Distinct loci in the CHRNA5/CHRNA3/CHRNB4 gene cluster are associated with onset of regular smoking.

Neuronal nicotinic acetylcholine receptor (nAChR) genes (CHRNA5/CHRNA3/CHRNB4) have been reproducibly associated with nicotine dependence, smoking behaviors, and lung cancer risk. Of the few reports that have focused on early smoking behaviors, association results have been mixed. This meta-analysis examines early smoking phenotypes and SNPs in the gene cluster to determine: (1) whether the most robust association signal in this region (rs16969968) for other smoking behaviors is also associated with early behaviors, and/or (2) if additional statistically independent signals are important in early smoking. We focused on two phenotypes: age of tobacco initiation (AOI) and age of first regular tobacco use (AOS). This study included 56,034 subjects (41 groups) spanning nine countries and evaluated five SNPs including rs1948, rs16969968, rs578776, rs588765, and rs684513. Each dataset was analyzed using a centrally generated script. Meta-analyses were conducted from summary statistics. AOS yielded significant associations with SNPs rs578776 (beta = 0.02, P = 0.004), rs1948 (beta = 0.023, P = 0.018), and rs684513 (beta = 0.032, P = 0.017), indicating protective effects. There were no significant associations for the AOI phenotype. Importantly, rs16969968, the most replicated signal in this region for nicotine dependence, cigarettes per day, and cotinine levels, was not associated with AOI (P = 0.59) or AOS (P = 0.92). These results provide important insight into the complexity of smoking behavior phenotypes, and suggest that association signals in the CHRNA5/A3/B4 gene cluster affecting early smoking behaviors may be different from those affecting the mature nicotine dependence phenotype.

Multiple independent loci at chromosome 15q25.1 affect smoking quantity: a meta-analysis and comparison with lung cancer and COPD.

Recently, genetic association findings for nicotine dependence, smoking behavior, and smoking-related diseases converged to implicate the chromosome 15q25.1 region, which includes the CHRNA5-CHRNA3-CHRNB4 cholinergic nicotinic receptor subunit genes. In particular, association with the nonsynonymous CHRNA5 SNP rs16969968 and correlates has been replicated in several independent studies. Extensive genotyping of this region has suggested additional statistically distinct signals for nicotine dependence, tagged by rs578776 and rs588765. One goal of the Consortium for the Genetic Analysis of Smoking Phenotypes (CGASP) is to elucidate the associations among these markers and dichotomous smoking quantity (heavy versus light smoking), lung cancer, and chronic obstructive pulmonary disease (COPD). We performed a meta-analysis across 34 datasets of European-ancestry subjects, including 38,617 smokers who were assessed for cigarettes-per-day, 7,700 lung cancer cases and 5,914 lung-cancer-free controls (all smokers), and 2,614 COPD cases and 3,568 COPD-free controls (all smokers). We demonstrate statistically independent associations of rs16969968 and rs588765 with smoking (mutually adjusted p-values<10(-35) and <10(-8) respectively). Because the risk alleles at these loci are negatively correlated, their association with smoking is stronger in the joint model than when each SNP is analyzed alone. Rs578776 also demonstrates association with smoking after adjustment for rs16969968 (p<10(-6)). In models adjusting for cigarettes-per-day, we confirm the association between rs16969968 and lung cancer (p<10(-20)) and observe a nominally significant association with COPD (p = 0.01); the other loci are not significantly associated with either lung cancer or COPD after adjusting for rs16969968. This study provides strong evidence that multiple statistically distinct loci in this region affect smoking behavior. This study is also the first report of association between rs588765 (and correlates) and smoking that achieves genome-wide significance; these SNPs have previously been associated with mRNA levels of CHRNA5 in brain and lung tissue.

Externalizing behaviors are associated with SNPs in the CHRNA5/CHRNA3/CHRNB4 gene cluster.

