Characterization of a Functional Polymorphism in the 3′ UTR of SLC6A4 and its Association With Drinking Intensity

Department of Psychiatry and Neurobehavioral Sciences, University of Virginia, 1670 Discovery Drive, Charlottesville, VA 22911, USA.
Alcoholism Clinical and Experimental Research (Impact Factor: 3.21). 12/2008; 33(2):332-9. DOI: 10.1111/j.1530-0277.2008.00837.x
Source: PubMed


The propensity for severe drinking is hypothesized to be regulated by differential expression of serotonin transporter gene (SLC6A4) in the human brain. The SLC6A4 promoter region 5-HTTLPR has been examined previously as a candidate polymorphic variant associated with severe drinking. In this study, we investigated whether other SLC6A4 single nucleotide polymorphisms (SNPs) are associated with drinking intensity among treatment-seeking alcoholics and whether these polymorphic variants result in differential SLC6A4 expression levels.
We analyzed associations of drinking intensity in 275 (78.5% male) treatment-seeking alcoholics of Caucasian and Hispanic origin, with 6 SLC6A4 polymorphisms. Next, to examine the functionality of the SNP that showed a significant association with drinking intensity, we transfected the 2 alleles of rs1042173 into HeLa cell cultures and measured serotonin transporter mRNA and protein expression levels by using qRT-PCR and western blotting techniques.
One of the 6 polymorphisms we examined, rs1042173 in the 3' untranslated region (3'-UTR) of SLC6A4, showed a significant association with drinking intensity. The G allele carriers for rs1042173 were associated with significantly lower drinking intensity (p = 0.0034) compared to T-allele homozygotes. In HeLa cell cultures, the cells transfected with G allele showed a significantly higher mRNA and protein levels than the T allele-transfected cells.
These findings suggest that the allelic variations of rs1042173 affect drinking intensity in alcoholics possibly by altering serotonin transporter expression levels. This provides additional support to the hypothesis that SLC6A4 polymorphisms play an important role in regulating propensity for severe drinking.

