We utilized a cohort of 828 treatment seeking self-identified white cigarette smokers (50% female) to rank candidate gene single nucleotide polymorphisms (SNPs) associated with the Fagerström Test for Nicotine Dependence (FTND), a measure of nicotine dependence which assesses quantity of cigarettes smoked and time- and place-dependent characteristics of the respondent’s smoking behavior. 1123 SNPs at 55 autosomal candidate genes, nicotinic acetylcholine receptors and genes involved in dopaminergic function, were tested for association to baseline FTND scores adjusted for age, depression, education, sex and study site. SNP P values were adjusted for the number of transmission models, the number of SNPs tested per candidate gene, and their intragenic correlation. DRD2, SLC6A3 and NR4A2 SNPs with adjusted P values < 0.10 were considered sufficiently noteworthy to justify further genetic, bioinformatic and literature analyses. Each independent signal among the top-ranked SNPs accounted for ~1% of the FTND variance in this sample. The DRD2 SNP appears to represent a novel association with nicotine dependence. The SLC6A3 SNPs have previously been shown to be associated with SLC6A3 transcription or dopamine transporter density in vitro, in vivo and ex vivo. Analysis of SLC6A3 and NR4A2 SNPs identified a statistically significant gene-gene interaction (P=0.001), consistent with in vitro evidence that the NR4A2 protein product (NURR1) regulates SLC6A3 transcription. A community cohort of N=175 multiplex ever smoking pedigrees (N=423 ever smokers) provided nominal evidence for association with the FTND at these top ranked SNPs, uncorrected for multiple comparisons.
dopamine transporter; Fagerström Test for Nicotine Dependence; single nucleotide polymorphism; candidate gene association scan; gene-gene interaction
Nicotine withdrawal symptoms are related to smoking cessation. A Rasch model has been used to develop a unidimensional sensitivity score representing multiple correlated measures of nicotine withdrawal. A previous autosome-wide screen identified a nonparametric linkage (NPL) log-likelihood ratio (LOD) score of 2.7 on chromosome 6q26 for the sum of nine withdrawal symptoms.
The objectives of these analyses are: a) to assess the influence of nicotine withdrawal sensitivity on relapse, b) conduct autosome-wide NPL analysis of nicotine withdrawal sensitivity among 158 pedigrees with 432 individuals with microsatellite genotypes and nicotine withdrawal scores, and c) explore family-based association of single nucleotide polymorphism (SNPs) at the mu opioid receptor (MOR) candidate gene (OPRM1) to nicotine withdrawal sensitivity in 172 nuclear pedigrees with 419 individuals with both SNP genotypes and nicotine withdrawal scores.
An increased risk for relapse was associated with nicotine withdrawal sensitivity score (odds ratio, OR=1.25, 95% confidence interval, 95%CI=1.10,1.42). A maximal NPL LOD score of 3.15, suggestive of significant linkage, was identified at chr6q26 for nicotine withdrawal sensitivity. Evaluation of 18 OPRM1 SNPs via the family based association test (FBAT) with the nicotine withdrawal sensitivity score identified eight tagging SNPs with global P-values<0.05 and false discovery rate Q-values<0.06.
An increased risk of relapse, suggestive linkage at chr6q26, and nominally significant association with multiple OPRM1 SNPs was found with Rasch modeled nicotine withdrawal sensitivity score in a multiplex smoking pedigree sample. Future studies should attempt to replicate these findings and investigate the relationship between nicotine withdrawal symptoms and variation at OPRM1.
Affordable early screening in subjects with high risk of lung cancer has great potential to improve survival from this deadly disease. We measured gene expression from lung tissue and peripheral whole blood (PWB) from adenocarcinoma cases and controls to identify dysregulated lung cancer genes that could be tested in blood to improve identification of at-risk patients in the future. Genome-wide mRNA expression analysis was conducted in 153 subjects (73 adenocarcinoma cases, 80 controls) from the Environment And Genetics in Lung cancer Etiology (EAGLE) study using PWB and paired snap-frozen tumor and non-involved lung tissue samples. Analyses were conducted using unpaired t-tests, linear mixed effects and ANOVA models. The area under the receiver operating characteristic curve (AUC) was computed to assess the predictive accuracy of the identified biomarkers. We identified 50 dysregulated genes in stage I adenocarcinoma versus control PWB samples (False Discovery Rate ≤0.1, fold change ≥1.5 or ≤0.66). Among them, eight (TGFBR3, RUNX3, TRGC2, TRGV9, TARP, ACP1, VCAN, and TSTA3) differentiated paired tumor versus non-involved lung tissue samples in stage I cases, suggesting a similar pattern of lung cancer-related changes in PWB and lung tissue. These results were confirmed in two independent gene expression analyses in a blood-based case-control study (n=212) and a tumor-non tumor paired tissue study (n=54). The eight genes discriminated patients with lung cancer from healthy controls with high accuracy (AUC=0.81, 95% CI=0.74–0.87). Our finding suggests the use of gene expression from PWB for the identification of early detection markers of lung cancer in the future.
