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. 2012 Dec 3;15(7):1190–1200. doi: 10.1093/ntr/nts245

The DRD4 Exon III VNTR, Bupropion, and Associations With Prospective Abstinence

Andrew W Bergen 1,✉, Harold S Javitz 1, Li Su 1,2, Yungang He 1,3, David V Conti 4, Neal L Benowitz 5, Rachel F Tyndale 6, Caryn Lerman 7, Gary E Swan 1
PMCID: PMC3682839  PMID: 23212438

Abstract

Introduction:

DRD4 Exon III Variable Number of Tandem Repeat (VNTR) variation was found to interact with bupropion to influence prospective smoking abstinence, in a recently published longitudinal analyses of N = 331 individuals from a randomized double-blind placebo-controlled trial of bupropion and intensive cognitive–behavioral mood management therapy.

Methods:

We used univariate, multivariate, and longitudinal logistic regression to evaluate gene, treatment, time, and interaction effects on point prevalence and continuous abstinence at end of treatment, 6 months, and 12 months, respectively, in N = 416 European ancestry participants in a double-blind pharmacogenetic efficacy trial randomizing participants to active or placebo bupropion. Participants received 10 weeks of pharmacotherapy and 7 sessions of behavioral therapy, with a target quit date 2 weeks after initiating both therapies. VNTR genotypes were coded with the long allele dominant resulting in 4 analysis categories. Covariates included demographics, dependence measures, depressive symptoms, and genetic ancestry. We also performed genotype-stratified secondary analyses.

Results:

We observed significant effects of time in longitudinal analyses of both abstinence outcomes, of treatment in individuals with VNTR long allele genotypes for both abstinence outcomes, and of covariates in some analyses. We observed non-significantly larger differences in active versus placebo effect sizes in individuals with VNTR long allele genotypes than in individuals without the VNTR long allele, in the directions previously reported.

Conclusions:

VNTR by treatment interaction differences between these and previous analyses may be attributable to insufficient size of the replication sample. Analyses of multiple randomized clinical trials will enable identification and validation of factors mediating treatment response.

INTRODUCTION

Bupropion hydrochloride is an antidepressant approved as an aid to smoking cessation treatment in its sustained release form. Bupropion’s efficacy was twice that of placebo in the first randomized controlled trial reporting results (Hurt et al., 1997) and in large meta-analyses (Fiore et al., 2008). Bupropion is rapidly metabolized by CYP2B6 to hydroxybupropion, an active metabolite (Damaj et al., 2004), and CYP2B6 polymorphisms significantly influence treatment effectiveness (Lee et al., 2007). Bupropion is a dual norepinephrine–dopamine reuptake inhibitor and a nicotinic acetylcholine receptor (nAChR) noncompetitive antagonist (Damaj et al., 2004; Learned-Coughlin et al., 2003). Catecholamine transporter and receptor genes and nAChR genes are candidate genes for pharmacogenetic investigation of bupropion effects on smoking cessation (Conti et al., 2008). The promise of pharmacogenetic analysis of nicotine addiction treatment includes the individualization of smoking cessation treatment leading to improved treatment outcomes (Lerman & Niaura, 2002).

Recently, Leventhal et al. (2012) reported a gene by treatment interaction in association with smoking abstinence with the DRD4 Exon III Variable Number of Tandem Repeat (VNTR) polymorphism (Van Tol et al., 1992). The analysis sample (N = 331) was drawn from a randomized double-blind placebo-controlled clinical trial of pharmacotherapy and behavioral therapy (Brown et al., 2007). The Brown et al. (2007) trial compared the effects of bupropion versus placebo and cognitive–behavioral treatment for depression added to standard cognitive–behavioral smoking cessation treatment versus standard cognitive–behavioral smoking cessation treatment on 7-day point prevalence abstinence, at four timepoints, in a sample of 524 smokers. Using longitudinal analyses (Zeger & Liang, 1986), Brown et al. (2007) reported a significantly increased effect of bupropion on abstinence (OR = 1.97, p = .03), consistent with previously observed bupropion effect sizes. In addition, Brown et al. (2007) reported significant negative effects of cigarettes smoked per week, time, and a treatment by time interaction on abstinence, but no additional significant interactions of pharmacotherapy nor significant or interactive effects of behavioral therapy. Brown et al. (2007) concluded that bupropion does not exhibit differential effects on abstinence among individuals with a history of depression or with elevated depressive symptoms, nor did specialized behavioral therapy for depression have significant effects on abstinence overall or among individuals with a depression history.

The DRD4 Exon III VNTR is a 48-base-pair coding region sequence exhibiting polymorphism in the number (Van Tol et al., 1992) and in the sequence of repeated units (Lichter et al., 1993). Two-, four-, and seven-repeat alleles are the most prevalent alleles, where the prevalence differs by ancestry (Chang, Kidd, Livak, Pakstis, & Kidd, 1996). In vitro evidence suggests that the seven-repeat receptor requires more dopamine to inhibit cyclic adenosine monophosphate (Asghari et al., 1995), and that the seven-repeat allele sequence suppresses transcription in engineered constructs (Schoots & Van Tol, 2003). The seven-repeat allele is somewhat less expressed in postmortem human brain samples (Simpson, Vetuz, Wilson, Brookes, & Kent, 2010). Haplotype and sequence analyses suggest that the seven-repeat allele has been subject to positive selection over the past 40- to 50,000 years (Ding et al., 2002; Wang et al., 2004).

Leventhal et al. (2012) utilized a sample of 331 individuals from the Brown et al. (2007) trial who consented to genotyping and who self-identified as White, genotyped the VNTR, and grouped individuals by treatment and by genotype to evaluate the effect of predictors and covariates on abstinence at four timepoints. Leventhal et al. (2012) utilized a dominant coding of alleles with seven or more 48-base-pair repeats and reported results from a series of unadjusted and adjusted longitudinal analyses. As expected (Brown et al., 2007), Leventhal et al. (2012) report significant effects of treatment (OR = 1.16, 95% CI: 1.07–1.26, p = .0004) and time (OR = 0.91, 95% CI: 0.89–0.92, p < .0001). They also report a significant gene by treatment interaction (OR = 1.24, 95% CI: 1.05–1.46, p = .014). The interaction reflects significantly increased abstinence observed in VNTR long allele carriers randomized to bupropion compared to placebo (OR [95% CI] of 5.70 [2.39–13.62], 5.50 [2.14–14.16], 10.36 [2.80–38.31], and 2.83 [0.89–9.00], respectively, for the four timepoints considered), compared to abstinence rates that do not significantly differ by treatment in individuals without the long allele, except at the first timepoint (1.79 [1.06–3.02], 1.58 [0.92–2.73], 1.12 [0.61–2.05], and 0.92 [0.48–1.74]).

