Global DNA promoter methylation in frontal cortex of alcoholics and controls

Abstract

To determine if ethanol consumption and alcoholism cause global DNA methylation disturbances, we examined alcoholics and controls using methylation specific microarrays to detect all annotated gene and non-coding microRNA promoters and their CpG islands. DNA was isolated and immunoprecipitated from the frontal cortex of 10 alcoholics and 10 age and gender-matched controls then labeled prior to co-hybridization. A modified Kolmogorov-Smirnov test was used to predict differentially enriched regions (peaks) from log-ratio estimates of amplified vs input DNA. More than 180,000 targets were identified for each subject which correlated with >30,000 distinct, integrated peaks or high probability methylation loci. Peaks were mapped to regions near 17,810 separate annotated genes per subject representing hypothetical methylation targets. No global methylation differences were observed between the two subject groups with 80% genetic overlap, but extreme methylation was observed in both groups at specific loci corresponding with known methylated genes (e.g., H19) and potentially other genes of unknown methylation status. Methylation density patterns targeting CpG islands visually correlated with recognized chromosome banding. Our study provides insight into global epigenetic regulation in the human brain in relationship to controls and potentially novel targets for hypothesis generation and follow-up studies of alcoholism.

Fig. 1

Roche NimbleGen Human DNA Methylation 2.1M Deluxe Promoter Array Analysis Protocol.

Fig. 2

Observed vs expected number of peaks by diagnostic category. Expected values are based upon the proportion of total peaks standardized by the relative length of the chromosome in base pairs. Simple Analysis of Variance found no significant differences between alcoholic and control subjects for any chromosome (p<0.05). A 2-sided t-test found a significant difference between observed and expected values (p<0.05) for all chromosomes in both groups except for chromosome 7.

Fig. 3

Selected plots of methylated DNA loci with chromosome banding patterns and DNA base pair assignments.

Fig. 4

Distribution and assortment of hypothetical gene targets based upon methylation level. Human DNA methylation data obtained using the Roche NimbleGen Human DNA Methylation 2.1M Deluxe Promoter Array grouped by diagnosis and level of methylation [low (2 to ≤3); medium (>3 to <4); high (≥4 to <6); extreme (≥6)] based upon mean peak score per gene. Labels indicate the level of gene methylation; number of genes per category; and percentage of genes. A total of 17,810 genes are reported for N = 10 subjects per group. Extreme methylation accounts for <1% of all methylated genes.