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Original Studies |
Division of Endocrinology, Department of Medicine (F.P.P, F.G., E.C., L.P., R.C.G.), Institute of Pharmacology (S.C., L.A.), University Hospital, 1011 Lausanne; and the Division of Biology of Growth and Reproduction, University Hospital (M.L.A.), 1211 Geneva 14, Switzerland
Address all correspondence and requests for reprints to: François P. Pralong, M.D., Division of Endocrinology, BH 19–707, CHUV, 1011 Lausanne, Switzerland. E-mail: francois.pralong{at}chuv.hospvd.ch
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Abstract |
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In conclusion, we report the first mutation of the GnRH-R gene that can induce a total loss of function of this receptor and is associated with a phenotype of complete hypogonadotropic hypogonadism.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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A functional receptor for GnRH (GnRH-R) is also crucial to both normal pubertal development and reproductive function. To elicit the cascade of events leading to sex steroid hormone biosynthesis and secretion as well as gametogenesis, hypothalamic GnRH secreted into the hypophysial portal blood has to interact with its high affinity GnRH-R expressed in the membranes of pituitary gonadotrope cells (15). The GnRH-R is a G protein-coupled receptor that activates phospholipase C leading to the intracellular increase in inositol phosphates (16). The GnRH-R gene is localized on human chromosome 4 (4q13) (17), and partially inactivating mutations of this gene (18, 19) have recently been described as a cause of familial isolated hypogonadotropic hypogonadism (20, 21, 22, 23). However, in all previously reported families, the phenotype of hypogonadism associated with inactivating mutations was present only when a given patient had inherited two different mutations (compound heterozygote).
In this study, we provide the first evidence of a completely inactivating mutation of the GnRH-R gene that was present in the homozygous state. The implications of our study are 2-fold. Firstly, our findings contribute to further elucidate the pathogenesis of hypogonadotropic hypogonadism and the molecular basis underlying the severity of the disease. Secondly, the identification of a new mutation might provide an interesting contribution to the study of the structure-function relationship of the GnRH-R.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The male patient presented in this study was first seen in our
clinic at the age of 19 yr, when he was referred for investigation of
delayed puberty. On initial questioning, he stated that he had a
conserved sense of smell, and this was confirmed by formal testing.
There was no family history of delayed puberty or infertility. He
appeared eunuchoid on physical examination with a height of 183 cm for
a pubis-feet length of 104 cm, and no facial or thoracic hair. Both
testes were cryptorchid. Biochemical work up included a serum
testosterone which was low (<0.7 nmol/L) in the face of low basal
gonadotropins (LH, <0.9 IU/L; FSH, < 0.4 IU/L) that failed to respond
to an acute GnRH test (0.1 mg, iv). Other tests of anterior pituitary
function were normal, as was a computed tomography scan of the
hypothalamo-pituitary region, thus leading to the diagnosis of isolated
hypogonadotropic hypogonadism. The patient underwent an initial
treatment with hCG (2000 IU, three times per week), followed after 4
months by a combination treatment with hCG and human menopausal
gonadotropin. He responded adequately to this regimen, with descent of
both testes and a rise in circulating testosterone levels (Table 1
).
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). This
regimen failed to induce any increase in testosterone secretion, and it
was therefore changed to exogenous gonadotropin injections (500 IU hCG
and 75 IU human menopausal gonadotropin, three times per week; Table 3). Gonadotropins almost immediately
induced a robust and sustained increase in testosterone secretion,
followed by testicular growth, and finally appearance of sperm in the
ejaculate after 14 months of treatment. Because of this primary failure
of pulsatile GnRH therapy, we hypothesized that this patient may
present a mutation in his GnRH-R gene.