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Available from: Weihua Huang, Apr 20, 2015
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    • "Thus, although is unclear as to whether one study sample better represents the genotype distribution for 5-HTTLPR among Non-Hispanic Whites versus another, the haplotype appears to provide a good approximation of the alleles. In contrast, when comparing the HRS Hispanic/Latino allelic variation (S = 11 %, L = 89 %) with other studies focused on participants from predominantly Spanishspeaking countries (range of values for S allele = 43– 60 %; (Schild et al. 2014; Seneviratne et al. 2009); for table of allele frequencies across studies see Supplemental material), the HRS sample clearly contained fewer S alleles . However, when comparing the HRS Hispanic/Latino sample to the Mexican–American sample from 1000 Genomes (S = 3 %; Consortium 2012), the frequencies are more similarly and predominantly comprised of Long alleles. "
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    ABSTRACT: Depending on genetic sensitivity to it, stress may affect depressive symptomatology differentially. Applying the stress-diathesis hypothesis to older adults, we postulate: (1) recent stress will associate with increased depressive symptoms levels and (2) this effect will be greater for individuals with at least one short allele of the serotonin transporter gene promoter region (5-HTTLPR). Further, we employ a design that addresses specific limitations of many prior studies that have examined the 5-HTTLPR x SLE relation, by: (a) using a within-person repeated-measures design to address fluctuations that occur within individuals over time, increase power for detecting GxE, and address GE correlation; (b) studying reports of exogenous stressful events (those unlikely to be caused by depression) to help rule out reverse causation and negativity bias, and in order to assess stressors that are more etiologically relevant to depressive symptomatology in older adults. The sample is drawn from the Health and Retirement Study, a U.S. population-based study of older adults (N=28,248; mean age = 67.5; 57.3% female; 80.7% Non-Hispanic White, 14.9% Hispanic/Latino, 4.5% African American; genetic subsample = 12,332), from whom measures of depressive symptoms and exogenous stressors were collected biannually (1994-2010). Variation in the 5-HTTLPR was characterized via haplotype, using two single nucleotide polymorphisms (SNPs). Ordered logit models were constructed to predict levels of depressive symptoms from 5-HTTLPR and stressors, comparing results of the most commonly applied statistical approaches (i.e., comparing allelic and genotypic models, and continuous and categorical predictors) used in the literature. All models were stratified by race/ethnicity. Overall, results show a main effect of recent stress for all ethnic groups, and mixed results for the variation in 5-HTTLPRxStress interaction, contingent upon statistical model used. Findings suggest there may be a differential effect of stressors and 5-HTTLPR on depressive symptoms by ethnicity, but further research is needed particularly when using a haplotype to characterize variation in 5-HTTLPR in population-based sample with a diverse ethnic composition.
    Behavior Genetics 08/2015; DOI:10.1007/s10519-015-9740-8 · 3.21 Impact Factor
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    • "We also selected two known functional polymorphisms in SLC6A4 to examine the association of variations of serotonin transporters with ND. They are rs1042173, located in the 3 -UTR region, and the 5 -serotonin transporter-linked polymorphic region (5 -HTTLPR), located in the promoter region of SLC6A4 (Lesch et al., 1999; Seneviratne et al., 2009). The 5 -HTTLPR long (L) and short (S) alleles were genotyped by PCR amplification of genomic DNA (15 ng) in a final volume of 20 ␮l containing 5 × MyTaq reaction buffer, Taq DNA polymerase, and 20 ␮M each of the forward ( "
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    ABSTRACT: Background: Pharmacologic studies implicate a significant role of genes encoding the serotonin transporter (SLC6A4) and the 5-HT3AB subunits HTR3A and HTR3B in nicotine dependence (ND). However, whether they are involved in ND remains largely unknown. Methods: Here, we examined the impact of variations in the three genes on ND in 1366 individuals from 402 African American (AA) and 671 individuals from 200 European American (EA) families. The ND of each smoker was assessed with smoking quantity (SQ), heaviness of smoking index (HSI), and Fagerström test for nicotine dependence (FTND). Results: Association analysis revealed marginal association of rs10160548 in HTR3A with SQ and HSI in AA, 5-HTTLPR in SLC6A4 with FTND in EA, and rs11606194 in HTR3B with SQ and FTND in the pooled sample. Haplotype-based association analysis revealed a few major haplotypes in HTR3A that were significantly associated with ND in the AA, EA, and pooled samples. However, none of these associations remained significant after correcting for multiple testing except for a haplotype G-C-C-T-A-T formed by SNPs rs1150226, rs1062613, rs33940208, rs1985242, rs2276302, and rs10160548 in HTR3A for the AA sample. Considering biological functions of the three genes, we examined interactive effects of variants in the three genes, which revealed significant interactions among rs1062613 and rs10160548 in HTR3A, rs1176744 in HTR3B, and 5-HTTLPR and rs1042173 in SLC6A4 in affecting ND in the three samples. Conclusions: We conclude that SLC6A4, HTR3A and HTR3B play a significant role in ND through genetic interactions.
    Drug and alcohol dependence 01/2013; 129(3). DOI:10.1016/j.drugalcdep.2012.12.007 · 3.42 Impact Factor
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    • "The word " craving " in the title implies a more inclusive definition, and, therefore, we did not mention both " urge " and " crave " in the title. These results supplement findings from our previous outpatient and in vitro studies (Seneviratne et al., 2009; Johnson et al., 2011) that showed lower 5-HTT expression levels and more intense alcohol drinking behavior associated with the TT genotype. From a biological and behavioral perspective, it could be argued that lower 5-HTT expression levels associated with the TT genotype may facilitate serotonergic and dopaminergic output in the reward pathways, thereby increasing the desire for more intense drinking , upon exposure to small priming doses. "
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    ABSTRACT: We previously have shown that cue-induced alcohol craving and propensity for higher drinking are modulated by allelic differences in SLC6A4. In an independent study, we characterized a functional polymorphism, rs1042173 (G/T), in the SLC6A4 3′-untranslated region (3′-UTR); the T allele was associated with lower mRNA and protein levels, and the alcohol-dependent (AD) individuals carrying the TT genotype showed higher drinking intensity compared with G-allele carriers. Building upon these findings, we hypothesized that the low-expressing TT genotype associated with intense drinking would predict higher craving for alcohol in AD individuals. In this pilot study, we sought to test our hypothesis by examining 34 Hispanic AD volunteers (mean age, 34.8 years) for rs1042173 genotype-based (i.e., TT vs. TG/GG (Gx)) differences in subjective response to alcohol. We employed a human laboratory paradigm and analyzed the data using a linear mixed-effects model to assess treatment, cue procedures, and genotype main effects as well as the two-way interaction effects between them. On subjective “urge to drink” and “crave for a drink”, we found a significant main effect of the cue experiment (p ≤ 0.01) and an interaction effect between genotype and cue effects (p < 0.05). TT genotype was associated with higher intensity of drinking and craving for alcohol. Our results not only support the hypothesis that rs1042173 is a genetic marker for cue-induced alcohol craving among AD males but also are suggestive of a neurobiological mechanism associated with the rs1042173-TT genotype that triggers a disproportionate craving in response to alcohol consumption, which in turn may lead to more intense drinking. Future studies with larger sample sizes are needed to characterize the interactive effects of the serotonin transporter-linked polymorphic region (5′-HTTLPR)-L-allele reported in our previous study and of the rs1042173-TT genotype on cue-induced alcohol craving.
    Frontiers in Psychiatry 02/2012; 3:6. DOI:10.3389/fpsyt.2012.00006
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