We selected a pharmacogenetic efficacy trial (Lerman et al., 2003) that randomized treatment-seeking smokers to active or placebo bupropion in a double-blind manner (the Lerman et al. (2003) trial), genotyped the VNTR, and assessed association of VNTR genotype, treatment, and genotype by treatment interaction with end-of-treatment (EOT), 6-month (6MO), and 12-month (12MO) point prevalence or continuous abstinence in multivariate logistic and longitudinal regression analyses. We performed genotype-stratified analyses as secondary analyses. We compared our results to those of Leventhal et al. (2012) and considered what participant characteristics, trial treatments, and analysis approaches might explain differences in significant findings.

METHODS

Participants

Lerman et al. (2003) recruited treatment-seeking smokers through advertisements in Washington, DC, and in Buffalo, NY, for a pharmacogenetic efficacy trial (Lerman et al., 2003). Research protocols were approved by Institutional Review Boards (IRBs) at Georgetown University and at the University at Buffalo, State University of New York. Inclusion criteria included ≥10 cigarettes smoked per day (CPD) for the past year, age ≥18 years, and informed consent for both genotyping and treatment. Exclusion criteria included pregnancy or lactation, uncontrolled hypertension, unstable angina, heart attack or stroke within the past 6 months, current treatment or recent diagnosis of cancer, drug or alcohol dependence, current diagnosis or history of a psychiatric disorder, seizure disorder, and current use of bupropion- or nicotine-containing products other than cigarettes. The trial randomized treatment-seeking smokers who were eligible to participate in bupropion or placebo treatment in double-blind fashion, and all participants received up to seven sessions of counseling that took place during clinic visits. Subjects started assigned medication (150mg the first week and 300mg thereafter) 2 weeks before, and continued with the medication for 8 weeks after, the target quit day. Abstinence was assessed via self-report at clinic visits and phone interviews using the calendar and timeline followback methods and verified by carbon monoxide testing and cotinine measurement. Blood was collected for pharmacogenetic analyses from all participants and approval for pharmacogenetic analyses was provided by IRBs at the University of Pennsylvania, the University of California San Francisco, and SRI International.

Genotyping of the VNTR and Ancestry Informative Markers

We interrogated the VNTR using primers A2 (5′-PET-GCTCATGCTGCTGCTCTACTGGGC-3′) and A1 (5′-CTGC GGGTCTGCGGTGGAGTCTGG-3′) (George, Cheng, Nguyen, Israel, & O’Dowd, 1993). The polymerase chain reaction mix contained 50ng of genomic DNA, 250nM of each primer, 0.5U of Failsafe™ DNA polymerase (Epicentre Technologies), 100 µM of 7-deaza-2′-deoxyguanosine 5′-triphosphate in 1X-G FailSafe™ buffer, in a total volume of 21 µl. Cycling conditions were initial denaturation at 95 °C for 10min, followed by 33 cycles of 60 s at 95 °C, 60 s at 66 °C, 1.5min at 72 °C, and a final elongation step for 4min at 72 °C. PCR products were assayed on an Applied Biosystems Prism® 3130xl Genetic Analyzer with GS600 LIZ size standard and Hi-Di™ Formamide (all Applied Biosystems) and analyzed using GeneMarker® v1.5 (SoftGenetics) software. Individuals with VNTR genotypes with 7R, 8R, or 9R alleles were coded as L+, while those without these alleles were coded as SS (Supplementary Table 1).

We genotyped single nucleotide polymorphisms (SNPs) at ancestry informative markers (AIMs), for analysis using STRUCTURE (Pritchard & Rosenberg, 1999) and Eigenstrat (Price et al., 2006), to identify coefficients of ancestry and principal components of population genetic variation and select individuals for analysis in association studies (Conti et al., 2008) with smoking-related phenotypes (Thomas et al., 2009). We selected self-identified and genetically confirmed individuals of European ancestry for this genotype–phenotype analysis as described (Conti et al., 2008).

Analysis of Predictors and Covariates

Multiple imputation by chained equations (Ambler, Omar, & Royston, 2007) was used to impute covariate missing values 20 times for education, marital status, CPD, Fagerström Test for Nicotine Dependence (FTND) score (Heatherton, Kozlowski, Frecker, & Fagerstrom, 1991), and the principal components of population genetic variation (four to seven individuals each). Using logistic regression, we performed univariate analyses of demographic variables, dependence measures, and depressive symptom associations with 7-day point prevalence abstinence (abstinence) at EOT, 6MO, and 12MO in the four categories defined by genotype and treatment. We performed multivariate analyses and longitudinal analyses using generalized estimating equations to analyze associations of genotype, treatment, time, genotype by treatment interaction, and with and without interaction with time (VNTR, treatment, and VNTR and treatment, with time, respectively), with abstinence. All analyses of abstinence used the intent-to-treat approach. Regression analyses were performed on each imputed dataset and the results were combined with adjustment to the variance of regression parameters to reflect the additional variance attributable to the imputations (Rubin, 1987). Each regression analysis included variables for the effects of demographics (age [age and age squared], education [presence or absence of college degree], gender, marital status [married or other]); dependence measures (FTND and CPD, coded as in the FTND) and interactions with demographic variables (CPD × age, CPD × gender, and FTND × gender); Center for Epidemiological Studies-Depression (CES-D) scores (Radloff, 1977), and the first 10 principal components of population genetic variation calculated from 45 AIMs. The demographic, dependence, and population genetic variables were chosen based on prior integrated analyses of eight randomized clinical trials of smoking cessation therapy (data not shown). Statistical power was estimated using QUANTO and the unmatched case–control gene by environment binary outcome model (Gauderman & Morrison, 2006). The threshold for declaring significance in all tests was an alpha of .05. Secondary analyses were performed using continuous abstinence as the outcome. Genotype-stratified longitudinal analyses of point prevalence abstinence and continuous abstinence were performed to evaluate treatment effects within VNTR genotype categories.

