Smokers of European ancestry (n=720) who participated in a double-blind, randomised, placebo-controlled trial of transdermal nicotine replacement therapy, were genotyped for two functional polymorphisms (variable number of tandem repeats (VNTR) and a C to T transition at position –521 (C-521T)) in the dopamine D4 receptor gene (DRD4) gene. Logistic regression models of abstinence at 12- and 26-week follow-ups were carried out separately for each polymorphism. For the DRD4 VNTR models, the main effect of treatment was significant at both 12-week (P=0.001) and 26-week (P=0.006) follow-ups, indicating an increased likelihood of successful cessation on active nicotine replacement therapy transdermal patch relative to placebo. The main effect of DRD4 VNTR genotype was associated with abstinence at 12-week follow-up (P=0.034), with possession of one or more copies of the long allele associated with reduced likelihood of cessation (17 vs 23%), but this effect was not observed at 26-week follow-up. For the DRD4 C-521T models, no main effect or interaction terms involving genotype were retained in the models at either 12- or 26-week follow-up. These data are consistent with observations from studies of the DRD2 gene that genetic variants related to relatively decreased dopaminergic tone in the mesocorticolimbic system are associated with increased risk for relapse to smoking following a cessation attempt.
Despite major public health achievements in the field of tobacco control over the last 50 years in reducing smoking prevalence, tobacco use remains a leading cause of mortality worldwide, in part because of the limited efficacy of pharmacologic and behavioural smoking cessation therapies.1, 2 Twin studies indicate that at least 50% of the variance in nicotine dependence can be explained by genetic factors.3, 4, 5, 6 Thus, the quest for more efficacious smoking cessation therapies has been augmented in recent years by the exploration of how variation in the human genome might influence treatment response to the two first-line pharmacotherapies for smoking cessation currently in widespread use, namely nicotine replacement therapy (NRT) and sustained-release bupropion.7
Converging evidence from animal and human studies indicates that nicotine dependence is under the influence of dopamine-dependent mesolimbic and mesocortical brain systems,8 and that nicotine stimulates burst firing of dopaminergic neurones when binding to nicotinic acetylcholine receptors in the midbrain tegmentum,9 which in turn results in enhanced release of dopamine in the outer shell of the nucleus accumbens and prefrontal cortex.10, 11 This cascade of events, including changes in post-synaptic signalling, is theorised to be positively reinforcing with hedonic and appetitive motivational properties.12, 13
Pharmacogenetic investigations of nicotine dependence, therefore, have focused largely on genetic variation in the dopamine pathway. Of the five subtypes of dopamine receptors, the dopamine D2 and D4 receptors have been the most widely investigated in genetic association studies of smoking-related phenotypes.14 Three polymorphisms (C957T, −141Cins/del and Taq1A or C32806T) in the dopamine D2 receptor gene (DRD2) appear to influence treatment response to NRT patch15, 16, 17 and/or bupropion for smoking cessation,7, 16, 18, 19, 20 although recent evidence indicates that the Taq1A polymorphism is, in fact, present in a protein kinase gene (ANKK1) located downstream of the DRD2 gene.21 However, to date, there are no published pharmacogenetic smoking cessation studies evaluating dopamine D4 receptor gene (DRD4) polymorphisms.
Dopamine D4 receptors are present in the shell of the nucleus accumbens and throughout the caudate nuclei and putamen.22 The dopamine DRD4 gene (11p15.5)23 is highly polymorphic, although research evaluating behavioural and pharmacogenetic phenotypes has focused almost entirely on a variable number of tandem repeats (VNTR) polymorphism located in exon III of the gene. This research has investigated the effects of the presence or absence of the 7-repeat (‘long’) allele of the VNTR, which is associated with decreased ligand binding,24 decreased gene expression in vitro and attenuation of cyclic AMP formation when dopamine is bound to the receptor25, 26, 27 compared with 6-repeat or fewer (‘short’) alleles. This polymorphism has been associated with externalising behaviours such as attention deficit hyperactivity disorder28, 29, 30, 31, 32, 33, 34 personality traits,35 including novelty-seeking36, 37, 38, 39, 40, 41, 42, 43 and reward dependence36, 37, 38, 39, 40, 41, 42, 43 executive functioning44 and risk of nicotine dependence.45, 46 More recently, polymorphisms in the promoter region of the DRD4 gene have been discovered and examined with respect to schizophrenia and personality traits.26, 27, 28, 29, 30, 31, 47, 48 One particular promoter polymorphism, a C to T transition at position −521 (C-521T), affects transcription, with the T allele reducing the transcriptional efficacy by 40% compared with the C allele.47 Only weak linkage disequilibrium (LD) has been reported between the VNTR and −521 locus.49
There are currently no published studies examining effects of the DRD4 VNTR or C-521T variants on smoking cessation or response to NRT for smoking cessation. We therefore sought to examine the influence of these functional polymorphisms in the DRD4 gene on smoking cessation outcomes, and to evaluate whether or not the DRD4 VNTR or C-521T polymorphisms would moderate NRT efficacy for smoking cessation in a placebo-controlled, randomised clinical trial of the NRT for smoking cessation. Consistent with the hypothesis that reduced dopaminergic tone represents a risk factor for substance use and dependence,50 and published pharmacogenetic studies of NRT patch15, 16, 17 and bupropion,7, 16, 18, 19, 20 we hypothesised that genetic variants resulting in decreased D4 receptor availability (DRD4 VNTR long and −521T alleles) would be associated with reduced likelihood of smoking cessation. Furthermore, we also hypothesised that participants with reduced activity DRD4 alleles would be more responsive to NRT.
