7.2.3 Genetic defects of the human somatotropic axis

Linear growth is controlled by complex interactions between genetic and environmental factors. Our understanding of the endocrine physiology of growth has been revolutionized over the last 20 years by the field of molecular genetics, which has identified many genes involved in inherited human growth disorders.

Genetic defects in the human somatotropic axis will be presented and are classified as those associated with:

The control of anterior pituitary development and cell lineage determination has been revealed by genetic studies in both mice and humans. During development, interaction between Rathke‘s pouch, the primordium of the anterior pituitary lobe, and the diencephalon influence the expression of transcription factors that control the differentiation of the hormone-secreting cells of the anterior pituitary. Examples of these transcription factors are LHX3, LHX4, POU1F1, and PROP1. Human defects of these genes result in genetic combined anterior pituitary hormone deficiency (1).

PROP1 is a pituitary-specific paired homeodomain transcription factor. The gene has three coding exons spanning 3.5 kb, and has been localized in mice to a region that exhibits linkage conservation with human chromosome 5q23–q35 (2). The gene was first identified as a result of genetic studies of the Ames dwarf mouse, which has pituitary hypoplasia and combined anterior pituitary hormone deficiencies. Ames mice have a point mutation in the Prop1 gene, which results in diminished binding of Prop1 to the POU1F1 promoter enhancer and, subsequently, decreased transcriptional stimulation. The Ames mice, therefore, fail to activate POU1F1 expression, which results in dysmorphogenesis of the anterior pituitary and combined hormone deficiencies.

Homozygous or compound heterozygous human mutations in PROP1 are associated with growth hormone, thyroid stimulating hormone (TSH), prolactin (PRL) and gonadotrophin deficiencies. Most patients present with early onset growth hormone deficiency (GHD) (1). The TSH deficiency is highly variable and may not be present from birth. The spectrum of gonadotropin deficiency is also variable ranging from severe hypogonadism to spontaneous puberty and infertility. Patients with PROP1 deficiency also demonstrate an evolving cortisol deficiency with age (3, 4). The mechanism for this is unknown because PROP1 is not expressed in corticotrophs (Fig. 7.2.3.1).

Most patients with PROP1 mutations have a small or normal-sized pituitary gland on MRI, however an enlarged pituitary resembling a tumour may be present (5), which can regress leading to complete involution of the gland (1).

POU1F1 (formally known at PIT1) is a member of the POU homeodomain transcription factor family. Its gene is located on chromosome 3 and has six exons, which encode two protein domains, the POU homeodomain and the POU-specific domain. These domains are necessary for high-affinity DNA binding and genetic studies have shown that POU1F1 binds and transactivates both growth hormone and prolactin genes, and autoregulates the expression of the growth hormone-releasing hormone (GHRH) receptor (6). POU1F1 is essential for the development of somatotrophs, lactotrophs, and thyrotrophs (Fig. 7.2.3.1). The importance of POU1F1 for anterior pituitary cell function came from studies of two dwarf mice with similar phenotypes. The Jackson dwarf mice have a large insertion mutation and Snell dwarf mice have a point mutation (W261C).

The spectrum of hormone deficiency varies widely in patients with POU1F1 mutations (1, 7). Patients with homozygous mutations usually present with early growth hormone and PRL deficiency, but the TSH deficiency is variable and may present later. A heterozygous R271W mutation has also been reported (7).

HESX1 is a homoeobox gene, which plays a crucial role in early determination and differentiation of the pituitary. In mice the targeted disruption of Hesx1 causes a phenotype similar to that of septo-optic dysplasia (SOD) in humans. SOD is a rare heterogeneous disorder characterized by the triad of midline forebrain abnormalities, optic nerve hypoplasia (ONH) and hypopituitarism. The degree of hypopituitarism may vary from isolated GHD to panhypopituitarism, sometimes involving posterior pituitary function. Neurological deficit is common varying from global retardation to epilepsy or hemiparesis (1). A homozygous missense mutation of the human HESX1 gene was first described in two siblings within a highly consanguineous family who had features of SOD, absence of the corpus callosum and panhypopituitarism (8). A subsequent homozygous mutation was reported in a patient with evolving pituitary deficiency (1, 9). Milder phenotypes of hypopituitarism have also been reported to be associated with heterozygous HESX1 mutations (10). These patients typically have isolated GHD with ectopic posterior pituitary.

