Growth Hormone & IGF Research 24 (2014) 227–232
Contents lists available at ScienceDirect
Growth Hormone & IGF Research journal homepage: www.elsevier.com/locate/ghir
A novel gross indel in the growth hormone releasing hormone receptor gene of Indian IGHD patients Shantanu Kale ⁎, Sweta Budyal, Rajeev Kasaliwal, Vyankatesh Shivane, Vijaya Raghavan, Anurag Lila, Tushar Bandgar, Nalini Shah Department of Endocrinology, Seth G.S. Medical College, K.E.M. Hospital, Parel, Mumbai 400012, India
a r t i c l e
i n f o
Article history: Received 17 July 2014 Accepted 29 July 2014 Available online 7 August 2014 Keywords: Novel gross indel GHRHR IGHD Indian
a b s t r a c t Context: Cohort specific mutations in the growth hormone (GH1) and growth hormone-releasing hormone receptor (GHRHR) genes have been reported worldwide in isolated growth hormone deficiency (IGHD) patients. However, limited data is available on ethnically diverse Indian IGHD patients. Objective: The aim of the study was to find GH1 and GHRHR gene mutations in Indian IGHD patients from two unrelated non-consanguineous families. Design: The 5′ and 3′ untranslated regions (UTRs) and coding regions with splice sites of the GH1 and GHRHR genes were sequenced for all patients (n = 6). Family members and 20 controls were evaluated for the sequence variants identified in the index patients. Online bioinformatics tools were used to confirm mutations and their pathogenicity. Results: GHRHR gene mutations were observed in all patients. Interestingly, a novel indel g.30999250_ 31006943delinsAGAGATCCA was observed in both the unrelated families. Three patients were homozygous for the novel indel, two were homozygous for the previously reported p.E72X mutation and one was compound heterozygous with both the mutations (indel and p.E72X) in the GHRHR gene. The novel indel has resulted in the loss of 5′ regulatory region and exon 1 of the GHRHR gene impairing the GHRHR expression. All the normal family members were heterozygous either for the indel or p.E72X mutation. None of the patients had GH1 gene mutations. Conclusions: We describe a novel gross indel in the GHRHR gene resulting in the loss of 5′ regulatory region and GHRHR exon 1 in four IGHD IB patients from two unrelated non-consanguineous Indian families. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Isolated growth hormone deficiency (IGHD) is the most common pituitary hormone deficiency. IGHD manifests due to defects in the synthesis or secretion of functional growth hormone (GH). Genetic aetiology may be found in up to 30% of IGHD patients. Mutations in the genes controlling hypothalamo-pituitary-GH axis have been identified as a cause of IGHD [1]. Based on the pattern of Mendelian inheritance, IGHD is classified into three categories: IGHD types IA and IB have autosomal recessive inheritance, while IGHD type II is autosomal dominant and IGHD type III is X-linked [2,3]. IGHD results from mutations in the GH1 and GHRHR genes. Mutations or rearrangements in the GH1 gene lead to the production of an aberrant GH molecule that either has partial function or generates immune tolerance. Although, the prevalence of GH1 gene mutations is very high in IGHD type IA (66.7%) and II (85%), it is only 1.7% in type IB patients [4,5]. This low frequency of GH1 mutations in familial IGHD ⁎ Corresponding author. Tel.: +91 2224162917; fax: +91 2224162883. E-mail address: [email protected] (S. Kale).
http://dx.doi.org/10.1016/j.ghir.2014.07.003 1096-6374/© 2014 Elsevier Ltd. All rights reserved.
IB suggested the importance of studying other genes involved in the secretion of GH, like the growth hormone releasing hormone receptor (GHRHR) which is required for the proliferation of somatotrophs and secretion of GH [5,6]. The human GHRHR being a G-protein-coupled receptor (GPCR) activates the stimulatory G-protein α-subunit (Gsα), leading to stimulation of adenylyl cyclase activity and increased intracellular cAMP concentrations in the somatotroph cells. These elevated cAMP leads to stimulation of GH synthesis and secretion from somatotrophs, as well as their proliferation and differentiation [7,8]. The GHRHR requires a transcription factor POU1F1 for its expression as evidenced by diminished GHRHR expression in pituitary cells of Pit-1-defective Snell dwarf mice. Two putative POU1F1-binding sites, P1 (-129 to -123) and P2 (-171 to -160) in the 5′ regulatory region of the human GHRHR are essential for its transcription [9–11]. Further, the translation into GHRHR protein gets arrested transiently after the binding of the signal recognition particle (SRP); a ribonucleoprotein, to the GHRHR signal peptide (first 22 amino acids) emerging from the exit site of the ribosome to form ribosome nascent chain (RNC)–SRP complex. The RNC–SRP complex then docks in a GTP-dependent manner with a
228
S. Kale et al. / Growth Hormone & IGF Research 24 (2014) 227–232
Fig. 1. a–c; Indian standard growth charts of patients S, T1 and T2 on GH replacement therapy, respectively, d–h; MRI-brain of patients S, M1, F1, T1 and T2 respectively.