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Genomic DNA of the proband as well as his parents and sisters was extracted from peripheral lymphocytes, using a commercially available kit (Nucleon Bacc 2, Amersham Pharmacia Biotech, Rainham, UK). The three exons of the GnRH-R gene (18, 19) were then amplified directly from genomic DNA by PCR, using overlapping primer pairs. Each complementary DNA (cDNA) was purified (Wizard PCR Preps, Promega Corp., Madison, WI), and both strands were submitted to cycle sequencing (Microsynth GmbH, Balgach, Switzerland). Once identified in the proband, the mutation was confirmed by sequencing of two additional cDNA fragments amplified independently. Both parents were also genotyped by direct sequencing.
The mutation identified introduces a new HinfI restriction enzyme site. This allowed us to screen the rest of the family (two phenotypically normal sisters) by digestion analysis and to confirm the homozygosity of the proband for this mutation.
In vitro mutagenesis
GnRH-R cDNA (1061-bp fragment) was amplified by PCR from a human pituitary gland cDNA library (Quick-Clone cDNA, CLONTECH Laboratories, Inc. Palo Alto, CA), and cloned into the pAlterMax plasmid (Promega Corp.). Primers used for the amplification were: 5'-GAGGAATTCAGCCTTGTGTCCTGGGAAAATAT-3' (sense) and 5'-TACGTCGACCCCTTCTTCATATGACTTCTTGT-3'. (antisense, underlined letters stand for the restriction sites EcoRI and SalI). Site-directed mutagenesis was then achieved by the megaprimer technique (24). Briefly, a PCR reaction was performed using the amplified GnRH-R cDNA as template, the antisense primer described above, and a mutagenic sense primer. After 15 cycles, the sense primer described above was added to the reaction, and 15 cycles of amplification were further performed. The product was gel purified, submitted to EcoRI and SalI digestion, and then subcloned into the expression vector pAlterMax. Mutant subclones were identified after PCR amplification by enzymatic digestion with HinfI. Both strands of wild-type and mutant subclones were confirmed by sequencing.
Construction of the receptor-green fluorescent protein (GFP) fusion protein
The EcoRI-SalI restriction fragment of the cDNA encoding the wild-type or mutated GnRH receptors was subcloned in the pEGFP-N1 plasmid (CLONTECH Laboratories, Inc.) encoding the GFP. After removal of the stop codon from the receptor cDNA by PCR site-directed mutagenesis, we obtained a receptor-GFP fusion protein containing 25 extra amino acids between the end of the receptor cDNA and the start codon of GFP. The fusion proteins were subcloned in pRK5.
Functional studies
Cell transfection. The cDNA encoding the wild-type GnRH-R, its mutant, or the receptor-GFP fusion proteins was subcloned into the pRK5 (25) and transfected into COS-7 cells by the diethylaminoethyl-dextran method. For ligand binding and inositol phosphate measurements, COS-7 cells (0.5 x 106) grown in six-well dishes were transfected with 2 µg DNA. For fluorescence microscopy, cells were grown and transfected on glass coverslips.
Ligand binding. Intact cell receptor binding was measured by incubating cell monolayers grown in 35-mm dishes with different concentrations of 125I-(D-Trp6-(N-Et)Pro9,desGly10)GnRH (provided by Dr. Jean Rivier, The Salk Institute, La Jolla, CA) in 0.6 ml DMEM at 4 C for 10 h. After binding, cells were washed three times with ice-cold PBS containing 0.1% BSA, scraped in water, and counted. GnRH (10-6 mol/L) was used to determine nonspecific binding, which was 25% of the total binding. Saturation analysis and competition curves were analyzed using Prism 2.0 (GraphPad Software, Inc., San Diego, CA).
