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CLINICAL CASE SEMINAR |
Department of Clinical Biochemistry (R.K.S., I.S.F., S.O.) and Department of Medical Genetics and Division of Renal Medicine (F.E.K.), Addenbrooke’s Hospital, University of Cambridge, Cambridge CB2 2QQ, United Kingdom; Department of Medicine and Institute of Child Health (J.C.A.), University College London, London WC1N 1EH, United Kingdom; Paradigm Therapeutics Ltd. (J.E., S.A.A.), Cambridge CB4 0WA, United Kingdom; Department of Oncology (S.A.A.), Hutchison Medical Research Council Centre, University of Cambridge, Addenbrookes CB2 2XZ, United Kingdom; and Great Ormond Street Hospital for Children (R.G.S.), London WC1N 3JH, United Kingdom
Address all correspondence and requests for reprints to: Robert Semple, Department of Clinical Biochemistry, Addenbrooke’s Hospital, Cambridge CB2 2QR, United Kingdom. E-mail: rks16{at}cam.ac.uk.
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Abstract |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Genetic defects in the hypothalamo-pituitary-gonadal axis are manifested clinically as hypogonadotropic hypogonadism (HH), with either primary failure to undergo puberty or severe delay in the process. Defects may broadly be categorized into those affecting embryonic migration of GnRH-secreting neurones, which lead to anosmia as well as HH (e.g. Kallmann syndrome due to KAL, or FGFR1 mutations), and those affecting the pulsatile release or function of GnRH from neurones that have migrated to their normal position. Patients suffering from the latter are normosmic. Around 40% of patients with autosomal recessive normosmic familial HH in one series were found to have mutations in the GnRH receptor gene, whereas only around 10–17% of sporadic cases are thought to harbor such mutations in GNRHR (2, 3).
In the last year, the G protein-coupled receptor (GPR)54 has been shown to be a gatekeeper gene for activation of the GnRH axis based on loss-of-function mutations in mice and humans. In humans, loss-of-function mutations in GPR54 have been described in two consanguineous pedigrees and two isolated subjects with HH (4, 5) (OMIM 604161). In tandem, mice bearing homozygous targeted disruptions of the Gpr54 gene have been found to exhibit a similar failure of sexual maturation (5). This shows that GPR54 is required for the normal function of this axis and suggests that the ligand, kisspeptin-1, may act as a novel neurohormonal regulator of the GnRH axis. The latter has recently received support from studies showing that peripheral and central administration of kisspeptin provokes GnRH release (6, 7, 8).
We now describe the genetic screening of the GPR54 gene as well as the KISS1 gene encoding its putative endogenous ligand, kisspeptin-1, in a cohort of 30 patients with normosmic HH, and report the clinical phenotype of a nonconsanguineous subject who shows compound heterozygosity for two novel missense mutations in the GPR54 gene. We have also documented the prevalence of single nucleotide polymorphisms (SNPs) in the GPR54 and KISS1 genes, one previously undescribed, in both the study population and a Caucasian control population.
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Subjects and Methods |
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Subjects for genetic screening were recruited from the clinics at Great Ormond Street Hospital (London, UK) and Addenbrooke’s Hospital (Cambridge, UK). Informed consent was obtained according to procedures approved by the local research ethics committee of each institution. Patient characteristics are outlined in Table 1
. Isolated HH was defined as inappropriately low gonadotropin levels together with prepubertal concentrations of sex steroids. It was deemed idiopathic in which no family history of the condition was apparent, and congenital in which undescended testes with or without small penis were noted at birth. Constitutional delay of puberty (CDP) was defined as the absence of signs of puberty at 14 yr of age in boys, this being described as severe if signs were still not apparent at 15 yr. All patients were reportedly normosmic, had otherwise normal anterior pituitary function and no midline facial defects, and showed no evidence of structural brain pathology, where assessed. In all but three cases, the coding sequence of the GnRHR gene had been sequenced and found to be normal.
