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Original Articles: Hormones and Reproductive Health |
Unidade de Endocrinologia do Desenvolvimento e Laboratório de Hormônios e Genética Molecular LIM/42, da Disciplina de Endocrinologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de Sao Paulo (E.M.F.C., B.B.M., I.J.P.A., A.C.L.), Sao Paulo 01060–970, Brazil; and Endocrine Hypertension Division, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School (U.B.K., G.Y.B.), Boston, Massachusetts 02115
Address all correspondence and requests for reprints to: Dr. Ana Claudia Latronico, Hospital das Clínicas, Faculdade de Medicina da Universidade de Sao Paulo, Disciplina de Endocrinologia, Universidade de Sao Paulo. Caixa Postal 3671, Sao Paulo 01060-970, Brazil. E-mail: anacl{at}usp.br
Abstract
Several point mutations in the GnRH receptor gene have been described in an autosomal recessive form of congenital isolated hypogonadotropic hypogonadism (HH). We investigated 17 Brazilian patients (10 males and 7 females) from 14 different families, with HH and normal olfaction. The diagnosis of HH was based on absent or incomplete sexual development after 17 yr of age associated with low or normal levels of LH in both sexes and low levels of testosterone in males and of estradiol in females. All patients presented with a normal sense of smell in an olfactory specific test.
The coding region of the GnRH receptor gene was amplified by PCR and directly sequenced. A novel missense mutation, Arg139His, located in the conserved DRS motif at the junction of the third transmembrane and the second intracellular loop of the GnRH receptor was identified in the homozygous state in one female with complete HH. The Arg139His mutation completely eliminated detectable GnRH-binding activity and prevented GnRH-induced stimulation of inositol phosphate accumulation in vitro.
In another family, a new compound heterozygous mutation (Asn10Lys and Gln106Arg) was identified in four siblings (two males and two females) with partial HH. The Gln106Arg mutation, located in the first extracellular loop, has been previously described, and in vitro analysis indicated that the mutant receptor was able to bind GnRH, but with a reduced affinity. The Asn10Lys mutation in the extracellular amino-terminal domain of the receptor also reduced the affinity for GnRH in vitro. In this family we also identified a previously described silent polymorphism at amino acid residue 151 in the second intracellular loop that segregated with the two inactivating mutations of the GnRH receptor. This polymorphism was also found in two unrelated patients with sporadic HH without GnRH receptor loss of function mutations. No mutations were identified in the remaining cases.
A good correlation between genotype and phenotype was found in our patients. The woman, who is homozygous for the completely inactivating Arg139His mutation, has complete HH with undetectable serum basal LH and FSH levels that failed to respond to GnRH stimulation. In addition, the affected patients who are compound heterozygotes for the Asn10Lys/Gln106Arg mutations, have partial HH with low serum basal LH levels that were responsive to GnRH stimulation.
No clinical or hormonal differences were found between HH patients with and without mutations in the GnRH receptor gene, indicating that these data do not contribute to the identification of HH patients with GnRH receptor mutations. In conclusion, we report the first naturally occurring mutation within the conserved DRS motif of the GnRH receptor in a female with complete HH and a novel compound heterozygous mutation (Asn10Lys and Gln106Arg) in a family with partial HH, increasing the repertoire of the inactivating mutations of the GnRH receptor.
CONGENITAL HYPOGONADOTROPIC hypogonadism (HH) is a genetically heterogeneous condition characterized by a functional deficit in hypothalamic GnRH production or action (1). Patients with HH have classically been divided into those with anosmia or hyposmia (Kallmann’s syndrome) and those with normal olfaction (idiopathic HH). Sporadic and familial cases with different modes of inheritance have been described. Mutations in two distinct genes located at the short arm of the X-chromosome, KAL-1 and DAX-1, are responsible for the X-linked forms of HH (1, 2, 3, 4). Autosomal inheritance has been implicated to date only in abnormalities of the GnRH receptor (GnRH-R) gene.
