Genome-wide association studies have identified common variation in the CHRNA5–CHRNA3–CHRNB4 and CHRNA6–CHRNB3 gene clusters that contribute to nicotine dependence. However, the role of rare variation in risk for nicotine dependence in these nicotinic receptor genes has not been studied. We undertook pooled sequencing of the coding regions and flanking sequence of the CHRNA5, CHRNA3, CHRNB4, CHRNA6 and CHRNB3 genes in African American and European American nicotine-dependent smokers and smokers without symptoms of dependence. Carrier status of individuals harboring rare missense variants at conserved sites in each of these genes was then compared in cases and controls to test for an association with nicotine dependence. Missense variants at conserved residues in CHRNB4 are associated with lower risk for nicotine dependence in African Americans and European Americans (AA P = 0.0025, odds-ratio (OR) = 0.31, 95% confidence-interval (CI) = 0.31–0.72; EA P = 0.023, OR = 0.69, 95% CI = 0.50–0.95). Furthermore, these individuals were found to smoke fewer cigarettes per day than non-carriers (AA P = 6.6 × 10−5, EA P = 0.021). Given the possibility of stochastic differences in rare allele frequencies between groups replication of this association is necessary to confirm these findings. The functional effects of the two CHRNB4 variants contributing most to this association (T375I and T91I) and a missense variant in CHRNA3 (R37H) in strong linkage disequilibrium with T91I were examined in vitro. The minor allele of each polymorphism increased cellular response to nicotine (T375I P = 0.01, T91I P = 0.02, R37H P = 0.003), but the largest effect on in vitro receptor activity was seen in the presence of both CHRNB4 T91I and CHRNA3 R37H (P = 2 × 10−6).

doi:10.1093/hmg/ddr498

PMCID: PMC3259016 PMID: 22042774

2. Multiple cholinergic nicotinic receptor genes affect nicotine dependence risk in African and European Americans

Genes, brain, and behavior 2010;9(7):741-750.

Several independent studies show that the chromosome 15q25.1 region, which contains the CHRNA5-CHRNA3-CHRNB4 gene cluster, harbors variants strongly associated with nicotine dependence, other smoking behaviors, lung cancer, and chronic obstructive pulmonary disease.

We investigated whether variants in other cholinergic nicotinic receptor subunit (CHRN) genes affect risk for nicotine dependence in a new sample of African-Americans (N = 710). We also analyzed this African-American sample together with a European-American sample (N=2062, 1608 of which have been previously studied), allowing for differing effects in the two populations. Cases are current nicotine-dependent smokers and controls are non-dependent smokers.

Variants in or near CHRND-CHRNG, CHRNA7, and CHRNA10 show modest association with nicotine dependence risk in the African-American sample. In addition, CHRNA4, CHRNB3-CHRNA6, and CHRNB1 show association in at least one population. CHRNG and CHRNA4 harbor SNPs that have opposite directions of effect in the two populations. In each of the population samples, these loci substantially increase the trait variation explained, although no loci meet Bonferroni-corrected significance in the African-American sample alone. The trait variation explained by three key associated SNPs in CHRNA5-CHRNA3-CHRNB4 is 1.9% in European-Americans and also 1.9% in African-Americans; this increases to 4.5% in EAs and 7.3% in AAs when we add six variants representing associations at other CHRN genes.

Multiple nicotinic receptor subunit genes outside of chromosome 15q25 are likely to be important in the biological processes and development of nicotine dependence, and some of these risks may be shared across diverse populations.

doi:10.1111/j.1601-183X.2010.00608.x

PMCID: PMC2970751 PMID: 20584212

genetic association; smoking; cholinergic nicotinic receptors; nicotinic acetylcholine receptors

3. Risk for nicotine dependence and lung cancer is conferred by mRNA expression levels and amino acid change in CHRNA5

Human Molecular Genetics 2009;18(16):3125-3135.

Nicotine dependence risk and lung cancer risk are associated with variants in a region of chromosome 15 encompassing genes encoding the nicotinic receptor subunits CHRNA5, CHRNA3 and CHRNB4. To identify potential biological mechanisms that underlie this risk, we tested for cis-acting eQTLs for CHRNA5, CHRNA3 and CHRNB4 in human brain. Using gene expression and disease association studies, we provide evidence that both nicotine-dependence risk and lung cancer risk are influenced by functional variation in CHRNA5. We demonstrated that the risk allele of rs16969968 primarily occurs on the low mRNA expression allele of CHRNA5. The non-risk allele at rs16969968 occurs on both high and low expression alleles tagged by rs588765 within CHRNA5. When the non-risk allele occurs on the background of low mRNA expression of CHRNA5, the risk for nicotine dependence and lung cancer is significantly lower compared to those with the higher mRNA expression. Together, these variants identify three levels of risk associated with CHRNA5. We conclude that there are at least two distinct mechanisms conferring risk for nicotine dependence and lung cancer: altered receptor function caused by a D398N amino acid variant in CHRNA5 (rs16969968) and variability in CHRNA5 mRNA expression.

doi:10.1093/hmg/ddp231

PMCID: PMC2714722 PMID: 19443489

4. Multiple Independent Loci at Chromosome 15q25.1 Affect Smoking Quantity: a Meta-Analysis and Comparison with Lung Cancer and COPD

PLoS Genetics 2010;6(8):e1001053.

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.

