ORIGINAL ARTICLE Endocrine Care A Recurrent Signal Peptide Mutation in the Growth Hormone Releasing Hormone Receptor with Defective Translocation to the Cell Surface and Isolated Growth Hormone Deficiency Michela Godi, Simona Mellone, Antonella Petri, Teresa Arrigo, Claudio Bardelli, Downloaded from https://academic.oup.com/jcem/article/94/10/3939/2597261 by guest on 26 September 2021 Lucia Corrado, Simonetta Bellone, Flavia Prodam, Patricia Momigliano-Richiardi, Gianni Bona, and Mara Giordano Laboratory of Human Genetics, Department of Medical Sciences and Interdisciplinary Research Center of Autoimmune Diseases (M.Go., S.M., L.C., P.M.-R., M.Gi.), and Unit of Pediatrics, Department of Medical Sciences (A.P., S.B., F.P., G.B.), University of Eastern Piedmont, 28100 Novara, Italy; Department of Pediatrics (T.A.), University of Messina, 98100 Messina, Italy; and Laboratory of Pharmacology (C.B.), Department of Medical Sciences, University of Eastern Piedmont, 28100 Novara, Italy Context: Mutations in the GHRH receptor (GHRHR) have been detected in the familial type-IB isolated GH deficiency (IGHD-IB) inherited as an autosomal recessive disorder and characterized by a low but detectable serum GH level and good response to substitutive GH therapy. Objective: The aim of our study was the identification of mutations in sporadic patients with a IGHD-IB phenotype. Subjects and Methods: The GHRHR gene was systematically screened by DHPLC in 134 IGHD pa- tients with no family history of the disorder or declared parental consanguinity. Results: We identified a novel variation, Val10Gly, within the signal peptide at the heterozygous state in three patients and in one of 1084 controls (P ϭ 0.004), suggesting that it might contribute to IGHD. The functional analysis showed that the signal peptide is not cleaved from the mutant GHRHR, which in turn is not translocated to the cellular surface, demonstrating that 10Gly dras- tically affects the receptor correct processing. Because 10Gly was also present in normal-stature relatives of the patients as well as in a control, it is likely that it exerts its effects in the context of other genetic and environmental susceptibility factors. Conclusion: At difference from previous papers reporting GHRHR mutations in familial cases with a clear recessive mode of inheritance, our study was conducted on a large sample of sporadic patients and allowed to discover a novel mechanism of the disease caused by a recurrent dominant mutation in the GHRHR signal peptide associated with incomplete penetrance. (J Clin Endocrinol Metab 94: 3939–3947, 2009) he GHRH receptor (GHRHR) is a seven-transmem- tracellular cAMP and consequently the opening of a volt- T brane G protein-coupled receptor of 423 amino acids age-gated calcium channel. The influx of calcium deter- (1). After binding GHRH, it activates a stimulatory G mines release of GH stored in secretory granules. The protein by catalyzing GTP binding to its ␣-subunit. This increase of cAMP also stimulates protein kinase A to ac- stimulates the adenylate cyclase-mediated increase of in- tivate cAMP response element binding protein transcrip- ISSN Print 0021-972X ISSN Online 1945-7197 Abbreviations: CHO, Chinese hamster ovary; ConA, concanavalin A; DHPLC, denaturing Printed in U.S.A. HPLC; ER, endoplasmic reticulum; GHRHR, GHRH receptor; HA, hemagglutinin; IGHD, Copyright © 2009 by The Endocrine Society isolated GH deficiency; PMSF, phenylmethylsulfonyl fluoride; SDS, SD score; SRP, signal doi: 10.1210/jc.2009-0833 Received April 20, 2009. Accepted July 10, 2009. recognition protein. First Published Online July 21, 2009 J Clin Endocrinol Metab, October 2009, 94(10):3939–3947 jcem.endojournals.org 3939 3940 Godi et al. A Novel Recurrent Mutation in the GHRHR J Clin Endocrinol Metab, October 2009, 94(10):3939–3947 tion. cAMP response element binding protein stimulates moment of diagnosis, 84% of the patients were prepubertal, the transcription of the pituitary transcription factor PIT-1, mean age was 8.4 Ϯ 4.1 SD, and the mean bone age was Ϫ1.5 Ϯ which in turns activates transcription of many pituitary 1.0 SD. None of the patients had consanguineous parents. Del- eterious mutations in the GH1 gene were excluded in all the genes, including GHRHR and GH1 (2, 3). patients. GHRHR mutations were detected in about 10% of the Italian individuals of normal height (university and hospital autosomal recessive forms of isolated GH deficiency (IGHD) staff), not tested for GH secretion levels, were used as controls. characterized by low but measurable serum GH levels and A written informed consent was obtained from the patients’ good response to exogenous GH therapy (IGHD-type IB): parents and from the normal-stature controls. seven missense mutations (4–8), two nonsense (9, 10), one impairing a PIT-1 binding site in the promoter (11), two Analysis of the GHRHR gene in the patients by microdeletions (5, 12), and five splicing mutations (10, 13– DHPLC Downloaded from