DNA methylation patterns in alcoholics and family controls

Table 1 Selected demographic and alcohol-use features at baseline

	Alcohol-dependent siblings (n = 25)	Non-alcoholic siblings (n = 22)
Age (yr) (SD, range)	40.7 (9.5, 24-54)	40.6 (11.4, 21-59)
Gender (M:F)	18:7	4:18
Current smoker	14	7
Race: EA, AA, NA	14, 10, 1	13, 8, 1
Hispanic Ethnicity	2	3
Past 12-mo drinking (mean ± SD)
Drinking days per week	3.91 ± 1.95b	0.95 ± 1.18
Drinks per drinking day	7.90 ± 4.01b	2.68 ± 1.34
Drinks per week	34.0 ± 30.8b	2.7 ± 3.6
1Heavy drinking days per week	2.53 ± 2.66b	0.18 ± 0.34

¹“Heavy drinking days” were defined as days in which men consumed more than 10 drinks and women more than 8 drinks.

^bt-test P < 0.001 vs non-alcoholic siblings. EA: European American; AA: African American; NA: Native American.

Table 2 Global methylation scores

	Alcohol-dependent siblings	Non-alcoholic siblings
Global methylation index for 1287 CpG sites
Mean methylation (SD)	0.282 (0.016)	0.282 (0.012)
Median methylation (SD)	0.082 (0.010)	0.079 (0.009)
Range	0.01-0.97	0.01-0.97
Sib pair difference in global methylation level at each of 1287 sites (alcoholic minus non-alcoholic sibling methylation level)
Mean difference (SD)	0.00005 (0.019)
Replicate pair difference in methylation level at each of 1287 sites (3 non-alcoholic and alcoholic siblings with replicate bisulfite treatment and methylation quantification)
Mean difference (SD)	0.0008 (0.006)	0.001 (0.003)

No global measures of methylation significantly differ between groups.

Table 3 Alcoholic minus non-alcoholic sib differences in methylation scores at 5 CpG sites with average difference in methylation frequency > 0.05

Gene symbol	Illumina CpG probe ID	Average Sib difference	Paired t-test statistic (2-tailed)	P value
LTA	820	0.083	2.46	0.021
CRK	3392	0.068	3.18	0.004
GSTM1	4902	0.054	1.82	0.081
HPN	4931	-0.084	-2.14	0.043
MSH3	2787	-0.052	-1.35	0.189

LTA: Lymphotoxin α precursor; CRK: v-crk sarcoma virus CT10 oncogene homolog isoform b; GSTM1: Glutathione S-transferase M1 isoform 1; HPN: Hepsin (transmembrane protease, serine 1); MSH3: MutS homolog 3.

Table 4 Global methylation score sib-pair differences for non-smokers vs sibship with alcoholic tobacco user (7 sib pairs)

	Sib pair concordance for smoking status
	Concordant		Discordant
	Both non-smokers	Both smokers	Proband smokes
Mean (SD) sib pair difference in methylation at each site (alcoholic minus non-alcoholic sibling methylation level)	+0.006 (0.018)	+0.010 (0.020)	-0.009 (0.025)

Mean sib pair difference for 1287 markers, ANOVA: F = 284 (df = 2), P < 0.0001. Among concordant non-smoking sib pairs, for 6 of 7 pairs alcoholic subject had higher methylation index among concordant. Five of 7 smoking sib pairs alcoholic subjects had higher methylation index. Among discordant pairs with an alcoholic smoker, 6 of 7 alcoholic subjects had a lower methylation index than non-alcoholic siblings.