The DRD2 Taqi-B Polymorphism and Its Relationship to Smoking Abstinence and Withdrawal Symptoms

The DRD2 TaqI-B polymorphism and its relationship to smoking abstinence and withdrawal symptoms

JD Robinson1, CY Lam1, The dopamine receptor D2 (DRD2) gene has polymorphisms that have been 1 2 linked to regulation of the dopamine system and to an increased prevalence JA Minnix , DW Wetter , of smoking. The present study examined the relationship of the DRD2 TaqI-A 3 4 GE Tomlinson , JD Minna , and -B polymorphisms with short-term clinical outcome (abstinence and TT-L Chen4 and PM Cinciripini1 withdrawal symptoms), collected from daily (14 pre-quit and 42 post-quit) diary data among smokers (n ¼ 116) treated with the nicotine patch plus 1Department of Behavioral Science, The either venlafaxine or placebo. The results showed that B1/B1 or B1/B2 University of Texas MD Anderson Cancer Center, smokers were slightly less likely to be abstinent on a given day than those Houston, TX, USA; 2Department of Health Disparities Research, The University of Texas MD homozygous for the TaqI-B2 allele. Significant DRD2 TaqI-B Â time interac- Anderson Cancer Center, Houston, TX, USA; 3The tions were found for several of the withdrawal scales, indicating that those Harold C Simmons Comprehensive Cancer smokers with the B1/B1 or B1/B2 genotypes tended to report more Center, Pediatric Hematology-Oncology Division, symptoms over time compared to those with the B2/B2 genotype. No The University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA and 4Hamon interactions or main effects were found for the DRD2 TaqI-A polymorphism. Center for Therapeutic Oncology Research, The The findings demonstrate that smokers homozygous for the TaqI-B2 allele Harold C Simmons Comprehensive Cancer experience progressive improvement in self-reported withdrawal symptoms Center, The University of Texas Southwestern while smokers with the TaqI-B1 allele showing little change. Medical Center at Dallas, Dallas, TX, USA The Pharmacogenomics Journal (2007) 7, 266–274; doi:10.1038/sj.tpj.6500427; Correspondence: published online 26 December 2006 Dr JD Robinson, Department of Behavioral Science, The University of Texas MD Anderson Keywords: DRD2; TaqI-A; TaqI-B; smoking; withdrawal; abstinence Cancer Center, PO Box 301439 – Unit 1330, Houston, TX 77230-1439, USA. E-mail: [email protected]

Introduction

Nicotine, along with a number of substances that share the potential for abuse, enhances dopamine activity in mesolimbic and mesocortical circuits thought to be important for behavioral reward and reinforcement.1 The role of genetic factors in these dopaminergic pathways are of interest in identifying risk factors for addiction (e.g.,2–4). Many genes are involved in the regulation of the dopamine system, and the DRD2 gene is one of the most frequently studied in addictive disorders. The human dopamine receptor D2 (DRD2) gene is located on chromosome 11q22–q23. Several polymorphisms in DRD2 have been described, including the TaqI-A restriction fragment length polymorphism (RFLP) located in the 30 flanking region 10 000 nucleotide pairs distal from the coding region of DRD2, and the TaqI-B RFLP, located in intron 1, 913 nucleotide pairs from the start codon in exon 2, and closer to the 50 regulatory region of the DRD2 than the more 30 TaqI-A.5 Recent findings, however, now place the TaqI-A polymorphism Received 6 June 2006; revised 2 October 2006; accepted 9 October 2006; published within an exon of an adjacent new protein kinase gene called ‘ankyrin repeat and online 26 December 2006 kinase domain containing 1’ (ANKK1), which may influence signal transduction DRD2 TaqI-B and withdrawal JD Robinson et al 267

