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Departments of Medicine (L.F.G.S., P.M.G.B., G.S.M.) and Virology (D.A.C.), Royal Free and University College Medical School, London; Department of Medicine (P.M.S.), Queen Elizabeth Hospital, Birmingham; and Department of Clinical Biochemistry (M.T.), Royal Free Hospital, London, United Kingdom
Address all correspondence and requests for reprints to: Dr. Pierre Bouloux, Director, Neuroendocrine Unit, Department of Medicine, Royal Free Hospital, Pond Street, London, United Kingdom. E-mail: . pmgb{at}rfhsm.ac.uk
Abstract
Mutations in the GnRH receptor (GnRHR) have been shown to be responsible for a significant number of autosomic recessive and, less commonly, sporadic cases of idiopathic hypogonadotropic hypogonadism. We describe a woman with complete GnRH resistance secondary to a novel homozygous GnRHR gene mutation, transmitted as an autosomal recessive trait. The propositus presented with primary amenorrhea and absent thelarche and pubarche. Dynamic tests demonstrated absent spontaneous gonadotropin pulsatility, and no response to either exogenous pulsatile (10 µg/pulse at 90-min intervals over 6 h) or acute (100 µg) GnRH administration. However, she responded to exogenous gonadotropin administration, with a resulting normal pregnancy. Genomic DNA extracted from peripheral blood was PCR amplified using amplimers spanning intron-exon boundaries for the three exons of GnRHR and revealed a homozygous splice junction mutation (G to A transversion) at the intron 1-exon 2 boundary. Her unaffected sister, with a totally normal phenotype, was heterozygous for this mutation. After lymphocyte Epstein-Barr virus transformation, RNA was extracted and subjected to RT-PCR, using primers located in the first and third exons. Results showed a transcript lacking all of exon 2 (exon 2 skipping), with splicing of exon 1 to exon 3. This created a frame shift, generating a coding sequence for three new amino acids, followed by a stop codon. Although it is not clear whether the mutant receptor is actually expressed, the resultant mRNA sequence was presumed to produce a truncated receptor with no binding or signaling capacity.
CONGENITAL IDIOPATHIC hypogonadotropic hypogonadism (IHH) is characterized by pubertal delay, low serum levels of sex steroids, and low or inappropriately normal levels of gonadotropins, in the absence of an anatomical cause (1). IHH is a heterogeneous condition, both clinically and genetically, that can be sporadic or inherited as either an X-linked or autosomal trait. It is commonly seen in association with other anomalies as in Kallmann syndrome (IHH and anosmia) or in adrenal hypoplasia congenita (IHH and adrenal insufficiency) (1). Although mutations in the X-linked loci responsible for the latter two conditions (KAL1 and DAX1, respectively) are well known (2, 3, 4, 5), until recently no genes involved in the autosomal forms of IHH had been described.
However, since 1997, GnRH receptor (GnRHR) mutations have been shown to be responsible for a significant proportion of autosomal recessive and, less commonly, sporadic cases of isolated IHH (6, 7, 8, 9, 10, 11, 12, 13, 14, 15). These patients present with variable degrees of hypogonadism, the majority of them comprising compound heterozygote mutations.
The GnRHR gene (4q13.1) (16) comprises three exons (17) and encodes a heptahelical transmembrane domain G protein-coupled receptor, lacking an intracellular carboxyl terminus normally seen in other members of this family (18, 19). Exon 1 encodes the receptor amino terminus, transmembrane domains 1, 2, and 3, and part of transmembrane domain 4. The rest of the fourth and the fifth transmembrane domains and part of the third intracellular loop are encoded by exon 2, the remainder of the receptor being encoded by exon 3 (17, 20). Ligand binding activates phospholipase C, increasing inositol triphosphate and resulting in mobilization of intracellular calcium, with release of LH and FSH (18).
In the present study, we describe a familial case of complete IHH associated with a novel homozygous mutation of the GnRHR gene. It is the first report of a splice site mutation in this gene, causing a transcriptional defect with the predicted generation of a severely truncated GnRHR receptor.
