S/BS), histomorphometry, 390 Osteocyte differentiation, 5 functional overview, 5e6 mechanosensing, 70e71 Osteogenesis imperfecta (OI) anesthesia precautions, 520 bone histomorphometry bisphosphonate treatment findings, 398e399 type I, 394e396 type III, 394e395 type IV, 394e395 type V, 395e396 type VI, 395e396 type VII, 396 bone turnover markers, 373e374 breech complications, 520 classification, 289e290, 511e513 clinical features Bruck syndrome, 517 ColeeCarpenter syndrome, 517 osteoporosisepseudoglioma syndrome, 444, 446, 516e517 type I, 514e515 type II, 513e514 type III, 514 type IV, 514 type V, 515e516 type VI, 516 type VII, 516

INDEX

diagnosis clinical features cardiovascular involvement, 518 dentinogenesis imperfecta, 520 endocrine changes, 519 neurological involvement, 517e518 ocular changes, 519e520 renal involvement, 518 respiratory problems, 519 connective tissue alterations, 519 differential diagnosis, 517e518 laboratory findings, 517 differential diagnosis, 291e292 idiopathic juvenile osteoporosis comparison, 450 life expectancy, 520e521 management bisphosphonates, 524e527 calcium, 526 growth hormone, 527e528 occupational therapy, 528e529 orthopedic management, 528 prospects, 529e531 vitamin D, 526 pathophysiology bone metabolic activity, 523e524 mineralization defects, 521e522 osteoblast function, 521, 523 prenatal radiology, 288, 290 radiographic findings, 289e292 Osteoid surface per bone surface (OS/BS), histomorphometry, 388e389 Osteomodulin, 19e20 Osteonecrosis, glucocorticoid induction, 485e485 Osteonectin gene, 22 knockout mouse, 22 structure, 22 Osteopathia striata with cranial sclerosis (OSCS), features and gene mutations, 548 Osteopathy of prematurity differential diagnosis, 302 grades, 300, 302 Osteopetrosis bone histomorphometry findings, 397 dental defects, 104 differential diagnosis, 295 radiographic findings, 294e295 types and features autosomal dominant osteopetrosis type II, 544e545 infantile osteopetrosis, 542e543 intermediate osteopetrosis, 543e544 osteoclast-poor osteopetrosis, 545e546 osteopetrosis with renal tubular acidosis, 544 Osteopoikilosis, features and gene mutations, 547 Osteopontin, 25e26 Osteoporosis

bone histomorphometry in idiopathic juvenile osteoporosis, 396e397 classification, 444 definition, 439 diagnosis bone mineral density scores, 440e441 differential diagnosis in children, 291e292 dual-energy X-ray absorptiometry, 439e440 overview, 439e440 fragility fracture radiological findings, 298e300 lactation, 236 mechanostat model and muscleebone interaction in bone growth and development, 441e443 pregnancy, 228 prevention and treatment, 486, 487e489 primary disease Bruck syndrome, 444 causes, 445 EhlerseDanlos syndrome, 446e447 homocystinuria, 448 idiopathic juvenile osteoporosis, 448e449 Marfan syndrome, 447e448 osteoporosisepseudoglioma syndrome, 444, 446 prospects for study, 489e490 secondary osteoporosis acute lymphoblastic leukemia treatment, 457e458 anorexia nervosa, 469e472 bulimia nervosa, 469e472 celiac disease, 462, 464e466 cerebral palsy, 451e454 cystic fibrosis, 466e469 diabetes, 473e475 Duchenne muscular dystrophy, 454e455 glucocorticoid therapy induction, 483e485 growth hormone deficiency, 476e478 hyperprolactinemia, 482e483 hyperthyroidism, 475e476 hypothyroidism, 476 immobilization, 455e457 inflammatory bowel disease, 461e464 juvenile dermatomyositis, 458, 461 juvenile idiopathic arthritis, 459 medication induction, 486e487 puberty disorders absence of puberty, 480e481 constitutional delay of puberty, 478, 480 overview of puberty importance in bone development, 478e479 precocious puberty, 480 Turner syndrome, 481e482 systemic lupus erythematosus, 461 Osteoporosisepseudoglioma syndrome (OPPG), features and gene mutations, 444, 446, 516e517

INDEX

Osteoprotegerin (OPG) bone resorption marker, 367 fetal bone development, 253 juvenile Paget’s disease mutations, 550 osteoclast formation role, 2, 68 Osterix, osteoblast differentiation and activity role in growth plate ossification, 47, 63e64 Oxford method, bone age assessment, 349e350

