The prevalence of gonadotropin-releasing hormone receptor mutations in a large cohort of patients with hypogonadotropic hypogonadism

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1 REPRODUCTIVE ENDOCRINOLOGY The prevalence of gonadotropin-releasing hormone receptor mutations in a large cohort of patients with hypogonadotropic hypogonadism Balasubramanian Bhagavath, M.D., a Metin Ozata, M.D., b I. C. Ozdemir, M.D., c Erol Bolu, M.D., c David P. Bick, M.D., d Richard J. Sherins, M.D., e and Lawrence C. Layman, M.D. a,f a Division of Reproductive Endocrinology, Infertility, and Genetics, Department of Obstetrics and Gynecology, Medical College of Georgia, Augusta, Georgia (currently Division of Reproductive Endocrinology, and Infertility, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, Texas); b GATA Haydarpasa Training Hospital, Camlica Askeri Hastanesi, Department of Endocrinology, Acibadem-Uskudar, Turkey; c Department of Endocrinology and Metabolism, Gulhane School of Medicine, Etlik-Ankara, Turkey; d Department of Pediatrics and Department of Obstetrics and Gynecology, Medical College of Wisconsin, Milwaukee, Wisconsin; e Genetics and IVF Institute, Fairfax, Virginia; and f Neurodevelopmental Biology Program, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia Objective: To determine the prevalence of GNRH receptor (GNRHR) gene mutations in a large cohort of patients with idiopathic hypogonadotropic hypogonadism (IHH). Design: Molecular analysis and genotype/phenotype correlations. Setting: University molecular reproductive endocrinology laboratory. Patient(s): North American and Turkish patients with IHH. Intervention(s): DNA from 185 IHH patients were subjected to denaturing gradient gel electrophoresis for exons and splice junctions of the GNRHR gene. Variant fragments were sequenced. Main Outcome Measure(s): GNRHR mutations were characterized and compared with the phenotype. The prevalence of GNRHR mutations was also determined. Result(s): Three of 185 (1.6%; confidence interval [CI] 0.3% 4.7%) total IHH patients demonstrated compound heterozygous GNRHR mutations. All three were identified from a cohort of 85 normosmic patients (3.5%, CI 0.73% 7.5%), and none were demonstrated in hyposmic or anosmic IHH patients. GNRHR mutations were identified in 1 of 15 (6.7%; CI 0.2% 32.0%) families with at least two affected siblings, and in 2 of 18 (11.1%; CI 1.4% 34.7%) normosmic females. None were found in presumably autosomal dominant families. Conclusion(s): GNRHR mutations account for approximately 3.5% of all normosmic and 7% 11% of presumed autosomal recessive IHH, suggesting that additional genes play an important role in normal puberty. We believe this to be the largest GNRHR gene mutation analysis performed to date in a population of IHH patients. (Fertil Steril 2005;84: by American Society for Reproductive Medicine.) Patients with idiopathic hypogonadotropic hypogonadism (IHH) have delayed or absent pubertal development and low serum gonadotropin levels, reflecting abnormalities of the hypothalamic-pituitary axis (1). IHH is genetically heterogeneous, having X-linked recessive, autosomal recessive, autosomal dominant, and apparent sporadic inheritance patterns (2, 3). The genetic basis for IHH was first described in X-linked recessive Kallmann syndrome, when KAL1 mutations were demonstrated in males with IHH and anosmia (4, 5). The Received December 9, 2004; revised and accepted April 8, Supported by National Institutes of Health grants HD33004 and HD (L.C.L.). Reprint requests: Lawrence C. Layman, M.D., Section of Reproductive Endocrinology, Infertility, and Genetics, Department of Obstetrics and Gynecology, Medical College of Georgia, th Street, Augusta, Georgia (FAX ; llayman@mcg.edu). known causes for IHH include mutations in genes inherited as X-linked recessive: Kallmann syndrome interval 1 (KAL1) and nuclear receptor subfamily 0, group B, member 1 (NR0B1); as autosomal recessive: GnHR receptor (GNRHR), leptin (LEP), leptin receptor (LEPR), proprotein convertase, subtilisin/kexin-type 1 (PCSK1), G-protein coupled receptor 54 (GPR54), and KISS1 metastin suppressor (KISS1); and as autosomal dominant: fibroblast growth factor receptor 1 (FGFR1) (3, 6). Combined pituitary deficiency due to mutations in homeobox gene expressed in ES cells (HESX1), prophet of PIT1 (PROP1), and Lim homeobox gene 3 (LHX3) may include IHH as one of the phenotypes (3, 6). The GNRHR gene was the first gene found to cause autosomal recessive IHH (7, 8). Subsequently, at least 18 different mutations have been described in IHH patients, a /05/$30.00 Fertility and Sterility Vol. 84, No. 4, October 2005 doi: /j.fertnstert Copyright 2005 American Society for Reproductive Medicine, Published by Elsevier Inc. 951