membrane-anchored SRP receptor (SR) on endoplasmic reticulum (ER) and the signal peptide is translocated across or integrated into the ER membrane through a channel called the translocon and then it gets cleaved by the signal peptidase. Later the SRP dissociates from the SR upon GTP hydrolysis that resumes further translation into the GHRHR protein. In the ER, GHRHR undergoes post-translational modifications prior to expression on the cell membrane [12,13]. Mutations in the GHRHR gene have been observed in 10% of familial and 3% of sporadic IGHD patients [14]. Cohort specific mutations have been identified in approximately 11% of patients with IGHD; this percentage can be higher in familial cases (34%) and in patients with a
height SD score (SDS) ≤ −4.5 (20%) [5,15]. To date, around 33 different GHRHR mutations have been described in IGHD IB patients (Human Genome Mutation Database; HGMD Professional 2013.4). Various homozygous and compound heterozygous mutations leading to loss of GHRHR function have been demonstrated in previous studies. A transversion (− 124ANC) within the P1 binding site in the promoter region of the GHRHR gene was reported to impair promoter activity and thus expected to obliterate GHRHR expression [16]. Nonsense and splicing mutations are expected to result in protein truncation or no protein expression [17–19]. Most pathogenic missense mutations appear to maintain protein trafficking and cell-surface
S. Kale et al. / Growth Hormone & IGF Research 24 (2014) 227–232
229
Table 1 Clinical parameters of families A and B. Agea (yrs)
BA (yrs)
BW wt (kg) (SDS)
Ht (cm) (SDSb)
BMI (kg/m2)
IGF1 (ng/mL)
Peak GH on stimulation test (ng/mL)
MRI pituitary
rhGH response
Family A F1 Sc M1 MU
38 8 30 35
NA 2.7 NA NA
NA 2.5 NA NA
127.5 (−9.6) 95.5 (−5.45) 123.5 (−7.7) 130 (−9.0)
18.45 11.93 17.44 31.95
b25.0 28.0 ND b25.0
0.050 1.78 0.102 0.098
HAP HAP HAP HAP
NR Good NR NR
Family B T1c T2c F2 M2
5 5 40 29
3 3 NA NA
2.1 2.5 NA NA
85.6 (−4.6) 83.6 (−5.09) 172.7 (0.66) 155.9 (−0.25)
15.41 15.30 21.54 26.25
b25.0 b25.0 NA NA
1.0 1.01 NA NA
HAP HAP NA NA
Good Good NR NR
BA: bone age; BMI: body mass index; IGF1: insulin like growth factor 1; MRI: magnetics resonance imaging; ND: not detectable, HAP; hypoplastic anterior pituitary; NA: not applicable; NR: not required. a Age at presentation. b As per Indian standards. c Index case.
expression, but show impaired ligand-binding and/or signalling properties [20]. We screened the GH1 and GHRHR genes in six idiopathic IGHD patients from two unrelated non-consanguineous families to identify pathogenic mutations. We describe for the first time a novel gross indel in the GHRHR gene of four IGHD patients.
2. Materials and methods Institutional ethics committee approval was obtained and written informed consent was obtained from all the subjects. Three index cases from two unrelated non-consanguineous families (A and B) presented to us for short stature.
a.
b. Fig. 2. a. PCR primers for GHRHR exon 1: four primer pair combinations (FP1/RP1, FP2/RP2, FP1/RP2 and FP2/RP1) were used to PCR amplify GHRHR exon 1 (highlighted red font). b. Gel electrophoresis: PCR products of GHRHR exon 1 (using different primer pairs) and exons 2–3 of patients (F1, M1, S, T1, T2) and controls (C1, C2) were electrophoresed with 100–3000 bp molecular marker (Std) (Thermo Scientific) on a 1% agarose gel.
230
S. Kale et al. / Growth Hormone & IGF Research 24 (2014) 227–232
a.
b. Fig. 3. a. Gel electrophoresis: LPCR products amplified by LFP1/LRP1 pair (product size: 9739 bp for wild type and 2054 bp for indel) and LFP1/LRP2 primer pair (product size: 8128 bp for wild type and 443 bp for indel) were electrophoresed in 1% agarose gel. Family A: F1, GF1, M1 and S; Family B: F2, M2, T1 and T2; Ctrl: Control. b. Schematic representation of GHRHR indel from g.30999250 to 31006943 (in red font) Horizontal 5′ to 3′ bold line, DNA; vertical thick blocks, exons; LFP1, long PCR forward primer; LRP1 and LRP2, long PCR reverse primers.
In one family (Family A) the 8 year old son (S) had a history of short stature. His height was 95.5 cm (− 5.45 SDS as per Indian standards) with a bone age of 2.7 yrs. He was born full term by LSCS with a birth weight of 2.5 kg. There was no history of neonatal hypoglycaemia or neonatal jaundice and had no dysmorphic features or microphallus. He presented a typical GHD facies with a high pitched voice. MRI revealed a hypoplastic anterior pituitary. The mid-parental height (MPH) was 132 cm (−8.14 SDS) suggesting short stature in the parents (F1 and M1) as well. They had typical GHD facies, high pitched voice and pituitary hypoplasia. The maternal uncle (MU) also exhibited the same phenotype. Patients S, F1, M1 and MU had GHD with peak GH less than 2 ng/mL on stimulation tests and IGF1 less than 25 ng/mL. Deficiencies of other hormones were not observed. In other family (Family B) 5 year old twin daughters (T1 and T2) had a history of short stature. Their heights were 85.6 cm (−4.6 SDS) and 83.6 cm (−5.09 SDS) respectively. They were born by preterm vaginal delivery. No history of neonatal hypoglycaemia or neonatal jaundice was present. Both had typical GHD facies and a small nail size. MRI revealed a hypoplastic anterior pituitary in both. The parents (F2 and M2) were staturally normal and hence not evaluated for GHD. The MPH was 157.8 cm (0.18 SDS). Both the twins had GHD with peak GH less than 1.0 ng/mL on stimulation tests and IGF1 less than 25.0 ng/mL. Other hormonal axes were intact. All the patients were subjected to routine endocrine workup and confirmed to have IGHD. Patients S, T1 and T2 responded well to GH replacement therapy (Fig. 1a–c). Typical IGHD IB phenotype (OMIM#612781) was thus observed in patients F1, M1, S, MU, T1 and T2. Their clinical parameters are summarised in Table 1. Genomic DNA was isolated from peripheral blood leukocytes by standard techniques. PCR primers were designed to amplify regions including the coding regions, intron-exon boundaries and 5′/3′ untranslated regions (UTRs) using Primer3 plus software (www.bioinformatics.nl/cgibin/primer3plus/primer3plus.cgi/). PCR reactions were standardised using HPSF (High Purity Salt Free) purified primers (Operon) and
GoTaq Green Master mix (Promega). Five exons of the GH1 gene were amplified in two overlapping PCR products and 13 exons of the GHRHR gene were obtained in 11 PCR products where exons 2 and 3 and exons 7 and 8 were clubbed (see diagrams in the Supplemental data). The GHRHR exon 1 could be amplified in a 430 bp product in patients S, M and all the controls except in patients F1, T1 and T2, using the first primer pair (FP1 and RP1) (Fig. 2a and b). Therefore a second set of primers designated FP2 and RP2 was designed to amplify the exon 1 in a larger fragment (854 bp) (Fig. 2a). These primers also amplified the exon 1 in patients S and M, and all the controls except in patients F1, T1 and T2. Further, several different combinations of primers (FP1, RP1, FP2 and RP2) (Fig. 2b) failed to amplify the exon 1 in patients F1, T1 and T2. The recurrent failure of this short range PCR to amplify GHRHR exon 1 in patients F1, T1 and T2, suggested a probable gross deletion upstream of the GHRHR exon 2. The recurrent failure of short range PCR to amplify exon 1 of the GHRHR using different primer sets has led us to attempt a long PCR (LPCR). Primers LFP1 (5′-AAGCCTGTAGCAGCAGACCATCCAC-3′) and LRP1 (5′-TGGATAGAGAGCCAGGAGCAGCAG-3′) were used to amplify the entire promoter region, exon 1 and exon 2 of the GHRHR gene in a 9739 bp product (Fig. 3a). Since GHRHR exons 2– 3 were amplified in all the patients (Fig. 2b), we included exon 2 in the LPCR product to assure annealing of the LRP1. Long PCR Enzyme Mix kit (Thermo Scientific) was used for amplification using the two step cycling protocol mentioned in its product information sheet. LPCR products were gel purified using GeneJET gel extraction kit (Thermo Scientific) and sequenced bidirectionally. Sequencing reactions were run in triplicate before evaluating results against controls and human genome. A second reverse primer LRP2 (5′-GATCTTGTGC TACACAGTGGCCATG-3′) was designed to confirm the identified deletion (Fig. 3b). The sequences obtained from the patients with deletion were screened by online software, Alibaba 2.1 (http://www.gene-regulation.