P

Paget’s disease, see Juvenile Paget’s disease Parathyroid gland chronic kidney disease bone and mineral disorder management with parathyroidecomy, 810e811 development, 254e255 tumors, see Hyperparathyroidism Parathyroid hormone (PTH), see also Hyperparathyroidism; Hypoparathyroidism analogs, 112e113 calcium homeostasis bone actions, 119e121 kidney actions, 118e119 chronic kidney disease bone and mineral disorder evaluation, 805 evolution, 113e114 fetal levels and functions, 249e250 functional overview, 109e111 gene expression regulation, 115 structure, 114, 557 knockout mouse, 265 lactation changes, 226 neonatal mineral homeostasis, 657 phosphate homeostasis, 146e149 secretion regulation by phosphate, 152e153 placental calcium transport regulation, 262e263 pregnancy changes, 226 receptor defects, see specific diseases ligand C-terminal receptors, 129 PTH1R gene, 125 ligand binding and activation mechanisms, 126e128 placental expression, 257 prolonged signaling-inducing ligands, 128e129 topology, 126 signaling, 123e125 structure, 124e126 tissue distribution, 558e559 types and phylogenetic relationships, 125, 558 renal phosphate handling regulation, 730e731 secretion regulation, 115e117

sequences, 111e112 synthesis and processing, 114e115, 557e558 X-linked hypophosphatemia findings, 703 Parathyroid hormone-related peptide (PTHrP) analogs, 112e113 chondrocyte proliferation and maturation role, 45e46, 58 evolution, 113e114 fetal levels and functions, 252e253 functional overview, 109, 121e123 gene, 121e122 knockout mouse, 265 lactation changes, 226e227 parathyroid tumor mutations in pathway, 575e576 placental calcium transport regulation, 260e262 pregnancy changes, 226e227 processing, 122 receptor ligand C-terminal receptors, 129 PTH1R gene, 125 ligand binding and activation mechanisms, 126e128 placental expression, 257 prolonged signaling-inducing ligands, 128e129 topology, 126 signaling, 123e125 structure, 124e126 types and phylogenetic relationships, 125 rickets findings, 637e638 sequence, 122 PBIB, osteogenesis imperfecta mutations, 290 PBM, see Peak bone mass PEA, see Phosphoethanolamine Peak bone mass (PBM) assessment, 190 biomechanical markers during puberty, 194e195 definition, 189 determinants calcium intake dietary recommendations, 210 geneeenvironment interactions, 209 individual differences of bone response, 208e209 interventional studies, 204e210 observational studies, 204 physical activity interactions, 206e207 pubertal maturation influences on supplementation effects, 205e206 skeletal site responsiveness, 205 energy intake and muscle mass development, 202 genetic factors, 195e197 growth hormone/IGF-1 axis, 198

849

mechanical factors age and optimal response to loading, 200 exercise and fracture prevention in later life, 200e201 protein intake bone acquisition effects, 211 bone metabolism effects, 210e211 CoffineLowry syndrome management, 213 dietary recommendations, 213 physical activity interactions, 212e213 pubertal timing, 198e199 sex hormones, 197e198 vitamin D status dietary recommendations, 203e204 interventional studies, 203 observational studies, 203 exercise effects geneeenvironment interactions, 202 intense exercise negative effects, 202e203 public health programs, 201 skeletal site specificity, 201 importance, 189e190 peripubertal transient fragility, 193 structural development, 190e191, 193 timing, 193e194 variability, 194 Peridontium, 95 Periodontal ligament function, 95 maturation, 95 peridontium, 95 PHEX, X-linked hypophosphatemia mouse model studies, 715e716 mutation, 703e704 pathophysiology, 704 Phosphate chondrocyte function, 143e144 chronic kidney disease bone and mineral disorder binding agents for treatment, 807e808 diagnostic evaluation, 804 distribution in body, 141e142 extrarenal soft tissue handling, 142 fetal metabolism, 248 fibroblast growth factor-23 secretion regulation, 153 functions, 141 homeostasis fibroblast growth factor-23, 146, 149e151 overview, 699e700 parathyroid hormone, 146e149 vitamin D, 149 1a-hydroxylase regulation, 153 intestinal absorption, 142 lactation dietary intake, 238e239 metabolism, 233 mitogen-activated protein kinase activation, 151e152