in the central nervous system (CNS).6 Among those of smokers by their withdrawal symptom trajectories and European ancestry, the TaqI-A polymorphism is in linkage variability over time, measured using daily diary data.22–25 disequilibrium (LD) with the TaqI-B5 and the DRD2 C957T,7 Also, analyses of withdrawal and abstinence data gathered although not with the À141C Ins/Del polymorphism.8 The daily, rather than retrospectively, may yield more informa- C957T and the À141C Ins/Del are functional alleles in the tion about the circumstances in which some genes may DRD2 gene that have been associated with mRNA stability exert their influence on smoking behaviors, given that the and expression. closer in time the assessment of smoking and withdrawal is Both the TaqI-A19–11 and -B110 alleles have been asso- to their actual occurrence, the greater the sensitivity at ciated with a decrease in D2 dopamine receptor density in discerning the relationships between the two.26 the striatum, presumably conferring a reward deficiency The current study used previously unpublished daily diary that might increase substance use in an effort to offset the data from a recent study in our laboratory27 investigating deficit. A recent meta-analysis suggested a significant role of the role of the DRD2 TaqI-A polymorphism in smoking the TaqI-A1 allele in smoking behavior, initiation of cessation. In that trial all smokers were provided with the smoking, persistence of smoking and cigarette consump- nicotine patch plus brief counseling and were randomized tion,12 whereas a second meta-analysis found a significantly to receive either placebo or venlafaxine, a non-selective higher prevalence of the TaqI-A1 allele in smokers vs non- serotonin reuptake inhibitor that inhibits the reuptake of smokers.13 In addition to the TaqI-A1 allele, the TaqI-B1 serotonin and norepinephrine. In this previous study, allele has also been linked to increased prevalence of average abstinence rates from the quit date to a one-year smoking. For example, several studies have reported higher post-treatment follow-up indicated that smokers carrying prevalence rates of the TaqI-A1 or TaqI-B1 allele in current the TaqI-A1 allele quit smoking significantly less often than and former smokers vs non-smokers.14–16 Pastorelli et al.17 those homozygous for the TaqI-A2, regardless of treatment noted that the TaqI-B1 allele was more common among condition. No interaction was observed between treatment non-alcoholic smokers than the matched non-alcoholic and genotype for abstinence. However, a genotype by non-smokers. An epidemiological study in Caucasian lung treatment interaction was observed for weekly ratings of cancer patients reported that ever-smokers had a fourfold negative affect, during treatment, which showed that only increase in the presence of the TaqI-B1 allele compared to smokers homozygous for the TaqI-A2 allele manifested a never-smokers in both the cancer patients and controls.16 reduction in retrospective ratings of negative mood, when Additionally, the age of onset of smoking behavior was receiving venlafaxine vs placebo. This suggests that examin- earlier in those patients with the TaqI-A1 or -B1 allele and ing withdrawal data, which have a significant affective they made fewer lifetime attempts to quit smoking than component,28 might provide additional data on cessation those patients without those alleles.16 Lastly, a study phenotypes, particularly if those assessments were measured involving Mexican-American and African-American partici- frequently, such as those obtained in daily diary ratings. pants reported greater frequency of smoking-related cancers For the present study, we focused on the relationship of among first-degree relatives of case subjects with the TaqI-A1 the DRD2 TaqI-A and -B polymorphisms with short-term or -B1 allele vs those without.18 daily treatment outcome and withdrawal symptoms, col- The above findings notwithstanding, the links between lected from diary data, among smokers treated with the TaqI-A1 and -B1 alleles and smoking have not been venlafaxine or placebo. We were interested in using daily consistently found. For example, higher prevalence rates of abstinence and withdrawal symptoms to identify potential the TaqI-A1 or -B1 alleles in current and former smokers vs endophenotypes of the DRD2 TaqI-A and the lesser-studied non-smokers were not found in a family-based analysis.19 DRD2 TaqI-B polymorphisms and expected that these daily Additionally, a case–control study comparing Mexican- measures would provide us with more sensitivity to detect American lung cancer patients and controls did not find the impact of this polymorphism on smoking behavior than any relation between the TaqI-B1 allele and smoking the weekly measures we previously reported.27 We hypothe- behavior in lung cancer patients, although they did report sized that smokers with the at-risk TaqI-A1 or -B1 allele an increased incidence of the TaqI-B1 allele in current vs would be less likely to abstain, as measured by a daily diary never-smokers in the control group.18 of cigarette use, than those homozygous for the TaqI-A2 or - Association studies have traditionally been the first step in B2 allele. We also hypothesized that those with the at-risk identifying candidate genetic markers for a particular allele (TaqI-A1 or -B1) would report greater withdrawal phenotype, although these have often resulted in hetero- symptoms measured daily than those without these alleles. geneous findings across studies and samples. These studies may differ because they often rely on gross characterizations of complex phenotypes and fail to capture behavioral Results variation among smokers with varying levels of dependence. Rather than focus on the presence or absence of a globally Allele frequency and genotype defined characteristic such as smoker vs non-smoker, Table 1 contains the frequencies for the TaqI-A and -B specific behavioral phenotypes (i.e., endophenotypes) can polymorphisms. The frequency of the TaqI-B genotypes met be formulated in relation to genetic markers (i.e.,20,21). criteria for Hardy–Weinberg equilibrium, w2(df ¼ 1) ¼ 0.46, Examples of specific endophenotypes include classifying P ¼ 0.50, and are consistent with the frequencies reported in