LHX3 and LHX4 are homoeobox genes that are expressed early in Rathke’s pouch. Endocrinologically, the phenotypes of patients with LHX3 mutations are similar to those of the PROP1 defect (growth hormone, TSH, luteinizing hormone, follicle-stimulating hormone (FSH), and PRL deficiencies). However an additional feature is the presence of a short rigid cervical spine with limited head rotation (11). A human LHX4 mutation was reported in a child with short stature, hypopituitarism, and unusual skull morphology (12).

Patients with X-linked mental retardation and hypopituitarism have been shown to have duplications of the Xq26–27 region, which could correspond to overdosage of the SOX3 gene, known to lie within this region (13). Two siblings with a duplication at Xq27 have also been reported with variable hypopituitarism, but no mental retardation (14). SOX3 would appear to be the strongest candidate gene for implication in this phenotype.

Genetic IGHD can be classified into types IA, IB, II and III (1). Their characteristics are as follows: Type IA (OMIM 262400) is caused by autosomal recessive mutations of the GH1 gene, and presents with severe short stature and development of anti-growth hormone antibodies on growth hormone therapy. Type IB (OMIM 612781) may be caused by recessive mutations of the GH1 or GHRH receptor (GHRHR) genes and has a less severe phenotype without anti-growth hormone antibodies. Type II (OMIM 173100) GHD is caused by autosomal dominant mutations in GH1 and typically has less severe short stature. Type III (OMIM 307200) describes an X-linked recessive disorder presenting with short stature and agammaglobulinaemia. Its genetic origin remains unknown.

Large homozygous deletions (6.7–45 kb within the GH1 gene) occurring in consanguineous families result in the typical phenotype of Type 1A IGHD (15). Microdeletions have also been described. The patients usually have complete GHD resulting in extreme short stature and may develop anti-growth hormone antibodies on growth hormone therapy. Some phenotypic heterogeneity has been described. Treatment with recombinant human (rh) insulin-like growth factor 1 (IGF-1) may be required due the development of growth hormone resistance. Homozygous splice-site mutations of GH1 typically cause a milder form of IGHD (Type 1B).

Dominantly inherited splice-site mutations, typically in intron III cause type II IGHD. These patients usually respond well to growth hormone therapy without antibody formation. In some recently studied patients, there is evolution of the endocrine features with development of TSH, PRL, gonadotropin, and ACTH deficiencies (16, 17).

The human GHRH receptor (GHRHR) is a seven transmembrane domain, G-protein coupled receptor with a high binding affinity for GHRH. The GHRHR is required for proliferation of somatotroph cells and is thus a key component of the growth axis. The first nonsense mutation (E72X) of the human GHRHR gene was reported in two consanguineous kindreds, one from India and one from Pakistan. The affected individuals had profound IGHD and were homozygous for this genetic defect, which resulted in a severely truncated receptor protein lacking all the transmembrane regions and the intracellular G-protein binding domain (18, 19). A large cohort of affected patients from Brazil has been described further characterizing the phenotype of GHD, which is less severe than seen in Type 1A IGHD due to GH1 mutations (20). Most patients appear to respond well to growth hormone therapy.

This syndrome was first described in the 1970s following the observation that some non-GHD short children with low serum IGF-1 levels showed a growth response and generation of IGF-1 following growth hormone therapy. The first convincing example of growth hormone bio-inactivity was reported in a child who had a heterozygous missense GH1 gene mutation (Arg77Cys), which is predicted to form an aberrant disulphide bond around site 1, one of the two growth hormone receptor binding sites of the growth hormone molecule (21). Another patient was reported with a heterozygous, missense mutation (D112G) adjacent to site 2 causing reduced growth hormone-induced tyrosine phosphorylation. (22). True growth hormone bio-inactivity causing abnormal growth and short stature appears to be very rare.

The human GHR gene (OMIM 600946) is located on chromosome 5, has 10 exons spread over 87 kb and encodes a protein of 638 amino acids (23). It is a member of the family of cytokine receptors, several interleukins, granulocyte-macrophage colony-stimulating factor, erythropoietin, leptin, and several interferons (24). The GHR gives rise to a soluble, high-affinity growth hormone binding protein (GHBP), derived from the extracellular domain of the receptor by proteolytic cleavage of the membrane-anchored receptor in man.

