CONTEMPORARY ENDOCRINOLOGY

Series Editor:P. Michael Conn, PhDOregon Health & Science UniversityBeaverton, OR, USA

For further volumes:http://www.springer.com/series/7680

Ken HoEditor

Growth Hormone Related Diseases and Therapy

A Molecular and Physiological Perspective for the Clinician

EditorKen HoCentres for Health ResearchPrincess Alexandra HospitalThe University of QueenslandBrisbane Australiak.ho@uq.edu.au

ISBN 978-1-60761-316-9 e-ISBN 978-1-60761-317-6DOI 10.1007/978-1-60761-317-6Springer New York Dordrecht Heidelberg London

Library of Congress Control Number: 2011931862

© Springer Science+Business Media, LLC 2011All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden.The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights.While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein.

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v

Preface

The molecular era ushered in the cloning of the growth hormone (GH) gene and the production of unlimited amounts of GH through recombinant technology. The con-tinuing momentum of research from basic science to clinical evaluation has brought unprecedented advances to the understanding of GH biology for the clinical endo-crinologist. This book endeavours to distill the new information of relevance to the endocrinologist spanning the last 20 years. It contains five sections covering physi-ology, molecular genetics, GH deficiency, acromegaly and pharmacotherapy.

The first section on physiology focuses on GH action. A review of the structure and function of the GH receptor is followed by a perspective on the regulatory role of ghrelin on GH secretion. Attention is drawn to the pattern of GH secretion as an important determinant of tissue action. The metabolic actions of GH are diverse affecting fat, carbohydrate and protein homeostasis in humans.

The second section on genetics covers pituitary function and adenomas. Transcription factors in pituitary cell type development and the disease phenotypes resulting from loss of function mutations causing isolated or combined GH defi-ciency are complemented by a timely review of associated structural abnormalities identifiable by modern day imaging. This section also presents new and fascinating information on familial pituitary adenomas, their genotype and phenotype.

The section on adult GH deficiency spans the epidemiology and diagnosis of GH deficiency with a strong reminder for the clinician that the transition period repre-sents a critical time of somatic maturation, occurring years after cessation of liner growth. Long-term global experience in replacement therapy has reconfirmed the safety and efficacy of GH in restoring body composition and fitness, with scant evidence for malignancy risk.

The section on acromegaly focuses on management, giving practical guides to the value of GH and IGF-1 measurements, the place of somatostatin analogues and of radiotherapy while reminding the reader as to why evaluating the quality of life is an important part of management. Compelling evidence is provided for clinicians to strive for tight control based on epidemiological evidence that mortality is returned to that of the general population when this is achieved.

vi Preface

The section on GH pharmacology takes the reader through innovative developments of long-acting GH formulations with some products on the threshold of clinical use. While there is much abuse of GH in the community, this section provides a balanced review of the effects of GH supplementation in ageing and in sports where recent data indicate an enhancing effect on a selective aspect of performance.

This book integrates a wealth of information for the paediatric endocrinologists, adult endocrinologists, endocrine scientists and internists interested in the human biology of GH.

Brisbane, Australia Ken Ho

vii

Contents

Part I Physiology

1 Growth Hormone Receptor in Growth ................................................ 3Vivian Hwa

2 Ghrelin in the Regulation of GH Secretion and Other Pituitary Hormones ............................................................. 17Fabio Lanfranco, Matteo Baldi, Giovanna Motta, Marco Alessandro Minetto, Filippa Marotta, Valentina Gasco, and Ezio Ghigo

3 Growth Hormone Pulsatility and its Impact on Growth and Metabolism in Humans .................................................................. 33Antonio Ribeiro-Oliveira Jr. and Ariel L. Barkan

4 Metabolic Actions of Growth Hormone ............................................... 57Morton G. Burt

Part II Genetics

5 Molecular Genetics of Congenital Growth Hormone Deficiency ............................................................................... 83Christopher J. Romero, Elyse Pine-Twaddell, and Sally Radovick

6 Structural Abnormalities in Congenital Growth Hormone Deficiency ............................................................................... 103Andrea Secco, Natascia Di Iorgi, and Mohamad Maghnie

7 Genetic Causes of Familial Pituitary Adenomas ................................. 137Silvia Vandeva, Sabina Zacharieva, Adrian F. Daly, and Albert Beckers

viii Contents

Part III Growth Hormone Deficiency

8 The Epidemiology of Growth Hormone Deficiency ............................ 153Kirstine Stochholm and Jens Sandahl Christiansen

9 Diagnosis of Growth Hormone Deficiency in Adults .......................... 169Sandra Pekic and Vera Popovic

10 Transition from Puberty to Adulthood ................................................ 187Helena Gleeson

11 Issues in Long-Term Management of Adults with Growth Hormone Deficiency ........................................................ 211Anne McGowan and James Gibney

