The seven GAL SNPs were polymorphic in all populations and interpopulation differences in allele frequencies were observed for most of the SNPs (). In general, allele frequencies were similar within both Caucasian populations. Likewise, apart from SNPs 5 and 6, there was little variation between the two American Indian tribes. A single haplotype block spanning GAL (at least 12 kb in all populations) was observed (). In the GAL haplotype block region, D’ averaged 82, 93, 90, 98 and 98 in the African Americans, US Caucasians, Finnish Caucasians, Plains Indians and Southwest Indians, respectively. Perhaps more importantly, the median D’ values within those five populations’ haplotype blocks were 79, 94, 93, 100 and 100, respectively, meaning that most of the SNPs were in very high LD. The greatest heterogeneity is to be expected in African Americans, the population studied with the highest contribution from the ancestral African population, and indeed this proved to be the case; there was some disruption of LD within the haploblock in African Americans ().
Haplotype frequencies in the five control populations are shown in . For each population, two to five common (≥0.05) haplotypes accounted for most of the total: 76% of African-American haplotype diversity and 92–98% of the two Caucasian and two American Indian populations’ haplotype diversity. The number of common (≥0.05) haplotypes in the region genotyped were: 5, 4, 4, 4 and 2 for African Americans, US Caucasians, Finnish Caucasians, Plains Indians and Southwest Indians, respectively. For each population, a smaller group of tag SNPs sufficient to maximize genetic information content within the GAL
region was selected from the larger panel of seven SNPs. The required number of tag SNPs varies according to the haplotype diversity of the region (and population) and the information content of the SNPs available. For GAL
, a total of four tag SNPs (SNPs 1, 2, 4 and 5) was generated by the SNPTagger program41
and used to investigate the association between GAL
and alcoholism/anxiety in Finnish Caucasians and Plains Indians.
Frequencies of haplotypes derived from seven GAL SNPs in 65 individuals from US Caucasians, Finnish Caucasians, African Americans, Plains Indians and Southwest Indians
There were four common haplotypes ( > 5% frequency) generated by the four tag SNPs (). Haplotype A was most abundant in both populations. Haplotype B differed by only one allele from haplotype A. Haplotypes C and D differed by only one allele from each other. Thus, haplotypes A and B derived from the same ancestral haplotype and similarly haplotypes C and D originated from a common ancestral haplotype (). Therefore, it was considered rational to analyze haplotypes A and B together as one group and haplotypes C and D together as another group where necessary because of small cell sizes.
(a—c) Frequencies of haplotypes derived from GAL tag SNPs (1,2, 4 and 5) in nonalcoholics and alcoholics
Haplotype and diplotype associations with alcoholism in men
There was a haplotype association with alcoholism in both the Finnish and Plains Indian men (Finns: χ2 = 23.4, 3 df, P < 0.001; Plains Indians: χ2 = 8.0, 3 df, P=0.045). Haplotypes A and B, differing only by SNP 5 alleles, were more common in alcoholics, whereas haplotypes C and D, differing only by SNP 2 alleles, were more common in nonalcoholics (, ). We compared haplotypes (A + B) with (C + D) across alcoholics and nonalcoholics (Finns: χ2 = 10.2, 1 df, P=0.001; Plains Indians: χ2 = 8.2, 1 df, P=0.004).
Differences in GAL haplotype frequencies between alcoholics and nonalcoholics. χ2 comparisons across all four haplotypes: Finnish men, n=996, P < 0.001; Plains Indian men, n=263, P=0.045; and Plains Indian women, n=357, P=0.254.
In the Finnish men, there was a significant association between the eight available diplotypes and alcoholism (χ2 = 27.8, 7 df, P=0.002). There were four common (≥5%) diplotypes (AA, AB, AC and AD) that accounted for 97% of the available diplotypes. The frequency of each common diplotype in alcoholics and nonalcoholics is given in ; diplotypes AA and AB were more abundant in alcoholics, whereas diplotypes AC and AD were more abundant in nonalcoholics (χ2 = 20.5, 3 df, P=0.0001). Both diplotypes AA and AB contributed to the significant effect when compared with diplotypes AC and AD: AA vs (AC + AD), χ2 = 8.4, 1 df, P=0.004; AB vs (AC + AD), χ2 = 8.0, 1 df, P=0.005.
(a—c) Frequencies of common GAL (≥5%) diplotypes in nonalcoholics and alcoholics
In the Plains Indian men, the seven available diplotypes were likewise significantly associated with alcoholism (χ2 = 13.2, 6 df, P=0.039). The three common (≥5% frequency) diplotypes (AA, AB and AC) accounted for 89% of all diplotypes and their frequencies are given in . As in the Finns, the frequencies of diplotypes AA and AB were increased in alcoholics, whereas diplotype AC was more abundant in nonalcoholics (χ2 = 9.8, 2 df, P=0.008). Once again, both diplotypes AA and AB contributed to the significant effect when compared with diplotypes AC: AA vs AC, χ2 = 8.6, 1 df, P=0.003; AB vs AC, χ2 = 6.3, 1 df, P=0.012.
