Association between Alcoholism and the Genetic Polymorphisms of the GABA_A Receptor Genes on Chromosome 5q33-34 in Korean Population

Subjects and clinical assessments

The study subjects consisted of 162 male alcoholics who were hospitalized in alcoholism treatment institutes (mean age=45.9 yr, SD=7.0 yr) and 172 control subjects (mean age=43.1 yr, SD=11.4 yr). The study was approved by the Institutional Review Board of Gyeongsang National University.

The 334 participants were interviewed by two psychiatrists. Individuals were diagnosed with alcohol dependence using DSM-IV criteria (15), and alcoholic subjects who had major psychiatric histories, including schizophrenia, major depressive disorders, bipolar disorders, anxiety disorder and organic mental disorder, or who had difficulties participating in the objective interview procedures due to cognitive and communication impairments, were excluded from the study.

The control subjects were recruited from the staff and the men who visited the Health Examination Center at Gyeongsang National University Hospital in Jinju City. All the subjects were asked by a physician to participate in the study, and a written informed consent was obtained from every subject. None of control subjects had a family history of alcoholism and a lifetime history of any other psychiatric, neurological or medical disorders. The control and alcoholic subjects were age-matched.

Patients were interviewed to collect demographic data, including age, gender, occupation, educational level, economic status, marital status and alcohol related questions that inquired about the subjects' alcohol drinking history and the alcohol-associated problematic history. Several standardized research scales were used for the clinical assessment of alcoholism, including the Alcohol Dependence Scale (ADS) (16), the Beck Depression Inventory (BDI) (17), and the Obsessive-Compulsive Drinking Scale (OCDS) (18). All of the above scales had been already standardized in Korean language.

Genomic DNA extraction

We collected a 3-4 mL venous blood sample, which were poured into 7.5% potassium-EDTA containing tubes, were extracted immediately or stored at -20℃ for later use. Genomic DNA was extracted by using QIAmp DNA blood midi kit according to the manufacturer's recommendations (QIAmp DNA blood midi kit, QIAGEN Inc., CA, U.S.A.). Briefly, 200 µL of Qiagen protease and 2.4 mL of buffer AL were added to 2 mL of specimen. Samples were incubated at 70℃ for 10 min after which time 2 mL of 100% ethanol was added to each. Each mixture was vortexed and pipetted into a QIAamp Spin column, which was then centrifuged at 1,850×g for 3 min at room temperature. Samples were washed by the adding 2 mL of AW1 to each spin column, followed by centrifugation at 4,500×g for 5 min. Washing was repeated with 2 mL of AW2, followed by centrifugation at 4,500×g for 5 min. Spin columns were then placed in clean centrifuge tubes, and a 300 µL volume of AE was added to elute the DNA. Elution was accomplished with a final centrifugation step at 4,500×g for 5 min. DNA yield is determined by measuring the concentration of DNA in the eluate by absorbance at 260 nm and 280 nm, an eluate containing 50-100 ng DNA/µL. Pure DNA has an A₂₆₀/A₂₈₀ ratio of 1.6-1.9.

Determination of GABA_A α1 receptor gene polymorphism

PCR was carried out with the primers, forward (5'-GCT ATG GAT TGG TTT ATT GCC GTG TG-3') and reverse (5'-ATA ATA TTG ATG TAC TAC AGG GAC-3'). A 50 µL total volume of the reaction mixture contained the following; 100 ng genomic DNA, 30 pmole of each primer, 1.25 mM dNTP, 2.5 mM MgCl₂, and 1 unit of DNA Taq polymerase. After an initial 5 min denaturation at 95℃, the reaction mix was processed by use of a Perkin Elmer thermocycler according to the following program; 40 cycles of 20 sec at 95℃, 30 sec at 55℃, 40 sec at 72℃, with final extension for 10 min at 72℃. The PCR product (15 µL) was then electrophoresed in 2% agarose gel and visualized with ethidium bromide and ultraviolet light. If the nucleotide 15 of the last intron of the GABA_A α1 is a G, the restriction enzyme cuts the 165 bp fragments into two fragments of 141 and 24 bp. If the nucleotide 15 is an A, the PCR fragment remains uncut.

Determination of the GABA_A α6 receptor gene polymorphism

Forward exon primer, 5'-GGA GGC ACC AGT AAA ATA GAC CAG-3'and reverse intronic primer, 5'-AAT ACT GAA CAA TGG AAG ACA AAA G-3', were used to amplify a specific region of 423 bp fragment. The reaction mixture contained PCR buffer, 2.5mM MgCl₂, 1 unit of Taq DNA polymerase, 1.25 mM dNTP, 30 pmole of each primer and 100 ng of genomic DNA. After an initial 5 min denaturation at 95℃, there followed 40 cycles of PCR (denaturation, 1 min 30 sec at 95℃; annealing, 30 sec at 56℃; polymerization, 40 sec at 72℃) with final extension for 10 min at 72℃ on a Perkin Elmer GeneAmp 9600 thermocycler. The PCR product was subjected to 2% agarose gel electrophoresis and stained with ethidium bromide for viewing. Two units of Nhe I (Roche, Mannhein, Germany) were digested with 22.3 µL of the PCR products at 37℃ for 4 hr. Electrophoresis of the digest was carried out with 3% agarose gel, and the bands were visualized by ethidium bromide staining. If the nucleotide 1,519 in the non-coding region of the the GABA_A α6 receptor gene is a T, the restriction enzyme cuts the 423 bp fragments into two fragments of 257 and 166 bp. If the nucleotide 1,519 is a C, the PCR fragment remains uncut.

