Jonathan Covault

Allelic and haplotypic association of GABRA2 with alcohol dependence.

Alcohol dependence is a highly prevalent disorder that is associated with serious morbidity and mortality. Because the GABAA neurotransmitter receptor is an important mediator for several behavioral effects of alcohol, genes encoding GABA‐related proteins are functional candidates to influence risk of alcohol dependence. Two genome‐wide scans showed linkage of alcohol dependence to a region on chromosome 4p, which contains a cluster of genes encoding GABAA receptor subunits. A recent effort to fine map that region showed a haplotypic association of alcohol dependence to the gene encoding the GABAA receptor α‐2 subunit (GABRA2). We examined 10 single nucleotide polymorphisms (SNPs) spanning the coding region of this gene in samples of European American subjects with alcohol dependence (n = 446), and controls (n = 334) screened to exclude substance use disorders. There was evidence of association to alcohol dependence for seven adjacent markers spanning 98,000 bp in the middle and 3′‐portion of the GABRA2 gene (range of P‐values = 0.008–0.03). When the subset of the alcohol‐dependent subjects excluding those with a diagnosis of cocaine or opioid dependence or major depressive episode (n = 198) was examined, the strength of the association was increased across these 7 SNPs (range of P‐values = 0.002–0.007). Two common haplotypes in this region accounted for 90.8% of chromosomes. The more common haplotype was present in 55.6% of control group chromosomes versus 48.2% of alcohol‐dependent subjects (P = 0.007) and 45.8% of subjects with alcohol dependence but no co‐morbid drug dependence or depression (P = 0.003). These findings replicate and extend recently reported findings, which together underscore the potential contribution of polymorphic variation at the GABRA2 locus to the risk for alcohol dependence.

Association Between Alcoholism and γ‐Amino Butyric Acid α2 Receptor Subtype in a Russian Population

Background: Two recent large genetic studies in the US population have reported association between genetic variation in γ-amino butyric acid α2 receptor subtype (GABRA2) and risk for alcohol dependence. The goal of this study was to test whether GABRA2 is associated with alcohol dependence in a sample of Russian alcohol-dependent men. Methods: A total of 113 Russian alcohol-dependent men and 100 male population control subjects were recruited in St. Petersburg and genotyped for seven GABRA2 single-nucleotide polymorphisms (SNPs) using real-time PCR (TaqMan). Six SNPs were located in a GABRA2 haplotype block previously associated with alcohol dependence (AD) in the US population. SNPs and haplotypes were tested for an association to AD using χ2 analysis and a likelihood ratio-based statistic implemented in the software COCAPHASE. Results: Significant associations between two SNPs and AD were observed (p < 0.05). In addition, a trend-level association was observed between AD and three adjacent SNPs (p < 0.1). Associated alleles were carried in a haplotype that was present at frequencies of 0.37 and 0.48 in the control and alcohol-dependent populations, respectively (p < 0.06). Tight linkage disequilibrium spanning from the central portion of the gene to the 3′ end was observed in this population. Comparison of the findings to the previously published studies in the US population revealed a highly similar linkage disequilibrium pattern in this population. Conclusions: These findings suggest that genetic variants of GABRA2 increase risk for AD in the Russian population and provide additional support to the hypothesis that polymorphic variation at the GABRA2 locus plays an important role in predisposing to AD at least in European-ancestry populations.