2 majority of which are compound heterozygotes (9 27). The GNRHR gene locus resides on chromosome 4q21.2 and consists of three exons and two introns (28 30). The GNRHR is a member of the large family of G-protein coupled receptors, and is expressed on the cell surface of pituitary gonadotropes (31). Pulsatile GNRH from the hypophysealportal system interacts with this receptor to initiate a cascade of events culminating in the synthesis and secretion of gonadotropins FSH and LH. Typically, following binding of GnRH to the receptor, via a G-protein, second messenger inositol trisphosphate (IP3) is produced which then triggers calcium release and subsequent release of both FSH and LH (31). There is evidence that the pulse frequency of GNRH plays a large role in determining which of the two gonadotropins is released (32). We first reported the prevalence of GNRHR mutations in a sample of IHH patients in 1998 (8). Compound heterozygous GNRHR mutations were found in a single proband (and affected siblings) from a total of 46 normosmic IHH patients with a prevalence of 2.2%. When only females were considered, then 1 of 14 (7.1%) were affected (8). In a larger study, 5 of 108 (4.6%) IHH patients had GNRHR mutations, including 5 of 48 (10.4%) with normosmic IHH, and none of 60 anosmic IHH patients (15). The objective of the present study was to determine the prevalence of GNRHR mutations in a larger sample of IHH patients, including those with presumptive autosomal recessive inheritance. MATERIALS AND METHODS Patients The diagnosis of IHH was based upon criteria published previously, which included absent or impaired puberty by age 17 in girls and age 18 in boys, low serum gonadotropins, no evidence of a pituitary tumor by radiologic imaging, and normal thyroid function, PRL, and cortisol (1). All males had a total T less than 100 ng/dl (normal 300 1,100 ng/dl), and all females had absent breast development (except two) and hypoestrogenic amenorrhea. Of the 151 males, 46 were anosmic, 11 hyposmic, 67 normosmic, and data were unavailable for 27 patients. Among the 34 females, 5 were anosmic, 3 hyposmic, 18 normosmic, and data were unavailable in 8 patients. This study was approved by the Human Assurance Committee of the Medical College of Georgia, and each patient signed an informed consent. Complete IHH was defined as absent breast development (Tanner stage 1) in females and testicular size 3 cm or less in males (33). Incomplete IHH suggested the presence of prior steroid production and was defined as breast development of Tanner 2 or greater in females and testes size 4 cm or greater in males. Molecular Analysis DNA was extracted from white blood cells in 185 patients with IHH using standard methods. GNRHR gene mutation analysis was performed with primers and cycling conditions as described previously (8, 34). Briefly, polymerase chain reaction (PCR) was performed on the protein-coding regions of all three exons and splice junctions of the GNRHR gene. One primer of each pair contained a 40-bp GC clamp. Exon 1 was amplified in two overlapping fragments, whereas exons 2 and 3 were each processed as single fragments. Each PCR also contained a negative control comprising of all reagents except DNA. The presence of the correct GCclamped PCR products was first confirmed by electrophoresis on 1.2% agarose gels stained with ethidium bromide and photographed. Mutation detection was determined by electrophoresis of the GC-clamped PCR products on 20% 80% denaturing gradient gels (100% denaturants 40% formamide and 7 mol/l urea). The two exon 1 fragments were electrophoresed for 700 VH and 800 VH, while exons 2 and 3 were electrophoresed at 800 VH and 750 VH, respectively (8). The gels were then stained with ethidium bromide and photographed. The DGGE was repeated for each of the variant fragments, and subjected to DNA sequencing on an ABI 310 automated DNA sequencer using Big Dye Sequencing kits (PE Bio Systems, Foster City, CA). DNA sequencing for abnormal fragments was repeated at least three times in both forward and reverse directions. RESULTS Agarose gel electrophoresis of GC-clamped PCR products from all 185 probands demonstrated the appropriately sized fragments for all three exons. No deletions or insertions were observed. Five different missense mutations (Arg262Gln, Tyr284Cys, Gln106Arg, Leu266Arg, and Ala129Asp) were identified in three IHH probands as compound heterozygotes (Table 1). Compound