Laron Syndrome - From Man to Mouse: Lessons from Clinical and Experimental Experience

Laron Syndrome - From Man To Mouse

Zvi Laron • John J. Kopchick (Editors)

Laron Syndrome - From Man to Mouse Lessons from Clinical and Experimental Experience

Editors Zvi Laron, MD Schneider Children’s Medical Center of Israel, Endocrine Research Unit, Kaplan Street 14, 49202 Petah Tikva, Sackler Faculty of Medicine, Tel Aviv University, Israel e-mail: [email protected]

John J. Kopchick, PhD Edison Biotechnology Institute and Department of Biomedical Sciences, Ohio University, 1 Water Tower Drive, The Ridges, Athens, OH 45701, USA e-mail: [email protected]

ISBN: 978-3-642-11182-2     e-ISBN: 978-3-642-11183-9 DOI: 10.1007/978-3-642-11183-9 Springer Heidelberg Dordrecht London New York Library of Congress Control Number: 2010937974 © Springer-Verlag Berlin Heidelberg 2011 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is ­concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer. Violations are liable to prosecution under the German Copyright Law. The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant ­protective laws and regulations and therefore free for general use. Product liability: The publishers cannot guarantee the accuracy of any information about dosage and application contained in this book. In every individual case the user must check such information by consulting the relevant literature. Cover design: eStudioCalamar, Figueres/Berlin Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

“I dedicate this book to the patients with Laron syndrome who heroically accepted many investigations to clarify their enigmatic disorder and subsequently to determine the therapeutic response to a new drug and to my devoted associates in our clinical and laboratory research” Zvi Laron “I would like to thank my ‘extended laboratory family, including undergraduate and graduate students, technicians, postdoctoral fellows, visiting scientists, and collaborators worldwide’” John J. Kopchick

Preface

It has been 50 years since the first family with Laron syndrome (LS, primary growth hormone (GH) insensitivity) was referred to the newly established Pediatric Endocrine Clinic at the Beilinson Hospital, Petah Tikva, Israel, a referral center now located at the Schneider Children’s Medical Center on the same campus. Since then, 64 patients have been diagnosed by the same team, investigated, and the majority followed by them, most at regular intervals, throughout childhood into adult age. This was the reason to base this book on the data accumulated from this cohort of patients forthwith called the Israeli cohort. It was also our privilege to be the first to determine its etiopathology and the first to study the effects of insulin-like growth factor-I (IGF-I) replacement therapy in these patients. The great amount of data accumulated during half a century in the large Israeli cohort of untreated and IGF-I treated LS patients is a unique source of knowledge which we thought should be shared and presented in a united manner. The clinical portion of this book represents a compendium of the Israeli team’s experience with LS from discovery, the struggle to define its pathogenesis, to determine the consequences if not treated by IGF-I, and the results of long-term IGF-I treatment. Reference to studies by other investigators are included when found appropriate. During this 50 year-long journey, LS has proven to be a unique model to learn the effects of congenital IGF-I deficiency, the pharmacology of IGF-I, and the GH-IGF-I interrelationships. The fact that LS is a condition in which the action of pituitary GH is excluded permits the comparison between isolated GH deficiency and IGF-I deficiency as well as the comparison between the responses to IGF-I and hGH replacement therapies. To enable the comparison of findings in the same patient, we have identified certain patients by their initials. In an attempt to generate an animal model of LS (GH resistance/insensitivity and IGF-I deficiency), the GH receptor gene disrupted the “knock-out” mouse (or Laron Mouse) was produced in the Kopchick laboratory in 1991. Experiments that could not be carried out in humans could now be advanced in this GH insensitive mouse. We are happy to review the results of studies using these mice on aging, adipose tissue, reproduction, metabolism, and cancer. Also, tissue-specific effects on the brain, heart, and bone are reviewed. Thus, this book is a combination of data obtained in man on the Israeli cohort and the GHR−/−mouse. In each scenario, the action of GH is attenuated resulting in low levels of IGF-I. Similarities and differences between the mouse and human data are pointed out in Chapter 59. The data are both of academic as well as of practical clinical importance.