Author Summary

Nicotine binds to cholinergic nicotinic receptors, which are composed of a variety of subunits. Genetic studies for smoking behavior and smoking-related diseases have implicated a genomic region that encodes the alpha5, alpha3, and beta4 subunits. We examined genetic data across this region for over 38,000 smokers, a subset of which had been assessed for lung cancer or chronic obstructive pulmonary disease. We demonstrate strong evidence that there are at least two statistically independent loci in this region that affect risk for heavy smoking. One of these loci represents a change in the protein structure of the alpha5 subunit. This work is also the first to report strong evidence of association between smoking and a group of genetic variants that are of biological interest because of their links to expression of the alpha5 cholinergic nicotinic receptor subunit gene. These advances in understanding the genetic influences on smoking behavior are important because of the profound public health burdens caused by smoking and nicotine addiction.

doi:10.1371/journal.pgen.1001053

PMCID: PMC2916847 PMID: 20700436

5. The CHRNA5-CHRNA3-CHRNB4 nicotinic receptor subunit gene cluster affects risk for nicotine dependence in African-Americans and in European-Americans

Cancer research 2009;69(17):6848-6856.

Genetic association studies have demonstrated the importance of variants in the CHRNA5-CHRNA3-CHRNB4 cholinergic nicotinic receptor subunit gene cluster on chromosome 15q24-25.1 in risk for nicotine dependence, smoking, and lung cancer in populations of European descent. We have now carried out a detailed study of this region using dense genotyping in both European- and African-Americans.

We genotyped 75 known single-nucleotide-polymorphisms (SNPs) and one sequencing-discovered SNP in an African-American (AA) sample (N = 710) and European-American (EA) sample (N = 2062). Cases were nicotine-dependent and controls were non-dependent smokers.

The non-synonymous CHRNA5 SNP rs16969968 is the most significant SNP associated with nicotine dependence in the full sample of 2772 subjects (p = 4.49×10−8, OR 1.42 (1.25–1.61)) as well as in AAs only (p = 0.015, OR = 2.04 (1.15–3.62)) and EAs only (p = 4.14×10−7, OR = 1.40 (1.23–1.59)). Other SNPs that have been shown to affect mRNA levels of CHRNA5 in EAs are associated with nicotine dependence in AAs but not in EAs. The CHRNA3 SNP rs578776, which has low correlation with rs16969968, is associated with nicotine dependence in EAs but not in AAs. Less common SNPs (frequency ≤ 5%) also are associated with nicotine dependence.

In summary, multiple variants in this gene cluster contribute to nicotine dependence risk, and some are also associated with functional effects on CHRNA5. The non-synonymous SNP rs16969968, a known risk variant in European-descent populations, is also significantly associated with risk in African-Americans. Additional SNPs contribute in distinct ways to risk in these two populations.

doi:10.1158/0008-5472.CAN-09-0786

PMCID: PMC2874321 PMID: 19706762

genetic association; smoking; cholinergic nicotinic receptors; nicotinic acetylcholine receptors

6. Smokers with the CHRNA Lung Cancer-Associated Variants are Exposed to Higher Levels of Nicotine Equivalents and a Carcinogenic Tobacco-Specific Nitrosamine

Cancer research 2008;68(22):9137-9140.

A locus at 15q24/15q25.1, which includes the nicotinic acetylcholine receptor A subunits 3 and 5 (CHRNA3, CHRNA5) genes, has recently been associated with lung cancer risk, self-reported number of cigarettes smoked per day and a nicotine-dependence scale. It is not clear whether the association with lung cancer is direct or mediated through differences in smoking behavior. We used urinary biomarkers to test whether two linked lung cancer risk variants in CHRNA3 (rs1051730) and CHRNA5 (rs16969968) are associated with intensity of smoking and exposure to a tobacco-specific carcinogenic nitrosamine per cigarette dose. We studied 819 smokers and found that carriers of these variants extract a greater amount of nicotine (p=0.003) and are exposed to a higher internal dose of NNK (p=0.03) per cigarette than non-carriers. Thus, smokers who carry the CHRNA3 and A5 variants are expected to be at increased risk for lung cancer, compared to smokers who do not carry these alleles even if they smoked the same number of cigarettes. Number of cigarettes per day, even if it could be accurately assessed, is not an adequate measure of smoking dose.

doi:10.1158/0008-5472.CAN-08-2271

PMCID: PMC2587068 PMID: 19010884

7. Variants in the Nicotinic Receptors Alter the Risk for Nicotine Dependence

The American journal of psychiatry 2008;165(9):1163-1171.

Objective

A recent study provisionally identified numerous genetic variants as risk factors for the transition from smoking to the development of nicotine dependence, including an amino acid change in the α5 nicotinic cholinergic receptor (CHRNA5). The purpose of this study is to replicate these findings in an independent dataset and more thoroughly investigate the role of genetic variation in the cluster of physically linked nicotinic receptors, CHRNA5-CHRNA3-CHRNB4, and the risk of smoking.

Methods

Individuals from 219 European American families (N=2,284) were genotyped across this gene cluster to test the genetic association with smoking. The frequency of the amino acid variant (rs16969968) was studied in 995 individuals from diverse ethnic populations. In vitro studies were performed to directly test whether the amino acid variant in the CHRNA5 influenced receptor function.

Results

A genetic variant marking an amino acid change showed association with the smoking phenotype (p=0.007). This variant is within a highly conserved region across non-human species, but its frequency varied across human populations (0% in African populations to 37% in European populations). Furthermore, functional studies demonstrated that the risk allele decreased response to a nicotine agonist. A second independent finding was seen at rs578776 (p=0.003), and the functional significance of this association remains unknown.

