Association of ADH1B and ALDH2 gene polymorphisms with alcohol dependence RESEARCH initial sensitivity to alcohol by virtue of their genetic make-up (ie the presence of the ALDH2*2 allele) are discouraged from drinking excessive amounts of alcohol.28 Assanangkornchai et al.29 found that ALDH2*2/*2 subjects are at minimum risk for developing AD and alcohol-related organ damage, compared with those with the ALDH2*1/*2 and ALDH2*1/*1 genotypes. A signiﬁcant variation in the allele frequency of the Glu487Lys polymorphism has been observed across different populations worldwide, with almost com- plete absence of the ALDH2*2 allele in Caucasians, Africans and Americans (single nucleotide poly- morphisms [SNP] database; National Center for Biotechnology Information) and a relatively higher frequency in East Asians. Studies on Chinese and Korean populations found that alcoholics are less likely to have the ALDH2*2 allele than con- trols.23,30,31 Pertinent to the East Asian population, approximately 2 – 12 per cent of Chinese and Korean individuals are homozygous for the ALDH2*2 allele (ALDH2*2/*2 genotype) and appear to be almost completely protected.23 The Glu487Lys polymorph- ism, however, is found to be monomorphic in Indian populations from Madhya Pradesh, Maharashtra and Andhra Pradesh.28 Due to the paucity of Indian data with regard to ADH1B/ALDH2 gene polymorphisms, the present study aimed to characterise the Arg47His and Glu487Lys polymorphisms in Indian subjects with AD and to establish the genotype/phenotype cor- relation (if any). Materials and methods Subjects In this study, 174 alcohol-dependent male subjects satisfying DSM 1V criteria derived from the Diagnostic Interview for Genetic Study (DIGS), and aged between 18 and 60 years of age, from the National Drug Dependence Treatment Center at the All India Institute of Medical Sciences, were recruited. Subjects with a history of any other sub- stance abuse/dependence were excluded. The study was carried out in accordance with the Declaration of Helsinki (2000) of the World Medical Association. Ethical committee clearance was obtained prior to recruitment of subjects. All sub- jects gave written informed consent to participate in the study. Clinical details, including ethnicity, family history, age at ﬁrst use of alcohol, quantity of alcohol consumed (g/day), duration of alcohol use, duration of AD, age at onset of dependence, presence/absence of delirium and any other psy- chiatric or physical illness were assessed and recorded. Serum proteins (albumin, bilirubin, glu- tamic oxaloacetic transaminase [SGOT] and gluta- mic pyruvic transaminase [SGPT]) were estimated on an autoanalyser, using biochemical kits from Boehringer Mannheim (Mannheim, Germany). Genomic DNA extraction and genotyping Peripheral blood (6 ml) was collected in sterile ethyl- ene diamine tetra-acetic acid (EDTA)-coated vacutai- ners and stored at 4ºC until processing for DNA extraction was carried out. Genomic DNA was extracted from peripheral blood leukocytes using the standard phenol – chloroform method. SNP genotyp- ing was done using the polymerase chain reaction (PCR) – restriction fragment length polymorphism (RFLP) approach. Forward and reverse primer sequences used for the ampliﬁcation of the ADH1B Arg47His SNP were 50-AATCTTTTCTGAA TCTGAACAG-30 and 50-GAAGGGGGGTCACCA GGTTGC-30, respectively; and primer pairs for ALDH2 Glu487Lys were 50-CAAATTACAGGGT CAAGGGCT-30 (forward) and 50-CCACACTCA CAGTTTTCTCTT-30 (reverse). After ampliﬁcation, the PCR product (20 ml) was divided into two equal parts. 10 ml of the ampliﬁed product was incubated at 378C for two hours with NmuCI and Mbo II restriction enzymes (RE) for ADH1B and ALDH2, respectively. DNA fragments in the RE digested and undigested mixture were analysed on 10 per cent polyacrylamide gel electrophoresis (PAGE) using one per cent tris-borate-EDTA (TBE) buffer and sized with the help of known DNA markers. For the ADH1B polymorphism, the presence of 95 base pair (bp) and 65 bp restriction fragments indicated the presence of wild-type Arg and mutant His alleles, respectively. The presence of 134 bp and 123 bp # HENRY STEWART PUBLICATIONS 1479–7364. HUMAN GENOMICS. VOL 3. NO 3. 213–220 APRIL 2009 215