The present study provides a comprehensive test of association between AD and haplotypes in the ADH gene cluster and two ALDH genes in four populations: Plains American Indians, SW American Indians, Finnish Caucasians and African Americans. Our study did not find evidence of any significant associations once the results were corrected for multiple comparisons. However there are several results that are of interest, particularly for ALDH1A1 and ADH4, since they replicate other findings in the literature.
Our study has shown an ALDH1A1 haplotype association with AD in SW American Indians. In ALDH1A1 haplotype block 3, one of the major yin yang haplotypes was more abundant in alcoholics whereas the other yin yang haplotype was more abundant in controls. Of the seven SNPs in this haplotype block, five were in allelic identity and associated with AD suggesting that this block might have been subjected to selective pressure. In the Plains Indians the same five SNPs were also in allelic identity unlike in the Finns and African Americans, suggesting that this selective pressure might be unique to American Indians. The ALDH1A1 block 3 yin-yang haplotype patterns in our four study populations are consistent with the phased haplotype patterns derived from the Asian, Caucasian and African HapMap populations ().
haplotype block 3 includes the promoter, the minimal promoter region (−91 to +53) that contains regulatory elements and the CCAAT box region intron1 (Yanagawa et al., 1995
). It is of interest that two low frequency promoter polymorphisms (0.012 – 0.035), a 17bp deletion (−416/−432) and a 3bp insertion (−524) (ALDH1A1*2
respectively) have previously been associated with AD in Southwest California Indians (Ehlers et al., 2004
) and Caribbean populations (Moore et al., 2007
). In secondary analyses we genotyped 91 yin-yin and 93 yang-yang SW American Indians for the ALDH1A1*2
variants but neither variant was detected.
We also found a yin yang haplotype association in Finnish Caucasians in haplotype blocks 1 and 2 that extends from intron 5 to the 3′ end of the gene. A recent study also in Finnish Caucasians found significant associations between AD and SNPs in intron 8 and the 3′ UTR (Lind et al., 2008
), the same locations as in our study. A study in European Americans found an association between rs8187974 (intron10), located in an ALDH1A1
splice site, and AD as well as max drinks / 24 hours (Sherva et al., 2009
). The closest SNP to rs8187974 in our study was rs63319 (at a distance of 2082bp), but rs63319 did not show any association with AD. Moreover, another study in which 1105 Irish Caucasians were genotyped for the same SNPs as in our study showed no association between AD and ALDH1A1
variation (Kuo et al., 2008
There are two distinct, major isozymes in mammalian liver: ALDH2 is mitochondrial in origin and has a high affinity for acetaldehyde whereas ALDH1 is cytosolic in origin and has a low affinity for acetaldehyde. Therefore it is not clear how ALDH1A1
may be implicated in AD in individuals with functioning ALDH2 enzymes. Nevertheless ALDH1 deficiency has been associated with sensitivity to alcohol (Agarwal, 1981
; Harada et al., 1981
) and with facial flushing (Harada et al., 1981
; Yoshida et al., 1989
) and fatty liver (Thomas et al., 1982
The class I enzymes encoded by ADH1A, ADH1B
contribute about 70% of the total ethanol oxidizing capacity. One minor haplotype in the ADH6-ADH1A-ADH1B
haplotype block was significantly more common in SW Indian controls than in AD subjects (p = 0.007) and showed a trend effect in the same direction in the African Americans (p = 0.11). This haplotype was not present in the other two groups. However, these associated haplotypes did not include the ADH1B*2
functional SNP rs1229984 (His48Arg). We found that rs1229984 was either not present or occurred at a low frequency (0.01) in our four samples and therefore we cannot comment on the potential influence of this functional polymorphism on AD with our current samples sizes. In contrast, Sherva et al were able to show an association between rs1229984 and AD in a sample of 1588 European Americans (Sherva et al., 2009
SNPs, rs698 and rs1693482, distinguish ADH1C*1
. The two SNPs are in very high LD, as shown in previous studies and in our study. Both ADH1C*1
are abundant in the four samples in this study. In individuals homozygous for the ADH1B*1
allele, as is the case for nearly all non-Asian individuals, the ethanol-oxidizing differences between the enzymes encoded by ADH1C*1
are low (Edenberg et al., 2006
; Lee et al., 2004
; Matsuo et al., 2007
; Wall et al., 2005
). This might explain the lack of association with AD in our datasets.