850

Phosphate (Continued) osteoblast function, 143e144 osteoclast function, 143 parathyroid hormone secretion regulation, 152e153 pregnancy dietary intake, 230 maternal fluxes from mother to offspring, 223e224 metabolism, 225 placental transport, 263e264 protein intake effects on bone metabolism, 210e211 renal transport, see Renal phosphate handling Phosphoethanolamine (PEA) hypophosphatasia levels, 779 tissue-non-specific alkaline phosphatase substrate, 785e786 PHP, see Pseudohypoparathyroidism Physical activity, see Exercise PICP, see Procollagen type I C-terminal propeptide PINP, see Procollagen type I N-terminal propeptide POH, see Progressive osseous heteroplasia PPi, see Pyrophosphate PRAD1, parathyroid tumor mutations, 572e573 Precocious puberty, osteoporosis association, 480 Pregnancy, see also Fetal bone development body build, diet, and lifestyle effects on offspring bone, 671 bone impact in later life, 228e229 bone turnover, 225e226 dietary effects on offspring bone calcium, 229e230 general nutrition, 231 magnesium, 230 phosphate, 230 vitamin D, 230e231 zinc, 230 epigenetic effects, 672e673 hormone levels calcitonin, 227 parathyroid hormone, 226 parathyroid hormone-related peptide, 226e227 vitamin D, 226 mineral fluxes from mother to offspring, 223e224 mineral metabolism calcium, 224 magnesium, 225 phosphorous, 225 zinc, 225 osteogenesis imperfecta and breech complications, 520s osteoporosis, 228 placental calcium transport fetal regulation parathyroid hormone, 262e263

INDEX

parathyroid hormone-related peptide, 260e262 vitamin D, 263 gene expression, 257e258 maternal regulation, 260 measurement, 258e260 overview, 256e257 placental structure, 257 placental magnesium transport, 263e264 placental phosphate transport, 263e264 skeletal changes, 227e228 vitamin D and calcium nutrition effects on offspring bone, 671 Prenatal bone growth, see Fetal bone growth Prenatal diagnosis hypophosphatasia, 784e785 osteogenesis imperfecta, 288, 290 skeletal dysplasia, 434e435 Preterm infants birth to hospital discharge bone mass, 661e662, 664 bone turnover, 664e666 fracture risk, 666 linear growth, 661 hospital discharge to two years bone mass, 666e667 bone turnover, 667 fracture risk, 667 linear growth, 666 mineral homeostasis at birth preterm infants, 658 term infants, 657e659 nutritional recommendations breast milk versus formula, 670 calcium, 669e670 formula nutrient content, 663e664 vitamin D, 670 osteopathy of prematurity differential diagnosis, 302 grades, 300, 302 rickets of prematurity biochemical findings, 661 clinical features, 661 history of study, 658, 660e661 radiological features, 661 two years onwards bone mass, 668e669 bone turnover, 669 fracture risk, 669 linear growth, 667e668 Procollagen type I C-terminal propeptide (PICP), bone formation marker, 365e366 Procollagen type I N-terminal propeptide (PINP), bone formation marker, 365e366 Progressive diaphyseal dysplasia, features and gene mutations, 549 Progressive osseous heteroplasia (POH) clinical features, 832e833 fibrodysplasia ossificans progressiva comparison, 835

GNAS functions, 834 mutations, 834 heterotopic ossification, 833e834 treatment, 835 Prolactin, hyperprolactinemia and osteoporosis, 482e484 Pseudohypoaldosteronism type I, 758e759 type II, 759 Pseudohypoparathyroidism (PHP) clinical features, 566 differential diagnosis, 293 GNAS mutations, 566e570 radiographic findings, 293 Pseudo-pseudohypoparathyroidism differential diagnosis, 293 radiographic findings, 293 PTH, see Parathyroid hormone PTHrP, see Parathyroid hormone-related peptide Puberty, see also Maturation; Peak bone mass bone turnover markers, 370 menarche, age at, 357e358 osteoporosis in disorders absence of puberty, 480e481 constitutional delay of puberty, 478, 480 overview of puberty importance in bone development, 478e479 precocious puberty, 480 Turner syndrome, 481e482 secondary sexual characteristics breast development stages, 355 clinical evaluation, 355e356, 358 genitalia development stages, 355 self-assessment of status, 356e357 sexual dimorphism in maturation, 344e345, 347 Pycnodysostosis, features and gene mutations, 546e547 PYD, see Pyridinoline Pyle disease, features and gene mutations, 553 Pyridinoline (PYD), bone resorption marker, 366 Pyridoxal phosphate (PLP) tissue-non-specific alkaline phosphatase substrate, 786 hypophosphatasia levels, 779 Pyrophosphate (PPi), hypophosphatasia levels, 779, 786e787