12 Quality of Life in Acromegaly and Growth Hormone Deficiency ............................................................................... 237Susan M. Webb, Eugenia Resmini, Alicia Santos, and Xavier Badia

Part IV Acromegaly

13 The Value of GH and IGF-I Measurements in the Management of Acromegaly....................................................... 253Pamela U. Freda

14 The Role of Somatostatin Analogues in Treatment of Acromegaly ......................................................................................... 271Haliza Haniff and Robert D. Murray

15 The Role of External Beam Radiation Therapy and Stereotactic Radiosurgery in Acromegaly .................................... 303Bruce E. Pollock

16 Mortality and Morbidity in Acromegaly: Impact of Disease Control ..................................................................... 317Ian M. Holdaway

17 GHR Antagonist: Efficacy and Safety .................................................. 339Claire E. Higham and Peter J. Trainer

ixContents

Part V Use of Growth Hormone

18 Long-Acting Growth Hormone Analogues .......................................... 361Alice Thorpe, Helen Freeman, Sarbendra L. Pradhananga, Ian R. Wilkinson, and Richard J.M. Ross

19 Growth Hormone Supplementation in the Elderly ............................. 375Ralf Nass and Jennifer Park

20 Growth Hormone in Sports: Is There Evidence of Benefit? .............. 389Anne E. Nelson, Ken Ho, and Vita Birzniece

Index ................................................................................................................ 405

xi

Contributors

Xavier Badia Health Economics and Outcomes Research, IMS Health, and CIBERER (Centro de Investigación Biomédica en Red en Enfermedades Raras), Barcelona, Spain

Matteo Baldi Department of Internal Medicine, Division of Endocrinology, Diabetology and Metabolism, University of Turin, Torino, Italy

Ariel L. Barkan Division of Metabolism, Endocrinology and Diabetes, Department of Neurosurgery, University of Michigan, Ann Arbor, MI, USA

Albert Beckers Department of Endocrinology, C.H.U. de Liège, University of Liège, Domaine Universitaire du Sart-Tilman, Liège, Belgium

Vita Birzniece Department of Endocrinology, Garvan Institute of Medical Research and St Vincent’s Hospital, Darlinghurst, NSW, Australia

Morton G. Burt Southern Adelaide Diabetes and Endocrine Services, Repatriation General Hospital and Flinders University, Adelaide, SA, Australia

Jens Sandahl Christiansen Department of Internal Medicine and Endocrinology, Aarhus University Hospital, Aarhus, Denmark

Adrian F. Daly Department of Endocrinology, University of Liège, Domaine Universitaire du Sart-Tilman, Liège, Belgium

Natascia Di Iorgi Department of Pediatrics, IRCCS, Giannina Gaslini – University of Genova, Genova, Italy

Helen Freeman Academic Unit of Diabetes, Endocrinology & Metabolism, Department of Human Metabolism, University of Sheffield, Royal Hallamshire Hospital, Sheffield, UK

Pamela U. Freda Department of Medicine, Columbia University, College of Physicians and Surgeons, New York, NY, USA

xii Contributors

Valentina Gasco Department of Internal Medicine, Division of Endocrinology, Diabetology and Metabolism, University of Turin, Torino, Italy

Ezio Ghigo Department of Internal Medicine, Division of Endocrinology, Diabetology and Metabolism, University of Turin, Torino, Italy

James Gibney Department of Endocrinology and Diabetes, Adelaide and Meath Hospital, Dublin, Ireland

Helena Gleeson Department of Endocrinology, Leicester Royal Infirmary, Leicester, UK

Haliza Haniff Department of Endocrinology, Leeds Teaching Hospitals NHS Trust, Leeds, UK

Claire E. Higham Department of Endocrinology, Christie Hospital, Manchester, UK

Ken Ho Centres for Health Research, Princess Alexandra Hospital and The University of Queensland, Brisbane, Australia

Ian M. Holdaway Department of Endocrinology, Greenlane Clinical Centre and Auckland Hospital, Auckland, New Zealand

Vivian Hwa Department of Pediatrics, Oregon Health & Science University, Portland, OR, USA

Fabio Lanfranco Department of Internal Medicine, Division of Endocrinology, Diabetology and Metabolism, University of Turin, Torino, Italy

Mohamad Maghnie Department of Pediatrics, IRCCS, Giannina Gaslini – University of Genova, Genova, Italy

Filippa Marotta Department of Internal Medicine, Division of Endocrinology, Diabetology and Metabolism, University of Turin, Torino, Italy

Anne McGowan Department of Endocrinology and Diabetes, Adelaide and Meath Hospital, Dublin, Ireland

Marco Alessandro Minetto Department of Internal Medicine, Division of Endocrinology, Diabetology and Metabolism, University of Turin, Torino, Italy