RESULTS

In the bupropion treatment (BUP) group, there were 59 and 164 individuals with a L+ and with a SS VNTR genotype, respectively, and with EOT, 6MO, and 12MO point prevalence abstinence, demographic, and dependence data available for analysis (Table 1). In the placebo treatment (PLA) group, the corresponding counts were 69 and 124, respectively. In univariate analyses across the four categories of treatment and genotype (Table 1), we observed no statistically significant differences in abstinence or differences in demographics, dependence measures, or depressive symptoms at EOT, 6MO, and 12MO. In multivariate analysis of point prevalence abstinence at EOT, 6MO, and 12MO (Table 2), there were no significant effects of genotype, treatment, their interaction, or covariates, with the exception of a significant effect of marital status being associated with increased abstinence at 12MO (p = .028). In longitudinal analyses of point prevalence abstinence (Table 3), time was significantly negatively associated with abstinence (p = .028 at 12MO), and more significantly so when interactions of time and treatment, time and genotype, and three way interactions were not included in the analysis (p = .002 and p < .001 at 6MO and 12MO, respectively). There were no other significant associations of genotype, treatment, their interaction, or covariates, with point prevalence abstinence.

Table 1.

Univariate Characteristics of the Lerman et al. Sample by Treatment and Genotype, Point Prevalence Abstinence

All BUP/L+ BUP/SS PLA/L+ PLA/SS p equality
N 416 59 164 69 124 –
Outcomes
    EOT point prevalence abstinence 0.274 0.390 0.299 0.203 0.225 .057
    6MO point prevalence abstinence 0.221 0.322 0.238 0.159 0.186 .109
    12MO point prevalence abstinence 0.171 0.220 0.171 0.145 0.161 .699
Demographics
    Age, years M (SD) 44.5 (11.5) 45.2 (10.8) 44.0 (11.9) 42.0 (11.3) 46.1 (11.0) .109
    BMI, M (SD)a 26.5 (4.7) 27.5 (5.6) 26.1 (4.5) 26.3 (4.7) 26.5 (4.3) .272
    Education (college degree) (%)b 46.4 (50.0) 50.0 (50.4) 47.2 (50.1) 47.8 (50.3) 42.7 (49.7) .786
    Female (%) 54.3 (49.9) 50.8 (50.4) 56.1 (49.8) 55.1 (50.1) 53.2 (50.1) .904
    Married (%)b 47.1 (50.0) 46.7 (50.3) 43.5 (49.7) 49.3 (50.4) 50.1 (50.2) .642
Dependence measures
    FTND, M (SD)c 5.1 (2.1) 5.2 (2.3) 5.1 (2.0) 4.8 (2.2) 5.4 (2.1) .363
    CPD, M (SD)b 21.8 (9.4) 22.4 (11.8) 21.4 (8.0) 20.9 (6.8) 22.5 (10.8) .582
Depressive symptoms
    CES-D scoreb 12.2 (8.4) 12.9 (8.3) 12.9 (9.0) 10.9 (7.7) 11.6 (8.0) .284
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Note. BMI = body mass index; BUP = bupropion; CES-D = Center for Epidemiological Studies-Depression; CPD = cigarettes smoked per day; EOT = end-of-treatment; FTND = Fagerström Test for Nicotine Dependence; 6MO = 6 months; 12MO = 12 months.

a N = 414, BUP/L+ N = 59, BUP/SS N = 163, PLA/L+ = 69, PLA/SS = 123.

b N = 412, BUP/L+ N = 58, BUP/SS N = 161, PLA/L+ = 69, PLA/SS = 124.

cN = 411, BUP/L+ N = 58, BUP/SS N = 160, PLA/L+ = 69, PLA/SS = 124.

Table 2.

Multivariate Logistic Analysis of Point Prevalence Abstinence at EOT, 6MO, and 12MO