microarray gene expression; peripheral blood; lung cancer; stage I
Genome-wide association studies have linked single-nucleotide polymorphisms (SNPs) in the CHRNA5/A3/B4 gene cluster with heaviness of smoking. The nicotine metabolite ratio (NMR), a measure of the rate of nicotine metabolism, is associated with the number of cigarettes per day (CPD) and likelihood of cessation. We tested the potential interacting effects of these two risk factors on CPD.
Pretreatment data from three prior clinical trials were pooled for analysis. One thousand and thirty treatment seekers of European ancestry with genotype data for the CHRNA5/A3/B4 SNPs rs578776 and rs1051730 and complete data for NMR and CPD at pretreatment were included. Data for the third SNP, rs16969968, were available for 677 individuals. Linear regression models estimated the main and interacting effects of genotype and NMR on CPD.
We confirmed independent associations between the NMR and CPD as well as between the SNPs rs16969968 and rs1051730 and CPD. We did not detect a significant interaction between NMR and any of the SNPs examined.
This study demonstrates the additive and independent association of the NMR and SNPs in the CHRNA5/A3/B4 gene cluster with smoking rate in treatment-seeking smokers.
Although previous investigations have indicated a role for genetic factors in smoking initiation, the underlying genetic mechanisms are still unknown. In 2,339 adolescents from a Chinese Han population in the Wuhan Smoking Prevention Trial (Wuhan, China, 1998–1999), the authors explored the association of 57 genes in the dopamine pathway with smoking initiation. Using a conservative approach for declaring significance, positive findings were further examined in an independent sample of 603 Caucasian adolescents followed for up to 10 years as part of the Children's Health Study (Southern California, 1993–2009). The authors identified 1 single nucleotide polymorphism (rs2298122) in the calcyon neuron-specific vesicular protein gene (CALY) that was positively associated with smoking initiation in females (odds ratio = 2.21, 95% confidence interval: 1.49, 3.27; P = 8.4 × 10−5) in the Wuhan Smoking Prevention Trial cohort, and they replicated the association in females from the Children's Health Study cohort (hazard rate ratio = 2.05, 95% confidence interval: 1.27, 3.31; P = 0.003). These results suggest that the CALY gene may influence smoking initiation in adolescents, although the potential roles of underlying psychological characteristics that may be components of the smoking-initiation phenotype, such as impulsivity or novelty-seeking, remain to be explored.
adolescent; dopamine; genetic association studies; smoking
This analysis is a follow-up to an earlier investigation of 182 genes selected as likely candidate genetic variations conferring susceptibility to anorexia nervosa (AN). As those initial case-control results revealed no statistically significant differences in single nucleotide polymorphisms, herein we investigate alternative phenotypes associated with AN. In 1762 females using regression analyses we examined: (1) lowest illness-related attained body mass index; (2) age at menarche; (3) drive for thinness; (4) body dissatisfaction; (5) trait anxiety; (6) concern over mistakes; and (7) the anticipatory worry and pessimism vs. uninhibited optimism subscale of the harm avoidance scale. After controlling for multiple comparisons, no statistically significant results emerged. Although results must be viewed in the context of limitations of statistical power, the approach illustrates a means of potentially identifying genetic variants conferring susceptibility to AN because less complex phenotypes associated with AN are more proximal to the genotype and may be influenced by fewer genes.
covariates; eating disorders; association studies; personality; genetic
The molecular drivers that determine histology in lung cancer are largely unknown. We investigated whether microRNA (miR) expression profiles can differentiate histological subtypes and predict survival for non-small cell lung cancer.
