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26.  Association of the Calcyon Neuron-Specific Vesicular Protein Gene (CALY) With Adolescent Smoking Initiation in China and California 
American Journal of Epidemiology  2011;173(9):1039-1048.
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.
PMCID: PMC3121219  PMID: 21415033
adolescent; dopamine; genetic association studies; smoking
27.  Association of Candidate Genes with Phenotypic Traits Relevant to Anorexia Nervosa 
European Eating Disorders Review  2011;19(6):487-493.
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.
PMCID: PMC3261131  PMID: 21780254
covariates; eating disorders; association studies; personality; genetic
28.  Specific common variants of the obesity-associated FTO gene are not associated with psychological and behavioral eating disorder phenotypes 
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.
PMCID: PMC3249222  PMID: 21438147
29.  Resequencing of Nicotinic Acetylcholine Receptor Genes and Association of Common and Rare Variants with the Fagerström Test for Nicotine Dependence 
Neuropsychopharmacology  2010;35(12):2392-2402.
Common single-nucleotide polymorphisms (SNPs) at nicotinic acetylcholine receptor (nAChR) subunit genes have previously been associated with measures of nicotine dependence. We investigated the contribution of common SNPs and rare single-nucleotide variants (SNVs) in nAChR genes to Fagerström test for nicotine dependence (FTND) scores in treatment-seeking smokers. Exons of 10 genes were resequenced with next-generation sequencing technology in 448 European-American participants of a smoking cessation trial, and CHRNB2 and CHRNA4 were resequenced by Sanger technology to improve sequence coverage. A total of 214 SNP/SNVs were identified, of which 19.2% were excluded from analyses because of reduced completion rate, 73.9% had minor allele frequencies <5%, and 48.1% were novel relative to dbSNP build 129. We tested associations of 173 SNP/SNVs with the FTND score using data obtained from 430 individuals (18 were excluded because of reduced completion rate) using linear regression for common, the cohort allelic sum test and the weighted sum statistic for rare, and the multivariate distance matrix regression method for both common and rare SNP/SNVs. Association testing with common SNPs with adjustment for correlated tests within each gene identified a significant association with two CHRNB2 SNPs, eg, the minor allele of rs2072660 increased the mean FTND score by 0.6 Units (P=0.01). We observed a significant evidence for association with the FTND score of common and rare SNP/SNVs at CHRNA5 and CHRNB2, and of rare SNVs at CHRNA4. Both common and/or rare SNP/SNVs from multiple nAChR subunit genes are associated with the FTND score in this sample of treatment-seeking smokers.
PMCID: PMC3055324  PMID: 20736995
Fagerström test for nicotine dependence; single-nucleotide polymorphism; candidate gene association scan; treatment-seeking smokers; addiction & substance abuse; clinical pharmacology; clinical trials; neurogenetics; acetylcholine
30.  MicroRNA expression differentiates histology and predicts survival of lung cancer 
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.
Experimental design
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.
PMCID: PMC3163170  PMID: 20068076
31.  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.
PMCID: PMC2800301  PMID: 19933698
alcohol drinking; case-control studies; ethanol; lung neoplasms; risk factors; smoking
32.  Association Study of 182 Candidate Genes in Anorexia Nervosa 
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.
PMCID: PMC2963154  PMID: 20468064
single nucleotide polymorphisms; probands; anorexia nervosa; bulimia nervosa
33.  Genes implicated in serotonergic and dopaminergic functioning predict BMI categories 
Obesity (Silver Spring, Md.)  2008;16(2):348-355.
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.
PMCID: PMC2919156  PMID: 18239643
Adolescents; Genetic Epidemiology; Serotonin; Neuro Transmitter
34.  Using DNA fingerprints to infer familial relationships within NHANES III households 
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.
PMCID: PMC2909633  PMID: 20664713
Forensics; allele sharing; population structure; CODIS; IBS; IBD
35.  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.
PMCID: PMC2693302  PMID: 18785207
pharmacokinetics; nicotine; dependence; linkage analysis
36.  A Partially Linear Tree-based Regression Model for Multivariate Outcomes 
Biometrics  2009;66(1):89-96.