Giovanna Motta Department of Internal Medicine, Division of Endocrinology, Diabetology and Metabolism, University of Turin, Torino, Italy

Robert D. Murray Department of Endocrinology, Leeds Teaching Hospitals NHS Trust, Leeds, UK

Ralf Nass Division of Endocrinology and Metabolism, University of Virginia, Charlottesville, VA, USA

xiiiContributors

Anne E. Nelson Pituitary Research Unit, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia

Jennifer Park Division of Diabetes, Endocrinology and Metabolism, University of California, San Francisco, CA, USA

Sandra Pekic Neuroendocrine Unit, Institute of Endocrinology, University Clinical Center, Belgrade, Serbia

Elyse Pine-Twaddell Department of Pediatrics, Division of Endocrinology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

Bruce E. Pollock Department of Neurological Surgery, Mayo Clinic College of Medicine, Rochester, MN, USA

Department of Radiation Oncology, Mayo Clinic College of Medicine, Rochester, MN, USA

Vera Popovic Neuroendocrine Unit, Institute of Endocrinology, University Clinical Center, Belgrade, Serbia

Sarbendra L. Pradhananga Academic Unit of Diabetes, Endocrinology & Metabolism, Department of Human Metabolism, University of Sheffield, Sheffield, UK

Sally Radovick Department of Pediatrics, Division of Endocrinology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

Eugenia Resmini Department of Endocrinology Medicine, Hospital Sant Pau, Universitat Autònoma de Barcelona and CIBERER (Centro de Investigación Biomédica en Red en Enfermedades Raras), Barcelona, Spain

Antonio Ribeiro-Oliveira Jr. Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil

Christopher J. Romero Department of Pediatrics, Division of Endocrinology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

Richard J.M. Ross Academic Unit of Diabetes, Endocrinology & Metabolism, Department of Human Metabolism, University of Sheffield, Royal Hallamshire Hospital, Sheffield, UK

Alicia Santos Department of Endocrinology Medicine, Hospital Sant Pau, Universitat Autònoma de Barcelona and CIBERER (Centro de Investigación Biomédica en Red en Enfermedades Raras), Barcelona, Spain

Andrea Secco Department of Pediatrics, IRCCS, Giannina Gaslini – University of Genova, Genova, Italy

Kirstine Stochholm Department of Internal Medicine and Endocrinology, Aarhus University Hospital, Aarhus, Denmark

xiv Contributors

Alice Thorpe Academic Unit of Diabetes, Endocrinology & Metabolism, Department of Human Metabolism, University of Sheffield, Royal Hallamshire Hospital, Sheffield, UK

Peter J. Trainer Department of Endocrinology, Christie Hospital, Manchester, UK

Silvia Vandeva Department of Endocrinology, C.H.U. de Liège, University of Liège, Domaine Universitaire du Sart-Tilman, Liège, Belgium

Clinical Center of Endocrinology and Gerontology, Medical University Sofia, Sofia, Bulgaria

Susan M. Webb Department of Endocrinology Medicine, Hospital Sant Pau, Universitat Autònoma de Barcelona and CIBERER (Centro de Investigación Biomédica en Red en Enfermedades Raras), Barcelona, Spain

Ian R. Wilkinson Academic Unit of Diabetes, Endocrinology & Metabolism, Department of Human Metabolism, University of Sheffield, Royal Hallamshire Hospital, Sheffield, UK

Sabina Zacharieva Clinical Center of Endocrinology and Gerontology, Medical University Sofia, Sofia, Bulgaria

Part IPhysiology

3K. Ho (ed.), Growth Hormone Related Diseases and Therapy: A Molecular and Physiological Perspective for the Clinician, Contemporary Endocrinology, DOI 10.1007/978-1-60761-317-6_1, © Springer Science+Business Media, LLC 2011

Abstract It has been approximately 20 years since the cloning and characteriza-tion of the human growth hormone (GH) receptor, GHR, gene. Cell-surface GHR binds circulating GH, which promotes postnatal growth by regulating the expres-sion of insulin-like growth factor (IGF)-I. Mutations in the GHR gene cause GH insensitivity (GHI) syndrome, also known as Laron syndrome, a syndrome charac-terized by severe postnatal growth retardation and low serum IGF-I concentrations in the presence of normal or elevated GH levels. Over 70 GHR mutations have been reported, with majority of the mutations found in exons encoding for the extracel-lular domain of the GHR. Inheritance of GHR mutations is predominantly auto-somal recessive. Evaluating the impact of identified mutations on GHR structure and function is important to understand the pathophysiology of the disease. Therapeutic options for patients carrying mutations in the GHR gene have recently expanded to include recombinant IGF-I therapy.

