When genetic load does not correlate with phenotypic spectrum: lessons from the GnRH receptor (GNRHR).

CONTEXT: A broad spectrum of GnRH-deficient phenotypes has been identified in individuals with both mono- and biallelic GNRHR mutations. OBJECTIVE: The objective of the study was to determine the correlation between the severity of the reproductive phenotype(s) and the number and functional severity of rare sequence variants in GNRHR. SUBJECTS: Eight hundred sixty-three probands with different forms of GnRH deficiency, 46 family members and 422 controls were screened for GNRHR mutations. The 70 subjects (32 patients and 38 family members) harboring mutations were divided into four groups (G1-G4) based on the functional severity of the mutations (complete or partial loss of function) and the number of affected alleles (monoallelic or biallelic) with mutations, and these classes were mapped on their clinical phenotypes. RESULTS: The prevalence of heterozygous rare sequence variants in GNRHR was significantly higher in probands vs. controls (P < 0.01). Among the G1-G3 groups (homozygous subjects with successively decreasing severity and number of mutations), the hypogonadotropic phenotype related to their genetic load. In contrast, subjects in G4, with only monoallelic mutations, demonstrated a greater diversity of clinical phenotypes. CONCLUSIONS: In patients with GnRH deficiency and biallelic mutations in GNRHR, genetic burden defined by severity and dose is associated with clinical phenotype. In contrast, for patients with monoallelic GNRHR mutations this correlation does not hold. Taken together, these data indicate that as-yet-unidentified genetic and/or environmental factors may combine with singly mutated GNRHR alleles to produce reproductive phenotypes.

Loss-of-function mutations in the genes encoding prokineticin-2 or prokineticin receptor-2 cause autosomal recessive Kallmann syndrome

Context: Physiological activation of the prokineticin pathway has a critical role in olfactory bulb morphogenesis and GnRH secretion in mice. Objective: To investigate PROK2 and PROKR2 mutations in patients with hypogonadotropic hypogonadism (HH) associated or not with olfactory abnormalities. Design: We studied 107 Brazilian patients with HH (63 with Kallmann syndrome and 44 with normosmic HH) and 100 control individuals. The coding regions of PROK2 and PROKR2 were amplified by PCR followed by direct automatic sequencing. Results: In PROK2, two known frameshift mutations were identified. Two brothers with Kallmann syndrome harbored the homozygous p. G100fsX121 mutation, whereas one male with normosmic HH harbored the heterozygous p. I55fsX56 mutation. In PROKR2, four distinct mutations (p. R80C, p. Y140X, p. L173R, and p. R268C) were identified in five patients with Kallmann syndrome and in one patient with normosmic HH. These mutations were not found in the control group. The p. R80C, p. L173R, and p. R268C missense mutations were identified in the heterozygous state in the HH patients and in their asymptomatic first-degree relatives. In addition, nomutations of FGFR1, KAL1, GnRHR, KiSS-1, or GPR54 were identified in these patients. Notably, the new nonsense mutation (p. Y140X) was identified in the homozygous state in an anosmic boy with micropenis, bilateral cryptorchidism, and high-arched palate. His asymptomatic parents were heterozygous for this severe defect. Conclusion: We expanded the repertoire of PROK2 and PROKR2 mutations in patients with HH. In addition, we show that PROKR2 haploinsufficiency is not sufficient to cause Kallmann syndrome or normosmic HH, whereas homozygous loss-of-function mutations either in PROKR2 or PROK2 are sufficient to cause disease phenotype, in accordance with the Prokr2 and Prok2 knockout mouse models.

Screening of autosomal gene deletions in patients with hypogonadotropic hypogonadism using multiplex ligation-dependent probe amplification: detection of a hemizygosis for the fibroblast growth factor receptor 1

P>Objective Congenital hypogonadotropic hypogonadism with anosmia (Kallmann syndrome) or with normal sense of smell is a heterogeneous genetic disorder caused by defects in the synthesis, secretion and action of gonadotrophin-releasing hormone (GnRH). Mutations involving autosomal genes have been identified in approximately 30% of all cases of hypogonadotropic hypogonadism. However, most studies that screened patients with hypogonadotropic hypogonadism for gene mutations did not include gene dosage methodologies. Therefore, it remains to be determined whether patients without detected point mutation carried a heterozygous deletion of one or more exons. Measurements We used the multiplex ligation-dependent probe amplification (MLPA) assay to evaluate the potential contribution of heterozygous deletions of FGFR1, GnRH1, GnRHR, GPR54 and NELF genes in the aetiology of GnRH deficiency. Patients We studied a mutation-negative cohort of 135 patients, 80 with Kallmann syndrome and 55 with normosmic hypogonadotropic hypogonadism. Results One large heterozygous deletion involving all FGFR1 exons was identified in a female patient with sporadic normosmic hypogonadotropic hypogonadism and mild dimorphisms as ogival palate and cavus foot. FGFR1 hemizygosity was confirmed by gene dosage with comparative multiplex and real-time PCRs. Conclusions FGFR1 or other autosomal gene deletion is a possible but very rare event and does not account for a significant number of sporadic or inherited cases of isolated GnRH deficiency.