Q

qCT, see Quantitative computed tomography Quantitative computed tomography (qCT), see also Bone mineral density fracture discrimination, 329 limitations in children bone length confounding effects, 328e329 overview, 326e327

851

INDEX

partial volume effects, 327 reference data, 327e328 scan protocols and multiple outcome measures, 326e327 overview, 283, 323e326 peak bone mass, 190 reference data, 329 Quantitative ultrasonography (QUS) fracture discrimination, 334 limitations, 332e333 overview, 329e332 peak bone mass, 190 reference data, 333e334 QUS, see Quantitative ultrasonography

R Radiogrammetry, bone mineral density, 312e313 Radiographic absorptiometry, bone mineral density, 313 Radiography bone age assessment, 284e285 bone imaging overview, 278 ionizing radiation dose and regulations, 285 skeletal survey, see Skeletal survey Radionuclide scanning (RNS), bone imaging overview, 279e281 Raine dysplasia, 542 RANKL bone resorption marker, 367 fetal bone development, 253 knockout mouse, 121 osteoclast formation role, 2e3, 67e68 osteopetrosis mutations, 546 Rb, parathyroid tumor mutations, 576 Remodeling, bone coupling principle, 69e70 overview, 1e2 Renal Fanconi’s syndrome, see Chronic kidney disease bone and mineral disorder Renal osteodystrophy, see Chronic kidney disease bone and mineral disorder Renal phosphate handling overview, 144e145, 727, 729 primary disorders of renal phosphate wasting, 728 regulation dietary phosphate, 730 fibroblast growth factor-23, 731e733 parathyroid hormone, 730e731 vitamin D, 731 rickets due to disorders autosomal dominant hypophosphatemic rickets, 737 autosomal recessive hypophosphatemic rickets, 737e738 Fanconi renotubular syndrome 2 defects, 733e735 hereditary hypophosphatemic rickets with hypercalciuria defects, 735 Klotho defects, 736

nephrolithiasis/osteoporosis, hypophosphatemic-2 defects, 735e736 overview, 733 renal Fanconi’s syndrome acquired, 742e743 Dent’s disease, 739e741 FanconieBickel syndrome, 739 hereditary, 738e742 Lowe’s syndrome, 741 mitochondrial cytopathies, 741e742 nephropathic cystinosis, 738e739 overview, 738 X-linked hypophosphatemic rickets, 736e737 transporters, 145e146, 729e730 Renal tubular acidosis (RTA) acidebase handling in kidney distal tubule, 754e755 proximal tubule, 753e754 distal renal tubular acidosis autosomal dominant disease, 756, 758 autosomal recessive disease, 758 overview, 756 hyperkalemic renal tubular acidosis, 758 mixed type, 758 proximal renal tubular acidosis, 755e756 pseudohypoaldosteronism type I, 758e759 type II, 759 Rickets biochemical changes in nutritional rickets, 636e641 bone histomorphometry hypophosphatemic rickets, 393e394 vitamin D-deficient rickets, 393 bone turnover marker effects, 375e376 calcium deficiency, 632e633 classification, 626e627 clinical presentation, 633e636 definition, 625e626 hereditary abnormalities 1,25-dihydroxyvitamin D resistant rickets clinical and laboratory features, 688 overview, 688 treatment, 693e694 vitamin D receptor defects, 688e693 1a-hydroxylase deficiency clinical features, 682e683 laboratory findings, 683 molecular genetics, 683e685 overview, 682 treatment, 687e688 hereditary disorders, see Hypophosphatemia; X-linked hypophosphatemia history of study, 627 overview, 625 preterm infants biochemical findings, 661 clinical features, 661

history of study, 658, 660e661 radiological features, 661 prevention, 646e647 radiological findings, 296e301, 641e644 renal phosphate handling disorders autosomal dominant hypophosphatemic rickets, 737 autosomal recessive hypophosphatemic rickets, 737e738 Fanconi renotubular syndrome 2 defects, 733e735 hereditary hypophosphatemic rickets with hypercalciuria defects, 735 Klotho defects, 736 nephrolithiasis/osteoporosis, hypophosphatemic-2 defects, 735e736 overview, 733 renal Fanconi’s syndrome acquired, 742e743 Dent’s disease, 739e741 FanconieBickel syndrome, 739 hereditary, 738e742 Lowe’s syndrome, 741 mitochondrial cytopathies, 741e742 nephropathic cystinosis, 738e739 overview, 738 X-linked hypophosphatemic rickets, 736e737 treatment, 644e646 vitamin D deficiency epidemiology, 627e630 induction by low calcium intakes, 631e632 Rieger syndrome, microdontia, 99 RIZ1, parathyroid tumor mutations, 576 RNS, see Radionuclide scanning RocheeWainereThissen technique, bone age assessment, 352 RTA, see Renal tubular acidosis Runx2 endochondral ossification role, 57 osteoblast differentiation and activity role in growth plate ossification, 47, 63e64