EOT 6MO 12MO
β LCI UCI p value β LCI UCI p value β LCI UCI p value
DRD4 L+ −0.26 −1.02 0.50 .501 −0.29 −1.12 0.53 .488 −0.19 −1.06 0.68 .665
BUP 0.33 −0.23 0.90 .248 0.30 −0.31 0.92 .330 0.06 −0.61 0.73 .859
Age 0.00 −0.25 0.26 .969 0.13 −0.14 0.40 .355 0.03 −0.26 0.33 .815
Age squared −0.12 −0.33 0.08 .238 −0.08 −0.29 0.13 .454 −0.03 −0.25 0.20 .811
Gender −0.22 −0.45 0.02 .070 −0.04 −0.29 0.21 .770 0.12 −0.16 0.40 .396
Education 0.07 −0.16 0.30 .546 0.05 −0.20 0.29 .708 0.08 −0.20 0.35 .586
Marital status 0.10 −0.13 0.34 .392 0.26 0.00 0.51 .051 0.32 0.03 0.60 .028
CES-D 0.00 −0.03 0.03 .914 0.01 −0.02 0.04 .715 0.01 −0.02 0.04 .589
CPD −0.07 −0.37 0.23 .654 −0.14 −0.46 0.18 .380 −0.16 −0.51 0.20 .388
FTND −0.17 −0.45 0.12 .255 −0.05 −0.36 0.25 .729 −0.08 −0.41 0.25 .639
CPD × age −0.06 −0.32 0.19 .634 −0.09 −0.36 0.17 .494 0.00 −0.29 0.28 .977
CPD × gender 0.08 −0.24 0.40 .627 0.15 −0.20 0.49 .398 0.31 −0.08 0.70 .124
FTND × gender −0.15 −0.45 0.14 .301 −0.06 −0.38 0.26 .704 −0.05 −0.40 0.29 .764
DRD4 × BUP 0.65 −0.35 1.65 .203 0.71 −0.37 1.78 .198 0.41 −0.76 1.58 .490
PC1 36.95 −24.85 98.76 .241 42.37 −24.45 109.18 .214 16.60 −58.72 91.93 .666
PC2 −5.00 −28.45 18.45 .676 −10.27 −35.31 14.77 .421 −4.42 −31.98 23.13 .753
PC3 −1.22 −15.93 13.49 .871 −2.01 −17.63 13.61 .801 −7.48 −24.93 9.97 .401
PC4 −4.19 −18.56 10.18 .568 0.75 −14.67 16.17 .924 −6.49 −23.43 10.45 .453
PC5 −3.45 −18.21 11.30 .646 −2.85 −18.49 12.79 .721 −2.20 −19.21 14.81 .800
PC6 5.64 −9.31 20.60 .460 4.95 −10.96 20.87 .542 2.29 −15.34 19.92 .799
PC7 −4.92 −18.76 8.92 .486 −4.90 −19.64 9.84 .515 −2.95 −19.36 13.46 .724
PC8 −0.99 −14.30 12.31 .884 −12.56 −26.97 1.85 .088 −7.34 −23.31 8.64 .368
PC9 −3.63 −17.74 10.48 .614 −3.74 −18.93 11.44 .629 2.61 −14.18 19.40 .761
PC10 7.37 −6.16 20.90 .285 9.36 −5.08 23.80 .204 18.67 2.70 34.64 .022
Constant −0.84 −1.57 −0.11 .024 −1.16 −1.93 −0.38 .003 −1.63 −2.49 −0.76 .000
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Note. BUP = bupropion; CES-D = Center for Epidemiological Studies-Depression; CPD = cigarettes smoked per day; EOT = end-of-treatment; FTND = Fagerström Test for Nicotine Dependence; LCI = 95% lower confidence interval; UCI = 95% upper confidence interval; 6MO = 6 months; 12MO = 12 months. Bold text represents a p value < .05.

Table 3.

Longitudinal Analysis of Point Prevalence Abstinence With and Without Interactions With Time

With interactions with time Without interactions with time
β LCI UCI p value β LCI UCI p value
DRD4 L+ −0.253 −1.005 0.498 .509 −0.272 −0.980 0.436 .451
6MO −0.254 −0.622 0.113 .175 −0.301 −0.492 −0.111 .002
12MO −0.427 −0.807 −0.047 .028 −0.639 −0.843 −0.436 .000
BUP 0.343 −0.219 0.905 .232 0.283 −0.244 0.810 .293
Age 0.053 −0.183 0.288 .662 0.055 −0.181 0.291 .648
Age squared −0.102 −0.290 0.086 .289 −0.101 −0.290 0.087 .291
Gender −0.111 −0.327 0.106 .316 −0.120 −0.337 0.097 .277
Education 0.069 −0.144 0.283 .525 0.063 −0.151 0.276 .566
Marital status 0.175 −0.046 0.396 .121 0.177 −0.044 0.399 .116
CES-D 0.004 −0.022 0.030 .754 0.004 −0.022 0.030 .757
CPD −0.095 −0.368 0.178 .494 −0.087 −0.360 0.187 .535
FTND −0.120 −0.386 0.146 .376 −0.127 −0.393 0.139 .348
CPD × age −0.071 −0.304 0.163 .554 −0.059 −0.292 0.175 .622
CPD × gender 0.114 −0.182 0.410 .450 0.124 −0.172 0.420 .412
FTND × gender −0.109 −0.381 0.163 .431 −0.128 −0.400 0.144 .355
DRD4 × BUP 0.634 −0.358 1.626 .210 0.639 −0.289 1.567 .177
BUP × 6MO −0.073 −0.544 0.397 .759
BUP × 12MO −0.335 −0.831 0.161 .185
DRD4 × 6MO −0.051 −0.686 0.584 .875
DRD4 × 12MO 0.006 −0.648 0.659 .986
DRD4 × BUP × 6MO 0.066 −0.767 0.900 .876
DRD4 × BUP × 12MO −0.096 −0.972 0.779 .829
PC1 32.676 −25.282 90.635 .269 34.983 −22.878 92.845 .236
PC2 −5.949 −27.723 15.825 .592 −5.745 −27.515 16.026 .605
PC3 −2.347 −15.983 11.289 .736 −1.734 −15.371 11.902 .803
PC4 −3.017 −16.371 10.337 .658 −3.275 −16.640 10.091 .631
PC5 −3.239 −16.868 10.389 .641 −2.364 −15.993 11.264 .734
PC6 5.856 −8.039 19.751 .409 5.085 −8.791 18.961 .473
PC7 −4.589 −17.477 8.299 .485 −4.379 −17.258 8.500 .505
PC8 −6.284 −18.710 6.142 .322 −5.864 −18.270 6.541 .354
PC9 −2.960 −16.113 10.193 .659 −2.509 −15.656 10.638 .708
PC10 9.766 −2.797 22.329 .128 9.647 −2.924 22.218 .133
Constant −0.909 −1.604 −0.214 .010 −0.863 −1.544 −0.182 .013
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Note. BUP = bupropion; CES-D = Center for Epidemiological Studies-Depression; CPD = cigarettes smoked per day; EOT = end-of-treatment; FTND = Fagerström Test for Nicotine Dependence; LCI = 95% lower confidence interval; UCI = 95% upper confidence interval; 6MO = 6 months; 12MO = 12 months. Bold text represents a p value < .05.

In univariate analyses of continuous abstinence, outcome data were available for 410 individuals, 59 and 164 individuals with a L+ and with a SS VNTR genotype randomized to BUP, and 69 and 121 individuals randomized to PLA, respectively (Table 3). In univariate analyses, we observed statistically significant differences in continuous abstinence prevalence at 6MO by treatment–genotype strata (p = .045), but no significant differences in continuous abstinence prevalence at EOT or 12MO. There were no significant differences in demographics, dependence measures, or depressive symptoms by treatment–genotype strata. In multivariate analyses of continuous abstinence at EOT, 6MO, and 12MO, we observed significant effects of gender on continuous abstinence at EOT and 6MO (p = .001 and p = .016, respectively), and depressive symptoms at baseline on continuous abstinence at 12MO (p = .049) (Table 4). In longitudinal analyses of continuous abstinence (Table 5), we observed highly statistically significant effects of time on continuous abstinence whether or not interactions between time, genotype, and/or treatment were included (p < .001 at 6MO and 12MO). We also observed a statistically significant effect of gender on continuous abstinence in longitudinal analysis (p = .001, with or without interactions with time). Two principal components of population genetic variation were observed to be significantly associated with continuous abstinence in multivariate analyses, and one of these was significant in longitudinal analyses.

