妊娠期糖尿病孕妇大网膜下脂肪组织全基因组DNA甲基化研究

Whole Genome DNA Methylation in the Visceral Omental Adipose Tissue of Patients with Gestational Diabetes Mellitus

背景大网膜下脂肪组织与妊娠期糖尿病(GDM)密切相关,目前,关于GDM孕妇大网膜下脂肪组织全基因组DNA甲基化研究较少。目的研究GDM孕妇和无GDM孕妇大网膜下脂肪组织全基因组DNA甲基化差异,为寻找GDM可能的致病基因提供线索。方法选取2012年1月—2014年5月于昆明医科大学第一附属医院妇产科门诊定期产前检查并入院分娩的GDM孕妇3例为病例组,同期健康孕妇3例为对照组。剖宫产术中剥离大网膜下脂肪组织,提取全基因组DNA。经变性和扩增后酶切扩增产物,将DNA片段与Illumina Methylation Bead Chip芯片进行杂交。根据Methylation Analysis Algorithms生成各样本每个位点的甲基化水平,经过偏差校正和位点过滤,得到甲基化水平。采用DAVID数据库对具有显著性差异的甲基化基因进行Gene Ontology(GO)和Kyoto Encyclopedia of Genes and Genomes(KEGG)信号通路分析。另分别选取同期GDM孕妇和无GDM孕妇各5例,验证候选基因启动子区域甲基化水平差异。结果病例组1 298个基因低甲基化,1 570个基因高甲基化,其中TMEM195、TCF7L2、IGF1、IGF1R甲基化可能与血糖调控密切相关,DGKG、NRXN3甲基化可能与体质指数(BMI)密切相关。GO功能分析显示,低甲基化基因参与的生物学过程主要是级联反应的活性调节、细胞凋亡调节及蛋白转运等,细胞成分主要为细胞外基质;高甲基化基因参与的生物学过程主要是抗原处理和呈递、主要组织相容复合体(MHC)Ⅱ参与的抗原处理和呈递,细胞成分主要为肌动骨架蛋白、MHC等。KEGG信号通路分析中,富集数>20.000的通路有抗原处理和呈递(富集数为23.142)、过氧化物酶体增殖物激活受体(PPAR)信号通路(富集数为22.068)。分别选取参与抗原处理和呈递、PPAR信号通路中的甲基化差异基因人类白细胞抗原G(HLA-G)、PPARGC1A进行验证。GDM孕妇大网膜下脂肪组织HLA-G启动子区域甲基化水平高于无GDM孕妇(t=4.968,P=0.001)。两者大网膜下脂肪组织PPARGC1A启动子区域甲基化水平比较,差异无统计学意义(t=0.929,P=0.380)。结论 GDM孕妇和健康孕妇大网膜下脂肪组织基因甲基化位点和水平存在显著差异,甲基化差异基因主要参与抗原处理和呈递及PPAR信号通路途径。

Background The visceral omental adipose tissues were closely associated with gestational diabetes mellitus（ GDM）.Till now,there had been few reports about genome-wide methylation profile in visceral omental adipose tissues of GDM.Objective To investigate the pathogenesis of GDM by measuring and comparing the genome-wide DNA methylation patterns in visceral omental adipose tissues between pregnant women with GDM and the normal pregnancies,in order to provide the clues for explaining the mechanism and process of GDM.Methods Pregnant women who accepted regular prenatal examination and hospital delivery in Department of Obstetrics and Gynecology,the First Affiliated Hospital of Kunming Medical University from January 2012 to May 2014 were enrolled.Three cases of GDM were enrolled as GDM group,while three cases of healthy pregnant women as control group.The visceral omental adipose tissues were obtained at time of term caesarean section.The genome-wide DNA of these tissues were extracted.The products were cleaved after degeneration and amplification.The DNA fragment were hybridized with Illumina Methylation Bead Chip chip.The methylation level of each gene sites were calculated according to Methylation Analysis Algorithms with deviation correction and infiltration.DAVID database was used to carry out Gene Ontology（ GO） and Kyoto Encyclopedia of Genes and Genomes（ KEGG） signaling pathway analysis in methylation gene with significant difference.Five cases of pregnant women with and without GDM in the same period were selected to verify the difference of methylation level of candidate gene promoter region.Results 1 298 genes were hypomethylated and 1 570 genes were hypermethylated in GDM group.Among them,the methylation of TMEM195,TCF7L2,IGF1 and IGF1 R might be closely associated with regulation of blood glucose level.The methylation of DGKG and NRXN3 might be closely associated with BMI.The GO function analysis supported that the genes with hypomethylation were mainly involved in the biological processes,such as the regulation of cascade reaction activity,the regulation of cell apoptosis and protein transport,the cellular components were mainly extracellular matrix.The biological processes involved in the hypermethylated genes were mainly antigen processing and presentation,and antigen processing and presentation participated by major histocompatibility complex（ MHC） Ⅱ.The main cellular components were muscle actin cytoskeleton proteins,MHC and so on.In the analysis of KEGG signal pathway,the pathway of enrichment 20.000 had antigen processing and presentation（ 23.142）,PPAR（ 22.068）.The different methylation gene HLA-G and PPARGC1 A were respectively selected from antigen processing and presentation and PPAR signal pathway to validate.The methylation level of HLA-G promoter region in the visceral omental adipose tissues of GDM pregnant women was higher than non GDM pregnant women（ t = 4.968,P = 0.001）.There was no statistically significant difference in the methylation of PPARGC1 A promoter region between GDM pregnant women and non GDM pregnant women（ t = 0.929,P= 0.380）.Conclusion There are statistically significant difference in gene methylation sites and levels between GDM and healthy pregnant women.The different methylation gene mainly involve in antigen processing and presentation and PPAR signaling pathway.