摘要：Objective To explore the specific methylation pattern changes based on the whole genome level of ovarian tissue in Uygur polycystic ovary syndrome (PCOS) patients.Methods From May 2014 to June 2016 in First People's Hospital of Kashgar Prefecture,a total of 60 cases PCOS ovarian tissues of Uygur PCOS patients (into PCOS group) and 60 cases of normal ovarian tissues of Uygur ovarian cysts patients (into control group) were obtained as research materials.The cytosine-phosphate-guanine (CpG) islands and promoter regions of whole genome of ovarian tissues in two groups were taken methylation scanning by methylated DNA immunoprecipitation-chip (MeDIP-chip)technology.Regions of MeDIP-chip results of peak scores ≥2 were taken as positive methylation regions to screening the methylation genes.The functional relevances of organism in which participated by specific hypermethylation genes with peak scores ≥5 were investigated by analysis of gene ontology (GO) and Pathway.The statistical methods of paired t test and chi-square test were used to compare the differences of ovarian tissue methylation spectrum between two groups.This study was in line with World Medical Association Declaration of Helsinki revised in 2013,and all participants were voluntary to participate in this study and signed the informed consents.Results ①The results of MeDIP-chip showed that the methylation rates of CpG islands and gene promoters in PCOS group were 12.9％ (3 633/28 226) and 3.8％ (687/18 028),respectively,and were 13.3％ (3 759/28 226) and 3.6％ (643/18 028) in control group,respectively.There were no significant differences between two groups of ovarian tissues in whole genome methylation levels and distributions of methylation fragments in genome regions (P＞ 0.05).② There were no significant differences between two groups of ovarian tissues in methylation levels in upstream regulatory regions and core promoter regions of genomic transcriptional regulatory regions (P＞0.05).The methylation level in gene coding regions of transcriptional regulatory regions of ovarian tissues in PCOS group was (3.14±0.21),which was lower than that in control group (3.96±0.23),and the difference was statistically significant (t=2.653,P=0.010).③There was significant difference between two groups of ovarian tissues whole genome in distribution of 3 types of hypermethylation promoters including high CpG promoters (HCP),intermediate CpG promoters (ICP) and low CpG promoters (LCP)(x2 =3.208,P=0.002),which were the highest HCP proportion (40.1％) in PCOS group and the lowest HCP proportion (13.3％) in control group.The HCP methylation level of ovarian tissues whole genome in PCOS group was (0.68±0.08),which was lower than that in control group (0.71±0.09),while the methylation levels of ICP and LCP were (0.74±0.13) and (0.76±0.11),and they were both higher than those in control group,which were (0.61±0.07) and (0.32±0.03),and all the differences were statistically significant (t=1.686,P=0.049;t=1.992,P=0.028;t=3.361,P=0.002).④The results of GO and Pathway analysis showed that the specific hypermethylation genes of Uygur PCOS patients' ovarian tissues were mainly participated in the progress of receptor binding,protein binding,hormone activity,transcriptional regulatory activity,transcription factor activity and DNA binding.Conclusions Compare with the Uygur female normal ovarian tissue,the Uygur PCOS patients ovarian tissue's methylation level in gene coding region was lower,the proportion of HCP was the highest with lower level of methylation,and the methylation level of ICP and LCP were both higher.All these specific methylation changes may response to the occurrence and development of PCOS in Uygur patients.%目的 在全基因组水平上,探讨维吾尔族多囊卵巢综合征(PCOS)患者卵巢组织的特异性甲基化谱改变.方法 选择2014年5月至2016年6月,于喀什地区第一人民医院进行诊治的60例维吾尔族PCOS患者的卵巢组织(PCOS组)及60例维吾尔族卵巢囊肿患者的正常卵巢组织(对照组)为研究对象.采用甲基化DNA免疫共沉淀结合芯片(MeDIP-chip)技术,对2组卵巢组织的基因组DNA进行全基因组胞嘧啶-磷酸-鸟嘌呤(CpG)岛和启动子区域甲基化扫描.将MeDIP-chip结果中,峰值得分≥2的区域,判定为阳性甲基化区域,以筛选甲基化基因;对PCOS卵巢组织峰值得分≥5的特异性高甲基化基因,进行基因本体(G())分析及Pathway分析,以探讨这些特异性高甲基化基因对机体相关功能的影响.对2组卵巢组织的甲基化谱差异,采用配对t检验及x 2检验进行统计学比较.本研究遵循的程序符合2013年修订的《世界医学协会赫尔辛基宣言》的要求,所有受试者均自愿参加本研究,并签署知情同意书.结果 ①MeDIP-chip结果显示:PCOS组全基因组CpG岛和启动子的甲基化率,分别为12.9％(3 633/28 226)和3.8％(687/18 028),对照组分别为13.3％(3 759/28 226)和3.6％(643/18 028).2组卵巢组织全基因组整体甲基化水平,以及甲基化片段在基因组各区的分布比较,差异均无统计学意义(P＞0.05).②2组卵巢组织全基因组转录调控区的上游调控区和核心启动子区域甲基化水平比较,差异均无统计学意义(P＞0.05).PCOS组转录调控区的基因编码区甲基化水平为(3.14±0.21),显著低于对照组的(3.96±0.23),并且差异有统计学意义(t=2.653,P=0.010).③2组卵巢组织全基因组高甲基化启动子的高-CpG含量启动子(HCP)、中-CpG含量启动子(ICP)及低-CpG含量启动子(LCP)的分布比较,差异有统计学意义(x2=3.208,P=0.002),其中PCOS组卵巢组织全基因组HCP比例最高(40.1％),对照组HCP比例则最低(13.3％).PCOS组卵巢组织全基因组HCP甲基化水平为(0.68±0.08),低于对照组的(0.71±0.09),而ICP及LCP甲基化水平分别为(0.74±0.13)和(0.76±0.11),则高于对照组的(0.61±0.07)和(0.32±0.03),并且差异均有统计学意义(t=1.686,P=0.049;t=1.992,P=0.028;t=3.361,P=0.002).④GO分析及Pathway分析结果显示:维吾尔族PCOS患者卵巢组织的特异性高甲基化基因,主要参与受体结合、蛋白质结合、激素活性、转录调节活性、转录因子活性和DNA结合等过程.结论 维吾尔族PCOS患者卵巢组织的基因编码区甲基化水平,较正常卵巢组织低;全基因组高甲基化启动子的HCP比例最高,但是甲基化水平较正常卵巢组织的低,ICP及LCP的甲基化水平,较正常卵巢组织的高.这些特异性甲基化改变,可能与维吾尔族女性的PCOS发生、发展有关系.