Interactive Effects of the Serotonin Transporter 5-HTTLPR Polymorphism and Stressful Life Events on College Student Drinking and Drug Use

BACKGROUND A common functional polymorphism, 5-HTTLPR, in the serotonin transporter gene has been associated with heavy drinking in college students. We examined this polymorphism as it interacted with negative life events to predict drinking and drug use in college students. METHODS Daily reports of drinking and drug use obtained using a daily web-based survey were combined with self-reports of past-year negative life events and 5-HTTLPR genotypes in a regression analysis of alcohol and nonprescribed drug use in a sample of 295 college students. RESULTS Genotype and negative life events significantly interacted in relation to drinking and drug use outcomes. Individuals homozygous for the short (s) allele who experienced multiple negative life events in the prior year reported more frequent drinking and heavy drinking, stronger intentions to drink, and greater nonprescribed drug use. In individuals homozygous for the long (l) allele, drinking and drug use were unaffected by past-year negative life events. Heterozygous subjects showed drinking outcomes that were intermediate to the two homozygous groups. CONCLUSIONS The 5-HTTLPR s-allele is associated with increased drinking and drug use among college students who have experienced multiple negative life events. The s-allele carriers may be at risk for a variety of adverse behavioral outcomes in response to stress.

GABRA2 alleles moderate the subjective effects of alcohol, which are attenuated by finasteride

GABAA receptors are involved in the subjective effects of alcohol. Endogenous neuroactive steroids interact with GABAA receptors to mediate several behavioral effects of alcohol in rodents. Based on a haplotypic association of alcohol dependence with the gene encoding the GABAA receptor α-2 subunit (GABRA2), we examined whether GABRA2 alleles are associated with the subjective response to alcohol. We also examined whether finasteride (a 5-α steroid reductase inhibitor), which blocks the synthesis of some neuroactive steroids, reduces the subjective response to alcohol. In all, 27 healthy social drinkers (15 males) completed a randomized, double-blind, placebo-controlled study of high-dose finasteride. After being pretreated with study drug, subjects consumed three alcoholic drinks. Subjective effects were measured repeatedly over the ascending blood alcohol curve. To examine the moderating role of genetic variation in GABRA2, a single-nucleotide polymorphism that was informative in association studies was included as a factor in the analysis. Subjects homozygous for the more common A-allele (n=7) showed more subjective effects of alcohol than did individuals with one or two copies of the alcohol dependence-associated G-allele (n=20, including two homozygotes). Among the A-allele homozygotes, there was a greater reduction in several subjective effects during the finasteride session compared to the placebo session. These findings provide preliminary evidence that the risk of alcoholism associated with GABRA2 alleles may be related to differences in the subjective response to alcohol. The effects of finasteride provide indirect evidence for a mediating role of neuroactive steroids in some of the subjective effects of alcohol.

A common polymorphism in serotonin receptor 1B mRNA moderates regulation by miR-96 and associates with aggressive human behaviors

Non-coding regulatory elements can transduce the human genomes response to environmental stimuli. Thus, there is a possibility that variation in non-coding regulatory elements may underlie some of the diversity in human behavior. However, this idea has remained largely untested due to the difficulty in accurately identifying regulatory elements in the 98% of the human genome that does not encode protein. The recent recognition that small trans-acting RNAs anneal to mRNA and regulate gene expression provides a means to identify and test such variants. Here, we show that microRNA-directed silencing of mRNA can be attenuated by a common human polymorphism. We have identified an element (A-element) within serotonin receptor 1B (HTR1B) mRNA that confers repression by miR-96. The repressive activity of this element is attenuated by a common human variant (G-element) that disrupts a nucleotide critical for its interaction with miR-96. Because deletion of the HTR1B gene leads to an aggressive phenotype in mice, we hypothesized an association between the A/G polymorphism and aggressive phenotypes in a sample of 359 college students. As predicted, individuals homozygous for the ancestral A-element reported more conduct-disorder behaviors than individuals with the G-element. Our studies suggest that such functional variants may be common and may help to refine the search for genes involved in complex behavioral disorders.

Serotonin transporter gene polymorphism (5-HTTLPR) and anxiety reactivity in daily life: a daily process approach to gene-environment interaction.

