PEDIATRIC BONE Biology and Diseases
This Page Intentionally Left Blank
PEDIATRIC BONE Biology and Diseases Editor-in-Chief
FRANCIS H. GLORIEUX Genetics Unit, Shriners Hospital McGill University Montreal, Quebec, Canada
Associate Editors II
JOHN M. PETTIFOR
HARALD lUPPNER
Mineral Metabolism Research Unit, Department of Pediatrics Chris Hani Baragwanath Hospital Soweto, Johannesburg, South Africa
Endocrine Unit, Department of Medicine and Pediatrics Massachusetts General Hospital and Harvard Medical Center Boston, Massachusetts
ACADEMIC PRESS A Harcourt Science and Technology Company
Amseterdam Boston Heidelberg London New York Oxford Paris San Diego San Francisco Singapore Sydney Tokyo
This book is printed on acid-free paper. @ Copyright 9 2003, Elsevier Science (USA). All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permissions may be sought directly from Elsevier's Science & Technology Rights Department in Oxford, UK: phone: (+44) 1865 843830, fax: (+44) 1865 853333, e-mail:
[email protected]. You may also complete your request on-line via the Elsevier Science homepage (http://elsevier.com), by selecting "Customer Support" and then "Obtaining Permissions." Academic Press An imprint of Elsevier Science 525 B Street, Suite 1900, San Diego, California 92101-4495, USA h ttp ://www. academicpress.com Academic Press 84 Theobald's Road, London WC1X 8RR, UK http://www.academicpress.com Library of Congress Catalog Card Number: 2003107900 International Standard Book Number: 0-12-286551-0 PRINTED IN THE UNITED STATES OF AMERICA 03 04 05 06 07 7 6 5 4 3 2 1
Table of Contents
Contributors xi Foreword xv Preface xvii
1. Structure of Growth Plate and Bone Matrix WILLIAM G. COLE
Introduction 1 Type I Collagen 1 Type V Collagen 6 Type II Collagen 8 Type IX Collagen 10 Type XI Collagen 11 Type X Collagen 14 Aggrecan 16 Cartilage Link Protein 1 16 Small, Leucine-Rich, Interstitial Proteoglycans Perlecan 20 Matrilins 21 Thrombospondins 22 Osteonectin 23 Osteocalcin 24 Matrix Gla Protein 25 Bone Sialoprotein 25 Bone Acidic Glycoprotein-75 26 Dentin Matrix Acidic Phosphoprotein-1 26 Osteopontin 26 References 27
Osteoprogenitor Cells and Regulation of Osteoblast Differentiation and Activity 50 Regulation of Osteoblast Differentiation and Activity 54 Osteocytes 58 Morphological Features of Osteoclasts 59 Mechanisms of Osteoclastic Bone Resorption 60 Origin of Osteoclasts 61 Regulation of Ostoclast Activity and Differentiation 61 Osteoclast Size: Multinucleation and Function 63 Stem Cell, Osteoblast, and Osteoclast Changes in Disease 63 Tissue Engineering and Stem Cell Therapy for Skeletal Diseases 64 References 65
17 3. P r e n a t a l B o n e D e v e l o p m e n t : O n t o g e n y and Regulation BENOITST-JACQUESANDJILLA. HELMS Introduction 77 Skeletogenesis 78 Skeletal Organization and Embryonic Origin of Bones 82 Molecular Regulation of Bone Formation 96 References 104
4. Postnatal B o n e Growth: Growth Plate 2. B o n e Cell Biology: O s t e o b l a s t s , O s t e o c y t e s , and O s t e o c l a s t s JANEE.AUBINANDJOHANN. M. HEERSCHE Introduction 43 Ontogeny of Osteoblasts and Control of Osteoblast Development 44
Biology, Modeling, and Remodeling GERARD KARSENTYAND HENRY M. KRONENBERG
Endochondral and Intramembranous Bone Formation 119 Growth Hormone and Insulin-Like Growth Factor- 1 121
vi
Table of Contents
Fibroblast Growth Factors 122 Thyroid Hormone 124 Estrogen and Androgen 124 Osteoblasts and Bone G r o w t h 124 Endocrine Regulation of Bone Formation References 130
References
9. P e a k B o n e M a s s a n d Its R e g u l a t i o n JEAN-PHILIPPEBONJOUR,THIERRYCHEVALLEY,SERGEFERRARI, AND RENF.RIZZOLI
126
5. P a r a t h y r o i d H o r m o n e a n d C a l c i u m Homeostasis GORDON J. STEWLER
Cellular and Extracellular Calcium Homeostasis Parathyroids and Secretion and Metabolism of Parathyroid Hormone 139 Assay of PTH 147 Parathyroid Hormone Action 148 References 160
135
6. P h o s p h a t e H o m e o s t a s i s R e g u l a t o r y Mechanisms JOSEPH CAVERZASIO, HEINI MURER, AND HARRIET S. TENENHOUSE
Introduction 173 Physiological Aspects 173 Cellular and Molecular Aspects 178 Pathophysiological Aspects 181 References 187
Definition and Importance of Peak Bone Mass 235 Characteristics of Peak Bone Mass Acquisition 235 Calcium-Phosphate Metabolism During Growth 236 Determinants of Bone Mass Gain 238 Conclusions 242 References 243
10. P r e g n a n c y a n d L a c t a t i o n ANN PRENTICE
Introduction 249 Mineral Fluxes from Mother to Offspring Pregnancy 250 Lactation 255 Summary 264 References 264
249
1 1. Fetal M i n e r a l H o m e o s t a s i s CHRISTOPHER S. KOVACS
7. Vitamin D Biology RENEST-ARNAUDANDMARIEB. DEMAY Metabolic Activation of Vitamin D 193 Mechanism of Action 198 Role of Vitamin D in Calcium Homeostasis Summary and Perspectives 208 References 209
201
8. O t h e r Factors C o n t r o l l i n g B o n e G r o w t h a n d D e v e l o p m e n t " C a l c i t o n i n , CGRP, O s t e o s t a t i n , Amylin, a n d A d r e n o m e d u l l i n JILLIANCORNISHANDTHOMASJOHNMARTIN Introduction 217 Calcitonin 217 Calcitonin Gene-Related Peptide 220 Parathyroid Hormone-Related Protein Amylin 225 Adrenomedullin 228
229
Fetal Adaptive Goals 271 Placental Calcium Transport 280 Placental Transport of Magnesium and Phosphate 288 Fetal Parathyroids 288 Calcium-Sensing Receptor 289 Thymus 290 Fetal Kidneys and Amniotic Fluid 290 Fetal Skeleton 291 Maternal Skeleton 292 Fetal Response to Maternal Hyperparathyroidism 293 Fetal Response to Maternal Hypoparathyroidism 293 Integrated Fetal Calcium Homeostasis 293 References 296
12. N o n i n v a s i v e T e c h n i q u e s for B o n e M a s s Measurement STEFANO MORA, LAURA BACHRACH, AND VINCENTE GILSANZ
223