vii

viii

Preface

Professor Laron acknowledges all the early collaborators in the clinical studies, especially Prof. Athalia Pertzelan, Prof. Rivka Kauli, Dalia Peled, RN, Dr. Beatrice Klinger and Avinoam Galatzer, MA, (deceased), and Prof. Liora Kornreich. The contributions of the collaborators in our laboratory were crucial in elucidating the pathophysiology of the disease: immunology (Sara Assa, PhD), GH (Ruth Keret, MSc), IGF-I, and GHBP (Aviva Silbergeld, MSc), GH receptor (Rina Eshet, PhD) as well as our more recent collaborators in genetics and cancer (Orit Shevah, MSc) and (Pearl Lilos, MA, statistician). William H. Daughaday provided invaluable help in the early IGF-I (somatomedin-A) measurements and in the diagnosis of LS. John S. Parks collaborated in the early genetic evaluation. Thanks also to our coauthors of the present text and Mrs. Gila Waichman and Mrs. Rachel Ronen (Endocrinology and Diabetes Research Unit) for their technical assistance in the preparation of the clinical manuscripts and Ms. Irit Lis, Ms. Shlomit Offman, and Mr. Howard Martel from the Medical Photography and Graphic Department, Rabin Medical Center, for their tremendous help during many years. We also wish to acknowledge the generous supply of IGF-I from Fujisawa Pharmaceuticals, Osaka, Japan. Professor Kopchick would like to cite his many colleagues over the years who helped with the work in the GH area. In particular, he is extremely proud of a young graduate student, Yihua Zhou, who first generated the mouse. Yihua went on to receive his PhD in Professor Kopchick’s laboratory and MD degree from Washington University in St. Louis. Additionally, Professor Kopchick would like to recognize his many students (both graduate and undergraduate), technicians, postdoctoral fellows, faculty colleagues, and visiting scientists who helped with the work. Also, through scientific collaborations involving this mouse, the Ohio University group has been able to make many international friends and colleagues, most of whom are cited in the following chapters. If for some reason we “missed” a publication, we are very sorry. Finally, Professor Kopchick would like to acknowledge the many funding sources that helped advance our studies including the Ohio Eminent Scholar Program (that includes a gift from Milton and Lawrence Goll), which provided funding for his endowed Professorship; and The Edison Biotechnology Institute, Molecular and Cellular Biology Program, Biomedical Sciences Department in the College of Medicine, Diabetes Research Initiative, and BioMolecular Innovation and Technology Partnership at Ohio University; NIH; USDA; and several corporate sponsors including Pfizer, Merck, Sensus, and DiAthegen. Last but not least, we wish to acknowledge the many people from Springer-Verlag GmbH, Berlin/Heidelberg, who assisted in the production of the book. Petah Tikva, Israel Athens, Ohio, USA December 2009

Zvi Laron John J. Kopchick

Contents

Part I  Clinical Aspects   1 History of the Israeli Cohort of Laron Syndrome Patients (1958–2009) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Zvi Laron

3

  2 Early Investigations: Characterizations of the Circulating Growth Hormone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Zvi Laron

9

  3 Clinical Evidence of Growth Hormone Resistance in Patients with Laron Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Zvi Laron

21

  4 Diagnosis of Laron Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Zvi Laron

27

  5 Genetic Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Orit Shevah and Zvi Laron

29

5.1  genetic investigations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Orit Shevah and Zvi Laron 5.2  Hypothesis of the Origin of Laron Syndrome . . . . . . . . . . . . . . . . . . Zvi Laron and Orit Shevah 5.3  Are the Pygmies Laron Syndrome Patients? . . . . . . . . . . . . . . . . . . . Zvi Laron

29

  6 Clinical Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Zvi Laron

53

  7 Perinatal Development in Laron Syndrome . . . . . . . . . . . . . . . . . . . . . . . Zvi Laron and Rivka Kauli

59

  8 Linear Growth Pattern of Untreated Laron Syndrome Patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Zvi Laron and Rivka Kauli



47 50

63

ix

x

Contents

  9 Head Shape, Size, and Growth of Untreated Patients with Laron Syndrome . . . . . . . . . . . . . . . . . . . . . . . . Zvi Laron and Rivka Kauli

