Metabolic programming of DNA methylation patterns by intrauterine exposure to gestational diabetes mellitus

工业化国家2型糖尿病和肥胖症的流行增加不能仅用营养过剩和缺乏运动来解释。越来越多的证据表明，产前因素，特别是妊娠期糖尿病(GDM)引起的高血糖，错误地影响了胎儿晚年的代谢，从而增加了发生复杂代谢疾病的风险。胎儿编程背后的机制在很大程度上仍然不清楚。表观遗传变化，即代谢基因的DNA甲基化，可能起到重要作用。在第一个无假设的方法中，我们将比较患有和不患有妊娠期糖尿病的母亲的新生儿的表观基因组。为此，我们将使用Illumina Infinium HumanMethylation450 BeadChips分析24例营养治疗的妊娠期糖尿病、24例胰岛素依赖型妊娠期糖尿病和48例非妊娠期糖尿病孕妇的脐带血和胎儿胎盘。生物信息学分析将确定显示组间显著差异的基因和途径。将考虑混杂因素的影响，特别是母亲肥胖的影响。来自全基因组甲基化分析的最佳候选基因(根据计算机优先排序)将在至少100名独立妊娠期糖尿病孕妇和100名对照的队列中得到验证。亚硫酸氢盐焦磷酸测序和实时RT-PCR将分别用于量化显著基因区域的甲基化(通过阵列分析)和由这些位点控制的转录本的表达。对于特别有趣的候选基因，例如已经确定的对脂肪细胞代谢至关重要的MEST基因，我们将对不同区域/转录本进行高分辨率甲基化和表达分析。这将在原代细胞类型(脐带血、胎盘、成人血液和脂肪组织)以及从脂肪来源的间充质干细胞分化的细胞类型中完成。5-羟甲基胞嘧啶在我们的候选基因代谢编程中的作用将通过氧化亚硫酸氢盐测序来阐明。总而言之，我们的研究将证明母亲糖尿病和/或肥胖对后代表观基因组的影响。

The epidemic increase in type 2 diabetes and obesity in industrialized countries cannot be explained by overnutrion and physical inactivity alone. Accumulating evidence suggests that prenatal factors, in particular hyperglycemia through gestational diabetes mellitus (GDM) mal-program the fetal metabolism for later life and thus, increase the risk for developing complex metabolic disorders. The mechanisms underlying fetal programming are still largely unclear. It is plausible that epigenetic changes, i.e. in DNA methylation of metabolic genes play an essential role. In a first hypothesis-free approach we will compare the epigenomes of newborns of mothers with and without GDM. To this end, we will analyze cord blood and fetal placenta of 24 pregnancies with dietetically treated GDM, 24 with insulin-dependent GDM and 48 without GDM, using Illumina Infinium HumanMethylation450 BeadChips. Bioinformatic analyses will identify genes and pathways showing significant between-group differences. The effects of confounding factors, in particular of maternal obesity will be considered. The best candidate genes (according to in silico prioritization) from genome-wide methylation analysis will be validated in cohorts of at least 100 independent GDM pregnancies and 100 controls. Bisulfite pyrosequencing and realtime RT PCR, respectively, will be used to quantify the methylation of conspicuous (by array analysis) gene regions and the expression of the transcripts controlled by these sites. For particularly interesting candidate genes, such as the already identified MEST gene, which is essential for adipocyte metabolism, we will perform high-resolution methylation and expression analyses of different regions/transcripts. This will be done in primary cell types (from cord blood, placenta, adult blood and fat tissue) as well as in cell types differentiated from adipose-derived mesenchymal stem cells. The role of 5-hydroxymethylcytosine in metabolic programming of our candidate genes will be elucidated by oxidative bisulfite sequencing. Collectively, our studies will demonstrate the effects of maternal diabetes and/or obesity on the epigenome of the offspring.