A Novel Splice-Disrupting Mutation and Study of Founder Effects

Original Paper

HOR MON E RE SE ARCH I N PÆDIATRIC S

Horm Res Paediatr 2012;78:165–172 DOI: 10.1159/000342760

Received: May 2, 2012 Accepted: August 15, 2012 Published online: October 10, 2012

GH-Releasing Hormone Receptor Gene: A Novel Splice-Disrupting Mutation and Study of Founder Effects Suemi Marui a, b Ericka B. Trarbach b Margaret C.S. Boguszewski c Marcela M. França a Alexander A.L. Jorge a Hiroshi Inoue e Mirian Y. Nishi a Luiz de Lacerda Filho c Manuel H. Aguiar-Oliveira d Berenice B. Mendonca a Ivo J.P. Arnhold a a

Unidade de Endocrinologia do Desenvolvimento, Laboratório de Hormônios e Genética Molecular LIM-42, and b Unidade de Tireóide, Laboratório de Endocrinologia Celular e Molecular LIM-25, Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo, São Paulo, c Department of Pediatrics, Federal University of Parana´, Curitiba, and d Department of Medicine, Federal University of Sergipe, Aracaju, Brazil; e University of Tokushima, Tokushima, Japan

Key Words GHRHR mutations ⴢ Isolated GH deficiency ⴢ Splicing mutations ⴢ Growth ⴢ Mutations

Abstract Background: Mutations in GH-releasing hormone receptor gene (GHRHR) are emerging as the most common cause of autosomal recessive isolated GH deficiency (IGHD). Objective: To search for GHRHR mutations in patients with familial or sporadic IGHD and to investigate founder effects in recurring mutations. Methods: The coding region of GHRHR was entirely amplified and sequenced from DNA of 18 patients with IGHD (16 unrelated) with topic posterior pituitary lobe on MRI. Haplotypes containing promoter SNPs and microsatellites flanking GHRHR were analyzed in patients with c.57+1G1A (IVS1+1G1A) mutation of our previously published kindred and also a Brazilian patient and 2 previously reported Japanese sisters with c.1146G1A (p.E382E) mutation. Results: A novel homozygous intronic GHRHR c.752-

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1G1A (IVS7-1G1A) mutation, predicting loss of the constitutive splice acceptor site, was identified in two siblings with IGHD. A compound heterozygous c.[57+1G1A];[1146G1A] and a heterozygous c.527C1T (p.A176V) were found in two sporadic cases. Haplotype analysis provided evidence for a founder effect for the c.57+1G1A mutation and independent recurrence for the c.1146G1A mutation. Conclusion: We report a novel splice-disrupting mutation in GHRHR in 2 siblings and provide evidence that all c.57+1G1A (IVS1+1G1A) mutant chromosomes have the same haplotype ancestor, indicating the occurrence of a founder effect in Brazilian patients with IGHD. Copyright © 2012 S. Karger AG, Basel

Introduction

Isolated GH deficiency (IGHD) can be congenital or acquired. A variable proportion of patients with congenital IGHD have consanguineous parents or an affected Suemi Marui Laboratório de Endocrinologia Molecular e Celular LIM-25 e Laboratório de Hormônios e Genética Molecular LIM-42 HCFMUSP Av. Dr. Arnaldo, 455, sala 4345, 01246-903 Sao Paulo (Brazil) E-Mail suemimar @ usp.br

relative, suggesting a genetic etiology [1, 2]. Familial IGHD has been traditionally classified according to the inheritance pattern in autosomal recessive (types IA and IB), autosomal dominant (type II) or X linked (type III). More recently, identification of genes that lead to IGHD allowed also a classification according to the underlying gene defect. Most patients with autosomal dominant IGHD have mutations in GH1, whereas most patients with autosomal recessive IGHD have mutations in GHreleasing hormone receptor gene (GHRHR) [1]. However, this relation might be influenced by the geographic region and founder effect of mutations. GHRHR is a G protein-coupled receptor with 423 amino acids containing an amino-terminal extracellular domain, seven transmembrane helices and a carboxy-terminal intracellular domain [3]. This receptor is highly expressed at anterior pituitary somatotroph cell membranes. Its activation by GH-releasing hormone (GHRH) results predominantly in Gs stimulation of cAMP leading to somatotroph proliferation, and synthesis and secretion of GH [3]. The gene that codes for GHRHR contains 13 exons and is located at chromosome 7p14 [4]. The first human GHRHR mutation (p.E72X) was described in 2 Indian cousins with IGHD born to consanguineous parents [5]. Subsequently, this mutation was identified in several different families from the Indian subcontinent, and a founder effect has been demonstrated [6]. In 1999, Salvatori et al. [7] identified the c.57 + 1G1A (IVS1 + 1G1A) GHRHR mutation in 22 patients with IGHD from a large kindred from the region of Itabaianinha in the state of Sergipe in Northeastern Brazil. In a previous study, we screened for this mutation in a cohort of 33 Brazilian patients (31 independent probands, 13 with normal and 18 with ectopic posterior pituitary lobe) with IGHD evaluated in São Paulo (more than 2,000 km from Itabaianinha) and detected the c.57 + 1G1A GHRHR mutation in homozygosity in 3 patients (2 brothers and another familial case), and in compound heterozygosity associated with a then novel c.365-2G1A (IVS4-2A 1G) GHRHR mutation in one sporadic case of IGHD [8]. All patients with GHRHR mutations who had been described previously had anterior pituitary hypoplasia on MRI, and we demonstrated for the first time that GHRHR mutations may occur in association with normal pituitary height; a finding later confirmed in other patients with GHRHR mutations [8, 9]. In contrast, the posterior pituitary lobe has had a consistently normal position in all patients with GHRHR mutations described so far [1]. Therefore, GHRHR was further analyzed only in patients with the posterior lobe in the normal position. 166