Conclusions

This study confirms that at least two independent variants in this nicotinic receptor gene cluster contribute to the development of habitual smoking in some populations, and it underscores the importance of multiple genetic variants contributing to the development of common diseases in various populations.

doi:10.1176/appi.ajp.2008.07111711

PMCID: PMC2574742 PMID: 18519524

8. Polymorphisms of CHRNA5-CHRNA3-CHRNB4 Gene Cluster and NSCLC Risk in Chinese Population1

Li, Zhijun | Bao, Suzhen | Xu, Xiaohong | Bao, Yejiang | Zhang, Yongjun

Translational Oncology 2012;5(6):448-452.

AIM: To explore the potential association between single-nucleotide polymorphisms (SNPs) and haplotypes of the CHRNA5-CHRNA3-CHRNB4 gene cluster and the non-small cell lung cancer (NSCLC) susceptibility in never-smoking Chinese. METHODS: A case-control study was conducted with 200 NSCLC patients and 200 healthy controls, matched on age and sex. Five SNPs distributed in CHRNA5-CHRNA3-CHRNB4 gene cluster were selected for genotyping. The association between genotype and lung cancer risk was evaluated by computing the odds ratio (OR) and 95% confidence interval (CI) from multivariate unconditional logistic regression analyses with adjustment for gender and age. RESULTS: For CHRNA3 rs578776 status, data were available in 199 NSCLC patients and 199 controls. The G/G homozygote in CHRNB4 rs7178270 had a reduced risk of developing NSCLC (OR = 0.553; 95% CI = 0.309–0.989; P = .0437), especially squamous cell carcinoma (SQC) (OR = 0.344; 95% CI = 0.161–0.732; P = .0043), compared with those who carry at least one C allele (C/C and C/G). The polymorphisms of rs578776, rs938682, rs17486278, and rs11637635 were not significantly different between controls and cases or between controls and histologic subgroups, adenocarcinoma and SQC, respectively. CONCLUSIONS: In our study, we found that the SNP of CHRNB4 rs7178270 is significantly associated with reduced risk of NSCLC, especially with reduced risk of SQC in never-smoking Chinese population.

PMCID: PMC3567724 PMID: 23397474

9. Variants Located Upstream of CHRNB4 on Chromosome 15q25.1 Are Associated with Age at Onset of Daily Smoking and Habitual Smoking

PLoS ONE 2012;7(3):e33513.

Several genome-wide association and candidate gene studies have linked chromosome 15q24–q25.1 (a region including the CHRNA5-CHRNA3-CHRNB4 gene cluster) with alcohol dependence, nicotine dependence and smoking-related illnesses such as lung cancer and chronic obstructive pulmonary disease. To further examine the impact of these genes on the development of substance use disorders, we tested whether variants within and flanking the CHRNA5-CHRNA3-CHRNB4 gene cluster affect the transition to daily smoking (individuals who smoked cigarettes 4 or more days per week) in a cross sectional sample of adolescents and young adults from the COGA (Collaborative Study of the Genetics of Alcoholism) families. Subjects were recruited from families affected with alcoholism (either as a first or second degree relative) and the comparison families. Participants completed the SSAGA interview, a comprehensive assessment of alcohol and other substance use and related behaviors. Using the Quantitative trait disequilibrium test (QTDT) significant association was detected between age at onset of daily smoking and variants located upstream of CHRNB4. Multivariate analysis using a Cox proportional hazards model further revealed that these variants significantly predict the age at onset of habitual smoking among daily smokers. These variants were not in high linkage disequilibrium (0.28

doi:10.1371/journal.pone.0033513

PMCID: PMC3306405 PMID: 22438940

10. Association of α4β2 nicotinic receptor and heavy smoking in schizophrenia

Journal of Psychiatry & Neuroscience : JPN 2007;32(6):412-416.

Introduction

Previously we suggested that the CHRNA7 polymorphism in nicotinic receptor genes, in particular the D15S1360 in CHRNA7, is associated with smoking in schizophrenia. Schizophrenia patients are usually heavy smokers. In this study we hypothesized that high-affinity nicotinic receptors are associated with smoking in such patients.

Objective

To investigate the role of α4 (Ch 20) and β2 (Ch 1) genes in conferring a risk for smoking and for smoking a large number of cigarettes daily in subjects with schizophrenia.

Methods

Our study sample consisted of 241 white European schizophrenia patients (157 smokers and 84 nonsmokers) from the Toronto area. Current smoking status was assessed by the medical history. We investigated 4 markers located in the CHRNA4 gene and 3 markers located in the CHRNB2 gene.

Results

There was no difference in age or ethnicity between the 2 groups and the population was not stratified (λ = 0.4527). We found a significant association between the CHRNA4 rs3746372 allele 1 and a large number of cigarettes smoked daily (p = 0.0203). The intragenic interaction between rs3787116 and rs3746372 (p = 0.0050) in CHRNA4 showed a significant interaction for the number of cigarettes smoked.

Conclusion

Although our findings suggest an association between rs3746372 allele 1 and heavy smoking, further study is warranted to investigate the relation between smoking and high-affinity nicotinic receptor genes in schizophrenia.

PMCID: PMC2077346 PMID: 18043764

nicotinic acetylcholine receptor alpha4 subunit; CHRNB2 protein, human; genetics; nicotine; receptors, nicotinic; schizophrenia

11. Associations between Variation in CHRNA5-CHRNA3-CHRNB4, Body Mass Index and Blood Pressure in the Northern Finland Birth Cohort 1966

PLoS ONE 2012;7(9):e46557.

Background

The CHRNA5-CHRNA3-CHRNB4 gene cluster on 15q25 has consistently been associated with smoking quantity, nicotine dependence and lung cancer. Recent research also points towards its involvement in cardiovascular homeostasis, but studies in large human samples are lacking, especially on the role of the gene cluster in blood pressure regulation.

