Analysis Of Aberrant DNA Methylation And Mechanism In Polycystic Ovary Syndrome

Background:In1935, American gynecologists Stein and Leventhal describe a series of symptoms, including amenorrhea, infertility, hirsutism, obesity and enlarged polycystic ovary. They describe these symptoms as Stein-Leventhal syndrome, which is also known as polycystic ovary syndrome (PCOS) nowadays. Along with the progress of modern medical research, our understanding of this ancient disease has been largely updated. Now, we have realized that PCOS is not just an reproductive endocrinopathy endangers women at reproductive age, PCOS also has significant impact on metabolism, cardiovascular system, skin and bone. PCOS jeopardizes a woman’s health throughout her entire lifespan.Polycystic ovary syndrome (PCOS) has a prevalence of5.6%among Chinese women of reproductive age and is regarded as the most common endocrinopathy in this age group. It is of great importance to decode the issues in treatment and management of PCOS.The etiology and pathogenesis of this syndrome remain unclear. The current etiology and PCOS can be mainly divided into genetic factors and non-genetic factors. Genetically,with the emergence of genome-wide association studies (GWASs), remarkable progress has been achieved in genetic studies of PCOS. However, those non-genetic factors were seldom researched. Epigenetic information in mammals can be transmitted in multiple forms, with methylation being the most common and extensively discussed. Methylation is the conversion of cytosine into5-methycytosine, which has been described as the fifth base in DNA. In vertebrates, this phenomenon typically occurs at CpG sites. Normal cellular function relies on the maintenance of epigenomic homeostasis; this is evidenced by an abundance of reported associations between epigenomic perturbations and human diseases, notably cancer and complex diseases. This part of research was aimed to investigate the candidate genes discovered by GWAS so that we can achieve preliminary knowledge about methylation status in women with PCOS.Target:The first PCOS GWAS identified luteinizing several loci as susceptibility genes for PCOS among the Han Chinese. We choose thyroid adenoma associated (THADA), luteinizing hormone/choriogonadotropin receptor (LHCGR) and thymocyte selection-associated high mobility group box (TOX3) as candidate genes for methylation status analysis of PCOS. Genome DNA was extracted from peripheral blood and DNA methylation level was measured by bisulfite sequencing.Method:Peripheral blood was collected from30women with PCOS and30age-matched healthy controls. DNA methylation level of THADA, LHCGR and TOX3was measured by bisulfite sequencing.Results:Methylation level of each CpG sites of the promoter region of LHCGR of PCOS subjects was largely lower than controls, but the overall methylation difference did not achieve statistical significance. Analysis of single CpG site showed that in PCOS subjects methylation level of CpG-141and+17were significantly lower (CpG-141:10.89%Vs.18.31%, P=0.023; CpG+17:2.02%Vs.8.45%, P=0.002). After FDR correction, the statistical significance of CpG+17remained. We did not discover methylation level disruption in TOX3and THADA among women with PCOS.Conclusion: After screening methylation level of the three PCOS candidate genes, aberrant methylation of LHCGR which is an essential gene in female reproduction was found. This finding paved the way to the theory of methylation disorder as the etiology of PCOS. Background:In the first part, we screened methylation level of the promoter regions of three PCOS candidate genes. And we found the promoter region of LHCGR was hypomethylated in women with PCOS. LHCGR, which is the receptor of luteinizing hormone and human chorionic gonadotropin, is mainly expressed in ovary and testicle. The activation of LHCGR plays a pivotal role in the hormonal function and folliculogenesis in human reproduction. Coincidently, LHCGR is demethylated in the ovarian tissue of dehydroepiandrosterone (DHEA)-induced PCOS mouse models. Previous research also has reported that the transcription of LHCGR is modulated by the methylation status of its promoter cytosine-phosphate-guanine sites. Because in different tissues or cells types, methylation level of a certain gene is different, In order to prove this hypothesis, it is not enough to obtain the data of peripheral blood only. Luteinized granulosa cells were collected from patients undergoing in vitro fertilization-embryo transfer, and methylation of LHCGR were detected in these cells. Also, expression of LHCGR was also measured. On the other hand, sporadic reports have indicated that activating mutations in affected women elicit hyperandrogenism, which is a key feature of PCOS. To ascertain all genetic variation and identify