Inositol phosphate determination. Cells were grown in 35-mm dishes and labeled with [3H]inositol at 5 mCi/ml for 15–18 h in inositol-free DMEM supplemented with 1% FBS. After labeling, cells were stimulated for 40 min with GnRH in the presence of 20 mmol/L LiCl. Inositol phosphates were extracted as previously described (18) and separated by Dowex AG1-X8 columns. Total inositol phosphates were eluted with 1 mol/L ammonium formate/0.1 mol/L formic acid.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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shows the results of
genomic DNA sequencing of the proband and his parents. A substitution
of thymine for adenine at nucleotide 504 of the GnRH-R gene was present
in the homozygote state in the proband and in the heterozygote state in
both parents. This single base substitution results in the mutation of
serine 168 into arginine (S168R) in the fourth transmembrane domain of
the GnRH-R. The mutated DNA sequence represents a restriction site for
HinfI that is absent in the wild-type DNA. Figure 1B also
shows the results of a HinfI digestion of a 196-bp DNA
fragment obtained by PCR amplification of the region of genomic DNA
encompassing the mutated base. The nondigested intact fragments
represent the wild-type alleles, whereas the digested fragments (170
bp) represent the mutated alleles. The mutated allele was present in
the homozygote state in the proband (P) and in the
heterozygous state in both parents (F and M) and one sister (S2). The
other sister (S1) was homozygote wild type.
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COS-7 cells transfected with the cDNA encoding the wild-type GnRH-R displayed specific binding of the GnRH analog 125I-(D-Trp6-(N-Et)Pro9,desGly10)GnRH. Saturation analysis using increasing concentrations of the unlabeled analog indicated that the binding capacity values ranged between 400–600 fmol/mg protein, and the Kd of the GnRH analog was 7 nmol/L.
In agreement with previous findings (18), the human GnRH-R
expressed in COS-7 cells could bind GnRH with high affinity
(IC50 of GnRH, 5 nmol/L). Stimulation of cells
expressing the wild-type GnRH receptor with a saturating concentration
of GnRH resulted in a 25-fold increase in inositol phosphate production
(Fig. 2). In contrast, cells transfected
with the cDNA encoding the mutated GnRH-R carrying the mutation of
serine 168 into arginine did not display any specific binding of
125I-(D-Trp6-(N-Et)Pro9,desGly10)GnRH
or GnRH-induced accumulation of inositol phosphates (Fig. 2).
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, the
GnRH-induced increase in inositol phosphates mediated by the wild-type
receptor-GFP was similar to that observed in cells expressing the
wild-type GnRH-R (Fig. 2
). This indicates that fusing the C-terminal
portion of the GnRH-R with GFP does not impair receptor-G protein
coupling. Fluorescence microscopy was used to visually look for
positive cells in a cohort of cells (
50,000 cells/dish) transfected
with different GFP constructs. In COS-7 cells transfected with the cDNA
encoding the GFP alone, the fluorescent signal was very bright and
diffused throughout the cytosol (Fig. 4A). In contrast, in cells transfected
with the cDNA encoding the wild-type GnRH-R-GFP (Fig. 4B) or the
S168R-mutated receptor-GFP (Fig. 4C) the fluorescence was not diffused
in the cytosol, but a large portion of it was localized at the cell
surface. Despite the fact that the comparison between the cells
expressing the wild-type and mutated receptor-GFP remains mainly
qualitative, both fusion proteins containing the wild-type or mutated
receptor seem to localize at the cell membrane. These findings strongly
suggest that the functional impairment of the GnRH-R carrying the
mutation of serine 168 into arginine does not result from the
mutation-induced lack of receptor expression.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Inactivating mutations of the GnRH-R gene have been previously described (20, 21). However, in these previously reported cases the phenotype was incomplete, and the resistance to GnRH stimulation was only partial. For example, in the study by de Roux et al. (20), the patient had descended testes (both measuring 8 mL), basal LH and FSH levels were both in the normal range, and the patient responded normally to an acute GnRH stimulation. In vitro studies demonstrated that these mutations could only partially inactivate the GnRH-R, resulting in no more than 50% impairment of the receptor-mediated maximal response. More recently, a novel family with complete hypogonadotropic hypogonadism caused by compound heterozygote mutations of the GnRH-R gene was reported (22). In this family, the previously described R262Q mutation of the third intracellular loop (20, 21) was associated with a novel A129D mutation in the third transmembrane domain. Although the latter mutation induced a complete loss of function of the receptor in vitro (22), the patients harboring this mutation as compound heterozygote were able to secrete LH pulses in response to exogenous GnRH stimulation.
