GENES and TOBACCO USE This module discusses the possible link between genes and tobacco use, a topic that has come to the forefront of contemporary tobacco research. With the trend toward slowed reduction in adult tobacco use in recent years has come a need for new insights into determinants of tobacco use. It has been hypothesized that genetic influences have an important impact on the remaining segment of the population that smokes (Pomerleau, 1995). Pomerleau OF. (1995). Individual differences in sensitivity to nicotine: Implications for genetic research on nicotine dependence. Behav Genet 25:161–177.

CAN GENES PREDICT WHO WILL… develop heart disease? develop lung cancer? become a smoker? be able to quit? As early as 1958, RA Fisher hypothesized that the link between smoking and lung cancer could be explained by shared genes that predisposed individuals to begin smoking as young adults and to develop lung cancer later in adulthood (Fisher, 1958). More recently, tobacco researchers have begun to uncover evidence suggesting that specific genetic factors do in fact contribute to tobacco use and dependence. However, the genes that increase risk for smoking behavior (e.g., initiation and dependence) are different from the genes that contribute to increased risk for lung cancer. Is it plausible that knowledge of one’s genetic code could enable accurate (or nearly accurate) prediction of one’s susceptibility for specific traits, disorders, or even behaviors, such as tobacco use? And if so, what are the implications of this on society and public health policy? Many questions remain unanswered with respect to genes and tobacco use, including (Sullivan & Kendler, 1999): Which genes predispose individuals to smoking initiation, and which to the development and maintenance of tobacco dependence? If such genes are identified, what are the biological mechanisms underlying their influence? Do the mechanisms work differently in different subpopulations, such as men versus women? Do the genes act by altering the pharmacokinetics or pharmacodynamics of nicotine, or both? Are the genes specific to nicotine, or do they also influence predisposition to other drugs of abuse? How do the genes interact with environmental or other factors? Fisher RA. (1958). Cigarettes, cancer and statistics. Centennial Review 2:151–166. Sullivan PF, Kendler KS. (1999). The genetic epidemiology of smoking. Nicotine Tob Res 1:S51–S57.

FACTORS CONTRIBUTING to TOBACCO USE Environment Tobacco advertising Conditioned stimuli Social interactions Physiology Genetic predisposition Coexisting medical conditions Tobacco Use As described in the Nicotine Pharmacology and Principles of Addiction module, nicotine is a powerful drug capable of inducing a variety of pharmacologic effects, including an alteration in brain chemistry. However, tobacco addiction is more than just a brain disease. It is a complex process involving the interplay of many factors (pharmacologic, environmental, and physiologic) that influence an individual’s decision to use tobacco (Benowitz, 1992; Hiatt & Rimer, 1999). The following slides discuss (1) the available evidence in support of a genetic link for tobacco use and dependence, (2) specific “candidate” genes that might influence onset of tobacco use and establishment of dependence, and (3) the societal and policy implications that correspond to genetic testing for tobacco dependence. Benowitz NL. (1992). Cigarette smoking and nicotine addiction. Med Clin N Am 76:415–437. Hiatt RA, Rimer BK. (1999). A new strategy for cancer control research. Cancer Epidemiol Biomarkers Prev 8:957–964. Lerman C, Patterson F, Berrettini W. (2005). Treating tobacco dependence: State of the science and new directions. J Clin Oncol 23:311–323. Leshner AI. (1999). Science-based views of drug addiction and its treatment. JAMA 282:1314–1316. Pharmacology Alleviation of withdrawal symptoms Weight control Pleasure

AVAILABLE EVIDENCE Adoption studies Twin studies Twins reared apart studies Linkage (family) studies Does tobacco use run in families? Studies of families consistently demonstrate that, compared to family members of nonsmokers, family members of smokers are more likely to be smokers also. A study demonstrating, for example, that persons with a nicotine-dependent sibling are 2.1–3.5 times more likely to also be dependent on nicotine than are those who do not have a nicotine-dependent sibling (Niu et al., 2000) might lead one to believe that there is a genetic component to tobacco use. However, it is important to consider environmental factors that promote tobacco use—siblings within the same family share the same environmental influences as well as the same genes. It can be difficult to tease apart these effects. To differentiate the genetic from the environmental influences, epidemiologists use adoption, twin, twins reared apart, and linkage study designs (Sullivan & Kendler, 1999). Niu T, Chen C, Ni J, et al. (2000). Nicotine dependence and its familial aggregation in Chinese. Int J Epidemiol 29:248–252. Sullivan PF, Kendler KS. (1999). The genetic epidemiology of smoking. Nicotine Tob Res 1:S51–S57.

