Cross-Generational Effects of Alcohol Dependence in Humans on HRAS and TP53 Methylation in Offspring

Figures & data

Table 1. First- and second-generation participant characteristics and recruitment source (n = 62).

Chracteristic	Alcohol dependence yes (n = 33)	Alcohol dependence no (n = 29)	Total
Age at DNA collection
– Mean	47.25	46.68	46.95
– SD	11.29	13.49	12.41
Gender (n)
– Male	33	18	51
– Female	0	11	11
Socioeconomic status (SES)^†
– Mean	38.59	46.85	42.37
– SD	12.48	11.93	12.82
Recruited risk status (n)
– High risk	33	10	43
– Low risk	0	19	19

^†SES information was available for 59 participants.

Table 2. Alcohol dependence and smoking status of second-generation parents of third generation.

	Mothers (n = 441)			Fathers (n = 441)
	Yes	No	Total	Yes	No	Total
Alcohol dependence^†	113	260	373	151	210	361
Smoker^‡	141	221	362	116	144	260

^†The AD status of 68 mothers and 80 fathers is unknown.

^‡The smoking status of 79 mothers and 181 fathers is unknown.

AD: Alcohol dependence.

Table 3. Number of relatives with alcohol dependence across generations.

	None	One	2 or >	Range	Mean (± SD)	Total
1st degree relatives with AD^†	224	162	51	0–2	0.60 (0.69)	437
2nd degree relatives with AD^†	159	66	207	0–12	1.81 (2.01)	432
Weighted relatives with AD^‡				0–8	1.49 (1.45)	441

^†First degree relatives are the offspring’s parents. Second degree includes grandparents, aunts and uncles.

^‡The weighted index counted each 1st degree relative as 1 with each 2nd degree relative counting as 0.5.

AD: Alcohol dependence.

Table 4. Number of parents and grandparents with alcohol dependence.

	None	Parent only	Parent and grandparent	Total
Male parent with AD^†	47	32	36	115
Female parent with AD^‡	124	73	29	226
Totals	171	105	65	341

^†Parent and grandparent with AD indicate father and paternal grandfather with AD.

^‡Parent and grandparent with AD indicate mother and maternal grandmother with AD.

AD: Alcohol dependence.

Table 5. Third generation participant characteristics and recruitment source (n = 441).

Age at DNA collection
– MEAN	13.4
– SD	5.6
Gender
– Male	221
– Female	220
Socioeconomic status^†
– Mean	41.0
– SD	11.8
Recruited risk status
– High risk	246
– Low risk	195
Study offspring from:
– Male adult AD proband pair	227
– Female adult AD proband pair	214

^†SES for the parents at the time of first evaluation was available for n = 440.

AD: Alcohol dependence.

Table 6. Personal use of substances prior to DNA collection for third generation participants (n = 441).

	Any use	No use	Percent without exposure	Median lifetime use for users
Alcohol^†	139	288	65.3	449.1^¶
Cigarettes^‡	65	354	80.3	365^#
Marihuana^†	60	367	83.2	106.4^††
Amphetamine^§	8	422	95.7	6.5^††
Cocaine^§	6	424	96.1	33.7^††
Opioids^§	7	423	95.9	63.7^††

^†Lifetime data not available for 14 participants who came into the study as young adults.

^‡Lifetime data not available for 12 participants.

^§Lifetime data not available for 11 participants.

^¶Number of drinks. A drink is one 6 oz glass of wine, a mixed drink with 1.5 ounces of hard liquor (e.g., gin, bourbon); or one 12 oz beer.

^#Number of packs (20 cigarettes to pack).

^††Number of separate occasions when drug was used.

Figure 1. A typical high risk pedigree is illustrated.

Black squares or circles indicate the presence of an alcohol dependence diagnosis. Gray indicates phenotype unknown. Squares indicate males; circles females.

Figure 2. The HRAS oncogene methylation in offspring without parents with alcohol dependence was compared with those with one or both parents with alcohol dependence.

Statistical analysis controlled for the age that DNA was obtained from offspring, prenatal use of substances by mothers (alcohol, drugs and cigarettes), and the personal use by the offspring up to the time that DNA was obtained. Hypomethylation of HRAS in offspring is seen in association with increased loading of parental AD (p = 0.022). This hypomethylation suggests increased expression of the oncogene.

AD: Alcohol dependence.

Figure 3. HRAS methylation in offspring was compared with those without a mother or grandmother with alcohol dependence, those with either a mother or grandmother and those having both.

Statistical analysis controlled for age at DNA collection, the mothers’ prenatal use of substances and the offspring’s personal use up to the time of DNA collection. Hypomethylation of HRAS CpG sites is seen in association with increased familial AD across generations (p = 0.003).

AD: Alcohol dependence.

Figure 4. The scatterplot illustrates the relationship between HRAS methylation and HRAS expression using RNA isolation and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR).

The parametric relationship showed an r of 0.38 with a marginally significant value, with the ordinal relationship showing good significance (p = 0.001).

Figure 5. TP53 methylation across generations.

TP53 methylation in offspring was compared with those without a father or grandfather with alcohol dependence, with those with either a father or grandfather and those having both. Statistical analysis controlled for age at DNA collection, the mothers’ prenatal use of substances and the offspring’s personal use up to the time of DNA collection. Hypermethylation of CpG sites is seen in association with increased familial AD across generations (p = 0.0068). This hypermethylation suggests lesser expression of the tumor suppressor gene.

AD: Alcohol dependence.