Horm Res Paediatr 2012;78:165–172

In the present study, we analyzed GHRHR in a cohort of patients with sporadic and familial forms of IGHD and a topic posterior pituitary, and identified one novel and two previously reported mutations. We also constructed haplotypes with GHRHR SNPs and microsatellites flanking GHRHR that indicated common ancestry for the c.57+1G1A (IVS1+1G1A) mutation in families originating from different parts of Brazil and independent mutational events for c.1146G1A. Subjects and Methods Informed parental consent, patient assent and approval by the Hospital Ethics Committee were obtained before initiating the genetic studies. Eighteen patients (16 independent probands, 10 boys) were diagnosed with IGHD in São Paulo (Southern Brazil) after failure of GH stimulation by clonidine and insulin-induced hypoglycemia (patients 1–18; table 1). Seven patients who had been previously screened only for the c.57 + 1G1A (IVS1 + 1G1A) mutation and were negative were now included for a more complete GHRHR study [8] (table 1). No patient had evidence of TSH, ACTH or gonadotropin deficiencies after basal thyroid hormone, cortisol and sex steroid concentrations, and TRH and GnRH stimulation tests at presentation and during follow-up [8]. Fourteen patients had no family history of GH deficiency and were considered as sporadic cases. Parental consanguinity was referred by 2 siblings and 3 sporadic cases with IGHD. Chronological ages ranged from 1.6 to 15.9 years (mean 8 SD, 8.9 8 4.4 year), and all patients had proportionate short stature with heights between –2.1 and –6.2 SD scores (SDS; –4.0 8 1.1). Most patients developed spontaneous puberty or menarche during follow-up. These patients were part of a previous study in which GH-1 and GHRH mutations had been excluded in all patients [10]. Pituitary MRI was performed in all patients and maximal height of the pituitary gland was measured perpendicular to the floor of the sella turcica and considered hypoplastic when !–2 SD compared to normal controls [11] (table 1). To investigate whether recurrence of the GHRHR c.57 + 1G1A (IVS1 + 1G1A) mutation was due to a founder effect, DNA of patient 3, of 4 patients with this mutation previously reported by us [8] (patients 19–22, table 1 and fig. 1b–d), and of 4 patients from Itabaianinha, Sergipe, Brazil, homozygous for c.57 + 1G1A (fig. 1c) were studied. Patients 1–22 were ascertained in São Paulo, more than 2,000 km from Itabaianinha. For the study of a founder effect of c.1146G1A, DNA samples of one new Brazilian patient (patient 3; fig. 1d) and of two siblings with this mutation, previously reported in Japan, were analyzed in Dr. Inoue’s laboratory [12]. DNA Analysis Genomic DNA was extracted from peripheral blood leukocytes by standard techniques. The 13 coding exons, exon-intron junctions, and a selected promoter region (–327 to –42) of the GHRHR gene (ENST00000326139) were amplified by PCR and sequenced, using primers and conditions previously described [7].

Marui et al.

Table 1. Clinical and biochemical characteristics of patients with isolated GH deficiency with and without GHRHR mutations

Patient

Sex

Age, years Height, SDS GH peak, ␮g/l AP, MRI GHRHR mutation

1a 2a 3 4 5 6 7 8 9 10b 11 12b 13 14 15 16 17 18 19 20c 21c 22

M F F F M M M F M M M F F F M M F M F M M F

10.1 3.5 7.1 10.3 7.6 15.9 15.7 12.5 9.8 1.6 13.7 2.5 4.5 10.7 2.9 10.8 10.7 14.8 12.1 7.5 6.0 4.7

–3.9 –2.5 –3.3 –4.5 –3.8 –5.0 –4.2 –4.1 –2.1 –5.2 –3.7 –4.0 –4.4 –2.6 –2.2 –4.8 –2.2 –4.4 –6.2 –4.7 –4.8 –4.4

2.6d 3.5d 0.1 0.7 0.6 3.6d 2.8 0.3 2.2 0.5 1.2 1.0 1.2 0.3 1.1 4.3d 2.6 1.6 0.2 2.0 0.6 1.9

