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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 7 3189-3198
Copyright © 2004 by The Endocrine Society

CLINICAL CASE SEMINAR

GNRHR Mutations in a Woman with Idiopathic Hypogonadotropic Hypogonadism Highlight the Differential Sensitivity of Luteinizing Hormone and Follicle-Stimulating Hormone to Gonadotropin-Releasing Hormone

Astrid U. Meysing, Haruhiko Kanasaki, Gregoy Y. Bedecarrats, James S. Acierno, Jr., P. Michael Conn, Kathryn A. Martin, Stephanie B. Seminara, Janet E. Hall, William F. Crowley, Jr. and Ursula B. Kaiser

Reproductive Endocrine Unit (A.U.M., J.S.A., K.A.M., S.B.S., J.E.H., W.F.C.) and Harvard-Wide Reproductive Endocrine Sciences Center (A.U.M., H.K., G.Y.B., J.S.A., K.A.M., S.B.S., J.E.H., W.F.C., U.B.K.), Massachusetts General Hospital, Boston, Massachusetts 02114; Division of Endocrinology, Diabetes and Hypertension (H.K., G.Y.B., U.B.K.), Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115; and Oregon National Primate Research Center and Department of Physiology and Pharmacology (P.M.C.), Oregon Health and Science University, Beaverton, Oregon 97006

Address all correspondence and requests for reprints to: Dr. Astrid Meysing, Reproductive Endocrine Unit, Bartlett Hall Extension 5, Massachusetts General Hospital, Boston, Massachusetts 02114. E-mail: ameysing{at}partners.org.


   Abstract
Top
 Abstract
Introduction
 Case Report
 Materials and Methods
 Results
 Discussion
 References
 
Mutations in the GnRH receptor gene (GNRHR) are a cause of idiopathic hypogonadotropic hypogonadism. We describe a normosmic female subject with congenital idiopathic hypogonadotropic hypogonadism in whom treatment with pulsatile GnRH resulted in an unusual response. The subject not only required an increased dose of pulsatile GnRH for ovarian follicular development, but LH secretion did not increase appropriately, estradiol levels remained low, and she did not ovulate spontaneously.

Sequencing of the GNRHR coding sequence revealed compound heterozygous mutations leading to amino acid substitutions [N10K+Q11K] and P320L. The introduction of the P320L mutation into the GnRH receptor led to failure of detectable ligand binding and failure of stimulation of inositol phosphate production and gonadotropin subunit gene promoter activity in response to GnRH in transiently transfected cells. The [N10K+Q11K] mutation resulted in reduced binding of a GnRH agonist to 25% of the wild-type receptor. In addition, the EC50 value for GnRH stimulation of inositol phosphate production was significantly increased, and the dose-response curves for stimulation of {alpha} gonadotropin subunit, LHß, and FSHß gene transcription by GnRH were similarly shifted to the right. Stimulation of FSHß gene transcription was more sensitive to GnRH than LHß for both wild-type and [N10K+Q11K] GnRH receptors, resulting in a greater loss of LHß stimulation than FSHß by the [N10K+Q11K] mutant at any given submaximal GnRH concentration.

We propose that the mutations in the GnRH receptor result in a rightward shift of the dose-response curves of gonadotropin responses to pulsatile GnRH in the subject and unmask the differential sensitivities of LH and FSH to GnRH, resulting in low LH and estradiol levels despite appropriate FSH secretion and follicular growth.


   Introduction
Top
 Abstract
 Introduction
Case Report
 Materials and Methods
 Results
 Discussion
 References
 
IDIOPATHIC HYPOGONADOTROPIC hypogonadism (IHH) is a genetically heterogeneous disorder that has been described in both males and females with delayed sexual development and low gonadotropin and sex steroid levels. Forty percent of autosomal recessive IHH cases and 16% of sporadic IHH cases are due to mutations in the GnRH receptor gene (GNRHR) (1, 2). In contrast to the predominantly male occurrence in congenital IHH, GNRHR mutations appear at a similar frequency in both sexes, as would be expected in an autosomal recessive disorder.

The phenotypic appearance of patients with GNRHR mutations is varied in both males and females (3) but can generally be characterized as partial or complete IHH, depending on the degree of the impairment of the hypothalamic-pituitary-gonadal axis. At puberty, some patients present with failure to undergo sexual maturation, whereas others undergo some pubertal development, including menses, that then ceases (4, 5, 6). Diagnosis of an incomplete form is made if there is evidence of some endogenous GnRH secretion as reflected by episodic LH secretion demonstrated by frequent LH sampling, some signs of sexual development, and secondary amenorrhea in women. Family members carrying the same GNRHR mutations can present with varying severity of phenotypic characteristics of IHH (7).