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.
PMCID: PMC2875329  PMID: 19432770
Generalized estimating equation; Genetic association study; Model selection; Multiple-comparison adjustment; Tree-based model
Pharmacogenetics and genomics  2009;19(5):388-398.
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.
PMCID: PMC2849278  PMID: 19300303
pharmacogenetics; nicotine; cotinine; metabolism; CYP2A6; twins; genetics; heritability
38.  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.
PMCID: PMC2790032  PMID: 19333968
Pathway analysis; genetic association study; permutation procedure
39.  Clinical trial participant characteristics and saliva and DNA metrics 
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.
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.
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.
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
PMCID: PMC2776600  PMID: 19874586
Behavioural pharmacology  2008;19(5-6):630-640.
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.
PMCID: PMC2743299  PMID: 18690117
nicotine; sensitivity; genetics; dopamine; reward; reinforcement
Behavioural pharmacology  2008;19(5-6):641-649.
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.
PMCID: PMC2717609  PMID: 18690118
smoking reward; reinforcement; mood; genetics; dopamine
42.  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.
PMCID: PMC2682568  PMID: 19479063
43.  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.
PMCID: PMC2644817  PMID: 19247474
44.  SLC6A3 and body mass index in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial 
BMC Medical Genetics  2009;10:9.
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.
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.
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).
Our results support a role of genetic variation at the dopamine transporter gene, SLC6A3, as a modifier of BMI.
PMCID: PMC2640369  PMID: 19183461
45.  Linkage analysis of anorexia and bulimia nervosa cohorts using selected behavioral phenotypes as quantitative traits or covariates 
To increase the likelihood of finding genetic variation conferring liability to eating disorders, we measured over 100 attributes thought to be related to liability to eating disorders on affected individuals from multiplex families and two cohorts: one recruited through a proband with anorexia nervosa (AN; AN cohort); the other recruited through a proband with bulimia nervosa (BN; BN cohort). By a multilayer decision process based on expert evaluation and statistical analysis, six traits were selected for linkage analysis (1): obsessionality (OBS), age at menarche (MENAR) and anxiety (ANX) for quantitative trait locus (QTL) linkage analysis; and lifetime minimum Body Mass Index (BMI), concern over mistakes (CM) and food-related obsessions (OBF) for covariate-based linkage analysis. The BN cohort produced the largest linkage signals: for QTL linkage analysis, four suggestive signals: (for MENAR, at 10p13; for ANX, at 1q31.1, 4q35.2, and 8q13.1); for covariate-based linkage analyses, both significant and suggestive linkages (for BMI, one significant [4q21.1] and three suggestive [3p23, 10p13, 5p15.3]; for CM, two significant [16p13.3, 14q21.1] and three suggestive [4p15.33, 8q11.23, 10p11.21]; and for OBF, one significant [14q21.1] and five suggestive [4p16.1, 10p13.1, 8q11.23, 16p13.3, 18p11.31]). Results from the AN cohort were far less compelling: for QTL linkage analysis, two suggestive signals (for OBS at 6q21 and for ANX at 9p21.3); for covariate-based linkage analysis, five suggestive signals (for BMI at 4q13.1, for CM at 11p11.2 and 17q25.1, and for OBF at 17q25.1 and 15q26.2). Overlap between the two cohorts was minimal for substantial linkage signals.
PMCID: PMC2590774  PMID: 16152574
Complex disease; endophenotype; liability; mixture model; regression
46.  Selection of eating-disorder phenotypes for linkage analysis 
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.
PMCID: PMC2560991  PMID: 16152575
Complex disease; endophenotype; liability; clinical judgment; covariate selection; mixture model; regression
47.  Nicotinic acetylcholine receptor β2 subunit gene implicated in a systems-based candidate gene study of smoking cessation 
Human Molecular Genetics  2008;17(18):2834-2848.
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.
PMCID: PMC2525499  PMID: 18593715
48.  Environment And Genetics in Lung cancer Etiology (EAGLE) study: An integrative population-based case-control study of lung cancer 
BMC Public Health  2008;8:203.