S SanjadeSakati syndrome, hypoparathyroidism, 565 Sclerosteosis, features and gene mutations, 551e552 Sclerostin (SOST) bone resorption marker, 367 mutation in disease, 551e552 SERPINH1, osteogenesis imperfecta mutations, 290 Sesame syndrome, 753 SIBLINGS, see Small integrin-binding ligand N-linked glycoproteins Skeletal dysplasia, see also specific diseases classification, 405e424 diagnosis cartilage histology, 431e433

852

INDEX

Skeletal dysplasia (Continued) laboratory studies, 431 medical history, 404, 425 molecular analysis, 433e434 physical examination, 425e426 prenatal diagnosis, 434e435 radiology, 426e430 epidemiology, 403 Skeletal survey anatomical localization, 286e287 bone characteristics, 287 characteristic findings, 287 child abuse, 302e303 complications, 287 diagnosis of skeletal dysplasia, 288e289 interpretation, 285e286 sites for radiographs, 285 soft tissue characteristics, 287 SLC34A3, hereditary hypophosphatemic rickets with hypercalciuria mutations, 710e711, 717 SLC4A genes, distal renal tubular acidosis defects, 756 SLE, see Systemic lupus erythematosus SLRPs, see Small, leucine-rich, interstitial proteoglycans Small integrin-binding ligand N-linked glycoproteins (SIBLINGS) bone sialoprotein, 24 dentin matrix acidic phosphoprotein-1, 24e25 functional overview, 23e24 mineralization role, 27e28 osteopontin, 25e26 Small, leucine-rich, interstitial proteoglycans (SLRPs) biglycan, 19 decorin, 19 fibromodulin, 19e20 functional overview, 10, 18e19 osteomodulin, 19e20 Sodium/phosphate co-transporter (NPT) Fanconi renotubular syndrome 2 defects, 733e735 hereditary hypophosphatemic rickets with hypercalciuria defects, 735 renal transport, 145e146, 729e730 SOST, see Sclerostin Structural hierarchy, bone, 9e10 Systemic lupus erythematosus (SLE), osteoporosis association, 461

T TannereWhitehouse method, bone age assessment, 284e285, 350e352 Tartrate-resistant acid phosphatase (TRAP), bone resorption marker, 366e367 Taurodontism, 99e100 TBCE, see Tubulin-specific chaperone TBX, DiGeorge syndrome mutations, 562 TBXAS1, Ghosal syndrome mutations, 553 TCIRG1, osteopetrosis mutations, 543 TDO, see Trichodento-osseous dysplasia

Testosterone, see Androgens TFIIB, vitamin D receptor interactions, 168 TGF-b, see Transforming growth factor-b b-Thalassemia, bone histomorphometry findings, 398 Thrombospondins cartilage oligomeric matrix protein, 20e21 knockout mice, 21e22 ligands, 21 structure, 21 types, 20 Thymus, development, 254 Thyroid hormone osteoporosis role hyperthyroidism, 475e476 hypothyroidism, 476 postnatal bone growth regulation, 73e74 TIO, see Tumor-induced osteomalcia Tissue-non-specific alkaline phosphatase (TNAP) defects, see Hypophosphatasia structure and function, 27, 771e773, 785, 787 substrates, 785e786 TNAP, see Tissue-non-specific alkaline phosphatase TNX, EhlerseDanlos syndrome defects, 446 Tooth bud cementum composition, 89e90 types and structural differences, 89 dental agenesis, 95e97 dentin cell and tissue organization cellular compartment, 88 dentin compartment, 88e89 predentin compartment, 88 circumpulpal dentin, 87e88 composition, 87 extracellular matrix composition, 89 outer peripheral dentin, 86e87 enamel composition, 86 formation, 85e86 structure, 85 root formation/eruption, 84e85 steps in tooth formation initial phase, 83 morphogenesis, 83e84 terminal differentiation, 84 Tooth eruption, see also Dental occlusion development defects, 92 dental follicle role, 90e91 genetic control, 91 mechanisms intraoral eruption, 91e92 intraosseous eruption, 91 overview, 90 prefunctional stage, 84 speed, 91 Trabecular number, histomorphometry, 387e388