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Genetic screening
Genomic DNA was isolated from whole blood using a QIAamp blood kit (Qiagen, Crawley, UK), and amplified using a GenomiPhi DNA amplification kit (Amersham Biosciences, Chalfont, UK). PCR was performed using BioTaq (Bioline, London, UK) and carried out as recommended by the manufacturer, with addition of dimethylsulfoxide where indicated. Thirty-five cycles (60 sec at 95 C, 60 sec at the annealing temperature, and 60 sec at 72 C) were performed using a PTC-225 Peltier thermal cycler (MJ Research, Watertown, MA). PCR products were verified electrophoretically and sequenced using ABI BigDye Terminator (version 3.1) reagents with electrophoresis on an ABI Prism 3100-Avant genetic analyzer (PE Applied Biosystems, Foster City, CA). Subsequent sequence analysis was performed using Sequencher software (Gene Codes, Ann Arbor, MI). Primers, annealing temperatures, and concentrations of MgCl2 and dimethylsulfoxide used for PCR are detailed in Table 2, together with sequencing primers where different.
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) were used to amplify exons 4 and 5 and the intervening intron using reaction conditions as for exon 5. The PCR product was then cloned into a pGEM-T Easy Vector (Promega, Southampton, UK), and 16 separate clones were sequenced. Creation of GPR54 mutant alleles and in vitro functional assays
Wild-type human GPR54 was amplified from human brain cDNA (Origene Technologies, Rockville, MD) using the GC-Rich PCR kit (Roche Diagnostics, Lewes, UK). The 5' primer TAATCAAAGCTTGCGATGCACACCGTGGCTACGTC and 3' primer ACATTAGGATCCTCACTTATCGTCGTCATCCTTGTAATCGAGAGGGGCGTTGTCCTCC (incorporating a 3' sequence encoding the FLAG epitope tag) were used. The resulting product was cloned into pCRBlunt (Invitrogen, Renfrew, UK) and sequence verified. Stable cell lines expressing the wild-type GPR54-FLAG or one of the two mutant species described were then created using the Flp-In system (Invitrogen). In brief, the GPR54-FLAG was subcloned into pcDNA5/FRT and the point mutations were introduced using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. All the resulting constructs were verified by direct sequencing. The Flp-In-293 cell line (Invitrogen) was maintained in DMEM supplemented with 10% Fetal Clone III (Hyclone, Cramlington, UK) and 2 mM L-glutamine. To create stable GPR54-expressing cell lines, Flp-In-293 cells were cotransfected with the pcDNA5/FRT-GPR54-FLAG constructs and the Flp recombinase-expressing plasmid pOG44 using polyfect (Qiagen), with subsequent selection by hygromycin B. Transcription of the inserted alleles was confirmed by RT-PCR (data not shown).
To assess signal transduction by the wild-type and mutant receptors, a sensitive calcium mobilization signaling assay was used as previously described (11, 12, 13), with calcium flux in response to kisspeptin-1 112–121 (Phoenix Pharmaceuticals, Belmont, CA) measured on a FlexStation using a FLIPR calcium assay kit (both from Molecular Devices, Sunnyvale, CA) in response to Kisspeptin-1 112–121 (Phoenix Pharmaceuticals). Data were analyzed using SoftMax Pro software (Molecular Devices). Experiments were performed in duplicate on 2 consecutive days, and SDs are indicated.
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Results |
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Sequencing of the coding regions of the GPR54 (GenBank accession no. AY253981) and KISS1 (GenBank accession no. AY117143) genes in all 30 patients revealed the presence of two previously described SNPs in the GPR54 gene, and three previously described SNPs, one novel SNP, and one previously described insertional polymorphism in the KISS1 gene. The details of these common variants, and their frequency in both the study population and 180 Caucasian control alleles are shown in Table 3.
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): the first, a transversion c.667T>C in exon 4, results in substitution of a cysteine near the cytoplasmic end of the fifth transmembrane 
-helix for arginine (C223R), whereas the second, a transversion c.891G>T in exon 5, leads to the substitution of an arginine in the third extracellular loop for leucine (R297L). Cloning a section of the GPR54 gene encompassing exons 4 and 5 and the intervening intron from the genomic DNA of the proband established that he was a compound heterozygote for these two changes. The cysteine at position 223 is highly conserved in mouse, rat, zebrafish, and pufferfish, whereas R297 is conserved in mouse and rat but not in zebrafish or pufferfish. Neither variant was detected in 180 Caucasian control chromosomes. The proband’s mother and father were of Turkish-Cypriot and Jamaican descent, respectively. Only his mother and younger brother were available for further study, and both were found to be heterozygous for the R297L variant but homozygous wild-type with respect to C223. The C223R variant was thus most likely inherited from his father, although de novo mutation cannot be ruled out. Screening of 100 control Afro-Caribbean alleles failed to reveal any further C223R changes, whereas R297L was not detected on screening 90 control Turkish alleles.
