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Context: Premature ovarian insufficiency (POI) is characterized by cessation of menstruation before 40 years of age and elevated serum level of FSH (>25 IU/L). Recent studies have found a few causative genes responsible for POI enriched in meiotic recombination and DNA damage repair pathways.Objective: To investigate the role of variations in homologous recombination genes played in POI pathogenesis.Methods: The whole exome sequencing was performed in 50 POI patients with primary amenorrhea. Functional characterizations of the novel variants were carried out in budding yeast and human cell line.Results: We identified 8 missense variants in 7 homologous recombination genes, including EXO1, RAD51, RMI1, MSH5, MSH2, MSH6, and MLH1. The mutation p.Thr52Ser in EXO1 impaired the meiotic process of budding yeast and p.Glu68Gly in RAD51-altered protein localization in human cells, both of them impaired the efficiency of homologous recombination repair for DNA double-stranded breaks in human cells.Conclusions: Our study first linked the variants of EXO1 and RAD51 with POI and further highlighted the role of DNA repair genes in ovarian dysgenesis. [ABSTRACT FROM AUTHOR]

Study Question: Are mutations and/or polymorphisms in the TP63 gene associated with human Müllerian duct anomalies (MDAs)?Summary Answer: The novel mutation c.*374 G > A in the TP63 gene resulted in decreased expression of TP63 by generating new binding sites with miR-1260a/miR-532-3p and revealed the potential association between TP63 and human MDAs.What Is Known Already: It has been shown that mice lacking Tp63 exhibit hypoplastic genitalia, a single cloacal opening, and persistence of columnar epithelium at lower genital tract sites. It has also been reported that a nonsense mutation in EMX2 results in decreased TP63 expression in a woman with MDAs. However, generally in humans the association between TP63 and MDAs is unknown.Study Design, Size, Duration: A total of 200 unrelated Chinese women with MDAs and 200 unrelated Chinese women with a normal uterus and vagina, as controls, were recruited in the Center for Reproductive Medicine of Shandong University. All participants had a normal karyotype (46, XX).Participants/materials, Setting, Methods: The 20 exons of the TP63 gene were sequenced in 200 cases and 200 controls. Putative binding sites for microRNAs were validated by dual luciferase activity assays. The role of microRNAs was further examined by western blot.Main Results and the Role Of Chance: Sequence analysis revealed 15 known single-nucleotide polymorphisms. Additionally, three novel heterozygous variants, c.387 G > C, c.*374 G > A and c.*749 G > A, were identified in three patients with MDAs, none of which were detected in controls. Variant c.*374 G > A, located in the 3' untranslated region, was highly conserved among mammals and predicted to create microRNAs binding sites, which was confirmed by dual luciferase activity assays. Western blot demonstrated that the binding with miR-1260a/miR-532-3p resulting from the variation c.*374 G > A decreased the expression of TP63.Large Scale Data: N/A LIMITATIONS, REASONS FOR CAUTION: Further study is needed to uncover the role of the EMX2-TP63 pathway in the development of the Müllerian duct.Wider Implications Of the Findings: This study revealed the possible association between TP63 and MDAs and suggested a potential contribution of microRNA-regulated expression of genes in the etiology of MDAs.Study Funding/competing Interests: This research was supported by National Basic Research Program of China (973 Program) (2012CB944700), the State Key Program of National Natural Science Foundation of China (81430029), the National Natural Science Foundation of China (81270662, 81471509), China Postdoctoral Science Foundation (2014M561939) and the Scientific Research Foundation of Shandong Province of Outstanding Young Scientists (BS2014YY013, 2014BSE27022). The authors have no competing interests. [ABSTRACT FROM AUTHOR]

