Hum Genet (2012) 131:857–876

DOI 10.1007/s00439-012-1143-9

REVIEW PAPER

Pharmacogenetics of smoking cessation: role of nicotine target and metabolism genes

Allison B. Gold · Caryn Lerman

Received: 19 July 2011 / Accepted: 19 January 2012 / Published online: 31 January 2012© Springer-Verlag 2012

Abstract Many smokers attempt to quit smoking but feware successful in the long term. The heritability of nicotineaddiction and smoking relapse have been documented, andresearch is focused on identifying speciWc genetic inXu-ences on the ability to quit smoking and response to speciWcmedications. Research in genetically modiWed cell lines andmice has identiWed nicotine acetylcholine receptor subtypesthat mediate the pharmacological and behavioral eVects ofnicotine sensitivity and withdrawal. Human genetic associ-ation studies have identiWed single nucleotide polymor-phisms (SNPs) in genes encoding nicotine acetylcholinereceptor subunits and nicotine metabolizing enzymes thatinXuence smoking cessation phenotypes. There is initialpromising evidence for a role in smoking cessation forSNPs in the �2 and �5/�3/�4 nAChR subunit genes; how-ever, eVects are small and not consistently replicated. Thereare reproducible and clinically signiWcant associations ofgenotypic and phenotypic measures of CYP2A6 enzymeactivity and nicotine metabolic rate with smoking cessationas well as response to nicotine replacement therapies andbupropion. Prospective clinical trials to identify associa-tions of genetic variants and gene–gene interactions onsmoking cessation are needed to generate the evidence basefor both medication development and targeted therapyapproaches based on genotype.

Introduction

Cigarette smoking continues to be the leading cause of pre-ventable deaths in the United States, accounting for 1 in 5deaths every year (CDC 2010). In 2009, 20.6% of USadults were current cigarette smokers (CDC 2010). Manysmokers are aware of the harmful eVects of smoking andabout 70% of smokers report wanting to quit completely(CDC 2010); however, only 4–7% of smokers are able toquit smoking without medication or formal treatment (Fioreet al. 2008). Yet, recent data also suggest that only about22% of smokers who attempted to quit used cessation med-ication (Cokkinides et al. 2005; Fiore et al. 2008). Amongthose smokers who do use pharmacotherapy, fewer thanone-third are able to stay smoke-free for at least 6 months(Fiore et al. 2008).

FDA-approved approaches for smoking cessationinclude nicotine replacement therapy (NRT), bupropion,and varenicline. NRTs include: transdermal patch, nicotinegum, inhalers, lozenges, and nasal spray. Meta-analyses ofclinical trial eYcacy data indicate that NRTs double theodds of successful quitting for at least 6 months comparedto placebo (Moore et al. 2009; Stead et al. 2008). Bupro-pion, an anti-depressant medication, is a norepinephrine-dopamine reuptake inhibitor (Learned-Coughlin et al.2003; Terry and Katz 1997) as well as a nicotinic acetyl-choline receptor antagonist (Slemmer et al. 2000). Bupro-pion has similar eYcacy to NRT and data from a meta-analysis revealed that bupropion doubles abstinence ratesfor at least 6 months compared to placebo (Hughes et al.2007). Varenicline, an �4�2* nicotinic acetylcholine recep-tor partial agonist, was developed to mimic the eVects ofnicotine by stimulating a moderate amount of dopaminerelease to prevent craving and withdrawal, while reducingthe reinforcing eVects of nicotine from cigarette smoking

A. B. Gold · C. Lerman (&)Center for Interdisciplinary Research on Nicotine Addiction, University of Pennsylvania, 3535 Market Street, Suite 4100, Philadelphia, PA 19104, USAe-mail: clerman@upenn.edu

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(Rollema et al. 2007). Meta-analysis data indicate thatvarenicline improves the odds of abstinence two to three-fold, relative to placebo (Cahill et al. 2011). Varenicline ismore eVective for smoking cessation than NRTs (Aubinet al. 2008; Wu et al. 2006b) or bupropion (Gonzales et al.2006). Yet, even with the most eYcacious pharmacothera-pies, the majority of smokers are unable to quit and main-tain abstinence (Schnoll and Lerman 2006).

Quitting smoking is very diYcult due to nicotine’saddictive properties. There is substantial variability in nico-tine addiction and relapse rates which is attributable, inpart, to heritable inXuences. Twin studies estimate that her-itable inXuences account for roughly 46–84% of the vari-ability in smoking initiation and smoking persistence and asmuch as 75% of the variability in nicotine dependence (Sul-livan and Kendler 1999; Vink et al. 2005). Other studieshave shown that heritable factors account for 29–53% ofthe variability in withdrawal symptoms and 51–54% of thevariance in quitting success (Lessov et al. 2004; Pergadiaet al. 2006; Xian et al. 2003, 2005).

Research has focused on identifying speciWc genetic inXu-ences on the ability to quit smoking. Such research can iden-tify novel molecular targets for medication development.Further, pharmacogenomics research focused on geneticinXuences on quitting success with speciWc medicationscould potentially lead to the development of targeted therapyapproaches that are more eYcacious. This review focuses onthe role in smoking cessation of genes coding for neuronalnicotinic acetylcholine receptors (nAChRs) and nicotinemetabolizing enzymes, the initial molecular targets for nico-tine’s eVects. First, we provide a brief overview of the neuro-biology of nicotine’s eVects in the brain, phenotyping ofsmoking cessation behaviors, and approaches for geneticstudies. Next, we review the biological plausibility and avail-able evidence for the association of speciWc cholinergic geneswith smoking cessation and therapeutic response. Lastly, wereview nicotine pharmacokinetics and the role of genetic var-iability in nicotine metabolism enzymes.

Neurobiology of nicotine

Nicotinic acetylcholine receptors

Neuronal nAChRs are heterogeneous cationic channels thatare widely distributed in the nervous system and non-neu-ronal tissues. The subunit composition and biophysicalproperties of nAChRs depend on the subtype and the areaof the brain in which they are found (Gaimarri et al. 2007;Gotti et al. 2007; Zoli et al. 2002). Nicotine diVuses readilyinto the brain and binds to ligand-gated nAChRs. After nic-otine binds to the nAChR, the channel opens and allowsentry of cations (Na+, Ca2+).

nAChRs are homopentameric or heteropentameric unitsthat are localized primarily at presynaptic or preterminalsites, where they modulate neurotransmitter release. Theyare also found on cell bodies and dendrites, where theymediate postsynaptic eVects. nAChRs have an importantrole in regulating transmitter release, cell excitability, andneuronal integration (Gotti and Clementi 2004; Hogg andBertrand 2004; Hogg et al. 2003).

nAChRs form a family of subtypes formed by Wve sub-units (Gotti and Clementi 2004; Gotti et al. 2006). The sub-units are encoded by nine (�2–�10) and three (�2–�4)subunit genes and the two major subfamilies are classiWedas either �Bgtx-sensitive or �Bgtx-insensitive. �Bgtx-sensi-tive nAChRs are mostly homopentameric and the �Bgtx-insensitive nAChRs are only heteropentameric, consistingof both � and � subunits. �Bgtx-insensitive nAChRs aremostly comprised of the �4�2* subtype and may include aprimary �6 or accessory �5 or �3 subunits. �5 and �3 donot form functional channels alone, but combined withother �-� combinations, they contribute to receptor tar-geting and localization in neuronal plasma membranedomains, and modify other receptor properties (Gotti et al.2006, 2007).