The GnRH receptor (GnRHR) belongs to the superfamily of G protein-coupled receptors. To date, 15 mutations in the coding sequence of the GNRHR gene have been found throughout the different domains of the receptor (1, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14). In addition, a mutation in the intronic region was reported recently (15), which resulted in splicing of exon 1 to exon 3, leading to transcripts lacking exon 2, presumed to produce a truncated receptor lacking binding and signaling capacity.

The mechanisms responsible for the loss of function of mutant GnRHRs are currently under investigation. Defects may occur in the synthesis of receptors, in trafficking to the cell membrane and/or in internalization, recycling, or degradation of receptors, in ligand binding, and/or in G protein coupling and signal transduction (15, 16, 17).

In vivo studies of baseline gonadotropin secretion and gonadotropin responses to exogenous pulsatile GnRH administration have demonstrated the impact of GNRHR mutations on LH, FSH, and sex steroid secretion. The previously reported female patients with GNRHR mutations and complete IHH did not respond to pulsatile GnRH with folliculogenesis and ovulation (4, 10), whereas patients with partial IHH demonstrated dose-dependent responses to pulsatile GnRH in terms of gonadotropin secretion and ovulation (18, 19).

Here, we report a newly identified case of a female subject with partial IHH who did not respond to increasing doses of GnRH with a normal sequence of follicular development, ovulation, and corpus luteum formation. Two previously unreported mutations in the coding sequence of the GNRHR gene were identified in this subject. The effects of these mutations on the hypothalamic-pituitary-ovarian axis were determined and correlated with their in vitro effects on receptor binding, signaling, and regulation of gonadotropin subunit (GSU) gene expression.


   Case Report
Top
 Abstract
 Introduction
 Case Report
Materials and Methods
 Results
 Discussion
 References
 
The propositus is a 26-yr-old Caucasian female, who came to the Reproductive Endocrine Unit of Massachusetts General Hospital seeking fertility. Her medical evaluation began at the age of 15 when she presented to a pediatrician with primary amenorrhea, lack of secondary sexual characteristics, and a normal sense of smell. Physical examination revealed Tanner stage III breasts, pubic hair growth, and axillary sebaceous gland development. A computerized tomography scan of the hypothalamic-pituitary region was normal. After endocrinological testing, the subject was diagnosed with isolated gonadotropin deficiency, and cyclic Premarin (1.25 mg) and Provera (10 mg) were initiated, which resulted in regular withdrawal bleeding. At age 16, a clomiphene citrate challenge test demonstrated no increase in LH, FSH, or estradiol levels. The subject continued Premarin and Provera until age 20, when Ortho Novum 1/50 was administered. At age 22 yr, she was referred to the Pediatric Endocrine Unit of Massachusetts General Hospital and underwent a GnRH stimulation test (200 µg bolus), which induced a marked increase in LH and FSH [baseline: LH, 0.9 IU/liter; FSH, 3.8 IU/liter; peak: LH, 19.3 IU/liter (30 min); FSH, 20.9 IU/liter (60 min)].

When presenting to the Reproductive Endocrine Unit at the age of 26, she was amenorrheic after discontinuing her oral contraceptives 3 months earlier and had a normal diet and no history of excessive exercise or stress. She had Tanner stage IV breasts and pubic hair, a height of 174.6 cm, and a weight of 58.6 kg (body mass index, 19.4 kg/m2). A pelvic examination and ultrasound imaging revealed a small uterus and ovaries. Her LH was 0.9 IU/liter, FSH was 2.6 IU/liter, estradiol was 15 pg/ml, and prolactin and TSH were within the normal range.

There was no family history of delayed puberty, although one aunt had been diagnosed with infertility of unknown cause. The sister of the propositus had conceived twice and delivered two healthy children. There was no indication of parental consanguinity.

Assessment of baseline gonadotropin secretion was performed. Exogenous pulsatile GnRH therapy was initiated to achieve fertility. The subject was begun on a dose of 75 ng/kg administered iv at a physiological frequency based on our previous normative data (20) that was then titrated up to 500 ng/kg over succeeding cycles. Subsequently, she received gonadotropin therapy and conceived healthy twins.


   Materials and Methods
Top
 Abstract
 Introduction
 Case Report
 Materials and Methods
Results
 Discussion
 References
 
In vivo studies

All studies were approved by the Institutional Review Board of Massachusetts General Hospital.

DNA extraction and sequencing. Blood samples were only available from the propositus and her mother. DNA was extracted using a commercially available extraction kit (Qiagen, Valencia, CA). The three exons of the GNRHR gene were amplified by PCR using sets of primers reported previously (1). Sequencing was performed using the AmpliTaq dye terminator sequencing kit and an ABI prism 377 DNA sequencer (Perkin-Elmer Corp., Foster City, CA).