Trabecular thickness, histomorphometry, 387e388 Transforming growth factor-b (TGF-b), latency-associated peptide mutations in progressive diaphyseal dysplasia, 549 TRAP, see Tartrate-resistant acid phosphatase Trichodento-osseous dysplasia (TDO), features and gene mutations, 101, 550 Tubulin-specific chaperone (TBCE), mutation in disease, 565 Tumor-induced osteomalcia (TIO) course, 712 evaluation, 711e712 overview, 711 pathology, 711 pathophysiology, 712e713 related syndromes, 713e714 treatment, 712 Turner syndrome dental defects, 99 osteoporosis association, 481e482

U

Ulcerative colitis, see Inflammatory bowel disease Ultrasonography (US) bone imaging overview, 281e282 quantitative, see Quantitative ultrasonography US, see Ultrasonography

V Van Buchem disease, features and gene mutations, 552 Vascular endothelial growth factor (VEGF) endochondral ossification role, 57 vascularization of cartilage and bone, 60e61 VDR, see Vitamin D receptor VEGF, see Vascular endothelial growth factor Vitamin D bone histomorphometry in deficiency, 393 calcium homeostasis 1a, 25-dihydroxyvitamin D actions, 170e178 vitamin D metabolites, 178e179 chronic kidney disease bone and mineral disorder management, 808e810 deficiency, see Rickets fetal levels and functions, 250e252 lactation changes, 226 status effects, 239 maternal nutrition effects on offspring bone, 671 metabolism 1a-hydroxylase, 164e167, 681e682 24-hydroxylase, 166e167, 681 25-hydroxylase, 163e164, 680e681

853

INDEX

knockout mouse studies, 172e178 overview, 163 rickets, 639e640 X-linked hypophosphatemia, 702e703 osteogenesis imperfecta management, 526 osteoporosis prevention, 488 parathyroid hormone expression regulation, 115 peak bone mass effects dietary recommendations, 203e204 interventional studies, 203 observational studies, 203 phosphate homeostasis, 149 placental calcium transport regulation, 263 pregnancy changes, 226 status effects, 230e231 preterm infant intake recommendations, 670 renal phosphate handling regulation, 731 umbilical venous cord blood markers insulin-like growth factor-1, 671e672 leptin, 671e672 Vitamin D binding protein (DBP), knockout mouse, 172 Vitamin D receptor (VDR) co-activators, 168 co-repressors, 168e169 domains, 167 epigenetic effects, 170 gene, 690 knockout mouse, 175e178 non-classical effects, 170 post-translational modifications, 167e168

response elements and target genes, 169e170 rickets defects clinical and laboratory features, 688 DNA-binding domain defects, 690e692 ligand-binding domain defects, 692e693 miscellaneous mutations, 693 overview, 688 treatment, 693e694 structure, 688e690 tissue distribution, 171

W Wall thickness, histomorphometry, 389 Williams syndrome, hyperparathyroidism, 578e579 Wnt chondrogenesis suppression, 45 osteoblast differentiation role, 48 osteoblast ossification differentiation and activity role, 62e63 WTX, osteopathia striata with cranial sclerosis mutations, 548

X X-linked hypophosphatemia (XLH) biochemical findings alkaline phosphatase activity, 703 hypophosphatemia evaluation, 702 parathyroid hormone, 703 vitamin D metabolism, 702e703 bone histomorphometry in hypophosphatemic rickets, 393 dental defects, 103e104, 701e702 growth, 701

heredity, 704 long-term sequelae, 703 mouse models bone and teeth phenotypes, 714 overview, 714 PHEX function, 715e716 phosphate wasting, 714 vitamin D metabolism, 714e715 PHEX mutation, 703e704 pathophysiology, 704 renal phosphate handling defects in rickets, 736e737 skeletal findings, 700e701 sporadic cases, 704 treatment adjunctive therapy, 706 complications, 707 dosing of calcitriol and phosphorous, 705e706 early childhood, 705 goals, 705 monitoring, 705 surgery, 706e707 XLH, see X-linked hypophosphatemia

Z Zinc lactation dietary intake, 238e239 metabolism, 233 pregnancy dietary intake, 230 maternal fluxes from mother to offspring, 223e224 metabolism, 225