We analyzed miR expression in 165 adenocarcinoma (AD) and 125 squamous cell carcinoma (SQ) tissue samples from the Environmental And Genetics in Lung cancer Etiology (EAGLE) study using a custom oligo array with 440 human mature antisense miRs. We compared miR expression profiles using t-tests and F-tests and accounted for multiple testing using global permutation tests. We assessed the association of miR expression with tobacco smoking using Spearman correlation coefficients and linear regression models, and with clinical outcome using log-rank tests, Cox proportional hazards and survival risk prediction models, accounting for demographic and tumor characteristics.
MiR expression profiles strongly differed between AD and SQ (global p<0.0001), particularly in the early stages, and included miRs located on chromosome loci most often altered in lung cancer (e.g., 3p21-22). Most miRs, including all members of the let-7 family, were down-regulated in SQ. Major findings were confirmed by QRT-PCR in EAGLE samples and in an independent set of lung cancer cases. In SQ, low expression of miRs down-regulated in the histology comparison was associated with 1.2 to 3.6-fold increased mortality risk. A 5-miR signature significantly predicted survival for SQ.
We identified a miR expression profile that strongly differentiated AD from SQ and had prognostic implications. These findings may lead to histology-based therapeutic approaches.
The authors investigated the relation between alcohol consumption and lung cancer risk in the Environment and Genetics in Lung Cancer Etiology (EAGLE) Study, a population-based case-control study. Between 2002 and 2005, 2,100 patients with primary lung cancer were recruited from 13 hospitals within the Lombardy region of Italy and were frequency-matched on sex, area of residence, and age to 2,120 randomly selected controls. Alcohol consumption during adulthood was assessed in 1,855 cases and 2,065 controls. Data on lifetime tobacco smoking, diet, education, and anthropometric measures were collected. Adjusted odds ratios and 95% confidence intervals for categories of mean daily ethanol intake were calculated using unconditional logistic regression. Overall, both nondrinkers (odds ratio = 1.42, 95% confidence interval: 1.03, 2.01) and very heavy drinkers (≥60 g/day; odds ratio = 1.44, 95% confidence interval: 1.01, 2.07) were at significantly greater risk than very light drinkers (0.1–4.9 g/day). The alcohol effect was modified by smoking behavior, with no excess risk being observed in never smokers. In summary, heavy alcohol consumption was a risk factor for lung cancer among smokers in this study. Although residual confounding by tobacco smoking cannot be ruled out, this finding may reflect interplay between alcohol and smoking, emphasizing the need for preventive measures.
alcohol drinking; case-control studies; ethanol; lung neoplasms; risk factors; smoking
We performed association studies with 5,151 SNPs that were judged as likely candidate genetic variations conferring susceptibility to anorexia nervosa (AN) based on location under reported linkage peaks, previous results in the literature (182 candidate genes), brain expression, biological plausibility, and estrogen responsivity. We employed a case–control design that tested each SNP individually as well as haplotypes derived from these SNPs in 1,085 case individuals with AN diagnoses and 677 control individuals. We also performed separate association analyses using three increasingly restrictive case definitions for AN: all individuals with any subtype of AN (All AN: n = 1,085); individuals with AN with no binge eating behavior (AN with No Binge Eating: n = 687); and individuals with the restricting subtype of AN (Restricting AN: n = 421). After accounting for multiple comparisons, there were no statistically significant associations for any individual SNP or haplotype block with any definition of illness. These results underscore the importance of large samples to yield appropriate power to detect genotypic differences in individuals with AN and also motivate complementary approaches involving Genome-Wide Association (GWA) studies, Copy Number Variation (CNV) analyses, sequencing-based rare variant discovery assays, and pathway-based analysis in order to make up for deficiencies in traditional candidate gene approaches to AN.
single nucleotide polymorphisms; probands; anorexia nervosa; bulimia nervosa
This study addressed the hypothesis that variation in genes associated with dopamine function (SLC6A3, DRD2, DRD4), serotonin function (SLC6A4), and regulation of monoamine levels (MAOA) may be predictive of BMI categories (obese and overweight + obese) in young adulthood and of changes in BMI as adolescents transition into young adulthood. Interactions with gender and race/ethnicity were also examined.
Research Methods and Procedures
Participants were a subsample of individuals from The National Longitudinal Study of Adolescent Health (Add Health), a nationally representative sample of adolescents followed from 1995 to 2002. The sample analyzed included a subset of 1584 unrelated individuals with genotype data. Multiple logistic regressions were conducted to evaluate associations between genotypes and obesity (BMI > 29.9) or overweight + obese combined (BMI > 25) with normal weight (BMI = 18.5–24.9) as a referent. Linear regression models were used examine change in BMI from adolescence to young adulthood.