Background: Primary ovarian insufficiency (POI) is characterized by marked heterogeneity, but with a significant genetic contribution. Identifying exact causative genes has been challenging, with many discoveries not replicated. It is timely to take stock of the field, outlining the progress made, framing the controversies and anticipating future directions in elucidating the genetics of POI.Methods: A search for original articles published up to May 2015 was performed using PubMed and Google Scholar, identifying studies on the genetic etiology of POI. Studies were included if chromosomal analysis, candidate gene screening and a genome-wide study were conducted. Articles identified were restricted to English language full-text papers.Results: Chromosomal abnormalities have long been recognized as a frequent cause of POI, with a currently estimated prevalence of 10-13%. Using the traditional karyotype methodology, monosomy X, mosaicism, X chromosome deletions and rearrangements, X-autosome translocations, and isochromosomes have been detected. Based on candidate gene studies, single gene perturbations unequivocally having a deleterious effect in at least one population include Bone morphogenetic protein 15 (BMP15), Progesterone receptor membrane component 1 (PGRMC1), and Fragile X mental retardation 1 (FMR1) premutation on the X chromosome; Growth differentiation factor 9 (GDF9), Folliculogenesis specific bHLH transcription factor (FIGLA), Newborn ovary homeobox gene (NOBOX), Nuclear receptor subfamily 5, group A, member 1 (NR5A1) and Nanos homolog 3 (NANOS3) seem likely as well, but mostly being found in no more than 1-2% of a single population studied. Whole genome approaches have utilized genome-wide association studies (GWAS) to reveal loci not predicted on the basis of a candidate gene, but it remains difficult to locate causative genes and susceptible loci were not always replicated. Cytogenomic methods (array CGH) have identified other regions of interest but studies have not shown consistent results, the resolution of arrays has varied and replication is uncommon. Whole-exome sequencing in non-syndromic POI kindreds has only recently begun, revealing mutations in the Stromal antigen 3 (STAG3), Synaptonemal complex central element 1 (SYCE1), minichromosome maintenance complex component 8 and 9 (MCM8, MCM9) and ATP-dependent DNA helicase homolog (HFM1) genes. Given the slow progress in candidate-gene analysis and relatively small sample sizes available for GWAS, family-based whole exome and whole genome sequencing appear to be the most promising approaches for detecting potential genes responsible for POI.Conclusion: Taken together, the cytogenetic, cytogenomic (array CGH) and exome sequencing approaches have revealed a genetic causation in ∼20-25% of POI cases. Uncovering the remainder of the causative genes will be facilitated not only by whole genome approaches involving larger cohorts in multiple populations but also incorporating environmental exposures and exploring signaling pathways in intragenic and intergenic regions that point to perturbations in regulatory genes and networks. [ABSTRACT FROM AUTHOR]

Premature ovarian failure (POF) is a rare, heterogeneous disorder characterized by cessation of menstruation occurring before the age of 40 years. Genetic etiology is responsible for perhaps 25% of cases, but most cases are sporadic and unexplained. In this study, through whole exome sequencing in a non-consanguineous family having four affected members with POF and Sanger sequencing in 432 sporadic cases, we identified three novel mutations in the fusion gene CSB-PGBD3. Subsequently functional studies suggest that mutated CSB-PGBD3 fusion protein was impaired in response to DNA damage, as indicated by delayed or absent recruitment to damaged sites. Our data provide the first evidence that mutations in the CSB-PGBD3 fusion protein can cause human disease, even in the presence of functional CSB, thus potentially explaining conservation of the fusion protein for 43 My since marmoset. The localization of the CSB-PGBD3 fusion protein to UVA-induced nuclear DNA repair foci further suggests that the CSB-PGBD3 fusion protein, like many other proteins that can cause POF, modulates or participates in DNA repair. [ABSTRACT FROM AUTHOR]

Purpose: To investigate whether mutations in the KISS1 gene are present in 170 Chinese patients with idiopathic hypogonadotropic hypogonadism (IHH). Methods: Mutational screening of the KISS1 gene was performed in 170 Chinese patients with IHH (133 male cases and 37 female cases) and 187 matched controls (94 males and 93 females). Results: Two known single-nucleotide polymorphisms (SNP), c. 58G > A in exon 1 and c. 242C > G in exon 2, were identified. However, no difference of genotype and allelic frequencies between cases and controls was observed. Conclusions: The results suggest that mutations in the coding sequence of KISS1 are not common in patients with IHH in this Chinese population. [ABSTRACT FROM AUTHOR]