Indications for Bone Mass Measurements 303 Challenges to Interpreting Bone Mass Measurements in Childhood and Adolescence 303 Noninvasive Measurement Techniques 304
~176
Table of Contents
Data Interpretation References 319
316
1 3. A s s e s s m e n t of M a t u r a t i o n " B o n e A g e a n d Pubertal Assessment NO[L CAMERON
Background 325 Initial Considerations 325 Methods of Assessment 329 Reliability 333 Comparability of the Atlas and Bone-Specific Methods 333 Secondary Sexual Development 334 References 337
14. B i o c h e m i c a l M a r k e r s o f B o n e Metabolism ECKHARDSCHONAUAND FRANKRAUCH
Introduction 339 Markers of Bone Formation 341 Markers of Bone Resorption 342 Bone Markers During Normal Development 344 Puberty 346 Bone and Collagen Markers in Metabolic Bone Diseases 346 Clinical and Research Value of Biochemical Markers of Bone Metabolism 352 References 353
15. B o n e H i s t o m o r p h o m e t r y FRANK RAUCH
Introduction 359 Methodology 359 Pediatric Bone Histomorphometry in Health and Disease 366 Indications for Bone Biopsy and Histomorphometry in Pediatric Bone Diseases 372 References 373 16. A D i a g n o s t i c A p p r o a c h t o S k e l e t a l Dysplasias SHEILA UNGER, ANDREA SUPERTI-FURGA, AND DAVID L. RIMOIN
Introduction 375 Background 375
VII
History and Physical Examination 376 Diagnostic Imaging 389 Biochemical Investigations 393 Cartilage Histology 395 Molecular Basis 396 Prenatal Detection of Suspected Skeletal Dysplasia 397 Conclusion 398 References 398
1 7. T h e S p e c t r u m of P e d i a t r i c Osteoporosis LEANNE M. WARD AND FRANCIS H. GLORIEUX
Abstract 401 Introduction 401 Definition and Diagnosis of Osteopenia/Osteoporosis in Pediatric Patients 401 The Role of the Mechanostat in the Pathogenesis of Pediatric Osteoporosis 403 The Scope of the Problem 405 Approach to Prevention and Intervention 426 Differentiating Child Abuse from Bone Fragility Conditions 428 Summary and Future Directions 430 References 431
18. O s t e o g e n e s i s I m p e r f e c t a HORACIAO PLOTKIN, DRAGAN PRIMORAC, AND DAVID ROWE
Introduction 443 Classification 444 Types of OI 445 Differential Diagnosis 449 General Clinical Findings 450 Pathophysiology 453 Therapy 457 References 463
19. S c l e r o s i n g B o n y D y s p l a s i a L. LYNDONKEY,JR.,AND WILLIAML. RIES
Introduction 473 Defects in Osteoclastic Bone Resorption 473 Skeletal Abnormalities Related to Overproduction of Bone: Involvement of Transforming Growth Factors 478 Summary 480 References 481
viii
Table of Contents
20. Parathyroid Disorders MURAT BASTEPE, HARALD JUPPNER, AND RAJESH V THAKKER
Introduction 485 PTH Gene Structure and Function 485 Hypocalcemic Disorders 488 Hypercalcemic Diseases 496 Conclusion 501 References 502
2 1 . Fibrous Dysplasia PAOLO BIANCO, PAMELA GEHRON ROBEY, AND SHLOMO WIENTROUB
Introduction 509 Clinical Features 510 Molecular Genetics 516 Determinants of Phenotypic Variability 518 Pathology 520 Pathogenesis 526 Management and Treatment 530 References 533
22. Nutritional Rickets JOHN M. PETTIFOR
Introduction 541 Definition of Rickets 541 Classification of Rickets 542 Nutritional Rickets 542 References 560
23.Metabolic Bone Disease of Prematurity NICK BISHOP AND MARY FEWTRELL
Introduction 567 In Utero Mineral Accretion and Bone Growth 567 Physiological Changes in Mineral Homeostasis at Birth 568 Metabolic Bone Disease 569 Skeletal Health in Preterm Infants 572 Suggested Guidelines for the Prevention of Metabolic Bone Disease in Preterm Infants 578 Conclusions 579 References 579
24. Rickets Due to Hereditary Abnormalities of Vitamin D Synthesis or Action ANTHONYA. PORTALE AND WALTER L. MILLER
Introduction
583
Biosynthesis of Vitamin D 583 Vitamin D Biosynthetic Enzymes 584 Vitamin D 25-Hydroxylase and 24-Hydroxylase 584 Vitamin D la-Hydroxylase 584 Rickets Due to Abnormalities of Vitamin D Metabolism 585 Rickets Due to Abnormalities of Vitamin D Action 592 References 598
25. Familial Hypophosphatemia and Related Disorders INGRID A. HOLM, MICHAEL J. ECONS. AND THOMAS 0. CARPENTER
Introduction 603 Clinical Description of Disease Entities 603 Treatment 613 Family/Genetic Studies 616 Molecules, Pathophysiology, and Lessons from Animal Models 617 Current Problems and Unresolved Questions 623 References 624
26. Rickets Due to Renal Tubular
Abnormalities RUSSELL W. CHESNEY AND DEBORAH I? IONES
Introduction 633 Fanconi Syndrome 633 Renal Magnesium Wasting 640 Hypercalciuria 642 Renal Tubular Acidosis 644 Conclusion 647 References 647
27. Hypophosphatasia DAVID E. C. COLE
Biology of Alkaline Phosphatase 651 Clinical Hypophosphatasia 661 References 672
28. Renal Osteodystrophy: Pathogenesis, Diagnosis, and Treatment BEATRIZ D. KUIZON AND ISIDRO B. SALUSKY
Introduction 679 The Spectrum of Renal Osteodystrophy 679 Pathogenesis of Renal Osteodystrophy 682
Table of Contents Clinical Manifestations 685 Biochemical Determinations 686 Histologic Manifestations 687 Radiographic Features of Renal Osteodystrophy 689 Long-Term Consequences 690 Treatment 691 References 695
2 9 . B o n e T u m o r s in C h i l d r e n MARCH. ISLERAND ROBERTE. TURCOTTE Introduction 703 General Principles of Treatement 703 Diagnosis 704 Classification and Nomenclature 706
ix
Back Pain and Spinal Neoplasms in Children 707 Fibrous Tumors of Bone 709 Cartilage-Forming Tumors 713 Bone-Forming Tumors 722 Hematopoietic 730 Vascular 731 Neurogenic 733 Adipose 734 Mixed 734 Notochord 735 Tumor-Like 735 Conclusion 740 References 740
Index
745
This Page Intentionally Left Blank
Contributors
Russell W. Chesney (633) University of Tennessee Health Science Center, Deparment of Pediatrics, Memphis, Tennessee 38103
Numbers in parentheses indicate the pages on which the authors' contribution begin.
