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1.  Pilot Study of CYP2B6 Genetic Variation to Explore the Contribution of Nitrosamine Activation to Lung Carcinogenesis 
We explored the contribution of nitrosamine metabolism to lung cancer in a pilot investigation of genetic variation in CYP2B6, a high-affinity enzymatic activator of tobacco-specific nitrosamines with a negligible role in nicotine metabolism. Previously we found that variation in CYP2A6 and CHRNA5-CHRNA3-CHRNB4 combined to increase lung cancer risk in a case-control study in European American ever-smokers (n = 860). However, these genes are involved in the pharmacology of both nicotine, through which they alter smoking behaviours, and carcinogenic nitrosamines. Herein, we separated participants by CYP2B6 genotype into a high- vs. low-risk group (*1/*1 + *1/*6 vs. *6/*6). Odds ratios estimated through logistic regression modeling were 1.25 (95% CI 0.68–2.30), 1.27 (95% CI 0.89–1.79) and 1.56 (95% CI 1.04–2.31) for CYP2B6, CYP2A6 and CHRNA5-CHRNA3-CHRNB4, respectively, with negligible differences when all genes were evaluated concurrently. Modeling the combined impact of high-risk genotypes yielded odds ratios that rose from 2.05 (95% CI 0.39–10.9) to 2.43 (95% CI 0.47–12.7) to 3.94 (95% CI 0.72–21.5) for those with 1, 2 and 3 vs. 0 high-risk genotypes, respectively. Findings from this pilot point to genetic variation in CYP2B6 as a lung cancer risk factor supporting a role for nitrosamine metabolic activation in the molecular mechanism of lung carcinogenesis.
doi:10.3390/ijms14048381
PMCID: PMC3645749  PMID: 23591849
CYP2B6; CYP2A6; CHRNA5-CHRNA3-CHRNB4; tobacco specific nitrosamines; lung cancer risk; genetic variation
2.  Rare missense variants in CHRNB4 are associated with reduced risk of nicotine dependence 
Human Molecular Genetics  2011;21(3):647-655.
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
3.  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
4.  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
5.  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
6.  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
7.  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
8.  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
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
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
Life sciences  2012;91(21-22):1103-1108.
Aims
Previous studies revealed association of lung cancer risk with single nucleotide polymorphisms (SNPs) in chromosome 15q25 region containing CHRNA5-CHRNA3-CHRNB4 nicotinic acetylcholine receptor (nAChR) subunit gene cluster. The genetic variations in other lung nAChRs remained unknown. In this study, we perform case-control analysis of CHRNA9 and CHRNA3 genes using 340 non-small cell lung cancer cases and 435 controls.
Main methods
All exons, 3’UTR, intron 1 and parts of other introns surrounding exons 2–5 of CHRNA9 gene as well as exons 2, 3 of CHRNA3 gene and parts of surrounding intronic regions were sequenced. The study was controlled for gender, age and ethnicity related differences. Each SNP in analyzed groups was assessed by allele frequency, genotype distribution and haplotype analysis.
Key findings
The case-control analysis revealed that an increased risk is associated with two SNPs in CHRNA9, rs56159866 and rs6819385, and one in CHRNA3, rs8040868. The risk was reduced for three SNPs in CHRNA9, rs55998310, rs56291234, and newly discovered ss410759555, and also in carriers of the haplotype NP_060051.2 containing ancestral N442 variant of α9.
Significance
The nonsynonymous substitutions can produce receptors exhibiting unique ligand-binding and downstream signaling characteristics, synonymous as well all intronic SNPs may affect protein production at the transcriptional and/or translational levels, or just manifest association with cancer by genetic linkage to other alleles. Elucidation of the mechanisms by which individual genetic variations in α9 affect predisposition to lung cancer may lead to development of personalized approaches to cancer prevention and treatment as well as protection against tobacco consumption.
doi:10.1016/j.lfs.2011.12.023
PMCID: PMC3341501  PMID: 22280835
lung cancer; CHRNA3; CHRNA9; α3 and α9 nicotinic acetylcholine receptors; single nucleotide polymorphisms
PLoS ONE  2013;8(7):e67664.
Background
CHRNA5-A3-B4, the gene cluster encoding nicotinic acetylcholine receptor subunits, is associated with lung cancer risk and smoking behaviors in people of European descent. Because cigarette smoking is also a major risk factor for esophageal squamous cell carcinoma (ESCC), we investigated the associations between variants in CHRNA5-A3-B4 and ESCC risk, as well as smoking behaviors, in a Chinese population.
Methods
A case-control study of 866 ESCC patients and 952 healthy controls was performed to study the association of polymorphisms (rs667282 and rs3743073) in CHRNA5-A3-B4 with cancer risk using logistic regression models. The relationships between CHRNA5-A3-B4 polymorphisms and smoking behaviors that can be quantified by cigarettes smoked per day (CPD) and pack-years of smoking were separately estimated with Kruskal-Wallis tests among all 840 smokers.
Results
CHRNA5-A3-B4 rs667282 TT/TG genotypes were associated with significantly increased risk of ESCC [adjusted odds ratio (OR) = 1.32, 95% confidence interval (CI) = 1.03 – 1.69, P = 0.029]. The increased ESCC risk was even higher among younger subjects (≤60 years) (OR = 1.44, 95% CI = 1.04 – 1.98, P = 0.024). These effects were not found in another polymorphism rs3743073. No evident association between the two polymorphisms and smoking behaviors was observed.
Conclusions
These results support the hypothesis that CHRNA5-A3-B4 is a susceptibility gene cluster for ESCC. The relationship between CHRNA5-A3-B4 and smoking behaviors in a Chinese population needs further investigation.
doi:10.1371/journal.pone.0067664
PMCID: PMC3699625  PMID: 23844051
Behavior genetics  2011;42(3):402-414.
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.
doi:10.1007/s10519-011-9514-x
PMCID: PMC3506120  PMID: 22042234
nicotinic receptor genes; externalizing behaviors; association study; disinhibition; drug behaviors
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
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
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
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
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
PLoS ONE  2013;8(11):e80204.
Variants within the gene cluster encoding α3, α5, and β4 nicotinic receptor subunits are major risk factors for substance dependence. The strongest impact on risk is associated with variation in the CHRNA5 gene, where at least two mechanisms are at work: amino acid variation and altered mRNA expression levels. The risk allele of the non-synonymous variant (rs16969968; D398N) primarily occurs on the haplotype containing the low mRNA expression allele. In populations of European ancestry, there are approximately 50 highly correlated variants in the CHRNA5-CHRNA3-CHRNB4 gene cluster and the adjacent PSMA4 gene region that are associated with CHRNA5 mRNA levels. It is not clear which of these variants contribute to the changes in CHRNA5 transcript level. Because populations of African ancestry have reduced linkage disequilibrium among variants spanning this gene cluster, eQTL mapping in subjects of African ancestry could potentially aid in defining the functional variants that affect CHRNA5 mRNA levels. We performed quantitative allele specific gene expression using frontal cortices derived from 49 subjects of African ancestry and 111 subjects of European ancestry. This method measures allele-specific transcript levels in the same individual, which eliminates other biological variation that occurs when comparing expression levels between different samples. This analysis confirmed that substance dependence associated variants have a direct cis-regulatory effect on CHRNA5 transcript levels in human frontal cortices of African and European ancestry and identified 10 highly correlated variants, located in a 9 kb region, that are potential functional variants modifying CHRNA5 mRNA expression levels.
doi:10.1371/journal.pone.0080204
PMCID: PMC3841173  PMID: 24303001
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
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
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
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
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
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

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