Lung cancer is the leading cause of cancer mortality worldwide. Tobacco smoking is its primary cause, and yet the precise molecular alterations induced by smoking in lung tissue that lead to lung cancer and impact survival have remained obscure. A new framework of research is needed to address the challenges offered by this complex disease.
We designed a large population-based case-control study that combines a traditional molecular epidemiology design with a more integrative approach to investigate the dynamic process that begins with smoking initiation, proceeds through dependency/smoking persistence, continues with lung cancer development and ends with progression to disseminated disease or response to therapy and survival. The study allows the integration of data from multiple sources in the same subjects (risk factors, germline variation, genomic alterations in tumors, and clinical endpoints) to tackle the disease etiology from different angles. Before beginning the study, we conducted a phone survey and pilot investigations to identify the best approach to ensure an acceptable participation in the study from cases and controls. Between 2002 and 2005, we enrolled 2101 incident primary lung cancer cases and 2120 population controls, with 86.6% and 72.4% participation rate, respectively, from a catchment area including 216 municipalities in the Lombardy region of Italy. Lung cancer cases were enrolled in 13 hospitals and population controls were randomly sampled from the area to match the cases by age, gender and residence. Detailed epidemiological information and biospecimens were collected from each participant, and clinical data and tissue specimens from the cases. Collection of follow-up data on treatment and survival is ongoing.
EAGLE is a new population-based case-control study that explores the full spectrum of lung cancer etiology, from smoking addiction to lung cancer outcome, through examination of epidemiological, molecular, and clinical data. We have provided a detailed description of the study design, field activities, management, and opportunities for research following this integrative approach, which allows a sharper and more comprehensive vision of the complex nature of this disease. The study is poised to accelerate the emergence of new preventive and therapeutic strategies with potentially enormous impact on public health.
PMCID: PMC2464602  PMID: 18538025
49.  Gene Expression Signature of Cigarette Smoking and Its Role in Lung Adenocarcinoma Development and Survival 
PLoS ONE  2008;3(2):e1651.
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.
Methodology/Principal Findings
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.
PMCID: PMC2249927  PMID: 18297132
50.  Linkage analysis of anti-CCP levels as dichotomized and quantitative traits using GAW15 single-nucleotide polymorphism scan of NARAC families 
BMC Proceedings  2007;1(Suppl 1):S107.
Rheumatoid arthritis is a clinically and genetically heterogeneous disease. Anti-cyclic citrullinated (anti-CCP) antibodies have a high specificity for rheumatoid arthritis and levels correlate with disease severity. The focus of this study was to examine whether analyzing anti-CCP levels could increase the power of linkage analysis by identifying a more homogeneous subset of rheumatoid arthritis patients. We also wanted to compare linkage signals when analyzing anti-CCP levels as dichotomized (CCP_binary), categorical (CCP_cat), and continuous traits, with and without transformation (log_CCP and CCP_cont). Illumina single-nucleotide polymorphism scans of the North American Rheumatoid Arthritis Consortium families were analyzed for four chromosomes (6, 7, 11, 22) using nonparametric linkage (NPL) (rheumatoid arthritis and CCP_binary), regress (CCP_cat and Log_CCP), and deviates (CCP_cont) analysis options as implemented in Merlin. Similar linkage results were obtained from analyses of rheumatoid arthritis, CCP_binary, and CCP_cont. The only exception was that we observed improved linkage signals and a narrower region for CCP_binary as compared to a clinical diagnosis of rheumatoid arthritis alone on chromosome 7, a region which previously showed variation in linkage results with rheumatoid arthritis according to anti-CCP levels. Analyses of CCP_cat and Log_CCP had little power to detect linkage. Our data suggested that linkage analyses of anti-CCP levels may facilitate identification of rheumatoid arthritis genes but quantitative analyses did not further improve power. Our study also highlighted that quantitative trait linkage results are highly sensitive to phenotype transformation and analytic approaches.
PMCID: PMC2367471  PMID: 18466447

Results 26-50 (58)