The human GnRH-R belongs to the G protein-coupled receptor (GPCR) family with seven transmembrane domains and an extracellular amino-terminus, but no intracellular carboxyl-terminus (5). The GnRH-R displays a variant of the conserved amino acid triplet (DRY motif) located at the junction of the third transmembrane domain and the second intracellular loop (6, 7). Unlike most GPCRs, the GnRH-R has a serine (S) residue instead of tyrosine (Y) in the conserved triplet. The aspartate (D) and arginine (R) residues of the amino acid triplet are highly conserved in almost all GPCRs (6). Activation of this receptor results in increased activity of phospholipase C and mobilization of intracellular calcium (5).
The human GnRH-R gene has been mapped to chromosome 4q13.2–3 and spans over 18.9 kb (8). It is comprised of three exons that encode a 328-amino acid protein (9, 10). To date, seven different mutations of the GnRH-R gene leading to partial or complete HH have been reported, and two mutations (Gln106Arg and Arg262Gln) were most frequently identified (11, 12, 13, 14, 15, 16). The aim of this study was to investigate the presence of mutations in the coding region of the GnRH-R gene in 17 Brazilian patients with partial or complete HH with normal olfaction.
Subjects and Methods
Patients
The study was approved by the ethical committee of Hospital das Clínicas, Faculdade de Medicina da Universidade de Sao Paulo. Written consent was obtained from all patients. The diagnosis of HH was based on absent or incomplete sexual development after 17 yr of age associated with low or normal levels of LH in both sexes and low levels of testosterone in males or estradiol in females. Seventeen Brazilian patients from 14 distinct families (10 males and 7 females, aged 17–54 yr) with isolated HH were selected for molecular analysis of the GnRH-R gene. One family had 4 affected members (2 males and 2 females).
Clinical and hormonal studies
The sense of smell was tested using the the Smell Identification Test Administration Manual, developed at University of Pennsylvania and commercialized by Sensonic, Inc. (www.smelltest.com), according to the manufacturer’s instructions (17).
Serum LH, FSH, estradiol, and testosterone were measured by immunofluorometric assays (Delfia, Wallac, Inc., Turku, Finland). The coefficient of variation was 5% or less for all assays. The lower limit of detection was 0.6 IU/L for LH, 1.0 IU/L for FSH, 47 pmol/L (13.6 pg/mL) for estradiol, and 0.6 nmol/L (14 ng/dL) for testosterone. The GnRH stimulation test (100 µg, iv) was performed in 15 of 17 patients. Serum LH and FSH levels were measured at -15, 0, 15, 30, 45, and 60 min after GnRH stimulation. The results were compared with normal values established in our laboratory (18). Olfactory bulbs and sulci and hypothalamic-pituitary structures were analyzed by magnetic resonance imaging.
DNA sequencing
Genomic DNA was isolated from peripheral blood cells using conventional procedures. DNA was amplified by PCR using six intronic primers based on the DNA sequences deposited in EMBL/GenBank data libraries under accession no. L03380. Products were purified and directly sequenced using BigDye terminator cycle sequencing ready reaction kit (PE Applied Biosystems, Foster City, CA) in an ABI PRISM 310 automatic sequencer (Perkin-Elmer Corp., Norwalk, CT).
Exon 1 of the GnRH-R gene from 50 normal controls and from members of a family with 4 affected subjects were digested with the restriction enzyme HinfI (Life Technologies, Inc., Gaithersburg, MD). Digested fragments were separated by electrophoresis on a 1% agarose gel.
Cell culture
All cell culture reagents were supplied by Life Technologies, Inc. COS-7 cells were cultured in low glucose DMEM containing 10% heat-inactivated FBS and 1% penicillin-streptomycin at 37 C in a humidified atmosphere of 5% CO2 in air.