Jane E. Aubin (43) Department of Medical Genetics and Microbiology, Faculty of Medicine, Toronto, Ontario M5S 1A8, Canada
Thierry Chevalley (235) Department of Internal Medicine, Division of Bone Diseases, World Health Organization Collaboration Center for Osteoporosis and Bone Diseases, University Hospital, CH 1211 Geneva, Switzerland
Laura Baehraeh (303) Division of Endocrinology, Stanford University School of Medicine, Stanford, California 94305
David E. C. Cole (651) Departments of Laboratory Medicine and Pathobiology, Medicine, and Genetics, University of Toronto, Toronto, Ontario MSG 1X8, Canada
Murat Bastepe (485) Endocrine Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston Massachusetts 02115
William G. Cole (1) Division of Orthopaedics and Research Institute, The Hospital for Sick Children, University of Toronto, Toronto, Ontario MSG 1X8, Canada
Paolo Bianeo (509) Department of Experimental Medicine and Pathology, La Sapienza University School of Medicine, 00161 Rome, Italy Nick Bishop (567) Academic Unit of Child Health, University of Sheffield, Sheffield Children's Hospital, Western Bank, Sheffield S10 2TH, United Kingdom
Jillian Cornish (217) Department of Medicine, University of Auckland, Auckland 1020, New Zealand Marie B. Demay (193) Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, Boston Massachusetts 02114
Jean-Philippe Bonjour (235) Department of Internal Medicine, Division of Bone Diseases, World Health Organization Collaborating Center for Osteoporosis and Bone Diseases, University Hospital, CH 1211 Geneva, Switzerland
Michael J. Econs (603) Indiana University School of Medicine, Indianapolis, Indiana 46202 Serge Ferrari (235) Department of Internal Medicine, Division of Bone Diseases, Worlth Health Organization Collaboration Center for Osteoporosis and Bone Diseases, University Hospital, CH 1211 Geneva, Switzerland
Noel Cameron (325) Human Biology Research Centre, Department of Human Sciences, Loughborough University, Loughborough, Leicestershire LEll 3TU, United Kingdom Thomas O. Carpenter (603) Yale University School of Medicine, New Haven, Conneticut 06510
Mary Fewtrell (567) MRC Childhood Nutrition Research Center, Institute of Child Health, London WC 1N 1EH, United Kingdom
Joseph Caverzasio (173) Division of Bone Diseases, Department of Internal Medicine, University Hospital of Geneva, CH-1211 Geneva, Switzerland
Vicente Gilsanz (303) Department of Radiology, Children's Hospital Los Angeles, University of Southern
xi
~176
Xll
Contributors
California School of Medicine, Los Angeles, California 90027 Francis H. Glorieux (401) Departments of Surgery, Pediatrics and Human Genetics, McGill University and the Shriners Hospital for Children, Montreal, Quebec, Canada H3A 2T5
Jill A. Helms (77) Department of Orthopedic Surgery, University of California San Francisco, San Francisco, California 94143-0514 Johan N. M. Herschel (43) Dental Research Institute, Faculty of Dentistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada lngrid A. Holm (603) Harvard Medical School, Boston Massachusetts 02115
Mare H. Isler (703) University of Montreal, Department of Orthopedic Oncology, Hopital Maisonneuve Rosemont and Hopital Sainte Justine, Montreal, Quebec, Canada H 1T 2M4 Deborah P. Jones (633) University of Tennessee Health Science Center, Deparment of Pediatrics, and Children's Foundation Research Center at Le Bonheur Children's Medical Center, Memphis, Tennessee 38103 Harald Jiippner (485) Endocrine Unit, Department of Medicine and Pediatrics, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02115 G6rard Karsenty (119) Department of Molecular and Human Genetics, Baylor College of Medicine, Houston Texas 77030
L. Lyndon Key, Jr. (473) Department of Pediatrics, Medical University of South Carolina, Charleston, South Carolina 29425 Christopher S. Kovacs (271) Faculty of Medicine, Endocrinology, Memorial University of Newfoundland, Health Sciences Centre, St. John's Newfoundland A1B 3V6, Canada Henry M. Kronenberg (119) Endocrine Unit, Massachusetts General Hospital, Boston, Massachusetts 02114 Beatriz D. Kuizon (679) Department of Pediatrics, UCLA School of Medicine, Los Angeles, California 90095 Thomas John Martin (217) St. Vincent's Institute of Medical Research, Melbourne, Victoria, Australia Walter L. Miller (583) Department of Pediatrics, University of California San Francisco, San Francisco, California 94143
Stefano Mora (303) Laboratory of Pediatric Endocrinology, Scientific Institute H San Raffaele, 20132 Milan, Italy Heini Murer (173) Institute of Physiology, University of Zurich, CH-8057 Zurich, Switzerland John M. Pettifor (541) MRC Mineral Metabolism Research Unit, Department of Pediatrics, University of the Witwatersrand and Chris Hani Baragwanath Hospital, Johannesburg, South Africa Horaeio Plotkin (443) Inherited Metabolic Diseases Section, Department of Pediatrics, University of Nebraska Medical Center and Children's Hospital, Omaha, Nebraska 98198 Anthony A. Portale (583) Department of Pediatrics, University of California San Francisco, San Francisco, California 94143 Ann Prentice (249) Medical Research Council Human Nutrition Research, Elsie Widdowson Laboratory, Cambridge CB 1 9NL, United Kingdom Dragan Primorae (443) Laboratory of Clinical and Forensic Genetics, Split University Hospital and School of Medicine, Split, Croatia Frank Raueh (339, 359) Genetics Unit, Shriners Hospital for Children, Montreal, Quebec H3G 1A6, Canada
William L. Ries (473) Department of Pediatrics, Medical University of South Carolina, Charleston, South Carolina 29425 David L. Rimoin (375) Medical Genetics Birth Defects Center, Cedars-Sinai Health System and Department of Pediatrics and Medicine, UCLA School of Medicine, Los Angeles, California 90048 Ren6 Rizzoli (235) Department of Internal Medicine, Division of Bone Diseases, Worlth Health Organization Collaboration Center for Osteoporosis and Bone Diseases, University Hospital, CH 1211 Geneva, Switzerland Pamela Gehron Robey (509) Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892 David Rowe (443) Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Conneticut 06030
lsidro B. Salusky (679) Department of Pediatrics, UCLA School of Medicine, Los Angeles, California 90095 Eckllard Sch6nau (339) Children's Hospital and Health Center, University of K61n, 50924 K61n, Germany
Contributors
xiii
Ren~ St-Arnaud (193) Genetics Unit, Shriners Hospital for Children, Montreal, Quebec H3G 1A6 Canada and Departments of Surgery and Human Genetics, McGill University, Montreal, Quebec H3A 2T5 Canada
Rajesh V. Thakker (485) Molecular Endocrinology Group, Nuttfield Department of Medicine, John Radcliffe Hospital Headington, Oxford OX3 9DU, United Kingdom
Benoit St-Jacques (77) Department of Human Genetics,
Robert E. Turcotte (703) University of Montreal and
McGill University and Genetics Unit, Shriners Hospital for Children, Montreal, Quebec H3G 1A6, Canada
McGill University, Department of Orthopedic Oncology, Hopital Maisonneuve Rosemont, Montreal, Quebec, Canada H 1T 2M4
Gordon J. Strewler (135) Walter Bradford Cannon Soci-
Sheila Unger (375) Division of Clinical and Metabolic
ety, Harvard Medical School, Boston, Massachusetts 02115
Genetics, Hospital for Sick Children, University of Toronto, Toronto, Canada M5G 1 X8
Andrea Superti-Furga (375) University of Lausanne, Div-
Leanne M. Ward (401) Department of Pediatrics, Div-
ision of Molecular Pediatrics, Centre Hospitalier Universitaire Vaudois, CH- 1011 Lausanne, Switzerland
ision of Endocrinology and Metabolism, University of Ottawa and the Children's Hospital of Eastern Ontario, Ottawa, Ontario, Canada K1H 8L1
Harriet S. Tenenhouse (173) Departments of Pediatrics
and Human Genetics, McGill University, Montreal Children's Hospital Research Institute, Montreal, Quebec H3Z 2Z3 Canada
Shlomo Wientroub (509) Department of Pediatric Orthopedic Surgery, Dana Children's Hospital, Tel Aviv Medical School, 64239 Tel Aviv, Isreal
This Page Intentionally Left Blank
Foreword
Harold Harrison's contributions to our understanding of pediatric bone disease (4). Chapters in the book, each one written by an authoritative member of the corresponding "academy," address particular topics, such as the cellular architecture of bone itself, how it develops, and how it remodels. The vast complexity of calcium and phosphate ion metabolism, and the complexity of their hormonal controls and of the corresponding nutritional dimensions are subjects of other chapters. There are chapters that describe how to measure the features of bone, and there are tables of mutations that point to online databases and the locus specific alleles that are causes of certain bone diseases. A series of twelve chapters address diseases of bone development (skeletal dysplasias), of bone density (osteoporosis), of tensile strength (osteogenetics imperfecta) and remodeling (osteopetrosis). Aberrations of hormonal control and ion homeostasis, are discussed. Acquired diseases of bone (e.g. tumors and renal osteodystrophy) are not forgotten. More important still, the patient with the problem is not forgotten. Between the covers of this book, there is a great deal of information from which the scientist and the clinician can acquire knowledge. More important the reader will gain wisdom about the precious skeleton of youthful Homo sapiens.
The human skeleton is a product of biological evolution; it accommodates an upright bipedal organism. It is still imperfect for the job it must do; there are speculations on how the human skeleton might be improved (1). Meantime, Homo sapiens has what it has. The healthy human skeleton and the organic matrices of which it is made are mysterious and wonderful structures. In the fetus, they are pliable and miniature, they accommodate both fetal growth and development and also the trauma of passage through the maternal pelvis. The infant's skeleton must then grow enormously in volume and span, remodeling itself all the while until adult dimensions are achieved. At the same time, it must be resisting the continuous action of gravity, while maintaining its integrity under physical trauma, on average for 8 decades! A truly mysterious and marvelous structure. This book, which reflects the vision of its outstanding editors, addresses the biology of bone, and the diseases that interfere with its structure and function. Industrialized societies of the northern hemisphere witnessed childhood rickets in epidemic proportions during the 19 th and early 20 th century. Rickets was an important pediatric disease. Then came the discovery of the anti-rachitic factor (vitamin D); awareness of the origins and importance of vitamin D, combined with improvements in living conditions, led to the virtual disappearance of nutritional infantile rickets, leaving the much rarer intrinsic biological forms of rickets to be recognized--the inherited forms of calcium or phosphate dishomeostasis. The first such report appeared in 1937 (2). It heralded the search for, and eventually the molecular definition of, heritable forms of rickets, and of metabolic and organic bone diseases in general in human societies (3). In the past half century, there has been an impressive growth of knowledge about cellular, ionic, organic, and hormonal aspects of bone metabolism, and of the diseases that interfere with the processes of development, growth, remodeling, and mineralization of bone. This book is a record of that understanding at the present time. It is the first of its kind, to my knowledge, to follow an earlier book honoring Helen and
(1)
Olshansky, S. J., Carnes, B. A., Butler, R. N. If humans were built to last. Sci. Am. 284:50-55, 2001.