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previous studies16; the TaqI-A genotypes also met criteria for Baseline participant characteristics Hardy–Weinberg equilibrium as described in Cinciripini et As summarized in Table 2, the sample was mainly white al.27 Owing to of the small number of B1/B1 genotypes, the participants in their 40s who smoked more than a pack a B1/B1 and B1/B2 genotypes were combined into a single day. No significant differences (P40.05) between genotype category of B1 carriers. A similar categorization was were found for any of the baseline measures in Table 2. As performed for the TaqI-A polymorphism. Smokers having excluding the small number of non-white participants the at-risk TaqI-A1 allele also had the TaqI-B1 allele 87.5% of (7.8%) did not affect gene frequency and did not alter the the time, whereas smokers homogonous for the TaqI-A2 results described below, we report results that include the allele were homogonous for the TaqI-B2 allele 78.1% of the entire sample. For the pre-quit baseline, there was no main time. effect of either TaqI-A or -B genotype on any of the withdrawal measures (P’s40.38). Table 1 Allele frequency and genotype

Allele n % DRD2 TaqI-A results The current study investigated the effects of the TaqI-A1 allele TaqI-A1 69 26 on withdrawal symptoms and abstinence rates for only the TaqI-A2 199 74 first 42 days post-quit, using daily assessments and a scale Total TaqI-A alleles 268 specifically developed to measure symptoms of nicotine withdrawal. Contrary to our hypotheses, smokers with the A1/A1 11 8 TaqI-A1 allele did not show decreased daily abstinence rates A1/A2 47 35 or decreased Wisconsin smoking withdrawal scale (WSWS) or A2/A2 76 57 Minnesota Withdrawal Symptom Checklist (MWSC) with- Total TaqI-A n 134 drawal symptoms during this period than those without the DRD2 Taq TaqI-B1 68 26 allele. We found no interactions between the I-A TaqI-B2 194 74 polymorphism and any of the fixed factors (i.e., treatment, Total TaqI-B alleles 262 abstinence, baseline cigarettes and time) for either the daily abstinence or daily withdrawal measures. B1/B1 3 2 B1/B2 62 47 DRD2 TaqI-B and abstinence B2/B2 66 50 Following standard procedures used in clinical trials Total TaqI-B n 131 research, we analyzed a more conservative intent-to-treat

Table 2 Demographics and baseline characteristics, by genotype

Characteristic Genotype

B2/B2 B1/B1/B2 A2/A2 A1/A1/A2 n (%) 66 50% 65 50% 76 57% 58 43% Female 39 59% 33 51% 43 58% 31 42%

Ethnicity (within genotype) White 60 91% 60 92% 71 93% 52 90% Other 6 9% 5 8% 5 7% 6 10%

Genotype (within ethnicity) White 60 50% 60 50% 71 58% 52 42% Other 6 55% 5 45% 5 45% 6 55% Positive depression history 14 21% 19 29% 18 23% 17 29% In drug group 29 44% 35 54% 36 47% 31 53%

Mean (s.d.) Age 46.94 10.28 44.43 10.59 46.33 10.05 45.00 11.12 BMI 25.32 5.09 25.06 5.17 25.16 5.00 25.06 5.33 CESD score 7.22 5.85 6.62 6.72 6.89 7.37 7.12 4.60 Cigarettes/day 26.95 10.81 26.31 12.10 27.42 11.70 25.60 11.09 FTND score 5.35 2.25 5.28 2.28 5.37 2.23 5.36 2.18 Years of smoking 27.08 10.20 25.89 10.70 26.26 10.41 26.48 11.28

Abbreviation: BMI, body mass index.

