Guangyu Li

A Genome-wide Association Study Reveals that Variants within the HLA Region Are Associated with Risk for Nonobstructive Azoospermia

A genome-wide association study of Han Chinese subjects was conducted to identify genetic susceptibility loci for nonobstructive azoospermia (NOA). In the discovery stage, 802 azoospermia cases and 1,863 controls were screened for genetic variants in the genome. Promising SNPs were subsequently confirmed in two independent sets of subjects: 818 azoospermia cases and 1,755 controls from northern China, and 606 azoospermia cases and 958 controls from central and southern China. We detected variants at human leukocyte antigen (HLA) regions that were independently associated with NOA (HLA-DRA, rs3129878, p(combine) = 3.70xa0× 10(-16), odds ratio [OR]xa0= 1.37; C6orf10 and BTNL2, rs498422, p(combine) = 2.43xa0× 10(-12), OR = 1.42). These findings provide additional insight into the pathogenesis of NOA.

Identification of YAP1 as a novel susceptibility gene for polycystic ovary syndrome

Background A previously reported genome-wide association study (GWAS) of polycystic ovary syndrome (PCOS) in Han Chinese found that several loci of p value around 10e-5 warrant investigation. Replication of the GWAS was applied in this study to determine whether gene YAP1 (yeast associated protein 1) is associated with PCOS. Methods An independent set of 1115 PCOS patients and 1137 controls were recruited; single nucleotide polymorphisms (SNPs) rs11225138, rs11225161, and rs11225166 from YAP1 were selected for the replication study. Real-time quantitative PCR was applied for genotyping by TaqMan-MGB probe assay. Results Meta-analysis showed that the allele frequency of rs11225161 (A/G) was significantly different between PCOS and controls at a GWA significance (Pmeta =3.98e-09). Genotype–phenotype correlation study found 30u2005min and 60u2005min glucose of the oral glucose tolerance test were higher in PCOS patients with rs11225161 risk allele A. The G allele of SNP rs11225138 (G/C) was a further risk factor for higher luteinising hormone level in PCOS patients (p=0.041). Conclusion YAP1 appears to be a new susceptibility gene for PCOS in Han Chinese women.

Hypomethylation of the LH/Choriogonadotropin Receptor Promoter Region Is a Potential Mechanism Underlying Susceptibility to Polycystic Ovary Syndrome

Our previous genome-wide association study identified LH/choriogonadotropin receptor (LHCGR) as a susceptibility gene for polycystic ovary syndrome (PCOS). The objective of this study was to determine whether the genetic or epigenetic components associated with LHCGR participate in the pathogenesis of PCOS. The exons and flanking regions of LHCGR were sequenced from 192 women with PCOS, and no novel somatic mutations were identified. In addition, the methylation statuses of 6 cytosine-phosphate-guanine (CpG) sites in the promoter region of LHCGR were measured by pyrosequencing using peripheral blood cells from 85 women with PCOS and 88 control women. We identified 2 hypomethylated sites, CpG -174 (corrected P = .018) and -111 (corrected P = .006). Bisulfite sequencing then was performed to replicate these findings and detect additional CpG sites in the promoter. CpG +17 was significantly hypomethylated in women with PCOS (corrected P = .02). Methylation statuses were further evaluated using granulosa cells (GCs), and the region described was hypomethylated as a whole (P = .004) with 8 significantly hypomethylated sites (CpG -174, -148, -61, -43, -8, +10, +17, and +20). Transcription of LHCGR was elevated in women with PCOS compared with that in control women (P < .01). These findings were consistent with the decreased LHCGR methylation status associated with PCOS. The tendency of LHCGR to be hypomethylated across different tissues and its corresponding expression level suggest that hypomethylation of LHCGR is a potential mechanism underlying susceptibility to PCOS. Further studies are needed to evaluate whether a causal relationship exists between LHCGR methylation status and PCOS.

Mutations in WNT4 are not responsible for Müllerian duct abnormalities in Chinese women.

The WNT4 gene plays a crucial role in sexual differentiation and female genital tract development. This study screened WNT4 for mutation in 189 Chinese women with Müllerian duct abnormalities (10 Mayer-Rokitansky-Küster-Hauser syndrome, five Müllerian aplasia and 174 incomplete Müllerian fusion) and detected no perturbation that would indicate a major role for WNT4. Only one novel synonymous mutation (c.1091G>A) in exon 5 and one known single-nucleotide polymorphism (rs16826648) in exon 2 were found. The results suggest that WNT4 might not contribute to the aetiology of Müllerian duct abnormalities in Chinese women.

CSB-PGBD3 Mutations Cause Premature Ovarian Failure

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.

Novel NR5A1 missense mutation in premature ovarian failure: detection in han chinese indicates causation in different ethnic groups.

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.