GHI syndrome (GHIS) or Laron syndrome (OMIM 262500) is a rare autosomal recessive condition, characterized clinically by hypoglycaemia in infancy and severe childhood growth failure, and biochemically by high levels of growth hormone, and low IGF-1, IGFBP-3, and acid-lablie subunit (ALS) (25). Craniofacial dysmorphic features include hypoplasia of the bridge of the nose, prominent forehead and decreased vertical dimension of the mid-face. The first GHR mutation in GHIS was reported in 1989 by Godowski et al. who described a homozygous deletion in the coding region of the growth hormone binding domain (26). The ease with which it is now possible to amplify the coding exons of GHR by PCR and sequence the amplification products, has led to the discovery of more than 60 mutations in the GHR gene of GHIS patients (23). They are almost all recessively inherited, either in homozygous or compound heterozygous forms, and range from exon deletions and splice mutations to missense, nonsense, and frame shift mutations. The majority of GHR defects occur in the region encoding the extracellular domain of the receptor. Patients with such mutations have absent or extremely low GHBP and typical features of Laron syndrome (25). More than 50 mutations in this domain have been reported, including large deletions and small deletions resulting in frame shifts and premature stop codons. Commonly point mutations result in a premature stop codon (nonsense), or altered amino acid (missense) and nucleotide substitutions resulting in activation of a cryptic splice site or creation of a new one.

Duquesnoy et al. reported several patients with an exon 6 D152H mutation and normal GHBP (27). A classical GHIS patient with a homozygous point mutation (IVS8ds + 1 G → C) in the splice donor site of intron 8 results in the skipping of exon 8. In this first description of a transmembrane mutation (28) the mutant GHR is released from cells and is measured as GHBP, lacking the ability to anchor to the cell surface. A point mutation (IVS7as-1 G → T) in the acceptor splice site of intron 7 results in the skipping of exon 8, giving rise to GHIS with high GHBP levels. GHIS may also result from mutations with the intracellular domain of the GHR: a homozygous 22bp deletion in exon 10 in two siblings with low, but detectable GHBP results in a GHR mutant with no binding site for the key signalling molecule STAT5 (29).

In 1997, Ayling et al. provided further insights into the genetics of GHIS, describing the first heterozygous mutation with a dominant negative effect. The mutation (IVS8as-1 G → C) was situated in the acceptor splice site of intron 8 resulting in the skipping of exon 9 and the production of a truncated GHR. The mutant GHR formed heterodimers with the wild-type GHR and exerted a dominant negative effect with markedly reduced growth hormone-induced signalling (30). A second mutation (IVS9ds+1 G → A) leading to the same consequence was described by Iida (31). Both patients have normal GHBP and normal facial appearance. A series of patients with a similar phenotype have been reported with a mutation causing the insertion of a pseudo-exon between exons 6 and 7 (32, 33). The 108 bp insertion caused the in-frame addition of 36 amino acids between codons 206 and 207. The exact mechanism of the receptor dysfunction is not yet clear.

Patients with idiopathic short stature (ISS) and normal growth hormone secretion have been evaluated for GHR abnormalities. Although more than 60 molecular defects in the GHR have been described, the majority of ISS patients have normal coding regions of the growth hormone receptor. In 1995 Goddard et al. studied a group of ISS patients with low serum concentrations of GHBP suggestive of partial GHIS (34). Four patients had heterozygous GHR mutations. In a compound heterozygote, the two deleterious mutations (E44K and R161C) would explain the patient’s short stature. In the other three cases there may have been a second unidentified mutation in the intracellular domain giving rise to the ISS because the transmembrane and intracellular domains of these patients were not sequenced. Further to this report, Goddard et al. studied 100 patients across the spectrum of ISS, resulting in the discovery of three more carriers of heterozygous extracellular mutations and one patient with a heterozygous mutation (A478T) in exon 10 (35). Several other publications have reported heterozygous GHR mutations. It is probable that up to 5% of patients with ISS have heterozygous GHR mutations, which may help explain their growth failure (23).