Site-directed mutagenesis
A hemagglutinin epitope (HA)-tagged human GnRH-R complementary DNA (cDNA) clone (19) was used as the wild-type human (h) GnRH-R and as template for generating hGnRH-R mutants by site-directed mutagenesis with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The Asn10Lys hGnRH-R mutant was generated by replacing Asn codon AAT with Lys codon AAA at nucleotide position 29 in the hGnRH-R cDNA (sense, 5'-GCCTCTCCTGAACAGAAACAAAATCACTGTTCAGCC-3'; antisense, 5'-GGCTGAACAGTCATTTTGTTTCTGTTCAGGAGAGGC-3'). The Arg139His hGnRH-R mutant was generated by replacing Arg codon CGC with His codon CAC at nucleotide position 416 in the hGnRH-R cDNA (sense, 5'-TGATCAGCCTGGACCACTCCCTGGCTATCACG-3'; antisense, 5'-CGTGATAGCCAGGGAGTGGTCCAGGCTGATCA-3'). The sequences of the mutant cDNAs were confirmed by bidirectional sequencing.
Immunofluorescence detection of tagged GnRH-R
COS-7 cells were plated on glass-bottomed 35-mm tissue culture dishes (MatTek Corp., Ashland, MA), and transiently transfected using GenePorter transfection reagent (Gene Therapy System, San Diego, CA) with 2 µg wild-type or mutant hGnRH-R cDNA constructs or control expression vector (pcDNA3). After 48 h of incubation at 37 C, cells were fixed for 30 min at room temperature with freshly prepared 4% formaldehyde solution, blocked with 1% BSA-PBS for 30 min at room temperature, and incubated overnight at 37 C with 5 µg/ml anti-HA- fluorescein antibody (clone 12CA5, Roche Molecular Biochemicals, Indianapolis, IN). Cells were then examined under an oil immersion objective (x40) using a Bio-Rad Laboratories, Inc. (MRC-1024 multiphoton system, Hercules, CA), confocal laser microscope.
Receptor binding assays
Buserelin
(D-tert-butyl-Ser6-des-Gly10-Pro9-ethylamide-GnRH)
used for binding assays was provided by Hoechst-Roussel Pharmaceuticals
(Somerville, NJ) and was iodinated as previously described
(20). COS-7 cells were plated in 60-mm tissue culture
dishes and transiently transfected with 3 µg wild-type or mutant
hGnRH-R cDNA constructs or control expression vector (pcDNA3) using
GenePorter transfection reagent. After 48 h of incubation at 37 C,
cells were rinsed with DMEM containing 0.1% BSA and further incubated
for 90 min at room temperature with 100,000 cpm
[125I]buserelin and serial concentrations (from
10-10–10-6
mol/L) of unlabeled GnRH (Sigma, St. Louis, MO) in
DMEM/0.1% BSA. Cells were rinsed twice with ice-cold PBS and lysed
with 2 mL 0.2 mol/L NaOH/0.1% SDS. The protein concentration in
lysates was calculated (Coomassie Plus protein assay reagent,
Pierce Chemical Co., Rockford, IL), and radioactivity was
measured in a 
-counter. The affinity (Kd) and
receptor number (Bmax) were calculated based on
Scatchard analysis. All assay points were performed in triplicate, and
experiments were repeated at least three times.