S. Kale et al. / Growth Hormone & IGF Research 24 (2014) 227–232
231
a.
b. Fig. 4. a. Pedigrees of two families: family A and B harbouring mutations in GHRHR gene. Squares, male; circles, females; full black and streaked symbols, subjects homozygous for the mutation; half white and half streaked symbols, subjects heterozygous for the mutation; the arrows indicate index cases. b. Chromatograms: patient F1: a nine base pair insertion AGAGATCCA — highlighted in a blue rectangle with deletion boundaries in red circles; patients M1 and MU: a homozygous c.214GNT (p.E72X) transversion; patient S: a heterozygous c.214G/T (p.E72X) mutation.
com/pub/programs/alibaba2/index.html), to predict transcription factor binding sites (TFBSs) in the genomic sequence upstream of the GHRHR exon 2. Open reading frames (ORFs) adjoining these TFBSs were detected by ExPASy Translate tool (http://web.expasy.org/translate/). 3. Results The primer pair LFP1/LRP1 amplified a product of b 2500 bp size in patients F1, T1 and T2 as against the expected size of 9739 bp (Fig. 3a) suggesting that these patients were homozygous for the deletion. We could determine the exact length (7694 bp) of this deletion through sequencing using the LFP1 primer since the deletion was close (312 bp) to 5′ end of the LPCR product (Fig. 3b). Interestingly an insertion of 9 bp in the deleted region was also observed. The exact size of the LPCR product obtained was thus 2054 bp confirming the gel predicted size. Considering limitations of the sequencing reaction it was impossible to confirm the deletion using the same reverse primer (LRP1) which was present 1733 bp downstream of the deletion (Fig. 3b). When the new primer pair LFP1/LRP2 was used the size of LPCR product was reduced to 443 bp in these patients and to 8128 bp in the controls (Fig. 3a). Therefore the reverse primer LRP2 was used for sequencing which confirmed the homozygous replacement of a 7694 bp fragment (g.30999250_ 31006943) by a 9 bp insertion (5′-AGAGATCCA-3) (Figs. 3b and 4b). Patient S, his paternal grandfather (GF1) and parents of the twin daughters (F2 and M2) were heterozygous for this deletion as two bands (8128 bp and 443 bp) were observed in the gel electrophoresis
of the respective LPCR products (Fig. 3a). Sequencing of these products confirmed the presence of both the wild type and indel GHRHR. (Data not shown.) Due to this gross deletion, the 5′ regulatory region (harbouring POU1F1 binding sites: P1 and P2) and the exon 1 of the GHRHR gene were absent in patients F1, T1 and T2 (Fig. 3a and b). Analysis carried out using Alibaba 2.1 indicated the presence of several TFBSs except POU1F1 binding sites (P1 and P2) in the genomic sequence upstream of the GHRHR exon 2 up to the Aquaporin 1 (AQP1) gene. The ORFs adjoining the predicted TFBSs could not code for the original signal peptide. In patient M1 and her brother (MU) from family A, the GHRHR gene analysis revealed the presence of a previously reported p.E72X mutation which resulted in the premature termination of the GHRHR, causing absence of transmembrane and intracellular domains. Her son (patient S) was compound heterozygous with both the indel and p.E72X (Fig. 4a and b). 4. Discussion In the current study we describe a unique gross indel (Accession No: RCV000050247.1; g.30999250_31006943delinsAGAGATCCA) in the GHRHR gene in Indian IGHD patients. An increasing frequency of GHRHR gene mutations has been reported during the last decade in familial IGHD patients. To date, 33 pathogenic mutations (HGMD Professional 2013.4) have been reported
232
S. Kale et al. / Growth Hormone & IGF Research 24 (2014) 227–232
following the discovery of the first human mutation in the GHRHR gene (p.E72X) [18,19]. Majority of these mutations are associated with autosomal recessive (AR) IGHD, the only exception being one missense mutation (V10G) reported by Godi et al. [21]. The indel observed in the current study has an AR mode of inheritance since its heterozygous carriers (GF1, F2 and M2) were normal. The novel indel led to the loss of 5′ regulatory region harbouring POU1F1 binding sites (P1 and P2) essential for GHRHR transcription as well as GHRHR exon 1 which codes for the signal peptide [9–11,21]. Bioinformatics prediction studies on the indel GHRHR showed binding sites for many transcription factors in the genomic sequence upstream of the GHRHR exon 2 up to the AQP1 gene except those for POU1F1 (P1 and P2 binding sites) [9–11]. ORFs downstream of these TFBSs did not code for the original signal peptide as coded by the signal sequence coding region (SSCR) present in the exon 1 [21]. As a result the co-translational translocation pathway of the GHRHR expression is probably completely impaired in patients F1, T1 and T2 and partially in the patient S being heterozygous for the indel. Patient S being heterozygous for the p.E72X mutation inherited from the mother thus had a complete loss of GHRHR activity (Fig. 4a and b) [18,19]. Our patients presented typical IGHD IB phenotype (OMIM#612781) caused by GHRHR gene mutations with severe short stature (− 6.9 mean SDS), low peak GH on stimulation test, good response to exogenous GH (in patients: S, T1 and T2), autosomal recessive (AR) mode of inheritance and hypoplastic anterior pituitary. Functional consequences of mutations in the region spanned by the indel were reflected in very few studies. Salvatori et al. reported a − 124ANC transversion in the P1 POU1F1 binding site in a 4-yr old IGHD IB patient that resulted in the impairment of POU1F1 dependent GHRHR expression [16]. Godi et al. reported a heterozygous signal peptide mutation (Val10Gly) in three unrelated patients with sporadic IGHD resulting in the reduced cell surface expression of the mutant receptor due to retention in the endoplasmic reticulum [21]. Thus the indel observed in our patients is likely to have a functional impact comparable to those reported by Salvatori et al. and Godi et al. [16,21]. We report for the first time a novel gross indel in the GHRHR gene to cause IGHD IB in four severely affected patients from two unrelated Indian non-consanguineous families. It would be interesting to study more idiopathic GHD patients to characterise mutations in the ethnically unique and diverse Indian population. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ghir.2014.07.003.