91

10 Sexual Development in Patients with Laron Syndrome . . . . . . . . . . . . . 101 Zvi Laron and Rivka Kauli 11 Sex Hormone Binding Proteins and Sex Hormones in Untreated and IGF-I Treated Patients with Laron Syndrome . . . . . . . . . . . . . . . . . 119 Zvi Laron and Rivka Kauli 11.1  Sex Hormone Binding Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 Zvi Laron 11.2  Sex Hormones in Untreated Patients with Laron Syndrome . . . . . 121 Zvi Laron and Rivka Kauli 12 The Adipose Tissue in Patients with Laron Syndrome . . . . . . . . . . . . . . 125 Zvi Laron 13 Investigations to Determine the Cause of Obesity in Patients with Laron Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 Zvi Laron, Shira Ginsberg, and Nahum Vaisman 14 Nonalcoholic Fatty Liver Disease (Nafld) in Patients with Laron Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 Zvi Laron 15 Serum Lipids in Patients with Laron Syndrome . . . . . . . . . . . . . . . . . . . 149 Zvi Laron 16 Adiponectin and Leptin in Laron Syndrome . . . . . . . . . . . . . . . . . . . . . . 157 Zvi Laron and Hannah Kanety 17 Muscle Force and Endurance in Untreated Adult and IGF-I Treated Children with Laron Syndrome . . . . . . . . . . . . . . . . 161 Zvi Laron 18 Bone Mineral Density in Untreated and IGF-I or Alendronate-Treated Patients with Laron Syndrome . . . . . . . . . . . . . 165 Zvi Laron 19 Muscle–Bone Relationship in Patients with Laron Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 Yoram Epstein, Amir Hadid, Zvi Laron, Daniel S. Moran, and Nahum Vaisman 19.1 Bone Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 Yoram Epstein, Amir Hadid, Zvi Laron, and Daniel S.Moran 19.2 Lean Body Mass (LBM) in Patients with Laron Syndrome . . . . . 173 Zvi Laron and Nahum Vaisman

Contents

xi

20 Imaging Findings in Laron Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 Liora Kornreich and Zvi Laron 21 Ocular Findings in Laron Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 Dan H. Bourla and Dov Weinberger 22 Hearing in Patients with Laron Syndrome . . . . . . . . . . . . . . . . . . . . . . . . 209 Zvi Laron, Omer Zarchi, and Joseph Attias 23 The Teeth in Patients with Laron Syndrome . . . . . . . . . . . . . . . . . . . . . . 213 Zvi Laron 24 Hair, Skin, and Nails in Patients with Laron Syndrome . . . . . . . . . . . . . 219 Zvi Laron 25 Hand, Foot, and Organ Size and Growth in Untreated and IGF-I Treated Patients with Laron Syndrome . . . . . . . . . . . . . . . . . 227 Zvi Laron and Aviva Silbergeld 25.1 Hand Size and Growth of Untreated and IGF-I Treated Laron Syndrome Patients . . . . . . . . . . . . . . . . 227 Zvi Laron 25.2 Foot Size and Growth in Untreated and IGF-I-Treated Patients with Laron Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231 Zvi Laron and Aviva Silbergeld 25.3 Organ Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233 Zvi Laron 26 Lifelong Serum Growth Hormone Levels in Patients with Laron Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 Zvi Laron and Orly Efros 27 IGF-I Binding Proteins in Laron Syndrome . . . . . . . . . . . . . . . . . . . . . . 241 Zvi Laron 28 Serum Prolactin in Untreated and IGF-I Treated Patients with Laron Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 Zvi Laron and Orly Efros 29 Thyroid Hormones in Untreated and IGF-I Treated Patients with Laron Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . 255 Zvi Laron and Rivka Kauli 30 Insulin Secretion and Carbohydrate Metabolism in Patients with Laron Syndrome: From Hypoglycemia to Diabetes Mellitus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259 Zvi Laron 31 Liver Enzymes in Patients with Laron Syndrome . . . . . . . . . . . . . . . . . . 273 Zvi Laron and Tsvia Karmon

xii

Contents

32 The Hematopoietic System in Patients with Laron Syndrome . . . . . . . . 283 Zvi Laron 33 Cardiovascular Aspects in Laron Syndrome Patients . . . . . . . . . . . . . . . 293 Mickey Scheinowitz, Micha S. Feinberg, Michael Shechter, Zvi Laron, and Rivka Kauli 33.1 Heart Functions in Untreated and IGF-I-Treated Patients with Laron Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mickey Scheinowitz, Micha S. Feinberg, and Zvi Laron 33.2 Endothelial Function in Adults with Laron Syndrome (Primary Growth Hormone Insensitivity) . . . . . . . . . . . . . . . . . . Michael Shechter and Zvi Laron 33.3 Intimal Thickness of the Extracranial Arteries in Untreated Adult Patients with Laron Syndrome . . . . . . . . . . . . . Zvi Laron 33.4 Cardiovascular Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Zvi Laron and Rivka Kauli