Significant associations were found between SLC6A4 5HTTLPR and categories of BMI, and between MAOA promoter VNTR among males and categories of BMI. Stratified analyses revealed that the association between these two genes and excess BMI was significant for males overall, and for White and Hispanic males specifically. Linear regression models indicated a significant effect of SLC6A4 5HTTLPR on change in BMI from adolescence to young adulthood.
Our findings lend further support to the involvement of genes implicated in dopamine and serotonin regulation on energy balance.
Adolescents; Genetic Epidemiology; Serotonin; Neuro Transmitter
Developing, targeting, and evaluating genomic strategies for population-based disease prevention require population-based data. In response to this urgent need, genotyping has been conducted within the Third National Health and Nutrition Examination (NHANES III), the nationally-representative household-interview health survey in the U.S. However, before these genetic analyses can occur, family relationships within households must be accurately ascertained. Unfortunately, reported family relationships within NHANES III households based on questionnaire data are incomplete and inconclusive with regards to actual biological relatedness of family members. We inferred family relationships within households using DNA fingerprints (Identifiler®) that contain the DNA loci used by law enforcement agencies for forensic identification of individuals. However, performance of these loci for relationship inference is not well understood. We evaluated two competing statistical methods for relationship inference on pairs of household members: an exact likelihood ratio relying on allele frequencies to an Identical By State (IBS) likelihood ratio that only requires matching alleles. We modified these methods to account for genotyping errors and population substructure. The two methods usually agree on the rankings of the most likely relationships. However, the IBS method underestimates the likelihood ratio by not accounting for the informativeness of matching rare alleles. The likelihood ratio is sensitive to estimates of population substructure, and parent-child relationships are sensitive to the specified genotyping error rate. These loci were unable to distinguish second-degree relationships and cousins from being unrelated. The genetic data is also useful for verifying reported relationships and identifying data quality issues. An important by-product is the first explicitly nationally-representative estimates of allele frequencies at these ubiquitous forensic loci.
Forensics; allele sharing; population structure; CODIS; IBS; IBD
Characterizing cotinine pharmacokinetics is a useful way to study nicotine metabolism because the same liver enzyme is primarily responsible for the metabolism of both, and the clearances of nicotine and cotinine are highly correlated. We conducted a whole-genome linkage analysis to search for candidate regions influencing quantitative variation in cotinine pharmacokinetics in a large-scale pharmacokinetic study with 61 families containing 224 healthy adult participants. The strongest linkage signal was identified at 135 cM of chromosome 9 with LOD=2.81 and P=0.0002; two other suggestive linkage peaks appear at 31.4 and 73.5 cM of chromosome 11 with LOD=1.96 (P=0.0013) and 1.94 (P=0.0014). The confidence level of the linkage between the three genome regions and cotinine pharmacokinetics is statistically significant with a genome-wide empirical probability of P=0.029.
pharmacokinetics; nicotine; dependence; linkage analysis
In the genetic study of complex traits, especially behavior related ones, such as smoking and alcoholism, usually several phenotypic measurements are obtained for the description of the complex trait, but no single measurement can quantify fully the complicated characteristics of the symptom because of our lack of understanding of the underlying etiology. If those phenotypes share a common genetic mechanism, rather than studying each individual phenotype separately, it is more advantageous to analyze them jointly as a multivariate trait in order to enhance the power to identify associated genes. We propose a multilocus association test for the study of multivariate traits. The test is derived from a partially linear tree-based regression model for multiple outcomes. This novel tree-based model provides a formal statistical testing framework for the evaluation of the association between a multivariate outcome and a set of candidate predictors, such as markers within a gene or pathway, while accommodating adjustment for other covariates. Through simulation studies we show that the proposed method has an acceptable type I error rate and improved power over the univariate outcome analysis, which studies each component of the complex trait separately with multiple-comparison adjustment. A candidate gene association study of multiple smoking-related phenotypes is used to demonstrate the application and advantages of this new method. The proposed method is general enough to be used for the assessment of the joint effect of a set of multiple risk factors on a multivariate outcome in other biomedical research settings.