Background: In premature ovarian failure (POF), cessation of menstruation occurs before the expected age of menopause. Approximately 1% of women are affected. FMR1 premutation was reported to be responsible for up to 3.3%–6.7% of sporadic POF and 13% of familial cases in Caucasians, while the data was absent in Chinese population. Therefore, the impact of FMR1 CGG repeat on ovarian reserve is needed to be investigated in large Chinese cohort. Methods: The number of FMR1 CGG repeat was determined in 379 Han Chinese women with well-defined 46, XX non-syndromic sporadic POF and 402 controls. The age of menopause onset in respect to CGG repeats was further analyzed. Results: The frequency of FMR1 premutation in Han Chinese POF was only 0.5% (2/379), although it was higher than that in matched controls (0%, 0/402), it was much lower than that reported in Caucasian with POF (3.3%–6.7%). The prevalence of intermediate FMR1 (41–54) was not increased significantly in sporadic POF than that in controls (2.9% vs. 1.7%, P = 0.343). However, POF patients more often carried a single additional CGG repeat in a single allele than did fertile women (allele-1: 29.7 vs. 28.8, P<0.001; allele-2: 32.6 vs. 31.5, P<0.001). POF patients with both alleles of CGG repeats outside (below or above) the normal range (26–34) showed an earlier age of cessation of menses than those with two alleles within normal range (hom-high/high vs. norm: 20.4±4.8 vs. 24.7±6.4, p<0.01; hom-low/high vs. norm: 18.7±1.7 vs. 24.7±6.4, p<0.01). Conclusions: FMR1 premutation seems to be an uncommon explanation for POF in Han Chinese. However, having both alleles with CGG repeats outside the normal range might still adversely affect ovarian aging. [ABSTRACT FROM AUTHOR]

Background: The etiology of most premature ovarian failure (POF) cases is usually elusive. Although genetic causes clearly exist and a likely susceptible region of 8q22.3 has been discovered, no predominant explanation exists for POF. More recently, evidences have indicated that mutations in NR5A1 gene could be causative for POF. We therefore screened for mutations in the NR5A1 gene in a large cohort of Chinese women with non-syndromic POF. Methods: Mutation screening of NR5A1 gene was performed in 400 Han Chinese women with well-defined 46,XX idiopathic non-syndromic POF and 400 controls. Subsequently, functional characterization of the novel mutation identified was evaluated in vitro. Results: A novel heterozygous missense mutation [c.13T>G (p.Tyr5Asp)] in NR5A1 was identified in 1 of 384 patients (0.26%). This mutation impaired transcriptional activation on Amh, Inhibin-a, Cyp11a1 and Cyp19a1 gene, as shown by transactivation assays. However, no dominant negative effect was observed, nor was there impact on protein expression and nuclear localization. Conclusions: This novel mutation p.Tyr5Asp, in a novel non-domain region, is presumed to result in haploinsufficiency. Irrespectively, perturbation in NR5A1 is not a common explanation for POF in Chinese. [ABSTRACT FROM AUTHOR]

We examined the PBX1 gene in 192 Chinese women with Müllerian duct abnormalities and revealed 2 known single nucleotide polymorphisms: c.61G＞A in exon 1 and c.998–1330A>G in intron 7. Future studies in large cohorts of different ethnic populations are warranted to establish definite associations between the PBX1 gene and Müllerian duct abnormalities. [ABSTRACT FROM AUTHOR]

The newborn ovary homeobox gene (NOBOX) is an oocyte-specific homeobox gene that plays a critical role in early folliculogenesis, thus representing an attractive candidate gene for nonsyndromic ovarian failure. We investigated whether perturbation in the homeodomain region of NOBOX was present in Chinese women with premature ovarian failure (POF). We sequenced 200 Chinese patients with POF, and discovered only two known single nucleotide polymorphisms: in intron 6 (c.1154+11 T>C and c.1155–22 G>A); neither offers plausible explanations for POF. Failing to find causative mutation contrasts with our previous study in a caucasian sample, in which we found plausible homeobox mutation in 1 of 96 POF cases. Mutations in the homeobox domain of NOBOX are not common explanations for POF in Chinese women. [Copyright &y& Elsevier]

We screened growth differentiation factor 9 coding regions for mutations in a Chinese sample of 100 women with premature ovarian failure and discovered four novel single-nucleotide polymorphisms: c.436C>T (p.Arg146Cys), c.588A>C (silent), c.712A>G (p.Thr238Ala), and c.1283G>C (p.Ser428Thr). Nonsynonymous single-nucleotide polymorphisms c.436C>T and c.1283G>C were also detected in the control population. The c.712A>G perturbation results in a missense mutation (p.Thr238Ala) and was not present in any of 96 controls. Substitution of the hydrophobic amino acid residue alanine for hydrophilic threonine may disrupt growth differentiation factor 9 function. [Copyright &y& Elsevier]

