Gene variants identified by whole-exome sequencing in 33 French women with premature ovarian insufficiency.

PURPOSE: To investigate the potential genetic etiology of premature ovarian insufficiency (POI). METHODS: Whole-exome sequencing (WES) was done on DNA samples from women diagnosed with POI. Mutations identified were analyzed by in silico tools and were annotated according to the guidelines of the American College of Medical Genetics and Genomics. Plausible variants were confirmed by Sanger sequencing. RESULTS: Four of the 33 individuals (12%) carried pathogenic or likely pathogenic variants, and 6 individuals carried variants of unknown significance. The genes identified with pathogenic or likely pathogenic variants included PMM2, MCM9, and PSMC3IP. CONCLUSIONS: WES is an efficient tool for identifying gene variants in POI women; however, interpretation of variants is hampered by few exome studies involving ovarian disorders and the need for trio sequencing to determine inheritance and to detect de novo variants.

Mutations and polymorphisms in FSH receptor: functional implications in human reproduction.

FSH brings about its physiological actions by activating a specific receptor located on target cells. Normal functioning of the FSH receptor (FSHR) is crucial for follicular development and estradiol production in females and for the regulation of Sertoli cell function and spermatogenesis in males. In the last two decades, the number of inactivating and activating mutations, single nucleotide polymorphisms, and spliced variants of FSHR gene has been identified in selected infertile cases. Information on genotype-phenotype correlation and in vitro functional characterization of the mutants has helped in understanding the possible genetic cause for female infertility in affected individuals. The information is also being used to dissect various extracellular and intracellular events involved in hormone-receptor interaction by studying the differences in the properties of the mutant receptor when compared with WT receptor. Studies on polymorphisms in the FSHR gene have shown variability in clinical outcome among women treated with FSH. These observations are being explored to develop molecular markers to predict the optimum dose of FSH required for controlled ovarian hyperstimulation. Pharmacogenetics is an emerging field in this area that aims at designing individual treatment protocols for reproductive abnormalities based on FSHR gene polymorphisms. The present review discusses the current knowledge of various genetic alterations in FSHR and their impact on receptor function in the female reproductive system.

Association of allelic combinations of FSHR gene polymorphisms with ovarian response.

During an IVF protocol, exogenous FSH is administered to women for ovulation induction. The ovarian response to gonadotrophin stimulation is variable and unpredictable in these women. The FSHR is the most studied gene in relation to ovarian response. The association of a FSHR gene polymorphism at position 680 (p.Asn680Ser) with ovarian response has been well documented. Recently, a polymorphism at position -29 in the 5'-untranslated region of FSHR (g.-29G>A) has been reported to be associated with poor ovarian response and reduced FSHR expression. The present study evaluated the combined effect of the polymorphisms at positions -29 and 680 of FSHR with type of ovarian response and receptor expression. The two FSHR gene polymorphisms together formed four discrete haplotypes and nine allelic combinations. Various clinical parameters revealed that 75% of the subjects with A/A-Asn/Asn genotype were poor ovarian responders (odds ratio 7.92; P=0.009). The relative FSHR mRNA expression in granulosa cells indicated that subjects with A/A-Asn/Asn genotype express significantly lower level of FSHR as compared to the subjects with G/G-Asn/Ser genotype (P=0.029). These results indicate that A/A-Asn/Asn genotype could be used as a potential marker to predict poor ovarian response. The action of FSH is mediated by its receptor (FSHR) present on the granulosa cells in the ovary. Any alterations in the hormone or its receptor are likely to disrupt its normal function, thus causing infertility. Several alterations (mutations/polymorphisms) of the FSHR gene have been reported in women with primary or secondary amenorrhoea. It has also been reported that FSHR gene polymorphisms are associated with variable ovarian response to FSH stimulation during IVF. Women may show poor or normal or hyperovarian response to FSH stimulation. It is well documented that the level of FSHR expression has a great effect on FSH action and is associated with ovarian response. In the present study, we screened normally menstruating women undergoing IVF due to tubal/male factor or unexplained infertility. We analysed two polymorphisms of FSHR, g-29G>A and p.Asn680Ser, in these women. In the subjects studied, 75% women with A/A-Asn/Asn genotype were observed to be poor ovarian responders to FSH stimulation. FSHR expression at the transcript level was observed to be significantly lower in women with A/A-Asn/Asn genotype as compared to women with G/G-Asn/Ser genotype. We also observed that women with A/A-Ser/Ser genotype were not present in the study population. These findings indicate the significance of A/A-Asn/Asn genotype as a predictive marker for poor ovarian response to FSH stimulation.

Genetics of Reproductive Aging from Gonadal Dysgenesis through Menopause.

