Genomic Dissection of Placental Lesions in Preeclampsia

项目总结 子痫前期(PE)影响3-8%的妊娠，是严重孕产妇发病率和 死亡率。PE的发病机制涉及EVT的异常分化和侵袭，从而导致重构失败 螺旋动脉受累，导致胎盘灌流不足，导致氧化应激。发展中 胎盘的异常表现为由这些缺陷引起的组织病理学损害，包括 母体血管灌注不良(MVM)，一系列大体和微观表现，代表 通过母体血管通道的异常血流。最近，普遍存在的胎盘马赛克和 在正常胎盘组织中发现了频繁突变的特征，这表明这是一种常见的特征。 胎盘发育的起源于不同的克隆扩张。这些突变在其中扮演的角色 组织病理损害，如MVM，胎盘功能障碍，母婴免疫耐受性差 明白了。我们假设，体细胞突变通过以下方式在先兆子痫的病因中起作用 影响滋养层细胞分化、增殖和免疫原性。我们将对功能 建立体细胞突变对体外滋养层细胞增殖和分化的影响 人胎盘诱导多能干细胞(IPSC)的滋养层干细胞(TSC)株及其应用 CRISPR-Cas9在每个IPSC系中敲入先前发现的与PE相关的功能丧失突变。我们 将表征滋养层细胞的增殖分化潜能和免疫原性 应用qRT-PCR、流式细胞术、免疫细胞化学、功能分析和免疫组织化学等方法检测同基因对照IPSC-TSC TSC(细胞滋养层/CTB)阶段和分化为合体滋养层细胞后的细胞因子阵列 (STB)和绒毛外滋养细胞(EVT)。接下来，我们将描述组织病理学的突变图景 重度早发胎盘和无胎盘早发胎盘的病变(MVM)。我们将进行全基因组测试 30倍覆盖率测序和单核苷酸、拷贝数和结构变异呼唤以鉴定种系 胎盘和胎盘病变内的(母体和胎儿)和体细胞突变。我们会计算出 突变负担，预测免疫原性，并执行临床浓缩分析以识别突变 相对于匹配的正常区域的胎盘病变和相对于正常的子痫前期 将确定与PE和MVM相关的突变，作为进一步功能测试的候选。为您的 胎盘突变在滋养层细胞分化、增殖和体外模拟中的功能作用 免疫原性将进一步加深我们对子痫前期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.