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1.  Dopamine genes and nicotine dependence in treatment seeking and community smokers 
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.
doi:10.1038/npp.2009.52
PMCID: PMC3558036  PMID: 19494806
dopamine transporter; Fagerström Test for Nicotine Dependence; single nucleotide polymorphism; candidate gene association scan; gene-gene interaction
2.  Nicotine withdrawal sensitivity, linkage to chr6q26, and association of OPRM1 SNPs in the SMOking in FAMilies (SMOFAM) sample 
Background
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1158/1055-9965.EPI-09-0960
PMCID: PMC3536862  PMID: 19959688
3.  Alcohol Consumption and Lung Cancer Risk in the Environment and Genetics in Lung Cancer Etiology (EAGLE) Study 
American Journal of Epidemiology  2009;171(1):36-44.
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.
doi:10.1093/aje/kwp332
PMCID: PMC2800301  PMID: 19933698
alcohol drinking; case-control studies; ethanol; lung neoplasms; risk factors; smoking
4.  Genome-wide Linkage of Cotinine Pharmacokinetics Suggests Candidate Regions on Chromosomes 9 and 11 
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.
doi:10.1002/ajmg.b.30859
PMCID: PMC2693302  PMID: 18785207
pharmacokinetics; nicotine; dependence; linkage analysis
5.  A Partially Linear Tree-based Regression Model for Multivariate Outcomes 
Biometrics  2009;66(1):89-96.
Summary
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.
doi:10.1111/j.1541-0420.2009.01235.x
PMCID: PMC2875329  PMID: 19432770
Generalized estimating equation; Genetic association study; Model selection; Multiple-comparison adjustment; Tree-based model
6.  GENETIC AND ENVIRONMENTAL INFLUENCES ON THE RATIO OF 3’HYDROXYCOTININE TO COTININE IN PLASMA AND URINE 
Pharmacogenetics and genomics  2009;19(5):388-398.
Objectives
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.
Methods
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.
Results
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.
Conclusions
Additive genetic factors are prominent in determining variation in plasma 3HC/COT variation but less so in determining variation in urine 3HC/COT.
doi:10.1097/FPC.0b013e32832a404f
PMCID: PMC2849278  PMID: 19300303
pharmacogenetics; nicotine; cotinine; metabolism; CYP2A6; twins; genetics; heritability
7.  Pathway analysis by adaptive combination of P-values 
Genetic epidemiology  2009;33(8):700-709.
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.
doi:10.1002/gepi.20422
PMCID: PMC2790032  PMID: 19333968
Pathway analysis; genetic association study; permutation procedure
8.  Clinical trial participant characteristics and saliva and DNA metrics 
Background
Clinical trial and epidemiological studies need high quality biospecimens from a representative sample of participants to investigate genetic influences on treatment response and disease. Obtaining blood biospecimens presents logistical and financial challenges. As a result, saliva biospecimen collection is becoming more frequent because of the ease of collection and lower cost. This article describes an assessment of saliva biospecimen samples collected through the mail, trial participant demographic and behavioral characteristics, and their association with saliva and DNA quantity and quality.
Methods
Saliva biospecimens were collected using the Oragene® DNA Self-Collection Kits from participants in a National Cancer Institute funded smoking cessation trial. Saliva biospecimens from 565 individuals were visually inspected for clarity prior to and after DNA extraction. DNA samples were then quantified by UV absorbance, PicoGreen®, and qPCR. Genotyping was performed on 11 SNPs using TaqMan® SNP assays and two VNTR assays. Univariate, correlation, and analysis of variance analyses were conducted to observe the relationship between saliva sample and participant characteristics.
Results
The biospecimen kit return rate was 58.5% among those invited to participate (n = 967) and 47.1% among all possible COMPASS participants (n = 1202). Significant gender differences were observed with males providing larger saliva volume (4.7 vs. 4.5 ml, p = 0.019), samples that were more likely to be judged as cloudy (39.5% vs. 24.9%, p < 0.001), and samples with greater DNA yield as measured by UV (190.0 vs. 138.5, p = 0.002), but reduced % human DNA content (73.2 vs. 77.6 p = 0.005) than females. Other participant characteristics (age, self-identified ethnicity, baseline cigarettes per day) were associated with saliva clarity. Saliva volume and saliva and DNA clarity were positively correlated with total DNA yield by all three quantification measurements (all r > 0.21, P < 0.001), but negatively correlated with % human DNA content (saliva volume r = -0.148 and all P < 0.010). Genotyping completion rate was not influenced by saliva or DNA clarity.
Conclusion
Findings from this study show that demographic and behavioral characteristics of smoking cessation trial participants have significant associations with saliva and DNA metrics, but not with the performance of TaqMan® SNP or VNTR genotyping assays.
Trial registration
COMPASS; registered as NCT00301145 at clinicaltrials.gov.
doi:10.1186/1471-2288-9-71
PMCID: PMC2776600  PMID: 19874586
9.  Phase I Metabolic Genes and Risk of Lung Cancer: Multiple Polymorphisms and mRNA Expression 
PLoS ONE  2009;4(5):e5652.
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.
doi:10.1371/journal.pone.0005652
PMCID: PMC2682568  PMID: 19479063
10.  Genome-Wide and Candidate Gene Association Study of Cigarette Smoking Behaviors 
PLoS ONE  2009;4(2):e4653.
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.
doi:10.1371/journal.pone.0004653
PMCID: PMC2644817  PMID: 19247474
11.  SLC6A3 and body mass index in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial 
BMC Medical Genetics  2009;10:9.
Background
To investigate the contribution of the dopamine transporter to dopaminergic reward-related behaviors and anthropometry, we evaluated associations between polymorphisms at the dopamine transporter gene(SLC6A3) and body mass index (BMI), among participants in the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial.
Methods
Four polymorphisms (rs6350, rs6413429, rs6347 and the 3' variable number of tandem repeat (3' VNTR) polymorphism) at the SLC6A3 gene were genotyped in 2,364 participants selected from the screening arm of PLCO randomly within strata of sex, age and smoking history. Height and weight at ages 20 and 50 years and baseline were assessed by questionnaire. BMI was calculated and categorized as underweight, normal, overweight and obese (<18.5, 18.5–24.9, 25.0–29.9, or ≥ 30 kg/m2, respectively). Odds ratios (ORs) and 95% confidence intervals (CIs) of SLC6A3 genotypes and haplotypes were computed using conditional logistic regression.
Results
Compared with individuals having a normal BMI, obese individuals at the time of the baseline study questionnaire were less likely to possess the 3' VNTR variant allele with 9 copies of the repeated sequence in a dose-dependent model (** is referent; OR*9 = 0.80, OR99 = 0.47, ptrend = 0.005). Compared with individuals having a normal BMI at age 50, overweight individuals (A-C-G-* is referent; ORA-C-G-9 = 0.80, 95% CI 0.65–0.99, p = 0.04) and obese individuals (A-C-G-* is referent; ORA-C-G-9 = 0.70, 95% CI 0.49–0.99, p = 0.04) were less likely to possess the haplotype with the 3'variant allele (A-C-G-9).
Conclusion
Our results support a role of genetic variation at the dopamine transporter gene, SLC6A3, as a modifier of BMI.
doi:10.1186/1471-2350-10-9
PMCID: PMC2640369  PMID: 19183461

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