In 2003, the first report was published of a defect in the growth hormone signalling cascade in patients with GHIS (36). Kofoed et al. reported a homozygous missense mutation in exon 15 of the STAT5b gene (OMIM 604260) and demonstrated that the mutant protein could not be activated by growth hormone, therefore failing to activate gene transcription (36). This child had features of severe GHI together with immunodeficiency consistent with a nonfunctional STAT5b protein. Several other case reports have confirmed the endocrine profile and phenotype of this interesting genetic defect (37, 38). Most cases are female and all but one has evidence of a significant immune deficiency, most often manifested by interstitial pneumonitis complicated by opportunistic infection.

IGF-1, the key growth hormone-dependent effector protein regulating human growth, circulates as a ternary complex consisting of IGF-1, IGFBP-3 and ALS (OMIM 147440, 146732 and 610489 respectively). An Als knockout (KO) animal model provided new insights in the role of ALS in the IGF-1 system, with growth deficits being seen 3 weeks after birth (39). Growth hormone levels were normal, however IGF-1 and IGFBP-3 were significantly decreased. In 2004, Domene et al. (40) reported the first human case with a homozygous inactivating ALS mutation. The defect was a guanine deletion at position 1338, resulting in a frame-shift and the appearance of a premature stop codon (1338delG, E35fsX120). The patient had minimal postnatal growth impairment, but basal growth hormone levels were increased, associated with severe reduction in IGF-1, IGFBP-3 and undetectable ALS, unresponsive to stimulation by growth hormone. Further reports have confirmed that the growth failure is not severe in this defect (41) suggesting that locally produced IGF-1 may be normal and thus be protective against severe short stature. An interesting additional feature is insulin resistance (42, 43).

The IGF1 gene (OMIM 147440) is located on chromosome 12q, spans more than 90 kb and consists of six exons with alternative splicing of exons 5 and 6 and alternative transcription start sites in exons 1 and 2 (44). IGF-1 is a 70 amino acid peptide, which mediates the majority of growth promoting effects of growth hormone after birth and is a major fetal growth factor. The first patient with a homozygous partial deletion of the IGF1 gene was reported by Woods et al. in 1996 (45) This patient had severe fetal and postnatal growth failure in association with microcephaly and mental retardation, sensorineural deafness, and dysmorphic features, including micrognathia, bilateral ptosis, and low hairline. Growth hormone levels were increased with normal GHBP, IGFBP-3, and ALS, but IGF-1 was undetectable. Insulin resistance was also present (46).

Sequencing of the patient’s cDNA revealed a homozygous partial IGF1 gene deletion, which predicts a markedly truncated protein. Both parents were confirmed to be heterozygotes. This case demonstrates that IGF-1 plays a critical role in human fetal and postnatal growth, and that absence of this gene can be compatible with life. Three further cases have been reported, one an adult with a similar phenotype to the first described case, but with elevated IGF-1 and a missense mutation causing a bio-inactive IGF-1 molecule. (47). Further studies in cohorts of patients born small for gestational age suggest that defects of the IGF-1 gene are likely only to be a rare cause of fetal growth failure.

The IGF-1 receptor (IGF-1R) is a tetramer consisting of a pair of disulphide-linked α and β subunits. It is a member of the tyrosine kinase receptor family and resembles the insulin receptor in structure. The gene for IGF1R is located on chromosome 15, consists of 21 exons and spans more than 100 kb. Two cases of a heterozygous IGF-1R mutation were reported in children with low birth weight and postnatal short stature (48). Serum IGF-1 levels were elevated or at the upper limit of normal consistent with IGF-1 resistance. Recently, a mother and daughter carrying the same heterozygous mutation were reported. Both had low birthweight and postnatal growth failure (49). Functional studies showed normal binding of IGF-1 to the IGF-1R, but reduction of autophosporylation and activation of the downstream signalling cascade, consistent with inactivation of one copy of the IGF-1R gene.

Molecular genetic analyses of children with pre- and postnatal growth failure have demonstrated an increasing array of genetic abnormalities in the somatotropic axis. As a general rule for the practising clinician, careful clinical assessment of the child with abnormal growth followed by delineation of an abnormal endocrine profile should precede molecular investigation. Certain gene defects can be predicted by clinical and endocrine characteristics. In the years to come further mutations will be described which together with their clinical and hormonal correlates will supplement our understanding of normal and abnormal childhood growth.