Keywords  Growth  hormone  insensitivity  •  Growth  hormone  receptor  •  IGF-I deficiency

Introduction

The growth-promoting effects of growth hormone (GH) are mediated primarily through regulating expression of insulin-like growth factor (IGF)-I, both circulating and peripheral, as demonstrated in rodent models and in case studies in humans. The critical importance of IGF-I for growth is highlighted by Igf1−/− null mice who

V. Hwa (*)Department of Pediatrics, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239-3098, USAe-mail: hwav@ohsu.edu

Chapter 1Growth Hormone Receptor in Growth

Vivian Hwa

4 V. Hwa

are severely growth retarded with most dying soon after birth from the consequences of muscular hypoplasia [1, 2]. In humans, homozygous IGF1 mutations are extremely rare, with only three convincing cases reported [3–5]. The observed intra-uterine growth retardation (IUGR) and severe postnatal growth failure (height SDS, HtSDS, below −4.9) in each case supported the importance of IGF-I for growth both in utero as well as postnatal. Microcephaly and mental retardation associated with the IGF1 mutation, furthermore, suggested IGF-I is critical for brain development, and sensorineural deafness was reported for two of the three cases [3, 4].

While it remains unclear how intrauterine IGF-I production is regulated, although both nutrition and insulin appear to play some role [6], postnatal production of cir-culating IGF-I is most dependent on GH. The clinical syndrome of GHI associated with an IGF-I deficiency (IGFD) and accompanied by severe growth retardation [7], in particular, has focused much attention on the pivotal role of the GH receptor (GHR) in this process.

Growth Hormone Insensitivity Syndrome

The clinical conditions of GHI and congenital GH deficiency (GHD) are characterized by minimal growth retardation in utero, profound postnatal growth retardation, infantile facial appearance, and markedly reduced serum concentrations of IGF-I. GHI is distinguished from GHD by demonstrated resistance to endogenous GH and exogenous (recombinant) GH in terms of growth, metabolic changes, or significant elevation of serum IGF-I [8, 9].

The condition of GHI was first reported in 1966 by Laron et al. [10], who described three siblings with clinical features of GHD (frontal bossing, hypoplasia of the midfacies and the nasal bridge, sparse hair, high-pitched voices, and blue scleria), but who had abnormally high levels of GH. The lack of response to GH was subsequently shown to be due to an absence of appropriate functional receptors for GH [11]. The molecular basis for this condition came with the eventual identifica-tion and cloning of the growth hormone receptor (GHR) gene [12, 13]. The patients in the reported study, who presented with severe growth failure (height standard deviations, Ht SDS, of −7.3 and −4.2), were shown to carry partial deletions in the GHR gene [13]. Since these first reports, the more than 70 GHR mutations identified in over 250 reported cases of GHI indicate a broader spectrum of phenotypic and biochemical abnormalities associated with GHI [14, 15].

The GH-IGF-I Axis in Postnatal Growth

The activation of the GH-IGF-I axis is initiated upon the interaction of pituitary-derived, circulating, GH with cell-surface GHR, a homodimeric transmembrane protein that belongs to the Type I class of the cytokine receptor superfamily. Like other members of the Type I family, which includes the prolactin receptor, the erythropoi-etin receptor, and a number of interleukin receptors, the GHR lacks the intrinsic

51 Growth Hormone Receptor in Growth

kinase activity necessary to initiate signal transduction. Instead, the GHR associates preferentially with cytosolic Janus kinase 2 (JAK2), and upon binding of one mol-ecule of GH to the dimeric GHR, conformational changes in the GHR induce the activation of JAK2 by auto-transphosphorylation [16]. The activated JAK2 subse-quently phosphorylates multiple tyrosines located on the intracellular domain of the GHR, which can serve as docking sites for cytosolic components of at least three distinct signaling pathways: the STAT (signal transducer and activator of transcription), the MAPK (mitogen-activated protein kinase), and the PI3K (phosphoinositide-3 kinase) pathways (Fig. 1.1). The signaling cascades culminate in the regulation of

Fig. 1.1 Schematic of the growth hormone (GH) – insulin-like growth factor (IGF)-I axis. The association of GH with the homodimeric GH receptor (GHR) complex induces JAK2 recruitment, transphosphorylation of JAK2, and subsequent activation of the MAPK/ERK, PI3K/AKT, and STAT5b pathways. JAK2 phosphorylates (P) intracellular GHR tyrosines (the seven tyrosines, Y, are depicted by purple lines), which leads to the recruitment and docking of STAT5b to the GHR phos-phorylated tyrosines. The recruited STAT5b is phosphorylated by JAK2, homodimerizes, and trans-locates to the nucleus where it binds to DNA and transcriptionally activates genes encoding for proteins such as IGF-I, IGF-binding protein (IGFBP)-3, and the acid labile subunits (ALS). IGF-I, IGFBP-3 and ALS are secreted and circulate in serum as a 150 kDa ternary complex, and exert endo-crine effects on growth. Solid purple arrows, signaling pathways activated by GHR-JAK2; solid green arrows, tyrosyl phosphorylation by JAK2; dashed blue arrows, translocation of indicated protein molecules. AKT AKT8 virus oncogene cellular homolog; ALS acid labile subunit; ERK extracellular signal-related kinase; GH growth hormone; IGF-I insulin-like growth factor-I; IGFBP-3 IGF-binding protein-3; JAK Janus-family tyrosine kinase; MAPK mitogen-activated protein kinase; PI3K phosphoinositide 3 kinase; STAT5b signal transducer and activator of transcription 5b