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The proband (46, XY, birth weight 2.98 kg) was born at term after an uneventful pregnancy. He was noted to have a micropenis and undescended testes at birth, and serum gonadotropins were undetectable at 2 months of age (LH < 0.5 mIU/ml, FSH < 0.5 mIU/ml). His penis grew reasonably in response to three injections of depot testosterone, but whereas his right testis was identified in the scrotum, his left testis remained undescended.
At 15 months of age, he was admitted for further investigation of anterior pituitary function. His height/length was progressing consistently along the 50th percentile and weight along the 25th percentile. He had a normal GH response to glucagon stimulation [2.1 (basal) to 30.5 ng/ml (peak) (6.2–91.5 mU/liter; North-East Thames Radio-Immuno Assay, cut-off for the diagnosis of GH deficiency, 11.7 ng/ml]. He had a normal cortisol response to glucagon stimulation [19.8–47.0 µg/dl (554–1317 nmol/liter)], normal free T4 [1.37 µg/dl (17.5 nmol/liter)] and normal TSH response to TRH stimulation (0.5–7.3 mU/liter). He had normal basal prolactin, and undetectable basal gonadotrophins (LH < 0.5 mIU/ml, FSH < 0.5 mIU/ml). Results of a 3-d human chorionic gonadotropin (hCG) stimulation test (1000 IU daily) showed a limited testosterone response to stimulation (Table 4A). An ultrasound scan at this time identified both testes and bilateral hydroceles. The right testis was soft, measured 9 x 6 x 10 mm, and was within the scrotal sac. The left testis had a maximum diameter of 7 mm and was identified at the left inguinal ring. A prolonged course of hCG stimulation produced some increase in penile size but failed to stimulate descent of the left testis. Therefore, herniotomy and orchidopexy of a very small left testis were performed.
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) and suboptimal testosterone response to 3 wk stimulation with hCG (1000 IU twice weekly) (Table 4C). He was therefore started electively on low-dose testosterone replacement to induce puberty. No neuroimaging was performed. The proband’s mother, who was heterozygous for R297L, had experienced menarche at 11 yr of age. She has regular menstrual cycles and detectable gonadotropins (LH 7.2 mIU/ml, FSH 8.3 mIU/ml). His father is believed to have normal reproductive function but was not available for testing. His younger brother, heterozygous for R297L but homozygous wild-type for C223 had normal genitalia and descended testes at birth. Of note, there was no known family history of hypogonadism or infertility in the father’s eight siblings.
Characterization of mutant GPR54 function
Using the proprietary Flp-In system, isogenic cell lines stably expressing either wild-type GPR54 or one of the two mutant GPR54s at the same level of transcription were generated. A sensitive fluorometric calcium mobilization assay was then used as described to assess the response of each GPR54 species to the kisspeptin 112–121 decapeptide, previously established to be a potent agonist for GPR54 (12, 14). In our hands (Fig. 2), this assay is highly sensitive for detecting GPR54 activity: calcium flux above baseline for wild-type GPR54 is detectable at subnanomolar concentrations of ligand, with maximal flux achieved at around 100 nM.
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). A one-sample t test suggests this EC50 difference is significant, with P < 0.0001 and t = 6.6. Kolmogorov-Smirnoff determination of skewedness and kurtosis shows the control data fit a normal distribution, so a parametric t test is appropriate. |
Discussion |
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Mouse and human studies to date suggest GPR54 has a role in the release of GnRH (5, 6), further weight having recently been added to this hypothesis by the demonstration that peripherally and centrally administered kisspeptins can provoke robust LH and FSH release (6, 7, 8). However, it is not yet clear whether kisspeptins subserve primarily autocrine, paracrine, or endocrine function. Intriguingly, the putative endogenous GPR54 ligand, an amidated proteolytic product of full-length kisspeptin-1, sometimes called metastin (12, 13, 14), is normally present only at low picomolar concentrations in plasma but rises some 10,000-fold by the end of pregnancy (15). Both kisspeptin-1 and GPR54 are widely expressed, with the highest GPR54 expression being reported in placenta, brain, pituitary, pancreas, and spinal cord, whereas the highest kisspeptin-1 expression is seen in placenta, with significant levels also in pancreas and testis (12, 13, 14). This widespread expression implies a more complex role for GPR54/kisspeptin-1 than appreciated at present, and studies of humans harboring loss-of-function mutations are likely to make a valuable contribution to further elucidation of its biology.