Mutations in MSH5 in primary ovarian insufficiency

Abstract Primary ovarian insufficiency (POI) is a genetically heterogeneous disorder that occurs in familial or sporadic fashion. Through whole exome sequencing in a Chinese pedigree with POI, we identified a novel homozygous missense mutation (ENST00000375755: c.1459Gu2009>u2009T, p.D487Y) in the MSH5 gene in two sisters with POI. The homologous mutation in mice resulted in atrophic ovaries without oocytes, and in vitro functional study revealed that mutant MSH5 impaired DNA homologous recombination repair. From sanger sequencing of MSH5 in 200 sporadic POI patients, we identified three heterozygous mutations (ENST00000375755: c.1057Cu2009>u2009A, p.L353M; c.1459Gu2009>u2009T, p.D487Y and c.2107 Au2009>u2009G, p.I703V). Considering the heterozygous p.D487Y carrier in the POI pedigree was fertile, the causality of the three heterozygous mutations in POI need more evidence. Our studies confirmed that perturbation of genes involved in DNA damage repair could lead to non-syndromic POI. The underlying mechanism-inability to repair DNA damage-will receive increasing attention with respect to POI.

Polycystic ovary syndrome susceptibility single nucleotide polymorphisms in women with a single PCOS clinical feature

STUDY QUESTIONnWhat is the direct genetic contribution of the polycystic ovary syndrome (PCOS) susceptibility single nucleotide polymorphisms (SNPs), identified by previous genome-wide association studies (GWAS) to the definitive clinical features of the syndrome?nnnSUMMARY ANSWERnEach single PCOS clinical feature had a specific genetic association, and rs4385527 in the chromosome 9 open reading frame 3 (C9orf3) conferred a particular risk to the three defined PCOS clinical features in this study, which suggested its fundamental role in the etiology of PCOS.nnnWHAT IS KNOWN ALREADYnPCOS is a heterogeneous disorder characterized by anovulation (OA), hyperandrogenism (HA) and polycystic ovary morphology (PCOM). Two previous GWAS in China have identified 15 independent susceptibility SNPs related to PCOS (PCOS-SNPs). However, little is known about the candidate gene of each clinical feature.nnnSTUDY DESIGN, SIZE, DURATIONnCase-control study. Three independent groups of women were recruited from 2010 to 2012: 746 subjects with OA only, 278 subjects with HA only and 536 subjects with PCOM only. A total of 1790 healthy women with none of the above pathological characteristics were also enrolled as control subjects during the same time period.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnAll participants were women of reproductive age. Genotype and allelic frequencies of 15 PCOS-SNPs were determined in all subjects using direct sequencing and Sequenom Arrays. The allelic frequencies of each case group were compared with the controls.nnnMAIN RESULTS AND THE ROLE OF CHANCEnAfter adjustment for age and BMI, variants in luteinizing hormone/choriogonadotropin receptor (LHCGR) (rs13405728), C9orf3 (rs4385527) and insulin receptor gene (INSR) (rs2059807) were strongly associated with OA (Padjust < 0.01, <0.001 and <0.05, respectively); rs4385527 in C9orf3 was strongly associated with HA (Padjust< 0.001); variants in the thyroid adenoma associated gene (THADA) (rs13429458 and rs12478601), DENN/MADD domain containing 1A (DENND1A)(rs10818854), and C9orf3 (rs4385527) were significantly associated with PCOM (Padjust < 0.01, <0.001, <0.05 and <0.001, respectively).nnnLIMITATIONS, REASONS FOR CAUTIONnThe sample size of some case groups was relatively small, which therefore limited the statistical power of the analysis to a certain extent.nnnWIDER IMPLICATIONS OF THE FINDINGSnThe present study indicates a potential common genetic basis of three PCOS clinical features. Other specific associated genes may play a synergistic role, leading to heterogeneous pathophysiological changes. Additionally, the increased frequency of PCOS-risk alleles in women with single PCOS clinical features suggests that these subjects have an elevated risk of developing the syndrome, although they cannot be currently diagnosed.nnnSTUDY FUNDING/COMPETING INTERESTSnThis research was supported by the National Basic Research Program of China (973 Program) (2012CB944700, 2011CB944502), the National Key Technology Research and Development Program(2011BAI17B00), the National Natural Science Foundation of China (81430029, 81201441, 81490743, 31371453), the Scientific Research Foundation of Shandong Province of Outstanding Young Scientist (2012BSE27089) and the Fundamental Research Funds of Shandong University(2014GN025). There were no competing interests.

FMR1 premutation is an uncommon explanation for premature ovarian failure in Han Chinese.

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%, Pu200a=u200a0.343). 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.

WNT9B in 542 Chinese women with Müllerian duct abnormalities: mutation analysis

The WNT9B gene is a common organizing signal regulating different segments of the mammalian urogenital system and plays a primary role in the development of the female reproductive tract. The aim of the present work was to examine the presence of WNT mutations in a population of women with Müllerian duct abnormalities (MDA) in order to elucidate whether mutations in WNT9B are causative for MDA in Chinese women. Initially, 191 Chinese MDA patients and 192 healthy individuals (controls) were recruited. All coding regions were amplified by PCR and sequenced to search for variants. To verify the initial results, the numbers of patients and ethnic-matched controls were expanded to 542 and 563, respectively. One known single-nucleotide polymorphism and four novel variants were identified in the first stage: two were synonymous; the other two were rare nonsynonymous novel variants (c.566G>A (p.Arg189Gln) and c.773G>A (p.Arg258His)). None of the four novel variants was found in controls. In the second stage, both novel nonsynonymous variants were detected in MDA cases and controls. The results indicate that mutations in the coding sequence of WNT9B are not responsible for MDA in the Chinese population.