Familial Growth Hormone Deficiency and Mutations in the GHRH Receptor Gene

7 Familial Growth Hormone Deficiency and Mutations in the GHRH Receptor Gene Maria Alba and Roberto Salvatori Department of Medicine, Division of Endocrinology Johns Hopkins University School of Medicine Baltimore, Maryland 21287

I. II. III. IV.

Introduction The GHRH Receptor and its Gene Mutations in the GHRHR Gene Clinical, Hormonal, and Radiological Phenotype of Patients with Bi-Allelic GHRHR Mutations V. Hormonal and Radiological Phenotype of Heterozygous Carriers VI. Conclusions References

Growth hormone (GH)-releasing hormone (GHRH) is necessary for the proliferation of the somatotropic cells of the anterior pituitary and the synthesis and secretion of GH. GHRH is released by the hypothalamus into the portal hypophysial circulation to bind to a membrane surface receptor [GHRH receptor (GHRHR)] expressed by the somatotropic cells. Because of the need of GHRH for GH secretion, it is to be expected that alterations in synthesis or action of GHRH would result in isolated GH deWciency (IGHD). Indeed, although GHRH gene mutations have Vitamins and Hormones Volume 69

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never been reported, mutations in the GHRHR gene (GHRHR) are emerging as a relatively common cause of inherited autosomal recessive IGHD. The Wrst human GHRHR mutations were discovered in families with a history of parental consanguinity. More recently, kindreds in which IGHD subjects are compound heterozygotes for two distinct mutations indicate that faulty GHRHR alleles may be prevalent and that these mutations may need to be suspected even in sporadic IGHD cases. Patients with two faulty GHRHR alleles have normal weight at birth. Growth failure becomes apparent during the Wrst year of life. Biochemical studies show low serum insulin-like growth factor-1 level, and absent or markedly reduced GH response to a variety of stimuli. Magnetic resonance imaging shows hypoplasia of the anterior pituitary. In this chapter, we describe the GHRHR mutations reported to date and the phenotype of aVected individuals. ß 2004 Elsevier Inc.

I. INTRODUCTION Growth hormone (GH) is a circulating protein that stimulates longitudinal somatic growth and controls numerous metabolic functions, including lipid, glucose, and protein metabolism, bone apposition, blood pressure, and cardiac muscle mass and function. GH acts on target tissues both directly and indirectly, with the mediation of insulin-like growth factor-1 (IGF-1, also called Somatomedin C), either circulating (of liver origin) or locally produced (Fig. 1). GH is secreted by the somatotropic cells of the anterior pituitary under the control of two hypothalamic factors: one stimulatory [GHreleasing hormone (GHRH)] and one inhibitory [somatostatin (SRIF)] (Ghigo et al., 2000). These two hormones act on speciWc G-protein-coupled receptors expressed on the surface of the somatotropic cells. Receptor activation increases (GHRH) or decreases (SRIF) intracellular cyclic adenosine monophosphate (AMP) (cAMP) production. Isolated growth hormone deWciency (IGHD) causes postnatal somatic growth retardation, which can range from mild to severe, depending on its degree. Because GH is not needed for intrauterine growth, children with congenital IGHD have normal size at birth. IGHD occurs with an incidence of 1/4000 to 1/10,000 live births (Lacey and Parkin, 1974; Lindsay et al., 1994; Rona and Tanner, 1977; Vimpani et al., 1997). Most of the cases are caused by a variety of acquired insults (ischemic, inXammatory, or neoplastic) to the hypothalamic–pituitary region. However, clear anatomical defects in this area have been found only in 12% of IGHD patients when examined by magnetic resonance imaging (MRI) (Cacciari et al., 1990), suggesting that genetic or functional, rather than structural, defects may be involved more frequently than commonly believed. The hypothesis of a signiWcant genetic inXuence is supported by the fact that 5–30% of IGHD