candidate causal variants, we sequenced the untranslated regions (UTRs), promoter regions, exons, and exon-intron boundaries of LHCGR in a cohort of Chinese women with PCOS.Objective:In the first part, we found the promoter region of LHCGR was hypomethylated in women with PCOS. In order to further investigate this phenomenon in another cell types,luteinized granulosa cells were collected from patients undergoing in vitro fertilization-embryo transfer, and methylation of LHCGR were detected in these cells. Also, expression of LHCGR was also measured. On the other hand, to ascertain all genetic variation and identify candidate causal variants, we sequenced the untranslated regions (UTRs), promoter regions, exons, and exon-intron boundaries of LHCGR in a cohort of Chinese women with PCOS.Methods:Luteinized granulosa cells were collected by the gradient centrifugation methods, of which12samples were from women with PCOS and12samples were from the age controlled healthy controls. The methylation level of LHCGR promoter region was sequenced by bisulfite sequencing. Real-time qPCR and western blotting were used in the measurement of LHCGR expression. We also sequenced the untranslated regions (UTRs), promoter regions, exons, and exon-intron boundaries of LHCGR in a cohort of Chinese women with PCOS.Results:Methylation statuses were further evaluated using granulosa cells (GCs), and the described region was hypomethylated as a whole (P=0.004) with eight significantly hypomethylated sites (CpG-174:14.00%vs.19.41%, corrected P=0.048; CpG-148:9.60vs.15.19%, corrected P=0.048; CpG-61:5.60%vs.10.13%, corrected P=0.048; CpG-43:5.60%vs.10.13%, corrected P=0.048; CpG-8:9.60%vs.14.77%, corrected P=0.046; CpG+10:6.00%vs.10.13%, corrected P=0.047; CpG+17:4.80%vs.10.13%, corrected P=0.050; CpG+20:4.80%vs.10.13%, corrected P=0.050). Expression of LHCGR was elevated in women with PCOS, compared with controls (P<0.01). These findings were consistent with the decreased LHCGR methylation status associated with PCOS. The exons and flanking regions of LHCGR were sequenced from192women with PCOS, and no novel somatic mutations were identified (except for rs2293275, rs11125179å'Œrs10176989).Conclusion:As an important candidate gene of PCOS, the mutations of LHCGR were not found in a large cohort of PCOS patients. A tendency toward LHCGR hypomethylation across different tissues and a correspondingly elevated LHCGR transcription level in PCOS cases, compared with controls. Hypomethylation of LHCGR is highly relevant, or even central, to the etiology of anovulation in PCOS. Our findings highlight the importance of epigenetic studies of PCOS. Background:In the first two parts, we have demonstrated hypomethylation of the promoter region of LHCGR in both peripheral blood and granulosa cells among women with PCOS. Hyper-expression of LHCGR was also elevated in granulosa cells in women with PCOS, compared with controls (P<0.01). The hyper-expression of LHCGRwere consistent with the decreased LHCGR methylation status associated with PCOS. In part III, the mechanism of LHCGR methylation level disorder will be researched. Previous genome wide association study has demonstrated rs13405728which is located in intronic region of LHCGR reached genomic statisticallevel. In the first two parts, because of the limited sample size and methods, we cannot evaluate the correlation between rs13405728and methylation level of LHCGR. In this part, qyrosequencing which is a more efficient method to measure methylation level was employed and more samples were enrolled. We also consider another the relationship between methylation level and another SNP, rs4073366, which is located in the5’UTR region of LHCGR. On the other hand, hormonal factors are also possible mechanism underlying LHCGR methylation disorder. Hyperandrogenemia is a key feature of PCOS. High circular LH level is also an essential characteristic of PCOS and included in the PCOS diagnosticcriteria of Japan. In the part of research, we will cultured granulosa cells from21mouse in culture media supplemented with different concentration of testosterone and LH, in order to observe if testosterone or LH has their effect on LHCGR methylation.Target:In this part, we will discuss the influence of genetic and hormonal factors on the methylation level of LHCGR. Firstly, we will sequence the genotype of rs13405728and rs4073366and measure methylation level of LHCGR by qyrosequencing in order to identify the possible allele specific methylation pattern. On the other hand, by culturing mouse granulosa cells in media supplemented with testosterone and luteinizing hormone, the effect of the two hormones on LHCGR methylation level will be investigated.Methods:Using the data and samples from our genome-wide association study, according to the genotype of rsl3405728,90PCOS