The Association Study Of YAP1Gene In The Pathogenesis Of Polycystic Ovary Syndrome

PART Ⅰ Association study of YAP1and PCOSBackground:Polycystic ovary syndrome (PCOS) is a highly complicated and heterogeneous endocrinopathy in women. It is characterized by the presence of the following features:menstrual cycle disturbance (chronic oligo-ovulation or anovulation), androgen excess (biochemical or clinical hyperandrogenism) and polycystic ovaries. Besides of the most common cause of anovulatory infertility, PCOS is also associated with endocrine-metabolic disorders such as obesity and insulin resistance. The long-term complications of PCOS such as atherosclerosis, insulin resistance, type2diabetes mellitus (T2DM) and endometrial cancer increase as long as age. In the previously conducted genome-wide association study on PCOS, following up studies among single nucleotide polymorphisms (SNPs) with P value less than10E-6were replicated and three susceptibility loci have been confirmed. However, loci of P value around10E-5are still intriguing and may also be the potential risks for PCOS. YAP1(yeast-associated protein1) is a pivotal transcriptional co-activator of Salvador-Warts-Hippo (SWH) pathway to control organ volume and to play an important role in tumors. In mammals, the YAP1is wildly expressed and its function domains are conservative and have been shown to bind to a number of signaling proteins. After reviewing the data of GWAS, SNPs in YAP1were selected for the enlarged replication study.Objective:Case-control association study of YAP1and PCOS has been applied to discover the relationship. As the susceptibility of YAP1gene to PCOS has been identified, the undergoing families-based study would help to eliminate the confounding factors of population stratification and confirm the susceptibility.Methods and materials:This study has been consisted of two parts. The first was case control association study, consisting of a total of2252Chinese Han women in which an independent set of1115PCOS patients and1137controls were recruited.. All subjects were from center for reproductive medicine, Shandong Provincial Hospital.SNPs rs11225138, rs11225161and rs11225166from YAP1were selected for the replication study. Real-time quantitative polymerase chain reaction was applied for genotyping by TaqMan-MGB probe assay. And the data were meta-analysis with GWAS data. As YAP1has been identified to be a new susceptibility gene for PCOS in Han Chinese women, family-based association study was performed to assess the over-transmission of risk alleles in YAP1SNPs.276unrelated PCOS patients have been recruited as the probands. And the nuclear family consisted of proband and her father and mother has been included in this family-based association study. TaqMan-MGB probe assay was applied for genotyping rs11225138and rs11225161by real-time quantitative polymerase chain reaction. And the transmitted disequilibrium test (TDT) has been applied for data analysis。Results:In the first case-control study,(1) The frequency of minor allele A of SNP rs11225161in PCOS is statistically higher than the controls (P=1.4E-4, OR=1.32). Meta analysis showed that the allele frequency of rs11225161(A/G) was significantly different between PCOS and controls at a genome-wide association significance (Pmeta=3.98E-09). After the adjustment of age and BMI, the difference was still significant (P=1.53E-04).(2) There was significant difference between the frequency of minor allele G in rs11225138between PCOS and the controls (P=6.2E-3, OR=1.21), and the difference has kept statistically different after the meta-analysis (P=1.16E-04, OR=1.22). But After the adjustment of age and BMI, the difference did not existed (P=0.09).(3) there was no difference of frequency of minor allele G in rs11225166between PCOS and controls (P=0.18). But after data combined with the GWAS, it was statistically different (P=0.0026)。In the second families based analysis, the genotypes of rs11225138and rs11225161were consistent with the Hardy Weinberg equilibrium. After the TDT, the risk allele G in rs11225138was over transmitted in families (Transmitted:NOT-Transmitted=52:119,χ2=26.25, P=2.99E-07). And the G allele in SNP rs11225161was over transmitted in families(Transmitted:NOT-Transmitted=40:100, χ2=25.71, P=3.96E-07). And the result did not differ after the exclusion of mother who has had irregular menstrual cycles.Conclusion:(1) YAP1appears to be a new susceptibility gene for PCOS in Han Chinese women. And the rs11225138and rs11225161were association SNPs.(2) In PCOS nuclear families, there were transmitted disequilibrium in rs11225138and rs11225161, which implies the association of YAP1and PCOS were independent of population stratification. Part Ⅱ Association study of susceptibility SNP of PCOS in YAP1and controlled ovulation outcomeBackground:The majority of PCOS patients with fertility problems can get successful pregnancy after weight control or clomiphene citrate ovulation. However, some of them would ask assisted reproductive technology for help. Most of the patients who undergo the controlled ovarian stimulation would response to a relatively low dose of gonadotropins (Gn) while get much more follicles for the next procedures. And this response to ovarian stimulation may due to the pathogenesis of PCOS per se.Objective:In order to further explore the mechanism of ovarian response and the function of YAP1in PCOS pathogenesis, we applied the SNP rs1894116to study the relationship between its genotype and controlled ovulation outcome, as well as the outcome of pregnancy.Methods and materials:As a retrospective study, subjects were from the center for reproductive medicine, Shandong Provincial Hospital. They were infertility and came for IVF/ICSI applying long-protocol, in which there were PCOS cases440and control subjects2254. And the relationship between genotype of rs1894116and controlled