Proband Characterization of baseline gonadotropin secretion.
Blood was sampled every 10 min for 12 h for assessment of baseline gonadotropin secretion; all samples were assayed for LH. Pulsatile hormone secretion was analyzed using a validated modification of the Santen and Bardin method (21).

Gonadotropin response to exogenous GnRH administration.
Pulsatile GnRH was administered as described previously (22). The frequency of GnRH secretion was adjusted during each cycle to mimic the frequency changes in GnRH secretion that occur during a normal menstrual cycle, as reported previously (20), beginning at every 90 min and increasing to every 60 min with development of a follicle greater than 11 mm. An initial dose of 75 ng/kg/bolus was administered iv because this dose has been shown previously to recreate the normal physiology of an ovulatory cycle in a large series of women with hypogonadotropic hypogonadism (23). However, due to a poor response, as reflected by low estradiol levels and lack of ovulation, the dose was progressively increased (75, 100, 150, 250, and 500 ng/kg/bolus) to a final dose of 500 ng/kg/bolus. Serial blood samples were obtained daily 45 min after a GnRH bolus for measurement of LH, FSH, estradiol, and progesterone during pulsatile GnRH therapy.

IHH controls.
Eight women with GnRH deficiency, aged 17–32 yr (mean, 23.6), were selected on the basis of the following criteria: primary amenorrhea and absence of spontaneous pubertal development; normal cranial imaging of the hypothalamic pituitary area; prolactin and thyroid-stimulating hormone within the normal range. All subjects had undergone a baseline frequent gonadotropin sampling study with blood sampled every 10 min for 12 h before pulsatile GnRH replacement to confirm the absence of pulsatile LH secretion. Subjects with the above characteristics and anosmia were considered to have Kallmann syndrome (n = 5). The other three IHH subjects had normal coding sequences when screened for GNRHR and DAX1 gene mutations.

All eight subjects underwent iv pulsatile GnRH therapy at a dose of 75 ng/kg/bolus. The frequency of GnRH administration was adjusted during the cycle to mimic the frequency changes that occur during the normal menstrual cycle (20, 23). Daily blood sampling was performed for LH, FSH, estradiol, and progesterone. Samples were drawn at the same time of the day and were collected 45 min after a GnRH bolus.

Hormone assays. All blood samples were measured in duplicate. Serum FSH and LH were assayed in specific ß- and dimer-directed RIAs, respectively, as described previously (23). Both hormones are expressed in international units per liter as equivalents of the Second International Reference Preparation of the human menopausal gonadotropin (World Health Organization, 71/223). The sensitivities of the assays were 0.8 IU/liter for LH and FSH. The intraassay coefficients of variation (CV) for LH and FSH were 6.1 and 7.0%, respectively, and the interassay CV were 8.5 and 11.6%, respectively. Serum estradiol was measured by the Abbott AxSYM system (Abbott Laboratories, Abbott Park, IL), which has an analytical sensitivity of 10 pg/ml, an intraassay CV of less than 6.4%, and an interassay CV of less than 10.6%. Progesterone was measured as described previously (24).

Data analysis. Data for responses to 75 ng/kg/bolus of pulsatile GnRH for the controls are presented as mean ± SE.

In vitro studies

Cell culture. All cell culture reagents were obtained from Life Technologies, Inc. (Gaithersburg, MD). COS-7 cells and GH3 cells were cultured as described previously (25).

Site-directed mutagenesis. Site-directed mutagenesis was used to introduce either [N10K+Q11K] or P320L into an expression vector encoding the wild-type human GnRHR (hGnRHR) fused to an N-terminal hemagglutinin (HA) epitope tag generously provided by Dr. Thomas Gudermann (Children’s University Hospital, Marburg, Germany) (26). For the [N10K+Q11K] hGnRHR mutant, the Asn codon AAT was replaced with the Lys codon AAA to mutate the nucleotide at position 30, and the Gln codon CAA was replaced with the Lys codon AAA to mutate the nucleotide at position 31 in the hGnRHR cDNA, using the primers 5'-GTGCCTCTCCTGAACAGAAAAAAAATCACTGTTCAGCCATC-3' (sense) and 5'-GATGGCTGAACAGTGATTTTTTTTCTGTTCAGGAGAGGCAC-3' (antisense). For the P320L mutant, the Pro codon CCA was replaced with the Leu codon CTA to mutate the nucleotide at position 959, using the primers 5'-AACCCATGCTTTGATCTACTTATCTATGGATA-3' (sense) and 5'-TATCCATAGATAAGTAGATCAAAGCATGGGTT-3' (antisense). Each hGnRHR mutant was generated using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA), and proper incorporation of the mutations was confirmed by bidirectional sequencing.