(2)
Albright, F., Butler, A. M., Bloomberg, E. Rickets resistant to vitamin D therapy. Am. J. Dis. Child 54:529-547, 1937
(3) Scriver, C. R., Tenenhouse, H. S. On the heritability of rickets, a common disease (Mendel, Mammals and Phosphate). Johns Hopkins Med. J. 149:179-187, 1981. (4)
DeLuca H. F., Anast C. S. Pediatric Diseases Related to Calcium. Elsevier. New York. 1980. Charles R. Scriver, M D C M Montreal Children's Hospital, Canada
XV
This Page Intentionally Left Blank
Preface
The description and investigation of bone diseases both in adults and in children have been, for a long time, part of the realms of Endocrinology, Nephrology, Rheumatology and also Orthopedics and Medical Genetics. In the past three decades, as new knowledge and technology rapidly developed, bone biology and its related diseases progressively became a well identified domain in medicine. Thus was published a series of important books in adult medicine dealing with the following: metabolic bone disease, diseases of connective tissue, osteoporosis (a field in itself), and bone biology. In some of these textbooks, chapters dealing with pediatric bone disease have been included. However, not since the book by Maroteaux (1) was published in 1974 in French, which contains a unique collection of radiographs, and not since the Proceedings of a Symposium held in 1980 to celebrate the careers of Harold and Helen Harrison (2), has there been a book attempting to integrate basic knowledge and clinical facts as they concern pediatric bone disease. When we started drawing up the outline for this book, we decided that its usefulness would be greatly enhanced by first providing detailed descriptions of the basic concepts underlying the development, structure, and homeostatic control of bone tissue. We also wanted to provide descriptions of the tools and methodologies now validated for the precise evaluation of most of the disturbances of bone and mineral metabolism in children. Thus the book opens with chapters on the structure of the growth plate and the bone matrix, bone cell biology, mineral ion homeostasis and prenatal and postnatal bone growth. This is followed by the descriptions of how to assess bone maturation by measuring biochemical bone markers in blood and urine, and of the unique information provided by bone histomorphometry. The next part comprises fourteen chapters discussing specific disorders, or groups of disorders, including basic and clinical aspects.
We hope that the book will be useful both to researchers interested in learning about the clinical expression of the biological processes they study at the bench, and to the practitioners who wish to better understand the molecular and genetic intricacies of the conditions they evaluate and treat in the clinical setting. We wish to express our gratitude to Tari Paschall and Jasna Markovac at Academic Press for their understanding and support. We also thank all of the contributors for their willingness to be a part of this large effort. Finally we hope that the summation of knowledge and observations under one single heading will give substance to the concept of "pediatric osteology," which has become an entity of its own and may need to be recognized as a speciality in itself. As such, it may become an important and exciting career opportunity for some of our younger colleagues. (1) Maroteaux, P. (1974). Les maladies osseuses de l'Enfant. Flammarion M6decine-Sciences, Paris. (2) DeLuca, H. F., and Anast, C. E. (Eds.) (1980). Pediatric Diseases Related to Calcium. Elsevier, North Holland, New York, NY. Francis H. Glorieux Harald W. Jueppner John M. Pettifor
m
XVII ~176
This Page Intentionally Left Blank
C
H
A
P
T
E
R
1 Structure of Growth Plate and Bone Matrix WILLIAM G. COLE Division of Orthopaedics and the Research Institute, The Hospital for Sick Children, and University of Toronto, Toronto, Ontario, Canada
INTRODUCTION This chapter focuses on the main macromolecules of the extracellular matrices of the growth plates and bone. These diverse macromolecules often perform critical biomechanical, physiological, and biochemical functions. The hyaline cartilage growth plates are highly organized structures responsible for the elongation of bones by endochondral ossification. The cellular events are described in detail in subsequent chapters. Hydrated hyaline cartilages contain approximately 65-80% water, 10-20% collagen, 4-7% aggrecan, and _ 5 . 0 -
0
E 800-
O"
e--
i
E
->.,- 7 0 0 -
0
8
3.0-
E if)
O|o
> o tl:l
.o_ 6 0 0 "0 tl:i t._ tl:i
1"1_
t" /
E
0 4000
9
0
8
,r 3 0 0 -
~ e-
0
o o
E 4.0:=1 0
more, kinetic studies in rats led to similar conclusions, with no evidence to suggest any increase in the active uptake of calcium by bone [12]. For example, when calcitonin was infused into rats that had been injected 12 hr previously with radiolabeled 45Ca, hormone treatment lowered plasma calcium without affecting plasma 45Ca levels (Fig. 1). Under these experimental conditions, the disappearance of radioactivity from the plasma reflected uptake of 45Ca by the skeleton. The failure of calcitonin to influence this reflects an action of the hormone to prevent calcium efflux from bone and is not consistent with active stimulation of calcium uptake by bone. Studies of the actions of hormones on isolated bone cell populations established that calcitonin acts directly on osteoclasts, with receptor autoradiography establishing osteoclasts as the only discernible bone cell targets [13]. Consistent with this are observations of its actions in organ culture, especially the demonstration that calcitonin-treated osteoclasts in cultured mouse calvaria rapidly lose their ruffled borders. A similar in vivo observation of loss of ruffled border in osteoclasts has been made in patients with Paget's disease, in whom bone biopsies were taken before and 30 min after an injection of calcitonin [14]. In the same clinical study, calcitonin was noted to decrease the number of osteoclasts in addition to altering their ultrastructure. Studies using isolated osteoclast preparations indicate a direct effect of calcitonin on the osteoclast, in which the hormone rapidly inhibits the activity of osteoclasts. In further experiments, it was also noted that although isolated osteoclasts remained quiescent in calcitonin as long as the hormone was present, they regained activity when osteoblasts were added to the culture [15]. This escape of osteoclasts from inhibition by calcitonin took place at a rate proportional to the number of osteoblasts
9
8
200
'
.
~> 100~ o
,,,~
~0 0
I1.
Q.
FIGURE 1 Effect of calcitonin infusion on plasma calcium, radioactivity, and specific activity in control (o) and in calcitonin-treated rats (.). 45Ca was injected 12 hr before beginning the 4-hr infusion of calcitonin or control solution [from Robinson, C. J., Martin, T. J., Matthews, E. W., et al. (1967). Mode of action of thyrocalcitonin. J. Endocrinol. 39, 71-77. Reproduced by permission of the Society for Endocrinology].