Expanding the phenotype and genotype of female GnRH deficiency.

Context: GnRH deficiency is a rare genetic disorder of absent or partial pubertal development. The clinical and genetic characteristics of GnRH-deficient women have not been well-described. Objective: To determine the phenotypic and genotypic spectrum of a large series of GnRH-deficient women. Design, Setting, and Subjects: Retrospective study of 248 females with GnRH deficiency evaluated at an academic medical center between 1980 and 2010. Main Outcome Measures: Clinical presentation, baseline endogenous GnRH secretory activity, and DNA sequence variants in 11 genes associated with GnRH deficiency. Results: Eighty-eight percent had undergone pubarche, 51% had spontaneous thelarche, and 10% had 1-2 menses. Women with spontaneous thelarche were more likely to demonstrate normal pubarche (P = 0.04). In 27% of women, neuroendocrine studies demonstrated evidence of some endogenous GnRH secretory activity. Thirty-six percent (a large excess relative to controls) harbored a rare sequence variant in a gene associated with GnRH deficiency (87% heterozygous and 13% biallelic), with variants in FGFR1 (15%), GNRHR (6.6%), and PROKR2 (6.6%) being most prevalent. One woman had a biallelic variant in the X-linked gene, KAL1, and nine women had heterozygous variants. Conclusions: The clinical presentation of female GnRH deficiency varies from primary amenorrhea and absence of any secondary sexual characteristics to spontaneous breast development and occasional menses. In this cohort, rare sequence variants were present in all of the known genes associated with GnRH deficiency, including the novel identification of GnRH-deficient women with KAL1 variants. The pathogenic mechanism through which KAL1 variants disrupt female reproductive development requires further investigation.

Responsiveness to a physiological regimen of GnRH therapy and relation to genotype in women with isolated hypogonadotropic hypogonadism.

CONTEXT: Isolated hypogonadotropic hypogonadism (IHH) is caused by defective GnRH secretion or action resulting in absent or incomplete pubertal development and infertility. Most women with IHH ovulate with physiological GnRH replacement, implicating GnRH deficiency as the etiology. However, a subset does not respond normally, suggesting the presence of defects at the pituitary or ovary. OBJECTIVES: The objective of the study was to unmask pituitary or ovarian defects in IHH women using a physiological regimen of GnRH replacement, relating these responses to genes known to cause IHH. DESIGN, SETTING, AND SUBJECTS: This study is a retrospective analysis of 37 IHH women treated with iv pulsatile GnRH (75 ng/kg per bolus). MAIN OUTCOME MEASURES: Serum gonadotropin and sex steroid levels were measured, and 14 genes implicated in IHH were sequenced. RESULTS: During their first cycle of GnRH replacement, normal cycles were recreated in 60% (22 of 37) of IHH women. Thirty percent of women (12 of 37) demonstrated an attenuated gonadotropin response, indicating pituitary resistance, and 10% (3 of 37) exhibited an exaggerated FSH response, consistent with ovarian resistance. Mutations in CHD7, FGFR1, KAL1, TAC3, and TACR3 were documented in IHH women with normal cycles, whereas mutations were identified in GNRHR, PROKR2, and FGFR1 in those with pituitary resistance. Women with ovarian resistance were mutation negative. CONCLUSIONS: Although physiological replacement with GnRH recreates normal menstrual cycle dynamics in most IHH women, hypogonadotropic responses in the first week of treatment identify a subset of women with pituitary dysfunction, only some of whom have mutations in GNRHR. IHH women with hypergonadotropic responses to GnRH replacement, consistent with an additional ovarian defect, did not have mutations in genes known to cause IHH, similar to our findings in a subset of IHH men with evidence of an additional testicular defect.

A genetic basis for functional hypothalamic amenorrhea.