– – N N N H H H N N N N H H N H N H H N N H

c.[752–1G>A];[ 752–1G>A] c.[752–1G>A];[ 752–1G>A] c.[57+1G>A];[1146G>A] c.[527C>T]; [=] none none none none none none none none none none none none none none c.[57+1G>A];[ 57+1G>A] c.[57+1G>A];[ 57+1G>A] c.[57+1G>A];[ 57+1G>A] c.[57+1G>A];[365-2G>A]

Family history No.e F, C F, C S S S, C S S, C S S F S, C F S S S S S S F, C F, C F, C S

new new new 11 new 12 new new new 10 new 9 13 new new 16 14 new 5 6 7 8

AP = anterior pituitary; H = hypoplastic; N = normal; F = familial; S = sporadic; C = consanguineous parents. a–c Siblings patients. d Measured by RIA. e Patients previously described in Osorio et al. [8] with the corresponding number.

All mutations were found on both strands and confirmed in two separate PCR reactions. Computational splice site prediction was performed using NetGene2 software (http://genome.cbs.dtu. dk/services/NetGene2/). All mutations were described according to HGSV nomenclature recommendations (www.hgvs.org/). For reference, the classical mutation nomenclature is provided in parentheses.

(rs2302019), c.–235C1T (rs230220), c.–45C 1T (rs2302021), c.975– 26G1A (rs4988504) and c.1146G1A by direct sequencing. Patient 3 and her parents were evaluated, and these results compared with previously published data from Japanese c.1146G1A mutated patients [12].

Results

Haplotype Analysis Microsatellites flanking the GHRHR gene in 7p15, the allele frequencies, the marker heterozygosity and the sequence-specific primers for the amplification of each microsatellite were retrieved from the UCSC Genome Browser (http://genome.ucsc.edu/). Markers analyzed were: D7S632, D7S526, AY179323 and AY179324, located respectively 193, 70, 33 kb distal (in the direction of the telomere) and 13 kb proximal (in the direction of the centromere) from the GHRHR locus. PCR conditions are available upon request. PCR products were submitted to capillary electrophoresis in an automatic sequencer and analyzed by GeneScan software. Two SNPs c.–261C1T (rs2302019) and c.–235C 1T (rs230220) in the promoter region of GHRHR were evaluated by direct sequencing. To determine whether the alleles carrying the c.1146G1A, p.E382E mutation in patient 3 and the affected Japanese sisters share common ancestry, we studied simultaneously seven microsatellites (D7S516, D7S2496, D7S2252, D7S484, D7S510, D7S691 and D7S2427) and 5 possible SNP/mutation sites c.–261C1T

Patients with IGHD and GHRHR Mutations Patients 1 and 2 with Homozygous GHRHR c.752-1G1A (IVS7-1G1A) In one boy (patient 1, family A, table 1 and fig. 1a) and his sister (patient 2), a novel c.752-1G1A (IVS7-1G1A) splicing mutation was identified in homozygous state. Their father (167 cm, SDS –1.2) and mother (156 cm, SDS –1.0) were first cousins who are both carriers of the same c.752-1G1A mutation (fig. 1a). Patient 1 was born after 39 weeks’ gestation weighing 2,620 g (SDS –0.8) and measuring 47 cm (SDS –2.3) in length. He sought medical attention at the age of 4.7 years due to short stature with a height of 88.4 cm (SDS –3.9) and weight of 11.1 kg (SDS –2.6). He had a large forehead,

GHRH Receptor Mutations


167

Patients 1 and 2, family A

Patients 20 and 21, family B NA

c.752-1G>A/wt

c.752-1G>A/wt

c.752-1G>A/752-1G>A

209 125 316 C C c.57+1G>A 332

c.752-1G>A/752-1G>A

a

b

Patient 19, family C

Itabaianinha patients

209 125 316 C C c.57+1G>A 332

209 125 316 C C c.57+1G>A 332

209 125 316 C C c.57+1G>A 332

213 121 322 T C wt 336

209 125 316 C C c.57+1G>A 332

217 121 322 C C wt 330

Patient 22, family D

213 209 121 125 322 316 T C C C wt c.57+1G>A 336 332

213 121 322 T C wt 336

Patient 3, family E

NA 209 125 316 C C c.57+1G>A 332

217 127 320 C C wt 330

209 125 316 C C c.57+1G>A 332

209 125 316 C C c.57+1G>A 332

209 125 ND C C c.57+1G>A ND

209 125 ND C C c.57+1G>A ND

215 127 318 C C wt 332

209 127 320 T T c.365-2A>G 330

217 121 316 C C c.57+1G>A 332

d

209 127 320 T T c.365-2A>G 330

217 121 316 C C c.57+1G>A 332

209 129 318 T C wt 332

209 125 322 T T wt 330

217 127 318 T T p.E382E 332

209 123 316 C C c.57+1G>A 340

207 121 316 T T wt 330

NA

c

209 125 316 C C c.57+1G>A 332

209 125 316 C C c.57+1G>A 332

209 125 316 C C c.57+1G>A 332

209 125 316 C C c.57+1G>A 332

209 125 316 C C c.57+1G>A 332

209 125 316 C C c.57+1G>A 332

217 127 318 T T p.E382E 332

209 123 316 C C c.57+1G>A 340

Fig. 1. a Familial segregation of the novel c.752-1G1A GHRHR mutation. b, c Haplotype analysis of cases with homozygous c.57 + 1G1A (IVS1 + 1G1A), including patients from Itabaianinha. d Haplotype analysis of non-familial patients with c.57 + 1G1A (IVS1 + 1G1A) in compound heterozygosis. Microsatellites/