ovulation outcome has been studied.Results:1) In PCOS cases, the initial dose of Gn (153.37±40.29IU) in PCOS groups was statistically lower than the controls, as well as Gn duration days and total of Gn (1945.81±870.37IU), but the number of oocytes (14.6±6.3) and the cleavage oocytes were higher (10.7±5.9). there were no differences in the rate of pregnancy and rate of abortion between the two groups.2) The dominant genotype model has been applied for the analysis of controlled ovulation outcome. The initial dose and total dose of Gn did not differ in the risk allele group (AA+AT) and protective allele group (TT). And there was no significant difference in the number of oocytes and cleavage oocytes between two groups. But the E2in HCG administration day was higher in risk allele group (6073.85±2986.91) than the protective allele group (5364.72±2766.58).3) In the controls, the same genetic model were applied for data analysis. In the risk allele groups, the E2in HCG administration day (3829.52±2357.78) were statistically higher than the protective groups (3641.75±2355.11). And the samiliar difference has been found in basal E2(41.10±17.21vs.39.10±15.95).4) In the analysis of neonates weight, the newborn weight of risk allele genotyped mother has been statistically higher than of the protective allele genotyped (3.48±0.66kg vs.3.31±0.79, P=0.03).Conclusion:The susceptibility SNPrs1894116of PCOS in YAP1has not related with the dose of Gn and the ovary response to PCOS. But the SNP rs1894116is associated with the E2secretion in controlled ovarian stimulation. And the neonate weight of mother of risk allele would be higher. Part Ⅲ Sequence YAP1in PCOS and the preliminary expression of YAP1in ovaryBackground:In previous studied, the replicated results of GWAS has identified the susceptibility gene YAP] to PCOS. And the results have been replicated in the second GWAS. However, a lot of more work need to explain the functional role of the susceptibility gene confirmed by GWAS or case-control association study. And the sequencing of exons of YAP1and preliminary position YAP1in ovary would facilitate the functional study.Objective:In order to clarify the function of YAP1in etiology of PCOS, the sequencing of all exons and un-translated regions (UTR) of YAP1in PCOS patients. And the expression of YAP1in target organ of PCOS, such as ovary needs confirmed.Material and methods:1) a total of192PCOS patients has been selected for the YAP1mutation screening. Direct sequence after the amplification of regular PCR has been applied for the sequencing of exons and (UTR).2) For the detection of YAP] expression in the reproductive system, we chose2week old mice,8weeks old mice and4months aged mice to sacrifice for the frozen section of ovary. Mouse ovary slides were then stained by immune-histo-chemical (IHC) of YAP1.3) Puncture granule cell specimens after oocyte retrieval of20cases of PCOS patients and12normal control in addition undergoing IVF/ICSI were collected. The mRNA of granulose cells has been extracted and real-time PCR has been applied for comparison of mRNA expression.Results:1) No pathogenic mutation of YAP] has been detected in PCOS patients.2) The IHC staining of YAP] in granular cells has been detected in2week old mice,8weeks old mice and4months aged mice. But the IHC-YAP1can be only found in oocytes of2week old mice. And it is shallow of granulosa lutein cells staining for YAP] comparing the granulosa cells.3) YAP1visible band could be found in gel electrophoresis. But there were no difference between the mRNA expression in controls and PCOS. Conclusion:Mutation or deletion in exons and UTR of YAP1do not devote to the pathogenesis of PCOS. The expression of YAP1in mice ovary has been found. And there may be timed expression. YAP1mRNA expression also has been detected in human granulose cells. Part Ⅳ Methylation and mRNA expression of YAP1in peripheral bloodBackground:The complex interactions between genetic background and environmental factor exist in the pathogenesis of PCOS. And the PCOS animal model have implied the fetal origin of the disorder. The possible explanation is the epigenetic traits of offspring such as loss of gene imprint and modification of DNA methylation would keep changing under influence of maternal intrauterine environment during the development of fetus. The changes of epigenetic would reflect the effect of environmental factor and the epigenetic itself would contribute to genes regulation.Objective:In order to explore the epigenetic regulation of YAP1function in PCOS, the methylation of YAP1has been studied in peripheral blood cells. Meanwhile, mRNA expression of YAP I in peripheral blood cells has been studied to valid the regulation function association of methylation.Method and materials:1) PCOS patients and age and BMI matched controls have been selected for DNA extraction. And then, the DNA has been translated by Bisulfite. PCR of Bisulfite conversion DNA was applied to achieve target sequence. The product was purified by gel and transfected into carriers after connected with plasmid. After incubation of carrier, the monoclone had been picked and further incubated. And then the plasmid has been extracted and sequenced to explore the methylation.2) Fresh blood samples from30PCOS and30control has been collected for the mRNA extraction and expression.Results:1) both in PCOS cases and controls, the target sequence of YAP1has been the demethylation. But the total methylation of controls (1.4%) was higher than the PCOS (0.7%).2) It is higher of the mRNA expression of controls, comparing with PCOS groups. But the difference was not statistically significant.Conclusion:It is mostly demethylation of YAP1in peripheral blood cells. And the demethylation of YAP1is in consistent with the expression characteristic of mRNA. The mathylation rate of YAP1is lower in PCOS than the controls, which maybe another trait of PCOS.