Genomic Dissection of Placental Lesions in Preeclampsia

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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.