T83. DIRECT DNA SEQUENCING REVEALS ALCOHOL USE DISORDER-ASSOCIATED ALTERATIONS IN DNA METHYLATION AND HYDROXYMETHYLATION IN HUMAN CAUDATE NUCLEUS

Alcohol Use Disorder (AUD) has a profound public health impact, yet our understanding of the underlying mechanisms contributing to its development and progression remains limited. Environmental factors, such as chronic drinking and stress, can cause long-lasting changes in neurocircuitry via epigenetic mechanisms. Here, we utilize Nanopore long-read sequencing to directly analyze DNA isolated from postmortem brain tissue, which allows detection of AUD-associated differences in the two most abundant types of epigenetic modifications in the brain: 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). We completed Nanopore long-read direct DNA-seq on caudate nucleus tissue of 144 decedents (75 cases, 69 controls). After simultaneous basecalling of both canonical and modified bases, we performed beta-binomial regression using the DSS (Dispersion Shrinkage for Sequencing data) program to detect differentially modified CpG sites and regions between cases and controls, adjusting for relevant covariates. Gene-set enrichment analyses were performed separately for genes annotated to both differentially methylated and hydroxymethylated regions. Previously characterized single nucleus multiome data from the same samples allowed us to link the detected epigenetic modifications with alterations in gene expression and chromatin accessibility. We identified 7,458 differentially methylated and 4,498 differentially hydroxymethylated regions associated with AUD, including those unique to only one type of modification. We detected significant enrichment of annotated genes in gene sets related to synapse organization, neurotransmission, and axonogenesis. Examining snRNA-seq data in the same brain samples demonstrated a strong correlation between gene body hydroxymethylation and neuronal gene expression. Our study presents a large-scale genome-wide atlas of AUD-associated methylation and hydroxymethylation changes in the caudate nucleus, indicating differential roles for 5mC and 5hmC and offering insight into the epigenetic mechanisms underlying AUD onset and progression.