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sample, where missing daily diary data points were coded as F(1,3853) ¼ 3.80, Po0.05, sadness, F(1,3854) ¼ 3.87, smoking. Differential attrition was not observed, as no Po0.05, and sleep, F(1,3857) ¼ 6.51, Po0.02 and the association was found between diary non-completion rates MWSC, F(1,3858) ¼ 9.24, Po0.003. Other interactions with and the presence of either the DRD2 TaqI-A (P ¼ 0.81) or the genotype were nonsignificant. The regression lines pre- TaqI-B (P ¼ 0.13) genotypes. sented in Figure 2 indicate that those smokers with the B2/ To evaluate whether the DRD2 TaqI-B genotype predicted B2 genotype tended to report fewer symptoms as a function abstinence, we examined whether genotype and its inter- of time. No interactions between TaqI-B genotype and either actions with time and treatment predicted daily abstinence, treatment or daily abstinence were found. whereas covarying baseline smoking rate (cigarettes/day), The main effects for depression history, treatment group depression history (positive vs negative), sex and race. We and daily abstinence on WSWS and MWSC withdrawal found a significant TaqI-B Â time interaction, F(1,4754) ¼ scales are presented in Table 3 for the DRD2 TaqI-B 4.00, Po0.05, for daily abstinence rates. Figure 1 indicates subsample. Overall, these main effects indicated that being that while both the B2/B2 and the at-risk B1 (B1/B1 or B1/ in the placebo group, having a history of depression, or B2) groups were less likely to report daily abstinence over relapsing led to greater absolute WSWS and MWSC scores the course of the 6-week post-quit period, those smokers during the 42 days post-quit compared to being in the drug with the at-risk TaqI-B1 allele were less likely to report daily group, having no history of depression, or abstaining from abstinence as the study progressed. No significant gene by smoking, respectively. Neither sex, race, nor baseline treatment interaction was found (P ¼ 0.79). Venlafaxine smoking rate predicted withdrawal symptoms. produced no appreciable increase in daily abstinence rates for either the B2/B2 (OR ¼ 0.75, 95% CI ¼ 0.31, 1.82) or the at-risk B1 (OR ¼ 0.90, 95% CI ¼ 0.36, 2.24) group, compared Discussion to placebo. We found that smokers carrying the DRD2 TaqI-B1 allele (i.e., B1/B1 or B1/B2) reported significantly more symptoms DRD2 TaqI-B and withdrawal symptoms of daily smoking withdrawal over time. Regardless of The mixed model multivariate regression approach was used treatment and daily abstinence status, smokers carrying to examine the impact of DRD2 TaqI-B genotype on daily the at risk TaqI-B1 allele reported significantly greater scores withdrawal symptoms as measured by the WSWS and the on the WSWS anger, anxiety, concentration, sadness, and MWSC. Raw data from these scales were Z-score transformed sleep scales and the MWSC withdrawal measure during the across the post-quit days 0–41 observations to facilitate first 42 days of smoking cessation compared to smokers comparison between scales. The DRD2 TaqI-B genotype and homozygous for the TaqI-B2 allele (i.e., B2/B2), whose its interactions with treatment, daily abstinence and time withdrawal symptoms significantly decreased over the same were examined, whereas covarying the corresponding mean period. These effects were significant even though we baseline (À14 to À1 days pre-quit) WSWS or MWSC score, covaried baseline withdrawal symptoms, baseline smoking baseline smoking rate (cigarettes/day), daily abstinence, rate, daily abstinence, depression history, sex and race. depression history (positive vs negative), sex and race. The findings suggest that smokers with different DRD2 Significant DRD2 TaqI-B Â time interactions were found for genotypes may exhibit distinct withdrawal trajectories, with several WSWS scales, including anger, F(1,3854) ¼ 6.32, smokers homozygous for the TaqI-B2 allele reporting pro- Po0.02, anxiety, F(1,3858) ¼ 8.45, Po0.004, concentration, gressive improvement in withdrawal symptoms while smokers with the TaqI-B1 allele showing little change. The examination of smoking withdrawal dynamics has received considerable attention in recent years and has yielded important findings relating withdrawal characteristics to smoking abstinence, lapse and relapse.22,24,29,30 Using withdrawal data from two clinical trials, Piasecki et al.22 empirically identified three different profiles of withdrawal symptoms. They found that many smokers exhibited a typical withdrawal profile in which they reported a steady improvement of withdrawal symptoms (Profile 1). However, other smokers reported an atypical profile in which they experienced either an initial reduction of symptoms followed by a return to the original level of distress (Profile 2) or a fairly constant level of strong withdrawal symptoms throughout Figure 1 DRD2-B Â time interaction for daily abstinence, using the the course of a cessation attempt (Profile 3). Although the intent to treat sample, while controlling for treatment (venlafaxine vs placebo) as a between-subject fixed effect and the covariates baseline symptom trajectories reported by smokers homozygous for smoking rate (cigarettes/day), depression history (positive vs negative), the TaqI-B2 allele is suggestive of a typical withdrawal profile, sex and race. The figure depicts abstinence probabilities that were the trajectories reported by smokers with the TaqI-B1 allele derived from predicted abstinence log odds. resembles that of an atypical, unremitting profile.

