Epigenetics of alcohol use disorder-A review of recent advances in DNA methylation profiling.

Alcohol use disorder (AUD) is a major contributor to morbidity and mortality worldwide. Although there is a heritable component, the etiology of AUD is complex and can involve environmental exposures like trauma and can be associated with many different patterns of alcohol consumption. Epigenetic modifications, which can mediate the influence of genetic variants and environmental variables on gene expression, have emerged as an important area of AUD research. Over the past decade, the number of studies investigating AUD and DNA methylation, a form of epigenetic modification, has grown rapidly. Yet we are still far from understanding how DNA methylation contributes to or reflects aspects of AUD. In this paper, we reviewed studies of DNA methylation and AUD and discussed how the field has evolved. We found that global DNA and candidate DNA methylation studies did not produce replicable results. To assess whether findings of epigenome-wide association studies (EWAS) were replicated, we aggregated significant findings across studies and identified 184 genes and 15 gene ontological pathways that were differentially methylated in at least two studies and four genes and three gene ontological pathways that were differentially methylated in three studies. These genes and pathways repeatedly found enrichment of immune processes, which is in line with recent developments suggesting that the immune system may be altered in AUD. Finally, we assess the current limitations of studies of DNA methylation and AUD and make recommendations on how to design future studies to resolve outstanding questions.

Epigenome-wide association study and multi-tissue replication of individuals with alcohol use disorder: evidence for abnormal glucocorticoid signaling pathway gene regulation.

Alcohol use disorder (AUD) is a chronic debilitating disorder with limited treatment options and poorly defined pathophysiology. There are substantial genetic and epigenetic components; however, the underlying mechanisms contributing to AUD remain largely unknown. We conducted the largest DNA methylation epigenome-wide association study (EWAS) analyses currently available for AUD (total N = 625) and employed a top hit replication (N = 4798) using a cross-tissue/cross-phenotypic approach with the goal of identifying novel epigenetic targets relevant to AUD. Results show that a network of differentially methylated regions in glucocorticoid signaling and inflammation-related genes were associated with alcohol use behaviors. A top probe consistently associated across all cohorts was located in the long non-coding RNA growth arrest specific five gene (GAS5) (p < 10-24). GAS5 has been implicated in regulating transcriptional activity of the glucocorticoid receptor and has multiple functions related to apoptosis, immune function and various cancers. Endophenotypic analyses using peripheral cortisol levels and neuroimaging paradigms showed that methylomic variation in GAS5 network-related probes were associated with stress phenotypes. Postmortem brain analyses documented increased GAS5 expression in the amygdala of individuals with AUD. Our data suggest that alcohol use is associated with differential methylation in the glucocorticoid system that might influence stress and inflammatory reactivity and subsequently risk for AUD.

Lipid profile dysregulation predicts alcohol withdrawal symptom severity in individuals with alcohol use disorder.

Alcohol withdrawal syndrome (AWS) is a serious medical condition of high variability in alcohol use disorder (AUD) after drinking cessation. Identification of clinical biomarkers capable of detecting severe AWS is needed. While alcohol consumption and withdrawal are linked with lipid profile dysregulation, the relationship between lipid levels (high-density lipoprotein cholesterol [HDL-C], low-density lipoprotein cholesterol [LDL-C], and triglycerides) and AWS is unknown. Therefore, this study investigated whether HDL-C, LDL-C, and triglycerides conferred risk for moderate-to-severe AWS symptoms in treatment-seeking individuals (n = 732) admitted to the National Institute on Alcohol Abuse and Alcoholism (NIAAA) alcohol treatment program. Lipid levels were measured upon admission, and the Clinical Institute Withdrawal Assessment of Alcohol Scale, Revised (CIWA-Ar) assessed AWS severity for generating a three-level AWS typology (none-to-mild, moderate, and severe). Multivariable multinomial logistic regression examined whether lipid levels were associated with risk for moderate-to-severe AWS. We found significant predictive relationships between AWS and HDL-C, LDL-C, and triglycerides. While extremely high HDL-C (≥100 mg/dL) conferred the highest odds for moderate (4.405, 95% CI, 2.572-7.546, p < 0.001) and severe AWS (5.494, 95% CI, 3.541-8.523, p < 0.001), the lowest odds ratios for moderate AWS (0.493, 95% CI, 0.248-0.981, p = 0.044) and severe AWS (0.303, 95% CI, 0.223-0.411, p < 0.001) were associated with high LDL-C (≥160 mg/dL). The present study demonstrates that altered lipid levels, measured upon admission for inpatient AUD treatment, may help to predict which individuals are at risk for medically relevant moderate-to-severe AWS. This suggests that further research into the role of lipid biomarkers in AWS may be beneficial for identifying biologically determined risk profiles in AUD.

