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The rs1051730 genetic variant within the CHRNA5-A3-B4 gene cluster is associated with heaviness of smoking and has recently been reported to be associated with likelihood of stopping smoking. We investigated the potential association of rs1051730 genotype with reduced likelihood of smoking cessation in 2 cohorts of treatment-seeking smokers in primary care in the United Kingdom.
Data were drawn from 2 clinical trials on which DNA was available. One sample was a randomized placebo-controlled trial of nicotine transdermal patch and the other sample an open-label trial where all participants received nicotine transdermal patch. Smoking status was biochemically verified. Logistic regression was used to assess evidence for association in each sample, and data were combined within a meta-analysis.
There was evidence of association of rs1051730 genotype with short-term (4-week) cessation in our open-label trial sample but not our placebo-controlled trial sample. When combined in a meta-analysis, this effect remained. There was no evidence of association at later follow-up intervals. Adjustment for cigarette consumption and tobacco dependence did not alter these results substantially.
Our data, taken together with previous recent studies, provide some support for a weak association between this variant and short-term smoking cessation in treatment-seeking smokers, which does not seem to operate only among those receiving nicotine replacement therapy. Moreover, the rs1051730 variant may not merely operate as a marker for dependence or heaviness of smoking.
Most smokers report a desire and intention to quit, but while nearly half attempt to quit in any given year, only 2%–3% of all smokers actually succeed. Quit attempts often fail within days (Hughes, 2003), even with treatment, so that better treatment strategies are needed. Smoking behavior is known to be under a degree of genetic influence (Munafo, Clark, Johnstone, Murphy, & Walton, 2004), and this may explain some of the interindividual variation in ability to stop smoking. Pharmacogenetic research has several aims, including determining whether the likelihood of successfully stopping smoking is influenced by inherited variation (Munafo, Shields, Berrettini, Patterson, & Lerman, 2005). A number of retrospective pharmacogenetic studies of nicotine replacement therapy (NRT) have been conducted (Dahl et al., 2006; David, Munafo, Murphy, Walton, & Johnstone, 2007; David et al., 2008; Johnstone et al., 2004, 2007; Lerman, Jepson, et al., 2006; Lerman, Tyndale, et al., 2006; Lerman et al., 2004; Malaiyandi et al., 2006; Munafo et al., 2006; Yudkin et al., 2004). Nicotine is the main addictive constituent of tobacco (Balfour, 2004; Pontieri, Tanda, Orzi, & Di Chiara, 1996), and therefore, genes that may influence sensitivity or response to nicotine are plausible candidates for pharmacogenetic investigation.
Some of the genetic influences on the ability to stop smoking are likely to overlap with those that influence tobacco dependence. Genome-wide association (GWA) studies have provided robust evidence that single nucleotide polymorphism (SNP) variants within the CHRNA5-A3-B4 gene cluster on chromosome 15q24 are associated with heaviness of smoking (Liu et al., 2010; Thorgeirsson et al., 2008, 2010; Tobacco-and-Genetics-Consortium, 2010). The first published study that highlighted this region identified the rs16966968 variant (Saccone et al., 2007), which was identified with genome-wide significance in a subsequent study (Berrettini et al., 2008). The rs1051730 variant is highly correlated with this SNP (r2 > .9) in samples of European ancestry, so that the two may be used interchangeably, and has also been shown to be associated with heaviness of smoking (Thorgeirsson et al., 2008). The rs1051730 SNP lies within the nicotinic acetylcholine receptor alpha 3 subunit gene (CHRNA3), in a linkage disequilibrium block containing two other candidate genes, encoding the nicotinic acetylcholine receptor alpha 5 (CHRNA5) and beta 4 (CHRNB4) subunit genes (Thorgeirsson et al., 2008). A recent study showed that the minor allele of the rs16969968 missense polymorphism, D398N, in CHRNA5 conferred a reduced response to a nicotinic agonist in vitro (Bierut et al., 2008). This polymorphism may therefore be the functional variant responsible for the association with smoking quantity.