position: D7S632 (209–217)/30,824,240 bp, D7S526 (121–129)/ 30,937,754 bp, AY179323 (316–322)/30,970,150 bp, AY179324 (330–340)/31,031,650 bp; GHRHR/31,003,638 bp; GHRHR SNPs: –261 C1T and –235C1T.

increased truncal fat and a high-pitched voice. Basal IGF1 level was 18 ng/ml, and peak GH measured by radioimmunoassay (RIA) was 1.3 ng/ml (1.3 ␮g/l) after a clonidine test, and 2.6 ng/ml (2.6 ␮g/l) after insulin-induced hypoglycemia. Treatment with recombinant GH was

started at 7.9 years with a good initial response, but supply of the medication was discontinuous. At the age of 11.9 years, with a height of 133 cm and weight of 33.3 kg, he developed spontaneous puberty. When he had a chronological age of 15.6 years and bone age of 16 years, he

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had attained 159.2 cm (SDS –1.4) and GH treatment was stopped. He was retested at the age of 20 years and had a basal IGF-1 level of 47 ng/ml and peak GH after insulininduced hypoglycemia of 0.08 ng/ml (0.08 ␮g/l). Patient 2 was born after a 38-week gestation with a birthweight of 2,960 g (SDS –0.2) and birth length of 49 cm (SDS –0.1). She had neonatal hypoxia without need of resuscitation, and did not have hypoglycemia or prolonged jaundice. Neuromotor development was normal. Patient 2 presented at the age of 3.0 years with a height of 82 cm (SDS –2.9) and weight of 11 kg (SDS –2.0). Peak GH measured by RIA was 3.49 ng/ml (3.49 ␮g/l) after clonidine, and 1.76 ng/ml (1.76 ␮g/l) after insulin-induced hypoglycemia. Treatment with recombinant GH (0.1 U/ kg.day) was started and resulted in a growth rate of 10.1 cm/years in the first year. Puberty started at 9.1 years with a height of 132.4 cm and weight of 34.4 kg, and menarche occurred at 10.9 years. GH replacement was stopped at 11.9 years, and she reached an adult height of 152 cm (SDS –1.7). GH peak during retesting at transition was 0.1 ng/ml (0.1 ␮g/l). c.752-1G1A is located in a canonical site of intron 7 and in silico analysis of this nucleotide substitution predicts loss of the constitutive splice acceptor site. This alteration was not found in 100 control alleles and had not been described in single-nucleotide polymorphism databases. Patient 3 Compound Heterozygous for GHRHR c.1146G1A p.E382E and c.57 + 1G1A In a girl with sporadic IGHD (patient 3, family E, fig. 1d), the synonymous p.E382E (c.1146G1A, exon 12) mutation was found in compound heterozygosity with the c.57 + 1G1A mutation. The father measured 172 cm (SDS –0.4) and was heterozygous for the c.1146G1A mutation. The unrelated mother was 164 cm tall (SDS +0.3) and was heterozygous for the c.57 + 1G1A mutation (family E; fig. 1d). There was no family history of short stature or consanguinity. Patient 3 was born after a 38-week gestation with a weight of 3,100 g (SDS –0.1) and a length of 47 cm (SDS –1.4). Her father measured 155 cm (SDS –3.0) and her mother 148 cm (SDS –2.4). At the age of 7 years she presented with short stature: her height was 101.6 cm (SDS –3.3) and weight 14.7 kg (SDS –2.3). Peak GH after two insulin-induced hypoglycemia tests was 0.1 ng/ml (0.1 ␮g/l). There was no evidence of other pituitary hormone deficiencies. At the age of 7.9 years (in the 1980s), therapy was started with pituitary-derived GH, but availability was discontinuous and she grew 6.5 cm in the first year GHRH Receptor Mutations