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Figure 2 DRD2-B Â time interactions for the WSWS scales (a) anger, (b) anxiety, (c) concentration, (d) sadness and (e) sleep, and the (f) MWSC, while controlling for daily abstinence (smoking vs abstinent) and treatment (venlafaxine vs placebo) as between-subject fixed effects and the covariates mean baseline WSWS score, baseline smoking rate (cigarettes/day), depression history (positive vs negative), sex and race.

Table 3 The main effects for treatment, daily abstinence, and depression history on WSWS scores and the WSC total score

Treatment Daily abstinence Depression history

Placebo Venlafaxine Smoking Abstinent Negative Positive

WSWS Anger 0.63 (0.20) 0.37 (0.20)* 0.62 (0.19) 0.38 (0.19)*** 0.34 (0.19) 0.66 (0.21)* Anxiety 0.44 (0.21) 0.22 (0.21) 1.12 (0.66) 0.48 (0.06)*** 0.21 (0.20) 0.45 (0.22) Concentration 0.43 (0.21) 0.25 (0.21) 0.75 (0.66) 0.49 (0.06)* 0.03 (0.20) 0.64 (0.22)*** Craving 0.47 (0.21) 0.50 (0.21) 0.66 (0.20) 0.31 (0.20)*** 0.30 (0.21) 0.67 (0.22)** Hunger 0.31 (0.21) 0.10 (0.22) 0.20 (0.21) 0.22 (0.21) 0.18 (0.21) 0.24 (0.23) Sadness 0.49 (0.18) 0.32 (0.18) 0.48 (0.17) 0.32 (0.17)** 0.26 (0.17) 0.54 (0.18)* Sleep 0.09 (0.19) 0.01 (0.19) 0.08 (0.18) 0.02 (0.18) À0.01 (0.19) 0.11 (0.20) MWSC 0.45 (0.21) 0.19 (0.21)* 0.38 (0.20) 0.26 (0.20)* 0.17 (0.20) 0.47 (0.22)*

Abbreviations: WSWS, Wisconsin smoking withdrawal scale; MWSC, Minnesota withdrawal symptom checklist. Z-score mean (s.d.). *Po0.05, **Po0.01, ***Po0.0001.