Characteristics of participants
Of the n=745 participants who were successfully genotyped for the DRD4 VNTR and C-521T polymorphisms, n=737 were of European ancestry. There were missing data on n=17 participants, resulting in a final sample for analysis of n=720 smokers (59% female) of European ancestry. The mean age of participants was 43 years (s.d.=10; range 25–65). Participants were older than non-participants (M=43 vs M=42 years; P=0.002), more likely to be female (59 vs 53%; P=0.010) and more likely to have quit for a year in the trial (14 vs 6%; P<0.001). Genotype frequencies did not deviate significantly from Hardy–Weinberg Equilibrium (VNTR: P=0.66; C-521T: P=0.45). Only weak LD was found between the VNTR and −521C/T polymorphism in our sample (D′=0.15; r2=0.004).
Analysis of smoking cessation outcomes
For the DRD4 VNTR models, the main effect of treatment was significant at both 12-week (P=0.001) and 26-week (P=0.006) follow-ups, indicating an increased likelihood of successful cessation on active NRT transdermal patch relative to placebo, as previously reported.51, 52 The main effect of DRD4 VNTR genotype was associated with abstinence at 12-week follow-up (P=0.034), with possession of one or more copies of the long allele associated with reduced likelihood of cessation, but this term was not retained in the model at 26-week follow-up. Age was positively associated with likelihood of abstinence at 12-week (P=0.006) and 26-week (P=0.007) follow-ups. Higher socioeconomic status (SES) was marginally associated with an increased likelihood of abstinence at 12-week follow-up (P=0.051), but was not retained in the model at 26-week follow-up. Finally, nicotine dependence was not retained in the model at 12-week follow-up, but was included at 26-week follow-up, indicating a trend for lower dependence to be associated with an increased likelihood of abstinence at 26-week follow-up (P=0.089).
For the DRD4 C-521T models, no main effect or interaction terms involving genotype were retained in the models at either 12- or 26-week follow-up (results are not presented in detail).
When these models were re-run using only data from those participants who attended at each follow-up, the results did not change substantially. The final DRD4 VNTR logistic regression models for abstinence at both 12- and 26-week follow-up are presented in Table 1. Smoking status frequencies by treatment group and DRD4 VNTR and C-521T genotypes at both 12-week (EOT) and 26-week follow-ups are presented in Table 2.
These data are broadly consistent with other pharmacogenetic analyses of smoking cessation outcomes in that functional polymorphisms related to relatively reduced mesostriatal dopamine receptor expression appear to be associated with lower abstinence rates following smoking cessation. To our knowledge, this is the first published study of genetic influences of the DRD4 genotype on smoking cessation with or without the use of NRT. We observed an effect of the VNTR functional polymorphism on likelihood of abstinence, with the presence of the long allele associated with a reduced likelihood of abstinence at 12-week follow-up, independent of treatment received, although this effect was no longer observed at 26-week follow-up. This polymorphism has been reported to be associated with reduced striatal dopamine D4 receptor availability, suggesting that decreased dopaminergic tone may reflect a risk factor for relapse to smoking following a smoking cessation attempt. If this polymorphism contributes to reduced dopamine set point, then one would anticipate that this polymorphism would also be associated with endophenotypes reflecting the aversive experience of nicotine withdrawal and craving.13, 28, 53 Interestingly, the VNTR polymorphism has also been associated with cue-induced craving for nicotine,54 as well as cue-induced craving for alcohol,55 heroin56 and food.57
In keeping with findings from pharmacogenetic studies of the DRD2 gene, we anticipated interactions between DRD4 and treatment response to the NRT transdermal patch for smoking cessation. However, no such associations were observed. Mechanistic explanations for why specific polymorphisms in DRD2 have previously been found to be associated with treatment response to NRT while no genotype × treatment interactions were observed for DRD4 would require speculation about the contribution of different dopamine receptor subtypes to nicotine dependence.