Inositol phosphate (IP) assay
The protocol used for measuring total IP accumulation has been described previously (21). COS-7 cells were transiently transfected by electroporation with 2 µg/well wild-type or mutant hGnRH-R cDNA constructs or control vector (pcDNA3), seeded into six-well tissue culture plates, and incubated at 37 C for 24 h. Subsequently, cells were incubated at 37 C in 1 mL inositol-free DMEM for 2 h, then in 1 mL of the same medium containing 2 µCi/well myo-[2-3H]inositol (NEN Life Science Products, Boston, MA) for 15 min, followed by the addition of 10 mmol/L LiCl. Cells were further incubated at 37 C for 14 h and stimulated with serial concentrations (from 10-10–10-6 mol/L) of GnRH for 45 min. Cells were then extracted twice on ice with 20 mmol/L formic acid, and lysates were neutralized to pH 7.5 with 7.5 mmol/L HEPES and 150 mmol/L KOH and centrifuged at 14,000 x g for 2 min. After the protein content was measured, supernatants were loaded onto 0.5-mL AG-X8 resin anion exchange columns (Bio-Rad Laboratories, Inc.) previously equilibrated with 2 mL 1 mol/L NaOH and 2 mL 1 mol/L formic acid and five times with 5 mL double distilled water. The columns were then washed with 5 mL double distilled water, followed by 5 mL 5 mmol/L borax and 60 mmol/L sodium formate, and IPs were eluted with 3 mL 0.9 mol/L ammonium formate and 0.1 mol/L formic acid. Eluates were counted in a scintillation counter and were corrected for protein content. Each data point was performed in triplicate, and each experiment was replicated at least three times.
Statistical analysis
All statistical analyses were performed using StatView (Abacus Concepts, Calabasas, CA). Changes in total binding and IP accumulation were analyzed using a nonparametric Mann-Whitney U test (P < 0.05).
Results
Clinical and hormonal data
All seven women presented with primary amenorrhea, three had no breast development, and the remaining four had spontaneous telarche at 13–18 yr of age. On physical examination, all presented with eunuchoid habitus, breast development at Tanner stage I–III, and pubic hair at Tanner stage I–V. All had small uterus and ovaries at pelvic ultrasonography.
All 10 males presented with eunuchoid habitus, micropenis (penis size, -2.0 SD or less) and small testes for adult men (testes length, 1.7–3.5 cm). Cryptorchidism was present in three. Pubic hair was Tanner stage I—III, and facial hair was absent in all patients.
All patients had a normal sense of smell, confirmed by the specific smell test. Magnetic resonance imaging of the olfactory bulbs and sulci as well as of the hypothalamic-pituitary area was normal in all patients.
The hormonal data for the 17 patients with HH are shown in Table 1
. Basal LH and FSH levels were below the
minimum detectable level in 12 and 9 patients with HH, respectively.
Basal LH levels were at pubertal levels in 3 patients. Two patients
(cases 6 and 10) exhibited GnRH-stimulated LH levels in the normal
adult pubertal range. The LH and FSH responses after GnRH stimulation
were completely absent in 1 female patient (case 13). Testosterone
levels were low in all males (range, 0.6- 4 nmol/L). Estradiol levels
were prepubertal in 6 of 7 female patients.
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Direct sequencing of the three exons of the GnRH-R gene revealed
mutations in one female patient (case 13; Table 1
) and in four siblings
(cases 14–17; Table 1). In a female (case 13) with complete HH, we
identified a homozygous replacement of guanine by adenine at nucleotide
416 within exon 1 of the GnRH-R gene. This resulted in the replacement
of the amino acid arginine at codon 139 by histidine in the conserved
DRS motif at the junction of the third transmembrane domain and the
second intracellular loop of the receptor.
Two mutations, both in the heterozygous state, and a previously described silent polymorphism in the homozygous state were found in four siblings (cases 14–17). The replacement of an adenine by a thymidine at nucleotide 30 in exon 1 yielded an Asn10Lys mutation in the N-terminal extracellular domain of the receptor. This mutation abolishes a HinfI recognition site in exon 1 of the GnRH-R gene. A second mutation characterized by replacement of an adenine for guanine at nucleotide position 317, yielded a Gln106Arg substitution in the first extracellular loop of the GnRH-R. The previously described polymorphism in amino acid residue 151 (16, 22) was also found in the second intracellular loop in these patients along with one of the two mutations. All affected members of this family were compound heterozygotes for the two missense mutations and homozygotes for the silent polymorphism. The unaffected heterozygous family members for each of the mutations were also heterozygous for the silent polymorphism.