There is strong evidence for shared genetic factors contributing to childhood externalizing disorders and substance abuse. Externalizing disorders often precede early substance experimentation, leading to the idea that individuals inherit a genetic vulnerability to generalized disinhibitory psychopathology. Genetic variation in the CHRNA5/CHRNA3/CHRNB4 gene cluster has been associated with early substance experimentation, nicotine dependence, and other drug behaviors. This study examines whether the CHRNA5/CHRNA3/CHRNB4 locus is correlated also with externalizing behaviors in three independent longitudinally assessed adolescent samples. We developed a common externalizing behavior phenotype from the available measures in the three samples, and tested for association with 10 SNPs in the gene cluster. Significant results were detected in two of the samples, including rs8040868, which remained significant after controlling for smoking quantity. These results expand on previous work focused mainly on drug behaviors, and support the hypothesis that variation in the CHRNA5/CHRNA3/CHRNB4 locus is associated with early externalizing behaviors.

Association of CHRN genes with "dizziness" to tobacco.

The neuronal nicotinic receptor genes (CHRN) have been implicated in a variety of smoking-related behaviors. Here we tested for association between an early subjective response phenotype, "dizziness," and 226 single nucleotide polymorphisms (SNPs) in CHRN genes. The sample included 789 nicotine-dependent cases and 811 controls, where early "dizziness" reports were significantly associated with case/control status (P < 0.0001). Multiple SNPs in the putative promoter region of the CHRNB3 gene were nominally associated with "dizziness" experience from the first few cigarettes (P < 0.01). Cell culture studies were conducted to examine the ability of different haplotypes in the CHRNB3 promoter to drive luciferase expression. Data from these experiments support the hypothesis that different alleles in the CHRNB3 upstream promoter region may lead to different levels of RNA expression. In addition, a novel finding of association between SNPs in the CHRNA10 gene reached experiment-wide empirical significance (P = 0.048), which implicates another CHRN gene as being involved in early subjective response to tobacco.

The effect of voluntary wheel running on 129/SvEvTac and C3H/Ibg alcohol consumption.

The mesolimbic dopaminergic reward pathway is activated by both alcohol and exercise, suggesting exercise as a possible treatment or preventative method for alcohol-use disorders (AUDs). Prior studies conducted in our lab have demonstrated the hedonic substitution of voluntary alcohol consumption for voluntary wheel running in female C57Bl/6Ibg mice, and a trend in male C57Bl/6Ibg mice. Given the important contribution of genetic background on AUDs, this study aims to assess the effects of voluntary wheel running on voluntary alcohol consumption in two moderate alcohol-consuming strains of mice, C3H/Ibg and 129/SvEvTac. Contrary to our previous studies conducted in C57Bl/6Ibg mice, 129/SvEvTac and male C3H/Ibg mice housed without a wheel consumed significantly more alcohol than mice housed with a free or locked wheel. This suggests that 129/SvEvTac and male C3H/Ibg mice are reducing their alcohol consumption due to an enriched environment and not exercise. Interestingly, the three groups of female C3H/Ibg mice (free wheel, locked wheel, no wheel) did not significantly differ in alcohol consumption, suggesting sex-specific differences in C3H/Ibg mice. In addition, genetic and sex effects were observed for running phenotypes in the presence of alcohol. Female 129/SvEvTac and C57Bl/6Ibg mice ran longer distances than male mice, whereas male and female C3H/Ibg mice did not differ in distance run. C3H/Ibg and female 129/SvEvTav mice with access only to water ran longer distances than mice with access to both alcohol and water. However, this effect was not observed in C57Bl/6Ibg or male 129/SvEvTac mice. The results of this mouse model highlight the importance of genetic background and sex on an animal's response to exercise as an enrichment to reduce voluntary alcohol consumption.

Test of association between 10 single nucleotide polymorphisms in the oxytocin receptor gene and conduct disorder.