Conflict of interest We have no conflicts of interest to disclose.
References [1] P.E. Mullis, Genetics of growth hormone deficiency, Endocrinol. Metab. Clin. North Am. 36 (2007) 17–36. [2] P.E. Mullis, Genetic control of growth, Eur. J. Endocrinol. 152 (2005) 11–31. [3] J.A. Phillips III, Inherited defects in growth hormone synthesis and action, in: C.R. Scriver, A.L. Beaudet, W.S. Sly, D. Valle (Eds.), 7th edition, The Metabolic and Molecular bases of Inherited Disease, vol. 2, McGraw-Hill, New York, 1995, pp. 3023–3044. [4] D. Fintini, R. Salvatori, S. Salemi, et al., Autosomal-dominant isolated growth hormone deficiency (IGHD type II) with normal GH-1 gene, Horm. Res. 65 (2006) 76–82. [5] J.K. Wagner, A. Eblé, P.C. Hindmarsh, P.E. Mullis, Prevalence of human GH-1 gene alterations in patients with isolated growth hormone deficiency, Pediatr. Res. 43 (1998) 105–110. [6] A. Solloso, L. Barreiro, R. Seoane, et al., GHRH proliferative action on somatotrophs is cell-type specific and dependent on Pit-1/GHF-1 expression, J. Cell. Physiol. 215 (2008) 140–150. [7] R. Salvatori, Growth hormone and IGF-1, Rev. Endocr. Metab. Disord. 5 (2004) 15–23. [8] K. Lin-Su, M.P. Wajnrajch, Growth hormone releasing hormone (GHRH) and the GHRH receptor, Rev. Endocr. Metab. Disord. 3 (2002) 313–323. [9] H. Inoue, N. Kangawa, A. Kinouchi, et al., Identification and functional analysis of novel human growth hormone releasing hormone receptor (GHRHR) gene mutations in Japanese Subjects with short stature, Clin. Endocrinol. (Oxf.) 74 (2011) 223–233. [10] G. Iguchi, Y. Okimura, T. Takahashi, et al., Cloning and characterization of the 5′flanking region of the human growth hormone-releasing hormone receptor gene, J. Biol. Chem. 274 (1999) 12108–12114. [11] S. Petersenn, A.C. Rasch, M. Heyens, Structure and regulation of the human growth hormone-releasing hormone receptor gene, Mol. Endocrinol. 12 (1998) 233–247. [12] K. Nagai, C. Oubridge, A. Kuglstatter, E. Menichelli, C. Isel, L. Jovine, Structure, function and evolution of the signal recognition particle, EMBO J. 22 (2003) 3479–3485. [13] D.T. Ng, J.D. Brown, P. Walter, Signal sequences specify the targeting route to the endoplasmic reticulum membrane, J. Cell Biol. 134 (1996) 269–278. [14] R. Salvatori, X. Fan, J.A. Phillips 3rd, et al., 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. 86 (2001) 273–279. [15] K.S. Alatzoglou, J.P. Turton, D. Kelberman, et al., 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. 94 (2009) 3191–3199. [16] R. Salvatori, X. Fan, P.E. Mullis, A. Haile, M.A. Levine, Decreased expression of the GHRH receptor gene due to a mutation in a Pit-1 binding site, Mol. Endocrinol. 16 (2002) 450–458. [17] R. Salvatori, X. Fan, J.D. Veldhuis, R. Couch, Serum GH response to pharmacological stimuli and physical exercise in two siblings with two new inactivating mutations in the GH-releasing hormone receptor gene, Eur. J. Endocrinol. 147 (2002) 591–596. [18] M.P. Wajnrajch, J.M. Gertner, M.D. Harbison, S.C. Chua Jr., R.L. Leibel, Nonsense mutation in the human growth hormone-releasing hormone receptor causes growth failure analogous to the little (lit) mouse, Nat. Genet. 12 (1996) 88–90. [19] H.G. Maheshwari, B.L. Silverman, J. Dupuis, G. Baumann, Phenotype and genetic analysis of a syndrome caused by an inactivating mutation in the growth hormone-releasing hormone receptor: dwarfism of Sindh, J. Clin. Endocrinol. Metab. 83 (1998) 4065–4074. [20] M. Alba, R. Salvatori, Naturally-occurring missense mutations in the human growth hormone-releasing hormone receptor alter ligand binding, J. Endocrinol. 186 (2005) 515–521. [21] M. Godi, S. Mellone, A. Petri, et al., 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. 94 (2009) 3939–3947.