ADOPTION STUDIES Adoption studies compare the similarities between Children who have been adopted and their biological parents versus children who have been adopted and their adoptive parents - OR - Adoptive sibling pairs versus biological sibling pairs Key to the adoption studies is the assumption that if a genetic link for tobacco use exists, then tobacco use behaviors (e.g., smoking status) will be more similar for persons who are related genetically (i.e., biologically) than for persons who are not related genetically. Hence, one would expect to observe greater similarities between children and their biological parents and siblings than would be observed between children and their adoptive parents or adopted siblings.

ADOPTION STUDIES (cont’d) Correlations between relatives for average reported cigarette consumption Relationship Correlation Coefficient Parent and offspring + 0.21 Identical (monozygotic) twins + 0.52 Fraternal (dizygotic) twins + 0.30 Siblings + 0.11 Adoptive parents and adoptive offspring – 0.02 Adoptive siblings + 0.05 The data in this slide show that, as hypothesized, stronger associations (i.e., higher correlation coefficients) exist between biologically related individuals, compared to nonbiologically related individuals, for the reported number of cigarettes consumed (Eysenck, 1980). In recent years, it has become more difficult to conduct adoption studies, because of the reduced number of intranational children available for adoption (Hall et al., 2002). Conducting adoption studies with children from other countries poses special ethical and logistical concerns. Additionally, delayed adoption (i.e., time elapsed between birth and entry into the new family) is common with international adoptions and might lead to an overestimation of genetic effects if early environmental influences are attributed to genetic influences (Rutter et al., 2001). Eysenck HJ. (1980). The Causes and Effects of Smoking. Beverly Hills, CA: Sage. Hall W, Madden P, Lynskey M. (2002). The genetics of tobacco use: Methods, findings and policy implications. Tobacco Control 11:119–124. Rutter M, Pickles A, Murray R, et al. (2001). Testing hypotheses on specific environmental causal effects on behavior. Psychol Bull 127:291–324. Biological Nonbiological Eysenck HJ, 1980.

Concordant or discordant? Estimated heritability for smoking = 0.53 TWIN STUDIES Twin studies compare the similarities between Identical (monozygotic) twins and fraternal (dizygotic) twins In twin studies, identical (monozygotic) twins and fraternal (dizygotic) twins are compared. Identical twins share the same genes; fraternal twins, like ordinary siblings, share approximately 50% of their genes. If a genetic link exists for the phenomenon under study, then one would expect to see a greater concordance in identical twins than in fraternal twins. Thus, in the case of tobacco use, one would expect to see a greater proportion of identical twins with the same tobacco use behavior than would be seen with fraternal twins. Statistically, twin studies aim to estimate the percentage of the variance in the behavior that is due to (1) genes (referred to as the “heritability”), (2) shared (within the family) environmental experiences, and (3) nonshared (external from the family) environmental experiences (Hall et al., 2002). A number of twin studies of tobacco use have been conducted in recent years. These studies have largely supported a genetic role (Hall et al., 2002; Hughes, 1986); higher concordance of tobacco use behavior is evident in identical twins than in fraternal twins. The estimated average heritability for smoking is 0.53 (range, 0.28–0.84; Carmelli et al., 1992; Hughes, 1986); approximately half of the variance in smoking is due to genetic factors. Carmelli D, Swan GE, Robinette D, Fabsitz R. (1992). Genetic influences on smoking—a study of male twins. N Engl J Med 327:829–833. Hall W, Madden P, Lynskey M. (2002). The genetics of tobacco use: Methods, findings and policy implications. Tobacco Control 11:119–124. Hughes JR. (1986). Genetics of smoking: A brief review. Behav Ther 17:335–345. Concordant or discordant? Higher concordance of tobacco use for identical than for fraternal twins Estimated heritability for smoking = 0.53

