Project Summary. This application is being submitted in response to PAR-16-355, Social Epigenomics Research Focused on Minority Health and Health Disparities (R01), and the overarching long-term goal of this research effort is to learn how to positively influence health trajectories, modify disease risk in racial/ethnic minorities, and reduce health disparities. This grant is designed to unravel the mechanisms by which social adversity confers risk for obesity in youth. Obesity was selected as the primary health outcome as it is a health measure with widening racial/ethnic disparities over the past three decades, and it is a potent predictor of a host of negative social, educational, vocational, and health outcomes later in life. In this study we propose to leverage two ongoing prenatal cohorts in Florida and North Carolina, and recruit a sample of 470 pregnant women and follow their children to 24 months of age. The sample will be enriched for adversity in pregnancy and approximately evenly divided among Caucasian, African American, and Hispanic subjects. We will assess the impact of mothers' prenatal stress on measures of DNA methylation in human umbilical vein endothelial cells (HUVEC), which are homogenous in terms of cellular composition, and widely accepted as an optimal choice for examining in utero responses to stressors. In the epigenetic analyses we will use both targeted and big data approaches, using pyrosequencing to measure methylation of 31 differentially methylated regions (DMRs) that regulate ~90 known imprinted genes, and the Illumina HumanMethylationEPIC (850k) bead chip for whole epigenome analyses. We will also assess maternal and child postnatal psychosocial stress exposure, and child growth in the first 24 months of life. Children who are overweight at any point during the first two years of life have an approximately six-fold increased risk for obesity at five years of age, and risk for chronic health problems across the lifecycle. The primary hypotheses of this study build on preliminary work from our group and include: 1) Maternal prenatal stress will be associated with increased DMRs regulating the expression of imprinted gene insulin-like growth factor 2 (IGF2) and maternally expressed gene 3 (MEG3) DMR, increased methylation in Calcium/calmodulin-dependent protein kinase type IV (CAMK4) gene, and methylation changes in novel CpG sites identified in the 850K analyses; 2) DNA methylation profile changes associated with prenatal stress will be related to alterations in gene expression; and 3) Methylation markers identified in Aim 1 will predict growth trajectories and measures of obesity at 24 months of age. Secondary analyses will examine several moderating factors, including: maternal Adverse Childhood Experiences (ACE), maternal social supports, maternal postnatal psychosocial distress, postnatal offspring ACE, genetic variants, and relevant confounding variables. The mediating role of inflammatory markers will also be explored, and saliva DNA specimens will be collected on the full cohort at 24-months and 50 children at birth, allowing for tests of comparability of DNA methylation between HUVECs and saliva, and the stability of markers over time.