Table 4.

Univariate Characteristics of the Lerman et al.* Sample by Treatment and Genotype, Continuous Abstinence

Cohort 3B All BUP/L+ BUP/SS PLA/L+ PLA/SS p equality
N 410 57 163 69 121 –
    EOT continuous abstinence 0.234 0.298 0.276 0.130 0.207 .056
    6MO continuous abstinence 0.163 0.246 0.196 0.101 0.116 .045
    12MO continuous abstinence 0.110 0.175 0.117 0.087 0.083 .276
Demographics
    Age, years M (SD) 44.6 (11.4) 45.5 (10.9) 44.1 (12.0) 42.0 (11.3) 46.2 (10.8) .082
    BMI, M (SD)a 26.5 (4.6) 27.6 (5.6) 26.1 (4.5) 26.3 (4.7) 26.4 (4.01) .180
    Education (%)b 46.1 (50.0) 48.2 (50.4) 47.5 (50.1) 47.8 (50.3) 42.1 (49.6) .786
    Female (%) 54.1 (49.9) 50.9 (50.4) 55.8 (49.8) 55.1 (50.1) 52.9 (50.1) .913
    Married (%)b 47.2 (50.0) 48.2 (50.4) 43.1 (49.7) 49.3 (50.4) 51.2 (50.2) .575
Dependence measures
    FTND, M (SD)c 5.2 (2.1) 5.4 (2.2) 5.1 (2.1) 4.8 (2.2) 5.3 (2.1) .363
    CPD, M (SD)b 21.8 (9.3) 22.7 (11.9) 21.3 (8.0) 20.9 (6.7) 22.5 (10.9) .525
Depressive symptoms
    CES-D scoreb 12.1 (8.4) 13.0 (8.4) 12.9 (9.0) 10.9 (7.7) 11.5 (7.9) .257
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Note. BMI = body mass index; BUP = bupropion; CES-D = Center for Epidemiological Studies-Depression; CPD = cigarettes smoked per day; EOT = end-of-treatment; FTND = Fagerström Test for Nicotine Dependence; 6MO = 6 months; 12MO = 12 months. Bold text represents a p value < .05.

a N = 408, BUP/L+ N = 57, BUP/SS N = 162, PLA/L+ = 69, PLA/SS = 120.

b N = 406, BUP/L+ N = 56, BUP/SS N = 160, PLA/L+ = 69, PLA/SS = 121.

c N = 405, BUP/L+ N = 56, BUP/SS N = 159, PLA/L+ = 69, PLA/SS = 121. *Lerman et al. (2003).

Table 5.

Multivariate Logistic Analysis of Continuous Abstinence at EOT, 6MO, and 12MO

EOT 6MO 12MO
β LCI UCI p value β LCI UCI p value β LCI UCI p value
DRD4 L+ −0.70 −1.57 0.18 .120 −0.13 −1.15 0.88 .796 0.22 −0.93 1.36 .711
BUP 0.43 −0.17 1.03 .160 0.70 −0.02 1.43 .057 0.49 −0.38 1.37 .269
Age 0.02 −0.26 0.31 .863 0.17 −0.16 0.50 .305 0.19 −0.23 0.60 .372
Age squared −0.16 −0.39 0.08 .192 −0.14 −0.40 0.13 .311 −0.15 −0.48 0.18 .364
Gender −0.42 −0.68 −0.16 .001 −0.36 −0.65 −0.07 .016 −0.30 −0.65 0.05 .093
Education 0.09 −0.16 0.33 .490 0.13 −0.15 0.41 .357 0.07 −0.27 0.41 .675
Marital status 0.09 −0.17 0.34 .499 0.02 −0.27 0.31 .885 0.28 −0.08 0.64 .122
CES-D 0.01 −0.02 0.04 .585 0.01 −0.02 0.04 .516 0.04 0.00 0.08 .049
CPD 0.01 −0.30 0.32 .954 −0.15 −0.50 0.20 .408 −0.14 −0.55 0.27 .495
FTND −0.18 −0.50 0.13 .249 −0.02 −0.38 0.34 .906 −0.03 −0.46 0.40 .888
CPD × age −0.13 −0.40 0.13 .325 −0.26 −0.57 0.06 .108 −0.15 −0.53 0.22 .416
CPD × gender −0.07 −0.40 0.27 .700 0.02 −0.36 0.40 .909 0.06 −0.40 0.52 .803
FTND × gender −0.05 −0.37 0.27 .760 −0.20 −0.57 0.17 .290 −0.15 −0.59 0.29 .504
DRD4 × BUP 0.76 −0.37 1.89 .186 0.34 −0.93 1.61 .600 0.05 −1.42 1.52 .946
PC1 −20.71 −92.84 51.41 .573 −20.60 −100.38 59.19 .613 −12.98 −107.58 81.62 .788
PC2 −7.58 −33.26 18.10 .563 1.52 −28.07 31.11 .920 −6.34 −41.50 28.83 .724
PC3 −12.32 −29.48 4.85 .160 −9.93 −29.12 9.25 .310 −11.61 −34.15 10.93 .313
PC4 0.16 −15.29 15.61 .984 −0.41 −17.90 17.09 .964 0.37 −20.58 21.32 .972
PC5 4.94 −11.15 21.04 .547 7.18 −10.90 25.25 .437 10.44 −11.18 32.07 .344
PC6 13.53 −3.47 30.53 .119 19.58 0.02 39.14 .050 20.54 −3.43 44.51 .093
PC7 3.13 −11.82 18.08 .682 −1.41 −18.54 15.72 .872 −2.30 −22.45 17.86 .823
PC8 4.67 −9.70 19.04 .524 0.16 −15.98 16.31 .984 −0.12 −19.34 19.11 .990
PC9 −18.98 −34.74 −3.23 .018 −10.52 −28.55 7.51 .253 2.83 −19.24 24.91 .801
PC10 5.63 −9.21 20.47 .457 20.25 3.26 37.24 .020 41.93 20.57 63.29 .000
Constant −1.48 −2.31 −0.65 .000 −2.32 −3.29 −1.35 .000 −3.20 −4.41 −2.00 .000
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Note. BUP = bupropion; CES-D = Center for Epidemiological Studies-Depression; CPD = cigarettes smoked per day; EOT = end-of-treatment; FTND = Fagerström Test for Nicotine Dependence; LCI = 95% lower confidence interval; UCI = 95% upper confidence interval; 6MO = 6 months; 12MO = 12 months. Bold text represents a p value < .05.

