Alcohol and Alcoholism Vol. 49, No. 1, pp. 1–9, 2014 Advance Access Publication 27 October 2013

doi: 10.1093/alcalc/agt163

A GABRA2 Variant Is Associated with Increased Stimulation and ‘High’ Following Alcohol Administration Albert J. Arias1,2, Jonathan Covault3, *, Richard Feinn4, Timothy Pond5, Bao-Zhu Yang1,2, Wenjing Ge1,2, Cheryl Oncken6 and Henry R. Kranzler5,7 1

Department of Psychiatry, Yale University School of Medicine, New Haven, CT, USA, 2VA Connecticut Healthcare System, West Haven, CT, USA, 3Department of Psychiatry, Alcohol Research Center, University of Connecticut School of Medicine, Farmington, CT, USA, 4Frank H. Netter School of Medicine, Quinnipiac University, North Haven, CT, USA, 5Department of Psychiatry, Perelman School of Medicine of the University of Pennsylvania, Philadelphia, PA, USA, 6 Department of Medicine, University of Connecticut School of Medicine, Farmington, CT, USA and 7VISN4 Mental Illness Research, Education, and Clinical Center, Philadelphia VAMC, Philadelphia, PA, USA *Corresponding author: Department of Psychiatry, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-1410, USA. Tel.:+1-860-679-7560; Fax: +1-860-679-1489; E-mail: [email protected] (Received 26 March 2013; first review notified 17 May 2013; in revised form 5 August 2013; accepted 1 October 2013)

INTRODUCTION Gamma-amino butyric acid (GABA) is the major inhibitory neurotransmitter in the human brain. GABAA receptors are pentameric, ligand-gated chloride channels that are expressed throughout the brain (Young and Chu, 1990). Ethanol acts on GABAA receptors to potentiate chloride ion flux by increasing the frequency and duration of channel opening (Young and Chu, 1990; Davies, 2003; Lobo and Harris, 2008). The modulation of GABA-ergic neurotransmission, and in particular GABAA receptors, by ethanol is thought to underlie many of alcohol’s pharmacologic effects in the brain, and is a major contributor to the pathophysiology of alcoholism (Krystal et al., 2006). Thus, genes encoding GABA-related proteins are functional candidates that could influence the risk of alcohol dependence (AD). Two genome-wide scans provided evidence of linkage of AD to chromosome 4p, in a region that includes a cluster of 4 GABAA receptor subunits (Long et al., 1998; Reich et al., 1998). Fine mapping of this region showed that single nucleotide polymorphisms (SNPs) throughout the 3 region of the gene encoding the GABAA alpha-2 subunit (GABRA2) and in the intergenic region between GABRA2 and GABRG1 (which encodes the GABAA gamma-1 subunit) were associated with AD in a large family-based study (Edenberg et al., 2004), while other members of the GABAA subunit gene cluster were not. Specifically, 24 of 39 SNPs in GABRA2, 6 of 8 in the intergenic region and 1 of 5 in GABRG1 were associated with AD, and haplotypic analysis showed that all 3-SNP haplotypes were significantly associated with the AD phenotype (Edenberg et al.,

2004). Covault et al. (2004) replicated this haplotypic association with GABRA2 in an independent sample. The most widely examined SNP in GABRA2, rs279858, was the only AD candidate marker that was at least modestly associated with AD in a genome-wide SNP analysis of AD (Olfson and Bierut, 2012). Taken together, these findings highlight a potential contribution of variation at the GABRA2 locus to the risk of AD. The functional significance of this association is underscored by animal studies, which have identified the GABAA alpha-2 subunit as the primary alpha subunit in limbic regions (Fritschy and Mohler, 1995; McKernan and Whiting, 1996). Further, the alpha-2 subunit is key among the predominant alpha subunits mediating the anxiolytic (Low et al., 2000) and myorelaxant (Crestani et al., 2001) effects of benzodiazepines and the hypnotic, but not sedative, effects of combined exposure to alcohol and benzodiazepines (Tauber et al., 2003). Molecular studies of the functional significance of these polymorphisms are lacking, but there is evidence supporting an association of these genetic variants with the acute subjective and physiologic responses to alcohol under laboratory conditions. In a study combining an alcohol challenge and administration of the medication finasteride or a placebo, the GABRA2 SNP rs279858 genotype moderated the subjective response to alcohol and interacted with the effect of finasteride in 27 healthy social drinkers (Pierucci-Lagha et al., 2005). This within-subject comparison did not include a placebo alcohol control. Subjects homozygous for the more common A allele showed significantly greater activation on central stimulant scores of the Alcohol Sensation Scale (SS) (Maisto et al., 1980) and greater stimulation on the Biphasic Alcohol

© The Author 2013. Medical Council on Alcohol and Oxford University Press. All rights reserved