The current report adds to the spectrum of loss-of-function mutations in GPR54. Previous studies reported five different mutations in four families or individuals with HH. Homozygosity for a 155-bp deletion spanning the splice acceptor site of the intron 4-exon 5 junction and part of exon 5 was detected in a consanguineous pedigree with five affected members (4). This homozygous deletion cosegregated with HH in this family and was inferred to result in loss of receptor function due to truncation of the coding sequence at residue 267. A second rare sequence variant, this time a missense mutation, L102P, immediately after the second transmembrane helix, was also found in one family on screening three other kindreds with familial HH. However, no cosegregation or functional studies of this mutant were reported (4). Another report of loss of function of GPR54 in patients with HH described both a homozygous L148S point mutation in a conserved region of the second intracellular loop in a consanguineous Saudi kindred and compound heterozygosity for two mutations, R331X and X399R, in an unrelated Afro-Caribbean patient, which were proven to result in significant loss of GPR54 signaling in vitro (5). In total, the published reports to date describe the screening of 68 unrelated probands with normosmic HH (five familial; 63 idiopathic) and normal GnRH receptor gene sequence, of whom three had two proven hypofunctional or nonfunctional GPR54 alleles (two familial; one idiopathic). This is consistent with our finding of one such patient in a further 17 studied (three familial; 14 idiopathic: Table 1). Cumulative results now suggest that, in those in which GnRHR coding sequence mutations have been ruled out, around 2–3% (two of 77 studied) of idiopathic normosmic HH may be accounted for by GPR54 loss-of-function mutations, whereas at present 25% (two of eight studied) of familial cases have been found to have such mutations. However, no GPR54 mutations were identified in patients or families with CDP alone.
It is highly likely, although not conclusively proven, that compound heterozygosity for the C223R and R297L variants is the cause of the idiopathic HH seen in our proband. C233R is severely defective in its signaling capability and is not seen as a common variant in the Afro-Caribbean population. In contrast, the effect of the R297L mutation on signaling is only modest, although highly statistically significant, when studied in vitro. However it is a highly conserved residue in mammalian GPR54 and was not found in an ethnically matched control population. It is likely that its effects in combination with the severe dysfunction of the C233R variant are sufficient to lead to a clinical phenotype.
Although the patient we describe is not yet beyond the age of normal puberty, he had sufficient clinical and biochemical evidence of HH to warrant induction of puberty at an age appropriate for his peer group. For example, bilateral cryptorchidism and micropenis were noted at birth; he had undetectable gonadotropins at 2 months of age [when the hypothalamic-pituitary-gonadal axis is usually relatively active (16)]; and his response to exogenous GnRH stimulation at the age of 10 yr was poor. In addition, his testosterone response to stimulation for 3 wk with hCG at this time was relatively low. Although this may reflect the tropic effect of prolonged gonadotropin insufficiency or delayed orchidopexy, it remains possible that the GPR54/kisspeptin-1 system could have a direct effect on testicular function, too. Of note, GPR54 mRNA has previously been detected, albeit at low levels, in human testis (13, 14). Thus, the effect of recombinant gonadotropin stimulation on the induction of fertility in patients with GPR54 gene mutations remains to be seen.