Immunocytochemistry. COS-7 cells were plated on glass-bottomed 35-mm tissue culture dishes (MatTek Corp., Ashland, MA) and transiently transfected by lipofection using GenePorter (Gene Therapy System, San Diego, CA) with 2 µg of each hGnRHR construct (wild type, [N10K+Q11K], or P320L) or the control expression vector (pcDNA3). After 48 h, cells were washed and fixed, as described previously (25). The presence of the wild-type and mutant hGnRHRs on the surface of intact, nonpermeabilized cells was examined under a x40 oil immersion objective using a confocal laser microscope (MRC-1024 multi-photon system; Bio-Rad, Hercules, CA) after incubation with an anti-[HA]-fluorescein antibody (Roche Molecular Biochemicals, Indianapolis, IN).

Receptor-binding assay. COS-7 cells were plated in 35-mm tissue culture dishes and transiently transfected by lipofection with 2 µg/well of each hGnRHR construct or pcDNA3. After 48 h of incubation at 37 C, cells were washed with DMEM containing 0.1% BSA and incubated for 90 min at room temperature with 100,000 cpm 125I-Buserelin (26). For specific binding measurements, 10–6 M GnRH of unlabeled GnRH (Sigma Chemical Co., St. Louis, MO) was added, whereas for displacement curve measurements, increasing concentrations (10–10 to 10–6 M) of GnRH were used. Cells were washed twice with ice-cold PBS and lysed with 1 ml of 0.2 M NaOH and 0.1% SDS. Protein content in the cell lysates was calculated (Coomassie Plus Protein Assay reagent; Pierce, Rockford, IL), and radioactivity was measured by a gamma counter. The binding affinity (Kd) and receptor number (Bmax) were calculated based on a nonlinear regression of homologous competition binding analysis using Prism 3.0 (GraphPad Software, San Diego, CA). All assay points were performed in triplicate, and each experiment was repeated at least three times.

Inositol phosphate (IP) assay. The protocol for measuring total IP production has been described previously (27). Briefly, COS-7 cells were transiently transfected by lipofection with 2 µg/well of each hGnRHR construct or pcDNA3 and plated into 6-well culture plates. After a 24-h incubation at 37 C, medium was replaced with 1 ml inositol-free DMEM for 2 h, then 1 ml of the same medium containing 1 µCi myo- [2-3H] inositol (NEN Life Science Products, Boston, MA), followed by the addition of 10 mM LiCl 15 min later. Cells were incubated at 37 C for an additional 14 h and stimulated with serial concentrations (10–10 to 10–6 M) of GnRH for 45 min. Cell lysates were prepared, protein content was measured, and IP was extracted as described previously (25). Radioactivity was quantified in a scintillation counter and corrected for protein content. All assay points were performed in triplicate, and each experiment was repeated at least three times.

Luciferase assays. Reporter constructs used were generated by fusing –797/+5 of the rat LHß gene, –2000/+698 of the rat FSHß gene, and –846/0 of the human {alpha}GSU gene, to the firefly luciferase (Luc) cDNA in pXP2, as described previously (28, 29, 30). GH3 cells have previously been shown to support GSU and GnRHR gene promoter activity and were, therefore, used for these studies. Cells were transiently transfected by electroporation with 2 µg/well of each hGnRHR construct or pcDNA3, 2 µg/well of {alpha}GSU-Luc, FSHß-Luc, or LHß-Luc, and 1 µg/well of Rous sarcoma virus-ß-galactosidase vector. Cells were then seeded into 6-well tissue culture plates, incubated for 48 h at 37 C, and stimulated for 4 h with increasing concentrations of GnRH (10–10 to 10–6 M). Cells were washed twice with ice-cold PBS and lysed with 125 mM Tris-HCl and 0.5% Triton. After centrifugation at 14,000 x g at 4 C, luciferase and ß-galactosidase activities were measured in the supernatants. Luciferase activity was normalized for ß-galactosidase activity to correct for transfection efficiency, and the results were expressed as fold of unstimulated controls. All assay points were performed in triplicate, and each experiment was repeated at least three times.

Data analysis. The data for each set of in vitro experiments were subjected to nonlinear regression analysis, and the EC50 value for each study was calculated using Prism 3.0 (GraphPad Software). All statistical analyses were performed using Instat 3.0 (GraphPad Software) and were based on nonparametric paired ANOVA (P < 0.05).