Methodology/Principal Findings

We studied the associations between 18 single nucleotide polymorphisms (SNPs) in CHRNA5-CHRNA3-CHRNB4 and systolic blood pressure (SBP), diastolic blood pressure (DBP), and body mass index (BMI) in 5402 young adults from the Northern Finland Birth Cohort 1966. We observed some evidence for associations between two SNPs and SBP and between six SNPs and BMI; the evidence for associations with DBP was weaker. The associations with the three phenotypes were driven by different loci with low linkage disequilibrium with each other. The associations appeared more pronounced in smokers, such that the smoking-increasing alleles would predict lower SBP and BMI. Each additional copy of the rs1948 G-allele and the rs950776 A-allele reduced SBP on average by −1.21 (95% CI −2.01, −0.40) mmHg in smokers. The variants associated with BMI included rs2036534, rs6495309, rs1996371, rs6495314, rs4887077 and rs11638372 and had an average effect size of −0.38 (−0.68, −0.08) kg/m2 per an additional copy of the risk allele in smokers. Formal assessments of interactions provided weaker support for these findings, especially after adjustment for multiple testing.

Conclusions

Variation at 15q25 appears to interact with smoking status in influencing SBP and BMI. The genetic loci associated with SBP were in low linkage disequilibrium with those associated with BMI suggesting that the gene cluster might regulate SBP through biological mechanisms that partly differ from those regulating BMI. Further studies in larger samples are needed for more precise evaluation of the possible interactions, and to understand the mechanisms behind.

doi:10.1371/journal.pone.0046557

PMCID: PMC3459914 PMID: 23029550

12. Genetic variation at CHRNA5-CHRNA3-CHRNB4 interacts with smoking status to influence body mass index

International Journal of Epidemiology 2011;40(6):1617-1628.

Background Cigarette smoking is associated with lower body mass index (BMI), and a commonly cited reason for unwillingness to quit smoking is a concern about weight gain. Common variation in the CHRNA5-CHRNA3-CHRNB4 gene region (chromosome 15q25) is robustly associated with smoking quantity in smokers, but its association with BMI is unknown. We hypothesized that genotype would accurately reflect smoking exposure and that, if smoking were causally related to weight, it would be associated with BMI in smokers, but not in never smokers.

Methods We stratified nine European study samples by smoking status and, in each stratum, analysed the association between genotype of the 15q25 SNP, rs1051730, and BMI. We meta-analysed the results (n = 24 198) and then tested for a genotype × smoking status interaction.

Results There was no evidence of association between BMI and genotype in the never smokers {difference per T-allele: 0.05 kg/m2 [95% confidence interval (95% CI): −0.05 to 0.18]; P = 0.25}. However, in ever smokers, each additional smoking-related T-allele was associated with a 0.23 kg/m2 (95% CI: 0.13–0.31) lower BMI (P = 8 × 10−6). The effect size was larger in current [0.33 kg/m2 lower BMI per T-allele (95% CI: 0.18–0.48); P = 6 × 10−5], than in former smokers [0.16 kg/m2 (95% CI: 0.03–0.29); P = 0.01]. There was strong evidence of genotype × smoking interaction (P = 0.0001).

Conclusions Smoking status modifies the association between the 15q25 variant and BMI, which strengthens evidence that smoking exposure is causally associated with reduced BMI. Smoking cessation initiatives might be more successful if they include support to maintain a healthy BMI.

doi:10.1093/ije/dyr077

PMCID: PMC3235017 PMID: 21593077

Smoking; BMI; SNP; genetic association; interaction

13. Interplay of Genetic Risk Factors (CHRNA5-CHRNA3-CHRNB4) and Cessation Treatments in Smoking Cessation Success

The American journal of psychiatry 2012;169(7):735-742.

Objective

Smoking is highly intractable and the genetic influences on cessation are unclear. Identifying the genetic factors affecting smoking cessation could elucidate the nature of tobacco dependence, enhance risk assessment, and support treatment algorithm development. This study tests whether variants in the nicotinic receptor gene cluster (CHRNA5-CHRNA3-CHRNB4) predict age of smoking cessation and relapse to smoking after a quit attempt.

Method

In a community-based, cross-sectional study (N=5,216) and a randomized comparative effectiveness smoking cessation trial (N=1,073), we used survival analyses and logistic regression to model relations between smoking cessation (self-reported quit age in a community study and point-prevalence abstinence at end-of-treatment in a clinical trial) and three common haplotypes in the CHRNA5-CHRNA3-CHRNB4 region defined by rs16969968 and rs680244.

Results

The genetic variants in the CHRNA5-CHRNA3-CHRNB4 region that predict nicotine dependence also predict a later age of smoking cessation in a community-based sample (X2=8.46, df=2, p=0.015). In the smoking cessation trial, these variants predict abstinence at end-of-treatment in individuals receiving placebo medication, but not amongst individuals receiving active medication. Genetic variants interact with treatment in affecting cessation success (X2=8.97, df=2, p=0.011).

Conclusions

Smokers with the high risk genetic variants have a three-fold increased likelihood of responding to pharmacologic cessation treatments, compared to smokers with the low risk genetic variants. The high-risk variants increase the risk of cessation failure, and this increased risk can be ameliorated by cessation pharmacotherapy. By identifying a high-risk genetic group with heightened response to smoking cessation pharmacotherapy, this work may support the development of personalized cessation treatments.

doi:10.1176/appi.ajp.2012.11101545

PMCID: PMC3433845 PMID: 22648373

14. Peer Smoking and the Nicotinic Receptor Genes: An Examination of Genetic and Environmental Risks for Nicotine Dependence

Addiction (Abingdon, England) 2010;105(11):2014-2022.