samples and90healthy controls were chosen. Methylation level of6CpG sites of LHCGR promoter region was measured by qyrosequencing. rs4073366as also genotyped by sequencing. On the other hand, by culturing mouse granulosa cells in media supplemented with testosterone and luteinizing hormone, the effect of the two hormones on LHCGR methylation level will be investigated.Results:In both PCOS samples and controls, the effect of genotype of rsl3405728on the methylation level of the promoter region of LHCGR was similar. The AA genotype could lower the methylation level, while the GG genotype was associated higher the methylation level. The allele specific methylation effect achieved statistical level (P<0.05). However, this allele specific methylation effect was not observed in rs4073366. Physical testosterone level (10-9M) could maintain methylation level of LHCGR in mouse granulosa cells, and high testosterone level could demethylate the promoter region of LHCGR.Conclusion:rsl3405728presented its allele specific methylation effect on the methylation level of LHCGR, but the phenomenon was not observed in rs4073366. High level of testosterone could result in demethylation of LHCGR. Genetic and hormonal factors both have their effects on methylation level of LHCGR. Background:Polycystic ovary syndrome (PCOS) is the most common endocrinopathy affecting6-8%women of reproductive age. The pathophysiology of PCOS is still shrouded in mystery. Genetic factors of PCOS have been the subject of intense investigation during the past decade. Though dozens of susceptibility loci have been identified and successfully replicated by recent genome wide association studies, these genetic findings are not sufficient to explain this highly heterogeneous and complex disorder. Currently, it is proposed that the combination and interaction of genetic, epigenetic and environmental factors contributes to the etiology of PCOS.Normal cellular function relies on the maintenance of epigenomic homeostasis, which is further highlighted by an increasing number of reported associations between epigenomic perturbations and human diseases, notably cancer and complex diseases. When it comes to PCOS, increasing interest is now emerging to explore the uncharted territory beyond genetics. Sporadic reports indicated associations between methylation disruption of candidate genes (LMNA, follistatin [FST] and androgen recptor [AR]) and PCOS. In a pilot study, no difference was found in total methylation of peripheral blood leukocyte DNA in PCOS patients versus matched controls. An epigenome-wide association study is desperately needed to generate a more comprehensive, unbiased, and discovery-driven data to unveil the epigenetic bases of PCOS.Objective:In the current study, using Infinium HumanMethylation450Beadchip (450K; Illumina Inc.), which measures CpG methylation at>470,000CpGs and covers99%RefSeq genes, we evaluated the relationship between DNA methylation disruption and PCOS in300PCOS patient blood samples and300matched controls.Methods:Three hundred PCOS blood samples300hundred age-matched controls were used for array-based methylation assays. Genomic DNA was abstracted The300samples and the300controls were randomly allocated into3PCOS groups and3control groups respectively. Equivalent amounts of DNA from each100PCOS participants or each100controls were assembled in DNA pools.A bisulfite conversion using the DNA pools was performed. Epigenome-wide methylation of the DNA pools was assessed using the Illumina HumanMethylation450BeadChip under contract by Shanghai biochip cooperation using the protocol specified by the manufacturer and the contractor. Methylation level of target CpG sites (IGF1R, UGR2b17, TNFa) were measured by bisulfite sequencing (BSP) using ten PCOS subjects and10age controlled participants. A|DiffScore|of≥20(equivalent to P<0.01) was considered statistically significant for DNA methylation array. Annotation and pathway enrichment analysis of the differently methylated genes were performed using an integrated online bioinformatics resource, Database for Annotation, Visualization and Integrated Discovery (DAVID).Results:The Illumina HumanMethylation450BeadChip covers over450,000CpG sites. We applied a stringent selection test (|DiffScore|of≥20[equivalent to P<0.01]), determining375differently methylated CpG sites, of which110sites (29.3%) were hypomethylated and265sites were (70.7%) hypermthylated. Notably, a large portion of the differently methylated CpG sites are located in CpG islands, shores and shelves which are of great importance in gene transcription control. The375differently methylated CpG sites covers314genes.Conclusion:Epigenome-wide scan reveals extensive DNA methylation change in polycystic ovary syndrome, which indicates its possible role in the pathogenesis of PCOS.