Neurotransmitter pathways and nicotine dependence

There is a well-supported role of nicotine as the pharmaco-logical agent that maintains dependence. The composition,anatomical localization, and physiological properties ofnAChRs contribute to various physiological and behavioraldiVerences (Govind et al. 2009; Marks et al. 1999).Nicotine and other nicotine agonists will elicit diVerentresponses of these receptors depending on the subtype itacts on (Brioni et al. 1997; Changeux 2010; Govind et al.2009). Nicotine stimulates release of dopamine in the mes-oaccumbens reward pathway (Champtiaux et al. 2003) anddependence is mediated, in part, by activation of speciWcsubtypes of nAChRs on dopamine neurons (Court et al.1998; Picciotto and Corrigall 2002). Dopamine release sig-nals a pleasurable experience and is a key component of thereinforcing eVects of nicotine and other drugs of abuse(Changeux 2010; Nestler 2005). Via nAChRs, nicotineactivates both dopamine and GABA neurons of the ventraltegmental area (VTA), resulting in dopamine release in thenucleus accumbens and contributing to the rewarding andmotivational eVects of nicotine (Corrigall et al. 1994;Laviolette and van der Kooy 2004; Mineur and Picciotto2008; Nisell et al. 1994). Further, molecular alterations incentral dopamine systems contribute to craving and modu-late the aversive eVects of nicotine withdrawal (Changeux2010; Laviolette and van der Kooy 2004; Mansvelder et al.2002; Mansvelder and McGehee 2002). While a singlecigarette smoked in the morning can saturate and desensitize

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roughly 80–90% of nAChRs for several hours, smokersmay continue to smoke throughout the day to avoid havingfree unbound receptors (Brody et al. 2006). The free, non-desensitized receptors may be responsible for cravings andcontinued smoking acts on these receptors to provide posi-tive reinforcement and sustained dopamine release to pre-vent withdrawal symptoms (Balfour 2004; Brody et al.2006).

Smoking cessation phenotypes

A well-deWned phenotype is crucial to the success of genet-ics research. Smoking cessation, the focus of this review, issimply deWned as the ability to maintain long-term absti-nence (Silagy et al. 2004). EVective cessation occurs byovercoming diVerent milestones to achieve persistent absti-nence, including a period of initial abstinence (24 h withoutsmoking), avoiding lapse (Wrst use after abstinence), andultimately avoiding relapse (proceeding from Wrst use afterabstinence back to regular daily use) (ShiVman et al. 2006).The inability to maintain abstinence is related to the sever-ity of withdrawal symptoms as well as genetic factors andinXuenced, in part, by psychological factors and contextualcues (Dawkins et al. 2009; Pomerleau et al. 2005; Xu et al.2008).

Nicotine dependence is associated with decreased rates ofinitial cessation and a higher risk of transitioning from lapseto relapse (Japuntich et al. 2011). Although pre-quit level ofnicotine dependence is related to relapse, it is not a consistentpredictor of quit attempts and smoking cessation, and thatassociation is highly dependent on which measurement isused (Etter 2005; Japuntich et al. 2011; Piper et al. 2006).For example, the widely used Fagerstrom Test for NicotineDependence (FTND) (Fagerstrom 1978; Heatherton et al.1991) does not consistently and robustly predict withdrawalsymptoms or quitting success (Borrelli et al. 2001; Piperet al. 2004; Silagy et al. 1994). The same is true for measuresof smoking heaviness predicting cessation (Fidler et al.2011). Together with evidence suggesting that nicotinedependence and smoking cessation each have some uniquegenetic contributions (Li et al. 2003; Xian et al. 2003), thesedata underscore the point that nicotine dependence andsmoking cessation represent distinct phenotypes. While somegenetic inXuences may be shared by these phenotypes, it can-not be assumed that genetic associations with nicotine depen-dence will translate to smoking cessation or vice versa.

Given the limited variability in successful quitting pre-dicted by smoking behavior and nicotine dependence,genetics studies are focusing increasingly on the smokingcessation phenotype. Retrospective cross-sectional mea-sures of cessation, comparing former smokers to currentsmokers, have been used with some success (Breitling et al.

2009a). A more reWned phenotype for smoking cessation isbased on ability to quit among treatment-seeking smokersfollowed prospectively in longitudinal observational stud-ies or clinical trials (Hughes et al. 2010). While retrospec-tive measures tend to be less precise than longitudinalmeasures, they can be collected more eYciently on largerpopulations.

Genetic research approaches

Nicotine dependence involves complex interactionsbetween multiple genes and environmental factors (Li2008). There are a wide range of research approaches tounderstand the genetic inXuences on nicotine dependence,ranging from transgenic animal models to population stud-ies. Pharmacological and molecular biology studies includein vitro receptor genetic mutations, as well as knock-out(KO) and point mutation knock-in animal models (Green-baum and Lerer 2009). These studies evaluate the pharma-cological and biochemical properties of speciWc genemanipulations to determine their contribution to nicotinedependence and/or nicotine withdrawal.

EVorts to discover chromosomal regions, and ultimatelygenes associated with a speciWc phenotype or disease likenicotine dependence, can utilize linkage analysis. Geneslocated near each other on the same chromosome will likelybe inherited together. Linkage analysis assesses the presenceof a phenotype in large, high-risk families, with the goal ofdetermining the location of the gene responsible for the phe-notype in relation to a known genetic marker. Linkage analy-sis studies can be very useful in identifying the location of asusceptibility disease gene, but there are several limitations.Largely, the results of linkage analyses of nicotine depen-dence have not been consistently replicated by more than twoindependent studies and there is a great deal of variability inthe measures that are used to assess nicotine dependence(Han et al. 2010; Li 2008). However, of the 13 regions on 11chromosomes that have been identiWed in nicotine depen-dence linkage analyses, regions on chromosomes 9, 10, 11,and 17 have been detected by the greatest number of studies;chromosomes 1, 5, 10, 11, 12, 16, 20, and 22 have also shownsome linkage signals (Li 2008). Overall, linkage analysishas identiWed several diVerent loci that are linked to nicotineaddiction and has been a starting point for identiWcation ofgenes that may inXuence nicotine dependence phenotypes(Han et al. 2010). The results of linkage analyses have, insome cases, led to candidate gene association studies ofnicotine dependence phenotypes.

Candidate gene association studies investigate singlenucleotide polymorphisms (SNPs) and other variants (e.g.,variable number tandem repeats) in genes suggested inlinkage analysis (Hernandez and Blazer 2006), and/or those

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with strong biological plausibility. These studies can com-pare unrelated groups (case–control) or members of familyunits (family-based) and may be useful in identifying genesthat play a minor role in nicotine dependence (Portugal andGould 2008; Risch and Merikangas 1996). Until recently,most studies have focused on a few select candidate generegions and it is possible that many of the loci that inXuencesmoking behavior may lie outside those previously studiedregions (Caporaso et al. 2009). Other limitations includefalse positive results (Sullivan et al. 2001), small samplesizes, diVerences in measures of smoking behavior, anddiVerences in ethnic and environmental backgrounds(Caporaso et al. 2009; Portugal and Gould 2008).

Pathway-based association studies are a useful tool tostudy the associations of multiple genes within a neurobio-logical pathway or system that has strong biological plausi-bility for association with a phenotype. If amply powered,such studies can account for multiple variants in interactingor related genes and may help to validate the role of SNPsor genes in the etiology of a nicotine dependence phenotype(Conti et al. 2008; Saccone et al. 2007; Wang et al. 2007).Wang and Li identiWed several pathways (calcium, dopa-mine receptor, and cAMP-mediated signaling) containinggenes associated with smoking behavior phenotypes,including initiation, dependence, and cessation. There isgenetic overlap in the main pathways identiWed for smok-ing behavior phenotypes, but the genes associated witheach phenotype within a pathway are diVerent (Wang andLi 2010). Many pathways important in addiction tococaine, alcohol, opioids overlap with those important innicotine addiction or smoking cessation, including gapjunction, LTP, and MAPK signaling (Li 2008). These Wnd-ings suggest that some but not all of the mechanisms under-lying nicotine dependence are common with othersubstances in addiction. There are likely many genetic tar-gets in other neuronal pathways that aVect smoking behav-ior (Munafo and Johnstone 2008); therefore a pathway-based approach may be restricted by the large number ofgenes that contribute to other disease phenotypes and thosefor which a biological function is still unknown (Elberset al. 2009; Munafo and Johnstone 2008; Wei et al. 2010).A pathway-based approach may help in the interpretationof genome-wide association data and lead to identiWcationof novel mechanisms and genes contributing to smokingbehavior (Wang et al. 2007; Wang and Li 2010).