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Abstract — Aims: Variation in genes encoding GABAA receptor subunits has been implicated in the risk of alcohol dependence (AD). We sought to replicate and extend previous findings of a moderating effect of single nucleotide polymorphisms (SNPs) in GABRA2 (which encodes the GABAA α-2 subunit) on the subjective effects of alcohol by examining SNPs in this and the adjacent GABRG1 gene on chromosome 4. Methods: Fifty-two European-Americans (22 males, 28 light drinkers and 24 heavy drinkers) completed 3 laboratory sessions, during which they drank low-dose, high-dose, or placebo alcohol prior to undergoing periodic assessments of stimulation, sedation and drug enjoyment. We genotyped subjects for three SNPs previously associated with AD: rs279858 in GABRA2, and rs7654165 and rs6447493 in GABRG1. Results: Two SNPs were associated with altered stimulatory effects of alcohol as measured on the Biphasic Alcohol Effects Scale, (rs279858: P = 0.0046; rs6447493: P = 0.0023); both effects were in the opposite direction of previous findings. Carriers of the rs279858 C allele experienced greater stimulation from alcohol. Further inspection of the rs6447493 interaction did not support a pharmacogenetic effect. The effects of rs279858 (but not the other two SNPs) on items from a secondary outcome measure, the Drug Effects Questionnaire (DEQ), were significant. Higher ratings by individuals with the C allele were observed on the DEQ items ‘feel the alcohol effect’ (P < 0.001), ‘like the alcohol effect’ (P < 0.001) and feel ‘high’ (P < 0.001). Conclusion: We did not find that the GABRG1 SNPs rs7654165 and rs6447493 moderated the effects of alcohol. Greater stimulatory and euphoric effects of alcohol in carriers of the rs279858 C allele may, in part, explain the previously reported association of this allele with AD.

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sedative effects following alcohol administration. We also examined the effects of two SNPs in the adjacent GABRG1 gene that have been associated with AD (Covault et al., 2008, Enoch et al., 2009). Finally, we explored a secondary measure of subjective response, i.e. the hedonic value of alcohol, hypothesizing that subjects with AD-associated alleles would report greater hedonic effects of alcohol. METHODS Study Design The study utilized a double-blind, within-subject design. Each subject received three different doses of alcohol ( placebo, low or high), one of each during three experimental sessions that took place, on average, 4 weeks apart. The high dose of alcohol was calculated by weight and sex to target a breath alcohol concentration (BrAC) of 100 mg% (0.10%), while the low dose targeted 40 mg% (0.04%). The volume of alcohol consumed at each session depended upon each subject’s body weight and sex (for the low- and high-dose sessions, respectively, men received 0.5 and 1.0 g/kg, and women received 0.4 and 0.8 g/kg). The order of beverages for each individual was determined using a balanced Latin Square design to avoid an effect of session order. Procedures Placebo drinks had a small volume of 1% ethanol floated on top to provide the odor of alcohol, and all drinks were served wrapped with alcohol-soaked gauze around the cup to disguise the placebo drinks. Drinks were mixed immediately before serving, so that subjects could taste and smell the alcohol floated on the placebo drink before it was diluted by the juice mixture. To control for expectancy effects, subjects were told that they might or might not be given alcohol (Martin and Sayette, 1993). Among women, experimental sessions were held during the follicular phase of their menstrual cycle (i.e. 5–9 days following the onset of menstruation). To ensure comparability across sex, the laboratory sessions were also separated by 1 month for men. The subjects fasted from solid foods beginning at midnight (for more consistent alcohol absorption), and were told to abstain from alcohol, nicotine and other drugs (caffeine not included) during the 24 h prior to each laboratory session. The subjects arrived at ~9:00 AM on the day of the lab session. At 10:00 AM, they ate a light breakfast and at 10:30 they completed the pre-alcohol subjective ratings and baseline physiologic measures were obtained. The first standard drink of alcohol or placebo beverage was administered at ~11:00 AM. Subjects had 36 min to consume the three beverages (i.e. one drink was administered every 12 min). Subjects The subjects were recruited by advertisements in the Greater Hartford, CT region, including at nearby colleges and universities. The Institutional Review Board of the University of Connecticut Health Center approved the study protocol and informed consent form (study #06-162S-1). All subjects gave informed consent to participate and were paid to participate. Because of our primary interest in rs279858, we oversampled