Background: Functional hypothalamic amenorrhea is a reversible form of gonadotropin-releasing hormone (GnRH) deficiency commonly triggered by stressors such as excessive exercise, nutritional deficits, or psychological distress. Women vary in their susceptibility to inhibition of the reproductive axis by such stressors, but it is unknown whether this variability reflects a genetic predisposition to hypothalamic amenorrhea. We hypothesized that mutations in genes involved in idiopathic hypogonadotropic hypogonadism, a congenital form of GnRH deficiency, are associated with hypothalamic amenorrhea. Methods: We analyzed the coding sequence of genes associated with idiopathic hypogonadotropic hypogonadism in 55 women with hypothalamic amenorrhea and performed in vitro studies of the identified mutations. Results: Six heterozygous mutations were identified in 7 of the 55 patients with hypothalamic amenorrhea: two variants in the fibroblast growth factor receptor 1 gene FGFR1 (G260E and R756H), two in the prokineticin receptor 2 gene PROKR2 (R85H and L173R), one in the GnRH receptor gene GNRHR (R262Q), and one in the Kallmann syndrome 1 sequence gene KAL1 (V371I). No mutations were found in a cohort of 422 controls with normal menstrual cycles. In vitro studies showed that FGFR1 G260E, FGFR1 R756H, and PROKR2 R85H are loss-of-function mutations, as has been previously shown for PROKR2 L173R and GNRHR R262Q. Conclusions: Rare variants in genes associated with idiopathic hypogonadotropic hypogonadism are found in women with hypothalamic amenorrhea, suggesting that these mutations may contribute to the variable susceptibility of women to the functional changes in GnRH secretion that characterize hypothalamic amenorrhea. Our observations provide evidence for the role of rare variants in common multifactorial disease. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and others; ClinicalTrials.gov number, NCT00494169.)

Clinical and molecular analysis of human reproductive disorders in Brazilian patients

Several genes that influence the development and function of the hypothalamic-pituitary-gonadal-axis (HPG) have been identified. These genes encode an array of transcription factors, matrix proteins, hormones, receptors, and enzymes that are expressed at multiple levels of the HPG. We report the experience of a single Endocrinology Unit in the identification and characterization of naturally occurring mutations in families affected by HPG disorders, including forms of precocious puberty, hypogonadism and abnormal sexual development due to impaired gonadotropin function. Eight distinct genes implicated in HPG function were studied: KAL, SF1, DAX1, GnRH, GnRHR, FSHß, FSHR, and LHR. Most mutations identified in our cohort are described for the first time in literature. New mutations in SF1, DAX1 and GnRHR genes were identified in three Brazilian patients with hypogonadism. Eight boys with luteinizing hormone- (LH) independent precocious puberty due to testotoxicosis were studied, and all have their LH receptor (LHR) defects elucidated. Among the identified LHR molecular defects, three were new activating mutations. In addition, these mutations were frequently associated with new clinical and hormonal aspects, contributing significantly to the knowledge of the molecular basis of reproductive disorders. In conclusion, the naturally occurring genetic mutations described in the Brazilian families studied provide important insights into the regulation of the HPG.

Participation of the endoplasmic reticulum protein chaperone thio-oxidoreductase in gonadotropin-releasing hormone receptor expression at the plasma membrane

Chaperone members of the protein disulfide isomerase family can catalyze the thiol-disulfide exchange reaction with pairs of cysteines. There are 14 protein disulfide isomerase family members, but the ability to catalyze a thiol disulfide exchange reaction has not been demonstrated for all of them. Human endoplasmic reticulum protein chaperone thio-oxidoreductase (ERp18) shows partial oxidative activity as a protein disulfide isomerase. The aim of the present study was to evaluate the participation of ERp18 in gonadotropin-releasing hormone receptor (GnRHR) expression at the plasma membrane. Cos-7 cells were cultured, plated, and transfected with 25 ng (unless indicated) wild-type human GnRHR (hGnRHR) or mutant GnRHR (Cys14Ala and Cys200Ala) and pcDNA3.1 without insert (empty vector) or ERp18 cDNA (75 ng/well), pre-loaded for 18 h with 1 µCi myo-[2-3H(N)]-inositol in 0.25 mL DMEM and treated for 2 h with buserelin. We observed a decrease in maximal inositol phosphate (IP) production in response to buserelin in the cells co-transfected with hGnRHR, and a decrease from 20 to 75 ng of ERp18 compared with cells co-transfected with hGnRHR and empty vector. The decrease in maximal IP was proportional to the amount of ERp18 DNA over the range examined. Mutants (Cys14Ala and Cys200Ala) that could not form the Cys14-Cys200 bridge essential for plasma membrane routing of the hGnRHR did not modify maximal IP production when they were co-transfected with ERp18. These results suggest that ERp18 has a reduction role on disulfide bonds in wild-type hGnRHR folding.