6 V. Hwa

multiple genes, including genes encoding for IGF-I, IGF-binding protein (IGFBP)-3, and acid labile subunit (ALS). Also upregulated are negative regulators, such as the SOCS family of proteins [17], which act in a feedback loop to dampen GH-GHR sig-nal transduction, thereby modulating IGF-I production.

The GH-induced, circulating IGF-I, IGFBP-3, and ALS are predominantly liver-derived and are found in the circulation as a ternary complex of 150 kDa. Reported aberrancies in the GHR [14] and the STAT5b genes [14, 18] significantly reduce the concentrations of serum IGF-I, IGFBP-3, and ALS, while defects in the IGF1 gene resulted in a deficiency in IGF-I only [3–5]. Mutations in the IGFALS gene, encod-ing for the ALS protein, were recently identified in a subset of clinical GHI cases with mild short stature [19]. The abnormally low serum IGF-I and IGFBP-3 levels associated with homozygous and compound heterozygous IGFALS mutations are incongruous with the mild short stature phenotype but are consistent with the criti-cal role of ALS in prolonging the half-life of both IGF-I and IGFBP-3. The mild effect(s) of ALS defects on growth has been attributed to a rapid clearance of IGF-I from the circulation [19]. Defects in IGFBP-3 have yet to be identified, suggesting that the other members of the IGFBP family may compensate for lack of a func-tional IGFBP-3, as has been demonstrated in rodent models for growth [20]. Indeed, circulating IGFBP-5 is also known to form a complex with IGF-I and ALS [21].

When serum GH levels are normal or elevated, but serum IGF-I, IGFBP-3, and ALS concentrations are below normal, and remain abnormally low upon GH therapy or in an IGF-I generation test (5–7 days of daily injections of recombinant GH, [22]), GHI is indicated and an aberrancy in the GH-IGF axis suggested. Measurements of serum levels of GH-binding protein (GHBP), which is the circulating extracel-lular domain of GHR (see below), furthermore, can indicate whether the defect is in the GHR gene.

The GHR Gene: Organization and Expression

The human GHR protein is encoded by a single GHR gene that spans 297.9 kilobases (kb), located on chromosome 5p13-p12. The gene consists of ten exons, from which a 4.4 kb mRNA is transcribed. Exons 2–10 encode for a prepeptide of 638 amino acid residues with the first 18 amino acids, the signal peptide, proteolytically removed upon the insertion of the receptor into the plasma membrane (Fig. 1.2). The mature GHR protein, 620 residues in length, is comprised of three domains: an extracellular, GH-binding domain encoded by exons 2–7 (246 amino acid residues), a short trans-membrane domain encoded by exon 8 (24 residues), and the intracellular portion of the GHR encoded by exons 9 and exon 10 (350 residues). Exon 10 also carries 2.4 kb of the 3¢ untranslated region (3¢UTR), a region that is usually necessary for stabilizing mRNA expression and can, therefore, potentially harbor detrimental mutations [23]. Posttranslational modification of the mature GHR produces a mono-mer of approximately 125 kDa in molecular weight, and the final GHR product translocates to the cell surface as a preformed homo-dimer [24].

71 Growth Hormone Receptor in Growth

The expression of full-length GHR (GHRfl) mRNA has been found to be widely distributed in human tissues with highest expression in the liver, fat, muscle, kidney, and heart [25]. Two GHR mRNA variants that utilize alternative splice sites located in exon 9 have also been detected in most tissues tested [26, 27]. Expression, how-ever, appeared to be considerably reduced compared to that of GHRfl mRNA [25]. Reconstitution studies have shown that the predicted peptides generated from these smaller mRNA isoforms could have serious biological consequences as the resultant truncated GHR 1–277 and 1–279 peptides lack the majority of the intracellular domain and are capable of exerting dominant-negative effects on GH signaling when coexpressed with GHRfl [25, 27]. Indeed, unique GHR splice site mutations that spliced out the entire exon 9 have been identified in heterozygous state and proved to be dominant-negative in two nonrelated, severely short-statured patients who were GHI and IGFD [28, 29].