Objective: To test whether individuals with at least one copy of the short (S) or long (L)G allele of the serotonin transporter polymorphism (5-HTTLPR) exhibit greater increases in anxiety, compared with LALA individuals, under periods of high daily stress. Although this common polymorphism in the serotonin transporter gene has been identified as a vulnerability factor for anxiety, findings in the literature are mixed. Discrepant findings could be explained by recent research showing that 5-HTTLPR is functionally triallelic (LA versus LG or S), rather than biallelic (L versus S). Mixed findings could also result from a lack of attention to diathesis-stress models, whereby genetic vulnerability is considered latent until activated by stress (gene-environment interplay). Based on this model, we argue that genotype differences in anxiety should be stronger in the presence of stress. Methods: A total of 350 college students recorded their daily stressors and mood for two 30-day periods, separated by 1 year. Results: Across both years, diathesis-stress patterns were observed for reports of anxious mood as a function of 5-HTTLPR. Individuals with at least one copy of the S or LG allele at 5-HTTLPR experienced elevated anxious mood on days with more intense stressors, as compared with those who were LA homozygotes. Genotype differences in anxiety were less apparent on low stress days. No consistent allelic association of 5-HTTLPR was observed with any other mood states, trait anxiety, or neuroticism. Conclusion: Our findings highlight the potential value of focusing on genetic vulnerability in the context of everyday environmental triggers. 5-HTTLPR = serotonin transporter gene promoter polymorphism; 5-HTT = serotonin transporter protein; fMRI = functional magnetic resonance imaging; STAI = State-Trait Anxiety Inventory.

The effects of exercise training of different intensities on neuromuscular junction morphology

SummaryLittle is known about the effects of exercise training on neuromuscular junction morphology in skeletal muscle. The objectives of this investigation were: 1) to determine if exercise training would elicit changes in neuromuscular junction morphology, 2) to determine if exercise training of different intensities would evoke specific changes in neuromuscular junction morphology, and 3) to determine whether changes in neuromuscular junction structure occur independently of changes in muscle fibre type and size. Twenty-four age and size matched male Sprague-Dawley rats were randomly assigned to three groups: high-intensity trained (HIT), low-intensity trained (LIT), or untrained. Neuromuscular junction morphology of the soleus muscle was determined via immunofluorescent staining. Presynaptic acetylcholine vesicles were visualized with SV-2 antibody in conjunction with fluorescein isothiocyanate labelled secondary antibody. Postsynaptic acetylcholine receptors were identified with rhodamine labelled α-bungarotoxin. Laser scanning microscopy was used to produce images of synapses, which were used to quantitate the following: total area of SV-2 and α-bungarotoxin staining, density of acetylcholine vesicles and receptors, structural complexity, and synaptic coupling. To visualize nerve terminal branching, a smaller number of neuromuscular junctions were stained with C-2 antibody, which reacts with a neurofilament epitope, in conjunction with fluorescein isothiocyanate labelled secondary antibody. Total length of branching, number of branches, average length of branches, and ratio of secondary to primary branches per neuromuscular junction were determined. Citrate synthase activity, fibre type composition and fibre cross-sectional areas of the soleus muscle were assessed to determine the presence of a training effect in that muscle. Results indicate that training did induce hypertrophy of the neuromuscular junction that was independent of muscle hypertorphy. Although the HIT and LIT groups exhibited similar hypertrophic responses of the neuromuscular junction, the HIT group displayed more dispersed synapses than the LIT group. Neither exercise training program, however, resulted in altered densities of acetylcholine vesicles or receptors, nor did training significantly change synaptic coupling. Nerve terminal branching was also affected by exercise training. Neuromuscular junctions from the HIT group demonstrated a greater total length of branching, average length per branch, and number of finer, or secondary, branches than those of the LIT group.

Topiramate treatment for heavy drinkers: moderation by a GRIK1 polymorphism.