Background

Peer smoking provides a socially reinforcing context of friends’ encouragement and approval that contributes to smoking behavior. Twin studies show correlations and interactions between peer substance use and genetic liability for substance use. However, none examined specific genes. Here we test the hypothesis that the nicotinic receptor genes CHRNA5 (rs16969968), CHRNA3 (rs578776), CHRNB3 (rs13277254), and CHRND (rs12466358) modify the risk for nicotine dependence (ND) associated with peer smoking.

Methods

Cases of current nicotine dependence (FTND ≥ 4) and smoking-exposed (smoked 100+ cigarettes lifetime), but non-dependent controls (lifetime FTND = 0) came from the Collaborative Genetic Study of Nicotine Dependence (n=2,038). Peer smoking was retrospectively assessed for grades 9–12.

Results

Peer smoking and the four SNPs were associated with ND. A statistically significant interaction was found between peer smoking and rs16969968 (p = 0.0077). Overall risk of ND was highest for the rs16969968 AA genotype. However, variance in ND attributable to peer smoking was substantially lower among those with the AA genotype at rs16969968 than the lower risk genotypes: AA = 2.5%, GA/AG = 11.2%, GG = 14.2%; p ≤ 0.004.

Conclusions

Peer smoking had a substantially lower effect on ND among those with the high risk AA genotype at the functional SNP rs16969968 (CHRNA5) than among those with lower risk genotypes. Such results highlight the possibility that given drug exposure those with specific genetic risks may be less affected by social contexts and intervention strategies focused on social factors could have less influence on those at highest genetic risk.

doi:10.1111/j.1360-0443.2010.03074.x

PMCID: PMC2970633 PMID: 20840187

nicotine dependence; peer smoking; gene-environmental interaction; nicotinic receptor genes; case control study

15. Variation in the Nicotinic Acetylcholine Receptor Gene Cluster CHRNA5–CHRNA3–CHRNB4 and Its Interaction with Recent Tobacco Use Influence Cognitive Flexibility

Zhang, Huiping | Kranzler, Henry R | Poling, James | Gelernter, Joel

Neuropsychopharmacology 2010;35(11):2211-2224.

Variants in the CHRNA5–CHRNA3–CHRNB4 gene cluster have been associated with nicotine dependence (ND) and ND-related traits. To evaluate a potential underlying mechanism for this association, we investigated the effects of 10 variants in this gene cluster and their interactive effects as a result of recent smoking on cognitive flexibility, a possible mediator of genetic effects in smokers. Cognitive flexibility of 466 European Americans (EAs; 360 current smokers) and 805 African Americans (AAs; 635 current smokers) was assessed using the Wisconsin Card Sorting Test. The main effects of variants and haplotypes and their interaction with recent smoking on cognitive flexibility were examined using multivariate analysis of variance and the haplotype analysis program HAPSTAT. In EAs, the major alleles of five variants (CHRNA5-rs3841324–22 bp-insertion-allele, CHRNA5-rs615470-C-allele, CHRNA3-rs6495307-C-allele, CHRNA3-rs2869546-T-allele, and CHRNB4-rs11637890-C-allele) were associated with significantly greater perseverative responses (P=0.003–0.017) and perseverative errors (P=0.004–0.026; recessive effect). Among EAs homozygous for the major alleles of each of these five variants, current smokers made fewer perseverative responses and perseverative errors than did past smokers. Significant interactive effects of four variants (rs3841324, rs615470, rs6495307, and rs2869546) and current smoking on cognitive flexibility were observed (perseverative responses (P=0.010–0.044); perseverative errors (P=0.017–0.050)). However, in AAs, 10 variants in this gene cluster showed no apparent effects on cognitive flexibility. These findings suggest that variation in the CHRNA5–CHRNA3–CHRNB4 gene cluster influences cognitive flexibility differentially in AAs and EAs and that current smoking moderates this effect. These findings could account in part for differences in ND risk associated with these variants in AAs and EAs.

doi:10.1038/npp.2010.95

PMCID: PMC3055317 PMID: 20631687

CHRNA5-CHRNA3-CHRNB4; cognitive flexibility; Wisconsin Card Sorting Test; gene × recent tobacco use; Neurogenetics; Cognition; Psychiatry & Behavioral Sciences; Learning & Memory; WCST; CHRNA5-CHRNA3-CHRNB4; cognitive flexibility; gene × recent tobacco use

16. Multiple Distinct Risk Loci for Nicotine Dependence Identified by Dense Coverage of the Complete Family of Nicotinic Receptor Subunit (CHRN) Genes

American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2009;150B(4):453-466.

Tobacco smoking continues to be a leading cause of preventable death. Recent research has underscored the important role of specific cholinergic nicotinic receptor subunit (CHRN) genes in risk for nicotine dependence and smoking. To detect and characterize the influence of genetic variation on vulnerability to nicotine dependence, we analyzed 226 SNPs covering the complete family of 16 CHRN genes, which encode the nicotinic acetylcholine receptor (nAChR) subunits, in a sample of 1050 nicotine-dependent cases and 879 non-dependent controls of European descent. This expanded SNP coverage has extended and refined the findings of our previous large scale genome-wide association and candidate gene study. After correcting for the multiple tests across this gene family, we found significant association for two distinct loci in the CHRNA5-CHRNA3-CHRNB4 gene cluster, one locus in the CHRNB3-CHRNA6 gene cluster, and a fourth, novel locus in the CHRND-CHRNG gene cluster. The two distinct loci in CHRNA5-CHRNA3-CHRNB4 are represented by the non-synonymous SNP rs16969968 in CHRNA5 and by rs578776 in CHRNA3, respectively, and joint analyses show that the associations at these two SNPs are statistically independent. Nominally significant single-SNP association was detected in CHRNA4 and CHRNB1. In summary, this is the most comprehensive study of the CHRN genes for involvement with nicotine dependence to date. Our analysis reveals significant evidence for at least four distinct loci in the nicotinic receptor subunit genes that each influence the transition from smoking to nicotine dependence and may inform the development of improved smoking cessation treatments and prevention initiatives.