GHR mutations associated with GHI have been identified in all coding exons (see below), except within exon 8 and encompass the normal spectrum of genetic variations: nonsense (single base change that alters an amino acid residue to a stop codon), missense (single base change that results in a substitution of amino acid residue for the normal residue), and nucleotide(s) insertions and deletions. Intronic polymorphisms are more common, but only those variants that directly impact

Fig. 1.2 Schematic of GHR primary structure. The exons encoding for the GHR domains are indicated. Box 1 and 2 in the intracellular domain are indicated as filled boxes. Mutations/variants identified within each domain are summarized [14, 15] and include more recently identified muta-tions (see text). The general effects of mutations on serum GHBP, IGF-I, IGFBP-3, and ALS concentrations are as indicated

8 V. Hwa

splicing events have been characterized [14]. The deletion of exon 3 (d3), reported in approximately 50% of the population, has turned out to be a common polymor-phism whose biological impact has remained controversial (see below).

It should be clarified at this time that reference to the position of the amino acid residues in the primary structure of the GHR protein differs between the established GHR literature and current, on-line, genetic and protein databases. The numbering of amino acid residues traditionally did not include the 18 residues of the GHR signal peptide, with only residues in the final, processed, mature GHR protein counted. However, the numeration now standardized in all databases includes the signal peptide and all amino acids that are translated from the mRNA. In this chapter, the amino acids’ positions indicated will be as they were published (i.e., mature peptide); where appropriate, the GHR prepeptide position will be indicated.

The Extracellular Domain of GHR Is Also the GH-Binding Protein

The extracellular domain of the dimeric, cell surface, GHR has been crystallized [30] and is considered to contain two functional subdomains of which subdomain 1, which comprised of the first 123 residues of the mature protein (exons 2–5), is involved in GH–GHR interaction, and subdomain 2, consisting of 6 beta-sheet regions that encompass residues 128–246 (exons 6–7), is involved in receptor dimerization and GH-induced rotation [16, 31]. Proteolytic cleavage at residues 242–244 releases the extracellular domain to circulate in plasma as GHBP, where it is believed to bind about 50% of circulating GH, thereby prolonging the half-life of GH, as well as modulating its bioactivity [32, 33]. Mutations identified in the extracellular domain can result in loss of detectable serum GHBP or render the GHR dysfunctional.

To date, almost all reported mutations are recessively inherited and are identified either in homozygous (the majority) or compound heterozygous forms. Nonsense and missense mutations predominate, with splicing and deletions (gross and small deletions) found with less frequency [14, 15].

GHBP-Negative, Classical GHI

The preponderance of mutations identified in GHI cases, including the first described cases, had abnormally low levels of circulating GHBP. These patients have been referred to as having classical GHI or Laron syndrome and present features typical of the first described cases [10].

The largest cohort of GHI patients who were GHBP-negative, IGFD, and presented with typical Laron features was identified in an inbred population in Ecuador [34]. All the affected individuals were homozygous for a sense mutation in exon 6 of the GHR gene that created a cryptic splice site [35]. The single nucleotide change did not alter the amino acid involved (E180), but induced the in-frame deletion of eight

91 Growth Hormone Receptor in Growth

residues (181–189). The resultant mutant GHR protein was originally predicted to be unstably expressed [36]. More recent evidence, based on reconstitution studies, suggested that, on the contrary, the aberrant GHR(E180sp) was stably expressed and had retained the ability to homodimerize independently of ligand binding [37], a process that occurs in the endoplasmic reticulum [16, 24]. The mutant protein, however, could not localize appropriately to the cell membrane [37].

The highly inbred Ecuador cohort ultimately numbered approximately 100 indi-viduals, and despite genetic homogeneity, displayed variable severity of GHI with final heights ranging from −12 to −5.3 SDS. Obesity was uniform in this group, typically 50% body fat, but diabetes mellitus was not observed [8]. The same muta-tion has since been identified in an Israeli patient of Moroccan origin [38], in 8 Brazilians [39], and in a Chilean family [40].

Mutations in the GHR Extracellular Domain That Are GHBP-Positive

Only a handful of defects in the extracellular domain that did not affect circulating GHBP concentrations but disrupted GHR functions have been described. These included a homozygous D152H missense mutation in exon 6 [41], a homozygous missense change in intron 6 that introduced a cryptic splice site and resulted in an insertion of a pseudoexon 6 [42], and C94S/H150Q compound heterozygous mutations identified in exon 5 and 6, respectively [43]. The D152H defect disrupted the structure of the extracellular domain sufficiently to inhibit the ability of the extracellular domain to dimerize [41]. The clinical features of the siblings who car-ried the C94S/H150Q compound heterozygous mutation were somewhat milder and atypical than classical Laron syndrome [43]: the elder child had normal hair, mild midfacial hypoplasia, a depressed nasal bridge, moderate frontal bossing, and no high-pitched voice; the younger child displayed a lack of Laron features. The small cohort of unrelated subjects (with overlapping ethnic background) who carried the homozygous insertion of a pseudoexon 6 (36 amino acid sequence inserted after residue 189) also displayed varying degrees of GHI, IGFD, and typical Laron features [44]. Interestingly, although the pseudoexon 6 and E180sp are both splicing muta-tions that cause alterations in the same region of the GHR protein, the deletion of eight amino acids (E180sp) resulted in more consistent clinical GHI features than the insertion of 36 residues [37].