8. Other Factors Controlling Bone Growth and Development
with which they were in contact. Calcitonin reduced the cytoplasmic spreading of isolated osteoclasts in a dosedependent manner [15]. PTH had no effect unless osteoblasts were cocultivated with the osteoclasts, in which case the addition of PTH resulted in a marked increase in cytoplasmic spreading of osteoclasts. It cannot be assumed that these phenomena reflect the responses of cells in bone in vivo, but this work provided for the first time some useful direct observations of actions of hormones on isolated bone cell preparations containing osteoclasts. The molecular mechanisms by which calcitonin decreases osteoclast function have yet to be fully defined. The rapid effects of the hormone may occur as a result of actions on a cytoskeletal function of osteoclasts, after initial events involving generation of intracellular second messengers. With the development of improved methods of studying isolated osteoclasts, it has been possible to establish that mammalian osteoclasts possess abundant, specific, high-affinity receptors for calcitonin and that calcitonin stimulates cAMP formation in a sensitive and dose-dependent manner as well as increasing intracellular calcium levels [13]. Although calcitonin inhibits bone resorption, it has been found in organ cultures that calcitonin inhibition is followed by "escape," which is defined as an increase in resorption in bones stimulated by a resorptive agent despite the continued presence of concentrations of calcitonin that initially were maximally inhibitory [16]. Furthermore, rats treated chronically with calcitonin become refractory to the hypocalcemic action of the peptide [17]. Data from in vitro experiments suggested that escape was due to a change in responsiveness of the bones rather than a loss of activity of the hormone. Calcitonin-induced homologous desensitization has been studied directly in osteoclasts, with results showing that treatment with the hormone rapidly leads to receptor loss, related to diminution in m R N A for the calcitonin receptor [18]. The mechanism of calcitonin-induced receptor m R N A loss appeared to be due principally to destabilization of receptor m R N A [19]. The 3' untranslated region of the mouse and rat calcitonin receptor m R N A contain four A U U U A motifs as well as other A/U-rich domains and a large number of poly-U regions. Such motifs, commonly found in cytokines and oncogenes, function as signals for rapid m R N A inactivation. We have noted that the calcitonin receptor behaves in a manner similar to the [32-adrenergic receptor. In the latter case, A/U-rich elements in the 3' untranslated region have been shown to bind to a number of cytosolic proteins, some of which have been characterized and which probably accelerate m R N A degradation. An additional interesting aspect regarding calcitonininduced receptor regulation is that glucocorticoid treat-
2_19
ment substantially prevented calcitonin receptor loss [19]. Glucocorticoid treatment was shown by nuclear run-on analysis to increase transcription of the calcitonin receptor gene. It is worth noting that clinical evidence suggests that glucocorticoids, given together with calcitonin, might prevent to some extent calcitonin-induced resistance to its own action [20]. A novel recent finding is that Katl antigen, a unique cell surface antigen on rat osteoclasts, provokes a marked stimulation of osteoclast formation in the presence of calcitonin but not in its absence [21]. It does so even in the presence of OPG, and its production in response to calcitonin could be related to the mechanism of calcitonin-induced resistance. Our concept of the physiological role of calcitonin is that it is an inhibitor of bone resorption whose function is to prevent bone loss at times of stress on calcium conservation, including pregnancy, lactation, and growth. When calcitonin was discovered, it seemed to provide the necessary explanation for the tight control of serum calcium, but events proved otherwise. Concepts of the role of bone in maintaining extracellular fluid calcium relied on observations made in the young, growing rat, in which it was clear that if accretion continued at the same rate and resorption was inhibited, the result would be a lowering of plasma calcium. The younger the animal, the more rapid the bone resorption rate. It would therefore be expected that the calcium-lowering effect of calcitonin should be greater in younger than in older animals. This was indeed the case in the rat, in which it was noted that in the biological assay of calcitonin, which depends on the calcium-lowering effect of the hormone, the response became less marked with increasing age of the animals [22] (Fig. 2). It should be noted, however, that the ability of calcitonin to counteract the effects of a calcium load was not impaired in older animals, at least in the rat [22]. This observation has not been explained and has not been extended to other species. In normal adult human subjects, even quite large doses of calcitonin have little effect on serum calcium levels. In those subjects in whom bone turnover is increased (e.g., in thyrotoxicosis and Paget's disease), calcitonin treatment acutely inhibits bone resorption and lowers the serum calcium [23]. Given that the acute effect of calcitonin on serum calcium is related to the prevailing rate of bone resorption, it is not surprising that calcitonin has little or no effect on calcium in the mature animal or human subject since the process of bone resorption is slow in maturity. It may be that the role of calcitonin in bone throughout life is that of a regulator of the bone resorptive process, whatever the overall rate of the latter. In the young or during pathological states of increased bone resorption in
2,2_0
Jillian Cornish and Thomas John Martin
CALCITONIN G EN E- RELATEDPEPTID E
11-
10 11 weeks old
E o 9-
-.m
0
o
E
v
IE 8 -
.m r
t3
E ~"
F-
Or)
6Ii /
/
0 -
O/__~N,,
I
13
I
26
I
52
I
104
Thyrocalitonin, MRC mU (log scale)
FIGURE 2 Decreasing hypocalcemic response to calcitonin with increasing age of the rat [from Cooper, C. W., Hirsch, P. F., Toverud, S. V., et al. (1967). An improved method for the biological assay of thyrocalcitonin. Endocrinology 81, 610-717. Reproduced by permission of The Endocrine Society].
maturity (e.g., Paget's disease and thyrotoxicosis), calcitonin inhibition of bone resorption can lower the serum calcium level, and there may even be a calcium homeostatic role for endogenous calcitonin in these circumstances. In a normal adult animal, however, when bone turnover is slow, even though calcitonin does not lower serum calcium, the physiological function of calcitonin in maturity may nevertheless be to regulate the bone resorptive process, in either a continuous or intermittent manner [8]. It follows that calcitonin should not necessarily be regarded as a calcium-regulating hormone in maturity but may yet be shown to be such in stages of rapid growth (e.g., in the young or in states of increased bone turnover). It is nevertheless important that bone resorption be regulated, and calcitonin could be capable of carrying out this function by a direct action on bone. Such a role might become more important in circumstances in which skeletal loss particularly needs to be prevented (e.g., in pregnancy and lactation). Evidence in support of such an important physiological role for endogenous calcitonin was provided by experiments showing that cancellous bone loss in thyroparathyroidectomized rats treated with PTH was greater than that in similarly treated sham-operated controls [24].
CGRP, a 37-amino acid peptide, has approximately 20% homology with calcitonin, and they have in common at the amino terminus a six-amino acid ring structure created by a disulfide bond as well as an amide group at the carboxyl terminus (Fig. 3). CGRP1 is generated by alternative processing of mRNA from the calcitonin gene, located on the short arm of chromosome 11. This gene has six exons, the first four of which produce mRNA for the precursor of calcitonin, preprocalcitonin. An alternative mRNA for the CGRP precursor, preproCGRP, is formed from exons 1-3,5, and 6. The alternative splicing of the calcitonin mRNA is tissue specific so that the predominant mRNA produced in the thyroid is that of calcitonin, whereas in the nervous system it is CGRP-1. A second form of CGRP, CGRP2, differs from CGRP by only three amino acids in the human and one amino acid in the rat. CGRP-2 is a product of a separate gene, also on the short arm of chromosome 11 [25]. CGRP is distributed throughout the nervous system and is one of the most abundant neuropeptides, with some data showing that CGRP circulates at concentrations of approximately 1 pmol/liter, levels that are increased by sex hormone replacement therapy in postmenopausal women. In the bone microenvironment, it is likely that CGRP concentrations are higher as a result of the local release of CGRP from nerve terminals. CGRPcontaining nerves develop when defects are created surgically in bone or following fractures. It is possible that CGRP aids bone growth through direct effects on osteoblast function. CGRP is a potent vasodilator, and because of the intimate association of the nerves with the blood vessels, CGRP may also have a role in regulating blood flow to sites of bone healing or growth. Soon after the discovery of CGRP [25] and its common origin with calcitonin, investigation of its effects on bone resorption was performed. CGRP lowers circulating calcium concentrations when the peptide is injected into intact animals [26]. In organ culture, CGRP was also demonstrated to lower radiolabeled 45Ca release from prelabeled neonatal mouse calvaria. The peptide inhibited both basal and stimulated bone resorption, but the half maximally effective concentration of CGRP was 500-fold higher than that of calcitonin [27]. A similar pattern of activity is seen with disaggregated isolated osteoclasts, where CGRP directly inhibits the activity of these cells. CGRP inhibits cell motility, probably via cAMP production; however, the osteoclast retraction seen with calcitonin is not produced by CGRP. CGRP inhibits the formation of TRAP-staining
m
r~..
O)
I--
i
I-
z
>
r3
(/)
,
I.-
Z
>
r3
r/)
,
~. - . - - L
Z:
......_ ~
,
,?,
m
u,, .......
m
a
Z
u)
Z
~
~
,.