293

298

300 303

34 Kidney Functions in Untreated and IGF-I Treated Patients with Laron Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . 307 Zvi Laron 35 Sleep and Sleep Disorders in Patients with Laron Syndrome . . . . . . . . 317 Zvi Laron, Rivka Kauli, and Eyal Rosenzweig 35.1 Sleep Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317 Zvi Laron and Rivka Kauli 35.2 Severe Obstructive Sleep Apnea (OSA) . . . . . . . . . . . . . . . . . . . . . 318 Zvi Laron 35.3 Additional Polysomnographic Examinations of Patients with Laron Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . 318 Eyal Rosenzweig and Zvi Laron 36 Neurological Aspects in Laron Syndrome . . . . . . . . . . . . . . . . . . . . . . . . 321 Zvi Laron 37 Orthopedic Problems in Laron Syndrome . . . . . . . . . . . . . . . . . . . . . . . . 323 Zvi Laron and Rivka Kauli 38 Psychological Aspects in Patients with Laron Syndrome . . . . . . . . . . . . 325 Zvi Laron 39 Adjustment and Rehabilitation Problems of Children, Adolescents, and Adults with Laron Syndrome . . . . . . . . . . . 335 Zvi Laron 40 Laron Syndrome Patients with Congenital IGF-I Deficiency Seem Protected from Malignant Diseases . . . . . . . . . . . . . . . 339 Zvi Laron, Rachel Steuerman, and Orit Shevah

Contents

xiii

41 Lifespan and Mortality of Patients with Laron Syndrome . . . . . . . . . . . 341 Zvi Laron 42 IGF-I Treatment of Patients with Laron Syndrome . . . . . . . . . . . . . . . . 343 Zvi Laron 43 IGF-I Stimulation of Head Growth in Patients with Laron Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381 Zvi Laron 44 Effects of Long-Term Administration of IGF-I on the Adipose Tissue and Carbohydrate Metabolism in Children with Laron Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389 Zvi Laron 45 Comparison of the Growth Promoting Response of IGF-I in Children with Laron Syndrome with that of hGH in Children with Isolated GH Deficiency . . . . . . . . . . . . . . 409 Zvi Laron 46 IGF-I Treatment of Adult Patients with Laron Syndrome . . . . . . . . . . . 413 Zvi Laron 47 Adverse Effects Encountered During IGF-I Treatment of Patients with Laron Syndrome . . . . . . . . . . . . . . . . . . . . . . 419 Zvi Laron 48 Summary of the Clinical History: The Laron Syndrome Clock . . . . . . . 425 Zvi Laron Part II The Growth Hormone Receptor “Null” Mouse or the Laron Mouse 49 The Laron Mouse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429 John J. Kopchick and John D. Blischak 50 Role of GH/IGF-I Deficiency in Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . 433 Edward O. List 51 Body Composition, Adipose Tissue, and Energy Balance . . . . . . . . . . . . 441 Darlene E. Berryman 52 Metabolism and Metabolic Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . 451 Lucila Sackmann-Sala, D. R. Bailey Miles, and John J. Kopchick 53 Skeletal Muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465 Juan Ding and John J. Kopchick

xiv

54 Cardiac Function in GHR−/− Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473 Diana Cruz-Topete and John J. Kopchick 55 Bone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 481 Shigeru Okada and Jacob Wright-Piekarski 56 GHR Knockout and the CNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489 Elahu S. Gosney and John J. Kopchick 57 Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495 Brian D. Bower and John J. Kopchick 58 Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 507 Bruce Kelder 59 Conclusions and Future Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513 John J. Kopchick, Hiroyoshi Tanda, Darlene Berryman, and Zvi Laron Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 521 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 525

Contents

Part Clinical Aspects

I

1

History of the Israeli Cohort of Laron Syndrome Patients (1958–2009) Zvi Laron

Core Message

›› A concise history of the discovery of the Laron

syndrome and the technological milestones enabling the elucidation of its etiopathology.

1.1 Introduction

Progressively more and more patients were referred to us comprising also of Moslem and Christian origin. It lasted 20 years from the first referrals until we were able to show that the pathogenicity of this disease is due to a lack of IGF-I (somatomedin A) generation (Daughaday et al. 1969; Laron et al. 1971) shown subsequently to be caused by a failure in the GH receptors in the liver (Eshet et  al. 1984). Further 5 years of advances in laboratory technology were needed to permit the identification of the molecular defects in the

In 1958, at a time when no radioimmunoassay for human growth hormone (hGH) was as yet available, three siblings (2 males, 1 female) from a consanguineous Yemenite Jewish family (the grandparents were first cousins) were referred to our newly established ­pediatric endocrine clinic. Their ages were 3.5, 1.5 years (Patients S.R. and SSi), and a newborn baby (SSh) (Fig. 1.1, Pedigree, see Chap. 5). They had five older siblings of normal stature (Laron et al. 1996). The clinical history, appearance, and laboratory findings resembled those found in children with isolated growth hormone deficiency (IGHD) (Laron 1983) (Fig.  1.2). When radioimmunoassays for GH became available (Glick et  al. 1963; Laron and Mannheimer 1966), we were astonished to find that their serum GH levels were very high. A short while thereafter, we were able to assemble 22 patients, all products of consanguineous Jewish families originating from mid-Eastern countries or North Africa (Laron et al. 1968) (Fig. 1.3). The striking finding was their typical appearance and the great resemblance between patients (Fig. 1.4).