Contents lists available at ScienceDirect
Growth Hormone & IGF Research journal homepage: www.elsevier.com/locate/ghir
A novel gross indel in the growth hormone releasing hormone receptor gene of Indian IGHD patients Shantanu Kale ⁎, Sweta Budyal, Rajeev Kasaliwal, Vyankatesh Shivane, Vijaya Raghavan, Anurag Lila, Tushar Bandgar, Nalini Shah Department of Endocrinology, Seth G.S. Medical College, K.E.M. Hospital, Parel, Mumbai 400012, India
a r t i c l e
i n f o
Article history: Received 17 July 2014 Accepted 29 July 2014 Available online 7 August 2014 Keywords: Novel gross indel GHRHR IGHD Indian
a b s t r a c t Context: Cohort specific mutations in the growth hormone (GH1) and growth hormone-releasing hormone receptor (GHRHR) genes have been reported worldwide in isolated growth hormone deficiency (IGHD) patients. However, limited data is available on ethnically diverse Indian IGHD patients. Objective: The aim of the study was to find GH1 and GHRHR gene mutations in Indian IGHD patients from two unrelated non-consanguineous families. Design: The 5′ and 3′ untranslated regions (UTRs) and coding regions with splice sites of the GH1 and GHRHR genes were sequenced for all patients (n = 6). Family members and 20 controls were evaluated for the sequence variants identified in the index patients. Online bioinformatics tools were used to confirm mutations and their pathogenicity. Results: GHRHR gene mutations were observed in all patients. Interestingly, a novel indel g.30999250_ 31006943delinsAGAGATCCA was observed in both the unrelated families. Three patients were homozygous for the novel indel, two were homozygous for the previously reported p.E72X mutation and one was compound heterozygous with both the mutations (indel and p.E72X) in the GHRHR gene. The novel indel has resulted in the loss of 5′ regulatory region and exon 1 of the GHRHR gene impairing the GHRHR expression. All the normal family members were heterozygous either for the indel or p.E72X mutation. None of the patients had GH1 gene mutations. Conclusions: We describe a novel gross indel in the GHRHR gene resulting in the loss of 5′ regulatory region and GHRHR exon 1 in four IGHD IB patients from two unrelated non-consanguineous Indian families. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Isolated growth hormone deficiency (IGHD) is the most common pituitary hormone deficiency. IGHD manifests due to defects in the synthesis or secretion of functional growth hormone (GH). Genetic aetiology may be found in up to 30% of IGHD patients. Mutations in the genes controlling hypothalamo-pituitary-GH axis have been identified as a cause of IGHD [1]. Based on the pattern of Mendelian inheritance, IGHD is classified into three categories: IGHD types IA and IB have autosomal recessive inheritance, while IGHD type II is autosomal dominant and IGHD type III is X-linked [2,3]. IGHD results from mutations in the GH1 and GHRHR genes. Mutations or rearrangements in the GH1 gene lead to the production of an aberrant GH molecule that either has partial function or generates immune tolerance. Although, the prevalence of GH1 gene mutations is very high in IGHD type IA (66.7%) and II (85%), it is only 1.7% in type IB patients [4,5]. This low frequency of GH1 mutations in familial IGHD ⁎ Corresponding author. Tel.: +91 2224162917; fax: +91 2224162883. E-mail address: [email protected] (S. Kale).
http://dx.doi.org/10.1016/j.ghir.2014.07.003 1096-6374/© 2014 Elsevier Ltd. All rights reserved.
IB suggested the importance of studying other genes involved in the secretion of GH, like the growth hormone releasing hormone receptor (GHRHR) which is required for the proliferation of somatotrophs and secretion of GH [5,6]. The human GHRHR being a G-protein-coupled receptor (GPCR) activates the stimulatory G-protein α-subunit (Gsα), leading to stimulation of adenylyl cyclase activity and increased intracellular cAMP concentrations in the somatotroph cells. These elevated cAMP leads to stimulation of GH synthesis and secretion from somatotrophs, as well as their proliferation and differentiation [7,8]. The GHRHR requires a transcription factor POU1F1 for its expression as evidenced by diminished GHRHR expression in pituitary cells of Pit-1-defective Snell dwarf mice. Two putative POU1F1-binding sites, P1 (-129 to -123) and P2 (-171 to -160) in the 5′ regulatory region of the human GHRHR are essential for its transcription [9–11]. Further, the translation into GHRHR protein gets arrested transiently after the binding of the signal recognition particle (SRP); a ribonucleoprotein, to the GHRHR signal peptide (first 22 amino acids) emerging from the exit site of the ribosome to form ribosome nascent chain (RNC)–SRP complex. The RNC–SRP complex then docks in a GTP-dependent manner with a
228
S. Kale et al. / Growth Hormone & IGF Research 24 (2014) 227–232
Fig. 1. a–c; Indian standard growth charts of patients S, T1 and T2 on GH replacement therapy, respectively, d–h; MRI-brain of patients S, M1, F1, T1 and T2 respectively.