It remains unclear, however, whether the rs1051730 variant is associated with other tobacco use phenotypes. GWA studies conducted to date, which typically rely on data from retrospective self-report, have failed to find evidence of association with smoking initiation (comparing ever- and never-smokers) or smoking cessation (comparing current and past smokers). One large cohort study, using prospectively assessed smoking status over the course of pregnancy, showed significant association of the rs1051730 variant with likelihood of smoking cessation, with the risk (T) allele associated with a reduced likelihood of stopping smoking (Freathy et al., 2009). This association remained after adjustment for heaviness of smoking, suggesting that the effect was not mediated solely through heaviness of smoking or tobacco dependence. A recent case–control study replicated this modest association (Thorgeirsson & Stefansson, 2010) in a reanalysis of a previous study (Thorgeirsson et al., 2008). Both studies indicated a per allele odds ratio (OR) of ~1.2 for persistence (or ~0.8 for cessation).
These studies were restricted to pregnant women, some of whom stopped smoking during the course of their pregnancy, assessed either prospectively (Freathy et al., 2009) or retrospectively (Thorgeirsson & Stefansson, 2010). It is unclear, therefore, whether these findings can be generalized to treatment-seeking smokers and whether any association with likelihood of cessation may be modified by the use of pharmacotherapies for smoking cessation, such as NRT. However, a further study has also shown association of a haplotype containing rs1051730 with increased likelihood of relapse to smoking in a sample of smokers making a quit attempt who received either bupropion pharmacotherapy or placebo (Baker et al., 2009). We therefore investigated the potential association of rs1051730 genotype with smoking cessation in two cohorts of treatment-seeking smokers. We aimed to test the hypothesis that the rs107130 risk (T) allele is associated with a reduced likelihood of smoking cessation.
Participants were eligible if they were aged between 25 and 65 years and smoked at least 15 cigarettes/day at the time of study entry and were recruited from general practices in Oxfordshire, UK. Of the n = 1,686 participants in the original trial, n = 1,532 were subsequently recontacted for DNA collection, while n = 154 were unavailable because they could not be located or were deceased. Blood samples were successfully collected from n = 755 (44%) participants. The full methods for recruitment, allocation, and randomization of the Patch study (Imperial-Cancer-Research-Fund, 1993) and the 8-year follow-up Patch II study (Yudkin et al., 2003) have been comprehensively described. Ethical approval was obtained from the Anglia and Oxford Multicentre Research Ethics Committee and from the Local Research Ethics Committees covering the areas of residence of the patients.
Participants were randomly assigned to wear active NRT transdermal patch of decreasing strength or placebo patch for 12 weeks. Active and placebo patches were identical as prepared by the manufacturer, and all investigators and patients were blinded to treatment allocation. Participants were assessed by a study nurse at 1, 4, 8, 12, 26, and 52 weeks, and smoking status was measured at every contact and verified by exhaled carbon monoxide (CO) monitoring (at 1, 4, 8, and 12 weeks) or salivary cotinine (at 12, 26, and 52 weeks). Confirmation of abstinence was defined as an expired CO reading less than 10 parts per million (ppm) or by a salivary cotinine level of less than 20 ng/ml.
Participants were eligible if they were aged 18 years or over and smoked 10 cigarettes/day or more at the time of study entry and were recruited from general practices in Buckinghamshire and Oxfordshire, UK. Physicians recruited patients attending for other reasons (n = 60, 7%) or patients volunteered having seen posters or heard about the study (n = 15, 2%), while in some practices, every registered smoker was contacted offering trial entry (n = 850, 92%). Blood samples were successfully collected, and DNA was extracted on n = 914 (99%) participants at trial entry. The full details and results of the main trial are reported elsewhere (Aveyard et al., 2007), while the lack of ethnic stratification in this sample, based on 1 M SNP multiple pool genotyping, has also been reported previously (Uhl et al., 2010). Ethical approval was obtained from the relevant Local Research Ethics Committee.
Participants were randomly assigned to one of two levels of smoking cessation behavioral support. Usual care provided behavioral support prior to cessation, in the first week and at four weeks. The other arm provided weekly support. In addition, all participants received 8 weeks of 15-mg NRT transdermal patch. Smoking status was measured at every contact and verified by exhaled CO monitoring. Participants were phoned at 12, 26, and 52 weeks from quit day to assess smoking status, and those claiming at least 7-day abstinence were posted a saliva collection device to subsequently measure salivary cotinine. Confirmation of abstinence was defined as an exhaled CO less than 10 ppm or salivary cotinine concentration less than 15 ng/ml on each occasion (Society-for-Research-on-Nicotine-and-Tobacco, 2002).