of treatment. Puberty developed spontaneously. After 7.8 years of GH replacement, at the age of 15.5 years, she reached a height of 151.1 cm (SDS –1.9). At 16.9 years, MRI revealed reduced pituitary height (5.0 mm, –3.6 SDS) and a topic posterior lobe. She was retested at the age of 20 years (before genetic studies): her basal IGF-1 level was !18 ng/ml, and during insulin-induced hypoglycemia, serum GH peaked at 0.1 ng/ml (0.1 ␮g/l). c.57 + 1G1A GHRHR mutation had been described in homozygous state in a large kindred with IGHD in the Northeast Brazil (Itabaianinha) [7]. c.1146G1A is located in the last nucleotide of exon 12, immediately upstream of the splice donor site. This mutation was previously reported [12]. Although it does not predict change of amino acids, in vitro splicing studies showed skipping of exon 12 [12]. Patient 4 Heterozygous for GHRHR c.527C1T p.A176V In another girl with sporadic IGHD (patient 4), the missense mutation p.A176V (c.527C1T, exon 6) was identified in heterozygous state. Patient 4 was born after 38 weeks’ gestation with 3,400 g (SDS +0.2) to non-consanguineous parents, and there was no family history of short stature. She presented at the age of 10.3 years with short stature (height 109.2 cm, SDS –4.5) and weight of 22 kg. Peak GH after stimulation was 0.7 ng/ml (0.7 ␮g/l). She was treated with GH and reached an adult height of 152 cm. At 14 years of age, MRI showed a normal anterior pituitary height (5.6 mm) and topic posterior pituitary lobe. In the other allele, no mutation was detected in the coding region, exon-intron junctions and promoter POU1F1 binding site in this patient. MLPA analysis also excluded whole exon deletions in GHRHR (data not shown). The p.A176V mutation was previously reported in the literature [13]. In the remaining 14 patients, the coding region and exon-intron boundaries of GHRHR had a normal sequence. GHRHR Haplotype Analysis Haplotype analysis of all individuals carrying the c.57 + 1G1A (IVS1 + 1G1A) mutation is shown in figure 1 (b–d). The 4 patients from Itabaianinha and patients 19– 21, all of them homozygous for the c.57 + 1G1A (IVS1 + 1G1A), shared the same 209-125-316-C-C-332 haplotype (fig. 1b, c), indicating that this mutation occurred in a unique chromosomal background (founder effect). Moreover, in patients 3 and 22, harboring the c.57 + 1G1A in Horm Res Paediatr 2012;78:165–172

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Table 2. GHRHR gene mutations identified in patients with IGHD

Mutation

Gene position Type

State

Functional consequence

Inheritance Reference

c.–124A>C p.Val10Gly

promoter exon 1

regulator missense

CH HT

S S

[14] [15]

c.57+1G>Aa c.57+2T>Ga p.Gln43X p.Glu72X c.268+1G>Aa p.Arg94Leu c.391delG c.366–2A>Ga p.His137Leu p.Leu144His p.Arg161Trp p.Ala176Val p.Ala222Glu p.Phe242Cys c.752+1G>Ca c.752-1G>A c.812+1G>Aa p.Trp273Ser p.Lys329Glu p.Arg357Cys c.1121–1124del p.Glu382Glu c.1146+2T>Aa c.1140–1144del

intron 1 intron 1 exon 2 exon 3 intron 3 exon 4 exon 4 intron 4 exon 5 exon 5 exon 6 exon 6 exon 7 exon 7 intron 7 intron 7 intron 8 exon 9 exon 11 exon11 exon 12 exon 12 intron 12 exon 13

splicing splicing nonsense nonsense splicing missense nonsense splicing missense missense missense missense missense missense splicing splicing splicing missense missense missense deletion synonymous splicing deletion

HM and CH HM CH HM and CH HT HM HM CH CH HM and CH CH HM HM CH HM HM HM HM HT HM HM HM and CH HM CH

lower GHRHR gene expression lower GHRHR gene expression at cellular surface splicing alterationb splicing alterationb truncated proteinb truncated proteinb splicing alterationb NR frameshift/truncatedb NR lower cAMP after GHRH stimulation lower cAMP after GHRH stimulation NR lower cAMP after GHRH stimulation lower cAMP after GHRH stimulation lower cAMP after GHRH stimulation splicing alterationb splicing alterationb splicing alterationb NR lower cAMP after GHRH stimulation lower cAMP after GHRH stimulation NR splicing alterationb splicing alterationb lower cAMP after GHRH stimulation

AR and S AR AR AR AR S AR S AR AR AR AR AR AR AR AR AR AR AR AR S AR AR AR

[7, 8]; this report [16] [17] [5, 18–21] [17] [19] [22] [8] [23] [24] [19] [13]; this report [19, 24] [24] [25] this report [26] [19] [14] [27] [28] [12]; this report [9] [23]

CH = Compound heterozygous; HT = heterozygous; HM = homozygous; AR = autosomal recessive; S = sporadic; NR = not reported. a c.57+1G>A (IVS1+1G>A); c.57+2T>G (IVS1+2T>G); c.268+1G>A (IVS3+1G>A); c.366–2A>G (IVS4–2A>G); c.752+1G>A (IVS7+1G>A); c.812+1G>A (IVS8+1G>A); c.1146+2T>A (IVS12+2T>A). b Even functional studies to nonsense mutation and at splicing consensus region were not performed, they are certainly deleterious, producing truncated protein and splicing errors, respectively.

compound heterozygosity, this mutation segregated with 209-123-316-C-C-340 and 217-121-316-C-C-332 haplotypes, respectively (fig. 1d). However, the 316-bp allele shared by these patients is present in only 5% of hundred alleles of a control Brazilian population, suggesting the occurrence of a founder mutation also in these patients with IGHD. In contrast, whereas the siblings from Japan exhibited the same haplotype 317-213-C-C-C-G-254-107-89-148224 [12], the allele carrying the c.1146G1A of patient 3 (313-232-T-T-C-G-256-107-85-150-248) did not share any of these markers, and only includes two of these SNPs, excluding a possible founder effect (online suppl. table 1, www.karger.com/doi/10.1159/000342760).