The class II enzyme encoded by ADH4
contributes about 30% the total ethanol oxidizing capacity. ADH4
has previously been associated with AD in European Americans (Luo et al., 2006a
). Our positive result for ADH4
is supported by the findings in the study from the Collaborative Study on the Genetics of Alcoholism (COGA) in which 110 SNPs across the ADH
gene cluster were genotyped in 1860 European American individuals from 218 families (Edenberg et al, 2006
). The pedigree disequilibrium test (PDT) showed that 12 SNPs spanning ADH4
were significantly associated with AD and one of the three haplotype tagging SNPs was rs3762894. In our study this SNP was associated with AD in the Plains Indians and showed a trend association in the African Americans. Although the COGA sample size was large (N = 1860), the best SNP association p value was 0.004. In Edenberg's study, the MAF was 0.17 for all European Americans. Our study detected the same frequencies in the Finnish Caucasians (0.16 in AD, 0.12 in controls) and this is similar to the frequency in HapMap European Americans (0.13). An effect size cannot be derived from the PDT and therefore we were unable to directly compare our results in the Finnish Caucasians with the European Americans in Edenberg et al, 2006
Kuo et al (2008)
tested the association between AD and the same set of SNPs as in our study, genotyped by the identical method (Hodgkinson et al., 2008
), in the Irish Affected Sib Pair Study of Alcohol Dependence (IASPSAD) Sample (n = 1105) (Kuo et al., 2008
). Likewise with the Caucasian sample in our study, there were few haplotype and SNP associations with AD. Out of all the SNPs tested in the Kuo et al study, two survived corrections for multiple testing: the ADH5
SNP rs1154414 and the ADH1B
SNP rs1353621. These findings were not replicated in our study. We performed power analyses based on the effect sizes for these two SNPs derived from the IASPSAD study. For ADH5
rs1154414 we calculated that we had 95% power to detect an effect in the Finnish Caucasian sample. The fact that we did not detect an association between rs1154414 and AD was due to the fact that the MAF differed between AD subjects in the IASPSAD study (0.09) and the Finnish Caucasians in our study (0.15) although the MAF in controls was the same in both studies (0.12). For ADH1B rs1353621, we calculated that we had 93% power to detect the same effect size in the Finnish Caucasians as had been detected in the IASPSAD study. However the MAF in the Finnish cases (0.30) and controls (0.33) were similar and differed from the MAF in cases (0.43) and controls (0.37) in the IASPSAD study.
shows no LD with other ADH genes. This is consistent with the findings of earlier studies (Edenberg et al., 2006
; Kuo et al., 2008
) and HapMap data. The intergenic haplotype block includes the 3′UTR and the nearby 3′ region of ADH7
, indicating that the haplotype association with AD that we found in SW Indians might implicate a regulatory locus. However, relatively high q values by FDR analysis showed that this association is likely to be due to chance. Osier et al reported a possible epistatic role of ADH7
in protection against alcohol abuse and/or AD by the haplotype rs1154458-rs1229984 (Osier et al., 2004
). However, in our study we found no evidence of LD between SNPs in AD7
It should be noted that there was considerable allele and haplotype frequency variation between the four populations for all genes, including variations between the two American Indian tribes. It could have been expected that of the four populations tested, the Plains Indians and SW Indians would be most likely to have alcohol metabolizing gene variants similar to Asians since their ancestors are thought to have migrated across the Bering Straits from Asia (Goebel et al., 2008
). However these two American Indian tribes do not have the Asian ADH and ALDH2 gene variants which is consistent with proposals of a population bottleneck at the time of entry to the Americas that would have eliminated much genetic variation in American Indian populations (Kitchen et al., 2008
). The considerable allelic variation could result in population stratification that can produce false-positive or false-negative results in case-control studies. However we included ethnic factor scores derived from 186 AIMs as covariates in our analyses when significant and therefore population stratification is unlikely to have influenced our results.
Yin yang haplotypes are defined as two high-frequency haplotypes with exactly opposite allelic configurations, for example the haplotypes 1222221 and 2111112 in ALDH1A1
block 3. It has been shown that the proportion of the genome spanned by yin yang haplotypes is approximately 75%-85% and their conservation across all populations suggests that they represent ancient chromosomal regions that are likely to predate the African diaspora (Zhang et al., 2003
). In accordance with HapMap data we found that yin yang haplotypes were present in all four populations although, as expected, at lower frequencies in the sample with African ancestry. At this point in time the significance of yin yang haplotypes is not clear. Using coalescence simulation, Zhang et al, 2003
have shown that yin yang haplotypes can be explained by strictly neutral evolution in a well-mixed population however another study indicates that yin yang haplotypes can be explained by the maintenance of ancient lineages by selection (Curtis and Vine, 2010
There are a few potential limitations to our study. Different diagnostic criteria (DSM-III-R and DSM-IV) and different psychiatric instruments were used in this study. However, the interviews of the Finnish Caucasians and African Americans were conducted by two experienced clinical psychiatrists and the interviews of the Plains and Southwestern Indians were conducted by two clinical social workers who were fully versed in tribal customs and cultures. Therefore we are confident in the validity of the AD diagnosis. The controls were standardized across all four samples in that they had no lifetime Axis 1 disorders however nicotine dependence was not an exclusion criteria since we only had smoking history for the Plains Indians. This resulted in effectively increasing the heterogeneity of the AD samples since the samples included AD alone and AD comorbid with other disorders. Two samples had significant comorbidity: cocaine and heroin dependence in the African Americans and ASPD in the Finnish Caucasians. Nevertheless the comorbid disorders are unlikely to have any associations with alcohol metabolizing genes. Because of the relatively small samples sizes across the four populations we were unable to determine potentially important rarer haplotype associations. Moreover, rare and uncommon haplotypes estimated using any method are subject to higher error because of increased sampling variance and genotyping errors. However we were able to analyze common haplotypic variation across these genes; for example the ALDH1A1 block 3 yin yang haplotypes are fairly abundant.
In conclusion, the systematic evaluation of common variation in alcohol metabolizing genes in four non East Asian populations has shown only modest associations with AD, largely for ALDH1A1 and ADH4. A concentration of signals for AD with ALDH1A1 yin yang haplotypes in several populations warrants further research. Our study is the first to provide a comprehensive analysis of alcohol metabolism genes in four independent populations. The fact that no strong genetic associations were found may reflect the heterogeneity of the disease and the large role that environmental factors play in the development of alcohol dependence.