A total of 305 male participants provided blood samples and completed the AUDIT questionnaire. The mean age of the study population was 45.1 ± 11.1 years, ranging from 21 to 75 years. While the average age of the treated alcoholics (44.3 years), problem drinkers (47.5 years) and control groups (44.2 years) at the time of interview did not differ significantly (p ≥ 0.05), the average weight (kg, p = 0.026) but not body mass index (kg/m2, p = 0.19) of the treated alcoholics (77.6 kg, 24.7 kg/m2) and control (81.3 kg, 25.3 kg/m2) groups did. Among the general population (n = 201), 15.9 per cent displayed hazardous patterns of alcohol consumption and 31.8 per cent scored ≥ 20 on the AUDIT and were classified in our analyses as problem drinkers (since they generally experienced both dependence symptoms and alcohol-related problems) and were also included in the problem/dependent group.
The problem drinkers and treated alcoholics had marked differences (p ≤ 0.0001) in their mean responses for all ten AUDIT items compared with the controls (Table ). Although all groups tended to consume alcoholic beverages on a weekly basis (item 1; 55 per cent of controls and an average of 77 per cent for the problem and dependent drinkers), the majority (82 per cent) of problem drinkers and treated subjects consumed an average of six or more drinks per session (item 2) compared with 13 per cent of controls (on average, the control group imbibed three or four drinks per session). This trend was similar for the frequency with which subjects consumed six or more drinks per session (item 3). While these significant differences in alcohol consumption behaviour (items 1-3) were observed between the control and problem/treated groups, an unambiguous distinction from the controls was seen with respect to those items related to dependence symptoms (items 4-6) and alcohol-related problems (items 7-10). For these seven items, the mean response was ≤0.5 and ≥2.2 for controls and problem/ treated groups, respectively, which on a 'occurs on a monthly or more' basis represents an affirmative response by ≤ 2 per cent and ≥ 63 per cent of the respective group subjects.
Alcohol Use Disorder Identification Test (AUDIT) response, mean and standard deviation per item, in the Finnish control drinkers, problem drinkers and treated alcohol-dependent groups, based on AUDIT score and/or alcohol treatment status
The physical locations of the eight SNPs across the ALDH1A1
gene are schematically presented in Figure . Frequencies of the ALDH1A1
genotypes were in general in Hardy-Weinberg equilibrium (HWE), except for rs595958 in intron 1 (p
= 0.0008), and are listed in Table . Minor allele frequencies within the control sample were similar to those observed in the Centre d'Etude du Polymorphisme Humain (CEPH) sample (data not shown) of the International HapMap Project [22
]. The extent of LD between the eight SNPs is presented in Figure . LD was strongest between markers rs610529 (intron 8) and rs13959 (exon 3), with one haplotype block structure detected by Haploview [19
] between these SNPs. The effective number of independent SNPs was 6.0, as determined by single nucleotide polymorphism spectral decomposition (SNPSpD) [23
Figure 1 A schematic representation of the human ALDH1A1 gene structure and linkage disequilibrium (LD). The gene structure of ALDH1A1 is shown with exons numbered and relative exon size denoted by the width of the vertical bars. The eight SNPs analysed in this (more ...)
Association analysis of eight cystolic aldehyde dehydrogenase (ALDH1A1) polymorphisms with AUDIT score
We observed nominally significant differences between the genotypic and/or allelic frequencies and mean AUDIT scores for three (rs348479 in the 3' UTR, rs610529 in intron 8 and rs348449 in intron 2) of the eight SNPs genotyped in ALDH1A1. The genotypic distribution and allelic frequencies of the three SNPs are presented in Table .