,,,I
z
:~
r,,=
--
~..
=i
12
Z
14,
(.g
,
Z
(.g
.
.---L
Z
9
,,(
r3
I
A
" "
=)
.-
W
~[
-
... ~'
..
Q
0
G)
0
~,
.I
-r
0
n,,,
.J
>.
-r,
(g
.J
-J
-,
..j
=i
(~
(,g
_J
I--
=~
..I
Z
U.
..j
I..-
m
..j . . . .
>
>
-r "0
4
"$
-2
2
|
*
:
Slope: -0.12 95.6% within +_ 2 cm
m
............. 5
m
5. Llk, j, "
,,,..
,,
91 t , mim mmmm 15
2o
m
~-4
i--"
@ "0-6 0,,_ 8
0
1'o
' 40
' 50
" 60
' 70
DPD/Cr at 4 weeks (nmoltmrnol)
FIGURE 10 Associationsbetweenurinary Gal-Hyl/Cr(r = 0.79)and DPD/Cr (r = 0.70) after 4 weeksof recombinant human GH treatment and height velocity + 12 (y axis). Reproduced, with permission, from Collagen markers deoxypyridinoline and hydroxylysine glycosides: pediatric reference data and use for growth prediction in growth hormone deficient children. Clin. Chem. 2002, 48(2), 315-322.
Observed HV (cm/yr)
FIGURE 1 1 Residuals of a prediction based on 3-month height velocity, relative bone age retardation before therapy, pretreatment IGF-1, and DPD measured after 1 month of therapy versus observed 12-month height velocity. Reproduced with permission from Schtinau, E., Rauch, F., Blum, W. F., (2001). Prediction of growth response to GH treatment: A reference guide. Abingdon: United Kingdom: TMG Healthcare Communications, Ltd.
351
14. Biochemical Markers of Bone Metabolism
in bone histomorphometry in adults with predialytic renal failure [16].
bone disease might be an indication to determine bonespecific ALP, which has been shown to correlate with histomorphometrically determined bone formation rate in adults [142]. The value of other bone markers in renal osteodystrophy remains to be elucidated. Urinary markers and serum assays of small molecules that are eliminated via the kidneys (such as osteocalcin and ICTP) are probably not useful when renal clearance is impaired. In contrast, serum levels of PICP are independent of renal function [16] and have been found to reflect dynamic parameters
Rickets Vitamin D deficiency rickets is the classical metabolic bone disease in childhood, which today is relatively rare in Western countries. Elevated total ALP activity is a characteristic finding and is very useful in monitoring the effect of treatment [4,59] (Fig. 12). The value of other markers is less certain. Because the contribution of the
A .j
40
60
m 35 :I.
ai 30
~
50
d
_J
r
N
~ 20 ffl
o 0
O j:: 15
Q.
O 5
80%) of CaSR mutations that result in a functional gain are located within the extracellular domain [85-91], which is different from findings in other disorders caused by activating mutations in different G protein-coupled receptors. Pseudohypoparathyroidism The term pseudohypoparathyroidism (PHP) describes patients with hypocalcemia and hyperphosphatemia due to PTH resistance rather than PTH deficiency [92]. Affected individuals show partial or complete resistance to biologically active, exogenous PTH as demonstrated by a failure to increase urinary cyclic AMP and urinary phosphate excretion in response to this hormone; this
condition is referred to as PHP type I [93-95]. If associated with other endocrine deficiencies and characteristic physical stigmata, collectively termed Albright's hereditary osteodystrophy (AHO), the condition is referred to as PHP type Ia. The latter syndrome is associated with heterozygous inactivating mutations in G N A S 1 located on chromosome 20q13.3. This gene gives rise to at least five differently spliced mRNAs, including Gs~. Heterozygous mutations in one of the exons 1-13 of G N A S 1 lead to an approximately 50% reduction in one of the Gs~ activity/protein, partially explaining the resistance toward PTH and other hormones that mediate their actions through G protein-coupled receptors [93-95]. A similar decrease in Gsu activity/protein is also found in patients with pseudo-pseudohypoparathyroidism (PPHP), who have the same physical appearance as individuals with PHP-Ia but lack endocrine abnormalities, including resistance to PTH. Thus, mutations in the Gsuspecific exons of G N A S 1 are thought to be necessary but not sufficient to fully explain either PHP-Ia or PPHP [93-98]. In fact, a retrospective analysis of numerous published cases with either PHP-Ia or PPHP indicated that both disorders are typically found within the same kindred but never within the same sibship [99]. Furthermore, hormonal resistance is paternally imprinted; that is, PHP-Ia occurs only if the defective gene is inherited from a female affected by either PHP-Ia or PPHP, and PPHP occurs only if the defective gene is inherited from a male affected by either of the two disorders [99,100]. Observations consistent with some of these findings in humans have recently been made in mice heterozygous for disruption of exon 2 of the Gnas gene. Animals that inherited the mutant allele from a female showed decreased blood calcium concentration due to resistance toward PTH. In contrast, offspring that obtained the mutant allele lacking exon 2 from a male showed no evidence of endocrine abnormalities [101]. Moreover, mice that carried the ablated allele on their maternal chromosome had undetectable Gs0c protein in the renal cortex but not in many other tissues. Tissue- or cellspecific Gs0~ expression from a single parental allele is thus almost certainly involved in the pathogenesis of PHP-Ia and PPHP, and it provides a reasonable explanation for the finding that heterozygous G N A S 1 mutations result in a dominant phenotype with regard to the hormonal resistance. Mutations in patients with PHP-Ia are spread throughout most of the Gsu coding exons of G N A S 1 . More than 35 different mutations have been identified, and a 4-bp deletion in exon 7 is the most frequently reported mutation [95,102]. No direct phenotypegenotype correlation has been established for patients with PHP-Ia, except for a missense mutation identified in two unrelated boys with PHP-Ia and testo-
20. Parathyroid Disorders
toxicosis [103]. This Ala-to-Ser substitution at codon 366 generates a temperature-sensitive Gs~ mutant that is constitutively active at cooler temperatures due to accelerated GDP release, but it results in a loss of function at ambient body temperature due to thermolability [103]. Heterozygous inactivating mutations of the Gs0ccoding GNAS1 exons or reduced Gs0~ levels have also been identified in some patients with progressive osseous heteroplasia (POH), a congenital disorder ofectopic bone formation that, unlike the ossification in AHO, affects deep connective tissue and skeletal muscle [104]. Initially, a case with severe plate-like osteoma cutis, a variant of POH, has been associated with a mutation found in several patients with PHP-Ia and PPHP, although no other AHO-like features or hormone resistance were documented [104]. Furthermore, a POH patient with a unique exon 1 mutation was reported who also had mild brachydactyly but lacked any additional features of AHO [105]. POH was also present in another case who presented with PHP-Ia and showed reduced Gs0~ levels [105]. Recent study of several familial cases of POH with Gs0~ mutations revealed an interesting paternal-exclusive inheritance pattern [106]. In a large three-generation kindred, affected individuals who inherited from their father the same 4-bp deletion in GNAS1 exon 7, which was previously found in patients with PHP-Ia and PPHP
493
[95,102], demonstrated extraskeletal ossification without developmental and hormonal abnormalities. In contrast, the affected children of female disease gene carriers had clinical features of AHO, but the maternal transmission of the mutation did not lead to hormonal resistance. These findings are surprising and unexpected, and it remains to be determined whether undefined genes that modify the actions of Gs0~may explain the variation in the degree of extraskeletal ossification among patients with the same mutations, and why maternal inheritance of GNAS1 mutations affecting the exons that encode Gs0~ do not invariably lead to PHP-Ia (i.e., AHO combined with hormonal resistance). The G N A S I gene was recently shown to be considerably more complex than previously thought because alternative promoter use and splicing result in several different mRNAs (Fig. 3). Some of these transcripts are derived from either the paternal or the maternal allele, whereas others show biallelic expression. It thus appears likely that the complexity of the G N A S I gene contributes to the unique phenotypic abnormalities in patients with PHP (Fig. 3). Gs0~is encoded by exons 1-13 of the G N A S I gene and mediates the biological functions of a large variety of G protein-coupled receptors, including the PTH/PTHrP receptor. The Gs0~ transcript shows a nonimprinted expression profile in most tissues [107-110].