Case Report
The propositus first came to medical attention at the age of 16 yr with primary amenorrhea and lack of secondary sexual characteristics. At physical examination, she had no pubic hair and her breast development was Tanner stage I. She had a normal sense of smell and no other associated condition. Hormonal evaluation revealed low serum estrogen levels and undetectable basal serum LH and FSH. Other tests of anterior pituitary function were normal, as was a subsequent magnetic resonance imaging scan of the hypothalamo-pituitary region, establishing the diagnosis of IHH.
She was treated with estrogen and progestin replacement, resulting in development of thelarche, pubarche, and withdrawal bleeding. At the age of 33, she was reevaluated for infertility and underwent ovulation-induction therapy. A successful pregnancy ensued after five cycles of exogenous gonadotropin injections.
The family tree is shown in Fig. 1
. The patient’s parents, as well as the unaffected siblings, had had normal spontaneous pubertal development. However, two brothers and one sister were affected with the same phenotype of complete hypogonadotropic hypogonadism, in a kindred of 11. The family was originally from India, and the parents were second-degree cousins. The condition was, therefore, inherited as an autosomal recessive trait.
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Analysis of gonadotropin secretion
Protocol. Dynamic tests to determine the pattern of gonadotropin secretion and response to GnRH were performed in the propositus 2 months after withdrawal of sex steroid replacement therapy. A GnRH test (100 µg, iv) was performed, and plasma LH and FSH levels measured at 0, 30, and 60 min. The pattern of endogenous LH and FSH release was evaluated by 20-min blood sampling over a 6-h period under basal conditions and then for an additional 6-h period during pulsatile GnRH administration (10 µg/pulse, every 90 min, iv), using a mini-infusion pump (Zyklomat; Ferring Pharmaceuticals Ltd., Kiel, Germany). A standard GnRH test was repeated after 15 h of pulsatile GnRH infusion.
Assays. Serum FSH and LH concentrations were measured by a two-step sandwich immunoassay, run on an Elecsys 2010 (Roche Diagnostics Ltd., East Sussex, UK). The interassay coefficients of variation for FSH were 10.5% at 7.1 IU/liter and 11.5% at 17.7 IU/liter whereas for LH they were 5.1% at 1.5 IU/liter and 4.7% at 14.2 IU/liter. The lower limit of detection was 0.1 IU/liter. for both FSH and LH.
Genomic DNA isolation and DNA sequencing
Genomic DNA of the propositus and of one of her phenotypically normal sisters was extracted from peripheral blood cells, using a commercially available kit (QIAamp DNA minikit; QIAGEN, West Sussex, UK). The three exons of the GnRHR gene were then amplified from genomic DNA by PCR using the following primer pairs: E1-F, 5'-GGCTTGAAGCTCTGTCCTGGG-3'; E1-R, 5'-CCAAGGTAACAGAACAGAGC-3'; E2-F, 5'-CACTAAGGAGCTTAGAAATTGC-3'; E2-R, 5'-GCCCACAAATGACACTAAC-3'; E3-F, 5'-GATGCTGTTTTCCTTTTTGTCCAC-3'; E3-R, 5'-TGAGGCCTCTGAAGACTG-3', based on the intron-exon boundaries, as described by Kottler et al. (21). Reactions were performed in a final volume of 50 µl containing 10 pmol of each primer, 150 ng template DNA, 200 µmol dNTPs, 2.5 U HotstarTaq polymerase (QIAGEN), 50 mmol/liter KCl, 10 mmol/liter Tris-HCl (pH 8.3), and 2 mmol/liter MgCl2 and carried out for 35 cycles: denaturation at 94 C for 30 sec, annealing at 59–62 C for 30 sec, extension at 72 C for 1 min, and a final extension for 15 min at 72 C after the last cycle. All PCR products were separated on 0.7% agarose gel electrophoresis and sequenced by automatic DNA sequencing using dye terminator chemistry in an ABI 377 sequencer (MWG, Ebersberg, Germany).