We used biochemically confirmed continuous abstinence at 4-, 12-, and 26-week follow-up as the main outcome measures. Prolonged abstinence typically allows a grace period, which in our study was up to the first visit providing this was within 14 days of the quit day, consistent with the guidelines of the Society for Research on Nicotine and Tobacco for the measurement of abstinence in clinical trials (Hughes et al., 2003) and the Russell Standard (West, Hajek, Stead, & Stapleton, 2005). Participants who were withdrawn (which was commonly due to reverting to smoking) or who were lost to follow-up (unless they moved to an untraceable address or had died) were counted as smokers, as is standard (West et al., 2005). Tobacco dependence was assessed using the Horn–Russell Tobacco Scale (Patch II) or the Fagerström Test for Nicotine Dependence (Patch in Practice).
Genotyping for rs1051730 was performed by primer extension and MALDI-TOF–based allele detection (Sequenom, San Diego, CA) using polymerase chain reaction (PCR) forward primer 5′-GGCTCTTCCATGAACCTCAA-3′, reverse primer 5′-GCCGGATGTACAGCGAGTAT-3′, and extension primer 5′-CATCATCAAAGCCCCAGGCTA-3′. Each PCR contains 1 μl genomic DNA (2.5 ng/μl), 0.4 μl deoxynucleotide triphosphates (dNTPs) mix (10 mM), 0.5 μl Amplitaq Gold PCR buffer (10×), 0.3 μl MgCl2 (1.5 mM), 0.2 μl each of forward and reverse primer (20 μM each), 0.025 μl of Amplitaq Gold DNA polymerase (0.025 U/μl) (Applied Biosystems Inc., Foster City, CA), and 2.38 μl double-distilled water (ddH2O). Thermal cycling was carried out as follows: (a) 10 min at 95 °C; (b) 35 cycles of 30 s at 94 °C, 30 s at 58 °C, and 45 s at 72 °C; and (c) a final 3 min at 72 °C extension followed by 4 °C. After PCR amplification, unincorporated dNTPs were removed by 20-min 37 °C incubation with shrimp alkaline phosphatase, and enzymes were inactivated by 5-min incubation at 85 °C according to manufacturer’s instructions. Primer extension was performed with 0.2 μl appropriate combination of deoxy dNTP, dideoxy ddNTP (2.25 mM), 0.054 μl extension primer (100 μM), 0.018 μl thermosequenase (32 U/μl), and 1.7 μl ddH2O. Thermal cycling was carried out as follows: (a) 94 °C for 2 min; (b) 40 cycles of 5 s at 94 °C, 5 s at 52 °C, and 5 s at 72 °C; and (c) 4 °C. Reactions were purified with SpectroClean resin (Sequenom), spotted in matrix on Sequenom arrays, and subjected to MALDI-TOF mass spectrographic analyses with automatic allele detection and manual allele confirmation.
We used logistic regression to test for the effects of rs1051730 genotype, coded as 0, 1, or 2 copies of the T (risk) allele, on smoking status at 4-, 12-, and 26-week follow-up. Analyses were performed with and without adjustment for age, sex, cigarette consumption, tobacco dependence, and treatment condition. The genotype × treatment condition was tested and removed if nonsignificant. Linear regression was used to test for the effects of rs1051730 genotype on cigarette consumption and tobacco dependence. Analyses were performed using SPSS version 16.0 (SPSS Inc., Chicago, IL) and Comprehensive Meta-Analysis version 2 (Biostat Inc., Newark, NJ), and an alpha level of .05 was maintained throughout.
A total of n = 1,686 participants were recruited into the original trial (Imperial-Cancer-Research-Fund, 1993) of whom n = 755 (45%) participants provided a DNA sample and n = 699 (42%) were successfully genotyped for the rs1051730 polymorphism. Data from n = 7 participants of non-European ancestry were excluded to prevent possible bias due to population stratification. Data were missing on cigarette consumption for one participant and tobacco dependence for one participant. Therefore, the final sample for analysis consisted of n = 692 (41%) participants and n = 690 (41%) on whom complete data were available. The allele frequencies for the rs1051730 variant were consistent with previous studies in similar populations (T allele: ~35% in both samples). Genotype was associated with cigarettes per day (p = .002) in the predicted direction but not with tobacco dependence (p = .35).
A total of n = 925 participants were recruited into the trial (Aveyard et al., 2007), of whom n = 914 (99%) participants provided a DNA sample and n = 882 (95%) were successfully genotyped for the rs1051730 polymorphism. Data from n = 65 participants of non-European ancestry were excluded to prevent possible bias due to population stratification. Data were missing on age for n = 3 participants, cigarette consumption for n = 18 participants, and tobacco dependence for n = 26 participants. Therefore, the final sample for analysis consisted of n = 817 (88%) participants and n = 778 (84%) on whom complete data were available. Genotype was associated with cigarettes per day (p = .015) and tobacco dependence (p = .004) in the predicted direction.