FIGURE 3 Intron/exon organization of the G N A S I gene and depiction of different mRNAs that are derived from alternative splicing. The mRNA encoding Gsu is thought to be expressed in most tissues from both alleles; however, in the renal cortex transcripts appear to be predominantly maternal in origin. The mRNAs encoding the splice variants XL~,s, AS, A/B, and NESP55 are derived either from the paternal allele or from the maternal allele (see text for details).
494
Murat Bastepe et al.
Nonetheless, its expression appears to occur only from the maternal allele in several tissues including the renal proximal tubule [101], although a recent study analyzing human fetal tissue demonstrated biallelic Gs~ expression at this site as well [111]. A second transcript, XL~s, comprises a novel first exon (XL) that splices onto exons 2-13. The encoded approximately 92-kDA protein shares amino acid sequence identity with Gsct in its carboxyl-terminal portion [112], and it appears capable of functioning as a stimulatory G protein in vitro [113,114]. The m R N A encoding XL~s is found at numerous sites; particularly high concentrations have been identified in endocrine and neuroendocrine cells [112,115], and in all investigated tissues it appears to be transcribed only from the paternal allele [107,108,110]. From the same XL~s m R N A , an additional protein with an electrophoretic mobility of approximately 48 kDA appears to also be translated using a second open reading frame; this protein, termed ALEX (alternative gene product encoded by XL-exon), does not share homology with Gs~ or XL~s [116]. The third transcript, NESP55 [117], expresses from the maternal allele only [108,110], and is encoded by another exon of the G N A S I gene that is located upstream of exon XL and the Gsu-specific exons 1-13. NESP55, which is a chromogranin-like neuroendocrine secretory protein [117], shares no amino acid sequence homology with either XL~s or Gsct, but its m R N A contains Gscz-specific exons in the 3' noncoding region. Another transcript with broad paternal-specific expression is A/B (also known as 1' or 1A), which uses a unique promoter and first exon located approximately 2.5 kb upstream of Gs~ [118-120]. The A/B transcript also shares exons 2-13 with the Gs0c transcript, but it is uncertain whether the former is translated into a protein. A recently identified transcript reads from the opposite strand of the GNAS1 gene and is hence termed the antisense transcript (AS). As with the A/B transcript, whether AS m R N A leads to a translated protein is unknown [121,122]. Consistent with the parentspecific expression profiles of its individual transcripts, GNAS1 shows allele-specific methylation. Although the promoter of Gs~ lacks methylation, promoters of the transcripts with parent-specific expression are methylated on the inactive, silenced allele. Because of the complexity of the G N A S I gene and the use of different allele- and strand-specific promoters, it appears plausible that mutations in the Gsct-specific exons 1-13 can affect not only the functional properties of Gsct but also those of XL~s, NESP55, and the A/B transcript [93-98,102]. Mutations in the exons encoding Gs~ have not been detected in PHP type Ib (PHP-Ib), a disorder in which affected individuals show PTH-resistant hypocalcemia and hyperphosphatemia but lack developmental defects and additional endocrine abnormalities [93-95].
Furthermore, individuals with PHP-Ib frequently show a normal osseous response to PTH or even biochemical and radiological evidence of increased bone turnover and osteoclastic bone resorption, indicating that the PTHdependent actions on osteoblasts are not impaired [93,94,123,124]. Moreover, PHP-Ib patients show no abnormalities in growth plate development and thus have normal longitudinal growth, indicating that the PTHrPdependent regulation of chondrocyte growth and differentiation is normal. These latter findings indicate that it is unlikely that defects in the PTH/PTHrP receptor can lead to PHP-Ib, and indeed many studies of the PTH/ PTHrP receptor gene and mRNA in PHP-Ib patients have failed to identify mutations [125-128]. In one study, however, a single amino acid deletion in the carboxyl-terminal region of the PTH/PTHrP receptor, de1382Ile, was demonstrated in three siblings with isolated PTH resistance [129]. This mutation appears to uncouple Gs~ from the PTH/PTHrP receptor only, leaving the function of several other Gs-coupled receptors intact. It may thus be the cause of an unusual variant of PHP-Ib in this kindred, although the advanced bone age documented for two of the affected children needs to be investigated further to disprove its association with the isolated PTH resistance. On the other hand, a genomewide scan in four unrelated kindreds mapped the PHP-Ib locus to chromosome 20q13.3, which contains the GNAS1 gene [130]. In this study, it was also shown that the genetic defect is paternally imprinted and is thus inherited in the same mode as the PTH-resistant hypocalcemia in kindreds with PHP-Ia and/or PPHP. The simplest explanation for these observations is that PHP-Ib is caused by a defect in a tissue- or cell-specific enhancer or promoter of the GNAS1 gene, which could directly or indirectly affect the expression levels of the Gs~-specific transcripts and/or the transcripts encoding XL~s and NESP55 (Fig. 3). Alternatively, PHP-Ib could be caused by a defect in a gene close to the GNAS1 locus that is transcribed only from the maternal allele and affects PTH/PTHrP receptor or Gs~ expression and/or function in some renal cells. Recent investigation of the methylation status of GNAS1 in nine sporadic and two familial cases of PHPIb demonstrated a specific loss of methylation at the exon A/B differentially methylated region [131]. This epigenetic defect, which was not present in healthy controls and patients with AHO, has also been found in affected individuals from a number of unrelated PHP-Ib kindreds in whom the genetic defect mapped to the previously defined locus on 20q13.3 [132]. These findings suggest that PHP-Ib is caused by a mutation in a putative cisacting element required for establishment and/or maintenance of the methylation imprint at GNAS1 exon A/B. Through more detailed haplotype analysis of one of the
20. Parathyroid Disorders
495
previously described kindreds, it was recently shown that this mutation is likely located at least 56 kbs centromeric of the abnormally methylated A/B region [132]. The importance of the epigenetic regulation of G N A S 1 in PHP-Ib has been further emphasized by findings in a child with paternal uniparental isodisomy of chromosome 20q who presented with PTH-resistant hypocalcemia at an early age [133]. This patient, who also had craniosynostosis, mild hypothyroidism, and a moderately elevated serum calcitonin level, but no evidence of A H O or Gs0~-specific mutations, demonstrated a methylation pattern at the G N A S 1 locus that is typically observed for the paternal allele. As a consequence, methylation at the A/B locus was lacking on both alleles, thus making it likely that loss of methylation in this region alone, presumably in the absence of any mutation, can be sufficient to lead to PTH resistance. Blomstrand's Disease Blomstrand's chondrodysplasia is an autosomal recessive disorder characterized by early lethality, dramatically advanced bone maturation, and accelerated chondrocyte differentiation [134]. Affected infants are typically born to consanguineous healthy parents (only in one case have unrelated healthy parents had two affected offspring) [135-139], show pronounced hyperdensity of the entire skeleton (Fig. 4) and markedly advanced ossification, and particularly the long bones are extremely short and poorly modeled. Recently, P T H / P T H r P receptor mutations that impair its functional properties have been identified as the most likely cause of Blomstrand's disease. One of these defects is a nucleotide exchange in exon M5 of the maternal PTH/ P T H r P receptor allele, which introduces a novel splice acceptor site and thus leads to the synthesis of a receptor mutant that does not efficiently mediate the actions of PTH or PTHrP, despite seemingly normal cell surface expression (Fig. 5). For unknown reasons, the patient's paternal P T H / P T H r P receptor allele is only poorly expressed [140]. In a second patient with Blomstrand's disease, the product of a consanguineous marriage, a proline residue located at position 132 was changed to leucine due to a missense mutation [141,142]. Despite reasonable cell surface expression, the resulting PTH/ P T H r P receptor mutant showed severely impaired binding of radiolabeled PTH and P T H r P analogs, greatly reduced agonist-stimulated cAMP accumulation, and no measurable inositol phosphate response. Additional loss-of-function mutations of the P T H / P T H r P receptor have been identified in three unrelated patients with Blomstrand's disease. Two of these mutations led to a frameshift and a truncated protein due to either a homozygous single nucleotide deletion in exon EL2 [143] or a
FIGURE 4 Radiological findings in a patient with Blomstrand's disease. A: Spine, AP view; B: Spine, lateral view; C: upper extremities; D: lower extremities. Note the markedly advanced ossification of all skeletal elements and the extremely short limbs, despite the comparatively normal size and shape of hands and feet. Furthermore, note that the clavicles are relatively long and abnormally shaped (reproduced with permission from Leroy et al. [136]).