In contrast to candidate gene and pathway-basedapproaches, genome-wide association studies (GWAS) arehypothesis-free and seek to identify loci for novel suscepti-bility genes. Such approaches have discovered genetic vari-ants in the �5/�3/�4 nAChR gene cluster as associated withnicotine dependence and heaviness of smoking (Berrettiniet al. 2008; Bierut et al. 2008; Thorgeirsson and Stefansson2008). These genes have attracted a signiWcant amount of

attention and the associations with smoking heaviness arerobust and important. However, studies attempting to con-Wrm associations of these SNPs with retrospective and pro-spective smoking cessation have yielded mixed results(Breitling et al. 2009a; Greenbaum and Lerer 2009; Rayet al. 2010). While GWAS is a very useful approach,genetic variants identiWed via GWAS account for a verysmall proportion of the variance in smoking behavior(<5%), and thus, the clinical utility of these results is notyet clear (Furberg et al. 2010). To date, few GWAS studiesof smoking cessation have been conducted (Uhl et al. 2007)in part due to the need for larger sample sizes than may befeasible to acquire from clinical trials. Below, we reviewgenetic studies of the smoking cessation phenotypes with afocus on the role of genes encoding nAChR subunits andthose implicated in nicotine metabolism.

Nicotinic acetylcholine receptor (nAChR) subunits

CHRNA5-CHRNA3-CHRNB4

The 15q24 region on chromosome 15 includes the genes(CHRNA5-CHRNA3-CHRNB4) that encode the �5, �3,and �4 nAChR subunits, which are in strong linkage dis-equilibrium (LD) with each other. Several recent GWASand pathway-based studies have identiWed SNPs in theCHRNA5-CHRNA3-CHRNB4 gene cluster as associatedwith heaviness of smoking and/or nicotine dependence(Berrettini et al. 2008; Bierut et al. 2007; Caporaso et al.2009; Saccone et al. 2007; TAG 2010; Thorgeirsson andStefansson 2008).

The �5 subunit does not form functional receptors whenexpressed alone, or co-expressed with a � subunit (Ramirez-Latorre et al. 1996), but when combined with an additional �subunit and either a �2 or �4 subunit it alters several pharma-cological properties of the receptor in response to nicotine(Gerzanich et al. 1998; Tapia et al. 2007). The �5 variantD398N, in the region encoding the �5, �3, and �4 nAChRsubunits, causes a change in the amino acid sequence and hasbeen shown to reduce the response to epibatidine of(�4�2)2�5 nAChRs, a subtype important in mediating theeVects of nicotine (Bierut et al. 2008). Kuryatov and othersexpressed this variant in X. laevis oocytes and observed thatthe �5 Asn 398 risk variant in (�4�2)2�5 nAChRs had lowerCa2+ permeability and greater short-term desensitization thanthe �5 Asp 398 variant when exposed to nicotine. The resultssuggest that this variation in the �5 subtype might reducenicotinic signaling, potentially resulting in heavier smoking(Kuryatov et al. 2011).

These in vitro studies are being extended in transgenicmouse model studies to clarify the behavioral mechanismsunderlying these associations. For example, �5 knock-out

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mice respond for higher doses of intravenously self-admin-istered nicotine infusions compared to wild-type mice, sug-gesting that the �5 subunit may be important in theexperience of toxicity associated with high nicotine doses(Fowler et al. 2011). In another study, �5 KO mice wereimplanted with osmotic mini-pumps containing nicotine for14 days, followed by precipitated withdrawal with meca-mylamine (Jackson et al. 2008). The �5 KO mice showed areduction in somatic signs only, suggesting that this sub-type is involved with the physical measures of withdrawalsymptoms but not aVective withdrawal signs (Jackson et al.2008). Salas and colleagues examined �5 KO mice chroni-cally treated with nicotine by osmotic mini-pumps or intheir drinking water, followed by mecamylamine precipi-tated withdrawal. Consistent with the results of Jackson andcolleagues, the lack of the �5 subunit abolished the somaticsymptoms of withdrawal to levels exhibited by saline-treated mice, suggesting that the �5 subunit mediates somephysical symptoms of nicotine withdrawal (Jackson et al.2008; Salas et al. 2009). However, because physical symp-toms are a less signiWcant component of the withdrawalsyndrome in human smokers compared to aVective andcognitive signs, the relevance in humans must be evaluated.

The �3 and �4 subunits are most commonly expressedtogether in the same receptor subtype, with or without �5,in the peripheral nervous system (Wang et al. 1996). The�4 subunit is also found co-expressed with other � subunitsin the central nervous system, with high expression in themedial habenula and interpeduncular nucleus (Salas et al.2003). �3 and �4 containing receptors are involved in nico-tine-induced seizures (Salas et al. 2004a) and �4 receptorsmay be important in anxiety-related behaviors (Salas et al.2003). Oocytes containing �4 subunit polymorphismsshowed varying diVerences in the EC50 for ACh activation(Liang et al. 2005). One variant, present in only the Cauca-sian sample, had a lower sensitivity to ACh, increased AChEC50, and the slowest onset of desensitization. The othervariants, found only in the African-American sample, had ahigher sensitivity to ACh and lower EC50 (Liang et al.2005). Mice null for the �4 nAChR subunit implanted with13-day osmotic mini-pumps displayed signiWcantlyreduced mecamylamine-induced somatic (shaking, groom-ing, scratching, chewing) and non-somatic (hyperalgesia)symptoms of withdrawal, suggesting a role for this subunitin the appearance of nicotine withdrawal symptoms (Salaset al. 2004b).

Evidence of the involvement of the �5, �3, and �4 subunitsin nACh receptor desensitization rates, calcium permeabil-ity, and response to nicotine (Gerzanich et al. 1998; Tapiaet al. 2007), as well as the association of the CHRNA5-CHRNA3-CHRNB4 gene cluster with nicotine dependencephenotypes, has led to studies of the role of key SNPs inwithdrawal symptoms and smoking cessation. In a case–

control study of 1,446 current versus former smokers, noevidence was found for associations of SNPs in theCHRNA5-CHRNA3-CHRNB4 gene cluster with retrospec-tive smoking cessation (Breitling et al. 2009a). Similarlytwo independent nAChR systems-based analyses found noevidence for associations of SNPs in this gene cluster withprospective smoking cessation in participants treated withtransdermal NRT or bupropion (Conti et al. 2008; Ray et al.2010). However, data published by Baker and others pro-vides evidence for association of CHRNA5-CHRNA3-CHRNB4 haplotypes with tolerance, craving, and loss ofcontrol, but only among individuals who began smokingearly in life, suggesting that the genetic risk may only occurwith early tobacco exposure (Baker et al. 2009). TheCHRNA5-CHRNA3-CHRNB4 haplotypes are also associ-ated with withdrawal severity and inability to stop smoking,but there was no interaction with age at daily smoking forthese phenotypes (Baker et al. 2009). Another study by Sarg-inson and colleagues identiWed CHRNA5 SNP rs680244 andCHRNB4 SNP rs12914008 as predictors of 7-day point-prev-alence at 52-week follow-up in Caucasians and the CHRNA3SNP rs8192475 as a predictor of craving after quitting andwithdrawal symptoms over 42 days (Sarginson et al. 2011).More recently, Munafo and colleagues found a weak associa-tion between the rs1051730 polymorphism and decreasedlikelihood of smoking cessation at follow-up after 4 weeks ofopen-label nicotine transdermal patch treatment, but not in asecond placebo-controlled clinical trial (Munafo et al. 2011).Recent data from King and colleagues show an association ofCHRNA5 SNP rs518425 with abstinence in smokers treatedwith the �4�2* partial agonist varenicline in a large pharma-cogenetic trial (King et al. 2011).