Z. Laron  Schneider Children’s Medical Center of Israel, Endocrine Research Unit, Kaplan Street 14, 49202 Petah Tikva, Israel e-mail: [email protected]

Fig. 1.1  Two of the 3 first patients with Laron syndrome (LS) referred to us in 1958. The girl (SR), 1.5 years old; and the boy (SSi), 3.5 years old. Note obesity, sparse hair, and saddle nose. Reproduced with permission from Laron (2004)

Z. Laron and J. Kopchick (eds.), Laron Syndrome - From Man to Mouse, DOI: 10.1007/978-3-642-11183-9_1, © Springer-Verlag Berlin Heidelberg 2011

3

4

Fig.  1.2  A 4-year-old boy with isolated congenital GH deficiency (IGHD) due to GH gene deletion. Note resemblance with boy in Fig.  1.1: dwarfism, protruding forehead, obesity, and small penis

Fig. 1.3  A group of LS patients (front row) with two fathers (extreme left and extreme right – second row) with four members of the medical team. The father on the right is himself a patient (MeS)

Z. Laron

Fig. 1.4  Typical appearance of a 4-year-old boy with LS. Note obesity, protruding forehead, and saddle nose (YG)

5

1  History of the Israeli Cohort of Laron Syndrome Patients (1958–2009)

hGH receptor gene (Amselem et al. 1989; Godowski et al. 1989). About the same time, IGF-I (somatomedin A) was synthesized (Niwa et al. 1986), and we were the first to administer IGF-I to children and initiate clinical trials in children with Laron syndrome (LS) with the new hormone (Laron et al. 1988; Laron et al. 1990; Laron et al. 1991; Laron et al. 1992). Over the years, further patients with this syndrome were referred to us. In 2009, our cohort consisted of 64 LS patients (33 males and 31 females) (Table 1.1 and Fig. 1.5), of these 60 living. This forthwith called the “Israeli cohort” consists of patients residing in Israel at time of referral, of patients from other countries examined and followed in our clinic, and of patients seen or consulted by Z.L. and followed in collaboration with local physicians. Ten (possibly 11) patients are married, and 9 married couples have 20 children. The countries of origin of the patients are shown in Table 1.2.

Table 1.1  The Israeli cohort of 64 patients with Laron syndrome (2009) Males Females Total numbera

33

31

Age over 30 years

12

17

Married

5

5

Have children

9

11

After our publications, patients with LS were diagnosed in many parts of the world, the majority of Medi­ terranean, Mid-Eastern or South Asian origin, or descendants from these geographical regions (Rosenfeld et al. 1994; Savage et al. 2006). Few patients have been diagnosed also in Japan (Iida et  al. 1998) and China (Chen et al. 2003). The estimated number of LS patients in the world ranges between 300 and 500.

Table 1.2  Countries of origin at referral of patients with Laron syndrome Female Male Total Israel

17

13

30

Palestine

1

4

5

Jordan

–

4

4

Lebanon

2

1

3

Iran

2

3

5

Malaya

–

2

2

Malta

1

–

1

Greece

1

–

1

Italy

4

–

4

Argentina

–

1

1

Ecuador

1

4

5

Peru

1

2

3

30

34

64

Including four deceased

a

The Israeli Cohort Age and Sex distribution of 64 patients with Laron Syndrome at referral for evaluation to our clinic (1958-2009) Males Female

Fig. 1.5  Age and sex at referral of 64 patients with LS to the BeilinsonSchneider Pediatric Endocrinology Clinic

4d-40

>20

>25

>25

>25

20 15 10 5 0

PATIENT No. SEX

3 4 6 7 9 12 13 14 15 16 17 18 19 21 22 23 24 25 F F M F F F M F M F M F M M F F M F

Fig. 2.6  Serum hGH response to arginine infusion in children with Laron syndrome. Reproduced with permission from Laron (1984)

concentrations of 10–68 ng/mL) was compared to that of serum hGH obtained from healthy children (stimulated serum hGH concentrations of 16–70  ng/mL), acromegalic patients (serum hGH 120–170  ng/mL), and healthy newborns (serum hGH concentrations of 16–70 ng/mL). The sera were tested with three different antisera (Eshet et  al. 1973). The anti-hGH sera were one prepared by immunization of guinea pigs in our laboratory denoted AS-1 and another received from Yalow (NIH anti-hGH-2-5-19) denoted AS-2 and the serum of a patient who developed high titer of

