Genetic influences of dopamine transport gene on alcohol dependence: A pooled analysis of 13 studies with 2483 cases and 1753 controls

Abstract

Previous genetic association studies have reported a possible role of the dopamine transporter (DAT, gene symbol: SLC6A3) gene in the etiology of alcohol dependence, but the results were conflicting with each other. We conducted a pooled analysis of published population-based case–control genetic studies investigating associations between polymorphisms in SLC6A3 and alcohol dependence. We also explored whether geographic area, ethnicity, gender, and diagnostic criteria moderated any association by using stratified analysis. Through combining 13 studies with 2483 cases and 1753 controls, the 40-base pair variable number tandem repeat (VNTR) in the 3′ un-translated region, the well studied polymorphism in SLC6A3, did not show any association with alcohol dependence in general or in stratified analyses according to geographic area, ethnicity, gender, and diagnostic criteria. Due to limited studies focused on polymorphisms in other regions of the SLC6A3 gene, we cannot rule out the role of the SLC6A3 gene in the involvement of the genetic risk of alcohol dependence. Further clarification of the genetic role of SLC6A3 in the susceptibility to alcohol dependence should be centered on other potential functional regions of the SLC6A3 gene.

Introduction

Alcohol dependence is one of the most prevalent mental health problems in the USA. The prevalence of lifetime and 12-month alcohol dependence in the USA was 12.5% and 3.8% (Hasin et al., 2007). Estimation of genetic effects on alcohol dependence is as high as 50 to 70% in both men and women, as is shown by numerous twin and adoption studies (Goldman et al., 2005, Pagan et al., 2006). In attempts to identify the genetic risks for alcohol dependence, genes involved in dopaminergic system have been paid much attention during the past twenty years. As is known, alcohol may activate the dopaminergic system, which in turn is associated with positive reinforcement (Berridge and Robinson, 1998). Therefore, genetic variants regulating genes coding for proteins involved in dopamine neurotransmission might account for different responses to alcohol or might contribute to individual variation in alcohol dependence (Persico et al., 1993).

Essential in dopamine regulation is the dopamine transporter (DAT1, gene symbol: SLC6A3). SLC6A3 is a 12-membrane domain Na⁺/Cl⁻-dependent transport protein, with the responsibility for the reuptake of extracellular synaptic dopamine into presynaptic neurons, and hereby termination of dopaminergic neurotransmission (Giros et al., 1992). As such, dopaminergic reward circuits are likely to function differently with different expression levels of SLC6A3 (Drgon et al., 2006), which implies the SLC6A3 gene is a potential candidate in clinical studies on alcohol dependence.

The biological mechanism under which SLC6A3 availability is regulated in the brain is still unclear. Brain imaging studies discover that SLC6A3 expression levels may be affected by alcohol consumption. Two studies reported that SLC6A3 levels were significantly lower in the striatum of alcohol-dependent humans and monkeys than in controls, but returned to normal levels after a period of abstinence (Laine et al., 1999, Mash et al., 1996). While two other studies did not find reductions in SLC6A3 levels in the brains of alcoholics when compared with controls (Heinz et al., 1998, Volkow et al., 1996), although this might be related to the fact that measurements were carried out up to several weeks after withdrawal of alcohol, in which the expression levels of SLC6A3 may have already returned to normal.

The availability of SLC6A3 in the brain may be dependent on genetic variation. SLC6A3 gene is mapped on chromosome 5p15.3 with 15 exons separated by 14 introns spanning more than 65 kb. The protein-coding portion begins within exon 2 and ends near the beginning of exon 15 (Banno et al., 2001). This coding region presents strong conservation and the polymorphisms within the coding region represent either silent nucleotide changes or rare conservative amino acid substitutions (Grünhage et al., 2000), indicating that individual differences in SLC6A3 expression must arise from regulatory sequences. Lin and Uhl (2003) observed the effects of protein-coding variants V55A and V382A in the human dopamine transporter gene on expression and uptake activities in vitro. Drgon et al. (2006) reported common human 5′ dopamine transporter haplotypes yield varying expression levels in vivo. Greenwood and Kelsoe (2003) identified a strong core promoter extending from − 251 to + 63 in the genomic sequence of the SLC6A3 gene and suggested the presence of repressor elements in the 5′-upstream region and within intron 1(Xu et al., 2010).

The genetic polymorphism of interest in most genetic association studies on alcohol dependence, however, is a variable number of tandem repeats (VNTR) of 40 bp in the 3′ un-translation region. For the VNTR, numbers ranging from 3 to 16 have been described, with the 9- and 10-repeat alleles being the most common variants (Banno et al., 2001, Vandenbergh et al., 1992). Several studies have demonstrated ethnic differences in SLC6A3 VNTR frequency distributions (Kang et al., 1999, Mitchell et al., 2000). The results of human genetic association studies investigating the relationship between this VNTR and alcohol dependence have been equivocal. Since the VNTR is located in the 3′-UTR of the gene, outside the open reading frame of the gene, allelic variants do not result in structural or functional differences in the SLC6A3 protein. Nonetheless, research suggests that the SLC6A3 VNTR is able to regulate specific gene functioning by influencing levels of expression. As such, SLC6A3 expression may be influenced by alteration in the length or sequence of the VNTR (Conne et al., 2000). It is also likely that this VNTR is in linkage disequilibrium with other susceptibility loci within the gene. A SNP in the 3′-UTR of SLC6A3 consists of a G to A mutation at position 2319 in SLC6A3 cDNA has also been investigated with the association of alcohol dependence by Ueno et al., 1999a, Ueno et al., 1999b, Choi et al., 2006a, Choi et al., 2006b, but the two studies with limited sample size reported conflicting results.

To identify a real association, we performed pooled analysis of multicenter genetic association studies to determine whether the failure to identify any real association is attributable to: 1) the low power of individual studies to detect a small effect; 2) etiological heterogeneity arose from potential confounding factors.