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FIGURE 1. Schematic representation of the stimulatory control and action of GH. has familial occurrence (Cogan and Philipps, 1998). It has to be noted that more recently, MRI studies have shown that, using age-adjusted pituitary measurements, a signiWcant percentage of IGHD patients has some degree of anterior pituitary hypoplasia (APH), often associated with the pituitary stalk agenesis (PSA) syndrome, which includes pituitary stalk transection and ectopic neurohypophysis. Based on clinical features, mode of inheritance, and response to exogenous GH therapy, four forms of familial IGHD have been described: Type IA: Autosomal recessive. AVected subjects have undetectable serum GH and often develop anti-GH antibodies after GH treatment, which cause blunted growth response to therapy. . Type IB: Autosomal recessive. This is the most common form of familial IGHD. Patients have low, but often detectable, serum GH levels and do not develop anti-GH antibodies in response to GH treatment. . Type II: Autosomal dominant. Patients usually have low but detectable serum levels and no development of anti-GH antibodies after GH treatment. . Type III: X-linked. This form is very rare; with complex and distinct clinical Wndings in diVerent families. .

Mutations in the gene encoding GH (GH1) can cause types IA, IB, and II phenotypes (Binder, 2002). However, although the prevalence of GH1

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mutations in children with IGHD IA is high (66.7%) (Wagner et al., 1998), it is only 1.7% in patients with type IB. Thus, in most cases IGHD IB is not caused by mutations in GH1. Considering the need of GHRH for GH secretion, IGHD could result from genetic defects that impair synthesis, secretion, or action of hypothalamic GHRH. Although the GHRH gene has been ruled out as a candidate gene in IGHD by linkage and direct gene analysis (Perez Jurado et al., 1994), mutations in the GHRH receptor (GHRHR) gene (GHRHR) are being reported with increasing frequency. This chapter describes the biology of the receptor, recapitulates the GHRHR mutations described so far, as well as the phenotype of both the aVected individuals and the heterozygous carriers.

II. THE GHRH RECEPTOR AND ITS GENE The GHRHR is a membrane-bound receptor that is coupled with GS protein and activates adenylyl cyclase, causing a rise in intracellular cAMP, which in turn induces activation of the protein kinase A pathway, resulting in cellular proliferation and GH secretion. A second pathway [mitogenactivated protein (MAP) kinase] is also activated by ligand binding, involved mostly in cell proliferation signaling (Pombo et al., 2000). The GHRHR belongs to family B, group III of the G-protein-coupled receptors superfamily, together with receptors for glucagon, glucagon-like peptide-1, secretin, vasoactive intestinal peptide, gastric inhibitory peptide, and pituitary adenylate cyclase-activating peptide. All of these receptors consist of a N-terminal extracellular domain, seven hydrophobic transmembrane domains, and a C-terminal intracellular domain. The GHRHR is initially translated as a 423-amino acid protein, and an N-terminal 22-amino acid signal peptide is cleaved during protein processing. The extracellular domain contains one glycosylation site. The C-terminal intracellular domain contains several potential phosphorylation sites and is thought to interact with GS protein. Although the N-terminal extracellular domain is essential for ligand binding, chimeric protein studies (DeAlmeida and Mayo, 1998) have shown that speciWcity for the ligand is provided by the transmembrane domains. Deletion of the extracellular domain impairs not only ligand binding, but also the transport of the receptor to the cell surface. The human GHRHR gene, located on chromosome 7p14, consists of 13 coding exons. Exons 1 through part of 5 encode for the extracellular domain, 5 through 12 for the seven transmembrane domains and extracellular and intracellular intervening loops, and part of 12 and 13 for the intracellular C terminus. Transcription is stimulated by glucocorticoids (Lam et al., 1996; Miller and Mayo, 1997; Nogami et al., 1999) and inhibited by estrogen (Lam et al., 1996). The pituitary-speciWc transcription factor

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Pit-1 simulates gene expression by binding to two speciWc sites identiWed in the proximal area of the promoter (Iguchi et al., 1999).