Generalized estimating equation; Genetic association study; Model selection; Multiple-comparison adjustment; Tree-based model
The ratio of trans-3’hydroxycotinine/cotinine (3HC/COT) is a marker of CYP2A6 activity, an important determinant of nicotine metabolism. This analysis sought to conduct a combined genetic epidemiologic and pharmacogenetic investigation of the 3HC/COT ratio in plasma and urine.
One hundred thirty nine twin pairs (110 monozygotic [MZ] and 29 dizygotic [DZ]) underwent a 30-minute infusion of stable isotope-labeled nicotine and its major metabolite, cotinine, followed by an 8-hour in-hospital stay. Blood and urine samples were taken at regular intervals for analysis of nicotine, cotinine, and metabolites. DNA was genotyped to confirm zygosity and for variation in the gene for the primary nicotine metabolic enzyme, CYP2A6 (variants genotyped: *1B, *1×2, *2, *4, *9, *12). Univariate biometric analyses quantified genetic and environmental influences on each measure in the presence and absence of covariates, including measured CYP2A6 genotype.
There was a substantial amount of variation in the free 3HC/COT ratio in plasma (6 hours post-infusion) attributable to additive genetic influences (67.4%, 95% CI = 55.9–76.2%). The heritability estimate was reduced to 61.0% and 49.4%, respectively, after taking into account the effect of covariates and CYP2A6 genotype. In urine (collected over 8 hours), the estimated amount of variation in the 3HC/COT ratio attributable to additive genetic influences was smaller (47.2%, 95% CI = 0–67.2%) and decreased to 44.6% and 42.0% after accounting for covariates and genotype.
Additive genetic factors are prominent in determining variation in plasma 3HC/COT variation but less so in determining variation in urine 3HC/COT.
pharmacogenetics; nicotine; cotinine; metabolism; CYP2A6; twins; genetics; heritability
It is increasingly recognized that pathway analyses—a joint test of association between the outcome and a group of single nucleotide polymorphisms (SNPs) within a biological pathway—could potentially complement single-SNP analysis and provide additional insights for the genetic architecture of complex diseases. Building upon existing P-value combining methods, we propose a class of highly flexible pathway analysis approaches based on an adaptive rank truncated product (ARTP) statistic that can effectively combine evidence of associations over different SNPs and genes within a pathway. The statistical significance of the pathway-level test-statistics is evaluated using a highly efficient permutation algorithm that remains computationally feasible irrespective of the size of the pathway and complexity of the underlying test-statistics for summarizing SNP- and gene-level associations. We demonstrate through simulation studies that a gene-based analysis, that treats the underlying genes, as opposed to the underlying SNPs, as the basic units for hypothesis testing, is a very robust and powerful approach to pathway-based association testing. We also illustrate the advantage of the proposed methods using a study of the association between the nicotinic receptor pathway and cigarette smoking behaviors.
Pathway analysis; genetic association study; permutation procedure
Genetic variation may influence initial sensitivity to nicotine (i.e. during early tobacco exposure), perhaps helping to explain differential vulnerability to nicotine dependence. This study explored associations of functional candidate gene polymorphisms with initial sensitivity to nicotine in 101 young adult nonsmokers of European ancestry. Nicotine (0, 5, 10 μg/kg) was administered via nasal spray followed by mood, nicotine reward (e.g. “liking”) and perception (e.g. “feel effects”) measures, physiological responses, sensory processing (pre-pulse inhibition of startle), and performance tasks. Nicotine reinforcement was assessed in a separate session using a nicotine vs. placebo spray choice procedure. For the dopamine D4 receptor (DRD4 VNTR), presence of the 7 repeat allele was associated with greater aversive responses to nicotine (decreases in “vigor”, positive affect, and rapid information processing; increased cortisol) and reduced nicotine choice. Individuals with at least one DRD4 7-repeat allele also reported increased “feel effects” and greater startle response, but in men only. Also observed in men but not women were other genetic associations, such as greater “feel effects” and anger, and reduced fatigue, in the dopamine D2 receptor (DRD2 C957T SNP) TT versus CT or CC genotypes. Very few or no significant associations were seen for the DRD2/ANKK1 TaqIA polymorphism, the serotonin transporter promoter VNTR or 5HTTLPR (SLC6A4), the dopamine transporter 3’ VNTR (SLC6A3), and the mu opioid receptor A118G SNP (OPRM1). Although these results are preliminary, this study is the first to suggest that genetic polymorphisms related to function in the dopamine D4, and perhaps D2, receptor may modulate initial sensitivity to nicotine prior to the onset of dependence and may do so differentially between men and women.