Familial segregation analysis, using restriction site enzymatic
digestion with HinfI followed by direct sequencing, revealed
the presence of the mutation Asn10Lys in the
mother, in all affected subjects (II:1, II:7, II:10, and II:13; Fig. 1) and in two normal siblings (II:3 and
II:12; Fig. 1).
|
). No
mutation was found in the remaining patients. Cell surface expression of mutant hGnRH-R
COS-7 cells were transiently transfected with mutant hGnRH-R cDNA
constructs and analyzed by immunocytochemistry for expression of the
receptors on the cell surface. Confocal fluorescent microscopic images
were obtained after incubating transfected cells with anti-HA antibody
conjugated to fluorescein (Fig. 2).
Fluorescence was detected at the periphery of the cells transfected
with wild-type hGnRH-R and mutant receptors cDNA, indicating normal
cellular expression and membrane localization. In contrast, no
fluorescence was detected on the surface of cells transfected with
pcDNA3.
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GnRH agonist binding by COS-7 cells transiently transfected with
either mutant hGnRH-R cDNAs or positive (wild-type hGnRH-R) or negative
(pcDNA3) controls was determined by incubating cells with 100,000 cpm
[125I]buserelin and increasing amounts of
unlabeled GnRH (Fig. 3A).
Asn10Lys hGnRH-R had reduced binding activity,
with a significantly higher calculated dissociation constant
(Kd, 9.72 ± 3.28 nmol/L) compared with
wild-type hGnRHR (Kd, 5.92 ± 1.47 nmol/L),
although the Bmax was similar to the wild-type
value (Table 2). The
Arg139His mutant failed to exhibit any detectable
binding.
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The ability of the mutant hGnRH-R to activate intracellular signal
transduction pathways was assessed by measuring IP accumulation, an
indicator of phospholipase C activity. Dose-response analyses were
performed by stimulating COS-7 cells transiently transfected with
either mutant hGnRH-R cDNAs or positive (wild-type hGnRH-R) or negative
(pcDNA3) controls with serial concentrations of GnRH (Fig. 3B). The
Asn10Lys mutant hGnRH-R response to GnRH had a
significantly higher ED50 (4.89 ± 1.04
nmol/L) compared with that of the wild-type receptor (2.12 ± 0.49
nmol/L), reflecting the reduced binding activity. The maximum response
was not significantly different from the wild-type response (Table 2).
Arg139His mutant hGnRH-R failed to respond to
GnRH at any concentration tested.
Discussion
Point mutations in the GnRH-R gene are a recently identified cause of the autosomal recessive form of congenital isolated HH (11, 12, 13, 14, 15, 16). The phenotypic spectrum of HH patients with GnRH-R gene mutations varies from partial to complete forms (11, 12, 13, 14, 15, 16). In addition, distinct families with the same molecular defect in the GnRH-R gene can exhibit different degrees of hypogonadism (15).
We report here a novel missense mutation at codon 139 of the GnRH-R in the homozygous state in a female patient with HH. This patient has a complete form of HH characterized by eunuchoid habitus, primary amenorrhea, lack of breast development, and infantile uterus at 18 yr of age. The total absence of a LH and FSH response after GnRH stimulation at 18 yr confirmed the clinical features of complete hypogonadism. She was treated with conjugated estrogen and progesterone for 7 yr and achieved full development of secondary sexual characteristics. GnRH stimulation was repeated at 25 yr, 1 month after sex steroid replacement therapy was withdrawn, and gonadotropin levels remained undetectable.
The Arg139His mutation is the first report of a naturally occurring mutation within the conserved DRS motif located at the junction of the third transmembrane domain with the second intracellular loop of the GnRH-R. The importance of this region to the structural integrity, expression, agonist-induced activation, and internalization of the GnRH-R was recently demonstrated by artificial mutagenesis at the conserved Asp and Arg residues in the DRS triplet (7). Mutants in which Arg139 was replaced by Gln, Ala, or Ser showed reduced internalization and GnRH-induced IP response (7). Consistent with this report and with the patient’s clinical presentation, the Arg139His mutation resulted in complete elimination of detectable ligand binding and complete loss of the ability to activate intracellular signal transduction pathways. The HA-tagged Arg139His mutant GnRH-R was present on the cell surface of transfected cells, indicating that the mutant receptor is expressed and trafficked through the cell appropriately, and that the primary defect is at the level of ligand binding.
