Genomic Dissection of Placental Lesions in Preeclampsia

项目摘要 先兆子痫（PE）影响3 - 8%的妊娠，是严重孕产妇发病的主要原因， mortality. PE的发病机制涉及异常的EVT分化和侵袭，导致重建失败 螺旋动脉破裂，导致胎盘灌注不足，引起氧化应激。发育 胎盘的异常表现为由这些缺陷引起的组织病理学病变，包括 母体血管灌注不良（MVM）是一组肉眼和显微镜检查结果， 通过母体血管通道的异常灌注。最近，广泛的胎盘镶嵌和 在正常胎盘组织中经常发生突变，表明这是一个共同特征 胎盘的发育是由不同的克隆扩张引起的。这些突变在 组织病理学病变如MVM、胎盘功能障碍、母胎免疫耐受差 明白我们假设体细胞突变通过以下方式导致先兆子痫的病因： 影响滋养层分化、增殖和免疫原性。我们将建立函数模型 体细胞突变对体外滋养层细胞增殖和分化的影响 来自人胎盘衍生的诱导多能干细胞（iPSC）的滋养层干细胞（TSC）系及其用途 CRISPR-Cas9敲入先前在每个iPSC系中鉴定的PE相关功能丧失突变。我们 将表征突变的滋养层细胞增殖和分化潜力以及免疫原性， 使用qRT-PCR、流式细胞术、免疫细胞化学、功能测定和免疫细胞化学方法， TSC（细胞滋养层/CTB）阶段和分化为合胞体滋养层后的细胞因子阵列 (STB)和绒毛外滋养层细胞（EVT）。接下来，我们将描述组织病理学的突变景观 重度早发性PE和无PE的胎盘中的MVM。我们将进行全基因组 以30 X覆盖率和单核苷酸、拷贝数和结构变异识别进行测序，以鉴定种系 （母体和胎儿）和胎盘内的体细胞突变和胎盘病变。我们将计算 突变负荷，预测免疫原性，并进行临床富集分析以鉴定突变 相对于匹配的正常区域在胎盘病变中富集，相对于正常区域在先兆子痫中富集， 将鉴定与PE和MVM相关的突变，作为进一步功能测试的候选者。建模 胎盘突变的功能作用与滋养层分化、增殖和 免疫原性的研究将进一步加深我们对先兆子痫中胎盘功能障碍的细胞动力学的理解。

PROJECT SUMMARY Preeclampsia (PE) affects 3-8% of pregnancies and is a leading cause of severe maternal morbidity and mortality. PE pathogenesis involves abnormal EVT differentiation and invasion, which leads to failed remodeling of spiral arteries, resulting in hypoperfusion of the placenta that causes oxidative stress. Developmental abnormalities in the placenta manifest as histopathological lesions that arise from these defects and include maternal vascular malperfusion (MVM), a constellation of gross and microscopic findings that represent abnormal perfusion through the maternal vascular channels. Recently, widespread placental mosaicism and frequent mutations have been characterized in normal placental tissue, suggesting that this is a common feature of placental development arising from distinct clonal expansions. The role that these mutations play in histopathological lesions such as MVM, placental dysfunction, maternal-fetal immune tolerance is poorly understood. We hypothesize that somatic mutations contribute to the etiology of preeclampsia by affecting trophoblast differentiation, proliferation and immunogenicity. We will model the functional consequences of somatic mutations on in vitro trophoblast proliferation and differentiation by establishing trophoblast stem cell (TSC) lines from human placenta-derived induced pluripotent stem cells (iPSC) and use CRISPR-Cas9 to knock-in previously identified PE-associated loss-of-function mutations in each iPSC line. We will characterize the trophoblast proliferation and differentiation potential and immunogenicity of mutated and isogenic control iPSC-TSC lines using qRT-PCR, flow cytometry, immunocytochemistry, functional assays, and cytokine arrays at the TSC (cytotrophoblast/CTB) stage and following differentiation into syncytiotrophoblasts (STB) and extravillous trophoblasts (EVT). Next, we will characterize the mutational landscape of histopathologic lesions (MVM) in placentas from severe early onset PE and without PE. We will perform whole genome sequencing at 30X coverage and single nucleotide, copy number and structural variant calling to identify germline (maternal and fetal) and somatic mutations within the placenta and placental lesions. We will calculate mutational burden, predict immunogenicity and perform a clinical enrichment analysis to identify mutations enriched in placental lesions relative to matched normal regions and in preeclampsia relative to normal, which will identify mutations associated with PE and MVM as candidates for further functional testing. Modeling the functional roles of placental mutations with in vitro modeling of trophoblast differentiation, proliferation and immunogenicity will further our understanding of the cellular dynamics of placental dysfunction in preeclampsia.