Fig. 2.7  Serum hGH, insulin, and glucagon response to intravenous somatostatin administration. Reproduced with permission from Laron (1984)

14

Z. Laron GROWTH HORMONE RESPONSE TO OGTT

65

50 40 30

60

22 20

55

18 16

PERCENT BOUND

Serum hGH (ng/mL)

24

14 12 10 8 6

C4 C7 NB1 N3 Hi4 Hi10 H4

50

45

4 2 0 No. 5 21 9 M M F PATIENT: SEX AGE, yrs 8 8 9

40 4 14 13 12 11 1 15 F F M F F F M 9 14 16 18 18 20 40

Fig.  2.8  Serum hGH response to an oral glucose load in 10 patients with Laron syndrome. Reproduced with permission from Laron (1984)

35

30

antibodies while on hGH therapy (kindly provided by Prader and Illig, Zurich) (AS-3). As all sera rendered similar results, we illustrate only the dilution curve with AS-1 (Fig.  2.9). Additional experiments used dilution curves of serum hGH of Laron syndrome patients in a hybrid system of RIA using 125hGH and antiserum against HCS (human chorionic somatotropin) compared to anti-hGH serum. All the experiments revealed no differences in the immunological behavior of the hGH from patients with Laron syndrome compared to that in the control sera (Eshet et al. 1974).

15 20 5 10 CONCENTRATION OF HGH, (ng/mL)

Fig. 2.9  Superimposition of dilution curves of serum hGH from Laron syndrome patients (Hi), compared with healthy children (C), newborns (NB), and acromegaly patients (A) on the standard curve using anti-hGH serum diluted 1:2 × 10–6. The hGH of the various samples tested was brought to the same concentration (Eshet et al. 1973). Reproduced with permission from Laron (1984)

2.3 Comparison Between Radioimmunoassays and Radioreceptor Assays to Measure Circulating High in Patients with Laron Syndrome

2.2.2 Conclusions The above findings were interpreted as suggestive that patients with Laron syndrome secrete hGH with a normal molecular structure. The next step to verify this assumption could be undertaken only when specific radioreceptor assays (RRA) for hGH became available.

Using a RRA for hGH (Tsushima and Friesen 1973) with a 100,000-g pellet of human liver tissue (obtained from a male donor of a kidney transplantation), serum hGH concentrations of 6 Laron syndrome patients, of healthy children, and of acromegalic patients were compared both by RIA and RRA (Eshet et al. 1985).

15

2  Early Investigations: Characterizations of the Circulating Growth Hormone

2.3.1 Methods The RRA assay employed was as follows:

2.3.1.1 Specific Binding of 125I-hGH to Human Livers in the Microsomal Fraction Specific binding of hGH was demonstrated in six human liver samples tested in 31 different binding assays. The specific binding of these six liver specimens ranged from 3.9 to 12% with a mean value of 7.2% per 6  mg/mL of microsomal fraction protein. The binding experiments were performed using the liver of a 13-year-old donor.

2.3.1.2 Radioreceptor Assay Precision. The specific binding of various amounts of 125 I-hGH to different concentrations of human liver microsomal receptors was tested. The most precise standard curve was obtained with 585  µg of microsomal fraction protein and 12.5 × 103 cpm of 125I-hGH (0.7 ng). Sensitivity. In seven separate RRA with human liver receptors using 585 µg/tube of microsomal protein and 0.7 ng of 125I-hGH, the total binding in the absence of unlabelled hormone was 17.79 ± 1.04%. Nonspecific binding, defined as the125I-hGH radioactivity bound in the presence of 5  µg/mL of unlabelled hormone, was 9.92 ± 0.57%. Specific binding, therefore, was 7.86 ± 0.82%.

Specificity. The 125I-hGH bound by human liver receptors was displaced only by hGH. Other species of GH, e.g., bovine and ovine GH as well as hPRL, ovine and bovine PRL, hPL, did not inhibit the binding of 125 I-hGH to the human liver receptors even at a concentration of 10 µg/mL. The results of the investigations are shown in Table 2.2. The hRRA/RIA ratio was better in the Laron syndrome patients than in the controls in whom the RIA values exceeded those found by the RRA. The dilution curves of the serum hGH from the Laron syndrome patients were parallel to the standard curve, denoting identical structure of the hGH at its binding sites.