NANOS3 encodes an RNA-binding protein and has a conserved function in germ cell development. Our objective was to investigate whether mutations in NANOS3 were present in Chinese and Caucasian women with premature ovarian failure. A known synonymous single-nucleotide polymorphism (rs 2016163) in exon 1 was identified through sequencing 80 Chinese and 88 Caucasian women with premature ovarian failure. No additional single-nucleotide polymorphisms or mutations were found in exons encoding for NANOS3. Our findings suggest that mutations in NANOS3 exons are rare in both Chinese and Caucasian women with premature ovarian failure. [Copyright &y& Elsevier]

Objective: To search for WNT7A gene mutations in a cohort of 191 Chinese Han patients with müllerian duct abnormalities (MDAs). Design: Phenotypic and mutational study. Setting: University hospital. Patient(s): A total of 191 Chinese Han patients with MDAs and 192 healthy control individuals. Intervention(s): Genomic DNA extracted from blood samples, all coding regions amplified by polymerase chain reaction (PCR) then directly sequenced to screen variants. Main Outcome Measure(s): Not applicable. Result(s): The sequence analysis revealed one novel synonymous variant and three known single-nucleotide polymorphisms (SNPs). Conclusion(s): The results indicate that mutations in the coding sequence of WNT7A are not responsible for müllerian duct abnormalities in the Chinese population. [ABSTRACT FROM AUTHOR]

Objective: To investigate whether LHX1 gene mutations exist in Han Chinese patients with müllerian duct abnormalities (MDAs). Design: Mutation screening. Setting: University hospital. Patient(s): Ninety-six MDA patients and 105 control subjects from a Han Chinese population. The parents of the patients carrying the genetic variation were also screened. Intervention(s): Gene sequencing. Main Outcome Measure(s): Karyotype, LHX1 gene sequencing. Result(s): We found no significant mutation in coding regions of LHX1. However, there is a new rare polymorphism of LHX1 gene, c.1070–1081del, found in 1 out of 77 incomplete müllerian fusion patients and 1 out of 105 control individuals in the Han Chinese population (thus affecting ∼1% of Han Chinese). Conclusion(s): No causative perturbation was identified in the LHX1 gene. Mutations in the coding regions of LHX1 may not be a common genetic etiologic factor involved in Han Chinese MDA patients. [ABSTRACT FROM AUTHOR]

Objective: To investigate whether mutations in the minichromosome maintenance complex component 9 (MCM9) gene were present in 192 patients with sporadic premature ovarian insufficiency (POI) of Chinese descent.Design: Genetic and functional study.Setting: University-based reproductive medicine center.Patient(s): A total of 192 patients with sporadic POI and 192 control women with regular menstruation.Intervention(s): Sanger sequencing performed in 192 sporadic POI patients, and potential pathogenic variants were excluded in matched controls. Functional effects of mutations on MCM9 were explored based on etoposide-induced DNA damage response, and DNA repair capacity was evaluated by histone H2AX phosphorylation level.Main Outcome Measure(s): Sanger sequencing and functional characteristics.Result(s): Three novel heterozygous mutations in MCM9, c.C1423T (p.L475F), c.T2921C (p.L974S), and c.G3388A (p.A1130T), were identified in three POI patients separately, which were absent in 192 controls. Functional studies showed that the human embryonic kidney 293 (HEK293) cells overexpressing mutant MCM9 presented with diminished DNA repair capacity compared with wild type.Conclusion(s): This study identified novel mutations in MCM9 that are potentially causative for sporadic POI in Chinese women and further highlighted the role of DNA repair capacity in maintenance of ovarian function. [ABSTRACT FROM AUTHOR]

Insights into common genetic susceptibility between primary ovarian insufficiency (POI) and natural or early menopause have delivered an innovative way of assessing the genetic mechanisms involved in POI. PRIM1 plays a crucial role in DNA replication by synthesizing RNA primers for Okazaki fragments. It is closely associated with age at natural menopause, early menopause and POI in European women. In this study, we aimed to investigate whether mutations in PRIM1 contribute to POI in Chinese women. All exons and exon–intron boundaries of PRIM1 gene were sequenced in 192 Han Chinese women with non-syndromic POI. No plausible mutations were identified. The results suggest that the perturbations in PRIM1 gene are not a common explanation for POI in Chinese women. [ABSTRACT FROM AUTHOR]

Exonuclease 1 (EXO1) is required for both DNA repair and meiosis. Inactivation of EXO1 gene in mice leads to infertility. This study aimed to investigate whether variants in the EXO1 gene contribute to human premature ovarian failure (POF). The coding region of EXO1 was sequenced in 186 Han Chinese patients with non-syndromic POF. No plausible mutation was detected. The results suggest that mutations in the coding region of EXO1 may not be responsible for POF in Han Chinese women. [ABSTRACT FROM AUTHOR]