doi:10.1002/ajmg.b.30828

PMCID: PMC2693307 PMID: 19259974

cholinergic nicotinic receptors; nicotinic acetylcholine receptors; smoking; genetic association

17. A common genetic variant in the 15q24 nicotinic acetylcholine receptor gene cluster (CHRNA5–CHRNA3–CHRNB4) is associated with a reduced ability of women to quit smoking in pregnancy

Human Molecular Genetics 2009;18(15):2922-2927.

Maternal smoking during pregnancy is associated with low birth weight and adverse pregnancy outcomes. Women are more likely to quit smoking during pregnancy than at any other time in their lives, but some pregnant women continue to smoke. A recent genome-wide association study demonstrated an association between a common polymorphism (rs1051730) in the nicotinic acetylcholine receptor gene cluster (CHRNA5–CHRNA3–CHRNB4) and both smoking quantity and nicotine dependence. We aimed to test whether the same polymorphism that predisposes to greater cigarette consumption would also reduce the likelihood of smoking cessation in pregnancy. We studied 7845 pregnant women of European descent from the South-West of England. Using 2474 women who smoked regularly immediately pre-pregnancy, we analysed the association between the rs1051730 risk allele and both smoking cessation during pregnancy and smoking quantity. Each additional copy of the risk allele was associated with a 1.27-fold higher odds (95% CI 1.11–1.45) of continued smoking during pregnancy (P = 0.0006). Adjustment for pre-pregnancy smoking quantity weakened, but did not remove this association [odds ratio (OR) 1.20 (95% CI 1.03–1.39); P = 0.018]. The same risk allele was also associated with heavier smoking before pregnancy and in the first, but not the last, trimester [OR for smoking 10+ cigarettes/day versus 1–9/day in first trimester = 1.30 (95% CI 1.13–1.50); P = 0.0003]. To conclude, we have found strong evidence of association between the rs1051730 variant and an increased likelihood of continued smoking in pregnancy and have confirmed the previously observed association with smoking quantity. Our data support the role of genetic factors in influencing smoking cessation during pregnancy.

doi:10.1093/hmg/ddp216

PMCID: PMC2706684 PMID: 19429911

18. In Vitro and Ex Vivo Analysis of CHRNA3 and CHRNA5 Haplotype Expression

PLoS ONE 2011;6(8):e23373.

Genome-wide association studies implicate variations in CHRNA5 and CHRNA3 as being associated with nicotine addiction (NA). Multiple common haplotypes (“risk”, “mixed” and “protective”) exist in Europeans; however, high linkage disequilibrium between variations in CHRNA5 and CHRNA3 makes assigning causative allele(s) for NA difficult through genotyping experiments alone. We investigated whether CHRNA5 or CHRNA3 promoter haplotypes, associated previously with NA, might influence allelic expression levels. For in vitro analyses, promoter haplotypes were sub-cloned into a luciferase reporter vector. When assessed in BE(2)-C cells, luciferase expression was equivalent among CHRNA3 haplotypes, but the combination of deletion at rs3841324 and variation at rs503464 decreased CHRNA5 promoter-derived luciferase activity, possibly due to loss of an SP-1 and other site(s). Variation within the CHRNA5 5’UTR at rs55853698 and rs55781567 also altered luciferase expression in BE(2)-C cells. Allelic expression imbalance (AEI) from the “risk” or “protective” haplotypes was assessed in post-mortem brain tissue from individuals heterozygous at coding polymorphisms in CHRNA3 (rs1051730) or CHRNA5 (rs16969968). In most cases, equivalent allelic expression was observed; however, one individual showed CHRNA5 AEI that favored the “protective” allele and that was concordant with heterozygosity at polymorphisms ∼13.5 kb upstream of the CHRNA5 transcription start site. Putative enhancer activity from these distal promoter elements was assessed using heterologous promoter constructs. We observed no differences in promoter activity from the two distal promoter haplotypes examined, but found that the distal promoter region strongly repressed transcription. We conclude that CHRNA5 promoter variants may affect relative risk for NA in some heterozygous individuals.

doi:10.1371/journal.pone.0023373

PMCID: PMC3155531 PMID: 21858091

19. Genome-wide association scan of tag SNPs identifies a susceptibility locus for lung cancer at 15q25.1

Nature genetics 2008;40(5):616-622.

To identify risk variants for lung cancer, we conducted a multistage genome-wide association study. In the discovery phase, we analyzed 315,450 tagging SNPs in 1,154 current and former (ever) smoking cases of European ancestry and 1,137 frequency-matched, ever-smoking controls from Houston, Texas. For replication, we evaluated the ten SNPs most significantly associated with lung cancer in an additional 711 cases and 632 controls from Texas and 2,013 cases and 3,062 controls from the UK. Two SNPs, rs1051730 and rs8034191, mapping to a region of strong linkage disequilibrium within 15q25.1 containing PSMA4 and the nicotinic acetylcholine receptor subunit genes CHRNA3 and CHRNA5, were significantly associated with risk in both replication sets. Combined analysis yielded odds ratios of 1.32 (P < 1 × 10−17) for both SNPs. Haplotype analysis was consistent with there being a single risk variant in this region. We conclude that variation in a region of 15q25.1 containing nicotinic acetylcholine receptors genes contributes to lung cancer risk.

doi:10.1038/ng.109

PMCID: PMC2713680 PMID: 18385676

20. Alpha-5 and -3 nicotinic receptor gene variants predict nicotine dependence but not cessation: Findings from the COMMIT cohort

Bousman, Chad A. | Rivard, Cheryl | Den Haese, Jason | Ambrosone, Christine | Hyland, Andrew

American Journal of Medical Genetics 2012;159B(2):227-235.

