Amnion as a surrogate tissue reporter of the effects of maternal preeclampsia on the fetus [RNA-Seq]

Background: Preeclampsia, traditionally characterized by high blood pressure and proteinuria, is a common pregnancy complication, which affects 2-8% of all pregnancies. Although children born to women with preeclampsia have a higher risk of hypertension in later life, the mechanism of this increased risk is unknown. DNA methylation is an epigenetic modification that has been studied as a mediator of cellular memory of adverse exposures in utero. Since each cell type in the body has a unique DNA profile, cell subtype composition is a major confounding factor in studies of tissues with heterogeneous cell types. The best way to avoid this confounding effect is by using purified cell types. However, the use purified cell types in large cohort translational studies is difficult. The amnion, the inner layer of the fetal membranes of placenta, is derived from the epiblast and consists of two cell types, which are easy to isolate from the delivered placenta. In this study, we demonstrate the value of using amnion samples for DNA methylation studies, revealing distinctive patterns between fetuses exposed to preeclampsia or hypertension and fetuses from normal pregnancies. Results: We performed a genome-wide DNA methylation analysis, HELP-tagging, on 62 amnion samples from placentas of uncomplicated, normal pregnancies, and those with complications of preeclampsia or hypertension. Using a regression model approach, we found 123, 85 and 99 loci with high confidence hypertension-associated, proteinuria-associated and hypertension and proteinuria-associated DNA methylation changes, respectively. We also found that these differentially methylated regions overlap loci previously reported as differentially methylated regions in preeclampsia. Conclusions: Our findings support prior observations that preeclampsia is associated with changes of DNA methylation near genes that have previously been found to be dysregulated in preeclampsia. We propose that amnionic membranes represent a valuable surrogate fetal tissue on which to perform epigenome-wide association studies of adverse intrauterine conditions. Directional RNA profiles of amnion membranes were generated by deep sequencing using Illumina HiSeq2500. Twenty-nine human amnion specimens were used: 12 control and 17 preeclampsia exposed.

背景：子痫前期（preeclampsia）传统上以高血压与蛋白尿为核心特征，是一类常见的妊娠并发症，累及全球2%~8%的妊娠人群。尽管子痫前期产妇所分娩的子代在成年后罹患高血压的风险显著升高，但其风险增加的具体机制迄今尚未阐明。DNA甲基化（DNA methylation）作为一种表观遗传修饰，此前被广泛研究作为子宫内不良暴露的细胞记忆介质。由于机体各细胞类型均拥有独特的DNA甲基化谱，细胞亚型组成是异质性细胞类型组织研究中的主要混杂因素。规避该混杂效应的最优方案是使用纯化的细胞类型，但在大型队列转化研究中应用纯化细胞类型存在诸多困难。羊膜（amnion）作为胎盘胎膜的内层结构，起源于上胚层，包含两种细胞亚型，且易于从娩出的胎盘中分离获取。本研究证实了羊膜样本在DNA甲基化研究中的应用价值，揭示了暴露于子痫前期或高血压的胎儿与正常妊娠胎儿之间存在显著差异的甲基化模式。
结果：我们对62份来自无并发症正常妊娠以及并发子痫前期或高血压的胎盘羊膜样本，开展了全基因组DNA甲基化分析——HELP-tagging。通过回归模型分析方法，我们分别鉴定出123个、85个和99个与高血压相关、蛋白尿相关以及高血压合并蛋白尿相关的高置信度DNA甲基化变化位点。此外，我们发现这些差异甲基化区域与既往报道的子痫前期差异甲基化区域存在重叠。
结论：本研究结果支持既往观察结论，即子痫前期与此前报道的子痫前期失调基因附近的DNA甲基化变化存在关联。我们提出，羊膜组织可作为开展子宫内不良暴露表观基因组全关联研究的极具价值的替代胎儿组织。本研究同时通过Illumina HiSeq2500平台进行深度测序，生成了羊膜组织的定向RNA转录组谱。本次研究共纳入29份人类羊膜标本，其中12份为对照样本，17份为子痫前期暴露样本。