New molecular variants of hypothalamus-pituitary-gonad axis genes and their association with early puberty phenotype in Bos taurus indicus (Nellore)

Endocrine system plays a major role in the control of reproductive functions which are regulated by the hypothalamus-pituitary-gonad axis and its interactions. FSH and LH receptor genes are expressed at the gonads and GnRH receptor gene is expressed at the anterior pituitary gland. Misense mutations of the FSH, LH or GnRH receptors, activating or inactivating their functions in mammals, are potentially useful to allow the understanding of the role of this group of gonadotropins in reproductive phenotypes as early puberty and birth interval length. In the present study, polymorphisms in bovine exon 11 and 3`UTR of LHR, exon 10 and 3`UTR of FSHR and GnRHR genes were characterized with some of them resulting in changes in the aminoacidic chain. These polymorphic sites were found in a Bos taurus indicus (Nellore) female population by means of PCR-SSCP and DNA sequencing. Association between nucleotidic/aminoacidic changes and early puberty were determined by Chi-square analysis. It was found association between FSHR 3`UTR polymorphisms at position 2181, 2248 and 2249 bp and early puberty phenotype (p < 0.05). The presence of these new molecular markers might be considered in further studies to validate its correlation with early puberty or other reproduction associated phenotypes in cattle breeds. (C) 2007 Published by Elsevier B.V.

New molecular variants of hypothalamus-pituitary-gonad axis genes and their association with early puberty phenotype in Bos taurus indicus (Nellore)

Endocrine system plays a major role in the control of reproductive functions which are regulated by the hypothalamus-pituitary-gonad axis and its interactions. FSH and LH receptor genes are expressed at the gonads and GnRH receptor gene is expressed at the anterior pituitary gland. Misense mutations of the FSH, LH or GnRH receptors, activating or inactivating their functions in mammals, are potentially useful to allow the understanding of the role of this group of gonadotropins in reproductive phenotypes as early puberty and birth interval length. In the present study, polymorphisms in bovine exon 11 and 3'UTR of LHR, exon 10 and 3'UTR of FSHR and GnRHR genes were characterized with some of them resulting in changes in the aminoacidic chain. These polymorphic sites were found in a Bos taurus indicus (Nellore) female population by means of PCR-SSCP and DNA sequencing. Association between nucleotidic/aminoacidic changes and early puberty were determined by Chi-square analysis. It was found association between FSHR 3'UTR polymorphisms at position 2181, 2248 and 2249 bp and early puberty phenotype (p < 0.05). The presence of these new molecular markers might be considered in further studies to validate its correlation with early puberty or other reproduction associated phenotypes in cattle breeds. (C) 2007 Published by Elsevier B.V.

AÇÃO REGULATÓRIA DO GnRH NO DESENVOLVIMENTO EMBRIONÁRIO PRECOCE E VITRIFICAÇÃO DE EMBRIÕES SUÍNOS PELO MÉTODO DE MICROGOTA (ROLE OF GnRH DURING EARLY EMBRYONIC DEVELOPMENT AND DEVELOPMENT OF SWINE EMBRYOS FOLLOWING VITRIFICATION BY MICRODROPLET METHOD)

