Posttraumatic stress disorder (PTSD) is a serious mental disorder that occurs in response to a traumatic event. In this project, we will examine DNA methylation, an epigenetic form of gene regulation, for association with PTSD. We will utilize data from state of the art methylation beadchips that measure more than 850,000 locations along the genome. First, we will perform an epigenome-wide association study (EWAS) of PTSD in blood. This will be followed by a candidate gene analysis of PTSD and glucocorticoid-responsive genes based on our recent genome-wide gene expression study of PTSD (Logue et al., 2015). That study found genes whose expression in blood was closely correlated with PTSD case/control status, including many glucocorticoid-responsive genes that have not been previously studied in relationship to PTSD. These analyses will utilize data from two U.S. Department of Veterans Affairs (VA) cohorts (total n>1000) made up primarily of US veterans with a high prevalence of PTSD. Replication of these findings will be sought from a large epigenetics consortium. Then, we will perform genome-wide and candidate-gene association studies of PTSD using tissue from 50 brains obtained from a recently formed PTSD brain bank. We will specifically analyze methylation in the hippocampus, amygdala, and hypothalamus—three regions implicated in PTSD, HPA axis functioning, and anxiety. The impact of PTSD-associated differential methylation on gene expression in the brain will be investigated using chromogenic in-situ hybridization that will provide a measure of gene expression as well as provide localization of that expression to particular cell types (e.g. neurons or glia). We will investigate the effects of these differences on neural integrity using a VA cohort with existing MRI data (n>400). In this way, we will link PTSD-associated DNA methylation differences in blood to DNA methylation differences in the brain, gene-expression differences in the brain, and morphological differences in the brain. This will yield clinically relevant biomarkers for PTSD, trauma exposure, and PTSD-related symptomatology and important information about the biology underpinning the observed associations.