Hypermethylation of Proopiomelanocortin and Period 2 Genes in Blood Are Associated with Greater Subjective and Behavioral Motivation for Alcohol in Humans

Abstract

Background: Epigenetic modifications of a gene have been shown to play a role in maintaining a long-lasting change in gene expression. We hypothesize that alcohol's modulating effect on DNA methylation on certain genes in blood is evident in binge and heavy alcohol drinkers and is associated with alcohol motivation.

Methods: Methylation-specific polymerase chain reaction (PCR) assays were used to measure changes in gene methylation of period 2 (PER2) and proopiomelanocortin (POMC) genes in peripheral blood samples collected from nonsmoking moderate, nonbinging, binge, and heavy social drinkers who participated in a 3-day behavioral alcohol motivation experiment of imagery exposure to either stress, neutral, or alcohol-related cues, 1 per day, presented on consecutive days in counterbalanced order. Following imagery exposure on each day, subjects were exposed to discrete alcoholic beer cues followed by an alcohol taste test (ATT) to assess behavioral motivation. Quantitative real-time PCR was used to measure gene expression of PER2 and POMC gene levels in blood samples across samples.

Results: In the sample of moderate, binge, and heavy drinkers, we found increased methylation of the PER2 and POMC DNA, reduced expression of these genes in the blood samples of the binge and heavy drinkers relative to the moderate, nonbinge drinkers. Increased PER2 and POMC DNA methylation was also significantly predictive of both increased levels of subjective alcohol craving immediately following imagery (p < 0.0001), and with presentation of the alcohol (2 beers) (p < 0.0001) prior to the ATT, as well as with alcohol amount consumed during the ATT (p < 0.003).

Conclusions: These data establish significant association between binge or heavy levels of alcohol drinking and elevated levels of methylation and reduced levels of expression of POMC and PER2 genes. Furthermore, elevated methylation of POMC and PER2 genes is associated with greater subjective and behavioral motivation for alcohol.

Keywords: Alcoholism; DNA Methylation; Period 2; Proopiomelanocortin.

Fig. 1.. Changes in alcohol consumption and craving rating in the moderate binge and heavy social drinkers during the laboratory sessions.

Across all three imagery provocation conditions and after presentation of the discrete alcoholic beer cue on each day, the binge and heavy social drinkers consumed significantly more beer during the alcohol taste test compared with the moderate social drinkers [F(2, 45) = 18.8, p=.0001], with moderate<binge<heavy< (p’s<.05). (Fig. A). Across all three imagery context sessions (stress, alcohol cue and neutral relaxing cue), alcohol craving ratings were significantly higher for the binge and heavy compared to the moderate social drinking groups, immediately following imagery (I) context manipulation [F(2, 45) = 9.01, p=.004; moderate<binge, p<.05; moderate<heavy, p<.01; Fig. B), and following presentation of discrete beer cues (R1b) [F(2, 45) = 11.38, p=.002; moderate<binge, p<.01; moderate<heavy, p<.01; Fig. C).

Fig. 2.. Changes in period 2 and proopiomelanocortin gene expression and DNA methylation in the moderate, binge and heavy social drinkers.

PER2 (A) and POMC gene expression (B) and PER2 (C) and POMC gene methylation (D, E) levels in each of the three drinking groups are shown. Gene expression levels were measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Gene methylation levels were measured by methylation specific PCR (MSP) and was represented as relative DNA methylation (C & D). POMC DNA methylation was additionally verified by pyrosequencing of the methylated DNA within the promoter area of the gene (Fig. E), Pyrosequencing assay for PER2 was not successful possibly because of high density of CpG residue. Data are represented as Mean ± SEM. Number of samples in each group is shown between brackets under the group heading on the X axis or within the figures. Statistically significant differences between groups are shown by lines with p values on the top of bar graphs.

Fig. 3.. Correlations between the hPER2 and POMC methylation with the A+B amount of alcoholic beer intake and the level of alcohol craving in the laboratory experiment across drinking groups.

Significant positive correlation of PER2 DNA methylation (met/unmet) (A) and POMC DNA methylation with A+B alcoholic beer intake across subjects (p’s<0.01) (B). Significant positive correlation of PER2 DNA methylation (C) and POMC DNA methylation (D) with provoked alcohol craving across I and R1B timepoints prior to alcohol taste test across all subjects (p’s<0.0001).