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Discussion

Here, we describe the novel c.752-1G1A (IVS7-1G1A) splicing GHRHR mutation in 2 siblings with IGHD. The patients’ clinical features are similar to those previously described in other patients with GHRHR mutations (table 2). By in silico analysis, this mutation was predicted to impair the normal splicing of transcript generating a truncated GHRHR protein or undergoing non-sensemediated mRNA decay and lead to the absence of protein production. Interestingly, the majority of GHRHR mutations were familial, and sporadic cases with GHRHR mutations have been rare [1]. The c.1146G1A, predicted to skip exon 12, was found in compound heterozygosity with c.57 + Marui et al.

1G1A in a patient with sporadic IGHD. This mutation had been recently reported in 2 sisters from Japan with IGHD [12]. Splicing in vitro studies confirmed the impairment of GHRHR mRNA splicing in p.E382E transcripts [12]. Haplotype analysis in the Brazilian and Japanese patients suggested no evidence for a founder effect but independent mutational events in different ethnic groups. The recurrent p.E382E (c.1146G1A) mutation is a G-to-A transition not located at CpG dinucleotides. Most recurring GHRHR mutations shared common ancestry, and c.1146G1A is only the second independently recurring mutation after the previously reported p.L144H [24]. The c.57 + 1G1A (IVS1 + 1G1A) mutation, initially described in patients from Northeastern Brazil was recurrent among Brazilian patients with IGHD studied up to 2,000 km apart [7, 8]. As high carrier rates are usually attributed to a founder effect, we investigated the origin of this mutation constructing the haplotypes of the c.57 + 1G1A alleles with intragenic SNPs and highly polymorphic markers flanking GHRHR. We found evidence for a founder effect for this mutation indicating that patients shared the same ancestry. The p.A176V was detected in heterozygosis in one female patient with sporadic IGHD. This same mutation had been described before in homozygosis in 2 male siblings born in Pakistan whose mother and unaffected siblings were carriers of the p.A176V [13]. Cells expressing the A176V receptor had a significantly reduced cAMP response to GHRH, despite appropriate expression on the cell surface [13]. Our patient had no evidence of Pakistani or Asian origin. Despite intense efforts, no additional mutation was found in GHRHR in our heterozygous p.A176V patient. However, the possibility of a pathogenic mutation in a not analyzed GHRHR region (for example intronic and 5ⴕ and 3ⴕ untranslated) cannot be ruled out. GH1 and GHRH genes had been screened and were normal. It is also possible that the patient does not carry a second GHRHR mutation but that a change in another gene or that other yet unidentified factors contribute to the phenotype and act synergistically. The phenotype of this patient was not different from that of those with homozygous or compound heterozygous GHRHR mutations. It is noteworthy that 4 patients bearing GHRHR mutations (patients 4, 20, 21 and 22) had normal anterior pituitary height [8]. Of note, normal pituitary height is based on a limited number of individuals in some age groups [8]. MRI was repeated in two patients after 3 years of follow-up, up to the age of 15 years, but pituitary size

did not change (data not shown). Variability of pituitary size among members of the same family with a GHRHR mutation had also been reported [9]. Therefore, normal anterior pituitary size does not exclude a possible GHRHR mutation in patients with IGHD. More than 20 distinct GHRHR mutations have been reported so far, including non-sense, splice site, microdeletions, missense and promoter mutations [1]. All of these mutations were demonstrated to impair normal receptor structure, function or expression (table 2). Of note, there appears to be no phenotype-genotype correlation. The phenotype of the patients has been uniformly that of GH deficiency with severe short stature, low GH levels and a topic posterior pituitary lobe, independently of the specific mutation [1]. Midfacial hypoplasia, neonatal hypoglycemia and micropenis, as found in patients with recessive GH1 mutations are rare [1]. Alatzoglou et al. [19] compared the phenotype of 26 patients with GH1 mutations with that of 15 patients with GHRHR mutations and found no significant differences. However, variable height deficit, variable peak GH levels and late development of other pituitary deficiencies, as has been described in patients with some autosomal dominant GH1 mutations, have not been found in patients with GHRHR mutations [1]. In conclusion, analysis of the GHRHR gene in our cohort of patients with IGHD and posterior pituitary lobe in the normal position revealed a novel c.752-1G1A (IVS7-1G1A) splice-disrupting mutation in 2 siblings, compound heterozygosity for c.57 + 1G1A (IVS7 + 1G1A) and c.1146G1A, p.E382E in a sporadic patient, and a founder effect for the c.57 + 1G1A mutation among 5 families.