In contrast, the patient described in our study displayed a phenotype of complete GnRH deficiency, as evidenced by bilateral cryptorchidism and undetectable levels of gonadotropins in the face of low testosterone values, associated with profound resistance to exogenous GnRH stimulation. The clinical phenotype observed is in excellent agreement with the results of our in vitro studies. In fact, when the S168R mutation was introduced in the wild-type GnRH-R, it resulted in a receptor mutant that totally failed to mediate the GnRH-induced stimulation of inositol phosphate production. Together, these results demonstrate a causal relationship between the mutation of serine 168 into arginine and the phenotype observed in vivo. In addition, the fact that the S168R mutation could entirely inactivate the receptor might explain the severity of the clinical phenotype observed.
In previous studies, spontaneous mutations of the GnRH-R have been found in the first extracellular loop, the third intracellular loop, and the third and the sixth transmembrane domains of the receptor. Studies investigating the structure-function relationship of the GnRH-R indicate that mutations in the first extracellular loop might affect ligand binding (20, 26, 27), whereas the third intracellular domain is probably crucially involved in receptor-G protein coupling (20).
In this study, we report that the integrity of serine 168 in the fourth transmembrane domain is crucial for receptor function. This was mainly demonstrated by the fact that the GnRH-R mutant carrying the mutation of serine 168 to arginine was impaired in its ability to bind GnRH as well as to mediate the agonist-induced response despite being normally expressed at the cell membrane. One hypothesis is that serine 168 is directly involved in docking GnRH despite the fact that there is no evidence supporting the role of the fourth transmembrane domain in ligand binding. Alternatively, the mutation of serine 168 into arginine has a disruptive effect on the receptor conformation, resulting in both undetectable ligand binding and the complete loss of receptor function. An additional novel finding of our study is that the S168R mutation results in the clinical phenotype when present in the homozygous state. In fact, the mutation was silent in the heterozygous state and therefore was inherited in an autosomal recessive manner, as previously described GnRH-R mutations (20, 21, 22). However, other mutations of the GnRH-R were never found in the homozygous state and did not result in a clinical phenotype unless combined with each other as compound heterozygotes. Here, we demonstrate that the S168R mutation results in severe hypogonadotropic hypogonadism when present alone in the homozygous state. This might result from the fact that the mutation of serine 168 into arginine can induce a complete loss of function of the GnRH-R, as demonstrated by the results of our in vitro studies.
The true prevalence of such mutations is difficult to assess because of the rarity of the syndrome of isolated GnRH deficiency. In their report, Layman and colleagues tentatively speculate that it may range between 2.2–7.5% in the families with hypogonadotropic hypogonadism that they could study. Despite the fact that two additional pedigrees have been described in the last year (22, 23), they probably will remain relatively uncommon because of the generally good clinical response of such patients to exogenous GnRH therapy. Therefore, finding the same mutation in the heterozygote state in both parents of the patient described here may suggest a founder effect in this family. However, this hypothesis was not tested and remains purely speculative.
In conclusion, inactivating mutations in the GnRH-R gene represent a recently described cause of isolated GnRH deficiency in the human. We have now demonstrated that the serine at position 168 of the GnRH-R is crucial for proper receptor function. Moreover, this report suggests that like other receptors of the same family [TSH (28) or LH (29) receptor], the GnRH-R can be affected by a variety of inactivating mutations ranging from partial to total loss of function. The GnRH-R gene is therefore the second gene, after KAL-1 (12, 13), responsible for the phenotype of isolated GnRH deficiency in the human.
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Acknowledgments |
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Footnotes |
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Received February 16, 1999.
Revised May 11, 1999.
Accepted June 30, 1999.
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