Most smoking behavior genetic studies limit theirsample populations to European ancestry, but associationstudies in diverse populations with diVerent linkage dis-equilibrium patterns and alternate phenotypes may enhancethe search for variants relevant to smoking behavior. Li andcolleagues reported data from the Wrst study of the associa-tion of SNPs in the CHRNA5-CHRNA3-CHRNB4 gene clus-ter with smoking behavior in a large Korean sample (Liet al. 2010). Smoking cessation, deWned based on smokingstatus as former versus current smoker, was associated withthe CHRNB4 SNP rs11072768 as well as with haplotypes inthis gene cluster (Li et al. 2010). Hamidovic et al. (2011)conducted a study in 1,710 African-American current orformer smokers. They evaluated smoking persistence,which accounts for periods of smoking cessation during alarger smoking timeframe. CHRNA5 SNP rs12915366 andCHRNA3 SNP rs12914385 were found to be associated withsmoking persistence (Hamidovic et al. 2011). BothSNPs are located in regions that have been associated withsmoking and nicotine dependence in European Americans(Liu et al. 2010; Saccone et al. 2009; TAG 2010), suggesting

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that there may be distinct variants which modulate smokingbehavior phenotypes in African-Americans. Smoking dur-ing pregnancy is a phenotype inXuenced by many factors,including genetic susceptibility to the addictive properties ofnicotine. Data from 2,474 European women, who smokedregularly before becoming pregnant, showed that the riskallele of the rs1051730 SNP in the CHRNA5-CHRNA3-CHRNB4 gene cluster is associated with continuing tosmoke during pregnancy (Freathy et al. 2009). Thorgeirssonand Stefansson (2010) replicated these Wndings, Wnding thers1051730 risk variant is also associated with continuedsmoking during pregnancy in their sample of 1,891 women.

The reviewed research on the CHRNA5-CHRNA3-CHRNB4 gene cluster in smoking cessation phenotypesfrom animal and human genetic data suggests that variabil-ity in these subunit genes may have some small contribu-tion to the physical withdrawal symptoms and the ability toabstain from smoking. The role of variation in theCHRNA5-CHRNA3-CHRNB4 gene cluster may be impor-tant in determining response to treatments for smoking ces-sation; however, this hypothesis has not been testedextensively. Ethnicity and smoking behavior phenotypesmay be critical factors in determining the signiWcance ofassociations between this gene cluster and smoking cessa-tion and cessation treatment response.

CHRNA6-CHRNB3

�6-containing nAChRs are expressed mainly in brain areasthat are involved in nicotine-stimulated dopamine release(Champtiaux et al. 2003; Lai et al. 2005; Salminen et al.2004), and have been implicated in mediating nicotinedependence (Laviolette and van der Kooy 2004). One of theWrst studies examining the pharmacology of �6-containingnAChRs generated mice lacking the �6 subunit gene (Cha-mptiaux et al. 2002). Null mutant mice had no high-aYnity�-conotoxin MII-sensitive (�6 selective antagonist) bindingsites (Champtiaux et al. 2002; Drenan et al. 2008; Yanget al. 2009). A majority of �3 subunits are assembled with�6 subunits (Champtiaux et al. 2003; Yang et al. 2009) andchronic nicotine treatment decreases �6-containing nAC-hRs in the striatum but does not change the density of �6nAChRs that contain �3 subunits (Perry et al. 2007). Jack-son and others measured physical (somatic and hyperalge-sia) and aVective [anxiety-related behavior and conditionedplace aversion (CPA)] nicotine withdrawal behaviors inC57BL/6J mice implanted with osmotic mini-pumps Wlledwith nicotine or saline for 14 days (28 days for CPA).Withdrawal was initiated after 14 days of nicotine infu-sion or by removal of the mini-pumps. On day 15, micewere injected intracerebrovascularly with �6-selectiveantagonist �-conotoxin MII(H9A;L15A) and withdrawalsigns were measured 10 min after injection. �-conotoxin

MII(H9A;L15A) blocked withdrawal-associated CPA andanxiety-related behaviors (elevated plus maze) compared tomice injected intracerebroventricularly with saline, sug-gesting a role for the �6 subunit in aVective nicotine with-drawal symptoms (Jackson et al. 2009). This study alsodemonstrated a role for the �6 subunit in nicotine reward,reXecting possible diVerences in nAChR subunit composi-tion (�4�6�2* vs. �6�2* vs. �4�6�5�2*), diVerent anatom-ical distribution of brain nAChRs, and/or diVerentintracellular mechanism in nicotine reward and withdrawal(Jackson et al. 2009). �6* nAChRs are located in both theVTA and locus coeruleus. The VTA has been implicated inboth nicotine reinforcement (Corrigall et al. 1992; Ponset al. 2008) and withdrawal (Bruijnzeel and Markou 2004)and the locus coeruleus has been implicated in withdrawaleVects from drugs of abuse (Dizgah et al. 2005), implyingthat the contributions of �6* nAChRs to nicotine reward aremediated by the VTA, but �6* nAChRs in the locus coeru-leus may be responsible for regulating nicotine withdrawal.

Genetic association studies have shown that SNPs in thegenes encoding the �6 and �3 subunits, CHRNA6 andCHRNB3, are associated with nicotine dependence and sub-jective response to nicotine (Bierut et al. 2007; Greenbaumet al. 2006; Saccone et al. 2007; Zeiger et al. 2008). Therole of CHRNA6 and CHRNB3 in smoking cessation wasexamined by Hoft et al. (2009). They used a family-basedapproach, genotyping 1,051 subjects (50% members of sib-ling pairs), to test for an association with quit attempts andoverall dependence as measured by DSM-IV dependencescale. The rs7004381, rs4950 and, rs13280604 SNPs in theCHRNB3 gene were associated with self-reported numberof unsuccessful quit attempts, while CHRNA6 SNPrs2304297 was associated with nicotine dependence. Repli-cation of associations between speciWc SNPs and smokingbehavior phenotypes has not been easy due to inconsisten-cies in the deWnition of a phenotype and the inclusion sam-ple. Hoft et al. (2009) performed secondary analyses withsets of individuals who reported having used tobacco everyday for at least 1 month in their lives and the entire sampleof 1,051 individuals. The found that analysis using diVerentsubsets of the sample did in fact show that sample selectionaVected the ability to detect an association (Hoft et al.2009).

It is necessary for future studies to understand the molec-ular and functional diVerences of various SNPs in both theCHRNA6 and CHRNB3 genes to be able to elucidate theirroles in nicotine dependence and determine if they contrib-ute to the ability to abstain from smoking.

CHRNA4

CHRNA4 is the gene that encodes the �4 nAChR subunit.The majority of �4 subunits form functional nAChR

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complexes with at least one �2 subunit and together theyaccount for more than 90% of the high-aYnity nicotinicreceptors in the brain (Whiting and Lindstrom 1986).�4�2* receptors are expressed in many CNS regions, and�4 mRNA is expressed in nearly every dopaminergic andGABAergic neuron in the VTA, the region aVecting dopa-mine release into the nucleus accumbens. The �4 subunitplays a role in the reinforcing eVects of nicotine (Klinket al. 2001), tolerance, and the modulation of mesolimbicdopamine function, all essential to the development of thenicotine dependence phenotype (Tapper et al. 2004).