Table 6.

Longitudinal Analysis of Continuous Abstinence With and Without Interactions With Time

With interactions with time Without interactions with time
β LCI UCI p value β LCI UCI p value
DRD4 L+ −0.738 −1.619 0.143 .101 −0.827 −1.716 0.063 .069
6MO −0.733 −1.109 −0.357 .000 −0.492 −0.673 −0.311 .000
12MO −1.123 −1.557 −0.689 .000 −0.988 −1.197 −0.779 .000
BUP 0.387 −0.212 0.985 .205 0.445 −0.144 1.035 .139
Age 0.019 −0.254 0.293 .889 0.034 −0.241 0.308 .810
Age squared −0.136 −0.360 0.088 .235 −0.128 −0.353 0.096 .262
Gender −0.442 −0.693 −0.191 .001 −0.441 −0.694 −0.187 .001
Education 0.125 −0.115 0.366 .308 0.103 −0.139 0.345 .406
Marital status 0.061 −0.188 0.311 .629 0.058 −0.193 0.309 .653
CES-D 0.006 −0.023 0.036 .671 0.002 −0.027 0.032 .878
CPD −0.005 −0.309 0.300 .976 −0.002 −0.309 0.304 .989
FTND −0.195 −0.502 0.113 .214 −0.210 −0.519 0.100 .184
CPD × age −0.153 −0.415 0.109 .253 −0.149 −0.411 0.113 .265
CPD × gender −0.058 −0.387 0.270 .728 −0.050 −0.381 0.281 .768
FTND × gender −0.065 −0.374 0.245 .682 −0.097 −0.408 0.214 .541
DRD4 × BUP 0.863 −0.268 1.993 .135 0.991 −0.132 2.114 .084
BUP × 6MO 0.239 −0.226 0.703 .314
BUP × 12MO −0.047 −0.592 0.498 .867
DRD4 × 6MO 0.439 −0.220 1.099 .192
DRD4 × 12MO 0.654 −0.062 1.370 .073
DRD4 × BUP × 6MO −0.226 −1.058 0.606 .595
DRD4 × BUP × 12MO −0.206 −1.120 0.708 .658
PC1 −37.867 −108.995 33.261 .297 −29.090 −100.208 42.028 .423
PC2 −6.313 −31.508 18.883 .623 −6.184 −31.458 19.089 .631
PC3 −12.074 −28.963 4.815 .161 −11.444 −28.345 5.457 .184
PC4 −0.526 −15.522 14.471 .945 −0.290 −15.369 14.790 .970
PC5 5.018 −10.706 20.742 .532 5.455 −10.380 21.289 .500
PC6 16.239 −0.406 32.884 .056 15.196 −1.499 31.891 .074
PC7 2.751 −11.882 17.384 .712 3.355 −11.397 18.107 .656
PC8 4.337 −9.729 18.403 .546 3.777 −10.323 17.877 .600
PC9 −19.304 −34.652 −3.957 .014 −19.348 −34.756 −3.939 .014
PC10 6.415 −8.104 20.934 .386 6.241 −8.320 20.803 .401
Constant −1.591 −2.413 −0.769 .000 −1.531 −2.350 −0.713 .000
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Note. BUP = bupropion; CES-D = Center for Epidemiological Studies-Depression; CPD = cigarettes smoked per day; EOT = end-of-treatment; FTND = Fagerström Test for Nicotine Dependence; LCI = 95% lower confidence interval; UCI = 95% upper confidence interval; 6MO = 6 months; 12MO = 12 months. Bold text represents a p value < .05.

In multivariate analyses at EOT, 6MO, and 12MO of each abstinence outcome stratified by genotype (Supplementary Tables 2–5), we observed significant association of (a) treatment with point prevalence abstinence in individuals with a VNTR L+ genotype at EOT (OR = 2.95, 95% CI: 1.21–7.21, p = .0.018) and 6MO (OR = 2.96, 95% CI: 1.08–8.08, p = .035), but not at 12MO (OR = 1.57, 95% CI: 0.51–4.86, p = .433), and with continuous abstinence at EOT (OR = 3.25, 95% CI: 1.15–9.16, p = .026) and 6MO (OR = 3.42, 95% CI: 1.01–11.50, p = .047), but not at 12MO (OR = 1.47, 95% CI: 0.33–6.60, p = .616); (b) gender at EOT with point prevalence and at EOT and 6MO with continuous abstinence, in individuals with a VNTR SS genotype; (c) marital status at 6MO and 12MO with point prevalence abstinence in individuals with a VNTR SS genotype; (d) CES-D score at 12MO with continuous abstinence in individuals with a VNTR L+ genotype; (e) age (age squared) at 6MO with point prevalence abstinence and at EOT with continuous abstinence in individuals with a L+ genotype; and (f) the interaction term CPD by gender at 12MO with both abstinence outcomes in individuals with a L+ genotype. We also observed two principal components of population genetic variation significantly associated with continuous abstinence in individuals with a SS genotype (ps < .024).