Objective To investigate the association between human empty spiracles homeobox 2 gene ( EMX2 ) and incomplete müllerian fusion (IMF). Design Case-control study. Setting University-based hospital. Patient(s) Cohort of 517 clinically well-characterized IMF cases and 563 control women. Intervention(s) None. Main Outcome Measure(s) In cases and control women, direct sequencing of EMX2 exons and further functional studies; for functional studies, wild-type and mutant EMX2 expression plasmids constructed; human embryonic kidney cells (HEK293FT) transfected with empty vector, wild-type EMX2, mutant EMX2, and 1:1 combination (wild-type/mutant plasmids) with additional functional studies performed to clarify the deleterious effect of the novel mutation detected. Result(s) A novel nonsense mutation p.E142X was detected in one woman with a didelphic uterus (1 of 517, 0.19%). The results of Western blot analysis confirmed that the mutation caused a truncated protein as predicted, and functional studies proved that it resulted in a dominant negative effect. Conclusion(s) The novel nonsense mutation we detected-EMX2, p.E142X- resulted in a dominant negative effect. The functional data were exemplified in HEK293FT cells. This reinforced the likelihood that EMX2 contributed to the pathophysiology of IMF. Although it is uncommon (0.19%), EMX2 is the first gene identified that if perturbed may cause isolated IMF. [ABSTRACT FROM AUTHOR]

Objective: To identify the potential FSHR mutation in a Chinese woman with primary ovarian insufficiency (POI).Design: Genetic and functional studies.Setting: University-based reproductive medicine center.Patient(s): A POI patient, her family members, and another 192 control women with regular menstruation.Intervention(s): Ovarian biopsy was performed in the patient. Sanger sequencing was carried out for the patient, her sister, and parents. The novel variant identified was further confirmed with the use of control subjects.Main Outcome Measure(s): Sanger sequencing and genotype analysis to identify the potential variant of the FSHR gene; hematoxylin and eosin staining of the ovarian section to observe the follicular development; Western blotting and immunofluorescence to detect FSH receptor (FSHR) expression; and cyclic adenosine monophosphate (cAMP) assay to monitor FSH-induced signaling.Result(s): Histologic examination of the ovaries in the patient revealed follicular development up to the early antral stage. Mutational screening and genotype analysis of the FSHR gene identified a novel homozygous mutation c.175C>T (p.R59X) in exon 2, which was inherited in the autosomal recessive mode from her heterozygous parents but was absent in her sister and the 192 control women. Functional studies demonstrated that in vitro the nonsense mutation caused the loss of full-length FSHR expression and that p.R59X mutant showed no response to FSH stimulation in the cAMP level.Conclusion(s): The mutation p.R59X in FSHR is causative for POI by means of arresting folliculogenesis. [ABSTRACT FROM AUTHOR]

Objective: To investigate whether mutations in the minichromosome maintenance complex component 8 (MCM8) gene were present in 192 patients with sporadic primary ovarian insufficiency (POI).Design: Retrospective case-control cohort study.Setting: University-based reproductive medicine center.Patient(s): A total of 192 patients with sporadic POI and 312 control women with regular menstruation (192 age-matched women and 120 women >45 years old).Intervention(s): Sanger sequencing was performed in patients with sporadic POI, and potentially pathogenic variants were confirmed in matched controls. DNA damage was induced by mitomycinC (MMC) treatment, and DNA repair capacity was evaluated by histone H2AX phosphorylation level.Main Outcome Measure(s): Sanger sequencing for MCM8 was performed in 192 patients with sporadic POI, and functional experiments were performed to explore the deleterious effects of mutations identified.Result(s): Two novel missense variants in MCM8, c. A950T (p. H317L), and c. A1802G (p. H601R), were identified in two patients with POI but absent in 312 controls (the upper 90% confidence limit for the proportion 2/192 is 2.24%). The HeLa cells overexpressing mutant p. H317L and p. H601R showed higher sensitivity to MMC compared with wild type. Furthermore, mutant p. H317L showed decreased repair capacity after MMC treatment with much more histone H2AX phosphorylation remaining after 2 hours of recovery.Conclusion(s): Our result suggests novel mutations p. H317L and p. H601R in the MCM8 gene are potentially causative for POI by dysfunctional DNA repair. [ABSTRACT FROM AUTHOR]