GHRH Receptor Mutations


Acknowledgments This work was supported by grants from Fundação de Amparo a Pesquisa do Estado de São Paulo 99/10692-8 (S.M.), 00/06677-2 (B.B.M.) and 00/14092-4, and Conselho Nacional de Pesquisa 475870/2009-3 and 301477/2009-4 (to A.A.L.J.), 301339/2008-9 (to B.B.M.) and 300982/2009-7 (to I.J.P.A.). The authors thank Ms. Yukiko Sakamoto and Prof. Mitsuo Itakura from the University of Tokushima, Japan, for the microsatellite studies regarding p.E382E; Anita H.O. Souza and Rafael A. Meneguz-Moreno from the Federal University of Sergipe, Aracaju, Brazil for the help in Itabaianinha studies, Dr. Roberto Salvatori from the Division of Endocrinology, Johns Hopkins University, Baltimore, Md., USA, for relevant suggestions, and Drs. Chin Jia Lin, Ângela Silva Barbosa, Ana E.C. Billerbeck from the University of São Paulo, São Paulo, Brazil, and Peter Kopp and Margrit Urbanek from the Division of Endocrinology, Metabolism and Molecular Medicine, Northwestern University Feinberg School of Medicine, Chicago, Ill., USA, for their assistance.

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References 1 Alatzoglou KS, Dattani MT: Genetic causes and treatment of isolated growth hormone deficiency – an update. Nat Rev Endocrinol 2010;6:562–576. 2 Rona RJ, Tanner JM: Aetiology of idiopathic growth hormone deficiency in England and Wales. Arch Dis Child 1977;52:197–208. 3 Mayo KE: Molecular cloning and expression of a pituitary-specific receptor for growth hormone-releasing hormone. Mol Endocrinol 1992; 6:1734–1744. 4 Vamvakopoulos NC, Kunz J, Olberding U, Scherer SW, Sioutopoulou TO, Schneider V, Durkin AS, Nierman WC: Mapping the human growth hormone-releasing hormone receptor (GHRHR) gene to the short arm of chromosome 7 (7p13-p21) near the epidermal growth factor receptor (EGFR) gene. Genomics 1994;20:338–340. 5 Wajnrajch MP, Gertner JM, Harbison MD, Chua SC, Leibel RL: Nonsense mutation in the human growth hormone-releasing hormone receptor causes growth failure analogous to the little (lit) mouse. Nat Genet 1996; 12:88–90. 6 Wajnrajch MP, Gertner JM, Sokoloff AS, Ten I, Harbison MD, Netchine I, Maheshwari HG, Goldstein DB, Amselem S, Baumann G, Leibel RL: Haplotype analysis of the growth hormone releasing hormone receptor locus in three apparently unrelated kindreds from the Indian subcontinent with the identical mutation in the GHRH receptor. Am J Med Genet A 2003;120A:77–83. 7 Salvatori R, Hayashida CY, Aguiar-Oliveira MH, Phillips JA, Souza AH, Gondo RG, Toledo SP, Conceicão MM, Prince M, Maheshwari HG, Baumann G, Levine MA: Familial dwarfism due to a novel mutation of the growth hormone-releasing hormone receptor gene. J Clin Endocrinol Metab 1999;84:917–923. 8 Osorio MG, Marui S, Jorge AA, Latronico AC, Lo LS, Leite CC, Estefan V, Mendonca BB, Arnhold IJ: Pituitary magnetic resonance imaging and function in patients with growth hormone deficiency with and without mutations in GHRH-R, GH-1, or PROP-1 genes. J Clin Endocrinol Metab 2002; 87: 5076–5084. 9 Alba M, Hall CM, Whatmore AJ, Clayton PE, Price DA, Salvatori R: Variability in anterior pituitary size within members of a family with GH deficiency due to a new splice mutation in the GHRH receptor gene. Clin Endocrinol (Oxf) 2004;60:470–475. 10 França MM, Jorge AA, Alatzoglou KS, Carvalho LR, Mendonca BB, Audi L, Carrascosa A, Dattani MT, Arnhold IJ: Absence of GHreleasing hormone (GHRH) mutations in selected patients with isolated GH deficiency. J Clin Endocrinol Metab 2011; 96:E1457– E1460.

