DNA甲基化异常预测子痫前期的可行性及机制研究

Background: Preeclampsia (PE) is the leading cause of maternal and fetal death. Its pathogenesis remains illusive and its predictive biomarkers are still unknown. Based on others' and our previous results,we suppose that aberrant DNA methylation in placenta might take an important role in PE and its presence in plasma might be the biomarkers in predicting PE..Objective: First:to screen and validate aberrant DNA methylation in placenta and plasma in patients with PE; second: to test the efficacy of aberrant DNA methylation in maternal plasma in predicting PE;third: based on the simple method we developed recently for high-throughput quantification of genome-wide DNA methylation by fluorescence polarization, we intend to develop the simple method in predicting PE according to the biomarkers we identified..Study design: We will conduct a nested-case control study, a total of 1500 pregnant women will be enrolled. Maternal blood samples will be withdrawn at 4 periods: gestational weeks 11-13, 17-20, PE and normal pregnant women on hospital admission, and 1 week within postpartum. Pleacenta will be collected after delivery. Based on 5% prevalence in PE, we will have 25 early onset PE and 25 late onset PE patients. .Methods: Genome-wide methylation analysis (Infinium Human Methylation 450 Bead Chip) to quantify methylation at over 450,000 sites will be performed on placenta and maternal plasma. Candidate sites will be prioritized by 1) False detection rate <5%; 2) magnitude of difference between group means >17% (as a measure of biological significance); 3) the detection of multiple altered CpGs associated with the same gene, and 4) potential role in trophoblast function and/or hypoxia response. Candidate sites will be validated and followed up using pyrosequencing. We will prioritize genes important for placental function and those known to be altered in PE along with what we identified in our previous cDNA array and the potential folate-related metabolism genes. RT-PCR, Western Blot will be used to analyze the transcription and translation of aberrantly methylated DNA. Differences in gene specific DNA methylation profiling between cases and controls will be compared. Bioinformatics such as DAVID analysis will be used to rank the biologic significance of gene function clusters or networks in PE..Statistical Analysis: Data from bisulfite pyrosequencing will be analyzed with two-tailed Student's t-test. Linear correlation will be used to analyze the correlation between DNA methylation and gene expression, as well as the correlation between data obtained from Illumina array and bisulfite pyrosequencing assays. We will also compare the aberrantly methylated DNA in case and control group in different gestational ages. Detection rate and false-positive rate will be calculated for each marker with a univariable receiver operating characteristic (ROC) curve.

子痫前期（PE）是导致母儿患病率和死亡率的主要原因。PE的发生机制及其预测标记物尚未阐明。我们前期研究发现口服含有叶酸的多种维生素可能通过调控胎盘基因甲基化降低PE的发生。进一步研究发现，PE患者的胎盘cDNA表达谱异常，可能是由于DNA甲基化异常所致。据此，我们提出假设，即胎盘DNA甲基化异常参与PE的发生，孕妇外周血中差异甲基化基因可能成为预测PE发生的新的标记物。为验证该假设，本项目拟采用巢式病例对照研究，利用甲基化芯片筛选、焦磷酸测序验证PE患者胎盘与外周血中差异甲基化DNA；检测差异甲基化DNA随孕周甲基化的变异程度，探讨预测PE的生物标记物；同时应用我们已经建立的简单、敏感、高通量检测全基因组甲基化水平的新方法，结合定量PCR技术，通过甲基化芯片筛选出可预测PE的有效生物学标记物，为子痫前期的防治及管理提供理论基础。

子痫前期（PE）是导致母儿患病率和死亡率的主要原因。PE的发生机制及其预测标记物尚未阐明。我们前期研究发现口服含有叶酸的多种维生素可能通过调控胎盘基因甲基化降低PE的发生。进一步研究发现，PE患者的胎盘cDNA表达谱异常，可能是由于DNA甲基化异常所致。据此，我们提出假设，即胎盘DNA甲基化异常参与PE的发生，孕妇外周血中差异甲基化基因可能成为预测PE发生的新的标记物。为验证该假设，本项目采用巢式病例对照研究，利用甲基化芯片筛选、焦磷酸测序验证PE患者胎盘与外周血中差异甲基化DNA；检测差异甲基化DNA随孕周甲基化的变异程度，探讨预测PE的生物标记物；并通过甲基化芯片筛选出可预测有效PE的生物学标记物。.本研究基本完成两个研究中心的入组实验对象征集，收集不同时期的母体外周血液标本。于早发型PE组，晚发型PE组及对照组各3例抽提其孕早期、孕中期及入院分娩时（孕晚期）血标本DNA（共27份样本）采用Illumina 450K甲基化芯片进行DNA甲基化程度进行测定，结果显示各组其均存在差异甲基化基因改变，根据芯片位点信息、基因功能及早期预测目的等，选取孕早期HOXA9、MTNR1A、PIWIL1进行了下一步验证。.本研究成功运用基因芯片分析了正常妊娠女性和PE患者母体外周血的DNA甲基化变化情况，进一步检验候选基因与PE的关系，发现HOXA9、MINR1A甲基差异化可作为潜在的早发型PE早期无创性预测指标，筛选出的激活差异的基因将为PE的防治及管理提供理论基础。下一步将继续对这些基因的筛选和验证。

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