Reproduction is essential for the survival of the species and is influenced by external factors such as smoking and exposure to chemotherapy as well as chronic disorders such as obesity and autoimmunity. Reproductive senescence, such as menopause, is also dependent on multiple intrinsic genetic factors. Reproductive aging is not isolated from an overall aging process, and several studies strongly support the link between the early age of menopause and mortality. The extreme form of reproductive aging is primary ovarian insufficiency (POI) with prevalence ranging from 1 to 5% of the female population. POI has been shown to have long-term consequences on overall health. POI and age of menopause have a significant hereditary component. The population-based genome-wide association studies have identified 44 genomic loci to associate with age of menopause, and 29 of 44 loci harbor DNA damage response genes. Recent application of whole exome sequencing on carefully selected families with POI has also revealed a significant contribution of DNA damage response genes. The inability to repair the DNA damage in both somatic and germ cells might be a predisposing factor for the link between reproductive and overall aging in a subset of individuals with POI. The aim of this review is to characterize recent advances in the genetics of POI and its link with overall health.

MCM8 and MCM9 Nucleotide Variants in Women With Primary Ovarian Insufficiency.

Objective: To assess the frequency of variants, including biallelic pathogenic variants, in minichromosome maintenance 8 (MCM8) and minichromosome maintenance 9 (MCM9), other genes related to MCM8-MCM9, and DNA damage repair (DDR) pathway in participants with primary ovarian insufficiency (POI). Design: MCM8, MCM9, and genes encoding DDR proteins that have been implicated in reproductive aging were sequenced among POI participants. Setting: Academic research institution. Participants: All were diagnosed with POI prior to age 40 years and presented with elevated follicle-stimulating hormone levels. Interventions: None. Main Outcome Measures: We identified nucleotide variants in MCM8, MCM9, and genes thought to be involved in the DNA damage response pathway and/or implicated in reproductive aging. Results: MCM8 was sequenced in 155 POI participants, whereas MCM9 was sequenced in 151 participants. Three of 155 (2%) participants carried possibly damaging heterozygous variants in MCM8, whereas 7 of 151 (5%) individuals carried possibly damaging heterozygous variants in MCM9. One participant carried a novel homozygous variant, c.1651C>T, p.Gln551*, in MCM9, which is predicted to introduce a premature stop codon in exon 9. Biallelic damaging heterozygous variants in both MCM8 and MCM9 were identified in 1 participant. Of a total of 10 participants carrying damaging heterozygous variants in either MCM8 or MCM9, 2 individuals carried heterozygous damaging variants in genes associated with either MCM8 or MCM9 or the DDR pathway. Conclusions: We identified a significant number of potentially damaging and novel variants in MCM8 and MCM9 among participants with POI and examined multiallelic association with variants in DDR and MCM8-MCM9 interactome genes.

Functional characterization of two naturally occurring mutations (Val514Ala and Ala575Val) in follicle-stimulating hormone receptor.

CONTEXT: Inactivating mutations have been reported in subjects with primary/secondary amenorrhea, whereas activating mutations are rare and seen only in women with ovarian hyperstimulation syndrome (OHSS). In the present study, we describe the functional characterization of the two mutations Val(514)Ala (novel mutation) and Ala(575)Val in FSH receptor (FSHR) identified in women with OHSS developed during in vitro fertilization and primary amenorrhea, respectively. OBJECTIVE: The objective of the investigation was to study the effect of mutations (514 and 575) on FSHR activity by in vitro functional studies. SETTING: The study was conducted at an academic research institute and a private in vitro fertilization clinic. METHODS: The site-directed mutagenesis was carried out to generate the mutations at position 514 and 575 in pSG5-FSHR construct. Stable cell lines expressing wild type or each of the mutant receptor were generated using Chinese hamster ovary cells. Functional characteristics of both the mutant receptors were assessed by a radioreceptor assay and a cAMP assay. RESULTS: The mutant receptor 514 showed increased cell surface expression as compared with the wild-type (WT) receptor. Although the hormone binding characteristics were similar to the WT receptor, its signaling activity was distinctly higher at lower dose of FSH as monitored by a cAMP assay. On the other hand, the mutant receptor 575 showed lower cell surface expression and higher internalized hormone receptor complex. Additionally, a dose-dependent increase in the cAMP accumulation was not observed in the case of this mutant as compared with WT. CONCLUSION: OHSS and primary amenorrhea observed in the two affected women, respectively, could be attributed to the functional characteristics of respective mutant FSHR.

The rs10993994 in the proximal MSMB promoter region is a functional polymorphism in Asian Indian subjects.