III. MUTATIONS IN THE GHRHR GENE The Wrst mutation in the GHRHR gene was discovered in rodents. Dwarf mice were noted at the Jackson laboratories in the early 1970s, and it was soon discovered that the phenotype, inherited as an autosomal recessive trait, was caused by GH deWciency (Eicher and Beamer, 1976). The mouse was named ‘‘little’’ (lit/lit) and became a widely used murine model of IGHD. About 20 years later, after the cloning of the mouse GHRHR gene, two groups independently discovered that the little phenotype is caused by a homozygous missense mutation that results in a single amino acid change (D60G) in the extracellular domain of the receptor (Godfrey et al., 1993; Lin et al., 1993). The mutated receptor cannot bind GHRHR (Gaylinn et al., 1999). The lack of GHRH action causes reduced somatotroph cell proliferation and hypoplasia of the anterior pituitary. Somatotropic cells are present in a limited area of the anterolateral section of the gland, but they are missing in the caudomedial area of the gland (Lin et al., 1993), indicating that their diVerentiation does not require GHRH, but their proliferation does. In 1996 the Wrst human GHRHR mutation was reported in two IGHD Indian Muslim cousins residing in New York born from consanguineous parents (Wajnrajch et al., 1996). The patients carried a homozygous G ! T transversion in exon 3 that introduces a stop codon at residue 72 (E72X) (also called E50X, if one starts counting amino acids after the removal of the 22-residue leader sequence). This mutation leads to a severely truncated receptor, lacking the whole transmembrane backbone. Two years later the same mutation was reported in a large Pakistani kindred (‘‘Dwarfs of Sindh’’) with 18 aVected IGHD individuals (Maheshwari et al., 1998), and in two siblings from Sri Lanka (Netchine et al., 1998). Although originating from the same subcontinent, the members of the three kindreds with the E72X mutation are of diVerent religion and are geographically very distant. However, linkage analysis studies have shown that they share the same ancestor (‘‘founder eVect’’) (Wajnrajch et al., 2003). In 1999 we reported the existence of a large group of individuals with autosomal recessive IGHD, residing in the northeastern Brazilian state of Sergipe, in a rural county called Itabaianinha (Salvatori et al., 1999). This is the largest kindred with IGHD described so far, with 105 dwarf individuals in seven generations, 70 presently alive. In this kindred all aVected individuals carry a homozygous donor splice GHRHR mutation (IVS1 þ 1G ! A) that predicts the retention of part or all of the large intron 1 (4.5 Kb) and the insertion of a premature stop codon 213 bases from the

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exon–intron junction (Salvatori et al., 1999). A founder eVect is present in this population as well. The high frequency of consanguineous marriages (about 25% of the unions) and the extremely low mobility of this population are the likely causes of the spread of this mutation in Itabaianinha. The timing of onset of the mutation and its origin are presently unknown. This mutation has later been found in other sporadic cases (Osorio et al., 2002), but never outside Brazil. Several other GHRHR mutations have been subsequently reported, with a prevalence of 10% in familial IGHD IB (Salvatori et al., 2001a). They include one promoter mutation (Salvatori et al., 2002a), two splice mutations (Roelfsema et al., 2001; Salvatori et al., 2002b), one nonsense mutation (Salvatori et al., 2002b), six missense mutations (Carakushansky et al., 2003; Salvatori et al., 2001a, 2002a,c), and two microdeletions (Horikawa, 2000; Salvatori et al., 2001b). The localization of the mutations reported to date (with the exception of the promoter mutation) is shown in Fig. 2. Each mutation is speciWc for a determinate geographical area, with the exception of the L144H, found in three diVerent continents (Europe, North America, and South America). Linkage analysis has revealed that this

FIGURE 2. Schematic representation of the GHRHR protein and localization of the mutations in the coding sequence and in the splice sites. The black bars represent the exon boundaries. The promoter mutation is not represented in this figure.