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To our knowledge, the current study is the first demonstra- past 7 days) used in the previous study may have captured tion of a possible association between the DRD2 TaqI-B more stable periods of abstinence than the daily, short-term polymorphism and withdrawal trajectories. However, because fluctuations in abstinence captured by the current study. the biological significance of the TaqI-B1 allele has not been Similarly, the PANAS questionnaire used in our previous fully explored, it is unclear what mechanisms may account study asks about mood during the ‘past week,’ whereas the for the relationship between genotype and withdrawal WSWS instructions ask about symptoms during the ‘last trajectory. Nevertheless, these results, if validated, may help 24 h.’ Although there is some similar content between the to explain earlier findings regarding the relationship between scales, the PANAS scales were designed to measure two the TaqI-B polymorphism and smoking. For instant, Spitz partially orthogonal affective dimensions,31 whereas the et al.16 found that smokers with the TaqI-B1 allele made fewer seven WSWS scales were designed to capture nicotine lifetime attempts to quit than those without the allele. If withdrawal symptoms and roughly correspond to six of smokers with the TaqI-B1 allele were more vulnerable to the eight symptoms listed in the Diagnostic and Statistical experience unremitting withdrawal symptoms during an Manual (DSM)-IV.32 initial attempt, they might be discouraged by their quit The lack of any significant gene by treatment group experience and became reluctant, should they relapse, to interaction, particularly for abstinence rate, is not surprising make new attempts in the future. Hence, B1 smokers may for several reasons. First, we found no gene (TaqI-A) by make fewer lifetime attempts to quit smoking. treatment group interaction in our previous study.27 Second, We also found a significant relationship between the TaqI- the sample size in this study was small, making it difficult to B polymorphism and daily smoking abstinence, with detect significant differences for the 2 Â 2 between-subjects smokers carrying the TaqI-B1 allele being increasingly less interaction. Third, while venlafaxine may influence dopa- likely to be abstinent on a given day than those carrying the mine at the D2 receptor site by inhibiting reuptake of homozygous TaqI-B2 allele. Given the association between serotonin (and norepinephrine), the magnitude and dura- the TaqI-B polymorphism and withdrawal profiles, the tion of this effect on the D2 receptor have not been reduced abstinence rate for TaqI-B1 smokers is consistent conclusively determined. Perhaps a marker for serotonin with previous findings that smokers with an atypical neuroregulation, such as the SERT (5-HTTPLR), may be a withdrawal trajectory are less likely to abstain from smok- better candidate for study with venlafaxine than the markers ing.22 That is, since smokers with the TaqI-B1 allele may not of dopaminergic function we used in this study. experience any improvement or relief of the negative This paper is limited by its single gene focus and the small withdrawal symptoms, they may become less successful in sample size. Smoking is likely to be polygenetically deter- achieving abstinence when they make a quit attempt. Taken mined, and isolation of a single gene provides only a limited together, the results suggest that, despite therapy (nicotine amount of information. However, the ability to test for patch, counseling and pharmacotherapy), at-risk B1 smokers multiple genes requires a significantly larger sample than experience a greater level of withdrawal symptoms over time that used here. Future research may partly overcome sample and are less likely to abstain from smoking. size issues by using within-subject designs and including Unlike our earlier findings,27 we failed to find a relation- other physiological (e.g., electroencephalographic) or cog- ship between TaqI-A genotype and either short-term daily nitive measures of nicotine withdrawal. abstinence or daily withdrawal symptoms. However, owing In summary, we showed that smokers with the TaqI-B1 to the different time periods sampled (42 days post-quit vs 1 allele are less likely to abstain from cigarettes during year post-quit) and the different measures used (WSWS vs. smoking cessation therapy. Given the strong LD between PANAS) between the two studies, the results are not the TaqI-A and -B polymorphisms, the current findings may necessarily contradictory. In our earlier study, we measured be viewed as complimentary to our earlier findings that abstinence and mood during weeks 1–6, 10, 18, 26, and 52 TaqI-A1 smokers may have a more difficult time quitting post-quit,27 using seven-day point prevalence, whereas in smoking.27 We also found that when TaqI-B1 smokers make the current study, measurement was taken daily for only the a quit attempt, they may be more likely to experience an first 42 days (6 weeks) post-quit. According to our earlier atypical withdrawal profile characterized by unremitting findings, the majority of the weekly abstinence assessments symptoms over time. The possible association between TaqI- during the first 6 weeks of the post-quit period showed only B genotypes and withdrawal trajectories is important and a small, possibly nonsignificant difference in abstinence rate warrants further examination. If confirmed, this association between those with and without the TaqI-A1 allele. The between genotypes and withdrawal profiles may have largest difference occurred at week 10 when over 55% of implications on the development of genotype-specific smokers homozygous for the TaqI-A2 allele remained treatment strategies. abstinent, compared to less than 40% of those carrying the TaqI-A1 allele. If smokers with and without the TaqI-A1 Materials and methods allele did not differ in their ability to quit until later in their post-quit period, our current analysis, given its short-term Participants focus, would not necessarily reveal a significant difference in Participants in this study were current smokers recruited for abstinence rates between the two groups. Furthermore, the a double-blind placebo controlled clinical trial of the longer duration and broader sampling points (i.e., over the antidepressant venlafaxine. Characteristics of the full