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Effects Scale (BAES) (Martin et al., 1993). Finasteride attenuated these effects to a greater extent in A-allele homozygotes than carriers of the G allele that had previously been associated with AD. (The GABRA2 gene has a reverse sense to the chromosome 4 reference forward strand. Some reports refer to the RNA base variation (A vs. G) while others refer to the chromosome 4 forward strand DNA sequence variation base (T vs. C) for rs279858. The RNA sense minor G allele is equivalent to the genomic forward strand minor C allele reported in this study, and both represent the alcohol dependence risk allele at rs279858.) Finasteride also attenuated effects on the anesthetic subscale of the SS more in A-allele homozygotes than G-allele carriers. Subjects homozygous for the A allele also had higher scores on the gastrointestinal subscale of the SS, and reported greater activation on the BAES stimulant subscale. Overall, G-allele carriers showed less sensitivity to the effects of alcohol, consistent with findings from population genetics studies showing this allele to be associated with AD. Roh et al. (2011) studied the effects of SNPs in GABRA2 and ALDH2 on the subjective response to alcohol in 110 Japanese subjects, consisting mostly of social drinkers. For 3 GABRA2 SNPs (rs279858, rs279869 and rs279837), the major alleles were associated with a greater subjective response to alcohol. The effects of rs279858 and rs279869, but not rs279837, were moderated by ALDH2 genotype. Uhart et al. (2012) examined the effect of GABRA2 variation on responses to an oral alcohol challenge in EuropeanAmericans. They found no association on their primary outcome measure (sedation on the BAES), but found that minor alleles of individual SNPs (including rs279858) associated with decreased effects of alcohol on secondary subjective outcomes. Further, a haplotype analysis showed significant moderating effects of GABRA2 on both primary and secondary outcome measures. Kareken et al. (2012) used a functional imaging paradigm to study the effects of the GABRA2 SNP rs279871. In their study, 36 subjects underwent alcohol cue exposure during alcohol intoxication or placebo alcohol conditions. Subjects homozygous for the A allele that was previously associated with AD reported significantly less ‘high’ and ‘intoxication’ with alcohol on the Subjective High Assessment Scale. Subjects homozygous for the A allele showed greater activation with an alcohol cue in the frontal and medial frontal cortical areas of the brain than did heterozygotes, while heterozygotes showed greater activation in the ventral tegmentum. The findings were interpreted as showing that GABRA2 variation moderates inter-individual differences in alcohol-related reward processing. Although the available evidence supports GABRA2 variation as contributing to the risk of AD, findings from alcohol challenge laboratory studies are inconsistent, and the contribution of GABRA2 variants to the pathophysiology of the disorder remains unknown. The findings from some, but not all, alcohol administration studies show that the GABRA2 variant rs279858 is associated with altered stimulation from alcohol ( possibly influencing the level of response to alcohol-induced conditioning) and alcohol-induced positive mood, i.e. the enjoyment of alcohol. In this study, we examined the moderating effects of rs279858 in GABRA2 on the subjective response to alcohol compared with a placebo drink. We hypothesized that carriers of the GABRA2 rs279858 allele previously associated with AD would show reduced subjective CNS stimulatory and

GABRA2 variation and the reinforcing effects of alcohol

subjects homozygous at this locus by enrolling approximately half of the heterozygous subjects and 100% of homozygotes. A total of 161 subjects were screened, of whom 53 were excluded based on GABRA2 genotype. An additional 42 subjects were screen failures or withdrew their participation. In total, 66 subjects completed at least 1 laboratory session and 58 subjects completed all 3 laboratory sessions. We limited the analysis described here to the 52 EuropeanAmerican completers (2 Hispanics, 30 women). Representation of other population groups (1 Asian, 5 African Americans) in the sample was insufficient to analyze the genetic associations with alcohol response, as the markers examined are tag SNPs for unknown functional variants associated with AD.

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AD (Covault et al., 2004, 2008). Rs279858 is a synonymous SNP in exon 5 of GABRA2; the C allele was associated with AD in European-American populations. Rs7654165 is 1 kb 5 of the GABRG1 transcription start site; the T allele was associated with AD in European-American populations (Covault et al., 2008; Enoch et al., 2009). We also genotyped subjects for the rs6447493 (C/T) SNP, which is in the 3 UTR region of GABRG1, the minor C allele at this locus was associated with AD (Enoch et al., 2009).

Genotyping Using primers and TaqMan probes described previously (Covault et al., 2008), subjects were genotyped at one tag SNP for each of the two haplotype blocks in the GABRA2/GABRG1 gene region, which we previously found to be associated with

Data analysis Because we oversampled homozygotes, we did not test for Hardy–Weinberg equilibrium. Linear mixed-effects models were used to detect the effect of genotype on BrAC and subjective responses. Six time points were included in the analysis

Fig. 1. Timeline of events in the laboratory sessions. VS, vital signs (heart rate and blood pressure); BrAC, breath alcohol concentration; SRM, subjective response measures.

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Inclusion/exclusion criteria Subjects were healthy volunteers, 21–45 years old, with a body mass index between 18.5 and 32.5, and body weight