Mice lacking the �4 subunit generated by Marubio et al.(1999) no longer express high-aYnity binding sites. �4 KOmice also show a reduced antinociceptive eVect of nicotineon the hot-plate test and diminished sensitivity to nicotinein the tail-Xick test. Tapper and colleagues generatedknock-in mice that express the Leu9�Ser mutation, express-ing a receptor hypersensitive to nicotine. They found thatselective activation of these receptors with low doses ofnicotine replicates tolerance and sensitization stimulated bychronic nicotine administration, suggesting a role for thissubunit in various smoking behavior phenotypes (Tapperet al. 2004). This mutant mouse model was also used byLabarca et al. (2001) and others who found that the hyper-sensitive mice display increased anxiety and poor motorlearning, which is eliminated by low levels of nicotineadministered i.p. QuantiWcation of nAChR subunit geneexpression indicates that posterior ventral tegmental area(pVTA) neurons express higher levels of �4 (as well as �6and �3) transcripts than anterior VTA neurons (Zhao-Sheaet al. 2011). Infusion of �-conotoxin MII into the VTAblocked activation of pVTA dopaminergic neurons in wild-type and �4* Leu9�Ala knock-in mice, indicating that nico-tine selectively activates dopaminergic neurons within thepVTA through �4�6* nAChRs (Zhao-Shea et al. 2011).

The A529T polymorphism in the CHRNA4 gene hasbeen shown to inXuence varying responses to nicotine andit was found that mice strains with the A529T genotypeconsumed less nicotine (in solution) and preferred solutionswith a lower concentration of nicotine (Butt et al. 2005;Stitzel et al. 2000; Tritto et al. 2002). Wilking and othersfound that CHRNA4 A529T knock-in mice exhibitedgreater sensitivity to the hypothermic eVects of nicotine,consumed less nicotine, and did not develop conditionedplace preference. A529T mice produced a smaller maximalfunctional response to acetylcholine (ACh) than T529A lit-termates and the percent of response to ACh by high sensi-tivity �4* nAChRs was signiWcantly greater for nAChRscontaining the A529T variant (Wilking et al. 2010).

In human studies, SNPs or haplotypes of the CHRNA4gene have been associated with nicotine dependence infamily-based analyses (Feng et al. 2004; Li et al. 2005), alarge-scale genetic association study (Breitling et al.

2009b), and in a study evaluating unrelated EuropeanAmericans and African-Americans (Han et al. 2011).GWAS studies have not identiWed the CHRNA4 gene as arisk locus for smoking behavior phenotypes (Berrettiniet al. 2008; Caporaso et al. 2009; Liu et al. 2009, 2010;TAG 2010; Thorgeirsson et al. 2010; Thorgeirsson and Ste-fansson 2008; Vink et al. 2009). However, in a candidategene study, the rs2236196 polymorphism was associatedwith smoking cessation outcomes (Hutchison et al. 2007).SpeciWcally, this SNP was associated with self-reportedabstinence for 7 days before the end of 8 weeks of treat-ment with either transdermal nicotine patch or nicotinenasal spray. An independent sample of smokers with thers2236196 polymorphism reported increased sensitivity tothe acute eVects of smoking after 8 h of abstinence and sub-jects treated with nasal spray maintained treatment gains at6 months follow-up (Hutchison et al. 2007). In addition,CHRNA4 SNP rs2236196, as well as rs3787138 andrs6062899, were associated with abstinence in smokerstreated with varenicline, consistent with varenicline’smolecular target (King et al. 2011). rs2236196 is located inthe 3� end of the untranslated region of the CHRNA4 geneand alters an mRNA biding site for the iron-responsive ele-ment (IRE). Nucleotide substitutions lead to a signiWcantloss of binding aYnity for IRE (Erlitzki et al. 2002; Hentzeet al. 2004; JaVrey et al. 1993) and IREs have been found toregulate translation of genes involved in CNS function(Rogers et al. 2002). The association of rs2236196 withchanges in sensitivity to the acute eVects of nicotine andwith response to nicotine therapy may be a result ofchanges at the protein level, which may lead to a change inCNS sensitivity to cholinergic stimulation (Hutchison et al.2007).

Evidence from animal and human studies supports a rolefor variation in the CHRNA4 gene in altering the sensitivityof �4-containing nAChRs and response to NRT. Initial evi-dence suggests that CHRNA4 variation may alter sensitivityto nicotine and quitting success with forms of NRT thatreach the CNS more quickly (nasal spray). Yet, these datahave yet to be replicated and more work is needed to iden-tify functional SNPs that may yield more robust and repro-ducible associations with cessation phenotypes.

CHRNB2

nAChRs that contain both �4 and �2 subunits are the mostabundant in the brain, and nicotine has the highest aYnityfor �4�2* receptors (Benowitz et al. 1989; Flores et al.1992; Wu et al. 2006a). Recent evidence suggests thatnAChRs containing the �2 subunit determines the sensitiv-ity to nicotine (Marubio et al. 1999; McCallum et al. 2006;Tritto et al. 2004) and are critical for many of the reinforcingproperties of nicotine (Nashmi and Lester 2006; Picciotto

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et al. 1998; Whiting and Lindstrom 1986), but may not playa role in nicotine withdrawal symptoms in rodents (Bessonet al. 2006). The VTA has been shown to mediate most ofthe positive motivational eVects of nicotine in rats (Lavio-lette and van der Kooy 2004) and most dopaminergic neu-rons in the VTA express �2* containing nAChRs (Klinket al. 2001) and upregulation of nAChRs by nicotine isrestricted to nAChRs containing the �2 subunit (Davila-Garcia et al. 2003; Flores et al. 1997, 1992; Mao et al.2008; Marks et al. 2004; McCallum et al. 2006; Nguyenet al. 2003).

Knockout mice that lack the �2 subunit do not self-administer nicotine and do not show nicotine-stimulateddopamine release in the ventral striatum, implicating itsrole in mediating nicotine dependence (Besson et al. 2006;Picciotto et al. 1998), however, the �2 KO mice exhibitnormal withdrawal symptoms (increased levels of rearingand jumping) (Besson et al. 2006). Marubio et al. (1999)showed that �2 KO mice display a reduction in high-aYn-ity binding sites, a reduced antinociceptive eVect to nicotineon the hot-plate test, and diminished sensitivity to nicotinein the tail-Xick test, supporting a role for this subunit in theantinociceptive eVects of nicotine.

The aVective and cognitive signs of nicotine withdrawalare believed to be an important factor in failed cessationattempts and continued use (Koob et al. 1993; Markou et al.1998). Jackson et al. (2008) measured aVective signs ofwithdrawal and found a loss of anxiety-related behaviorand loss of aversion in v2 KO mice. Cognitive withdrawalsymptoms, as assessed by the contextual fear conditioningparadigm, were examined in �2 KO mice by Portugal et al.(2008). v2 KO mice withdrawn from nicotine showed nowithdrawal-related deWcits on contextual fear conditioning(Portugal and Gould 2008) compared to deWcits exhibitedin wild-type mice. Further, nicotine acting on intact hippo-campal �2-containing nAChRs may alter hippocampalfunction accounting for the withdrawal deWcits in contex-tual fear conditioning (Davis and Gould 2007). Subse-quently, Raybuck and Gould found that nicotinewithdrawal in �2 KO mice did not produce the trace fearconditioning deWcits observed in wild-type mice. Evidencefrom these studies suggests an important role for �2 con-taining nAChRs in withdrawal-related deWcits in learningand memory (Raybuck and Gould 2009).

There is evidence to support the role of the �2 subunit insmoking cessation in humans. rs2072661 in the CHRNB2gene has been associated with smoking cessation successand time to relapse in a nAChR systems-based analysis ofdata from a bupropion placebo-controlled randomized clin-ical trial (Conti et al. 2008). Smokers who carry the minorallele had lower abstinence rates and more severe with-drawal severity than those heterozygous for wild-type allele(Conti et al. 2008). Perkins et al. (2009) examined the asso-

ciation of this polymorphism with ability to quit during aweek of nicotine versus placebo patch use. Consistent withthe prior study (Conti et al. 2008), smokers who carried theminor allele were less successful maintaining abstinence onNRT versus placebo compared to those with the wild-typegenotype (Perkins et al. 2009). However, the function ofthis speciWc variant is unknown. More recently, King et al.(2011) reported CHRNB2 SNPs rs3811450 and rs4262952were associated with abstinence in smokers treated withvarenicline. There is also evidence that CHRNB2 interactswith CHRNA4 in aVecting nicotine dependence (Li et al.2008). Thus, future studies of smoking cessation that aresuYciently powered to detect gene–gene interactions couldextend this Wnding.