https://academic.oup.com/jcem/article/94/10/3939/2597261 by guest on 26 September 2021 15). The missense mutations (His137Leu, Leu144His, GHRHR was amplified by PCR from genomic DNA (Sup- Ala176Val, Ala222Glu, Phe242Cys, and Lys329Glu) do not plemental Methods and Supplementary Table S1, published as alter the receptor surface expression but interfere with ligand supplemental data on The Endocrine Society’s Journals On- line web site at http://jcem.endojournals.org). Search for mu- binding, thus affecting GHRH signaling (16). Nonsense and tations was performed by denaturing HPLC (DHPLC) scanning splicing mutations are expected to lead to the synthesis of a on an HPLC instrument (Wave; Transgenomic, Glasgow, UK). severely truncated receptor or to the absence of the protein. To allow heteroduplex formation, PCR products were subjected With the exception of Leu144His, which is recurrent and has to 3 min 95 C denaturation followed by a gradual reannealing been found in different continents (6), each mutation is spe- from 95 C to 41 C in 30 min. The temperature required for cific for a particular geographical region and is likely to de- successful resolution of heteroduplex molecules was determined using the DHPLC Melt Program (http://insertion.stanford.edu/ rive from a common ancestor. Most of the reported patients melt.html) (Supplemental Table S1). All of the samples were run belonged to consanguineous pedigrees and were homozy- at the predicted temperatures (RTm) and at RTm ϩ 2 C as rec- gous. In a minority of the families, the affected members ommended (20). All detected heteroduplex fragments were sub- carried two distinct GHRHR mutations (4, 5). sequently sequenced using the Big-dye terminator reaction kit The aim of our study was the identification of GHRHR and an ABI Prism 3100 genetic analyzer (Applied Biosystems, causal mutations in a large cohort of unrelated patients Foster City, CA). (n ϭ 134) with no family history of IGHD, with no evi- dence of parental consanguinity, and with the IGHD-IB Genotyping phenotype. SNPs were genotyped by SnaPshot (Applied Biosystems) We identified a novel missense substitution in the signal following the manufacturer’s instructions. Eight fragments peptide at the heterozygous state in three patients with a containing nine SNPs were amplified in a single PCR using the QIAGEN Multiplex PCR Kit (QIAGEN Inc., Valencia, CA) drastic effect on GHRHR processing and translocation to with the same primers used for DHPLC analysis (see Supple- the cell surface. mental Methods and Supplemental Table S2). Plasmid constructs and transfection Subjects and Methods A wild-type GHRHR cDNA cloned in pcDNA3 was kindly donated by Professor Kelly Mayo. Nucleotides encoding the Subjects FLAG epitope tag (DYKDDDDK) were inserted within the A total of 134 Italian patients (92 males and 42 females) with GHRHR N-terminal domain at residue 37 downstream of the sig- short stature consequent to IGHD were recruited from different nal peptide. The hemagglutinin (HA) epitope tag (YPYDVPDYA) centers. Their mean height SD score (SDS) was Ϫ2.4 Ϯ 0.9, and was inserted at the C terminal in a separate construct. Both plas- height velocity over 1 yr was less than Ϫ1.5 SDS. The patients mids were used as templates into which the T position ϩ29 was were considered sporadic because none of them had relatives substituted with a G (corresponding to the change 10ValϾGly) with a demonstrated GH deficit, although some had short-stat- using the Quik Change Site-Directed Mutagenesis Kit (Strat- ured parents. The mean target corrected height of the patients agene, La Jolla, CA). was Ϫ1.6 Ϯ 1.3. IGHD was defined as a peak GH response below Wild-type and mutant constructs were transiently expressed 10 ng/ml after two consecutive classical provocative tests (with in Chinese hamster ovary (CHO) cells not expressing endoge- arginine or clonidine or insulin) (17) or below 20 ng/ml after one nous GHRHR. double stimulus with GHRH ϩ arginine (18) and normal serum Cells were grown to 70–80% confluency in six-well plates in level of other pituitary hormones. The patients had a mean se- Ham’s F12 medium with 10% heat-inactivated fetal bovine se- cretion peak of 4.2 Ϯ 2.2 ng/ml after the single stimuli (n ϭ 113) rum, 100 U/ml penicillin, 100 ␮g/ml streptomycin, and 2 mM or 9.2 Ϯ 5.7 ng/ml after the double provocative test (n ϭ 21). L-glutamine (Sigma Aldrich, St. Louis, MO). Transfections were Patients with a known cause of acquired hypopituitarism were performed using 2 ␮g of each construct with Fugene 6 (Roche excluded. Skeletal maturation was estimated as bone age (radius, Diagnostics, Indianapolis, IN) following the manufacturer’s ulna, and short bone) according to Tanner et al. (19). At the instructions. J Clin Endocrinol Metab, October 2009, 94(10):3939–3947 jcem.endojournals.org 3941 GHRHR mRNA detection Statistical analysis Total RNA was extracted 24 h after transfection using the Significance was evaluated from 2 ϫ 2 contingency tables by RNeasy kit (QIAGEN).