membrane-anchored SRP receptor (SR) on endoplasmic reticulum (ER) and the signal peptide is translocated across or integrated into the ER membrane through a channel called the translocon and then it gets cleaved by the signal peptidase. Later the SRP dissociates from the SR upon GTP hydrolysis that resumes further translation into the GHRHR protein. In the ER, GHRHR undergoes post-translational modifications prior to expression on the cell membrane [12,13]. Mutations in the GHRHR gene have been observed in 10% of familial and 3% of sporadic IGHD patients [14]. Cohort specific mutations have been identified in approximately 11% of patients with IGHD; this percentage can be higher in familial cases (34%) and in patients with a
height SD score (SDS) ≤ −4.5 (20%) [5,15]. To date, around 33 different GHRHR mutations have been described in IGHD IB patients (Human Genome Mutation Database; HGMD Professional 2013.4). Various homozygous and compound heterozygous mutations leading to loss of GHRHR function have been demonstrated in previous studies. A transversion (− 124ANC) within the P1 binding site in the promoter region of the GHRHR gene was reported to impair promoter activity and thus expected to obliterate GHRHR expression [16]. Nonsense and splicing mutations are expected to result in protein truncation or no protein expression [17–19]. Most pathogenic missense mutations appear to maintain protein trafficking and cell-surface
S. Kale et al. / Growth Hormone & IGF Research 24 (2014) 227–232
229
Table 1 Clinical parameters of families A and B. Agea (yrs)
BA (yrs)
BW wt (kg) (SDS)
Ht (cm) (SDSb)
BMI (kg/m2)
IGF1 (ng/mL)
Peak GH on stimulation test (ng/mL)
MRI pituitary
rhGH response
Family A F1 Sc M1 MU
38 8 30 35
NA 2.7 NA NA
NA 2.5 NA NA
127.5 (−9.6) 95.5 (−5.45) 123.5 (−7.7) 130 (−9.0)
18.45 11.93 17.44 31.95
b25.0 28.0 ND b25.0
0.050 1.78 0.102 0.098
HAP HAP HAP HAP
NR Good NR NR
Family B T1c T2c F2 M2
5 5 40 29
3 3 NA NA
2.1 2.5 NA NA
85.6 (−4.6) 83.6 (−5.09) 172.7 (0.66) 155.9 (−0.25)
15.41 15.30 21.54 26.25
b25.0 b25.0 NA NA
1.0 1.01 NA NA
HAP HAP NA NA
Good Good NR NR
BA: bone age; BMI: body mass index; IGF1: insulin like growth factor 1; MRI: magnetics resonance imaging; ND: not detectable, HAP; hypoplastic anterior pituitary; NA: not applicable; NR: not required. a Age at presentation. b As per Indian standards. c Index case.
expression, but show impaired ligand-binding and/or signalling properties [20]. We screened the GH1 and GHRHR genes in six idiopathic IGHD patients from two unrelated non-consanguineous families to identify pathogenic mutations. We describe for the first time a novel gross indel in the GHRHR gene of four IGHD patients.
2. Materials and methods Institutional ethics committee approval was obtained and written informed consent was obtained from all the subjects. Three index cases from two unrelated non-consanguineous families (A and B) presented to us for short stature.
a.
b. Fig. 2. a. PCR primers for GHRHR exon 1: four primer pair combinations (FP1/RP1, FP2/RP2, FP1/RP2 and FP2/RP1) were used to PCR amplify GHRHR exon 1 (highlighted red font). b. Gel electrophoresis: PCR products of GHRHR exon 1 (using different primer pairs) and exons 2–3 of patients (F1, M1, S, T1, T2) and controls (C1, C2) were electrophoresed with 100–3000 bp molecular marker (Std) (Thermo Scientific) on a 1% agarose gel.
230
S. Kale et al. / Growth Hormone & IGF Research 24 (2014) 227–232
a.
b. Fig. 3. a. Gel electrophoresis: LPCR products amplified by LFP1/LRP1 pair (product size: 9739 bp for wild type and 2054 bp for indel) and LFP1/LRP2 primer pair (product size: 8128 bp for wild type and 443 bp for indel) were electrophoresed in 1% agarose gel. Family A: F1, GF1, M1 and S; Family B: F2, M2, T1 and T2; Ctrl: Control. b. Schematic representation of GHRHR indel from g.30999250 to 31006943 (in red font) Horizontal 5′ to 3′ bold line, DNA; vertical thick blocks, exons; LFP1, long PCR forward primer; LRP1 and LRP2, long PCR reverse primers.
In one family (Family A) the 8 year old son (S) had a history of short stature. His height was 95.5 cm (− 5.45 SDS as per Indian standards) with a bone age of 2.7 yrs. He was born full term by LSCS with a birth weight of 2.5 kg. There was no history of neonatal hypoglycaemia or neonatal jaundice and had no dysmorphic features or microphallus. He presented a typical GHD facies with a high pitched voice. MRI revealed a hypoplastic anterior pituitary. The mid-parental height (MPH) was 132 cm (−8.14 SDS) suggesting short stature in the parents (F1 and M1) as well. They had typical GHD facies, high pitched voice and pituitary hypoplasia. The maternal uncle (MU) also exhibited the same phenotype. Patients S, F1, M1 and MU had GHD with peak GH less than 2 ng/mL on stimulation tests and IGF1 less than 25 ng/mL. Deficiencies of other hormones were not observed. In other family (Family B) 5 year old twin daughters (T1 and T2) had a history of short stature. Their heights were 85.6 cm (−4.6 SDS) and 83.6 cm (−5.09 SDS) respectively. They were born by preterm vaginal delivery. No history of neonatal hypoglycaemia or neonatal jaundice was present. Both had typical GHD facies and a small nail size. MRI revealed a hypoplastic anterior pituitary in both. The parents (F2 and M2) were staturally normal and hence not evaluated for GHD. The MPH was 157.8 cm (0.18 SDS). Both the twins had GHD with peak GH less than 1.0 ng/mL on stimulation tests and IGF1 less than 25.0 ng/mL. Other hormonal axes were intact. All the patients were subjected to routine endocrine workup and confirmed to have IGHD. Patients S, T1 and T2 responded well to GH replacement therapy (Fig. 1a–c). Typical IGHD IB phenotype (OMIM#612781) was thus observed in patients F1, M1, S, MU, T1 and T2. Their clinical parameters are summarised in Table 1. Genomic DNA was isolated from peripheral blood leukocytes by standard techniques. PCR primers were designed to amplify regions including the coding regions, intron-exon boundaries and 5′/3′ untranslated regions (UTRs) using Primer3 plus software (www.bioinformatics.nl/cgibin/primer3plus/primer3plus.cgi/). PCR reactions were standardised using HPSF (High Purity Salt Free) purified primers (Operon) and
GoTaq Green Master mix (Promega). Five exons of the GH1 gene were amplified in two overlapping PCR products and 13 exons of the GHRHR gene were obtained in 11 PCR products where exons 2 and 3 and exons 7 and 8 were clubbed (see diagrams in the Supplemental data). The GHRHR exon 1 could be amplified in a 430 bp product in patients S, M and all the controls except in patients F1, T1 and T2, using the first primer pair (FP1 and RP1) (Fig. 2a and b). Therefore a second set of primers designated FP2 and RP2 was designed to amplify the exon 1 in a larger fragment (854 bp) (Fig. 2a). These primers also amplified the exon 1 in patients S and M, and all the controls except in patients F1, T1 and T2. Further, several different combinations of primers (FP1, RP1, FP2 and RP2) (Fig. 2b) failed to amplify the exon 1 in patients F1, T1 and T2. The recurrent failure of this short range PCR to amplify GHRHR exon 1 in patients F1, T1 and T2, suggested a probable gross deletion upstream of the GHRHR exon 2. The recurrent failure of short range PCR to amplify exon 1 of the GHRHR using different primer sets has led us to attempt a long PCR (LPCR). Primers LFP1 (5′-AAGCCTGTAGCAGCAGACCATCCAC-3′) and LRP1 (5′-TGGATAGAGAGCCAGGAGCAGCAG-3′) were used to amplify the entire promoter region, exon 1 and exon 2 of the GHRHR gene in a 9739 bp product (Fig. 3a). Since GHRHR exons 2– 3 were amplified in all the patients (Fig. 2b), we included exon 2 in the LPCR product to assure annealing of the LRP1. Long PCR Enzyme Mix kit (Thermo Scientific) was used for amplification using the two step cycling protocol mentioned in its product information sheet. LPCR products were gel purified using GeneJET gel extraction kit (Thermo Scientific) and sequenced bidirectionally. Sequencing reactions were run in triplicate before evaluating results against controls and human genome. A second reverse primer LRP2 (5′-GATCTTGTGC TACACAGTGGCCATG-3′) was designed to confirm the identified deletion (Fig. 3b). The sequences obtained from the patients with deletion were screened by online software, Alibaba 2.1 (http://www.gene-regulation.