The objective of this study was to investigate the role of GnRH on the preimplantation development of mouse embryos in vitro. GnRH-I, GnRH-II, and GnRH agonists: Des-Gly, Des-Trp and histrelin did not improve embryo development. However, treatment with the specific GnRH antagonist SB-75 blocked embryo development at morula stage. The inhibition of embryo development by SB-75 could be rescued by the addition of histrelin. To determine which intracellular signaling cascade is involved following binding of GnRH to the GnRHR, embryos were cultured in the presence of specific PKC (GFX) or PKA (SQ22536) inhibitors. The PKC inhibitor blocked embryo development at a similar stage as SB-75, whereas SQ22536 had an inhibitory effect, diminishing blastocyst formation and hatched rates. There are evidences that GnRH has an essential autocrine effect on mouse embryonic development via GnRHR, probably by activating PKC signaling cascade while the inhibition of the GnRH signaling does not activate apoptotic mechanisms involving caspase-3. In another experiment, development in vitro of embryos from Chinese Meishan (M) and occidental white crossbred (WC) females were investigated after improving the vitrification protocol for pig embryos. Efficient cryopreservation of zona pellucida-intact porcine embryos and studies of the difference among breeds could greatly impact the swine industry. The percentage of embryos surviving 24 h after cryopreservation without lysis or degeneration was higher for M (72%) than WC (44%). However, in vitro development of embryos that survived cryopreservation was not different between M and WC at the expanded (64%) or hatched (22%) blastocyst stages. Developmental rates were significantly higher for control embryos than frozen embryos from both breeds at expanded blastocyst stage, but not at hatched blastocyst stage. Rates of expanded blastocyst formation did not differ between M and WC control embryos (98 and 95%, respectively). With a new procedure to warm vitrified pig embryos, the survival rates may be improved. The optimal stages to vitrify pig embryos using the microdroplet method ranges from late compact morula to early expanded blastocyst. The results suggest that M embryos have a higher capacity to survive the vitrification process than WC embryos. O objetivo do presente estudo foi investigar a importância do GnRH no desenvolvimento embrionário precoce em camundongos. GnRH-I, GnRH-II e os GnRH agonistas: Des-Gly, Des-Trp e histrelina não incrementaram o desenvolvimento embrionário. Entretanto, o tratamento com SB-75, um antagonista específico do GnRH, bloqueou o desenvolvimento embrionário no estádio de mórula. A inibição do desenvolvimento embrionário pelo SB-75 pôde ser revertida com a adição de histrelina. Para determinar a cascata do sinal intracelular desencadeada pela ligação do GnRH com o seu receptor, embriões foram cultivados na presença de inibidores específicos da PKC (GFX) e da PKA (SQ22536). O inibidor da PKC bloqueou o desenvolvimento embrionário em estádio similar ao bloqueio mediado pelo SB- 75, enquanto o SQ22536 teve efeito inibitório diminuindo a formação de blastocisto e taxas de eclosão. Os resultados sugerem que o GnRH tem um efeito autócrino essencial no desenvolvimento embrionário através do GnRHR, provavelmente, ativando a cascata da PKC. Por outro lado, a inibição do sinal do GnRH não ativa mecanismos apoptóticos que involvam caspase-3. Em outro experimento, foi investigado o desenvolvimento in vitro de embriões da raça Meishan (M) e branco cruzado (WC) após vitrificação pelo método microgota. O desenvolvimento de protocolos eficientes para criopreservação de embriões suínos com a zona pelúcida intacta e a avaliação das diferenças entre raças pode ter um significativo impacto na suinocultura. A percentagem de embriões que sobreviveram à criopreservação depois de 24 h foi maior na M (72%) do que na WC (44%). No entanto, o desenvolvimento in vitro dos embriões que sobreviveram à criopreservação não foi diferente entre M e WC nos estádios de blastocisto expandido (64%) ou eclodido (22%). Os índices de desenvolvimento foram significativamente mais altos para os embriões controle do que para os embriões vitrificados nas duas raças no estádio de blastocisto expandido, porém não foram diferentes para o estádio de blastocisto eclodido. A formação de blastocisto expandido não diferiu entre os embriões controle M e WC (98 e 95%, respectivamente). Com o novo procedimento (“hot warm”) para descongelar embriões vitrificados pelo método de microgota, pode-se aumentar dos índices de sobrevivência. Os melhores estádios embrionários para a vitrificação de embriões suínos variam de mórula compacta tardia até blastocisto expandido inicial. Os resultados sugerem que embriões M têm mais capacidade de sobreviver ao processo de vitrificação do que embriões WC.

A mechanism for the differential regulation of gonadotropin subunit gene expression by gonadotropin-releasing hormone.

The hypothalamic hormone gonadotropin-releasing hormone (GnRH) is released in a pulsatile fashion, with its frequency varying throughout the reproductive cycle. Varying pulse frequencies and amplitudes differentially regulate the biosynthesis and secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by pituitary gonadotropes. The mechanism by which this occurs remains a major question in reproductive physiology. Previous studies have been limited by lack of available cell lines that express the LH and FSH subunit genes and respond to GnRH. We have overcome this limitation by transfecting the rat pituitary GH3 cell line with rat GnRH receptor (GnRHR) cDNA driven by a heterologous promoter. These cells, when cotransfected with regulatory regions of the common alpha, LH beta, or FSH beta subunit gene fused to a luciferase reporter gene, respond to GnRH with an increase in luciferase activity. Using this model, we demonstrate that different cell surface densities of the GnRHR result in the differential regulation of LH and FSH subunit gene expression by GnRH. This suggests that the differential regulation of gonadotropin subunit gene expression by GnRH observed in vivo in rats may, in turn, be mediated by varying gonadotrope cell surface GnRHR concentrations. This provides a physiologic mechanism by which a single ligand can act through a single receptor to regulate differentially the production of two hormones in the same cell.