Animal and human studies have implicated oxytocin in affiliative and prosocial behaviors. We tested whether genetic variation in the oxytocin receptor (OXTR) gene is associated with conduct disorder (CD). Utilizing a family-based sample of adolescent probands recruited from an adolescent substance abuse treatment program, control probands and their families (total sample, n=1750), we conducted three tests of association with CD and 10 single nucleotide polymorphisms (SNPs) in the OXTR gene: (a) family-based comparison utilizing the entire sample; (b) within-Whites, case-control comparison of adolescent patients with CD and controls without CD; and (c) within-Whites case-control comparison of parents of patients and parents of controls. Family-based association tests failed to show significant results (no results P<0.05). While strictly correcting for the number of tests (α=0.002), adolescent patients with CD did not differ significantly from adolescent controls in genotype frequency for the OXTR SNPs tested; similarly, comparison of OXTR genotype frequencies for parents failed to differentiate patient and control family type, except a trend association for rs237889 (P=0.004). We concluded that in this sample, 10 SNPs in the OXTR gene were not significantly associated with CD.

The α6 nicotinic acetylcholine receptor subunit influences ethanol-induced sedation.

Alcohol and nicotine are often co-used and data from human and animals studies have demonstrated that common genes underlie responses to these two drugs. Recently, the genes that code for the subunits of the nicotinic acetylcholine receptors have been implicated as a common genetic mediator for alcohol and nicotine responses. The mammalian genes that code for the α6 and ß3 subunits of the nicotinic acetylcholine receptor (Chrna6 and Chrnb3, respectively) are located adjacent to each other on human and mouse chromosome 8. These subunits have gained attention as potential regulators of drug behaviors because of their expression in the striatum where they have been shown to modulate dopamine release. Human genetic studies have shown that variation in these genes is associated with alcohol phenotypes. In the current experiments, mice lacking the Chrna6 or Chrnb3 gene were tested for three ethanol behaviors: choice ethanol consumption, ataxia, and sedation. Wildtype (WT), heterozygous (HET), and knockout (KO) mice of each strain went through a standard 2-bottle choice drinking paradigm, the balance beam, and the Loss of Righting Reflex (LORR) paradigm. No genotypic effects on any of the 3 behavioral tasks were observed in Chrnb3 animals. While the Chrna6 gene did not significantly influence ethanol consumption (g/kg) or ataxia, mice lacking the α6 subunit took significantly longer to recover their righting reflex than WT animals. These data provide evidence that receptors containing this subunit modulate the sedative effects of ethanol. Further work examining other models of ethanol consumption and behavioral responses to ethanol is needed to fully characterize the role of these receptor subunits in modulating ethanol responses.

Reduced alcohol consumption in mice with access to a running wheel.

Studies of the behavioral effects of alcohol in humans and rodent models have implicated a number of neurological pathways and genes. Separate studies have shown that certain regions of the brain are involved in behavioral responses to exercise. The aim of this study was to determine whether mice which normally voluntarily consume high amounts of alcohol (C57BL/6 strain) would exhibit reduced alcohol consumption when given access to a running wheel under two different models of voluntary consumption: unlimited access two-bottle choice and limited access drinking in the dark (DID). Under the two-bottle choice model, the animals voluntarily consumed less alcohol when a wheel was present in their cage. However, sex-specific differences emerged because female mice voluntarily consumed less alcohol when they have the opportunity to exercise on a running wheel, whereas male mice consumed less alcohol even if the running wheel was locked. There were no significant differences observed in alcohol metabolism or food consumption. Under the DID protocol, no differences in alcohol consumption were observed in the presence of a running wheel. These results suggest that exercise may be a useful approach to consider for treatment for some types of chronic human alcohol problem behaviors, but may be less applicable to human binge drinking.

Genetic association of the CHRNA6 and CHRNB3 genes with tobacco dependence in a nationally representative sample.