It is apparent from the animal and human studies whichreviewed that CHRNB2 is an important gene in maintainingnicotine dependence and contributes to signiWcant symp-toms of nicotine withdrawal. Smoking cessation therapiestargeted to individuals with speciWc alterations in this genemay provide relief from the aVective symptoms that pro-mote relapse and continued use. However, additionalresearch is necessary to provide the evidence base for thisclinical approach.

CHRNA7

�7 nACh receptors are homomeric, comprising of Wve �7subunits. Along with �4�2* nAChRs, �7 nAChRs are themost widely expressed nAChRs in the central nervous sys-tem (Dani and De Biasi 2001; Hogg et al. 2003). �7 nAC-hRs are activated by acetylcholine and blocked by �Bgtxwith high calcium permeability and a rapid desensitizationrate (Gotti and Clementi 2004). Presynaptic �7 nAChRsfacilitate neurotransmitter release (Alkondon et al. 2000)and have been implicated in several eVects of nicotine,including aversion and reward (Laviolette and van derKooy 2003), anxiety-like behavior (Tucci et al. 2003), andworking memory (Levin 2002). �7 KO mice have impairedattentional performance (Hoyle et al. 2006; Young et al.2007) and �7 antagonists impair spatial working memory(Levin et al. 2003), while �7 agonists signiWcantly improvelearning, memory, and attentional function in rodents (Bit-ner et al. 2007; Boess et al. 2007; Levin et al. 1999), andreverse aging-induced cognitive impairments (Beracocheaet al. 2008; Marighetto et al. 2008), but there is less evi-dence for the role of �7 nAChRs in nicotine reinforcement(Smith et al. 2007).

The role of the �7 subunit, encoded by the CHRNA7gene, in nicotine dependence or withdrawal is not fullyunderstood, and studies have found few diVerences in theeVects of nicotine between �7 KO and wild-type mice(Davis and Gould 2007; Walters et al. 2006). Damaj et al.(2003) found that �7 nAChRs contribute to some nicotine

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withdrawal signs, but Grabus et al. (2005) and Markou andPaterson (2001) found that methyllycaconitine (MLA), an�7 nAChR antagonist, did not precipitate withdrawal.

�7 KO mice have been widely studied to explore the roleof this subunit in nicotine’s eVects. Grabus et al. (2005)examined withdrawal after chronic oral nicotine adminis-tration in �7 KO mice by replacing nicotine solutions withwater. Withdrawal-induced hyperalgesia was attenuated in�7 KO compared to wild-type mice, but there was no diVer-ence in other aspects of nicotine withdrawal, such as thesomatic signs (paw tremors, backing, head shakes). Simi-larly, Jackson et al. (2008) found that mecamylamine(MEC)-precipitated nicotine withdrawal in �7 KO miceimplanted with osmotic mini-pumps resulted in a loss ofhyperalgesia. No diVerences in the somatic signs of with-drawal were observed between nicotine-dependent wild-type �7 KO mice compared to saline-treated wild-type and�7 KO mice. In contrast, using a nicotine withdrawal proto-col similar to Jackson et al. (2008) mice lacking the �7 sub-unit exhibited decreases in the somatic signs of MEC-precipitated withdrawal and exhibited normal MLA-precip-itated nicotine withdrawal, compared to wild-type mice(Salas et al. 2007). The opposing results from Jackson et al.(2008) and Salas et al. (2007) indicate a clearly undeWnedrole of the �7 subunit in nicotine withdrawal, suggestingthe possibility that there may be �7* nACh receptors thatassemble with other subunits (Khiroug et al. 2002; Palmaet al. 1999) that respond diVerently to the nAChR antago-nists used to precipitate withdrawal. MEC is highly selec-tive for �3�4* nAChRs, has a very low aYnity for �7nAChRs (Papke et al. 2001), and has been shown to be themost eVective nAChR antagonist at precipitating both thesomatic and non-somatic signs of withdrawal (Damaj et al.2003; David et al. 2007; Malin et al. 1998). MLA, how-ever, is a selective antagonist for �7 nAChRs (Roegge andLevin 2006) and has demonstrated contrasting results innicotine withdrawal. MLA did not induce somatic signs ofwithdrawal after prolonged nicotine exposure in rats(Markou and Paterson 2001), but consistent with Salaset al. (2007), Damaj et al. (2003) showed that MLA wasable to precipitate withdrawal, suggesting that MLA mayhave speciWcity for nAChR subunits other than �7 and theobserved symptoms of withdrawal may only partially dueto the �7 subunit.

There are very few human genetic studies examining therole of the CHRNA7 gene with smoking behavior pheno-types. The results from a study of Jewish females indicatethat CHRNA7 SNPs are associated with severity of nicotinedependence in females (Greenbaum et al. 2006) but there isno known association of CHRNA7 with smoking cessation(Conti et al. 2008; Ray et al. 2010). In a systems-basedgenetic study of smoking cessation, 1,295 SNPs in 58 geneswithin the neuronal nAChR and dopamine systems were

investigated for their role in cessation within a bupropionplacebo-controlled randomized clinical trial (Conti et al.2008). A follow-up study on an independent sample ofsmokers treated with transdermal nicotine also assessedgenes involved in acetylcholine synthesis and transport (Rayet al. 2010). Neither study identiWed signiWcant SNPs in theCHRNA7 gene that contributed to smoking cessation pheno-types. However, King et al. (2011) recently reported that, intreatment-seeking smokers randomized to varenicline,CHRNA7 SNP rs6494212 was associated with abstinence.

Acetylcholine synthesis

Choline acetyltransferase (ChAT)

ChAT is responsible for the synthesis and modulation ofendogenous acetylcholine (Whittaker 1988). ChAT inXu-ences several cholinergic-dependent functions includingcognitive performance (Bacciottini et al. 2001) and hasbeen shown to mediate modulatory eVects cholinergicreceptors on mesocorticolimbic dopaminergic pathways(Gronier et al. 2000). Chronic nicotine administration inadult rats has been shown to increase ChAT enzyme activ-ity (Hernandez and Terry 2005) and ChAT enzyme activityis altered during nicotine withdrawal in adolescent animals(Slotkin et al. 2008). Molecules that alter ChAT enzymeactivity have been tested clinically. Galantamine, an acetyl-cholinesterase inhibitor that increases the concentration ofacetylcholine, may reduce smoking behavior (Diehl et al.2006) suggesting that ChAT is a biologically plausible can-didate gene for smoking behavior phenotypes.

In a sample of treatment-seeking smokers and an inde-pendent sample of family-based non-treatment seekers con-sisting of either African-Americans or EuropeanAmericans, signiWcant ChAT SNP and haplotype associa-tions were identiWed for multiple measures of nicotinedependence (Ray et al. 2010; Wei et al. 2010). With respectto smoking cessation, Ray and colleagues performed a sys-tems-based genetic association analysis in a discovery sam-ple of 472 treatment-seeking smokers of Europeanancestry, assessing smoking relapse following 8 weeks oftransdermal nicotine therapy. A cluster of SNPs in ChAThaplotype block 6 was signiWcantly associated with relapse(Ray et al. 2010). These results are consistent with Heitjanet al. (2008) who found that ChAT SNP rs1917810 in hap-lotype 6 was associated with response to bupropion andsmokers with the minor allele had higher abstinence rateson placebo.

To date, few human genetic association studies haveexamined the role of ChAT in smoking cessation. Futurestudies are needed for further characterization of ChAT andidentiWcation of functional variants that may alter expression

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levels or enzymatic function. The initial Wndings of geneticassociations of ChAT make ChAT an interesting candidatefor future research.