Adverse Childhood Experiences are Associated with High-Intensity Binge Drinking Behavior in Adulthood and Mediated by Psychiatric Disorders.

AIM: High-intensity binge drinking (HIBD), defined as two or more times the gender-specific binge threshold, is rapidly increasing in the USA; however, the underlying contributing factors are poorly understood. This study investigated the relationship of adverse childhood experiences (ACEs) and HIBD. METHODS: Two independent, cross-sectional samples were analysed: (a) past 12-month drinkers in the National Epidemiological Survey on Alcohol and Related Conditions-III (NESARC-III; n = 25,552) and (b) the National Institute on Alcohol Abuse and Alcoholism (NIAAA) clinical sample (n = 1303). Multinomial logistic regressions were utilized to estimate adjusted odds ratios (AORs) of ACEs on HIBD. Mediation analysis was performed to examine the relationship between the past 12-month psychiatric disorders, ACEs, and HIBD. RESULTS: In the NESARC-III sample, prevalence of ACEs increased across all binge levels with the highest prevalence in extreme HIBD; ACEs were associated with higher odds for HIBD (level II, odds ratio (OR) = 1.2-1.4; P = 0.03-0.001; level III, OR = 1.3-1.9; P < 0.001). Prevalence of DSM-5 diagnoses also increased across all binge levels. Substance use disorders (SUD), mood, personality and post-traumatic stress disorders (PTSD) conferred the highest odds with extreme HIBD (SUD: OR = 21.32; mood: 1.73; personality: 2.84; PTSD: 1.97; all Ps < 0.001). Mediation analyses showed that the association between ACEs and HIBD was fully mediated through SUD (proportion mediated: 70-90%) and partially through other psychiatric disorders (20-80%). In the NIAAA sample, ACEs were 2-5 times more prevalent in extreme HIBD with higher odds (ORs = 3-8, P < 0.001) compared with non-bingers. CONCLUSION: ACEs were associated with significantly increased odds of HIBD and the relationship may be mediated by psychiatric disorders.

Epigenetic aging is accelerated in alcohol use disorder and regulated by genetic variation in APOL2.

To investigate the potential role of alcohol use disorder (AUD) in aging processes, we employed Levine's epigenetic clock (DNAm PhenoAge) to estimate DNA methylation age in 331 individuals with AUD and 201 healthy controls (HC). We evaluated the effects of heavy, chronic alcohol consumption on epigenetic age acceleration (EAA) using clinical biomarkers, including liver function test enzymes (LFTs) and clinical measures. To characterize potential underlying genetic variation contributing to EAA in AUD, we performed genome-wide association studies (GWAS) on EAA, including pathway analyses. We followed up on relevant top findings with in silico expression quantitative trait loci (eQTL) analyses for biological function using the BRAINEAC database. There was a 2.22-year age acceleration in AUD compared to controls after adjusting for gender and blood cell composition (p = 1.85 × 10-5). This association remained significant after adjusting for race, body mass index, and smoking status (1.38 years, p = 0.02). Secondary analyses showed more pronounced EAA in individuals with more severe AUD-associated phenotypes, including elevated gamma-glutamyl transferase (GGT) and alanine aminotransferase (ALT), and higher number of heavy drinking days (all ps < 0.05). The genome-wide meta-analysis of EAA in AUD revealed a significant single nucleotide polymorphism (SNP), rs916264 (p = 5.43 × 10-8), in apolipoprotein L2 (APOL2) at the genome-wide level. The minor allele A of rs916264 was associated with EAA and with increased mRNA expression in hippocampus (p = 0.0015). Our data demonstrate EAA in AUD and suggest that disease severity further accelerates epigenetic aging. EAA was associated with genetic variation in APOL2, suggesting potential novel biological mechanisms for age acceleration in AUD.

Association of High-Intensity Binge Drinking With Lipid and Liver Function Enzyme Levels.