Neuronal nicotinic acetylcholine receptors are activated by both endogenous acetylcholine and exogenous nicotine, making sequence variations in these receptors likely candidates for association with tobacco phenotypes. Previous studies have found evidence for significant association between single nucleotide polymorphisms (SNPs) in the genomic region containing the CHRNA6 and CHRNB3 genes and tobacco behaviors. In this study, we provide support for an association between these genes and tobacco dependence in the National Youth Survey Family Study wave 10, a nationally representative sample of households. Eight SNPs in the CHRNA6 and CHRNB3 genomic region were genotyped in 1051 subjects, approximately half of whom are members of sibling pairs. Genetic association with DSM-IV dependence was assessed using a family-based approach as implemented in the statistical package PBAT. Individual SNPs were tested for association with quit attempts and overall dependence. Variation in CHRNA6 was found to be associated with tobacco dependence (p=0.007 in Caucasians). SNPs in CHRNB3 were found to be associated with the number of quit attempts (p=0.0024). Together these results further implicate the region downstream of CHRNA6 and the region upstream of CHRNB3 in risk of nicotine dependence.

The genetic components of alcohol and nicotine co-addiction: from genes to behavior.

Co-occurrence of alcohol and nicotine addiction in humans is well documented and there is good evidence that common genes may contribute to both disorders. Although genetic factors contributing to tobacco and alcohol problem use have been well established through adoption, twin and family studies, specific genes remain to be identified and their mode of action elucidated. Recent work from human genetics studies has provided evidence that neuronal nicotinic acetylcholine receptors (nAChR) genes may have a role in mediating early behaviors that are risk factors for alcohol and nicotine dependence, such as age of initiation and early subjective responses to the drugs. Converging evidence suggests that the dopaminergic system is likely to be important in mediating the pleasurable feelings of reward when activated by nicotine and/or alcohol consumption. The nAChRs are important components of the dopaminergic reward system because some of the receptors have been shown to activate the release of dopamine, and mice lacking genes for specific nAChR gene subunits show altered behavioral responses to nicotine and alcohol. Furthermore, complex interactions between other neurotransmitter circuits including GABA, glutamate and serotonin may be modulated by nAChRs, leading researchers to study genes involved in neurobiology shared by different drugs. Future studies aimed at understanding the variation among these genes, and their corresponding functional implications, will help elucidate how natural variants in nicotinic receptor genes contribute to these common co-morbid disorders.

The CHRNA5/A3/B4 gene cluster variability as an important determinant of early alcohol and tobacco initiation in young adults.

BACKGROUND: One potential site of convergence of the nicotine and alcohol actions is the family of the neuronal nicotinic acetylcholine receptors. Our study examines the genetic association between variations in the genomic region containing the CHRNA5, A3, and B4 gene cluster (A5A3B4) and several phenotypes of alcohol and tobacco use in an ethnically diverse young adult sample. Significant results were then replicated in a separate adult population-representative sample. METHODS: In a selected sample, nine single nucleotide polymorphisms (SNPs) were tested for association with various nicotine and alcohol phenotypes, including age of initiation and measures of frequency, quantity, and subjective responses to the substances. Analysis was conducted with the statistical genetics program WHAP in the full sample (1075 subjects) including ethnicities as covariates and within each ethnic group sub-sample. Replication of the significant results in a separate population-based sample was carried out with the PBAT statistical genetics program. RESULTS: Two linked SNPs (rs8023462 and rs1948) located in a conserved region of the A5A3B4 gene cluster significantly predicted early age of initiation for tobacco with a hazard ratio (HR) of 1.35 (95% confidence interval [CI]1.08-1.70) for the CC genotype of rs8023462 and a HR of 1.29 (95% CI 1.01-1.63) for the TT genotype of rs1948 [corrected]. These findings were then replicated in a separate population-representative sample, showing rs1948 and rs8023462 to be associated with age of initiation for both tobacco and alcohol use (p < .01 and p < .001). CONCLUSIONS: Variations in A5A3B4 genes might influence behaviors that promote early age of experimentation with drugs.