Nevertheless, there is evidence from this line of investigation that confirms animal research in demonstrating substantial variation in distribution of D2 and D4 receptors. Genetic variation in DRD2 appears to influence dorsal striatum expression of D2 receptors, but the DRD4 gene appears to influence expression predominantly in the prefrontal cortex.22, 58 The ventral striatum and prefrontal cortex appear to have integrated and differentiated roles in the process of reward signalling and aversive affects of nicotine administration and withdrawal.59, 60, 61, 62 However, whether or not the differentiation in processing rewarding and aversive drug experiences varies among dopamine receptor subtype, or how this might translate to lack of apparent pharmacogenetic effects on smoking cessation of DRD4 genotype compared with DRD2 genotype, would require further study.
As noted above, participants (contributing DNA) were slightly older and more likely to be abstinent than non-participants and age was associated with abstinence outcomes at 12 and 28 weeks. However, while it is conceivable that age might confound observed associations between treatment and abstinence outcomes, we have no reason to expect—nor do we have the means to assess—whether or not age was a confounder to observed main effects of DRD4 genotype on abstinence outcomes.
In summary, in this pharmacogenetic analysis of a randomised, placebo-controlled clinical trial of the NRT transdermal patch for smoking cessation, we observed a significant main effect of the DRD4 exon III VNTR polymorphism on likelihood of abstinence in the first 3 months of follow-up, but not on likelihood of abstinence at 6 months. No association was observed for the DRD4 C-521T polymorphism on smoking cessation, and there were no significant genotype × treatment interactions for either locus. These data are consistent with the dopamine hypothesis proposed by Lerman and co-workers, and prevailing models of nicotine dependence based upon extensive animal research.8, 13, 63 These results do not support the application of genetically tailored smoking cessation therapy using NRT patch with the DRD4 gene, although they may have translational application in the potential to identify subgroups of smokers who are more prone to relapse early in the course of smoking cessation therapy, independent of NRT usage. However, the feasibility of using a VNTR polymorphism in a clinical setting would be limited with currently available technologies, as such variants are less amenable to high-throughput genotyping or multiplex platforms. Future work would benefit from the identification of single nucleotide variants in high-LD with the VNTR that may be more tractable for inclusion in a pharmacogenetic panel.
Materials and methods
Participants in the original study included n=1686 patients from general practice (GP) surgeries in Oxfordshire, UK, who participated in a double-blind, randomised, placebo-controlled trial of the nicotine transdermal patch between June 1991 and March 1992 (the Patch study).51, 52 The inclusion criteria for this study were that participants smoked at least 15 cigarettes a day and were aged between 25 and 65 years.
In 1999–2000, n=1532 participants were recontacted and invited to enter the study. Of the n=1686 participants enrolled in the Patch study, n=154 subjects were unavailable because they could not be located (moved, emigrated or untraceable) or were deceased. Invitation letters were sent to remaining participants and those interested in joining the study were given an appointment with a nurse at each participant's GP surgery, during which a short questionnaire was given and a 10 ml blood sample was collected. Blood samples were successfully collected from n=755 (49%) participants. The methods for recruitment, allocation and randomisation of the Patch study51, 52 and the 8-year follow-up (Patch II study)15, 17, 64 have been comprehensively described.
In the Patch study (1991–1992), participants were randomly assigned to wear active nicotine patches or placebo patches for 12 weeks by prior random allocation of study numbers to each intervention group and sequential allocation of a study number to patients on entry. Participants were assessed by a study nurse at 1, 4, 8, 12, 26 and 52 weeks. Active and placebo patches were identical as prepared by the manufacturer and all investigators and patients were blinded to treatment allocation. The main outcome measure reported was abstinence, which was confirmed with salivary cotinine and exhaled carbon monoxide (CO) measurement.
Ethical approval was obtained from the Anglia and Oxford Multicentre Research Ethics Committee and from the 86 Local Research Ethics Committees covering the areas of residence of the patients.
Abstinence at 1, 4 and 8 weeks was confirmed by an expired CO reading ⩽10 parts per million (p.p.m.), and at 12, 26 and 52 weeks by a salivary cotinine level ⩽20 ng/ml (89% of cases) or expired CO reading ⩽10 p.p.m. Salivary cotinine was assayed by gas chromatography at the Department of Preventive Medicine at St Bartholomew's Medical College (London, UK).