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mutation has arisen at least twice (Salvatori et al., 2002c), making it the only recurring GHRHR mutation reported to date. This mutation introduces a DraIII restriction site, making it relatively easy to detect by digestion of polymerase chain reaction (PCR)-ampliWed genomic DNA. Therefore, it should be the Wrst mutation searched in GHRHR analysis (on kindreds not originating from the Indian subcontinent). All the missense mutations change amino acids that are highly conserved in all the GHRHR genes cloned so far, including human, mouse, rat, ovine, porcine, and bovine. They have all been proven to cause receptor malfunction in expression studies in eukaryotic cells. Both the Del 1140–1144 and Del 1121–1124 cause a frame shift in the coding sequence. In addition, Del 1140–1144 introduces a stop codon 67 bp downstream of the deletion. The splice mutations are predicted to cause retention of intronic sequences in mRNA, causing disruption of protein sequence. Of particular interest is a mutation (A ! C transversion) in position 124 of the GHRHR promoter region. This occurs in one of the two promoter areas that bind the pituitary-speciWc transcription factor Pit-1. This base change reduces transcription by impairing Pit-1 binding, proving the importance of the Pit-1 transcription factor in the expression of the GHRHR (Salvatori et al., 2002a). All of the GHRHR mutations are inherited as autosomal recessive traits. The only proposed exception is the Del 1121–1124, described in a Japanese boy. The receptor expressing this deletion has a ‘‘dominant negative’’ eVect in vitro, altering signal transduction by the normal receptor (Horikawa, 2000). This mechanism should result in an autosomal dominant mode of transmission. However, the patient who is homozygous for this deletion is much shorter than his heterozygous parents, making the in vivo consequences of heterozygosity for this particular mutation less clear. In four kindreds with mutated GHRHR, the aVected members are compound heterozygotes (each chromosome carries a distinct mutation). Therefore, faulty GHRHR alleles might be rather prevalent in the general population. Clinicians should suspect mutations in this gene in IGHD children, even in families without any history of short stature or consanguinity.

IV. CLINICAL, HORMONAL, AND RADIOLOGICAL PHENOTYPE OF PATIENTS WITH BI-ALLELIC GHRHR MUTATIONS All of the patients with bi-allelic GHRHR mutations present a similar phenotype. At birth, they have normal weight, and no micropenis or neonatal hypoglycemia has ever been reported. Severe growth retardation

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appears evident during the Wrst year of life. Untreated adult individuals have a high-pitched voice, mild frontal bossing, mild hypercholesterolemia, increased abdominal obesity, and, despite increased systolic blood pressure, reduced cardiac mass (Barreto-Filho et al., 2002). Biochemical proWle invariably shows low serum IGF-1. With modern monoclonal antibody-based assays, serum GH is usually measurable, albeit low, with absent or blunted response to a variety of stimuli (GHRH, hypoglycemia, arginine, clonidine, propranolol, and physical exercise). Even 1 week of bi-daily priming with subcutaneous GHRH does not restore GHRH responsiveness in patients with the IVS1 þ 1G ! A mutation (Salvatori et al., 1999). It is possible that this may not be the case in patients with missense mutations in whom the mutated receptor may maintain some biological function. To date, all of the cases reported had severe GH deWciency, possibly due to the selection of the patients whose DNA was studied. It is conceivable that in the future mutations that allow the receptor function to be partially maintained will be discovered in subjects with a milder form of IGHD. Even within the mutations discovered to date, however, it is very diYcult to establish genotype–phenotype correlation, as a result of diVerent genetic backgrounds (genetic height targets may vary greatly), diVerent GH stimulation tests used, and diVerent timing and dosing of exogenous GH therapy. As one would expect, the pituitary function of patients with mutated GHRHR is otherwise normal. They often have mildly delayed puberty, but both sexes have normal fertility. Because of past episodes of obstetric complications related to cephalic–pelvic disproportion (normal-size babies from dwarf mothers), for many years dwarf women of Itabaianinha had been advised against childbirth. More recently, the use of caesarean section has circumvented this problem, and several normal babies have been born from dwarf mothers without complications, and mothers are able to breast-feed. MRI studies in most cases show isolated APH with normal neurohypophysis and without PSA (Carakushansky et al., 2003; Murray et al., 2000; Netchine et al., 1998; Oliveira et al., 2003; Osorio et al., 2002; Salvatori et al., 2001a, 2002b,c). Although pathological studies have not yet been performed in humans, APH is likely the expression of hypoplasia of the somatotropic cells, similar to what is observed in the little mouse. Because MRI studies have never been performed in children younger than 4 years of age, it is unknown if APH is present at birth or during the earlier years of life. However, the lack of APH in a GHD child who is 4 years of age or older makes a bi-allelic GHRHR mutation extremely unlikely. Patients with GHRHR mutations have good growth response to exogenous GH therapy. If therapy is instituted in a timely fashion, they reach their genetic target height. No study on the development of GH