nicotine; sensitivity; genetics; dopamine; reward; reinforcement
Negative mood increases smoking reinforcement and risk of relapse. We explored associations of gene variants in the dopamine, opioid, and serotonin pathways with smoking reward (“liking”) and reinforcement (latency to first puff, total puffs) as a function of negative mood and expected vs. actual nicotine content of the cigarette. Smokers of European ancestry (n=72) were randomized to one of four groups in a 2 × 2 balanced-placebo design, corresponding to manipulation of actual (0.6 mg vs. 0.05 mg) and expected (told nicotine, told denicotinized) nicotine “dose” in cigarettes during each of two sessions (negative vs. positive mood induction). Following mood induction and expectancy instructions, they sampled and rated the assigned cigarette, and then smoked additional cigarettes ad lib during continued mood induction. The increase in smoking amount due to negative mood was associated with: DRD2 C957T (CC>TT or CT), SLC6A3 (presence of 9 repeat > absence of 9), and among those given a nicotine cigarette, DRD4 (presence of 7 repeat > absence of 7) and DRD2/ANKK1 TaqIA (TT or CT > CC). SLC6A3 and DRD2/ANKK1 TaqIA were also associated with smoking reward and smoking latency. OPRM1 (AA > AG or GG) was associated with smoking reward, but SLC6A4 VNTR was unrelated to any of these measures. These results warrant replication but provide the first evidence for genetic associations with the acute increase in smoking reward and reinforcement due to negative mood.
smoking reward; reinforcement; mood; genetics; dopamine
Vulnerability to anorexia nervosa (AN) and bulimia nervosa (BN) arise from the interplay of genetic and environmental factors. To explore the genetic contribution, we measured over 100 psychiatric, personality and temperament phenotypes of individuals with eating disorders from 154 multiplex families accessed through an AN proband (AN cohort) and 244 multiplex families accessed through a BN proband (BN cohort). To select a parsimonious subset of these attributes for linkage analysis, we subjected the variables to a multilayer decision process based on expert evaluation and statistical analysis. Criteria for trait choice included relevance to eating disorders pathology, published evidence for heritability, and results from our data. Based on these criteria, we chose six traits to analyze for linkage. Obsessionality, Age-at-Menarche, and a composite Anxiety measure displayed features of heritable quantitative traits, such as normal distribution and familial correlation, and thus appeared ideal for quantitative trait locus (QTL) linkage analysis. By contrast, some families showed highly concordant and extreme values for three variables — lifetime minimum Body Mass Index (lowest BMI attained during the course of illness), concern over mistakes, and food-related obsessions — whereas others did not. These distributions are consistent with a mixture of populations, and thus the variables were matched with covariate linkage analysis. Linkage results appear in a subsequent report. Our report lays out a systematic roadmap for utilizing a rich set of phenotypes for genetic analyses, including the selection of linkage methods paired to those phenotypes.
Complex disease; endophenotype; liability; clinical judgment; covariate selection; mixture model; regression
Although the efficacy of pharmacotherapy for tobacco dependence has been previously demonstrated, there is substantial variability among individuals in treatment response. We performed a systems-based candidate gene study of 1295 single nucleotide polymorphisms (SNPs) in 58 genes within the neuronal nicotinic receptor and dopamine systems to investigate their role in smoking cessation in a bupropion placebo-controlled randomized clinical trial. Putative functional variants were supplemented with tagSNPs within each gene. We used global tests of main effects and treatment interactions, adjusting the P-values for multiple correlated tests. An SNP (rs2072661) in the 3′ UTR region of the β2 nicotinic acetylcholine receptor subunit (CHRNB2) has an impact on abstinence rates at the end of treatment (adjusted P = 0.01) and after a 6-month follow-up period (adjusted P = 0.0002). This latter P-value is also significant with adjustment for the number of genes tested. Independent of treatment at 6-month follow-up, individuals carrying the minor allele have substantially decreased the odds of quitting (OR = 0.31; 95% CI 0.18–0.55). Effect of estimates indicate that the treatment is more effective for individuals with the wild-type (OR = 2.14, 95% CI 1.20–3.81) compared with individuals carrying the minor allele (OR = 0.83, 95% CI 0.32–2.19), although this difference is only suggestive (P = 0.10). Furthermore, this SNP demonstrated a role in the time to relapse (P = 0.0002) and an impact on withdrawal symptoms at target quit date (TQD) (P = 0.0009). Overall, while our results indicate strong evidence for CHRNB2 in ability to quit smoking, these results require replication in an independent sample.