Rare cases of partial GHI have been described in which serum GHBP concentra-tions have been abnormally high, up to 100-fold greater than normal [28, 45–50]. The unusual biochemistry has been attributed to splicing defects identified in intron 7 and intron 8 of the GHR gene (see below). The predicted end result of these mutations was the stable expression of mutant GHR variants, which could not anchor to the cell surface due to the splicing out of exon 8 [49]. The consequence of the massive serum concentrations of GHBP was hypothesized to compete with membrane-attached GHRs for physiologically secreted GH and led to the observed partial GHI.

10 V. Hwa

The genetic-phenotype correlations that are perhaps the most difficult to reconcile are reports where the mutations identified in the GHR extracellular domain are heterozygous and are not obviously dominant-negative, yet appears to correlate to idiopathic short stature (ISS) and some degree of GHI [51]. For example, the heterozy-gous Arg211His, R211H (or R229H of the GHR prepeptide) was believed to be the etiology for the severe growth retardation in a subject who had a height SDS of −5.1 [51]. Regeneration of the R211H variant in the extracellular portion of the GHR only [51] demonstrated poor expression and the hypotheses proposed was that R211H functioned as a dominant-negative or that the second allele was poorly expressed, therefore accounting for the short stature and undetectable serum GHBP levels in the subject [51]. An in silico program that predicts possible impact of an amino acid substitution on protein structure and function of a human protein (Polymorphism Phenotyping: http://genetics.bwh.harvard.edu/pph2/) suggests that the R229H (prepeptide) substitution was probably damaging. However, in the most recent SNP database (http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?locusId=2690), heterozygous R211H (prepeptide designation, R229H, in the database) was found with a frequency of 0.023, suggesting this is a polymorphism that is more common than should be associated with severe growth retardation. Indeed, we have identified the same heterozygous variant in family members who are of normal stature as well as in respective probands with growth retardation (unpublished; [52]). The conun-drum of heterozygous GHR variants on growth phenotype awaits better under-standing of the GHR structure and function. The possibility of a coexisting defect(s) should also be considered.

Mutations in the Transmembrane and GHR Intracellular Domain

Unlike the extracellular domain, no crystal structures of the GHR transmembrane or intracellular domains are available, although several regions critical to GHR functions have been characterized. Two regions, a proline-rich motif-designated Box 1 (resi-dues 279–287, ILPPVPVPK, encoded by exon 9) and a region designated Box 2 (residues 325–338), are critical for interacting with JAK2, and a 10-amino acid motif (residues 322–331, DSWVEFIELD, exon 10), which overlaps with Box 2, has been demonstrated in vitro to be necessary for ubiquitin-dependent endocytosis of GH-bound GHR [53]. The human GHR also contains seven tyrosines which are believed to be phosphorylated by JAK2 upon ligand binding.

Mutations have not been identified within the short transmembrane domain of GHR, encoded by exon 8, although three splicing mutations (two homozygous and one heterozygous) which effectively excise exon 8 have been described [46, 47, 49]. The transcriptional fusion of exon 7 to exon 9 upon excision of exon 8 resulted in a frameshift and premature termination of protein expression. These nonfunctional GHR variants were unable to anchor to the cell surface, and as a consequence, serum GHBP levels were dramatically elevated by up to 110-fold (see above). The two subjects carrying homozygous splicing mutations had features typical of Laron

111 Growth Hormone Receptor in Growth

syndrome, with height SDS below −5 and low serum levels of IGF-I and IGFBP-3 consistent with a nonfunctioning GHR [46, 47]. Subjects who were heterozygous for the splicing mutations also had supranormal levels of GHBP, but serum IGF-I and IGFBP-3 concentrations were normal, and subjects were either modestly short [49] or had heights within low-normal range [47, 49]. Altogether, these observations suggested that the presence of one wild-type GHR allele was sufficient to permit some normality in GH-induced actions.