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sample (N ¼ 166) as well as treatment outcome data related administered interview that assesses the DSM-IV Axis I to the effects of venlafaxine on smoking cessation have been disorders.37 The affective disorders section of the SCID was described in another report.33 The current paper involves an administered before randomization to assess current and assessment of the effects of genotype on smoking cessation past history of major depressive disorder. and smoking withdrawal symptoms, assessed daily, among the participants from the larger sample for which DRD2 Baseline nicotine dependence and smoking behaviors. The TaqI-A (n ¼ 134) and DRD2 TaqI-B (n ¼ 131) genetic informa- Fagerstro¨m test of nicotine dependence (FTND) is a 6-item tion was available. All smokers were volunteers recruited measure used to assess nicotine dependence.38 Baseline daily from the Houston, Texas, metropolitan area. To be included smoking rate was also collected by this instrument. It was in this trial, smokers were required to smoke at least 10 administered before randomization. cigarettes per day at baseline and to be 18–75 years of age. Smokers participating in any other smoking cessation Abstinence status. Participants recorded their daily cigarette treatment, currently taking other psychoactive medication intake each night in a diary before going to bed for 56 or those having any uncontrolled systemic illness, contra- consecutive days, 14 pre- and 42 post-quit. The smokers indications for taking venlafaxine or the nicotine patch, were instructed to fill out the diaries before going to bed current substance abuse or other psychiatric disorders were each evening, recording their cigarette use over the past excluded. This research was approved by the University of 24 h, counting even a puff as a cigarette. The smoking diaries Texas MD Anderson Cancer Center IRB. were collected by the staff during the weekly sessions that occurred during this period. Daily abstinence was calculated Procedure based on this smoking diary, with zero cigarettes defined as Participant screening, treatment and assessment procedures abstinent and all other responses, including missing, have been detailed in earlier reports27,33 but will be briefly defined as non-abstinent. Seven-day point prevalence summarized here. Smokers who met all inclusion criteria abstinence was verified before weekly treatment sessions were stratified by gender and history of depression and using expired CO (p10 p.p.m.). Measures were obtained on randomly assigned to receive 20 weeks of either active four occasions during the post-quit diary period, including venlafaxine (Effexor; Wyeth Pharmaceuticals, Madison, NJ, the quit date and during post-quit weeks 1, 3, and 6. When USA) or placebo. All participants also received brief smoking CO abstinence was compared to the daily smoking diary cessation counseling and nicotine replacement therapy entry for each of the corresponding days, we found that a (Prostep Nicotine Patch; Lederle Laboratories, Pearl River, diary-reported claim of abstinence did not correspond to CO NY, USA). Antidepressant therapy began 2 weeks before 5.86% of the time. As leaving out these observations or quitting, at an initial dose of 75 mg/day (37.5 mg/day b.i.d.). recoding them as smoking days did not alter the abstinence The dose was titrated for both active and placebo groups up results presented below, we decided to retain them in our to 150 mg/day in the week just before quitting. In each analyses without alteration. Weekly mood and abstinence subsequent week, the dose was raised for both groups in assessments, collected before the treatment session, were 37.5 mg increments up to a maximum 225 mg/day. An conducted at weeks 1–6, 10, 18, 26, and 52 post-quit and are increase in dosage was recommended for either group, if reported elsewhere.27,33 participants reported no side effects on their current dose and were either unable to remain abstinent and/or reported Wisconsin smoking withdrawal scale. The WSWS is a self- significant feelings of negative affect or other symptoms of report measure of nicotine withdrawal. It consists of the nicotine withdrawal. Two weeks before the end of treatment following seven subscales: anger, anxiety, concentration, (i.e., at 16 weeks post-quit), participants began tapering the craving, hunger, sadness and sleep. The WSWS mood and medication, decreasing the dose by 37.5 mg every 2–3 days. craving scales demonstrate increases as a function of The medication cycle was completed 18 weeks after quit- nicotine abstinence.32 The WSWS was administered for 56 ting. All participants also used the nicotine patch (Prostep, consecutive days along with the smoking diary. Participants Lederle Laboratories, Pearl River, NY; 22 mg) for 6 weeks, were given a packet of these questionnaires at each of the beginning on their quit-date. weekly sessions during this period and instructed to fill A smoking cessation counseling treatment was provided them out before going to bed each evening, rating their concurrent with antidepressant therapy. Following the symptoms over the past 24 h. baseline visit, all smokers received six in-person and three telephone counseling sessions over the next 9 weeks. Minnesota withdrawal symptom checklist. The MWSC consists The content of the counseling sessions was a cognitive- of a 9-item list of tobacco withdrawal symptoms (i.e., desire behavioral approach similar to that used in our previous to smoke (craving), anger/irritability/frustration, anxiety/ studies (e.g.,34,35), and applied a standard coping skills nervousness, difficulty concentrating, impatience/ training approach36 to cessation and relapse prevention. restlessness, hunger, awakening at night and depression).39 Items were rated on a 0–4 scale (none-severe) of symptom Assessments severity and are summed to create a withdrawal score. Scheduled clinical interview for DSM-IV. The scheduled clinical Participants completed the MWSC on a daily basis for 56 interview for DSM-IV (SCID) is a widely used clinician days as described above for the WSWS.