   Results
Top
 Abstract
 Introduction
 Case Report
 Materials and Methods
 Results
Discussion
 References
 
DNA sequencing

Direct DNA sequencing of GNRHR revealed two heterozygous mutations in the coding sequence of the propositus (Fig. 1AGo). On one allele, an adenine was substituted for thymine at nucleotide position 30 (relative to the translational start site) and an adenine was substituted for cytosine at position 31 in exon 1, resulting in a two-amino acid substitution, lysine for asparagine and lysine for glutamine ([N10K+Q11K]) in the N terminal extracellular domain (Fig. 1BGo). On the other allele, a thymine was substituted for a cytosine at nucleotide position 959 in exon 3, resulting in substitution of leucine for proline (P320L) in the seventh transmembrane domain of the GnRHR (Fig. 1BGo). The mother of the propositus was analyzed by the same procedures and was shown to be heterozygous for only the [N10K+Q11K] mutation.



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  		FIG. 1. Sequence traces and amino acid changes of the proband. A, Forward sequence traces of the proband. Nucleotide changes are indicated by arrows. M, Mutant; WT, wild-type. B, The changes in amino acids resulting from the changes in nucleotides are presented for the mutants compared with the wild-type and are presented within a GnRHR model.

 
In vivo studies

Characterization of baseline gonadotropin secretion. Results of the frequent blood sampling study to assess baseline gonadotropin secretion and determine the presence of spontaneous pulses indicated that the subject had LH levels that ranged from 1.3–2.3 IU/liter, with a mean LH of 1.78 IU/liter. A single LH pulse of low amplitude was identified (data not shown), whereas no LH pulses were identified in any of the IHH controls.

Gonadotropin and steroid response to exogenous GnRH administration and comparison with IHH controls. To determine the responsiveness of the subject to exogenous GnRH, increasing doses of GnRH were administered, as described above. Results are summarized in Table 1Go. At 75 ng/kg GnRH/pulse, the first dose used, there was evidence of folliculogenesis, but estradiol levels did not increase and ovulation did not occur. Results of LH, FSH, and estradiol levels between d 2–5 of administration of 75 ng/kg/pulse of iv pulsatile GnRH for the subject were compared with those of the IHH controls (Table 1Go; Fig. 2Go). As demonstrated in Fig. 2AGo, the LH levels in the subject were consistently below the levels of the IHH controls. In contrast, the mean FSH levels (Fig. 2BGo) were similar in subjects and controls. The resultant LH/FSH ratio for d 2–5 in our subject were consistently below those of the IHH controls (Fig. 2CGo; Table 1Go), reflecting the lower levels of LH relative to FSH in our subject. These lower levels of LH were associated with a minimal increase in estradiol (Fig. 2DGo).


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  		TABLE 1. Hormonal responses to pulsatile GnRH in the subject compared with IHH controls

 


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  		FIG. 2. Comparison of LH (A), FSH (B), LH/FSH ratio (C), and estradiol (D) levels of the proband (•) with eight IHH controls ({circ}) on d 2–5 of 75 ng/kg/pulse iv GnRH administration. Bars indicate SEs for the data of the IHH controls.

 
A dose of pulsatile GnRH of 100 ng/kg/bolus in the subject resulted in growth of a follicle to 16 mm, but again there was an inadequate increase in estradiol (Table 1Go). In the next cycle, the dose of GnRH was increased to 150 ng/kg/pulse. At this dose, FSH and LH increased within the first 5 d of treatment, although the LH response was below that of the IHH controls. Follicular development was somewhat delayed in comparison to the response in the IHH control group, with two follicles of 12 mm seen after 11 d of stimulation and a single dominant follicle of 25 mm after 17 d with a peak estradiol of 256 pg/ml. The peak estradiol attained was below the 10th percentile for the control IHH women (Table 1Go). The subject was given human chorionic gonadotropin (hCG) on d 18 of therapy as follicle size had plateaued. Ovulation occurred, but the subject did not conceive. At a subsequent dose of 250 ng/kg/pulse, there was again an increase in follicle size to 18 mm, but no associated increase in estradiol or spontaneous ovulation. Finally, at a dose of 500 ng/kg/pulse, two follicles developed, and there was an increase in estradiol to a peak of 126 pg/ml, but ovulation again failed to occur (Table 1Go).

In vitro studies

Cell surface expression of mutant hGnRHRs. To ensure that the mutant hGnRHRs were expressed appropriately, COS-7 cells were transiently transfected with HA-tagged wild-type or HA-tagged mutant hGnRHRs and analyzed by immunocytochemistry for detection of receptors on the surface of fixed, intact, nonpermeabilized cells. Fluorescence was detected at the periphery of COS-7 cells transfected with either the wild-type hGnRHR (Fig. 3AGo) or the mutant (Fig. 3Go, B and C) receptors, confirming cellular expression and membrane localization. No fluorescence was detected in cells transfected with pcDNA3, used as a negative control (Fig. 3DGo).



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  		FIG. 3. Confocal microscopy images of intact, nonpermeabilized COS-7 cells transfected with HA-tagged hGnRHR constructs encoding wild-type (A), [N10K+Q11K] (B), P320L (C), and pcDNA3 (D). The wild-type hGnRHR and both mutants are detected on the cell surface. Each image is representative of three independent experiments.

