Our findings provide evidence that rs1042173, a SNP in the 3’ UTR of the SLC6A4 gene, was associated with intensity of drinking among Caucasians dependent on alcohol. By using a site-directed mutagenesis approach, we further showed that rs1042173 is a functional polymorphism that resulted in a difference in 5-HTT expression levels in HeLa cell cultures, with G allele associated with higher 5-HTT mRNA and protein expression levels than the T allele. Of multiple approaches used to determine whether a polymorphism is a function one, a direct comparison of expression level between two alleles through an in vitro expression system as used in this study represents one of the most convenient molecular techniques in the field. We expect such an approach will become more popular as more and more potential causative polymorphisms are being identified through association study.
Alcohol-dependent individuals who were G-allele carriers for rs1042173 showed less intensity of drinking compared with those who were homozygous for the T allele. Importantly, the average intensity of drinking for both of these allelic groups exceeded the threshold for heavy drinking (i.e., ≥5 and ≥4 standard drinks/day for men and women, respectively), and all were dependent on alcohol. At the time of entry, subjects in both allelic groups were not statistically significantly different in average chronological age and duration of alcohol dependency. It is, therefore, reasonable to propose that alcohol-dependent individuals with the TT genotype might constitute a subtype of more intense drinkers among heavy-drinking alcoholics of European descent.
To our knowledge, this is the first study to investigate the function of the rs1042173 SNP in an alcohol-dependent population. The rs1042173 polymorphism is not only located at a putative polyadenylation signal site in the 3’ UTR of the 5-HTT gene but also near a potential binding site for microRNA miRNA-135 according to a bioinformatics prediction with PicTar program (Chen et al., 2006
). It has been hypothesized that a variant at this location may change expression levels by affecting the stability of mRNA (Battersby et al., 1999
; Beaudoing et al., 2000
; Chen et al., 2006
). Therefore, our finding that those homozygous for the T allele, compared with their G-allelic counterparts, have lower mRNA and protein expression levels, providing support evidence for this hypothesis although the mechanism(s) by which these mRNA and protein expression levels change remains to be further explored in the future. Our findings have been further supported by two recent reports. The first study reported by Vallender et al. (2008)
revealed that a functional haplotype containing T allele of rs1042173 was associated with higher mRNA expression in HEK293 cells compared to the haplotype consisting of G allele. Another study reported by Lim et al. (2006)
showed that G-allele had increased allelic expression imbalance (AEI) in Epstein-Barr virus transformed lymphoblast cells while human pons tissue showed a decreased AEI for G-allele. Although the expression levels associated with each allele of rs1042173 are inconsistent among these studies (likely due to different reporter genes and/or cell lines used among them), they all reveal that rs1042173 is a functional one.
It is, therefore, tempting to speculate as to the mechanism whereby alcohol-dependent individuals who are homozygous for the T allele drink more severely than carriers of the G allele. Plausibly, those homozygous for the T allele compared with their Gallele-carrying counterparts will have lower expression of 5-HTT mRNA and protein levels and, therefore, a relatively higher intrasynaptic 5-HT level. Because 5-HTTs in the raphe nuclei are somatodendritic (Little et al., 1998
), a reduction in their numbers will be associated with reduced 5-HT firing rates due to increased self-inhibition and, consequently, an upregulation of postsynaptic 5-HT receptors mediating the reinforcing effects of alcohol. Hence, chronic alcohol intake, which generally increases 5-HT turnover (LeMarquand et al., 1994
), might be serving as a neuroadaptive response to “normalize” serotonergic function in the raphe nuclei.
We considered the possibility that a potential confound to our results was that the associations of the T and G alleles at the 3’ UTR SNP rs1042173 with intensity of drinking were being influenced by allelic differences in the promoter region of the 5-HTT gene; hence, these associations could be spurious. However, a lack of significant linkage disequilibrium and interaction between 3’ UTR SNP rs1042173 and 5-HTTLPR region L/S alleles excluded this possibility.
We took into consideration the potential for ethnicity and gender to confound our results by performing independent statistical tests to show that these factors did not influence our findings; thus, our cohort was relatively homogenous. The finding of no association between rs1042173 genotype and intensity of drinking in Hispanics, which differed from that of an association among Caucasians, while the allelic frequencies for T and G alleles in Caucasians and Hispanics were not significantly different, does suggest the possibility of differential regulation of gene expression by ethnic group. Due to the relatively small sample size of the cohort, such a premise needs to be treated as preliminary and confirmed by larger studies.
Another caveat to our results is that our cohort was a population of treatment-seeking, alcohol-dependent individuals who were motivated to decrease or cease their alcohol consumption. Therefore, this cohort may be more motivated and perhaps less dense in drinking pathophysiology compared with alcohol-dependent individuals derived from community samples (Wrase et al., 2006
We also considered the possibility that the specified time point of 90 days prior to enrollment for measuring the level of drinking intensity might be an arbitrary point at which to perform comparative analyses with the genotype. We were, however, able to show that the association between the intensity of drinking and the genotype remained significant in the same manner even if we varied the drinking period prior to enrollment within a range of 14 to 90 days (data not shown). The consistency of these results strengthened our findings.
In summary, we detected that a subgroup of treatment-seeking adult Caucasian male and female alcohol-dependent individuals who were homozygous for the T allele of the 3’ UTR SNP rs1042173 in SLC6A4 gene were more severe drinkers than their G-allele-carrying counterparts. Using an in vitro approach, we demonstrated that the T allele, which is associated with greater intensity of drinking, expressed lower mRNA and 5-HTT protein levels compared with the G-allele carriers of rs1042173 SNP. Taken together, these findings suggest the possibility that two different subgroups of treatment-seeking alcoholics with allelic differences at the 3’ UTR SNP rs1042173 can differ in their intensity of drinking, an effect that might be associated with underlying differences in expression of 5-HTT. Further studies with a large sample size are needed to confirm our findings, and to determine whether alcohol-dependent individuals with these allelic differences at the 3’ UTR SNP rs1042173 would vary in response to different types of specific serotonergic medication.