Smoking many cigarettes per day (CPD) and short interval to first cigarette (TTF) after waking are two of the most heritable smoking phenotypes and comprise the Heavy Smoking Index (HSI). These phenotypes are often used as proxies for nicotine dependence (ND) and are associated with smoking cessation outcomes. Case-control and genome-wide association studies have reported links between single nucleotide polymorphisms (SNPs) in the alpha-5 and -3 nicotinic receptor subunit (CHRNA5 and CHRNA3) genes and CPD but few have examined TTF or cessation outcomes. In this study we longitudinally assessed 1301 European-American smokers at four time-points from 1988 to 2005. One CHRNA5 (rs16969968) and two CHRNA3 (rs1051703, rs6495308) SNPs were examined for their ability to predict smokers who ‘ever’ reported ND based on three phenotypic classifications: 1) 25+ CPD, 2) TTF < 10 minutes, and 3) HSI ≥ 4. In a subsample of 1157 quit attempters, we also examined each SNP’s ability to predict ‘ever’ quitting for a period of >6 months. Demographically adjusted logistic regressions showed significant allelic and genotypic associations between all three SNPs and CPD but not TTF, HSI, or smoking cessation. Carriers of both the rs16969968-AA and rs6495308-TT genotypes had approximately two-fold greater odds for ND defined using CPD or TTF. Results suggest nicotinic receptor variants are associated with greater odds of ND according to CPD and to a lesser extent TTF. Research examining the effect of nicotinic receptor genetic variation on ND phenotypes beyond CPD is warranted.

doi:10.1002/ajmg.b.32019

PMCID: PMC3262775 PMID: 22223462

Cholinergic; Nicotinic; Allele; Dependence; Cessation

21. Nicotinic Receptor Gene Variants Influence Susceptibility to Heavy Smoking

Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 2008;17(12):3517-3525.

Heavy smoking is a strong predictor of nicotine dependence, which is a major impediment to smoking cessation. Although both heavy smoking and nicotine dependence are highly heritable, previous attempts to identify genes influencing these phenotypes have been largely unsuccessful until very recently. We studied 1,452 heavy smokers (defined as smoking at least 30 cigarettes per day for at least 5 years) and 1,395 light smokers (defined as smoking <5 cigarettes per day for at least 1 year) to investigate the association of common variants in nicotinic receptor subunit genes with smoking behavior. Compared to the most common allele, two separate groups of SNPs in the CHRNA5-CHRNA3-CHRNB4 gene cluster were associated with heavy smoking with a very high statistical significance. One group of eight SNPs, that included a nonsynonymous SNP in the CHRNA5 gene, was in strong linkage disequilibrium and associated with increased risk of heavy smoking. A second group of SNPs not strongly correlated with the first was associated with decreased risk of heavy smoking. Analyses that combined both groups of SNPs found associations with heavy smoking that varied by more than two-fold. Our findings identify two loci in the CHRNA5-CHRNA3-CHRNB4 gene cluster that predict smoking behavior and provide strong evidence for the involvement of the α5 nicotinic receptor in heavy smoking.

doi:10.1158/1055-9965.EPI-08-0585

PMCID: PMC2614129 PMID: 19029397

22. Nicotinic Acetylcholine Receptor Region on Chromosome 15q25 and Lung Cancer Risk Among African Americans: A Case–Control Study

JNCI Journal of the National Cancer Institute 2010;102(15):1199-1205.

Genome-wide association studies of white persons with lung cancer have identified a region of extensive linkage disequilibrium on chromosome 15q25.1 that appears to be associated with both risk for lung cancer and smoking dependence. Because studying African American persons, who exhibit lower levels of linkage disequilibrium in this region, may identify additional loci that are associated with lung cancer, we genotyped 34 single-nucleotide polymorphisms (SNPs) in this region (including LOC123688, PSMA4, CHRNA5, CHRNA3, and CHRNB4 genes) in 467 African American patients with lung cancer and 388 frequency-matched African American control subjects. Associations of SNPs in LOC123688 (rs10519203; odds ratio [OR] = 1.60, 95% confidence interval [CI] = 1.25 to 2.05, P = .00016), CHRNA5 (rs2036527; OR = 1.67, 95% CI = 1.26 to 2.21, P = .00031), and CHRNA3 (rs1051730; OR = 1.81, 95% CI = 1.26 to 2.59, P = .00137) genes with lung cancer risk reached Bonferroni-corrected levels of statistical significance (all statistical tests were two-sided). Joint logistic regression analysis showed that rs684513 (OR = 0.47, 95% CI = 0.31 to 0.71, P = .0003) in CHRNA5 and rs8034191 (OR = 1.76, 95% CI = 1.23 to 2.52, P = .002) in LOC123688 were also associated with risk. The functional A variant of rs1696698 in CHRNA5 had the strongest association with lung cancer (OR = 1.98, 95% CI = 1.25 to 3.11, P = .003). These SNPs were primarily associated with increased risk for lung adenocarcinoma histology and were only weakly associated with smoking phenotypes. Thus, among African American persons, multiple loci in the region of chromosome 15q25.1 appear to be strongly associated with lung cancer risk.

doi:10.1093/jnci/djq232

PMCID: PMC2914761 PMID: 20554942

23. Association of the CHRNA5-A3-B4 Gene Cluster With Heaviness of Smoking: A Meta-Analysis

Ware, Jennifer J. | van den Bree, Marianne B. M. | Munafò, Marcus R.