Pharmacokinetics of nicotine

Cigarette smoking produces a rapid distribution of nicotinethrough the bloodstream and nicotine crosses the blood–brain barrier, with drug levels peaking in the brain within10 s of inhalation (Le Houezec 2003). Smoking produceshigh concentrations of nicotine in the brain that are compa-rable to concentrations observed after intravenous adminis-tration (Hukkanen et al. 2005). Various studies have shownthat smokers will try to maintain their plasma nicotine con-centration within a narrow range by modulating their smok-ing behavior. This may include altering the number ofcigarettes they smoke per day or how they smoke a ciga-rette (e.g., depth of inhalation) (Benowitz 2008).

The elimination half-life of nicotine is around 2 h.Among several nicotine metabolites, the major metaboliteis cotinine. In humans, 70–80% of nicotine is converted tocotinine, mediated largely by the liver enzyme cytochromeP450 CYP2A6. Approximately 33–40% of cotinine is con-verted to its primary metabolite, 3�-hydroxycotinine (3HC),also by CYP2A6. Cotinine and 3�-hydroxycotinine aremetabolized at a much slower rate than nicotine, and theirrates of elimination are not signiWcantly aVected bychanges in liver blood Xow (Hukkanen et al. 2005; Mweni-fumbo and Tyndale 2007).

Due to the slow rate of elimination, cotinine and itsmetabolite 3HC are useful in assessing nicotine exposure andmetabolism. The ratio of 3HC to cotinine has been found tobe a reliable marker for CYP2A6 activity and for individualdiVerences in the rate of nicotine metabolism (Dempsey et al.2004). Metabolism is faster in woman than in men, andreduced metabolism is found more frequently among Asiansand African-Americans than among Caucasians and Hispan-ics (Benowitz et al. 2006a). Rate of nicotine metabolism hasbeen associated with genetic polymorphisms in CYP2A6(Benowitz et al. 2006b; Bloom et al. 2011; Malaiyandi et al.2005; Mwenifumbo and Tyndale 2007); however, twin stud-ies suggest that known CYP2A6 variants account for only aportion of the heritability of nicotine clearance rates (Rayet al. 2009; Swan et al. 2005).

CYP2A6

Several reduced and null activity alleles of the CYP2A6gene have been identiWed and shown to alter enzyme activ-ity and in vivo nicotine metabolism (Fernandez-Salgueroet al. 1995; Goodz and Tyndale 2002; Mwenifumbo et al.2008; Mwenifumbo and Tyndale 2007). The CYP2A6*1

allele is the “normal” activity (wild-type) variant andCYP2A6*1B is an increased activity allele (faster nicotineclearance). The CYP2A6*2 and *4 alleles are null activityalleles, occurring in about 20% of Asians and 1% of Cauca-sians (Nakajama et al. 2006; Rao et al. 2000; Schoedelet al. 2004). The CYP2A6*9 and *12 are found more com-monly in Caucasian populations, about 9% of Caucasianscarry one or more of these alleles (Malaiyandi et al. 2005;Schoedel et al. 2004). These alleles have reduced activity(slower nicotine clearance) (Mwenifumbo et al. 2008;Mwenifumbo and Tyndale 2007; Ray et al. 2009).

Genetic variability in CYP2A6 has been associated witha variety of smoking behavior phenotypes. Reduced or nullactivity CYP2A6 alleles (CYP2A6*9, CYP2A6*12,CYP2A6*2, or CYP2A6*4) are more prevalent in non-smokers than smokers (Iwahashi et al. 2004; Malaiyandiet al. 2005; Mwenifumbo and Tyndale 2007; Pianezza et al.1998). Smokers with reduced or null activity smoke fewercigarettes (Audrain-McGovern et al. 2007; Fujieda et al.2004; Malaiyandi et al. 2005, 2006; Minematsu et al. 2003;Mwenifumbo et al. 2007; Rao et al. 2000; Schoedel et al.2004; Tyndale et al. 1999) and tend to be less dependent onnicotine (Kubota et al. 2006; Malaiyandi et al. 2006) thansmokers with normal activity alleles. With respect to smok-ing initiation, adolescents with normal activity alleles mayprogress to nicotine dependence more quickly than slowermetabolizers (CYP2A6*9, CYP2A6*12, CYP2A6*2, orCYP2A6*4) (Audrain-McGovern et al. 2007). A recentGWAS conducted in 31,266 smokers found an associationof the rs4105144 SNP with reduced smoking quantity,assessed as cigarettes per day (Thorgeirsson et al. 2010).The rs4105144 SNP is in linkage disequilibrium with theCYP2A6*2 reduced activity allele.

A few studies have reported associations of CYP2A6with smoking cessation and response to treatment. Smokerswith the null activity allele, CYP2A6*2, are twice as likelyto quit smoking as smokers who do not possess that allele(Gu et al. 2000). Further, smokers with high activity alleles(CYP2A6*1/*1B) report more serious withdrawal symp-toms during smoking cessation (Kubota et al. 2006). Thecorrelation between CYP2A6 genotypes and withdrawalsymptoms was more notable in smokers who used NRT,such as transdermal nicotine patch in which a high dose ofnicotine is administered initially with a gradual decrease indosage. Smokers with high CYP2A6 activity, treated withNRT, might maintain a low level of nicotine that desensi-tizes/inactivates a larger number of receptors than smokerswith low activity.

Nicotine metabolite ratio

Variation in nicotine metabolism rate can also be determinedby the ratio of the nicotine metabolites (3-HC/cotinine) that is

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derived from cigarette smoking (Benowitz et al. 2003). Theratio of 3-HC/cotinine is generally referred to as the nico-tine metabolite ratio and can be measured reliably in salivaor plasma (Dempsey et al. 2004) and is independent ofsmoking patterns or time since last cigarette, among regularsmokers (Levi et al. 2007). Null or reduced activityCYP2A6 alleles (CYP2A6*2, *4, *9, and *12) are associ-ated with lower nicotine metabolite ratios and slowermetabolism (Dempsey et al. 2004; Johnstone et al. 2006;Malaiyandi et al. 2006). At present, use of the nicotinemetabolite ratio may be more preferable to assessingCYP2A6 genotype, given the large number of CYP2A6 alle-les and the presence of non-genetic inXuences on nicotinemetabolism rates (Ray et al. 2009).

Lerman and colleagues assessed 480 treatment-seekingsmokers who were randomized to 8 weeks of transdermalnicotine or nicotine nasal spray. The nicotine metabolismratio predicted the eVectiveness of transdermal nicotinetreatment at the end of treatment and at 6-month follow-up;however, the nicotine metabolite ratio was not a predictorof abstinence with use of nicotine nasal spray, presumablybecause smokers were able to titrate the dose of nasal spraybased on diVerential metabolism (Lerman et al. 2006). Thenicotine metabolite ratio was validated as a predictor ofquitting success in an independent sample of smokers after8 weeks of transdermal nicotine treatment (Schnoll et al.2009). Based on these results, Lerman and others examinedthe eYcacy of extended (6-month) transdermal nicotinetherapy versus the standard 8-week therapy in 471 Cauca-sian smokers with either normal or reduced rates of nicotinemetabolism. Reduced metabolizers were found to beneWtmore from extended therapy than standard 8-week therapycompared to normal metabolizers (Lerman et al. 2010).

To determine whether the predictive validity of the nico-tine metabolite ratio is speciWc for transdermal nicotinetherapy, another study evaluated this biomarker among 414smokers in a 10-week placebo-controlled randomized trialof bupropion (Patterson et al. 2008). Faster metabolizershad signiWcantly lower quit rates than slower metabolizersif treated with placebo, but had equivalent quit rates toslower metabolizers if treated with bupropion (Pattersonet al. 2008). In a clinical trial of African-American lightsmokers, slower metabolizers based on the nicotine metab-olite ratio had higher quit rates than faster metabolizerstreated with nicotine gum or placebo (Ho et al. 2009).