Importance: The prevalence of high-intensity binge drinking (HIBD), defined as consuming 2 or more times the binge threshold defined by the National Institute on Alcohol Abuse and Alcoholism (NIAAA), is rapidly increasing in the United States. While the relationship between alcohol consumption and lipid and liver function enzyme (LFT) biomarkers has been previously examined, the associations of HIBD with those biomarkers remain unknown. Objective: To examine associations of HIBD with lipid and LFT levels in a cross-sectional sample enriched with participants who engage in HIBD. Design, Setting, and Participants: Cross-sectional study using data from the NIAAA clinical sample collected from March 3, 2005, to August 21, 2017, with participants recruited for either the NIAAA screening protocols or inpatient alcohol treatment program. For this study, participants were stratified by self-reported alcohol consumption into 4 sex-specific binge levels: nonbinge and 1, 2, and 3 or more times the binge threshold (levels I, II, and III). Multivariable analyses examined the odds of clinically high levels of lipids and LFTs across binge levels. Analyses were performed from December 3, 2018, to January 30, 2019. Main Outcomes and Measures: Serum levels of high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol, total cholesterol, triglycerides, alanine aminotransferase, aspartate aminotransferase, and γ-glutamyltransferase. Results: A total of 2065 participants underwent protocol screening; 1519 with data available on alcohol consumption, body mass index, lipid levels, and LFT levels were included in the final analyses. Mean (SD) age was 39.7 (12.1) years; mean (SD) body mass index was 26.6 (5.1); 978 (64.4%) were male; 718 (47.3%) were white; and 578 (31.1%) consumed alcohol at the nonbinge level, 321 (21.2%) at level I, 239 (15.7%) at level II, and 318 (25.1%) at level III. High-intensity binge drinking was associated with 2- to 8-fold increased odds for clinically high levels of HDL-C, total cholesterol, triglycerides, and all LFTs (eg, for HDL-C: level III odds ratio [OR], 8.65; 95% CI, 4.75-15.77 and for γ-glutamyltransferase: level III OR, 8.21; 95% CI, 5.90-11.43). Increased HIBD frequency (days consuming at levels II and III) was associated with increased odds for clinically high levels of HDL-C, total cholesterol, and all LFTs (per unit increase in days consuming at the respective binge level) (eg, for HDL-C: level II OR, 1.025; 95% CI, 1.014-1.036 and level III OR, 1.033; 95% CI, 1.019-1.047 and for γ-glutamyltransferase: level II OR, 1.028; 95% CI, 1.019-1.037 and level III OR, 1.033; 95% CI, 1.019-1.047). Conclusions and Relevance: High-impact binge drinking was significantly associated with increased odds for clinically high levels of lipids and LFTs. Given that HIBD is increasingly common among US adults, targeted interventions aimed at reducing HIBD may have important health benefits.

PCSK9 is Increased in Cerebrospinal Fluid of Individuals With Alcohol Use Disorder.

BACKGROUND: Recent studies have shown that alcohol use affects the regulation and expression of proprotein convertase subtilisin/kexin 9 (PCSK9). While a major role of PCSK9 in hepatic function and lipid regulation has been clearly established, other pleiotropic effects remain poorly understood. Existing research suggests a positive association between PCSK9 expression in the brain and psychopathology, with increased levels of PCSK9 in the cerebrospinal fluid (CSF) of individuals with dementia and epigenetic modifications of PCSK9 associated with alcohol use disorder (AUD). In this study, we hypothesized that chronic alcohol use would increase PCSK9 expression in CSF. METHODS: PCSK9 levels in CSF were measured in individuals with AUD (n = 42) admitted to an inpatient rehabilitation program and controls (n = 25). CSF samples in AUD were assessed at 2 time points, at day 5 and day 21 after admission. Furthermore, plasma samples were collected and measured from the individuals with AUD. RESULTS: PCSK9 in CSF was significantly increased in the AUD group at day 5 and day 21 compared to the controls (p < 0.0001). Plasma PCSK9 levels were correlated positively with CSF PCSK9 levels in AUD (p = 0.0493). CONCLUSIONS: Our data suggest that PCSK9 is elevated in the CSF of individuals with AUD, which may indicate a potential role of PCSK9 in AUD. Additional studies are necessary to further elucidate the functions of PCSK9 in the brain.