heterozygosity for Arg262Gln/Tyr284Cys, described previously (8, 35), was observed in a family with four affected siblings all having complete IHH with normosmia. The Ala129Asp/Arg262Gln also described previously (20) was identified in a single male with complete IHH and normal sense of smell. Both parents were deceased, but presumably were heterozygotes. He had several brothers and sisters who were not affected but were unable to be studied. In addition, a new compound heterozygous mutation Gln106Arg/Leu266Arg was identified in a normosmic female with complete IHH. Unfortunately, no additional clinical information was available. These three compound heterozygous genotypes were identified in one male and two female probands (Table 1). Functional effects of the mutations are shown in Table 2. It should also be noted that the Leu266Arg impaired GNRHR signaling but also was not able to stimulate gonadotropin subunit or GNRHR promoter activity in vitro (23). The Gln106Arg mutation diminished GNRH ligand binding and the Arg262Gln impaired signaling, but both also reduced the sensitivity of the FSHB promoter to GNRH (24). 952 Bhagavath et al. Prevalence of GNRHR mutations Vol. 84, No. 4, October 2005

3 TABLE 1 The five different GNRHR mutations identified are shown on the left and genotype/phenotype correlation is shown on the right. Mutations Genotype Phenotype Affected Arg262Gln Arg262Gln/Tyr284Cys Complete IHH M, F Tyr284Cys Gln106Arg Ala129Asp/Arg262Gln Complete IHH M Ala129Asp Leu266Arg Gln106Arg/Leu266Arg Complete IHH F Note: M male; F female. Bhagavath. Prevalence of GNRHR mutations. Fertil Steril Of all 185 IHH patients (anosmic/hyposmic and normosmic) analyzed for mutations in GNRHR gene, only three had mutations (1.6%; confidence interval [CI] 0.3% 4.7%). All GNRHR mutations occurred in normosmic IHH patients with complete IHH. No mutations were identified in any of the 65 IHH patients who had anosmia or hyposmia. If only the 85 patients with documented normosmia were considered, the prevalence of GNRHR mutations in our sample was 3 of 85 (3.5%, CI 0.73% 7.5%). Because untreated IHH affects fertility, families typically do not contain a large number of affected individuals. We considered that families with at least two affected siblings (in the absence of vertical transmission), and families in which the proband was a female could provide some indication of autosomal recessive inheritance. In our patient population, there were 15 families with two or more affected siblings, suggesting the likelihood of autosomal recessive inheritance. Only 1 of 15 (6.7%; CI 0.2% 32.0%) families with at least two affected siblings was affected by mutations in the GNRHR gene. As stated above and reported previously, the single female proband had three other siblings (two female and one male) with IHH (8, 35). All family members were affected by the same Arg262Gln/Tyr284Cys mutation. Although all had complete IHH, the gonadotropin TABLE 2 All reported human GNRHR mutations to date, including mutation location in the gene and protein, the number of times the allele has been reported, and the effects upon binding and signaling. No. Author Mutation Exon Allele, n Protein Binding Signaling 1 (7, 12, 15 18) Gln106Arg 1 15 ECL1 Decreased Decreased 2 (7 9, 12, 15, 20) Arg262Gln 3 7 ICL3 Normal Decreased 3 (8) Tyr284Cys 3 1 TMD7 Normal Decreased 4 (11) Ser168Arg 1 2 TMD4 Decreased Decreased 5 (10) Ser217Arg 2 1 TMD5 Decreased Decreased 6 (9, 20) Ala129Asp 1 2 TMD3 Decreased Decreased 7 (13) Leu314X 3 1 TMD7 Decreased Decreased 8 (15) Thr32Ile 1 1 ECD Not tested Decreased 9 (15) Cys200Tyr 2 1 ECL2 Not tested Decreased 10 (15, current study) Leu266Arg 3 2 ICL3 Not tested Decreased 11 (15) Cys279Tyr 3 2 TMD6 Not tested Decreased 12 (17, 25) Arg139His 1 4 TMD3/ICL2 Absent Decreased 13 (17) Asn10Lys 1 1 ECD Decreased Decreased 14 (14, 19) Glu90Lys 1 2 TMD2 Decreased Decreased 15 (21) IVS1, G-A, 1 IVS1 2 Exon2 skip Not tested Not tested 16 (26) Ala171Thr 1 1 TMD4 Absent Decreased 17 (27) Asn10Lys Gln11Lys 1 1 ECD Decreased Decreased 18 (27) Pro320Leu 3 1 TMD7 Absent Decreased Note: ECL extracellular loop; TMD transmembrane domain; ICL intracellular loop; ECD extracellular domain. Bhagavath. Prevalence of GNRHR mutations. Fertil Steril Fertility and Sterility 953