Withdrawal symptoms interfere with smoking cessation(ShiVman et al. 2006) and faster metabolizers of nicotinemay experience more intense withdrawal symptoms due toa more rapid decline in blood and brain nicotine concentra-tions after smoking a cigarette. In adolescent light smokers,faster metabolizers reported greater withdrawal symptomsafter 24 h and described themselves as more highlyaddicted than slow metabolizers (Rubinstein et al. 2008). In

adult treatment-seeking smokers, higher nicotine metabo-lite ratios predicted more severe cravings for cigarettesafter 1 week of treatment with transdermal nicotine (Ler-man et al. 2006); however, there was no signiWcant associa-tion of the nicotine metabolite ratio with withdrawalsymptoms or nicotine craving in a second study of transder-mal nicotine (Schnoll et al. 2009) or in the bupropion pla-cebo-controlled trial (Patterson et al. 2008). It is possiblethat diVerences in very early withdrawal symptoms exist,but were not captured by the measurement points used inthe clinical trials.

CYP2B6

The role of the CYP2B6 enzyme in nicotine metabolism isunclear. There is some evidence for a small role in themetabolism of nicotine to cotinine (Dicke et al. 2005;Yamazaki et al. 1999). CYP2B6 may contribute to nicotinemetabolism that is not mediated by CYP2A6 (Messinaet al. 1997). The CYP2B6*6 haplotype (Q172H and K262Rvariants) is associated with faster nicotine and cotinineclearance (Benowitz et al. 2006b; Ring et al. 2007) and theCYP2B6*4 allele is associated with higher 3-HC/cotinineratios (Johnstone et al. 2006) among individuals withreduced CYP2A6 activity.

The CYP2B6 enzyme is the primary enzyme involved inthe metabolism of the bupropion (Faucette et al. 2000) andthe CYP2B6*5 and *6 variants are associated with slowerbupropion metabolism (Hesse et al. 2004; Lang et al. 2001;Loboz et al. 2006). CYP2B6 genotype was examined as apredictor of relapse in a placebo-controlled trial of bupro-pion for smoking cessation (Lerman et al. 2002). 426 par-ticipants of European ancestry were genotyped for thefunctional C1459T (CYP2B6*5) variant and receivedbupropion or placebo for 10 weeks. Female smokers carry-ing the decreased activity allele reported greater increasesin cravings following the target quit date and had higherrelapse rates on placebo, which were reversed with bupro-pion (Lerman et al. 2002). David and others examined asample of 291 smokers of European ancestry who partici-pated in a double-blind randomized 12-week trial of bupro-pion versus placebo. Individuals who carried both theCYP2B6*5 allele and the dopamine D2 receptor DRD2-Taq1A allele demonstrated the highest long-term absti-nence rates with bupropion (David et al. 2007).

The CYP2B6*6 haplotype is deWned as having both theQ172H and K262R variants and is the most commonCYP2B6 haplotype (Lee et al. 2007). Smokers carrying theCYP2B6*6 genotype exhibited greater quitting successwith bupropion compared to placebo at the end of treatmentand 6-month follow-up. However, the genotype by treat-ment interaction was driven by diVerential quit rates bygenotype on placebo; the two genotype groups had equiva-

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lent quit rates on bupropion suggesting that this associationmay be mediated by nicotine metabolism rather than bupro-pion metabolism (Lee et al. 2007). Recently, severalCYP2B6 polymorphisms were associated with abstinenceamong treatment-seeking smokers randomized to bupro-pion therapy (King et al. 2011), although their function isnot known.

The reviewed studies suggest that individual variability inthe rate of nicotine metabolism inXuences smoking behaviorand response to smoking cessation treatments. Given thelarge number of CYP2A6 alleles, as well as the inXuence ofenvironmental factors on nicotine metabolism, a phenotypicbiomarker of CYP2A6 activity (3HC/cotinine) appears to bea more robust predictor of cessation than genotype.

Conclusions

This review examined the evidence for the association ofcholinergic and nicotine metabolism genes with smokingcessation and therapeutic response. Although SNPs in theCHRNA5-CHRNA3-CHRNB4 gene cluster have reproduc-ible associations with smoking heaviness, evidence sup-porting the role of the CHRNA5-CHRNA3-CHRNB4 genecluster in smoking cessation is mixed. There is initial evi-dence for the role of the CHRNB2 gene in smoking cessa-tion. Smokers who carry the minor allele of rs2072661 hadlower abstinence rates in two independent trials; however,the function of this CHRNB2 variant is unknown (Contiet al. 2008; Perkins et al. 2009). Human genetic studiesexamining the ChAT gene have also reported initial associ-ations with smoking cessation (Heitjan et al. 2008; Rayet al. 2010). The function of the SNPs in ChAT is unknownand further evidence of replication is important. There is noreplicated evidence yet supporting a role of CHRNA6-CHRNB3, CHRNA4, and CHNRA7 genes with smokingcessation as yet.

The research on genetic variation in nicotine metabolismdemonstrates reproducible and clinically signiWcant eVects ofnicotine metabolism rate on smoking cessation. Smokerswith reduced or null activity alleles of CYP2A6 metabolizenicotine more slowly, and may experience less severe with-drawal symptoms and be more likely quit smoking (Gu et al.2000; Kubota et al. 2006). The nicotine metabolite ratio is areliable phenotypic measure to examine the rate of nicotinemetabolism in smokers (Ray et al. 2009). Five independentstudies show signiWcant associations of this pre-treatmentmarker with smoking cessation and response to pharmaco-therapy (Ho et al. 2009; Lerman et al. 2010; Lerman et al.2006; Patterson et al. 2008; Schnoll et al. 2009). There is alsosome support for CYP2B6 genotypes predicting smokers’response to bupropion treatment for smoking cessation(David et al. 2007; Lee et al. 2007; Lerman et al. 2002).

Despite initial encouraging data, there are several limita-tions of the smoking cessation genetic studies conducted todate. Many of the clinical trials have relatively small sam-ple sizes (300–500 subjects). A large portion of the smok-ing population, such as ethnic minorities, are not accountedfor in these studies. Larger sample sizes and studies thattarget various ethnic groups may help to identify novelgenetic associations with smoking cessation. Another limi-tation is that the function of many of the gene variantsexamined is still unknown. At least for the genotype mark-ers, eVect sizes tend to be small and the studies are notpowered to detect gene–gene and gene–environment inter-actions important in cessation.

It will be important to understand the mechanistic basisof the association of genetic variants with smoking cessa-tion. Nicotinic receptors are involved in many neuronalfunctions, including diVerentiation and synaptic plasticity,which are the basis for learning and memory. Responseinhibition, attention, and working memory are inXuenceddirectly by nAChRs or through nAChR interactions withother neurotransmitter systems (Rezvani and Levin 2001).The cognitive signs of nicotine withdrawal are believed tobe an important factor in failed cessation attempts and con-tinued use (Koob et al. 1993; Markou et al. 1998; Pattersonet al. 2010). The nAChR CHRNA4 SNP rs1044396 con-tributes to individual diVerences in visuospatial attention(Greenwood et al. 2005) and another study showed a strongassociation of rs6090384 with severe inattention (Toddet al. 2003). Recent tobacco use improved cognitive Xexibilityin European American smokers with variants in theCHRNA5-CHRNA3-CHRNB4 gene cluster, implying thatnicotine can enhance various aspects of cognitive process-ing by activation of nicotinic receptors and may modulatethe eVect of genetic factors on cognitive Xexibility (Marchantet al. 2008; Zhang et al. 2010) .

Overall, there is still much unknown about the role ofgenetic variability in cholinergic and nicotine metabolismgenes in smoking cessation. Research examining the cellu-lar and molecular mechanisms of SNPs and haplotypes ingenes associated with smoking cessation phenotypes canfacilitate the development of novel pharmacotherapies andthe delivery of personalized smoking cessation treatment.

Acknowledgments Funding for this research was provided by P50-CA143187 (National Institutes of Health/National Cancer Institute)and U01-DA020830 (National Institutes of Health/National Instituteon Drug Abuse).

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