There was no evidence of association between the T allele of the rs1051730 polymorphism and decreased likelihood of smoking cessation at 4- (OR = 0.84, 95% CI 0.66–1.08, p = .18), 12- (OR = 0.89, 95% CI 0.67–1.18, p = .43), or 26-week (OR = 0.78, 95% CI 0.57–1.07, p = .12) follow-up. These results were not altered substantially when adjusted for age, sex, cigarette consumption, tobacco dependence, and treatment condition. The genotype × treatment condition interaction was not significant and therefore not retained in the model. These results are presented in Table 1.
There was weak evidence of association between the T allele of the rs1051730 polymorphism and decreased likelihood of smoking cessation at 4-week follow-up (OR = 0.77, 95% CI 0.60–0.99, p = .040), but this association attenuated at 12-week (OR = 0.80, 95% CI 0.59–1.09, p = .15), and 26-week (OR = 0.90, 95% CI 0.64–1.26, p = .53) follow-up. These results were not altered substantially when adjusted for age, sex, cigarette consumption, tobacco dependence, and treatment condition. The genotype × treatment condition interaction was not significant and therefore not retained in the model. These results are presented in Table 1.
We combined unadjusted allelic ORs from the two samples in a meta-analysis, within a fixed-effects framework using inverse variance methods (Munafo & Flint, 2004). This indicated weak evidence of association of the T allele with reduced likelihood of smoking cessation at 4-week follow-up (OR = 0.81, 95% CI 0.68–0.96, p = .015). The strength of this association was similar at 12- (OR = 0.85, 95% CI 0.69–1.04, p = .12) and 26-week (OR = 0.83, 95% CI 0.66–1.05, p = .12) follow-up but no longer statistically significant. For all analyses, there was no between-study heterogeneity (I2s = 0%), and these results were not altered substantially when adjusted allelic ORs were used. Genotype frequencies by smoking cessation status at 4-, 12-, and 26-week follow-up for the Patch II and Patch in Practice studies are presented in Table 2.
While our data on their own are inconclusive with respect to any association between variation in the CHRNA5-A3-B4 gene cluster and short-term smoking cessation in treatment-seeking smokers, it is notable that the magnitude of the effect we observed is consistent with recent evidence from a cohort study of pregnant women (Freathy et al., 2009), a community study of pregnant women (Thorgeirsson & Stefansson, 2010), and a clinical trial of treatment-seeking men and women (Baker et al., 2009). Taken together, these data are suggestive of a weak effect of genetic variation in this region on smoking cessation. Given the lack of a genotype × treatment interaction in the Patch II sample, which employed a placebo-controlled design, it is unlikely that any effect operates only among those receiving NRT. Moreover, adjustment for cigarette consumption and tobacco dependence did not alter these results substantially, suggesting that any effect on cessation may not be mediated through dependence or heaviness of smoking. The observed per allele effect is equivalent to ~0.5% of phenotypic variance in quit success, which is clearly modest (although consistent with expectation for common variants and complex behavioral phenotypes). However, any effect on cessation may be cumulative over the multiple attempts to stop smoking any one individual might make.
Previous studies have observed that this variant and other nearby variants on chromosome 15 are associated with heaviness of smoking among current smokers but not smoking status (i.e., ever-smoker vs. never-smoker or current smoker vs. ex-smoker), indicating no association with either smoking initiation or cessation. However, large-scale case–control studies typically rely on self-report point prevalence smoking status and typically lack biochemical verification of current smoking status. This will reduce the ability to detect these associations. The data in the present study are derived from the prospective assessment of treatment-seeking smokers, where smoking status was verified with biochemical assessment, which may allow for a more precise measurement of smoking cessation.
It is also worth noting that the participants in both samples were selected to be heavy smokers actively seeking treatment to stop smoking. There may be important differences between this population and the wider population of smokers who attempt to stop smoking, most of whom do so spontaneously and without behavioral or pharmacological support. However, together with previous evidence (Baker et al., 2009; Freathy et al., 2009; Thorgeirsson & Stefansson, 2010), there is now evidence from five independent samples indicating that the rs1051730 variant may be weakly associated with the short-term ability to stop smoking. This may be independent of both heaviness of smoking and tobacco dependence and whether or not the individual is using NRT. While we provide statistical evidence of association with smoking cessation, the clinical significance of this association (if genuine) is modest, given the small effect size and the lack of a specific effect among individuals receiving NRT. Nevertheless, it is larger than would be predicted from an effect on heaviness of smoking equivalent to 1 cigarette/day, which suggests it may operate via some unmeasured attribute related to cigarette smoking.