FIGURE 5 Schematicrepresentation of the human PTH/PTHrP receptor. The approximate locations of heterozygous missense mutations that lead to constitutive receptor activation in patients with Jansen's disease are indicated by open circles. Mutations identified in patients with Blomstrand's disease are indicated by closed circles or boxes (see text for details). H, histidine; R, arginine; T, threonine; P, proline; I, isoleucine; L, leucine; X, termination codon.
496
Murat Bastepe et al.
27-bp insertion between exons M4 and EL2 [144]. The other defect was a nonsense mutation at residue 104 and thus resulted in a truncated receptor protein [144]. As in Jansen's disease, the identification of mutant PTH/ PTHrP receptors has provided a plausible explanation for the severe abnormalities in endochondral bone formation in patients with Blomstrand's chondrodysplasia. The disease is lethal, but it is likely that in addition to striking skeletal defects, affected infants show abnormalities in other organs, including secondary hyperplasia of the parathyroid glands presumably due to hypocalcemia. In addition, analyses of fetuses with Blomstrand's disease have revealed abnormal breast development and tooth impaction, highlighting the involvement of the PTH/PTHrP receptor in the normal development of breasts and teeth [145].
HYPERCALCEMIC DISEASES M a n i f e s t a t i o n s of h y p e r c a l c e m i a a n d t r e a t m e n t of h y p e r c a l c e m i c d i s o r d e r s Mild hypercalcemia (>11 mg/dl) can be associated with little or no symptoms. More severe hypercalcemia (>13 mg/dl) can be accompanied by failure to thrive, anorexia, nausea, abdominal pain (vomiting), somnolence, stupor, constipation, muscle weakness, polydipsia, and polyuria. The complications of long-standing hypercalcemia can include nephrocalcinosis, renal stones, and renal failure.
Acute treatment of severe hypercalcemia (> 15.0mg/ dl) requires forced diuresis with intravenous normal saline (1.5 times maintenance) and, after achieving adequate hydration, furosemide (1 mg/kg every six hours); peritoneal dialysis or hemodialysis with low calcium concentration in dialysate may be necessary. Since hypercalcemia is usually caused by excessive bone resorption, treatment with a bisphosphonate (for example, pamidronate 0.5-2.0 mg/kg body weight i.v. over 4 hours; usually a single treatment is sufficient to normalize serum calcium concentration within 24-48 hours) should be considered to reduce osteoclast activity; calcitonin (salmon calcitonin, 4 IU/kg body weight every 12 hours IM or SC) may also be effective. For treatment of persistent mild hypercalcemia dietary restriction of calcium (to 4 mg/kg/24 hr or, alternatively, as a random urine calcium:creatinine ratio >0.20. Urinary calcium excretion is higher in infants and toddlers younger than 2 years of age. In children, IH presents with hematuria (either gross or microscopic) or as urolithiasis. Occasionally, IH may manifest as enuresis, dysuria, or flank pain. Laboratory investigation for IH includes a timed urine for calcium, creatinine, and sodium. This should be performed when the child is well, on a regular diet, and not during an acute stone episode. High salt intake is associated with elevated urinary calcium excretion. Therefore, reduction of dietary sodium is often the initial recommendation. In some children, dietary salt restriction (2 or 3 g per day) alone results in normalization of urinary calcium excretion and resolution of hematuria. Stapleton recommends dietary calcium restriction (no milk products) as the next diagnostic step in the evaluation of IH. If hypercalciuria continues, then a trial of hydrochlorothiazide (1 or 2 mg/kg/day) for 4 weeks followed by measurement of urinary calcium excretion is useful in proving that the urinary symptoms (i.e., hematuria or lower tract symptoms) are related to the hypercalciuria. Secondary conditions, such as renal tubular acidosis, Dent's disease, hyperparathyroidism, hypervitaminosis D, and systemic disorders such as juvenile rheumatoid arthritis, should be excluded. Stapleton no longer recommends establishing the subtype of IH (absorptive or renal leak) by oral calcium loading test, although this may be of use in research studies. Bone D i s e a s e A s s o c i a t e d with Hypercalciuria
Clinical Characteristics As mentioned previously, IH in children most often presents as hematuria or during the evaluation ofnephrolithiasis. Of 83 children with IH and no history ofurolithiasis or urinary tract infection, hypercalciuria was present
643
in 27% [52]. Gross hematuria and a positive family history of renal stones were more likely in the group with IH. The condition has been reported in children throughout the world but is relatively uncommon in African American children. Frequency/dysuria, incontinence/enuresis, and abdominal/back pain are associated symptoms of hypercalciuria in some series [53]. Among the hypercalciuric children reported by Alon and Berenbom [53], the urine calcium:creatinine ratio ranged from 0.22 to 0.45 (mean, 0.32) and the 24-hr urine calcium excretions ranged from 4.49 to 9.2mg/kg (mean, 5.64 mg/kg). At follow-up of 33 children after an average of 4.6 years, no child had a renal stone or recurrent macroscopic hematuria. Pharmacologic treatment was not used, and approximately one-third of the children were said to adhere to a relatively salt-restricted diet. Half of the children remained hypercalciuric on the first urine sample, and one-fourth remained hypercalciuric on the second sample. Only 1 child demonstrated a renal calculus on ultrasound examination. Of those children with persistent hypercalciuria, all responded to a low-sodium and highpotassium diet with normalization of urinary calcium.
Alterations in Bone Mineral Density Decreased bone mineral density has been found among stone-forming patients as well as hypercalciuric patients with both types of hypercalciuria, although findings are conflicting, with some studies failing to demonstrate decreased bone density among the absorptive type [54,55]. When placed on a low-calcium diet, subjects who responded with normalization of urinary calcium tended to have normal bone density, whereas those who continued to excrete excess calcium had decreased vertebral mineral density [56]. Some children with hypercalciuria have decreased bone mineral density as measured by bone densitometry on the lumbar spine. Perrone et al. [57] found that 4 of 20 children with IH had decreased bone mineral density early in their diagnosis and the osteopenia appeared to be progressive in those children with sustained hypercalciuria. Seventy-three Spanish children with IH underwent analysis; 22 children (30%) had osteopenia as defined by a z score o f - 1 or less [58]. Weisenger [42] found decreased lumbar spine bone mineral density in 21 hypercalciuric children with a mean age of 9.3 years. Cortical and total bone mineral density were not decreased.
Histology Although limited, bone biopsy data are available. Malluche et al. [59] performed bone biopsies on 15 recurrent
644
Russell W. Chesney and Deborah P. Jones
stone-formers with hypercalciuria. There was increased osteoid volume and decreased osteoblastic activity and mineralizing osteoid seams. The authors summarized their findings as decreased mineralization of osteoid with no evidence of increased bone resorption. This was supported by the work of Pak and colleagues [60]. Other groups noted increased bone resorption [61,62]. Pathogenesis The possibility that disordered bone metabolism might result in IH has been considered. Increased expression of interleukin- 1 (IL- 1) by monocytes from patients with renal leak and not absorptive IH was reported; however, due to the age of the population, some postmenopausal women may have been included (increased IL-1 production has been proposed to contribute to postmenopausal osteoporosis) [63]. Weisinger et al. [64] found that IL-10~ production had an inverse correlation with bone mineral density in 29 stone-forming hypercalciuric adults, all of whom displayed diminished trabecular bone mineral density. There was no significant increase in the nonstimulated production of IL01[3, IL-6, and tumor necrosis factor