BACKGROUND: The microseminoprotein gene encoding prostate secretory protein of 94 amino acids (PSP94) harbours a potential risk allele (rs10993994) for prostate cancer (PCa) in its promoter region. However, studies on rs10993994 have been sparse in Asian Indians. METHODS: The present study recruited a sample population of 44 benign prostatic hyperplasia patients, 33 PCa patients and 60 healthy participants, of which, participants without other confounding risk factors for PCa were retained. The serum PSP94 (sPSP94) levels were measured by a serum-based ELISA in an earlier study. A novel RFLP technique was developed to screen for rs10993994 which was validated with direct sequencing. RESULTS: Sequencing showed additional 4 SNPs (rs41274660, rs141211965, rs12770171, rs10669586) and 2 novel variants (GenBank accession nos. KM265191 and KM265192). In silico DNA topographical studies predicted that KM265192 would have higher cleavage intensity and more accessibility for binding of transcription factors. Even though, similar frequencies were observed for all the variants in all the three study groups, the risk allele 'T' (rs10993994) was seen to be associated with reduced PSP94 expression both at mRNA and protein level. Further, mRNA expression as studied by real-time PCR correlated positively with sPSP94 levels. Interestingly, CC genotype of rs10993994 showed highest sPSP94 levels in all the three study groups and was associated with Gleason score ≤7 in PCa patients. In contrast, TT genotype of rs10993994 was associated with lesser sPSP94 levels and with aggressiveness of PCa. CONCLUSION: rs10993994 was found to be a functional SNP in the studied Asian Indian population.

Development of an ELISA for sPSP94 and utility of the sPSP94/sPSA ratio as a diagnostic indicator to differentiate between benign prostatic hyperplasia and prostate cancer.

BACKGROUND: The serum PSA (sPSA) test has low specificity for prostate cancer (PCa), since sPSA also rises in benign prostatic hyperplasia (BPH). Serum PSP94 (sPSP94), a major secreted prostate protein, is indicated as a PCa marker. The potential of sPSP94 and sPSA in conjunction with each other to improve specificity of diagnostic test for PCa needs to be evaluated. METHODS: PCa patients (n=33), BPH patients (n=44) and healthy controls (n=50) were recruited. A serum-based sandwich ELISA was developed to measure sPSP94 concentrations. Utility of sPSP94 in improving specificity of sPSA test was evaluated by studying sPSP94/sPSA ratios of study participants. RESULTS: Considerable decrease in overlap among sPSP94/sPSA ratio values of BPH and PCa patients was observed, as compared to sPSP94 or sPSA alone. For differentiating between BPH and PCa patients, this ratio had a maximum area under the curve (AUC) of 0.859 (P=0.0132) and had a comparable sensitivity (90.91%) to sPSA with an increased specificity of 70.45%. Further, decision curve analysis (DCA) showed that sPSP94/sPSA ratio had a superior net benefit in identifying PCa, in patients opting for biopsy. CONCLUSION: The sPSP94/sPSA ratio can be a better differentiating marker between BPH and PCa, than sPSP94 or sPSA alone.

Follicle-stimulating hormone receptor polymorphism (G-29A) is associated with altered level of receptor expression in Granulosa cells.

CONTEXT: Polymorphisms of the FSHR gene are associated with variable ovarian response to FSH stimulation in subjects undergoing in vitro fertilization (IVF) treatment. The type of ovarian response is correlated with the level of FSH receptor (FSHR) expression on granulosa cells. OBJECTIVE: We investigated whether the polymorphism at position -29 in the promoter of the FSHR gene may contribute in altered receptor expression. DESIGN AND PATIENTS: FSHR polymorphism at position -29 was studied in 100 subjects undergoing IVF treatment. Association of this polymorphism with level of FSHR expression was retrospectively analyzed. SETTING: The study was conducted at an academic research institute and private IVF clinic. METHODS: The genotype at position -29 of the FSHR gene was studied in IVF subjects by PCR-restriction fragment length polymorphism. Total RNA and protein was extracted from granulosa cells. The relative FSHR mRNA expression was carried out by real-time PCR. The receptor protein expression was evaluated by Western blot and confocal microscopy. RESULTS: The clinical and endocrinological parameters revealed that almost 72% of subjects with the AA genotype at position -29 of FSHR gene were poor ovarian responders (odds ratio 8.63, 95% confidential interval 1.84-45.79; P = 0.001). The lower cleavage intensity predicted by in silico analysis for A allele as compared with the G allele suggest the difference in the DNA-protein binding affinity. The relative expression of FSHR at mRNA and protein level was significantly reduced in subjects with AA genotype as compared with the GG genotype. CONCLUSION: Poor ovarian response observed in subjects with the AA genotype at position -29 of the FSHR gene is due to reduced receptor expression.