Genotypic and allelic analysis of the ALDH1A1 rs348479, rs6l0529 and rs348449 polymorphisms in Finnish controls, problem drinkers and clinically treated alcoholic subjects
The rs348479 T allele was more frequent in subjects with higher AUDIT scores, where the mean AUDIT scores observed for the rs348479 G and T alleles were 17.8 and 19.7, respectively (Table ) and the frequency of the T allele was 82 per cent in controls compared with 85 per cent (Table ) in the treated alcoholic drinkers. In the analysis of the entire sample (n = 305), the association of the T allele with the AUDIT score approached nominal significance (p = 0.08 for general association) and the estimated genotypic AUDIT score means were suggestive of a dominant genetic model (p = 0.025) with respect to the T allele, where the AUDIT score is highest in those individuals with one or two copies of the T allele compared with individuals with two copies of the G allele (mean AUDIT score of 11.0, 19.5 and 19.3 with zero, one and two rs348479 T alleles, respectively). The rs348479 genotypic frequencies were significantly different between the controls and treated alcoholics (p = 0.019); however, case-control odds ratio calculations did not indicate significant allele frequency differences between the controls and treated alcoholics (Table ). Furthermore, the absence of significant allelic and genotypic differences between the controls and problem drinkers may indicate that rs348479 is involved in risk for alcohol dependence rather than increased or problem alcohol consumption. Nonetheless, the results of this study should also be interpreted in the context of multiple testing, since, correcting for the number of independent SNPs alone, a Bonferroni-corrected p-value of 0.0083 (0.05/6) would be required for an association to be declared significant.
The rs610529 polymorphism in intron 8 was associated (p = 0.027 and p = 0.013 for the general and best model of association, which is based upon a recessive mode of inheritance for the G allele) with the AUDIT score. The genotypic AUDIT score means were 20.5, 19.1 and 14.7 for zero, one and two rs610529 G alleles, respectively (Table ). Although there were no significant differences in allelic frequencies between control and problem drinkers (OR = 0.92, 95 per cent CI 0.58-1.44), treated alcoholics were approximately 1.5 times less likely to have the G allele (OR = 0.65, 95 per cent CI 0.43-0.98) and three times more likely to be either a homozygous AA or a heterozygous AG (OR = 0.32, 95 per cent CI 0.12-0.84) (Table ). As with rs348479, these analyses provide some evidence for an association between rs6105249 and risk for alcohol dependence rather than problematic alcohol consumption behaviour.
The rs348449 polymorphism was nominally associated (p = 0.043 for the dominant model of inheritance; p = 0.13 for general association) where the AUDIT score genotypic means were 18.9, 24.4 and 22.0 for zero, one and two G alleles, respectively (Table ). Significant differences in both the rs348449 genotypic and allelic frequencies were detected between the controls and the problem drinkers (Table ). The frequency of the minor allele G was 1 per cent in controls compared with 7 per cent in the problem drinkers and 3.4 per cent in treated alcoholics. No G/G homozygotes were observed in the control and treated alcoholics groups. Problem drinkers were 7.8 times more likely to have the rs348449 G allele (OR = 7.87, 95 per cent CI 1.67-37.01, p = 0.003) and 7.4 times more likely to have the GG or GA genotype (p = 0.017). By contrast, the allelic and genotypic distributions in the treated alcoholics and control groups were not significantly different. Therefore, rs348449 may play a role in the consumption of high levels of alcohol (which can lead to dependence symptoms and alcohol-related problems) rather than the development of alcohol dependence.
Since the age of the control subjects ranged between 24 and 69 years, it is possible that a subset of the younger controls could develop alcohol dependence later in life. Therefore, we divided the controls into an older cohort, aged over 40 years (n = 66), and repeated the allelic and genotypic analyses. With each SNP, we observed the same direction of, and similar levels of, association (data not shown).
Two- and three-locus haplotypic association between ALDH1A1 SNP genotypes and the quantitative AUDIT score is graphically presented in Figure . Using Qtphase and a sliding window approach, the significance of association increased from p = 0.014 for the single rs610529 SNP analysis to 0.0015 (likelihood ratio statistic [LRS] = 15.47, degrees of freedom [DF] = 3) for rs610529-rs2288087 haplotype analysis, where the two SNPs are in high LD (D' = 0.97, r2 = 0.60). Three common haplotypes (≥1 per cent frequency) were observed, totalling 99.4 per cent of the observed haplotypes. Two common haplotypes, '1-1' (10.4 per cent frequency) and '1-2' (35.9 per cent frequency) had mean AUDIT scores of 23.42 (χ2 = 8.37, p = 0.0038) and 17.64 (χ2 = 7.3, p = 0.0070), respectively. The third common haplotype ('2-1', 53.1 per cent) was not significantly associated (χ2 = 0.4, p = 0.55) with the AUDIT score (mean score = 19.59). The addition of a third neighbouring SNP, rs13959, did not increase the strength of association (p = 0.0024).
Plot of minus log of p-value for Qtphase single-point and sliding window haplotype analysis of association between eight ALDH1A1 SNPs and the AUDIT score, a quantitative measure of alcohol consumption behaviour.