2.3.1.3 Discussion Previous reports of quantitative comparison of the hGH values obtained with the RIA and RRA in acromegalic and normal subjects using rabbit liver receptors showed a systematic overestimation by RIA (Herington et al. 1974; Sneid et al. 1975). In our study with hRRA, we made the same observation (Table 2.2). Testing of the serum of Laron syndrome patients, however, showed a higher value of hGH in the RRA in four of the six patients. It is of note that Jacobs et al. (1976), who prior to this study had used a rabbit liver assay to test the serum of another seven of our Laron syndrome patients, found also an overestimation of the hGH values by RRA in two of these patients. These findings may be due to the fact that Jacobs et al. (1976) used a pregnant rabbit liver RRA which also binds lactogenic

Table  2.2  Comparison of hGH concentrations in serum as measured by RIA and human liver hRRA in patients with Laron syndrome, acromegaly, and a healthy control Subjects Laron syndrome patients

Acromegaly Normal controla Standard curve

Sample number

RIA (ng/mL)

hRRA (ng/mL)

hRRA/RIA ratio, %

Slope of regression curve

F statistics

1 2 3 4 5 6

105.0 12.0 90.0 80.0 17.5 20.0

18.0 20.0 140.0 130.0 40.0 23.0

20 170 150 150 200 100

−2.645 −4.631 −1.255 −1.453 −1.226 −3.517

0.47

12

352.0 168.0

50.0 45.0

1

58.0a

22.4

14.6 25 50 −1.765

The hGH values of each sample are expressed as the mean of three dilutionsModified from Eshet et al (1985) a The values given for the normal subject is the peak value in a clonidine stimulation test

16

hormones in contradistinction to the human liver RRA we used and which was found specific for hGH (Eshet et al. 1985). The physiological meaning of discrepant findings is not clear and needs further investigation.

2.3.1.4 Conclusions The immunological studies of hGH from Laron syndrome patients were interpreted as showing that the structure of the circulating pituitary GH of these patients is normal and that the RRA using human liver proved that their GH is biologically active.

2.4 Evidence that the Etiology of Laron Syndrome is a GH-Receptor Defect 2.4.1 Introduction Having shown that the pituitary GH secreted by Laron syndrome patients has a normal immunological behavior and binds to human GH receptors, we disproved one of the early expressed possibilities that Laron syndrome patients secrete an abnormal GH. Thus, the second possibility of a GH resistant (insensitive) state had to exist (Laron et al. 1980). To prove this assumption, we convinced the mother of one 4-year-old Laron syndrome patient and a young adult Laron syndrome patient to agree to undergo an open liver biopsy to enable the preparation of GH receptors from Laron syndrome livers (Eshet et al. 1984). Control liver tissue was obtained from kidney donors immediately after pronouncement of clinical death by traumatic causes (there were six donors, aged 13–47 years with a mean age of 24 years). The investigation was approved by the Ethical Hospital Committee.

2.4.2 Radioreceptor Assays Using Liver of Laron Syndrome Patients 2.4.2.1 Methods The preparation of microsomes bearing hGH receptors was done as described byTsushima and Friesen (1973). The liver tissue was homogenized in a 0.25  mM

Z. Laron

sucrose solution and then centrifuged at 10,000 g for 10  min; the supernatant was then centrifuged at 100,000 g for 90 min yielding the microsomal pellet. One gram of liver yielded 18 mg/mL of microsomal fraction protein. For the assay, 1.2 mg/100 µL of this liver microsomal pellet fraction was incubated in duplicate with 0.7 ng/100 µL of labeled hGH or insulin (specific activity 80  µCi/ µg and 90  µCi/ µg, respectively) in a 25 mM tris-HCl + 10 mM MgC2 buffer, pH 7.4 and 0.1% bovine serum albumin in a final volume of 0.5  mL. This mixture was incubated at 4°C with constant shaking for 48 h. Incubation was terminated by adding 2 mL ice-cold 0.1% bovine serum albumin tris/magnesium buffer. The fractions containing receptor-bound and free radioactivity were separated by centrifugation at 2,000 g for 30 min at 4°C. Radioactivity was measured in a 5260 Autogamma Scintillation Spectrometer (Packard, USA). Parallel incubations were made in the presence of excess (5 µg/mL) unlabeled hormone. Specific binding is the difference between radioactivity bound in the absence (total binding) and in the presence (nonspecific binding) of excess unlabeled hormone, (in this case hGH) and is expressed as a percentage of the total radioactivity in the incubation mixture. The receptor binding studies made with the liver tissue from the 2 Laron syndrome patients were carried out concomitantly with those of the control liver tissue of a 13-year-old boy. The microsome preparations from the liver tissue of the six healthy control subjects were studied repeatedly. 2.4.2.2 Results The liver microsomes from the Laron syndrome patients showed almost no specific binding of 125IhGH, (Table  2.3 and Fig.  2.10) whereas those from the healthy liver tissue showed specific binding ranging from 7.9 to 24% (average 14.5 ± 3.2%) (Tables 2.3 and 2.4). Liver tissue from the 13-year-old control boy assayed concomitantly with that of the 2 Laron syndrome patients showed a specific binding of 28% for hGH. The microsomes of the Laron syndrome also showed binding of 125I-insulin, which was comparable to those observed in healthy individuals. The serum levels of hGH prior to biopsy were 45 ng/mL in Patient 1 and 20  ng/mL in Patient 2; serum insulin was undetectable in Patient 1 and 48  mU/mL in Patient 2.