Tobacco smoking is responsible for over 90% of lung cancer cases, and yet the precise molecular alterations induced by smoking in lung that develop into cancer and impact survival have remained obscure.
We performed gene expression analysis using HG-U133A Affymetrix chips on 135 fresh frozen tissue samples of adenocarcinoma and paired noninvolved lung tissue from current, former and never smokers, with biochemically validated smoking information. ANOVA analysis adjusted for potential confounders, multiple testing procedure, Gene Set Enrichment Analysis, and GO-functional classification were conducted for gene selection. Results were confirmed in independent adenocarcinoma and non-tumor tissues from two studies. We identified a gene expression signature characteristic of smoking that includes cell cycle genes, particularly those involved in the mitotic spindle formation (e.g., NEK2, TTK, PRC1). Expression of these genes strongly differentiated both smokers from non-smokers in lung tumors and early stage tumor tissue from non-tumor tissue (p<0.001 and fold-change >1.5, for each comparison), consistent with an important role for this pathway in lung carcinogenesis induced by smoking. These changes persisted many years after smoking cessation. NEK2 (p<0.001) and TTK (p = 0.002) expression in the noninvolved lung tissue was also associated with a 3-fold increased risk of mortality from lung adenocarcinoma in smokers.
Our work provides insight into the smoking-related mechanisms of lung neoplasia, and shows that the very mitotic genes known to be involved in cancer development are induced by smoking and affect survival. These genes are candidate targets for chemoprevention and treatment of lung cancer in smokers.
This study evaluated association between common and rare sequence variants in 10 nicotinic acetylcholine receptor subunit genes and the severity of nausea 21 days after initiating the standard, FDA-approved varenicline regimen for smoking cessation. Included in the analysis were 397 participants from a randomized clinical effectiveness trial with complete clinical and DNA resequencing data (mean age = 49.2 years; 68.0% female). Evidence for significant association between common sequence variants in CHRNB2 and nausea severity was obtained after adjusting for age, gender, and correlated tests (all PACT<.05). Individuals with the minor allele of CHRNB2 variants experienced less nausea than did those without the minor allele, consistent with previously reported findings for CHRNB2 and the occurrence of nausea and dizziness as a consequence of first smoking attempt in adolescents, and with the known neurophysiology of nausea. As nausea is the most common reason for discontinuance of varenicline, further pharmacogenetic investigations are warranted.
varenicline; nausea; smoking cessation; adherence
Mood disorders may affect lung cancer risk. We evaluated this hypothesis in two large studies.
We examined 1,939 lung cancer cases and 2,102 controls from the Environment And Genetics in Lung cancer Etiology (EAGLE) case-control study conducted in Italy (2002–2005), and 82,945 inpatients with a lung cancer diagnosis and 3,586,299 person-years without a lung cancer diagnosis in the U.S. Veterans Affairs Inpatient Cohort (VA study), composed of veterans with a VA hospital admission (1969–1996). In EAGLE, we calculated odds ratios (ORs) and 95% confidence intervals (CI), with extensive adjustment for tobacco smoking and multiple lifestyle factors. In the VA study, we estimated lung cancer relative risks (RRs) and 95% CIs with time-dependent Poisson regression, adjusting for attained age, calendar year, hospital visits, time within the study, and related previous medical diagnoses. In EAGLE, we found decreased lung cancer risk in subjects with a personal history of mood disorders (OR: 0.59, 95% CI: 0.44–0.79, based on 121 lung cancer incident cases and 192 controls) and family history of mood disorders (OR: 0.62, 95% CI: 0.50–0.77, based on 223 lung cancer cases and 345 controls). The VA study analyses yielded similar results (RR: 0.74, 95% CI: 0.71–0.77, based on 2,304 incident lung cancer cases and 177,267 non-cancer person-years) in men with discharge diagnoses for mood disorders. History of mood disorders was associated with nicotine dependence, alcohol and substance use and psychometric scales of depressive and anxiety symptoms in controls for these studies.
The consistent finding of a relationship between mood disorders and lung cancer risk across two large studies calls for further research into the complex interplay of risk factors associated with these two widespread and debilitating diseases. Although we adjusted for smoking effects in EAGLE, residual confounding of the results by smoking cannot be ruled out.