To date, only two mutations of the GnRH-R in homozygous state were identified: Ser168Arg and Arg139His, both associated with phenotype of complete HH. The Ser168Arg mutation in the fourth transmembrane domain of the GnRH-R was described in a 19-yr-old male patient who displayed a conserved sense of smell, eunuchoid habitus, no facial or thoracic hair, bilateral cryptorchidism, and undetectable levels of gonadotropins in the face of low testosterone levels associated with resistance to exogenous GnRH stimulation (13).
We also describe two other mutations(Asn10Lys and Gln106Arg) in the GnRH-R in a family with four affected siblings. All affected members were compound heterozygotes for these mutations. The mother and three additional siblings were heterozygotes for the Asn10Lys mutation and had a normal phenotype. The disease is transmitted as an autosomal recessive trait. The propositus of this family (case 16) was a 20-yr-old woman with eunuchoid habitus, primary amenorrhea, Tanner stage III breast development, and prepubertally sized uterus and ovaries. She was treated with conjugated estrogen and progesterone and achieved complete pubertal development. A GnRH stimulation test, performed before treatment at 20 yr of age, induced a prepubertal level of LH release; this test was repeated at 36 yr, 4 weeks after the withdrawal of steroid treatment, disclosing a similar LH response. Her younger affected brother (case 17 and subject II:13) was a 17-yr-old man with sexual infantilism. He had no facial and body hair, Tanner stage II pubic hair, micropenis, and topic prepubertal-sized testes. He was treated with testosterone enanthate, achieving complete sexual development. A GnRH stimulation test performed at 31 yr, 8 weeks after the withdrawal of steroid replacement treatment, induced a pubertal LH response. The clinical and hormonal findings of these two siblings indicate partial hypogonadism and suggest that the compound heterozygous mutations (Asn10Lys and Gln106Arg) did not completely inactivate GnRH-R function. Limited clinical and hormonal data were available on the other two affected members (cases 14 and 15). Case 14 was a 54-yr-old female with primary amenorrhea and infertility who had spontaneous pubarche and thelarche at the age of 15 and 18 yr, respectively. Case 15 developed spontaneous pubarche at the age of 15 yr and had sparse facial hair. He was treated with exogenous testosterone, but complied irregularly. At 43 yr, he is undervirilized and has been married for 6 yr without conceiving children. The other five siblings had normal pubertal development and regular menstrual cycles, and all of them have children.
Asn10 is a nonconserved residue in the N-terminal extracellular domain of the receptor. The functional studies of the Asn10Lys mutant receptor revealed a 2-fold reduction in ligand binding affinity in transiently transfected COS-7 cells. Similarly, a 2-fold increase in ED50 was evident for stimulation of IP accumulation by GnRH. Levels of the mutant receptor expressed on the cell surface of transfected cells were indistinguishable from those of the wild-type receptor, as evident from confocal microscopic studies and from Scatchard analyses and calculation of Bmax. These studies indicate a partial reduction of function for the mutant receptor, correlating with the partial phenotype of the affected subjects.
The Gln106Arg mutation in the first extracellular loop of the GnRH-R is one of the most frequent mutations found in this receptor in patients with congenital isolated HH (11, 16). This mutation was previously described in combination with Arg262Gln or Leu314Stop mutations in patients with partial or complete HH, respectively (11, 16). Functional studies of the Gln106Arg receptor mutant revealed almost complete abolition of GnRH binding in association with reduced activation of intracellular signaling pathways in response to GnRH (11). Interestingly, the allelic combination of mutations with different biological activities appears to be important in the determination of phenotypic differences (16).