 
Receptor binding assays. The detection of receptors by immunocytochemistry was not quantitative and did not necessarily indicate normal levels of cell surface expression or proper conformation of the mutant receptors. Therefore, to determine whether the mutations affected the ability of receptors to bind ligand with high-affinity, ligand binding assays were performed in transiently transfected cells (Fig. 4Go). Specific binding of 125I-Buserelin by the [N10K+Q11K] mutant receptors was 25% of the wild-type hGnRHR, whereas the P320L mutant receptors failed to exhibit any detectable specific binding (Fig. 4AGo). To further characterize the binding characteristics of the [N10K+Q11K] hGnRHR in comparison with the wild-type hGnRHR, displacement analysis was performed, and Kd and Bmax were calculated (Fig. 4Go, B and C). Bmax of the [N10K+Q11K] mutant was significantly reduced compared with the wild-type receptor (1.2 ± 0.8 vs. 8.9 ± 1.7, respectively), but there was no change in the Kd. These results indicate that the [N10K+Q11K] mutation reduces cell surface receptor expression but has no effect on ligand-binding affinity, whereas the P320L mutation renders the receptor entirely unable to bind ligand in transiently transfected cells.



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  		FIG. 4. Binding analysis of wild-type and mutant receptors. A, COS-7 cells were transiently transfected with wild-type or mutant hGnRHRs, and specific binding of 125I-Buserelin was measured. B, Displacement curve measurements were performed by incubating the cells for 90 min at room temperature with 100,000 cpm 125I-Buserelin and increasing concentrations of GnRH (10–10 to 10–6 M). All assay points were performed in triplicate, and each experiment was repeated at least three times. Values are mean ± SE. *, P < 0.01 vs. wild-type hGnRHR; #, P < 0.05 vs. pcDNA3. C, After nonlinear regression analysis, Kd and Bmax were calculated.

 
IP accumulation in response to GnRH. To determine the effects of the hGnRHR mutations identified in the patient on GnRH-induced intracellular signaling, we measured the ability of the mutant hGnRHRs to stimulate IP accumulation in response to increasing concentrations of GnRH (Fig. 5Go). The P320L mutant hGnRHR was unable to stimulate any detectable increase in IP production (data not shown). In contrast, the [N10K+Q11K] mutant GnRHR was able to mediate GnRH-stimulated IP production (Fig. 5Go). Interestingly, the maximal IP responses for the [N10K+Q11K] mutant were the same as for the wild-type receptor, despite the reduced receptor numbers. However, the dose-response curve was shifted to the right, resulting in an EC50 value for IP stimulation that was significantly increased, by 3.7-fold, when compared with the wild-type hGnRHR (11.5 ± 5.4 vs. 3.1 ± 1.2, respectively; P < 0.01). These results suggest that in addition to reducing receptor number, the [N10K+Q11K] mutation also reduces efficacy of coupling of the GnRHR to intracellular signal transduction pathways.



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  		FIG. 5. Stimulation of GnRH-mediated IP production by wild-type and [N10K+Q11K] receptors. Values for each GnRHR are expressed relative to IP levels in untreated cells. The dose-response curves were used to calculate the EC50 values. Data are presented as mean ± SE of three independent experiments, each performed in triplicate. *, P < 0.01 vs. wild type.

 
Stimulation of GSU gene promoter activity by GnRH. It has been shown that the effects of hGnRHR mutations on IP induction do not always correlate with the effects on GSU gene expression (30). Therefore, because our ultimate interest is to understand the effects of the GnRHR mutations on gonadotropin production and to correlate with in vivo observations, we studied the effects of the [N10K+Q11K] and P320L hGnRHR mutations on GnRH-stimulated {alpha}GSU, FSHß, and LHß gene transcription. The P320L mutant was not able to stimulate any of the luciferase reporters ({alpha}Luc, FSHßLuc, and LHßLuc) even at high concentrations of GnRH (data not shown), in keeping with its failure to bind ligand or stimulate IP production. Dose-response analyses were performed for the [N10K+Q11K] mutant and compared with the wild-type GnRHR (Fig. 6Go, Table 2Go).



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  		FIG. 6. Effect of GnRH on stimulation of GSU gene transcription by wild-type and [N10K+Q11K] hGnRHRs. GH3 cells were transiently cotransfected with hGnRHRs, Rous sarcoma virus-ß-galactosidase, and {alpha}GSU-Luc (A), LHß-Luc (B), and FSHß-Luc (C). Forty-eight hours after transfection, cells were stimulated with increasing concentrations of GnRH (from 10–10 to 10–6) for 4 h, and cell extracts were assayed for luciferase activity and normalized for ß-galactosidase activity. All luciferase activities are expressed relative to levels in untreated cells transfected with pcDNA3. Values are presented as means ± SE. All assay points were performed in triplicate, and each experiment was repeated at least three times. *, P < 0.01 vs. wild type.