17

2  Early Investigations: Characterizations of the Circulating Growth Hormone

Table 2.3  Specific binding of 125I-hGH and 125I-insulin to liver microsomes from two patients with Laron syndrome and from one healthy control subject Liver Sex Age Binding of 125I-hGH (%) Binding of 125I-insulin (%) a microsomes (year) Total Nonspecific Specific Total Nonspecificb Specific Laron syndrome Patient 1

M

4

7.0

6.5

0.5

47.3

13.9

33.4

Patient 2

M

26

7.0

6.9

0.1

11.2

4.1

7.1

Healthy control subject

M

13

35.1

6.8

28.3

17.3

9.6

7.7

Liver tissue was tested per 12 mg/mL of microsomal fraction protein Reprinted with permission from Eshet et al. (1984) hGH human growth hormone; M male a With 5 mg/mL hGH b With 10 mg/mL insulin % binding of 125-I hGH 30

Insulin binding

Table  2.4  Specific binding of 125I-hGH to liver microsomes taken from six normal-kidney donors Sex Age Number of assays Specific (year) performed binding (%)

25

30

20

20

Male

13

10

24.1 ± 4.3a

15

10

Male

16

7

13.4 ± 3.0a

Male

19

2

15.6 & 10.8

Female

20

5

16.2 ± 4.5a

Male

30

2

7.3 & 8.6

Male

47

5

12.2 ± 3.2a

Control LTD1 LTD2

10 5 0

Normal range Control (n = 6)

LTD1

LTD2

Fig. 2.10  Lack of 125hGH binding to liver microsomes prepared from 2 Laron syndrome patients compared to liver microsomes from healthy controls. In contradistinction 125labeled insulin bound to the liver microsomes from Laron syndrome patients

2.4.2.3 Discussion Having shown that the liver of Laron syndrome patients is insensitive to hGH, the stimulus to generate IGF-I and the fact that the liver is the major source of IGF-I explain the lack of IGF-I secretion in this syndrome (Daughaday et al. 1969; Laron et al. 1971). This concept concurs with the findings of Golde et al. (1980) that erythroid colonies grown in  vitro from 2 Laron syndrome patients were unresponsive to exogenous hGH in contrast to colonies from control subjects and patients with primary hGH deficiency. Further evidence found by us was that the addition of normal serum to cultures of fibroblasts from Laron syndrome patients enhanced the incorporation of 3H-thymidine (Nevo et al. 1977; Laron et al. 1980) (Table 2.5), whereas the addition of serum from Laron

Liver tissue was expressed per 12  mg/mL of microsomal fraction protein hGH human growth hormone Reproduced with permission from Eshet et al (1984) a Values are means ± SD

Table  2.5  Effect of sera (10%) from patients with Laron syndrome or, isolated GH deficiency (IGHD) and normal serum on the growth, in culture, of fibroblasts derived from 2 patients with Laron syndrome aged 10 and 12 years 3 Number Protein Diagnosis H-thymidine of sera per plate (source of (cpm/ (mg ± SD) serum) plate ± SD) Laron syndrome

2

117 ± 13

927 ± 74

IGHD

7

105 ± 17

652 ± 168

Normal

Two pools

169 ± 6

1,251 ± 36

8 ± 4

38 ± 21

Without serum

All the trials were performed in triplicate. Cells were allowed to grow for 72 h before harvest. Statistical evaluation Protein per plate: LS vs. control, p