4 response to single and repeated GNRH varied among these individuals. Interestingly, the affected male also had a ring chromosome 21 and a sister with Down syndrome (8, 35). Eye abnormalities were observed in these affected IHH patients. Another potential indicator of an autosomal recessive disease would be to determine the prevalence of GNRHR mutations in all 34 unrelated females in the study population. Of the 34 female IHH patients, two (5.9%) were affected, making the prevalence slightly higher in female patients. If only normosmic females were considered, 2 of 18 (11.1%; CI 1.4% 34.7%) had GNRHR mutations. No GNRHR mutations were found in any of the presumed autosomal dominant or X-linked recessive families. DISCUSSION Idiopathic hypogonadotropic hypogonadism constitutes a very heterogeneous disorder at both the clinical and the molecular level. Mutations in KAL1 appear to account for about 10% of the cases of X-linked recessive Kallmann syndrome, whereas NR0B1 mutations appear to occur mostly in patients with adrenal hypoplasia congenita and IHH (3). Autosomal dominant Kallmann syndrome is caused by FGFR1 (KAL2) mutations in approximately 10%, according to one study (36). Although autosomal recessive gene mutations have been reported in at least six genes (GNRHR, LEP, LEPR, PCSK1, GPR54, and KISS1), only GNRHR mutations appear to be very common. Mutations in the other five genes have been described in only a few affected individuals (6, 37). Mutations in the GNRHR gene constituted the first known autosomal recessive molecular cause of IHH (7, 8). In our initial description of unrelated, normosmic IHH patients, 1 of 46 (2.2%) had mutations of the GNRHR gene, and the prevalence was higher if only female probands were considered (1/14, or 7.1%) (8). The purpose of the present study was to determine the prevalence of GNRHR mutations from a larger cohort of IHH patients. We identified three Caucasian IHH probands who had GNRHR mutations, two in females and one in a male (Table 1). All three patients were normosmic and had complete IHH, in which there was no evidence of steroid production as manifested by a completely prepubertal phenotype. One proband s family demonstrated an obvious autosomal recessive inheritance pattern, as there were four total affected siblings (8, 20). Although GNRHR mutations are inherited in an autosomal recessive fashion, this was not apparent from their pedigrees because no additional family members were affected. No mutations were present in any autosomal dominant families, as has been reported for the growth hormone gene, in which mutations may cause autosomal dominant or autosomal recessive disease. As is common in many genetic diseases, clinical heterogeneity in phenotype was observed within families with GNRHR mutations. Although all siblings had a complete absence of pubertal development, the response to GNRH varied among the four affected members in the family with the Arg262Gln/Tyr284Cys compound heterozygous mutations (20). This occurred even though we demonstrated that both mutations markedly impaired IP3 signal transduction (20-fold decrease for Tyr284Cys and 10-fold decrease for Arg262Gln) (8). When a second dose of GNRH was given to two of these patients, all FSH and LH levels were higher than the responses after the initial dose, suggesting that some priming and function of the receptor occurs with these mutant GNRHRs (20). Other investigators have reported successful priming of the GNRHR and even ovulation and pregnancy with exogenous GNRH (12, 18) or spontaneous pregnancy (18). However, neither of these pregnant patients had a term delivery, suggesting perhaps that GNRH signaling effects may not be entirely normal and could be necessary for pregnancy following implantation. To date, at least 18 different GNRHR mutations, 17 of which are missense mutations, have been identified in 16 different unrelated IHH probands (Table 2). All mutations occurred in exons 1 or 3, except for one in exon 2 and one in intron 1 that affects splicing. Although concentrated in two exons, the mutations seem to be spread throughout different regions of the protein: the transmembrane domains, extracellular, and intracellular loops. Nevertheless, two mutations appear to be particularly common, even among different ethnic groups: Gln106Arg represents 15 of 46 (32.6%) mutant alleles and Arg262Gln constitutes 7 of 46 (15.2%) mutant alleles (Table 2). In vitro analysis has demonstrated reduced GNRH binding in 11 of 18 (61%) GNRHR mutations, and the others affect signaling. In addition, some mutations were studied for their effects upon gonadotropin subunit and GNRHR promoter activity. Of the 16 different probands with GNRHR mutations, 13 of 16 (81%) had compound heterozygous mutations, and the remaining ones demonstrated homozygosity (Table 3). About 60% of the probands with GNRHR mutations did not exhibit any previous evidence of functional, pulsatile