Allele frequencies differed significantly between alcoholics and nonalcoholics in all four tag SNPs in the Finns (SNP 1: χ2 = 6.2, P=0.013; SNP 2: χ2 = 17.8, P < 0.0001; SNP 4: χ2 = 12.1, P=0.0005; SNP 5: χ2 = 4.9 P=0.027 (all 1 df)), and in tag SNPs 1 and 4 in the Plains Indian men (SNP 1: χ2 = 5.9, P=0.015; SNP 2: χ2 = 0.8, P=0.384; SNP 4: χ2 = 6.7, P=0.0098; SNP 5: χ2 = 2.5 P=0.114 (all 1 df)).
Haplotype and diplotype associations with HA
In the Finnish alcoholics, there was a trend for HA variation between the four common haplotypes: A and B tended to be associated with lower HA (mean HA of A = 17.4±6.4 and B = 17.1±7.2) and haplotypes C and D tended to be associated with higher HA (mean HA of C = 18.7±6.0 and D = 24.0±8.5) (F(1,481) = 2.2, P=0.142). The association between the diplotypes and HA in alcoholics reflected the haplotype relationship (F(1,217) = 3.3, P=0.072). There was no similar haplotype or diplotype association in nonalcoholics (mean HA: A = 10.9±5.3, B = 13.1±7.6, C = 11.3±5.1 and D = 11.8±5.8). There was no significant relationship between haplotypes or diplotypes and HA in the Plains Indians.
Our previous study in these two populations has shown that alcoholics with high HA, defined as ≥ the population mean (taken as the mean HA for nonalcoholics), and low HA, defined as < the population mean, may be distinct subtypes.31
In the current study, we only found a nonsignificant trend for the association of HA with GAL
haplotypes and diplotypes in alcoholics. Nevertheless, because our study has no precedent, we decided to pursue exploratory analyses of the HA association. We therefore divided the alcoholics into high and low HA groups, as described previously. Mean (s.d.) HA for nonalcoholics was 11.1 (5.3) in the Finns and 12.1 (5.7) in the Plains Indians. The high HA alcoholics had mean HA scores of 19.1±5.2 (Finns) and 16.9±4.2 (Plains Indians). The low HA alcoholics had mean HA scores of 7.2±2.5 (Finns) and 7.6±2.5 (Plains Indians). High and low HA alcoholics did not differ significantly in the prevalence of ASPD, MD, anxiety disorders, drug dependence, novelty seeking or age of onset of heavy drinking (Tables , ).
In Finns, distribution of the four haplotypes (χ2 = 37.2, 6 df, P < 0.0001) and the four common diplotypes (χ2 = 30.0, 6 df, P < 0.0001) differed between high HA alcoholics, low HA alcoholics and nonalcoholics (Tables , , ). High HA alcoholics had an excess of haplotype A and diplotype AA relative to low HA alcoholics and nonalcoholics. Low HA alcoholics were distinguished by a sizeable excess of haplotype B (three-fold) and diplotype AB (four-fold) compared with high HA alcoholics and nonalcoholics (). Haplotype D was almost exclusively confined to nonalcoholics. There was no significant difference in haplotype or diplotype distribution between nonalcoholics with high and low HA.
Figure 3 GAL haplotype and diplotype frequencies in Finnish men: alcoholics with high harm avoidance (HA) ( > mean HA of nonalcoholics), alcoholics with low HA ( < mean HA of nonalcoholics) and nonalcoholics. χ2 comparisons of the three (more ...)
In the Plains Indian men, haplotype and diplotype frequencies did not differ between high HA and low HA alcoholics.
Haplotype/diplotype associations with alcoholism and HA in women
No significant associations were found in the women.
There were no significant associations between haplotypes or diplotypes and anxiety disorders in either population.
Phylogenic analysis of GAL SNPs
Among the seven genotyped SNPs, SNP 1 (rs4930241) showed the highest overall sequence conservation in the nearby region among the five examined species (). The sequence conservation surrounding the seven genotyped SNPs decreased from the 5′-end to the 3′-end of GAL. For three genotyped human SNPs (SNP 4 (rs3136540), SNP 5 (rs3136541) and SNP 7 (rs2513304)), both alleles were found in some of the five examined vertebrate species. Therefore, these SNPs might have originated before speciation. In contrast, the corresponding dog sequence for human SNP 3 (rs1546309) is A, which differs from the human C or T alleles.
Figure 4 Conservation of GAL SNPs across five mammalian species (from top to bottom: human, chimp, mouse, rat, dog). Sequence alignment surrounding SNPs 1–7. The SNP numbers and positions are marked above the aligned sequences for five species. Dashed (more ...)