DNA METHYLATION SIGNATURES OF CHRONIC ALCOHOL DEPENDENCE IN PURIFIED CD3+ T-CELLS OF PATIENTS UNDERGOING ALCOHOL TREATMENT

Background Alcohol Dependence (AD) is a severe disorder that has long-lasting detrimental consequences, resulting in considerable health, economic and societal burden. According to the World Health Organization, alcohol related diseases account for approximately 3.3 million deaths per year (WHO, 2014). The pathogenesis of AD is complex, as the disease arises from the interaction of genetic as well as environmental factors. One of the most compelling candidate mechanisms for the mediation of such gene x environment effects is epigenetic regulation, among which DNA methylation is the most frequently studied mechanism. In fact, several recent studies have shown an association of alcohol dependence with DNA methylation (DNAm), suggesting that environmentally-induced changes on epigenomic variation may play an important role in alcohol dependence. Methods In the present study, we assessed genome-wide DNAm profiles of purified CD3+ T-cells of a well-characterized cohort of long-term chronic AD patients participating in a clinical 3-week alcohol treatment program, along with the profiles of healthy controls closely matched for sex, age, ethnicity and smoking behaviour. 42,43 Furthermore, by comparing the patients before and after 3 weeks of participating in a clinical alcohol treatment program, we sought to identify differentially methylated sites that may play a potential role in alcohol withdrawal and early recovery. In order to test whether our findings were robust, we validated four of our top-ranked hits by pyrosequencing, replicated the top-ranking hits in an independent second cohort of AD patients and matched controls and additionally confirmed the top-ranking hits in whole blood DNA of our cohort samples. Results We identified 59 differentially methylated CpG sites comparing patients prior to treatment with healthy controls and were able to confirm 8 of those sites in additional analyses for differentially methylated regions. Comparing patients before and after a 3-week alcohol treatment program we revealed another unique set of 48 differentially methylated CpG sites. Additionally, we found that the mean global DNAm was significantly lower in patients prior to treatment compared to controls, but reverted back to levels similar to controls after treatment. We validated top-ranked hits derived from the epigenome-wide analysis by pyrosequencing and further replicated two of them in an independent cohort and confirmed differential DNAm of HECW2 and SRPK3 in whole blood. Discussion The reduction in mean global DNAm observed in AD patients and our finding that DNAm in patients reverts back to levels comparable to those in controls after alcohol treatment are supported by previous studies. However, the top hits of differentially methylated sites derived from T-cells did not overlap with previously reported associations of AD with DNAm.This can at least in part be explained by the use of heterogeneous biological material (i.e. whole blood, PBMCs), differences in the cohorts used or in the strategies applied to match patients and controls as well as by varying methodologies for DNAm measurement, with reduced or discordant coverage of CpG sites in previous studies. Still, our top-ranking hits in HECW2 and SRPK3 might contribute to reveal mechanisms that may play a role in AD. This study is the first to show widespread DNAm variation in a disease-relevant blood cell type and implicates HECW2 and SRPK3 DNAm as promising blood-based candidates to follow up in future studies.