AbstractAlcohol misuse is common in many societies worldwide and is associated with extensive morbidity and mortality, often leading to alcohol use disorders (AUD) and alcohol-related end-organ damage. The underlying mechanisms contributing to the development of AUD are largely unknown; however, growing evidence suggests that alcohol consumption is strongly associated with alterations in DNA methylation. Identification of alcohol-associated methylomic variation might provide novel insights into pathophysiology and novel treatment targets for AUD. Here we performed the largest single-cohort epigenome-wide association study (EWAS) of alcohol consumption to date (N = 8161) and cross-validated findings in AUD populations with relevant endophenotypes, as well as alcohol-related animal models. Results showed 2504 CpGs significantly associated with alcohol consumption (Bonferroni p value < 6.8 × 10⁻⁸) with the five leading probes located in SLC7A11 (p = 7.75 × 10⁻¹⁰⁸), JDP2 (p = 1.44 × 10⁻⁵⁶), GAS5 (p = 2.71 × 10⁻⁴⁷), TRA2B (p = 3.54 × 10⁻⁴²), and SLC43A1 (p = 1.18 × 10⁻⁴⁰). Genes annotated to associated CpG sites are implicated in liver and brain function, the cellular response to alcohol and alcohol-associated diseases, including hypertension and Alzheimer’s disease. Two-sample Mendelian randomization confirmed the causal relationship of consumption on AUD risk (inverse variance weighted (IVW) p = 5.37 × 10⁻⁰⁹). A methylation-based predictor of alcohol consumption was able to discriminate AUD cases in two independent cohorts (p = 6.32 × 10⁻³⁸ and p = 5.41 × 10⁻¹⁴). The top EWAS probe cg06690548, located in the cystine/glutamate transporter SLC7A11, was replicated in an independent cohort of AUD and control participants (N = 615) and showed strong hypomethylation in AUD (p < 10⁻¹⁷). Decreased CpG methylation at this probe was consistently associated with clinical measures including increased heavy drinking days (p < 10⁻⁴), increased liver function enzymes (GGT (p = 1.03 × 10⁻²¹), ALT (p = 1.29 × 10⁻⁶), and AST (p = 1.97 × 10⁻⁸)) in individuals with AUD. Postmortem brain analyses documented increased SLC7A11 expression in the frontal cortex of individuals with AUD and animal models showed marked increased expression in liver, suggesting a mechanism by which alcohol leads to hypomethylation-induced overexpression of SLC7A11. Taken together, our EWAS discovery sample and subsequent validation of the top probe in AUD suggest a strong role of abnormal glutamate signaling mediated by methylomic variation in SLC7A11. Our data are intriguing given the prominent role of glutamate signaling in brain and liver and might provide an important target for therapeutic intervention.