S. Kale et al. / Growth Hormone & IGF Research 24 (2014) 227–232
231
a.
b. Fig. 4. a. Pedigrees of two families: family A and B harbouring mutations in GHRHR gene. Squares, male; circles, females; full black and streaked symbols, subjects homozygous for the mutation; half white and half streaked symbols, subjects heterozygous for the mutation; the arrows indicate index cases. b. Chromatograms: patient F1: a nine base pair insertion AGAGATCCA — highlighted in a blue rectangle with deletion boundaries in red circles; patients M1 and MU: a homozygous c.214GNT (p.E72X) transversion; patient S: a heterozygous c.214G/T (p.E72X) mutation.
com/pub/programs/alibaba2/index.html), to predict transcription factor binding sites (TFBSs) in the genomic sequence upstream of the GHRHR exon 2. Open reading frames (ORFs) adjoining these TFBSs were detected by ExPASy Translate tool (http://web.expasy.org/translate/). 3. Results The primer pair LFP1/LRP1 amplified a product of b 2500 bp size in patients F1, T1 and T2 as against the expected size of 9739 bp (Fig. 3a) suggesting that these patients were homozygous for the deletion. We could determine the exact length (7694 bp) of this deletion through sequencing using the LFP1 primer since the deletion was close (312 bp) to 5′ end of the LPCR product (Fig. 3b). Interestingly an insertion of 9 bp in the deleted region was also observed. The exact size of the LPCR product obtained was thus 2054 bp confirming the gel predicted size. Considering limitations of the sequencing reaction it was impossible to confirm the deletion using the same reverse primer (LRP1) which was present 1733 bp downstream of the deletion (Fig. 3b). When the new primer pair LFP1/LRP2 was used the size of LPCR product was reduced to 443 bp in these patients and to 8128 bp in the controls (Fig. 3a). Therefore the reverse primer LRP2 was used for sequencing which confirmed the homozygous replacement of a 7694 bp fragment (g.30999250_ 31006943) by a 9 bp insertion (5′-AGAGATCCA-3) (Figs. 3b and 4b). Patient S, his paternal grandfather (GF1) and parents of the twin daughters (F2 and M2) were heterozygous for this deletion as two bands (8128 bp and 443 bp) were observed in the gel electrophoresis
of the respective LPCR products (Fig. 3a). Sequencing of these products confirmed the presence of both the wild type and indel GHRHR. (Data not shown.) Due to this gross deletion, the 5′ regulatory region (harbouring POU1F1 binding sites: P1 and P2) and the exon 1 of the GHRHR gene were absent in patients F1, T1 and T2 (Fig. 3a and b). Analysis carried out using Alibaba 2.1 indicated the presence of several TFBSs except POU1F1 binding sites (P1 and P2) in the genomic sequence upstream of the GHRHR exon 2 up to the Aquaporin 1 (AQP1) gene. The ORFs adjoining the predicted TFBSs could not code for the original signal peptide. In patient M1 and her brother (MU) from family A, the GHRHR gene analysis revealed the presence of a previously reported p.E72X mutation which resulted in the premature termination of the GHRHR, causing absence of transmembrane and intracellular domains. Her son (patient S) was compound heterozygous with both the indel and p.E72X (Fig. 4a and b). 4. Discussion In the current study we describe a unique gross indel (Accession No: RCV000050247.1; g.30999250_31006943delinsAGAGATCCA) in the GHRHR gene in Indian IGHD patients. An increasing frequency of GHRHR gene mutations has been reported during the last decade in familial IGHD patients. To date, 33 pathogenic mutations (HGMD Professional 2013.4) have been reported
232
S. Kale et al. / Growth Hormone & IGF Research 24 (2014) 227–232
following the discovery of the first human mutation in the GHRHR gene (p.E72X) [18,19]. Majority of these mutations are associated with autosomal recessive (AR) IGHD, the only exception being one missense mutation (V10G) reported by Godi et al. [21]. The indel observed in the current study has an AR mode of inheritance since its heterozygous carriers (GF1, F2 and M2) were normal. The novel indel led to the loss of 5′ regulatory region harbouring POU1F1 binding sites (P1 and P2) essential for GHRHR transcription as well as GHRHR exon 1 which codes for the signal peptide [9–11,21]. Bioinformatics prediction studies on the indel GHRHR showed binding sites for many transcription factors in the genomic sequence upstream of the GHRHR exon 2 up to the AQP1 gene except those for POU1F1 (P1 and P2 binding sites) [9–11]. ORFs downstream of these TFBSs did not code for the original signal peptide as coded by the signal sequence coding region (SSCR) present in the exon 1 [21]. As a result the co-translational translocation pathway of the GHRHR expression is probably completely impaired in patients F1, T1 and T2 and partially in the patient S being heterozygous for the indel. Patient S being heterozygous for the p.E72X mutation inherited from the mother thus had a complete loss of GHRHR activity (Fig. 4a and b) [18,19]. Our patients presented typical IGHD IB phenotype (OMIM#612781) caused by GHRHR gene mutations with severe short stature (− 6.9 mean SDS), low peak GH on stimulation test, good response to exogenous GH (in patients: S, T1 and T2), autosomal recessive (AR) mode of inheritance and hypoplastic anterior pituitary. Functional consequences of mutations in the region spanned by the indel were reflected in very few studies. Salvatori et al. reported a − 124ANC transversion in the P1 POU1F1 binding site in a 4-yr old IGHD IB patient that resulted in the impairment of POU1F1 dependent GHRHR expression [16]. Godi et al. reported a heterozygous signal peptide mutation (Val10Gly) in three unrelated patients with sporadic IGHD resulting in the reduced cell surface expression of the mutant receptor due to retention in the endoplasmic reticulum [21]. Thus the indel observed in our patients is likely to have a functional impact comparable to those reported by Salvatori et al. and Godi et al. [16,21]. We report for the first time a novel gross indel in the GHRHR gene to cause IGHD IB in four severely affected patients from two unrelated Indian non-consanguineous families. It would be interesting to study more idiopathic GHD patients to characterise mutations in the ethnically unique and diverse Indian population. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ghir.2014.07.003.