In this family we also identified a previously described silent polymorphism at amino acid residue 151 in the second intracellular loop that segregated with the two inactivating mutations (Asn10Lys and Gln106Arg) of the GnRH-R. This polymorphism was also found associated with Gln106Arg in a woman with complete HH (16, 22).
A low frequency (2.2%) of GnRH-R gene mutations was found in the screening of 46 unrelated patients with HH (32 males and 14 females) without anosmia (12). We found a higher frequency of such mutations in this Brazilian series (14%), but the etiology of the vast majority of the patients with congenital isolated HH and normal sense of smell remains unknown. These findings suggest that different autosomal genes implicated in GnRH regulation and/or action might account for this disorder.
We observed a good correlation between genotype and phenotype in our patients with GnRH-R mutations. The female patient, who is homozygous for the completely inactivating Arg139His mutation, has complete HH with undetectable serum basal LH and FSH levels that failed to respond to GnRH stimulation. The affected patients, who are compound heterozygous for the Asn10Lys/Gln106Arg mutations, have partial HH with low serum basal LH levels that were responsive to GnRH stimulation. There was no difference between the clinical and hormonal data of patients with and without mutations in the GnRH-R gene, indicating that these data do not contribute to the identification of HH patients harboring GnRH-R mutations.
In conclusion, we report the first naturally occurring inactivating mutation in the conserved DRS motif of the GnRH-R in a female with complete HH. Our study also describes a novel compound heterozygous mutation, Asn10Lys/Gln106Arg, in the GnRH-R as the cause of partial hypogonadism in another Brazilian family. Mutations in GnRH-R gene were found at a low frequency in patients with HH with normal olfaction, suggesting that another gene(s) is involved in the etiology of this heterogeneous disorder.
Acknowledgments
We thank Katja Linher for her technical assistance, and Dr. P. Michael Conn, Oregon Health Sciences University (Beaverton, OR), for providing us with [125I]buserelin.
Footnotes
1 This work was supported in part by Fundacáo de Amparo
à Pesquisa do Estado de Sáo Paulo Grant 1999/06469-1
and Conselmo Nacional de Desenvoluimento Cientifico e
Technológico Grant 300151/96 (to A.C.L.) and 301246/95-5 (to
B.B.M.); Harvard-wide Reproductive Endocrine Sciences Center (NIH Grant
U54-HD-28138), and NIH Grant RO1-HD-19938 (to U.B.K.). 
Received December 20, 2000.
Revised February 12, 2001.
Accepted February 22, 2001.
References
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G. Y. Bedecarrats, K. D. Linher, and U. B. Kaiser Two Common Naturally Occurring Mutations in the Human Gonadotropin-Releasing Hormone (GnRH) Receptor Have Differential Effects on Gonadotropin Gene Expression and on GnRH-Mediated Signal Transduction J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 834 - 843. [Abstract] [Full Text] [PDF]
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J. A. Janovick, G. Maya-Nunez, and P. M. Conn Rescue of Hypogonadotropic Hypogonadism-Causing and Manufactured GnRH Receptor Mutants by a Specific Protein-Folding Template: Misrouted Proteins as a Novel Disease Etiology and Therapeutic Target J. Clin. Endocrinol. Metab., July 1, 2002; 87(7): 3255 - 3262. [Abstract] [Full Text] [PDF]
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L. F. G. Silveira, P. M. Stewart, M. Thomas, D. A. Clark, P. M. G. Bouloux, and G. S. MacColl Novel Homozygous Splice Acceptor Site GnRH Receptor (GnRHR) Mutation: Human GnRHR "Knockout" J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2973 - 2977. [Abstract] [Full Text] [PDF]
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 E. R. Norwitz, S. Xu, K.-H. Jeong, G. Y. Bedecarrats, L. D. Winebrenner, W. W. Chin, and U. B. Kaiser Activin A Augments GnRH-Mediated Transcriptional Activation of the Mouse GnRH Receptor Gene Endocrinology, March 1, 2002; 143(3): 985 - 997. [Abstract] [Full Text]
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