Extensive population-based genome-wide association studies have identified an association between the FTO gene and BMI; however, the mechanism of action is still unknown. To determine whether FTO may influence weight regulation through psychological and behavioral factors, seven single nucleotide polymorphisms (SNPs) of the FTO gene were genotyped in 1085 individuals with anorexia nervosa (AN) and 677 healthy weight controls from the international Price Foundation Genetic Studies of Eating Disorders. Each SNP was tested in association with eating disorder phenotypes and measures that have previously been associated with eating behavior pathology: trait anxiety, harm-avoidance, novelty seeking, impulsivity, obsessionality, compulsivity, and concern over mistakes. After appropriate correction for multiple comparisons, no significant associations between individual FTO gene SNPs and eating disorder phenotypes or related eating behavior pathology were identified in cases or controls. Thus, this study found no evidence that FTO gene variants associated with weight regulation in the general population are associated with eating disorder phenotypes in AN participants or matched controls.
Polymorphisms in genes coding for enzymes that activate tobacco lung carcinogens may generate inter-individual differences in lung cancer risk. Previous studies had limited sample sizes, poor exposure characterization, and a few single nucleotide polymorphisms (SNPs) tested in candidate genes. We analyzed 25 SNPs (some previously untested) in 2101 primary lung cancer cases and 2120 population controls from the Environment And Genetics in Lung cancer Etiology (EAGLE) study from six phase I metabolic genes, including cytochrome P450s, microsomal epoxide hydrolase, and myeloperoxidase. We evaluated the main genotype effects and genotype-smoking interactions in lung cancer risk overall and in the major histology subtypes. We tested the combined effect of multiple SNPs on lung cancer risk and on gene expression. Findings were prioritized based on significance thresholds and consistency across different analyses, and accounted for multiple testing and prior knowledge. Two haplotypes in EPHX1 were significantly associated with lung cancer risk in the overall population. In addition, CYP1B1 and CYP2A6 polymorphisms were inversely associated with adenocarcinoma and squamous cell carcinoma risk, respectively. Moreover, the association between CYP1A1 rs2606345 genotype and lung cancer was significantly modified by intensity of cigarette smoking, suggesting an underling dose-response mechanism. Finally, increasing number of variants at CYP1A1/A2 genes revealed significant protection in never smokers and risk in ever smokers. Results were supported by differential gene expression in non-tumor lung tissue samples with down-regulation of CYP1A1 in never smokers and up-regulation in smokers from CYP1A1/A2 SNPs. The significant haplotype associations emphasize that the effect of multiple SNPs may be important despite null single SNP-associations, and warrants consideration in genome-wide association studies (GWAS). Our findings emphasize the necessity of post-GWAS fine mapping and SNP functional assessment to further elucidate cancer risk associations.
The contribution of common genetic variation to one or more established smoking behaviors was investigated in a joint analysis of two genome wide association studies (GWAS) performed as part of the Cancer Genetic Markers of Susceptibility (CGEMS) project in 2,329 men from the Prostate, Lung, Colon and Ovarian (PLCO) Trial, and 2,282 women from the Nurses' Health Study (NHS). We analyzed seven measures of smoking behavior, four continuous (cigarettes per day [CPD], age at initiation of smoking, duration of smoking, and pack years), and three binary (ever versus never smoking, ≤10 versus >10 cigarettes per day [CPDBI], and current versus former smoking). Association testing for each single nucleotide polymorphism (SNP) was conducted by study and adjusted for age, cohabitation/marital status, education, site, and principal components of population substructure. None of the SNPs achieved genome-wide significance (p<10−7) in any combined analysis pooling evidence for association across the two studies; we observed between two and seven SNPs with p<10−5 for each of the seven measures. In the chr15q25.1 region spanning the nicotinic receptors CHRNA3 and CHRNA5, we identified multiple SNPs associated with CPD (p<10−3), including rs1051730, which has been associated with nicotine dependence, smoking intensity and lung cancer risk. In parallel, we selected 11,199 SNPs drawn from 359 a priori candidate genes and performed individual-gene and gene-group analyses. After adjusting for multiple tests conducted within each gene, we identified between two and five genes associated with each measure of smoking behavior. Besides CHRNA3 and CHRNA5, MAOA was associated with CPDBI (gene-level p<5.4×10−5), our analysis provides independent replication of the association between the chr15q25.1 region and smoking intensity and data for multiple other loci associated with smoking behavior that merit further follow-up.