RNA isolation and RT-PCR for the GnRHR
Lymphocytes were isolated from the patient’s peripheral blood and from a healthy sex- and age-matched control using the Ficoll-Hypaque method, as described previously by Chen et al. (22). Total RNA was extracted using a commercially available kit (RNeasy midi kit; QIAGEN) and reverse transcribed using the First Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech, Buckinghamshire, UK), according to the manufacturer’s instructions. The resultant cDNA was submitted to nested PCR for GnRHR. Human pituitary cDNA was used as a positive control. The first product was amplified using two pairs of primers (GnRHR-F1 and GnRHR-R1) located in exons 1 and 2 of the GnRHR (22). The nested reaction used 5 µl of the first reaction product as template and primers GnRHR-F2 and GnRHR-R2, resulting in a sequence spanning the last 67 bp of exon 1 and the first 145 bp of exon 2 (Fig. 2). Both PCR reactions were carried out for 40 cycles: denaturation at 94 C for 1 min, annealing steps at 57 C and 56 C for 40 sec for each reaction, respectively, and extension at 72 C for 1 min, followed by a final extension for 15 min at 72 C after the last cycle.
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). The first step PCR reaction was performed with GnRHR-F1 and GnRHR-R3 primers using 4 µl cDNA as template. Five microliters of the first reaction products were used as templates for the second step reaction, using primers GnRHR-F2 and GnRHR-R4. Each PCR reaction was carried out for 40 cycles: denaturation at 94 C for 1 min, annealing at 55 C for 40 sec, extension at 72 C for 1 min, and a final extension for 15 min at 72 C after the last cycle. The quality and predicted size of each PCR product was assessed by electrophoresis through a 0.7% agarose gel. Nested PCR products were cloned using the TOPO/TA cloning vectors (pcDNA3.1/V5/His-TOPO 5.5 kb; Invitrogen, Groningen, The Netherlands) and transfected into Top10 competent cells (Invitrogen). After an overnight culture in LB Broth medium containing ampicillin, recombinant DNA was extracted using a commercially available kit (QIAprep miniprep; QIAGEN) and sequenced using dye terminator chemistry in an ABI 377 sequencer (MWG).
Results
Analysis of gonadotropin secretion
Spontaneous gonadotropin secretion. Evaluation of spontaneous gonadotropin secretion in the propositus showed suppressed plasma LH and FSH levels and a complete absence of pulses. During the 6-h basal profile, LH and FSH serum levels were less than 0.1 IU/liter and 0.1 IU/liter, respectively, in all samples.
Response to pulsatile GnRH administration. LH and FSH remained very low during the 6-h pulsatile GnRH administration, with all the LH levels less than 0.1 IU/L. FSH levels were 0.2 IU/liter in 2 samples and 0.1 IU/liter in the remaining 15 samples.
GnRH test. GnRH tests, performed under basal conditions and after 15-h pulsatile GnRH infusion, showed a complete lack of response, with LH and FSH serum levels remaining less than 0.1 IU/liter and 0.1 IU/liter, respectively, in the 0, 30, and 60 min samples in both tests.
DNA sequencing
Figure 3 shows the results of genomic DNA sequencing of the GnRHR gene in the propositus and her unaffected sister. A G to A transversion in the acceptor splice site at the intron 1-exon 2 boundary was identified in the homozygous state in the propositus. The patient’s unaffected sister was heterozygote for this mutation.
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Lymphocytes have been shown to express GnRHR mRNA (22). Lymphocyte RNA was used to identify possible GnRHR transcripts originating from the mutated gene. Because the mutation was located in the acceptor splice site of exon 2, causing loss of the consensus acceptor splicing sequence (AG
AA), we predicted an abnormal transcription product. Using primers spanning the last 67 bp of exon 1 and the first 145 bp of exon 2 (Fig. 4), a PCR product corresponding to the expected sequence of GnRHR was identified from pituitary and control lymphocyte cDNA, but not from the patient’s lymphocyte cDNA, suggesting that at least the first 145 bp of exon 2 were deleted in the transcript from the mutated gene.
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and 4). The sequence was identical to the wild type, except for a deletion corresponding to the 220 bp of exon 2. In the sequence obtained, exon 1 splices directly on exon 3. The deletion of exon 2 causes a frame shift in the open reading frame, thus generating a new coding sequence after abnormal splicing, creating a stop codon 10 bp downstream. Therefore, the mutated gene encodes a 177 amino acid peptide, instead of the wild-type 328 amino acids long GnRHR. Discussion
In this study, we report a novel inactivating homozygous mutation of the GnRHR gene in a familial case of complete IHH. The severity of the phenotype is indicated by the clinical history of absent spontaneous secondary sexual development and confirmed by completely suppressed basal and stimulated levels of gonadotropins in all dynamic tests performed in the patient. In this pedigree, the heterozygous state is not associated with a phenotype, as shown in one unaffected sister of the propositus. The history of consanguinity between the parents and the pedigree with 4 affected siblings in 11 corroborates the autosomal recessive mode of inheritance.