Determination of the GABA_A β2 receptor gene polymorphism

Forward exon primer, 5'-AAG CAC AAT GCT AGC CTA TGG TGC-3'and reverse intronic primer, 5'-GTT CAC ATA ATA AAG CCA ATA GAC GAT-3', were used to amplify a specific region of 253 bp fragment. The reaction mixture contained PCR buffer, 1.5 mM MgCl₂, 1 unit of Taq DNA polymerase, 1.25 mM dNTP, 30 pmole of each primer and 100 ng of genomic DNA. After an initial 5 min denaturation at 95℃, the reaction mix was processed by use of a Perkin Elmer thermocycler according to the following program; 40 cycles of 30 sec at 95℃, 30 sec at 56℃, 40 sec at 72℃, with final extension for 10 min at 72℃. The PCR product was subjected to % agarose gel electrophoresis and stained with ethidium bromide for viewing. Two units of Nhe I (Roche, Mannhein, Germany) were digested with 22.3 µL of the PCR products at 37℃ for 4 hr. Electrophoresis of the digest was carried out with 3% agarose gel, and the bands were visualized by ethidium bromide staining. If the nucleotide 1,412 in the last exon before the non-coding region of the GABA_A β2 receptor gene is a C, Ban I digests the 253 bp fragments into two fragments of 229 and 24 bp. If the nucleotide 1,412 is a T, the PCR fragment remains uncut.

Determination of the GABA_A γ2 receptor gene polymorphism

Forward exon primer, 5'-AAA GAT AAA AAG AAG AAA AAC CCT-3'and reverse intronic primer, 5'-CAC AGA AAA TAG AAA CAG ACT TGA-3', were used to amplify a specific region of 224 bp fragment. The reaction mixture contained PCR buffer, 1.5 mM MgCl₂, 1 unit of Taq DNA polymerase, 1.25 mM dNTP, 30 pmole of each primer and 100 ng of genomic DNA. After an initial 5 min denaturation at 95℃, the reaction mix was processed by use of a Perkin Elmer thermocycler according to the following program; 40 cycles of 30 sec at 95℃, 30 sec at 50℃, 40 sec at 72℃, with final extension for 10 min at 72℃. The PCR product was subjected to 2% agarose gel electrophoresis and stained with ethidium bromide for viewing. Two units of Nhe I (Roche, Mannhein, Germany) were digested with 22.3 µL of the PCR products at 37℃ for 4 hr. Electrophoresis of the digest was carried out with 3% agarose gel, and the bands were visualized by ethidium bromide staining. If the nucleotide 123 in the upstream intron of 24 bp exon of the GABA_A γ2 is an A, the restriction enzyme cuts the 224 bp fragments into two fragments of 103 and 99 bp. If C is present at this position, the PCR fragment does not digest.

Statistical analysis

For the case-control genetic comparisons, differences in genotype and allele frequencies between groups were evaluated by using the chi-square test or Fisher's exact test, where appropriate. Univariate comparisons of clinical data between groups were conducted with independent t-tests or ANOVA were used. The Duncan test was used for the post-hoc analysis. Since we compare genotypic and allelic distributions of four independent polymorphic markers between healthy comparison subjects and alcoholism, a Bonferroni correction was applied to correct for multiple testing level of significant was set to alpha<0.05/4=0.0125). The Hardy-Weinberg equilibrium was tested for by the chi-square test. All significance levels were set at p<0.05 and the statistical analyses were run on SPSS software, version 11.0 (SPSS, Chicago, IL, U.S.A.).

Genotype and allele frequencies of the GABA_A receptor gene polymorphisms

The genotype frequency of the polymorphisms did not deviate from that expected according to Hardy-Weinberg equilibrium (GABA_A α1, p=0.893; GABA_A α6, p=0.892; GABA_A β2, p=0.650; GABA_A γ2, p=0.918). When compared with the controls, an association between polymorphisms of the GABA_A α1 and α6 receptor gene was with alcohol dependence. Allelic distribution of polymorphisms of the GABA_A α1 and α6 receptor gene was associated with alcohol dependence. These associations remained statistically significant even after Bonferroni correction. However, there were no significant associations between alcohol dependence and the genetic polymorphisms of the GABA_A β2 and GABA_A γ2 receptor genes (Table 1).

Table 1
Genotype and allele frequencies of the GABA_A receptor gene polymorphisms on chromosome 5q33-34 in alcoholic and control subjects

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Association of the clinical characteristics with genetic polymorphisms of the GABA_A receptor genes

Genetic polymorphisms of the GABA_A α1 receptor gene were associated with the mean onset age of alcoholism and the alcohol withdrawal symptoms and mean score of the ADS. The GG genotype of the GABA_A α1 receptor gene was associated with the early onset of alcoholism and the alcohol withdrawal symptoms. The ADS mean score was significantly higher for alcoholics with the GG genotype than for the alcoholics with the AA and AG genotypes of the GABA_A α1 receptor gene. However, the GABA_A α1 receptor gene polymorphisms were not associated with the scores of the BDI and OCDS. There were no associations found between the other GABA_A receptor gene polymorphisms and the clinical characteristics of the alcoholics (Table 2).

Table 2
Clinical characteristics and genetic polymorphisms of the GABA_A receptor genes on chromosome 5q33-34

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