 

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  		TABLE 2. Effect of varying concentrations of GnRH on the stimulation of GSU gene transcription (fold induction; mean ± SEM) in wild-type and mutant hGnRHRs

 
As for IP stimulation, the dose-response curves for stimulation of {alpha}GSU, FSHß, and LHß promoter activities were all shifted to the right for the [N10K+Q11K] mutant GnRHR compared with the wild-type receptor. As a result, the EC50 values for all three GSU genes were significantly increased (by 1.9-fold for {alpha}GSULuc, by 3.0-fold for LHßLuc, and by 3.5-fold for FSHßLuc) (Fig. 6Go). Table 2Go presents the absolute values for the fold induction for each of the subunit gene promoters in response to varied doses of GnRH, for both wild-type and [N10K+Q11K] hGnRHRs. As for IP stimulation, the maximal induction of {alpha}GSU-, FSHß-, and LHß-Luc activity at high concentrations of GnRH was the same for the [N10K+Q11K] mutant as for the wild-type hGnRHR. The same effect on the dose-response curves and on the EC50 values was observed even when the receptor number was modified by transfecting varying amounts of the GnRHR plasmids (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Case Report
 Materials and Methods
 Results
 Discussion
References
 
In women, FSH has been recognized as the key driver of ovarian follicle growth and maturation (31). LH, in turn, acts on theca cells to induce the production of androgen substrate. Androgens then are aromatized to estrogen under the control of FSH (32, 33). Treatment of female patients with complete GnRH deficiency with recombinant FSH only (i.e. in the absence of LH) allows multiple follicles to develop but produces inadequate estradiol concentrations and reduces the occurrence of ovulation in response to hCG (33, 34, 35). These results are consistent with the requirement for LH to produce androgen substrate for aromatization to estradiol. Our subject presented a similar clinical response to pulsatile GnRH. She developed follicles up to a size of 25 mm in diameter, but with low estradiol levels despite the use of high pharmacological doses of pulsatile GnRH, and failed to ovulate in the absence of exogenous hCG.

The amount of LH activity required for a physiological cycle is not known [but is likely to be very low because <1% of follicular LH receptors need to be occupied to allow normal steroidogenesis (32)]. We compared the follicular phase response of our proband to 75 ng/kg pulsatile GnRH to that of a group of eight GnRH-deficient women using the same dose of GnRH replacement. The LH levels and the LH/FSH ratios of our subject on d 2–5 of therapy were clearly below the values of the control group and reflect the low LH, but normal FSH, secretion in our subject.

The results in the current subject stand in contrast to a patient previously described by our group (19), with the mutations Gln106Arg and Arg262Gln in the GnRHR, who not only responded to iv pulsatile GnRH by developing a single dominant follicle but also conceived three times. This patient was able to reestablish a physiological cycle pattern with appropriate LH and FSH secretion in a dose-dependent manner. In this patient, high doses of pulsatile GnRH were able to overcome the receptor defects (19). In contrast, in the current subject, doses of pulsatile GnRH ranging from 75–500 ng/kg were neither able to completely overcome the effects of the mutant receptors nor were they able to normalize the LH/FSH ratio to reestablish a physiological pattern of LH and estradiol secretion.

The finding that the EC50 values for stimulation of {alpha}GSU, FSHß, and LHß by GnRH were similarly affected by the GnRHR mutants appeared to be at variance with our subject’s differential response in FSH and LH to iv administration of pulsatile GnRH. However, although the fold changes in EC50 values for GnRH stimulation of all three GSU genes were comparable, the absolute EC50 values were different for the different subunits for both wild-type and [N10K+Q11K] hGnRHRs. In particular, transcriptional activation of the FSHß gene was more sensitive to GnRH than the LHß gene (EC50 values for wild-type GnRHR: 3.5 ± 1.7 vs. 9.6 ± 1.0 for FSHß vs. LHß, respectively; 12.4 ± 5.1 vs. 28.8 ± 4.5 for [N10K+Q11K] hGnRHR). This differential sensitivity of FSHß and LHß to GnRH has been observed previously (30). The net effect of this differential sensitivity is that the [N10K+Q11K] mutation in the GnRHR may result in a greater loss of LHß induction than of FSHß induction by a given concentration of GnRH. For example, at 10–8 M GnRH, stimulation of LHß Luc activity was 5-fold for wild-type GnRHR but only 2.5-fold for the [N10K+Q11K] mutant, whereas stimulation of FSH ßLuc activity was 7-fold for wild-type hGnRHR and remained quite highly responsive (~6-fold) for the [N10K+Q11K] mutant. Differences in LHß and FSHß subunit gene stimulation by GnRH are more apparent for the [N10K+Q11K] mutant receptors at submaximal concentrations of GnRH. These differences may underlie the differential responses of LH and FSH secretion to GnRH observed in vivo. At high GnRH doses, this differential effect is diminished. Indeed, our subject responded to a very high pharmacological dose of 200 µg (3400 ng/kg) GnRH with both LH and FSH secretion and an LH/FSH ratio of 0.92.