Intracellular Domain Mutations

Surprisingly, few GHR mutations identified in GHI patients are located in the intra-cellular domain. Five of these mutations involved deletions – a gross chromosomal deletion in one allele that included loss of the GHR exons 4–10 [54] or small GHR deletions [55–58]. Two splice site mutations [28, 29] leading to the excision of exon 9 have also been described, and recently, a heterozygous single nucleotide duplication in Box 1 (c.899dupC) was identified in a young boy, age 2.8 years, with a height SDS of −4.07 [52]. With the exception of the gross deletion [54], the remaining seven reported mutations induced frameshifts with subsequent premature protein termination. The truncated GHR variants were expressed, as serum GHBP levels were relatively normal in the respective patients. The two splice site mutations, IVS8-1G > C and IVS9 + 1G > A, generated truncated GHRs that exerted dominant-negative effects on normal GHR [28, 29]. Phenotypically, the majority of the subjects resembled those with Laron syndrome.

It is of note that neither homozygous nonsense nor homozygous missense muta-tions in the GHR intracellular domain have been identified to date. Only three heterozygous missense variants are reported to be associated with short stature and an IGF deficiency: C422F [59], A478T [60], and P561T [59, 61]. However, evalua-tion of C422F (prepeptide, C440F) in reconstitution systems [62] and P561T (pre-peptide, P579T) in a population study [63] indicated that neither variant appeared to be responsible for short stature, and each variant, in fact, has been found with a heterozygous frequency of 0.098 and 0.093, respectively, according to the SNP database (http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?locusId=2690). Further, these two SNPs appear to be found only in those of Asian decent, with recent analysis suggesting that the polymorphisms were correlated to mandibular height [64, 65]. Polymorphisms associated with codon A478 (prepeptide, A496), to date, have not been reported. It remains unclear whether an A478T change has bio-logical significance, although the in silico Polymorphism Phenotyping program (http://genetics.bwh.harvard.edu/pph2/) predicted that an A496T (prepeptide) sub-stitution would likely to be damaging to GHR structure/function.

The activation of the GHR tyrosines on the intracellular domain is crucial to the recruitment and activation of the STAT5b signaling pathway, a pathway that is responsible for regulating IGF-I production. The handful of mutations identified in the intracellular domain of GHR that are clearly implicated in IGFD and GHI (see above) involve frameshifts due to deletions, duplications, or splicing mutations, all

12 V. Hwa

of which resulted in either premature protein truncations that abrogated most of the tyrosines, or destabilizes the entire GHR protein structure. For the human GHR, recent investigations suggested that the critical tyrosines appear to be Y534, Y566, and Y627, and the inactivation of all three tyrosines is necessary to abrogate, or significantly reduce, STAT5b signal transduction [66]. This redundancy of tyrosine usage by STAT5b, which is consistent among all mammalian GH receptors ana-lyzed, could explain why a simple, homozygous, missense mutation, even within one of the critical tyrosines, has yet to be identified in GHI subjects. Such a muta-tion would be predicted to have minimal impact on STAT5b signaling unless the mutation significantly compromised the structure of the GHR.

Exon 3-Deleted GHR

In addition to identified, specific, GHR mutations, it has been recently reported that a common polymorphism of the GHR is associated with increased responsiveness to GH [67]. Approximately half of western Europeans are hetero- or homozygous with respect to an allele encoding an isoform of the GHR gene that is lacking exon 3 (d3-GHR). Evaluation of two cohorts of children of European descent with heights < −2SD, carrying a diagnosis of either ISS or small for gestational age (SGA) and receiving treatment with GH, suggested that children carrying one or both d3-GHR alleles grew almost twice as well as children who were homozygous for the full-length isoform. These observations have been confirmed in some, but not all, studies, leading to continued controversy as to whether the d3-GHR isoform actually confers increased GH responsiveness. In an attempt to put all such studies into perspective, a recent meta-analysis of prepubertal children with short stature concluded that the d3-GHR does appear to be associated with an increased baseline height in response to 1 year of GH therapy only in GHD, but not in non-GHD children with short stature [68]. The magnitude of this growth response, however, was only 0.5 cm/year, suggesting that genotyping for d3-GHR may have limited value as a predictor of therapeutic results.

Perspective and Summary

It has been approximately 20 years since the cloning and characterization of the human GHR gene. Mutations in the GHR gene are the cause of GHI syndrome, with inheritance predominantly autosomal recessive. Evaluating the impact of identified mutations on GHR structure and function is important to understanding the pathophysiology of the disease. Therapeutic options for patients carrying mutations in the GHR gene have recently expanded to include recombinant IGF-I therapy; Chernausek et al., for example, reported an average baseline of 2.8–8.0 cm/year during the first year of IGF-I treatment, and lower height velocities, but above

131 Growth Hormone Receptor in Growth

baseline, during subsequent years [69]. Based on studies demonstrating the efficacy and safety of IGF-I therapy, the US Food and Drug Administration has approved rhIGF-I therapy for children with height SD score of −3 or less, serum IGF-I score of −3 or less (<2.5th percentile in the European Union), and normal or elevated GH [70]. Optimizations of doses are currently under investigations [70].

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