GNRH and had complete IHH. Most of the remainder had incomplete IHH, and the phenotype was unknown in one. Although a variety of compound heterozygotes constitute the most common genotype, the two most common genotypes are Gln106Arg/Arg262Gln (n 3) and homozygosity for Gln106Arg (n 3). Our family with Arg262Gln/Tyr284Cys is the only one described in the literature, but our male with Ala129Asp/Arg262Gln mutations and our female with the Gln106Arg/Leu266Arg have been described once each (9, 15). Although both reported males with the Ala129Asp/ Arg262Gln mutations had complete IHH, it is interesting that the phenotype of the Gln106Arg/Leu266Arg mutation may be either complete or incomplete IHH. However, as is true in many different genetic diseases, the phenotype may vary even if the same genotype is present. Certainly mutations in other modifying genes, epistatic events, and/or environmental factors may contribute to the phenotype. 954 Bhagavath et al. Prevalence of GNRHR mutations Vol. 84, No. 4, October 2005

5 TABLE 3 Genotype/phenotype correlations are shown, the number of times the specific genotype has been reported, the sex of the proband, and ethnicity. No. Author Genotype Phenotype n Sex Ethnicity 1 (7, 12, 15) a Gln106Arg/Arg262Gln Incomplete 3 M, F NA Incomplete F NA Complete F NA 2 (8) Arg262Gln/Tyr284Cys Complete 1 M, F Caucasian 3 (10) Arg262Gln/Gln106Arg Incomplete 1 M, F NA Ser217Arg 4 (11) Ser168Arg (HMZ) Complete 1 M NA 5 (9, 20) Ala129Asp/Arg262Gln Complete 2 M, F NA Complete M Caucasian 6 (13) Leu314X/Gln106Arg Complete 1 F NA 7 (15) Thr32Ile/Cys200Tyr Complete 1 M NA 8 (15, current study) Gln106Arg/Leu266Arg Incomplete 2 F NA Complete F Caucasian 9 (15) Cys279Tyr (HMZ) Unknown 1 M NA 10 (15, 16, 18) b Gln106Arg (HMZ) Incomplete 3 M NA Incomplete F NA Incomplete M NA 11 (17, 25) Arg139His (HMZ) Complete 2 F Brazilian Complete M NA 12 (17) Asn10Lys/Gln106Arg Incomplete 1 M, F Brazilian 13 (14, 19) Glu90Lys (HMZ) Complete 1 M Mexican-mestizo 14 (21) IVS1, G-A, 1 Complete 1 F Indian 15 (26) Ala171Thr/Gln106Arg Complete 1 M Caucasian 16 (27) Asn10Lys Gln11Lys/Pro320Leu Incomplete 1 F Caucasian Note: M male; F female; NA not available. a Pregnant with exogenous GnRH. b Spontaneous pregnancy (ref. 18). Bhagavath. Prevalence of GNRHR mutations. Fertil Steril Our study of a large cohort of IHH patients provides a more representative indication of how common GNRHR mutations are in IHH patients. Denaturing gradient gel electrophoresis of GC-clamped PCR products has been shown to detect more than 95% of mutations (38). Since our initial prevalence report of 46 IHH patients (8), Beranova et al. (15) screened 108 IHH patients for mutations using temperature gradient gel electrophoresis, and overall 5 of 108 (4.6%) patients had GNRHR mutations. If only normosmic patients were included, 5 of 48 (10.4%) patients had GNRHR mutations. Similarly to our current study, they found no GNRHR mutations in patients with anosmia or hyposmia. These investigators reported that GNRHR mutations occurred in 40% of autosomal recessive families, however this was based upon only five families (15). Differences in prevalence between our study and theirs are likely due to different populations of IHH families. Our study reported here is currently the largest conducted to assess the prevalence of GNRHR mutations in IHH patients. Not surprisingly, the prevalence of GNRHR mutations in IHH patients is low overall at 1.6% (both anosmic and normosmic IHH). Of all normosmic patients, 3 of 85 (3.5%) had GNRHR mutations. Because the mode of inheritance is difficult to predict in most families, we considered those families with at least two affected siblings and those with a female proband to potentially represent autosomal recessive inheritance. Only 1 of 15 (6.7%) unrelated probands from families with two or more affected siblings (therefore more likely to be autosomal recessive) demonstrated GNRHR gene mutations, which is less than the 2 of 5 (40%) reported by Beranova et al. (15). However, in both studies, the number of autosomal recessive families is too small to make broad generalizations. Another potential indicator of an autosomal recessive disease would be to determine the prevalence of GNRHR mutations in all unrelated females, and in particular, normosmic females in the study population. When only female probands are considered, 2 of 34 (5.9%) had GNRHR mutations, and if only normosmic females are considered, 2 of 18 (11.1%) had GNRHR mutations. In summary, our findings from a large cohort Fertility and Sterility 955

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