Beranova et al. (13) suggest that GnRHR gene mutations may be more frequent than previously suspected, being present in about 17% of the sporadic and in 40% of the autosomal recessive cases of IHH. No mutation in this gene has been found in the autosomal dominant and X-linked cases. To date, several different mutations have been described in the GnRHR gene (6, 7, 8, 9, 10, 11, 12, 13, 14, 15), resulting in a broad phenotypic spectrum, varying from mild forms, as in the fertile eunuch syndrome (24), to complete GnRH resistance (10, 12, 14). This heterogeneity seems to be related, at least in part, to the site of mutation and, therefore, to the degree of impaired biological activity of the receptor, although patients with different phenotypes and the same mutations have been described (8). In the case described here, the striking severity of GnRH resistance is consistent with complete loss of receptor function.
Of the previously reported mutations, all are point mutations causing one amino acid substitution, except one, Leu314X, that encodes a stop codon at the end of exon 3, causing a deletion of the 10 last amino acids of the receptor, completely impairing ligand binding (11). Two of them, Gln106Arg and Arg262Gln, can be considered hot spots, reported six and four times in unrelated cases, respectively, both leading to mild defects (partial receptor function). Most patients displaying GnRHR mutations are compound heterozygotes and show some degree of response to a standard GnRH test. Interestingly, patients displaying homozygous mutations tend to exhibit a more severe phenotype.
GnRHR has been shown to be expressed outside the central nervous system, in a variety of endocrine (23, 25) and nonendocrine (26, 27) tissues. Chen et al. (22) demonstrated GnRHR mRNA expression in peripheral lymphocytes, which was up regulated by GnRH exposure in vitro, suggesting a role for GnRH in the immune system. In this study, we used lymphocyte cDNA to identify the transcripts originating from the mutated gene. Using lymphocytes from a healthy control, we were able to amplify the GnRHR cDNA, with a sequence identical to their pituitary counterparts, confirming the findings of Chen et al. (22). In the patient’s lymphocyte cDNA, however, a markedly truncated sequence was amplified.
The mutation described here affects the acceptor splice site in the first intron-exon 2 boundary, where the consensus AG sequence is replaced for AA. This impairs normal splicing, leading to the deletion of the whole exon 2, with exon 1 splicing directly on exon 3. The deletion of the 220 bp of exon 2 causes a frame shift, creating a stop codon 10 bp downstream in exon 3, leading to a premature translational termination. Thus, the mutated cDNA is predicted to encode a truncated receptor, with only four transmembrane domains. The importance of exons 2 and 3 is also illustrated by studies done with the human GnRHR splice variant, described by Grosse et al. (28). The alternative splicing caused the deletion of the first 128 bp of exon 2, generating a frame shift in the open reading frame and an abnormal coding sequence for an additional 75 amino acids. The resultant truncated receptor showed reduced plasma membrane insertion when compared with the wild-type receptor, and it was totally incapable of ligand binding or signal transduction in vitro.
A second subtype of GnRHR, GnRHR2, has been described recently that has approximately 40% homology with GnRHR and is broadly distributed in the central nervous system. Although it has high affinity for GnRH2, its physiological role is not yet understood (29, 30). The case described here suggests that, even if it has any role in the hypothalamic-pituitary-gonadal axis modulation, its action is not enough to compensate for GnRHR1 loss of function.
In summary, we have described a woman with complete IHH caused by a novel homozygous loss of function mutation in the GnRHR gene, in a family presenting with an autosomal recessive pattern of inheritance. A good correlation between phenotype and genotype was observed in this case. This is the first time a splice site mutation has been described, leading to complete deletion of exon 2 and a predicted markedly truncated receptor, lacking domains that are critical for ligand recognition and signal transduction (31, 32, 33). Furthermore, the heterozygote was phenotypically unaffected, confirming that a gene disruption affecting one allele can be completely compensated for.
Acknowledgments
Footnotes
Abbreviations: GnRHR, GnRH receptor; IHH, idiopathic hypogonadotropic hypogonadism.
Received October 31, 2001.
Accepted January 28, 2002.
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