Nicotine & Tobacco Research 2011;13(12):1167-1175.

Introduction:

Variation in the CHRNA5-A3-B4 gene cluster is a promising candidate region for smoking behavior and has been linked to multiple smoking-related phenotypes (e.g., nicotine dependence) and diseases (e.g., lung cancer). Two single nucleotide polymorphisms (SNPs), rs16969968 in CHRNA5 and rs1051730 in CHRNA3, have generated particular interest.

Methods:

We evaluated the published evidence for association between rs16969968 (k = 27 samples) and rs1051730 (k = 44 samples) SNPs with heaviness of smoking using meta-analytic techniques. We explored which SNP provided a stronger genetic signal and investigated study-level characteristics (i.e., ancestry, disease state) to establish whether the strength of association differed across populations. We additionally tested for small study bias and explored the impact of year of publication.

Results and Conclusions:

Meta-analysis indicated compelling evidence of an association between the rs1051730/rs16966968 variants and daily cigarette consumption (fixed effects: B = 0.91, 95% CI = 0.77, 1.06, p < .001; random effects: B = 1.01, 95% CI = 0.81, 1.22, p < .001), equivalent to a per-allele effect of approximately 1 cigarette/day. SNP rs1051730 was found to provide a stronger signal than rs16966968 in stratified analyses (pdiff = .028), although this difference was only qualitatively observed in the subset of samples that provided data on both SNPs. While the functional relevance of rs1051730 is unknown, it may be a strong tagging SNP for functional haplotypes in this region.

doi:10.1093/ntr/ntr118

PMCID: PMC3223575 PMID: 22071378

24. CHRNB2 Promoter Region: Association with subjective effects to nicotine and expression differences

Hoft, Nicole R. | Stitzel, Jerry A. | Hutchison, Kent E. | Ehringer, Marissa A.

Genes, Brain, and Behavior 2010;10(2):176-185.

Smoking behavior is complex, and includes multiple stages in the progression from experimentation to continued use and dependence. The experience of subjective effects, such as dizziness, euphoria, heart pounding, nausea, and high, have been associated with varying degrees of persistence and subsequent abuse/dependence of marijuana, cocaine, tobacco and alcohol (Grant et al., 2005, Wagner & Anthony, 2002). Previous studies have reported associations between neuronal nicotinic receptor (CHRN) genes and subjective effects to nicotine. We sought to replicate and expand this work by examining eight SNPs in a sample of adult smokers (n=316) who reported subjective effects following cigarette smoking in a controlled laboratory environment. Two SNPs each in the CHRNB2, CHRNB3, CHRNA6 and CHRNA4 genes were examined. A significant association was found between two SNPs and physical effects reported after smoking the first experimental cigarette. SNP rs2072658 is upstream of CHRNB2 (p value = 0.0046) and rs2229959 is a synonymous change in exon 5 of CHRNA4 (p value = 0.0051). We also examined possible functional relevance of SNP rs2072658 using an in vitro gene expression assay. These studies provided evidence that the minor allele of rs2072658 may lead to decreased gene expression, using two separate cell lines, P19 and SH-SY5Y cell lines (18% p<0.001 and 26% p<0.001 respectively). The human genetic study and functional assays suggest that variation in the promoter region of CHRNB2 gene may be important in mediating levels of expression of the β2 nicotinic receptor subunit, which may be associated with variation in subjective response to nicotine.

doi:10.1111/j.1601-183X.2010.00650.x

PMCID: PMC3403289 PMID: 20854418

Nicotinic receptors; SNP; Genetic association; Tobacco use; Subjective effects

25. An Exploratory Study on the CHRNA3-CHRNA5-CHRNB4 Cluster, Smoking, and Parkinson's Disease

Neuro-Degenerative Diseases 2011;8(5):296-299.

Background

Smokers have a lower risk of Parkinson's disease (PD). Recent genome-wide association studies (GWAS) have consistently linked several single nucleotide polymorphisms (SNPs) in the CHRNA3-CHRNA5-CHRNB4 cluster on chromosome 15.q25 to smoking behaviors and nicotine dependence. Investigations into these SNPs may help explain the nature and mechanisms of the smoking-PD relationship. Objective: To examine whether the genetic variations that were consistently associated with smoking or nicotine dependence in recent GWAS also predict the risk of PD.

Methods

This is a population-based case-control study of 788 physician-diagnosed PD patients and 911 controls, all non-Hispanic Whites. Seven SNPs were selected based on findings from recent GWAS on smoking and nicotine dependence, all from the nicotinic acetylcholine receptor subunits (CHRN) A3-A5-B4. Odds ratios (ORs) and 95% confidence intervals were derived from logistic regression models under the assumption of logit-additive allelic effects.

Results

Four SNPs in linkage disequilibrium from the CHRNA3-CHRNA5-CHRNB4 cluster were associated with smoking duration (OR >1.3, p < 0.05). However, none of the SNPs from this cluster was associated with PD risk in the overall analysis or after stratifying on smoking status.

Conclusion

This preliminary analysis does not support a relationship between these smoking-related GWAS SNPs and PD.

doi:10.1159/000323190

PMCID: PMC3121544 PMID: 21228559

Parkinson's disease; Smoking; CHRNA3-CHRNA5-CHRNB4 cluster; Nicotine dependence

Results 1-25 (556360)

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