OBJECTIVE Topiramate has been shown to reduce drinking and heavy drinking in individuals with alcohol dependence whose goal was to stop drinking. The authors evaluated the efficacy and tolerability of topiramate in heavy drinkers whose treatment goal was to reduce drinking to safe levels. METHOD A total of 138 individuals (62.3% men) were randomly assigned to receive 12 weeks of treatment with topiramate (N=67), at a maximal daily dose of 200 mg, or matching placebo (N=71). Both groups received brief counseling to reduce drinking and increase abstinent days. It was hypothesized that topiramate-treated patients would be better able to achieve these goals, and it was predicted that based on prior research, the effects would be moderated by a single nucleotide polymorphism (rs2832407) in GRIK1, encoding the kainate GluK1 receptor subunit. RESULTS The rate of treatment completion was 84.9% and equal by treatment group. Topiramate treatment significantly reduced heavy drinking days and increased abstinent days relative to placebo. Patients receiving topiramate also had lower concentrations of the liver enzyme γ-glutamyl transpeptidase and lower scores on a measure of alcohol-related problems than the placebo group. In a European American subsample (N=122), topiramates effect on heavy drinking days was significantly greater than that for placebo only in rs2832407 C-allele homozygotes. CONCLUSIONS These findings support the use of topiramate at a daily dose of 200 mg to reduce heavy drinking in problem drinkers. The moderator effect of rs2832407, if validated, would facilitate the identification of heavy drinkers who are likely to respond well to topiramate treatment and provide an important personalized treatment option. The pharmacogenetic findings also implicate the kainate receptor in the mechanism of topiramates effects on heavy drinking.

Markers in the 5′-Region of GABRG1 Associate to Alcohol Dependence and are in Linkage Disequilibrium with Markers in the Adjacent GABRA2 Gene

Following an initial report, there have been multiple replications of an association of alcohol dependence (AD) to markers within a haplotype block that includes the 3′-half of the gene encoding the GABAA α-2 subunit (GABRA2), on chromosome 4p. We examined the intergenic extent of this haplotype block and the association to AD of markers in the adjacent 5′ haplotype block in GABRG1, which encodes the GABAA receptor γ-1 subunit. We genotyped 15 single nucleotide polymorphisms in the GABRG1-GABRA2 interval as well as at 34 ancestry informative markers in three samples: 435 AD and 635 screened control subjects from Connecticut and 812 participants from a multicenter AD treatment trial. We observed two large haplotype blocks in the GABRG1-GABRA2 intergenic interval with a region of increased recombination midway between the two genes. Markers in the two haplotype blocks were in moderate linkage disequilibrium. Compared with markers in the GABRA2 haplotype block, markers in the 5′ GABRG1 haplotype showed greater allelic, genotypic and haplotypic association with AD in European Americans from both AD samples. Logistic regression analysis indicated that genetic elements in the GABRG1 haplotype block likely contribute to AD risk in an additive manner, whereas those in the GABRA2 haplotype block may act in a dominant manner in relation to risk of AD.

Endurance and resistance exercise induce muscle fiber type specific responses in androgen binding capacity

This study examined the effects of different exercise training programs on androgen receptor content and receptor affinity to dihydrotestosterone in fast glycolytic (FG) and slow oxidative (SO) skeletal muscle fibers in rats. Twenty-four male Sprague-Dawley rats were equally divided into three groups: control, endurance exercise trained and resistance exercise trained. After the exercise programs were completed, the extensor digitorum longus (EDL), predominantly a FG muscle, and the soleus, predominantly a SO muscle, were isolated, weighted and both androgen receptor content and affinity to dihydrotestosterone were determined. Resistance training evoked a significant (P < 0.05) hypertrophic response in the soleus but not the EDL. Endurance training was not associated with any significant hypertrophy in either the soleus or the EDL. Neither the endurance nor the resistance training program resulted in changes in androgen receptor affinity to dihydrotestosterone. However, alterations in androgen receptor content were noted. The endurance training program resulted in a significant increase in androgen receptor content in the soleus, but no significant difference in the EDL. The resistance training program elicited a significant decrease in androgen receptor content in the soleus, and a significant increase in the EDL. These results indicate that different exercise stimuli induce changes in androgen receptor content that are specific to skeletal muscle fiber type.