In addition to GnRH pulse amplitude, another regulatory component in the control of gonadotropin secretion is the number of GnRHRs on the cell surface of gonadotropins. GnRH has been shown to stimulate the expression of its own receptor (36), and an effect of the GnRHR mutants on GnRHR gene promoter stimulation might result in changes in cell surface receptor number (25). Ten of the 15 reported GnRHR mutants have been shown to be expressed on the cell surface by fluorescence methods using green fluorescent protein or a HA tag (37). However, these methods are not quantitative and may not reflect appropriate targeting of the receptor to the cell membrane in normal numbers and in physiologically active conformations. The number of receptors on the cell surface has been determined for some mutants by in vitro binding studies and is frequently reduced in comparison to wild-type (37). As we used HA-tagged constructs for our in vitro experiments, we cannot exclude the possibility that the presence of the HA tag affects the expression of receptors on the cell surface.

Our proband had a 7.4-fold reduction in the number of [N10K+Q11K] mutant receptors present on the cell surface compared with wild-type. Mutations in the receptors may affect the rates of synthesis and/or degradation of the GnRHR (37). It has been suggested that in different cell types as much as 30% of synthesized proteins never attain their correct structure and are degraded (38). According to Leanos-Miranda et al. (37), the misfolding of receptors can be viewed as the causative event for loss of function in the majority of naturally occurring GnRHR mutants because recovery of function can be achieved pharmacologically by the addition of small molecular weight compounds that "rescue" GnRHR mutants and increase cell surface receptor expression. Studies by Conn and co-workers (16, 39) indicated that 11 of 13 mutant GnRHRs tested could be rescued in terms of their cell surface expression, signaling, and binding function. Their hypothesis is that these "pharmacoperones" correct protein misfolding created by the mutations (16, 39).

Recently, Karges et al. (40) demonstrated in a molecular model of the Ala171Thr GnRHR mutant that agonist binding and receptor signaling are dependent on conformational changes in the ligand-receptor interaction. These conformational changes become critical for the biological response to a given ligand in the proposed multistate model of receptor activation by Schwartz et al. (41). In this receptor model, the signaling of the GnRHR is highly dependent on the GnRH dose. Differential effects of conformational receptor changes on LH and FSH biosynthesis have not been investigated. The findings of the current study suggest that this would be a fruitful area for additional studies. Conformational changes in response to different GnRH doses could underlie the clinical presentation and gonadotropin response to GnRH in our subject.

In summary, this report identifies two previously unreported mutations in the GNRHR gene as an autosomal recessive cause for IHH. In our subject, the mutations led to a striking phenotype with a rightward shift of the dose-response curves of gonadotropin responses to pulsatile GnRH and differential sensitivities of LH and FSH to GnRH stimulation. These mutant receptors unmask the physiological dose-dependent regulation of LH and FSH by GnRH. The administration of pulsatile GnRH is a tool that can be used to elucidate the impact of GnRHR mutations on the hypothalamic-pituitary-gonadal axis in far more detail than a GnRH bolus stimulatory test.


   Acknowledgments
 
We gratefully acknowledge Patrick M. Sluss, Ph.D., for assistance with the gonadotropin assays and Nelly Pitteloud, M.D., and Patricia Smith for contributions to this manuscript.


   Footnotes
 
This work was supported in part by the National Institute of Child Health and Human Development/National Institutes of Health through Cooperative Agreement U54 HD28138 as part of the Specialized Cooperative Centers Program in Reproduction Research (to W.F.C. and U.B.K.) and by National Institutes of Health Grant HD19899 (to P.M.C.), the Deutsche Forschungsgemeinschaft (to A.U.M.), and the Lalor Foundation (to G.Y.B.).

A.U.M. and H.K. contributed equally to this work.

Abbreviations: GnRHR, GnRH receptor; GSU, gonadotropin subunit; HA, hemagglutinin; hCG, human chorionic gonadotropin; hGnRHR, human GnRHR; IHH, idiopathic hypogonadotropic hypogonadism; IP, inositol phosphate.

Received October 16, 2003.

Accepted February 26, 2004.


   References
Top
 Abstract
 Introduction
 Case Report
 Materials and Methods
 Results
 Discussion
 References
 

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