The mechanism by which genetic variation in the CHRNA5-A3-B4 gene cluster influences heaviness of smoking remains unclear, and it is therefore difficult to speculate on how it influences smoking cessation. One possibility is that individuals with one or more copies of the T allele may be more resistant to the aversive side effects of nicotine, as shown in recent studies of nAChR α5 knockout mice (Jackson et al., 2010), and may therefore be able to tolerate higher doses of NRT. Conceivably, nicotine derived from combinations of smoking and NRT, or different combinations of NRTs, might thus provide different dose–response relationships for individuals with nAChRs encoded by haplotypes with these variants at this locus. However, NRT is generally well tolerated at high doses, and in the studies reported here, there was no variation in dose offered. Moreover, this explanation is also not consistent with our failure to observe a differential effect in the placebo and active arms of the Patch II study and the effect of rs1051730 reported in other studies (Baker et al., 2009; Freathy et al., 2009; Thorgeirsson & Stefansson, 2010). A second possibility is that it might be associated with cessation because it influences heaviness of smoking or tobacco dependence. In particular, it may in fact be the case that dependence mediates any association between this variant and likelihood of smoking cessation success, but error variance in the measurement of dependence or heaviness of smoking may obscure this. In addition to measurement error, there is considerable interindividual variability in number of puffs taken per cigarette, depth of inhalation, and other aspects of smoking topography, meaning that cigarettes per day is a relatively poor indicator of typical nicotine consumption. This would be supported if adjusting for precessation cotinine levels (which more accurately assay nicotine consumption) attenuates the association of genotype with cessation success. Both of these possibilities should be investigated in future laboratory and clinical studies.
There are several limitations to this study, which should be considered when interpreting these results. First, the sample size was small by current standards of genetic epidemiology. However, this is perhaps offset to some degree by the greater precision afforded by the prospective assessment of biochemically verified smoking status (Phillips & Smith, 1993). Second, the observed association only achieved statistical significance in one sample and would not survive Bonferonni correction for multiple tests across three outcome measures. However, the effect size estimates were comparable in both samples, and the overall effect on short-term abstinence was significant in the meta-analysis, with no evidence of between-study heterogeneity. Third, we did not genotype the rs16966968 variant, which has been proposed as the putative functional variant within this region, or other nearby variants identified in recent genome-wide analyses. However, the strong prior evidence for association with heaviness of smoking suggests that the rs1051730 is an acceptable marker at this locus. Fourth, the imprecision in self-report assessments of heaviness of smoking and tobacco dependence may have reduced our ability to determine whether the rs1051730 variant is simply acting as a marker for these measures compared with more direct measures of use and exposure, such as precessation levels of cotinine. For example, while we observed the predicted association between rs1051730 and cigarettes per day, we only observed an association with tobacco dependence in the Patch in Practice sample and not the Patch II sample, most likely due to differences in the measure of dependence used (Fagerström Test for Nicotine Dependence vs. Horn–Russell Tobacco Scale, respectively). This possibility can be investigated in future laboratory and clinical studies.
In conclusion, our data, in conjunction with previous studies, provide some support for a small effect of the rs1051730 on short-term smoking cessation, independently of heaviness of smoking, tobacco dependence, or the use of NRT. Future studies attempting to replicate this finding should attempt to do so in large samples of prospectively assessed smokers whose smoking status can be confirmed biochemically.
This research was supported in part by a Cancer Research UK programme grant (C53/A6281) and by the National Institutes of Health intramural research program (National Institute on Drug Abuse) Department of Health and Human Services, USA. PA is supported by the National Institute of Health Research.
MRM and PA are members of the UK Centre for Tobacco Control Studies, a U.K. Clinical Research Collaboration Public Health Research Centre of Excellence. Funding from the Economic and Social Research Council, the British Heart Foundation, Cancer Research UK, the Department of Health, and the Medical Research Council, under the auspices of the UK Clinical Research Collaboration, is gratefully acknowledged. The authors are grateful for the support of the Patch Study Team and the general practices which participated in the trial.