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antibody has been performed, but the good response to therapy makes the presence of such antibodies unlikely. Patients lacking functional GHRHR represent a model to study the GHRH-independent regulation of GH secretion, albeit limited by the somatotropic cell hypoplasia. These patients maintain normal timing of GH pulses (with greatly diminished amplitude), suggesting that natural pulsatility is an expression of changes in somatostatinergic tone rather than GHRH pulses (Maheshwari et al., 2002). In addition, they have a small but signiWcant GH response to GH-releasing peptides (GH-RPs) and to physical exercise, proving that these stimuli can act, at least in part, directly on the somatotropic cells without need of GHRH (Gondo et al., 2001; Roelfsema et al., 2001; Salvatori et al., 2002b). The GH response is, however, barely detectable, and is still consistent with the IGHD diagnosis.

V. HORMONAL AND RADIOLOGICAL PHENOTYPE OF HETEROZYGOUS CARRIERS We and others have hypothesized that heterozygous carriers of GHRHR mutations may present a mild phenotype that would be intermediate between homozygous normal and GHD patients (Hayashida et al., 2000; Maheshwari et al., 1998). Indeed, heterozygous subjects do have lower serum IGF-1 than homozygous normal subjects from the same area (Hayashida et al., 2000; Oliveira et al., 2003). Whether this reXects in shorter stature or in other phenotypical diVerences is presently being investigated in the Itabaianinha kindred, estimated to include more than 3000 heterozygotes.

VI. CONCLUSIONS GHRHR mutations are a relatively common cause of familial IGHD. They should be suspected in a child who presents with early growth failure (starting within the Wrst year of life), clear evidence of GHD (low serum IGF-1 and blunted or absent GH response to stimulation), and otherwise normal pituitary function. MRI evidence of APH is usually present in children that are 4 years of age or older, but it is not known if it is present in younger children. Parental consanguinity or a positive family history of IGHD should increase the suspicion for one of these mutations. However, the lack of these historical features does not exclude it. One important caveat: subjects with homozygous mutations in genes encoding for the pituitary-speciWc transcription factors Pit-1 (POU-1) and prophet of Pit-1 (PROP-1) (both causing multiple pituitary hormone deWciencies) may initially present to clinical attention with IGHD

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(Parks et al., 1999), and may develop other pituitary hormone deWcits later in life. Therefore, attention must be paid to signs or symptoms of additional pituitary hormone deWcits. Given the large size of the GHRHR and the heterogeneity of the mutations reported to date, no easy screening for GHRHR mutations is available, and their detection is limited to research laboratories. However, knowledge of the genetic cause is important for family counseling and helps in predicting the phenotype and the response to GH therapy.

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