In longitudinal analyses of each abstinence outcome stratified by genotype (Supplementary Tables 6 and 7), we observed significant association of (a) treatment in individuals with a VNTR L+ genotype for both abstinence outcomes (point prevalence abstinence [OR = 2.74, 95% CI: 1.14–6.59, p = .025] and continuous abstinence [OR = 3.15, 95% CI: 1.12–8.88, p = .030] respectively); (b) time in individuals with a VNTR SS genotype at 6MO with point prevalence abstinence and at 6MO and 12MO with continuous abstinence; (c) gender in individuals with a VNTR SS genotype with continuous abstinence; and (d) age (age squared) in individuals with a VNTR L+ genotype with continuous abstinence. We also observed three principal components of population genetic variation significantly associated with continuous abstinence in individuals with a SS genotype (ps < .033).

The effect sizes of treatment in individuals with a VNTR SS genotype in multivariate (EOT, 6MO, and 12MO) and longitudinal analyses of point prevalence and continuous abstinence outcomes were nonsignificant: OR = 1.39, 95% CI: 0.78–2.48, p = .262; OR = 1.29, 95% CI: 0.69–2.41, p = .428; OR = .96, 95% CI: 0.48–1.90, p = .896; OR = 1.48, 95% CI: 0.80–2.77, p = .215; OR = 1.92, 95% CI: 0.91–4.06, p = .087; OR = 1.42, 95% CI: 0.58–3.47, p = .441; OR = 1.38, 95% CI: 0.78–2.44, p = .27; and OR = 1.47, 95% CI: 0.78–2.75, p = .23, respectively.

The power to detect the gene by environment effect reported by Leventhal et al. (2012) with the abstinent (case) and nonabstinent (control) sample sizes, dominantly coded genotype prevalence, and treatment prevalence of the Lerman et al. (2003) sample, and the average placebo abstinence rate of 13.8% from 80 placebo arms (Fiore et al., 2008), was 11.5%. Under the same assumptions, a sample size of 3,362 individuals would be required to detect the gene by treatment interaction effect reported by Leventhal et al. (2012) with 80% power.

DISCUSSION

In analyses of the relations between bupropion treatment and VNTR genotype in N = 416 self-identified White treatment-seeking smokers, we observed statistically significant effects of treatment in multivariate analyses of both abstinence outcomes at EOT and at 6MO, and in longitudinal analyses of both abstinence outcomes, but only in individuals with a VNTR L+ genotype. We did not observe statistically significant associations of genotype or genotype by treatment interaction. Time was observed to be significantly associated with both abstinence outcomes, as expected. There were a few covariate associations with both abstinence outcomes, including principal components of population genetic variation. The magnitude of the statistically significant association of active treatment with both abstinence outcomes was not significantly different from the magnitude of effects previously observed in samples not stratified by DRD4 VNTR genotype (Brown et al., 2007; Hurt et al., 1997), and in those stratified by DRD4 VNTR genotype (Leventhal et al., 2012).

The most parsimonious explanation for the lack of a statistically significant genotype by treatment interaction effect is insufficient sample size, as indicated by our power analyses. Effective increases in sample size due to longitudinal modeling may have improved statistical power, but the sample size estimated by the power analysis required to observe the genotype by treatment interaction is almost three-fold larger in size than the effective sample size in the longitudinal analysis performed by Leventhal et al. (2012) and here in the Lerman et al. (2003) sample. We did observe nonsignificant effects of treatment, genotype, and their interaction in the directions observed by Leventhal et al. (2012) suggesting that the concordance between studies could reflect similar interactions between VNTR genotype, treatment, and abstinence, but with the reduced effect size typically observed in studies conducted after the initial discovery is reported (Ioannidis, 2008; Kraft, 2008). If the concordance in the directionality of effects between the studies is not due to chance or to the typical reduction in effect size observed, what participant, trial, or analytic methodology characteristics might account for the observed differences in VNTR by treatment effect sizes?

We note that Brown et al. (2007) excluded individuals with current DSM-IV substance abuse diagnoses (other than nicotine dependence), major depression, or other Axis I disorder, while Lerman et al. (2003) excluded individuals with DSM-IV drug or alcohol dependence, and a current diagnosis or lifetime history of an Axis I disorder. Given the exclusions in both trials, effects of current psychiatric disorders are very unlikely to have influenced the differences in treatment by genotype association observed between the trials.

Major depression has negative effects on cessation and positive effects on relapse status among current smokers and former smokers in large population-based samples (Weinberger, Pilver, Desai, Mazure, & McKee, 2012). Depressive symptoms do not always have negative effects on abstinence in clinical trials (Hall et al., 2006; Niaura et al., 2001), and where there is a relation, the relations between depressive symptoms and abstinence may be influenced by additional covariates, for example, ethnicity and social status in longitudinal series of patients undergoing smoking cessation treatments (Castro et al., 2011; Reitzel et al., 2010). Major depression and/or depressive symptoms do not have effects on abstinence in randomized clinical trials of bupropion versus placebo (Brown et al., 2007; Lerman et al., 2003).

A history of major depression, which was modestly prevalent in the Brown et al. (2007) trial participants, and absent in the Lerman et al. (2003) trial participants, might contribute to observed gene by treatment interaction association differences, if individuals with a history of depression and with the VNTR long allele were more responsive to bupropion treatment than individuals without a history of depression and with the VNTR long allele. The VNTR has previously been associated with mood disorders in a meta-analysis (Lopez Leon et al., 2005), although the association described was with the two repeat VNTR allele and unipolar mood disorders. Individuals with long VNTR alleles are more likely to smoke more puffs of a cigarette after negative affect induction (Perkins et al., 2008), and to respond to smoking cues (Hutchison, LaChance, Niaura, Bryan, & Smolen, 2002; Munafo & Johnstone, 2008), suggesting that individuals with a depression history and long VNTR alleles may benefit more from bupropion than individuals without both characteristics.

The Brown et al. (2007) trial sample and the Lerman et al. (2003) trial sample differ in the prevalence of individuals with a substance dependence diagnosis in the same fashion as with depression history. McGeary reviewed the VNTR association with substance dependence literature and concluded that there was some evidence for association with intermediate phenotypes, but a failure to consistently identify associations with substance dependence diagnostic measures (McGeary, 2009). This suggests that drug dependence prevalence differences have not contributed to the VNTR by treatment association differences observed by Leventhal et al. (2012) and in the current analysis of the Lerman et al. (2003) sample.