Conflict of interest We have no conflicts of interest to disclose.
References [1] P.E. Mullis, Genetics of growth hormone deficiency, Endocrinol. Metab. Clin. North Am. 36 (2007) 17–36. [2] P.E. Mullis, Genetic control of growth, Eur. J. Endocrinol. 152 (2005) 11–31. [3] J.A. Phillips III, Inherited defects in growth hormone synthesis and action, in: C.R. Scriver, A.L. Beaudet, W.S. Sly, D. Valle (Eds.), 7th edition, The Metabolic and Molecular bases of Inherited Disease, vol. 2, McGraw-Hill, New York, 1995, pp. 3023–3044. [4] D. Fintini, R. Salvatori, S. Salemi, et al., Autosomal-dominant isolated growth hormone deficiency (IGHD type II) with normal GH-1 gene, Horm. Res. 65 (2006) 76–82. [5] J.K. Wagner, A. Eblé, P.C. Hindmarsh, P.E. Mullis, Prevalence of human GH-1 gene alterations in patients with isolated growth hormone deficiency, Pediatr. Res. 43 (1998) 105–110. [6] A. Solloso, L. Barreiro, R. Seoane, et al., GHRH proliferative action on somatotrophs is cell-type specific and dependent on Pit-1/GHF-1 expression, J. Cell. Physiol. 215 (2008) 140–150. [7] R. Salvatori, Growth hormone and IGF-1, Rev. Endocr. Metab. Disord. 5 (2004) 15–23. [8] K. Lin-Su, M.P. Wajnrajch, Growth hormone releasing hormone (GHRH) and the GHRH receptor, Rev. Endocr. Metab. Disord. 3 (2002) 313–323. [9] H. Inoue, N. Kangawa, A. Kinouchi, et al., Identification and functional analysis of novel human growth hormone releasing hormone receptor (GHRHR) gene mutations in Japanese Subjects with short stature, Clin. Endocrinol. (Oxf.) 74 (2011) 223–233. [10] G. Iguchi, Y. Okimura, T. Takahashi, et al., Cloning and characterization of the 5′flanking region of the human growth hormone-releasing hormone receptor gene, J. Biol. Chem. 274 (1999) 12108–12114. [11] S. Petersenn, A.C. Rasch, M. Heyens, Structure and regulation of the human growth hormone-releasing hormone receptor gene, Mol. Endocrinol. 12 (1998) 233–247. [12] K. Nagai, C. Oubridge, A. Kuglstatter, E. Menichelli, C. Isel, L. Jovine, Structure, function and evolution of the signal recognition particle, EMBO J. 22 (2003) 3479–3485. [13] D.T. Ng, J.D. Brown, P. Walter, Signal sequences specify the targeting route to the endoplasmic reticulum membrane, J. Cell Biol. 134 (1996) 269–278. [14] R. Salvatori, X. Fan, J.A. Phillips 3rd, et al., 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. 86 (2001) 273–279. [15] K.S. Alatzoglou, J.P. Turton, D. Kelberman, et al., 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. 94 (2009) 3191–3199. [16] R. Salvatori, X. Fan, P.E. Mullis, A. Haile, M.A. Levine, Decreased expression of the GHRH receptor gene due to a mutation in a Pit-1 binding site, Mol. Endocrinol. 16 (2002) 450–458. [17] R. Salvatori, X. Fan, J.D. Veldhuis, R. Couch, Serum GH response to pharmacological stimuli and physical exercise in two siblings with two new inactivating mutations in the GH-releasing hormone receptor gene, Eur. J. Endocrinol. 147 (2002) 591–596. [18] M.P. Wajnrajch, J.M. Gertner, M.D. Harbison, S.C. Chua Jr., R.L. Leibel, Nonsense mutation in the human growth hormone-releasing hormone receptor causes growth failure analogous to the little (lit) mouse, Nat. Genet. 12 (1996) 88–90. [19] H.G. Maheshwari, B.L. Silverman, J. Dupuis, G. Baumann, Phenotype and genetic analysis of a syndrome caused by an inactivating mutation in the growth hormone-releasing hormone receptor: dwarfism of Sindh, J. Clin. Endocrinol. Metab. 83 (1998) 4065–4074. [20] M. Alba, R. Salvatori, Naturally-occurring missense mutations in the human growth hormone-releasing hormone receptor alter ligand binding, J. Endocrinol. 186 (2005) 515–521. [21] M. Godi, S. Mellone, A. Petri, et al., 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. 94 (2009) 3939–3947.