Blood samples were separated and frozen on the day of receipt, or stored overnight at 4°C. Plasma and buffy coat lymphocytes were stored at −80°C until required for analysis. Participants were genotyped at the Cancer Research UK General Practice Research Group Laboratory at the Radcliffe Infirmary (Oxford, UK) using methods previously described and briefly summarised here.
The DRD4 VNTR was amplified by polymerase chain reaction (PCR) using primers and methods previously described.26, 65 After separation by electrophoresis for 3 h on a 2.5% agarose gel, the PCR products (2–8- or 10-repeat units) were sized using a 50 bp ladder. To genotype the DRD4 C-521T polymorphism, PCR was carried out with an allele-specific tetra primer system based on the method of Ronai,66 with slight modifications. Briefly, the reaction mixture contained 1 μ M each primer (M3, M4, SN0 and MS0),66 approximately 30 ng DNA, 200 μ M dNTPs, 1.5 mM MgCl2 and 1 M betaine in a final volume of 25 μl. After an initial denaturation step at 95°C for 15 min, thermocycling consisted of 40 cycles of 1 min at 95°C, 1 min at 65°C and 1 min at 72°C, then a final extension step of 72°C. PCR products (C allele: 405 bp product; T allele: 235 bp product) were separated on a 2.5% gel at 140 V for 2.5 h next to a 50 bp ladder.
Biochemically verified point-prevalence 7-day abstinence, at 12-week (EOT) and 26-week follow-ups, were the primary outcome measures. Participants lost to follow-up were assumed to have relapsed to smoking and coded as such in outcome analyses (i.e., intent to treat analyses). This resulted in n=47 participants at 12-week follow-up and n=213 participants at 26-week follow-up being designated as smokers.
Separate models of outcome at 12- and 26-week follow-ups were generated within a logistic regression framework, because pharmacotherapy was available only during the treatment phase (i.e., to evaluate the pharmacogenetic effects of genotype during active treatment). The full models included age, sex, socioeconomic status and nicotine dependence at baseline, treatment group (active patch, placebo patch), genotype (see below) and interaction terms for treatment × sex, treatment × genotype, sex × genotype and treatment × sex × genotype. Terms were entered using the backwards conditional method, with term removal conditional on P>0.10. Initial analyses were carried out for DRD4 VNTR and C-521T genotypes in separate models. DRD4 VNTR alleles were classified as long (L) if they consisted of seven or more repeats, and short (S) if they consisted of six or fewer repeats, consistent with previous studies.30, 45, 46, 54 For comparisons involving DRD4 VNTR genotype, genotype was entered as a categorical variable, with SS as the reference group (SS, SL+LL). For comparisons involving DRD4 C-521T genotype, genotype was entered as an ordinal variable, with TT as the reference group based on allele dosing of the C allele (TT, TC and CC). DRD4 VNTR SL and LL genotypes were grouped in order to increase statistical power, given the small number of LL genotypes (n=33), to maximise comparability with previous studies30, 45, 46, 54 and on the basis of in vivo evidence, for dominant effects on functional activity.59
All analyses were performed using the Statistical Package for the Social Sciences (v. 12.0). An α level of 0.05 was maintained throughout the analysis.
Of the n=720 participants in the final study population, n=362 received active patch and n=358 received placebo patch. The sample size was adequate to detect a risk ratio of 1.6 at 12-week follow-up and 1.8 at 26-week follow-up, with a power of 0.80, for a main effect of genotype on cessation.
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The authors thank Michael Churchman for assistance with assay development. In addition, we acknowledge Kate Hey, Sarah Roberts and Sarah Welch, who undertook data collection. This study was funded by a Cancer Research UK programme grant. Personal funding was provided to SPD by United States Public Health Service grant 1K08 DA14276-04 and the Robert Wood Johnson Foundation.
Duality Of Interest
Dr Walton has been the Chief Scientific Officer of G-Nostics Ltd. since October 2004.
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David, S., Munafò, M., Murphy, M. et al. Genetic variation in the dopamine D4 receptor (DRD4) gene and smoking cessation: follow-up of a randomised clinical trial of transdermal nicotine patch. Pharmacogenomics J 8, 122–128 (2008). https://doi.org/10.1038/sj.tpj.6500447
- smoking cessation
- nicotine replacement therapy
The VNTR 48 bp Polymorphism in the DRD4 Gene Is Associated with Higher Tobacco Smoking in Male Mexican Mestizo Smokers with and without COPD
Pharmacotherapy for smoking cessation: effects by subgroup defined by genetically informed biomarkers
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