The most robust associations of the VNTR with a psychiatric diagnosis assessed in a meta-analysis are with a diagnosis of attention deficit hyperactivity disorder (ADHD) (Gizer, Ficks, & Waldman, 2009). A meta-analyses of five placebo-controlled trials showed that bupropion is significantly more effective than placebo for treatment of ADHD (Maneeton, Maneeton, Srisurapanont, & Martin, 2011). Self-reported inattentive and hyperactive/impulsive symptoms have been associated with increased prevalence of regular smoking in a large adolescent sample (Kollins, McClernon, & Fuemmeler, 2005), childhood ADHD diagnosis has been associated with reduced abstinence in a clinical trial (Humfleet et al., 2005), and highly nicotine-dependent individuals with a combined inattentive and hyperactive/impulsive ADHD subtype diagnosis responded to pharmacological treatment for smoking cessation with increased abstinence (Covey et al., 2011). This suggests that inattentive and hyperactive/impulsive symptom severity differences in the absence of a history of an ADHD diagnosis may account for some of the VNTR by treatment association differences observed.

Genetic differences between the samples or genetically based analytic differences could play a role in differences observed between the analysis of Leventhal et al. (2012) and the current analysis. In addition to analytic confirmation of self-identified European ancestry (Conti et al., 2008), we included principal components of population genetic variation as covariates in the analyses reported here. We did observe several principal components significantly associated with abstinence. Gizer et al. noted significant heterogeneity in association of the VNTR with ADHD diagnosis, including differences in the association direction and strength due to genetic ancestry differences (Gizer et al., 2009). Inclusion of covariates of genetic ancestry will correct for continental and some subcontinental population genetic ancestry differences, but will not address genetic differences due to rare variation that have arisen in the recent expansion of the human population (Keinan & Clark, 2012). Rare-linked variants have been associated with ADHD diagnosis (Grady et al., 2003, 2005; Tovo-Rodrigues et al., 2012). CYP2B6 exhibits extensive coding and noncoding variation within and between continental population groups that influence CYP2B6 expression and function (Lang et al., 2001). Sampling effects within analysis categories may result in differences in relevant pharmacogenetic variation (Lee et al., 2007).

Differences in treatment between the trials encompass the different behavioral treatments, with the Brown et al. (2007) trial focused on evaluating differences between effects of cognitive–behavioral treatment for depression added to standard cognitive–behavioral smoking cessation treatment (CBTD) versus standard cognitive–behavioral smoking cessation treatment (CBT), while the behavioral treatment in the Lerman et al. (2003) trial consisted of cognitive–behavioral therapy in a group counseling setting. While no differences were observed between CBTD and CBT on point prevalence abstinence in the Brown et al. (2007) trial (Brown et al., 2007), both behavioral treatments in the Brown et al. (2007) trial were more intensive (in the number and length of counseling sessions) than the treatments in the Lerman et al. (2003) trial. The Lerman et al. (2003) sample, as reported in this analysis, reports lower abstinence (point prevalence and continuous abstinence) prevalence than the Brown et al. (2007) sample reported in Leventhal et al. (2012) for approximately 75% of the 24 comparisons (four genotype by pharmacotherapy treatment categories, three timepoints, two abstinence outcomes). Differences in behavioral treatment intensity between the trials might account for some of these differences. Differences in the efficacy of behavioral treatment might also influence relative efficacy by pharmacotherapy and genotype, and account for some of the treatment by genotype interaction association differences observed between the trials.

Differences in the values of some behavioral or genetic characteristics, genetically based covariates, or relative treatment efficacy by genotype, might explain a portion of the differences in association results observed between the results of Leventhal et al. (2012) and our analyses, although the role of chance, in either previously reported findings or in differences between analysis results, cannot be ignored. The analysis of multiple randomized clinical trials in an integrated data analysis framework that considers these different behavioral and genetic variables will be necessary to resolve whether VNTR by treatment interaction in association with abstinence occurs in treatment-seeking smokers (Ioannidis, 2009). The sample sizes that power analyses suggest will be necessary to detect the previously reported finding are available within randomized clinical trials curated by the Pharmacogenetics of Nicotine Addiction Treatment consortium (http://pgrn.org/display/pgrnwebsite/PNAT+Profile), although additional trials would be required to focus exclusively on trials randomizing individuals to active bupropion and placebo treatment. Analysis of a double-blind, randomized, placebo-controlled trial of transdermal nicotine replacement therapy has reported a main effect of the VNTR on abstinence at one timepoint (David et al., 2008), suggesting that nicotine replacement therapy trials should be included in further analyses. Multiple treatment meta-analyses of a sufficient number of trials to test main and interactive effects of the VNTR and response to multiple treatments may improve our knowledge of the influence of the DRD4 Exon III VNTR on prospective abstinence, and potentially, guide therapeutic strategies (McDonagh, Whirl-Carrillo, Garten, Altman, & Klein, 2011).

SUPPLEMENTARY MATERIAL

Supplementary Tables 1–7 can be found online at http://www.ntr.oxfordjournals.org

FUNDING

This work was supported by the National Institutes of Health of the U.S. Department of Health and Human Services (U01 DA020830 to RFT and CL, and P50 CA143187 to CL). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the Department of Health and Human Services.

DECLARATION OF INTERESTS

Dr. Bergen has received research support, in the past, through institutional collaboration agreements between Medco Health Solutions, Affymetrix, and SRI International, and between Perlegen Sciences and SRI International. Dr. Benowitz has been a consultant for several pharmaceutical companies that market smoking cessation medications.

Supplementary Material

Supplementary Data
supp_15_7_1190__index.html (915B, html)

ACKNOWLEDGMENTS

We gratefully acknowledge the bupropion versus placebo clinical trial participants for their contributions to research. We thank Faith Allen (UCSF), Chris Jepson (University of Pennsylvania), Ruth Krasnow, Martha Michel, Huaiyu Mi, and Denise Nishita (SRI International) for curation and management of clinical and genotype data, and for management of biospecimens and generation and management of genotyping data, respectively.

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