Mapping the epigenomic landscape of post-traumatic stress disorder in human cortical neurons

Abstract Most epigenetic research on post-traumatic stress disorder (PTSD) has primarily focused on DNA methylation (5mC) in peripheral tissues, particularly at CpG sites. DNA hydroxymethylation (5hmC) has been found to be highly enriched in the mammalian brain, while 5mC at non-CpG sites shows high enrichment in neurons. However, little is known about their role in PTSD. Here, we characterize genome-wide differential 5mC and 5hmC at both CpG and non-CpG sites in postmortem orbitofrontal neurons from PTSD cases and controls. Utilizing reduced-representation oxidative bisulfite sequencing, we found that genome-wide significant (GWS) differential CpGs were primarily hyper-5mC/5hmC, whereas GWS differential non-CpGs were hypo-5mC/5hmC. Compared with 5mC, we show that 5hmC is a more sensitive epigenetic mark in PTSD, with a higher number of differential 5hmC sites and a stronger significance in enriched pathways. Integrating other -omics data highlighted developmental processes as significant convergent pathways and revealed overlap of our GWS 5hmC findings with 50 previously reported PTSD-associated genes, including potential therapeutic targets such as CRHR1 and DRD4. This study underscores the importance of evaluating 5hmC in the human brain and our multi-omics integration provides insights into potential target genes for future therapeutic interventions in PTSD. Graphical abstract The study conducted a comprehensive genome-wide analysis of differential 5mC and 5hmC modifications at both CpG and non-CpG sites in postmortem orbitofrontal neurons from 25 PTSD cases and 13 healthy controls. It was observed that PTSD patients exhibit a greater number of differential 5hmC sites compared to 5mC sites. Specifically, individuals with PTSD tend to show hyper-5mC/5hmC at CpG sites, particularly within CpG islands and promoter regions, and hypo-5mC/5hmC at non-CpG sites, especially within intragenic regions. Functional enrichment analysis indicated distinct yet interconnected roles for 5mC and 5hmC in PTSD. The 5mC marks primarily regulate cell-cell adhesion processes, whereas 5hmC marks are involved in embryonic morphogenesis and cell fate commitment. By integrating published PTSD findings from central and peripheral tissues through multi-omics approaches, several biological mechanisms were prioritized, including developmental processes, HPA axis regulation, and immune responses. Based on the consistent enrichment in developmental processes, we hypothesize that if epigenetic changes occur during early developmental stages, they may increase the risk of developing PTSD following trauma exposure. Conversely, if these epigenetic changes occur in adulthood, they may influence neuronal apoptosis and survival mechanisms. Competing Interest Statement John H. Krystal, MD served as a consultant for Aptinyx, Inc., Biogen, Idec, MA, Bionomics, Limited (Australia), Boehringer Ingelheim International, Epiodyne, Inc., EpiVario, Inc., Janssen Research & Development, Jazz Pharmaceuticals, Inc., Otsuka America Pharmaceutical, Inc., Spring Care, Inc., Sunovion Pharmaceuticals, Inc., Freedom Biosciences, Inc., Biohaven Pharmaceuticals, BioXcel Therapeutics, Inc. (Clinical Advisory Board), Cerevel Therapeutics, LLC, Delix Therapeutics, Inc., Eisai, Inc., EpiVario, Inc., Jazz Pharmaceuticals, Inc., Neumora Therapeutics, Inc., Neurocrine Biosciences, Inc., Novartis Pharmaceuticals Corporation, PsychoGenics, Inc., Takeda Pharmaceuticals, Tempero Bio, Inc., Terran Biosciences, Inc., Biohaven Pharmaceuticals, Freedom Biosciences, Spring Health, Inc., Biohaven Pharmaceuticals Medical Sciences, Cartego Therapeutics, Damona Pharmaceuticals, Delix Therapeutics, EpiVario, Inc., Neumora Therapeutics, Inc., Rest Therapeutics, Tempero Bio, Inc., Terran Biosciences, Inc., and Tetricus, Inc. Funding Statement This study was supported by the National Institute on Drug Abuse R21DA050160 and DP1DA058737 (J.L.M.O.); the U.S. Department of Veterans Affairs via the National Center for Posttraumatic Stress Disorder, Veterans Affairs Connecticut (J.L.M.O.); 1IK2CX002095-01A1 (J.L.M.O.), and the Kavli Institute for Neuroscience at Yale University Kavli Postdoctoral Award for Academic Diversity (J.J.M.M.). This publication was made possible in part by the Yale Center for Brain and Mind Health, which is sponsored by the Yale School of Medicine (D.L.N.R.). Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Samples used here were collected from the National Posttraumatic Stress Disorder Brain Bank, which is part of the US Department of Veterans Affairs (VA). As part of the VA, any local VA facility to be a participating site in a research project that is reviewed by the VA Central IRB, the local facility must also enter into a Memorandum of Understanding (MOU) with the VHA Central Office Human Research Protections Program (HRPP), and rewrite its SOPs to incorporate the processes for using the VA CIRB. Both ORO and ORD must approve changes in the facilities IRB relationships, so the MOU will be reviewed by each. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data Availability All raw data produced in the present study are available upon reasonable request to the authors. Summary stats of the analysis will be publicly available in supplementary tables