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Genotyping assay. DNA was extracted from whole-blood continuous dependent variable, genotype (B1/B1 or B1/B2 samples for polymerase chain reaction (PCR) analyses using vs B2/B2), daily abstinence (smoking vs abstinent) and standard phenol extraction methods. Genotyping for the treatment (venlafaxine vs placebo) as between-subject fixed DRD2 TaqI-A and -B polymorphisms followed the PCR effects, and time (post-quit days 0–41) as the repeated method reported in one of our previous studies.16 measures factor. Covariates included the corresponding mean baseline (À14 to À1 days pre-quit) WSWS score, Statistical analysis baseline smoking rate (cigarettes/day), daily abstinence, The primary analytic strategy used a mixed model approach depression history (positive vs negative), sex and race. to examine the effects of genotype on repeated measures of Subject was modeled as a random effect. The residual error abstinence and daily withdrawal. The mixed model ap- variances over time were modeled as a heteroscedastic proach provides a generalization to the classic linear random effect, using an autoregressive function, as a regression model, using likelihood functions instead of least likelihood ratio test indicated that it improved model fit squares to estimate effects.40 The mixed model approach is over the homoscedastic model. ideally suited for analysis of repeated measures data (i.e., The basic model for this analysis included genotype, growth curve modeling) in that it allows for estimates of the treatment, time and their 2-way and 3-way interaction correlation structure of the residuals and efficiently handles terms. Significant main effects and interactions related to unbalanced designs and missing data, without excluding genotype were further explored using a least-squares mean participants or imputing values.41,42 We used PROC MIXED procedure to contrast pairwise differences among selected (SAS Version 9.1, SAS Institute Inc Cary, NC, USA) to means participating in the effect. Each WSWS scale was estimate and test the models involving the measures of analyzed separately. Similar analyses were performed on the withdrawal. When abstinence was the dependent variable, MWSC scores. Nonlinear functions of time (e.g., quadratic we used SAS PROC GLMMIX, an adaptation of PROC MIXED and cubic functions) were modeled but were found to be that is suitable for the evaluation of a dichotomous outcome nonsignificant and are not reported here. Separate analyses variable (abstinence). To facilitate comparisons between were conducted to evaluate the effects of the DRD2 TaqI-A scales, WSWS scale and MWSC scores were standardized (A1/A1 or A1/A2 vs A2/A2) and –B (B1/B1 or B1/B2 vs B2/B2) using a Z-scores transformation across all observations. polymorphisms.

Effect of genotype on abstinence. The effect of genotype on Analysis of demographic and baseline variables abstinence was evaluated using a model that included daily Student’s t-tests (continuous variables) and w2-tests (discrete abstinence as the binary-dependent variable, genotype and variables) were used to examine differences on baseline treatment (venlafaxine vs placebo) as between-subject fixed demographic characteristics and mean WSWS and MWSC effects, and time (post-quit days 0–41) as the repeated scale scores during the pre-quit baseline, by genotype. measures factor. Covariates included baseline smoking rate (cigarettes/day), depression history (positive vs negative), Acknowledgments sex and race. Subject was modeled as a random effect. Separate models were tested to evaluate the effects of the Support for this research was provided by grants from the MD DRD2 TaqI-A (A1/A1/A2 vs A2/A2) and -B (B1/B1/B2 vs B2/ Anderson Cancer Center (PRS), the National Cancer Institute B2) polymorphisms. The analyses for the effects of (SPORE P50CA70907), and the National Institute on Drug Abuse venlafaxine treatment (without regard to genotype) on (R01DA1182-01) to Paul M Cinciripini and MD Anderson Education smoking cessation have been previously reported for the Program in Cancer Prevention Postdoctoral Fellowship Grants (R25 CA57730) to Jason D Robinson, Cho Y Lam and Jennifer A Minnix. larger sample and are not duplicated here.33 Study medication and nicotine patches were provided by Wyeth- A standard approach to model building was followed that Ayerst Laboratories. Portions of this research were presented at the included an initial assessment of main effects due to annual meeting of the Society for Research on Nicotine and genotype, time (week of assessment) and treatment, fol- Tobacco, Orlando, Florida, February 2006. lowed by evaluation of the 2-way and 3-way interaction terms. Main effects were evaluated on the first iteration (e.g., genotype and time controlling for treatment) followed by Duality of Interest the 2-way and 3-way interactions. F-statistics for all None declared. significant effects are reported, and for comparisons involving genotype and daily abstinence, odds ratios and References confidence intervals (95% CI’s) are also included. 1 Wise RA, Rompre PP. Brain dopamine and reward. Ann Rev Psychol Effects of genotype on daily withdrawal 1989; 40: 191–225. A multivariate regression mixed models approach was used 2 Blum K, Noble EP, Sheridan PJ, Finley O, Montgomery A, Ritchie T et al. to evaluate the effects of genotype on withdrawal symptoms Association of the A1 allele of the D2 dopamine receptor gene with during smoking cessation.43 The effects of genotype on severe alcoholism. Alcohol 1991; 8: 409–416. 3 Comings DE, Comings BG, Muhlemann D. The dopamine D2 receptor withdrawal measures were simultaneously evaluated using a locus as a modifying gene in neuropsychiatric disorders. JAMA 1991; model that included WSWS and MWSC scale scores as the 266: 1793–1800.

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