The present report uses a GPR54 signaling assay that is substantially more sensitive than that used to evaluate previous pathogenic mutations. The assay reported originally was based on transient expression of GPR54, stimulation with kisspeptin-1 112–121 decapeptide, and determination of the generation of inositol triphosphate. Stimulation of inositol triphosphate levels was seen only at around 10 nM kisspeptin-1 112–121 for wild-type GPR54, with maximal activity around 100 µM (3). In contrast, this study employed real-time measurements of calcium flux, similar in principle to those previously used in the identification of kisspeptin as a bona fide GPR54 ligand (12, 13, 14). As seen in Fig. 2, this assay detects signaling at subnanomolar concentrations of the kisspeptin decapeptide, and activity is maximal at around 100 nM, some 2–3 orders of magnitude lower than the ligand concentration required to maximize inositol triphosphate generation in the previous assay.
In the absence of functional assessment of the GPR54 L102P variant, GPR54 C223R and R297L are the second- and third-point mutations after L148S reported to result in impaired GPR54 function because the 155-bp deletion, R331X, and X399R are all likely to have resulted in loss of function largely through impaired expression of mRNA and/or protein product. The new mutations may thus prove more informative with respect to understanding of the molecular determinants of GPR54 signaling. The dramatic loss of function of GPR54 C223R is unsurprising in view of likely disruption of the fifth transmembrane helix. Indeed, it will be of interest to establish whether receptor protein is expressed at wild-type level at the plasma membrane and, if so, whether ligand binding is preserved. R297L is situated in the final extracellular loop of GPR54 in a slightly less conserved region, and its effect on signaling is consequently relatively mild in vitro. It remains to be determined whether its additional loss of function in vivo is due to inefficient receptor synthesis or processing. Nevertheless, the dose-response characteristics of this mutation raise the possibility that supraphysiological doses of exogenous kisspeptin or an analog could be used to restore GnRH pulsatility and possibly fertility in this patient. A similar approach, using high doses of GnRH, has been used to induce ovulation in a woman with a partial loss-of-function mutation in the GnRH receptor (17). Furthermore, because we have not been able to establish whether the father of the propositus is indeed heterozygous for the C223R variant, we cannot conclude on phenotypic grounds that heterozygosity for this variant is not clinically expressed. Thus, dominant negativity of GPR54 C223R, or an interaction between C223R and R297L, cannot be formally excluded. However, although there are precedents for dominant-negative GPR mutations, often based on receptor misrouting or G protein sequestration, the dominant negativity is often more apparent in vitro than in vivo (18), and we believe it more likely in this case that it is the cumulative loss of function of the two variants that is responsible for the clinical phenotype.
In addition to identifying and characterizing two rare sequence variants in the GPR54 gene, we have also confirmed two previously described SNPs (Table 3A). One of these is nonsynonymous, with an alternative histidine to leucine change encoded in the cytoplasmic tail. A third SNP recorded in the dbSNP database was not confirmed (1155G>A; rs3746147). Analysis of KISS1 revealed four single nucleotide polymorphisms, three nonsynonymous, and one not previously recorded. In addition, an insertional polymorphism that leads to truncation of the kisspeptin-1 protein product by seven amino acids was also confirmed (Table 3B). Once again, a further SNP recorded in the dbSNP database was not confirmed (287C>G; rs1132112). All three nonsynonymous KISS1 sequence variants lead to amino acid changes in the amino-terminal part of the molecule. This is not directly involved in signaling but is likely to contribute to the regulation of processing and secretion of the active peptide. We have documented the frequency of occurrence of these variants in our study population and also in 180 Caucasian control chromosomes. This information could be used in future to guide case control studies looking at either indices of reproductive function or fertility or possibly indices of tumor invasion or metastasis in patients with malignancy, in view of the considerable interest in KISS1 as a metastasis-suppressing gene.
In summary, we have described two novel missense mutations in the G protein-coupled receptor GPR54 that result in impaired kisspeptin-1 stimulation of calcium flux. Compound heterozygosity for these mutations is associated with HH, detected at birth in the proband due to bilateral cryptorchidism and micropenis. Although not common, loss-of-function mutations in GPR54 appear to be a significant cause of HH in patients from diverse ethnic backgrounds.
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Acknowledgments |
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Footnotes |
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First Published Online December 14, 2004
Abbreviations: CDP, Constitutional delay of puberty; GPR, G protein-coupled receptor; hCG, human chorionic gonadotropin; HH, hypogonadotropic hypogonadism; SNP, single nucleotide polymorphism.
Received July 25, 2004.
Accepted December 7, 2004.
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