172

11 Argyropoulou M, Perignon F, Brunelle F, Brauner R, Rappaport R: Height of normal pituitary gland as a function of age evaluated by magnetic resonance imaging in children. Pediatr Radiol 1991;21:247–249. 12 Inoue H, Kangawa N, Kinouchi A, Sakamoto Y, Kimura C, Horikawa R, Shigematsu Y, Itakura M, Ogata T, Fujieda K, Consortium JGG: Identification and functional analysis of novel human growth hormone-releasing hormone receptor (GHRHR) gene mutations in Japanese subjects with short stature. Clin Endocrinol (Oxf) 2011;74:223–233. 13 Carakushansky M, Whatmore AJ, Clayton PE, Shalet SM, Gleeson HK, Price DA, Levine MA, Salvatori R: A new missense mutation in the growth hormone-releasing hormone receptor gene in familial isolated GH deficiency. Eur J Endocrinol 2003;148:25–30. 14 Salvatori R, Fan X, Mullis PE, Haile A, Levine MA: Decreased expression of the GHRH receptor gene due to a mutation in a Pit-1 binding site. Mol Endocrinol 2002; 16: 450–458. 15 Godi M, Mellone S, Petri A, Arrigo T, Bardelli C, Corrado L, Bellone S, Prodam F, Momigliano-Richiardi P, Bona G, Giordano M: A recurrent signal peptide mutation in the growth hormone releasing hormone receptor with defective translocation to the cell surface and isolated growth hormone deficiency. J Clin Endocrinol Metab 2009; 94: 3939–3947. 16 Hilal L, Hajaji Y, Vie-Luton MP, Ajaltouni Z, Benazzouz B, Chana M, Chraïbi A, Kadiri A, Amselem S, Sobrier ML: Unusual phenotypic features in a patient with a novel splice mutation in the GHRHR gene. Mol Med 2008; 14:286–292. 17 Salvatori R, Fan X, Veldhuis JD, Couch R: Serum GH response to pharmacological stimuli and physical exercise in two siblings with two new inactivating mutations in the GHreleasing hormone receptor gene. Eur J Endocrinol 2002;147:591–596. 18 Netchine I, Talon P, Dastot F, Vitaux F, Goossens M, Amselem S: Extensive phenotypic analysis of a family with growth hormone (GH) deficiency caused by a mutation in the GH-releasing hormone receptor gene. J Clin Endocrinol Metab 1998; 83:432–436. 19 Alatzoglou KS, Turton JP, Kelberman D, Clayton PE, Mehta A, Buchanan C, Aylwin S, Crowne EC, Christesen HT, Hertel NT, Trainer PJ, Savage MO, Raza J, Banerjee K, Sinha SK, Ten S, Mushtaq T, Brauner R, Cheetham TD, Hindmarsh PC, Mullis PE, Dattani MT: Expanding the spectrum of mutations in GH1 and GHRHR: genetic screening in a large cohort of patients with congenital isolated growth hormone deficiency. J Clin Endocrinol Metab 2009;94:3191–3199.


20 Baumann G, Maheshwari H: The Dwarfs of Sindh: severe growth hormone (GH) deficiency caused by a mutation in the GH-releasing hormone receptor gene. Acta Paediatr Suppl 1997; 423:33–38. 21 Sıklar Z, Berberoğlu M, Legendre M, Amselem S, Evliyaoğlu O, Hacıhamdioğlu B, Erdeve SS, Oçal G: Two siblings with isolated GH deficiency due to loss-of-function mutation in the GHRHR gene: successful treatment with growth hormone despite late admission and severe growth retardation. J Clin Res Pediatr Endocrinol 2010;2:164–167. 22 Shohreh R, Sherafat-Kazemzadeh R, Jee YH, Blitz A, Salvatori R: A novel frame shift mutation in the GHRH receptor gene in familial isolated GH deficiency: early occurrence of anterior pituitary hypoplasia. J Clin Endocrinol Metab 2011;96:2982–2986. 23 Salvatori R, Fan X, Phillips JA, Prince M, Levine MA: Isolated growth hormone (GH) deficiency due to compound heterozygosity for two new mutations in the GH-releasing hormone receptor gene. Clin Endocrinol (Oxf) 2001;54:681–687. 24 Salvatori R, Fan X, Phillips JA, EspigaresMartin R, Martin De Lara I, Freeman KL, Plotnick L, Al-Ashwal A, Levine MA: Three new mutations in the gene for the growth hormone (GH)-releasing hormone receptor in familial isolated GH deficiency type IB. J Clin Endocrinol Metab 2001;86:273–279. 25 Roelfsema F, Biermasz NR, Veldman RG, Veldhuis JD, Frölich M, Stokvis-Brantsma WH, Wit JM: Growth hormone (GH) secretion in patients with an inactivating defect of the GH-releasing hormone (GHRH) receptor is pulsatile: evidence for a role for nonGHRH inputs into the generation of GH pulses. J Clin Endocrinol Metab 2001; 86: 2459–2464. 26 Wang Q, Diao Y, Xu Z, Li X, Luo XP, Xu H, Ouyang P, Liu M, Hu Z, Wang QK, Liu JY: Identification of a novel splicing mutation in the growth hormone (GH)-releasing hormone receptor gene in a Chinese family with pituitary dwarfism. Mol Cell Endocrinol 2009;313:50–56. 27 Haskin O, Lazar L, Jaber L, Salvatori R, Alba M, Kornreich L, Phillip M, Gat-Yablonski G: A new mutation in the growth hormone-releasing hormone receptor gene in two Israeli Arab families. J Endocrinol Invest 2006; 29: 122–130. 28 Horikawa R: Isolated GH deficiency due to inactivating mutation of GHRH receptor (in Japanese). Nihon Rinsho 2002;60:297–305.

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