TWINS REARED APART STUDIES Combine aspects of adoption and twin studies Separate the effects of genetics from the effects of environment Have found that 60% of the variance in regular smoking in men and women born after 1940 is attributable to genetic factors (Kendler et al., 2000) The twins reared apart study design is powerful, in that it combines elements of both adoption and twin studies (Hall et al., 2002). Because identical twins reared apart share the same genes but a different environment, this design makes it possible to differentiate the two effects. Observation of a greater concordance in identical than in fraternal twins reared apart would provide evidence for a genetic component to tobacco use. A handful of studies of smoking have been conducted with twins reared apart. Perhaps the most methodologically sound study, published by Kendler and colleagues (2000), reported that 60% of the variance in regular smoking in men and women born after 1940 is attributed to genetic factors. Hall W, Madden P, Lynskey M. (2002). The genetics of tobacco use: Methods, findings and policy implications. Tobacco Control 11:119–124. Kendler KS, Thornton LM, Pedersen NL. (2000). Tobacco consumption in Swedish twins reared apart and reared together. Arch Gen Psychiatry 57:886–892.

LINKAGE STUDIES Use human genome mapping to enable researchers to identify genes associated with traits or disorders Examine family pedigrees to determine modes of inheritance of disorders Are more difficult when multiple genes have a role Recent advances in the mapping of the human genome have enabled researchers to search for genes associated with specific disorders, including tobacco use. Using a statistical technique called linkage analysis, it is possible to identify genes that predict a trait or disorder. This process is not based on prior knowledge of a gene’s function, but rather it is determined by examining whether the trait or disorder is coinherited with markers found in specified chromosomal regions. Typically these types of investigations involve collection of large family pedigrees, which are studied to determine inheritance of the trait or disorder. This method works well when a single gene is responsible for the outcome; however, it becomes more difficult when multiple genes have an impact, such as with tobacco use.

LINKAGE STUDIES (cont’d) Linkage studies of smoking Identify families with affected individuals (i.e., tobacco users) Genotype two or more affected siblings and biological parents Conduct linkage analysis to determine whether affected siblings are likely to share the same gene as the parents In linkage studies of smoking, it is common for investigators to identify families, ideally with two or more biologically related relatives that have the trait or disorder under study (referred to as affected individuals, in this case, smokers) and other unaffected relatives. For example, data from affected sibling pairs with parents is a common design in linkage analysis. A tissue sample (typically blood) is taken from each individual, and the sample undergoes genotyping to obtain information about the study participant’s unique genetic code. If a gene in a specific region of a chromosome is associated with smoking, and if a genetic marker is linked (i.e., in proximity), then the affected pairs (such as affected sibling pairs) will have increased odds for sharing the same paternal/maternal gene (Hall et al., 2002). Hall W, Madden P, Lynskey M. (2002). The genetics of tobacco use: Methods, findings and policy implications. Tobacco Control 11:119–124.

“CANDIDATE” GENES Candidate genes are genes hypothesized to contribute to the susceptibility for a trait or disorder. Two current lines of research in the area of candidate genes for smoking: Genes affecting nicotine pharmacodynamics Genes affecting nicotine pharmacokinetics As genetic research moves forward, new clues provide insight into which genes might be promising “candidates” as contributors to tobacco use and dependence. Currently, there are two general lines of research in the area of candidate genes for smoking. One examines genes that affect nicotine pharmacodynamics (the way that nicotine affects the body) and the other examines genes that affect nicotine pharmacokinetics (the way that the body affects nicotine). The next four slides describe key mechanisms by which genes are being explored as factors that increase the likelihood of smoking or the development and maintenance of nicotine dependence.

Dopamine transporter (SLC6A3) Metabolism Catechol-O -methyltransferase P h a r m a c o l o g y Pharmacodynamics Pharmacokinetics DOPAMINE Synthesis Tyrosine hydroxylase Receptor activation DRD1, DRD2, DRD3, DRD4, DRD5 Reuptake Dopamine transporter (SLC6A3) Metabolism Catechol-O -methyltransferase MAO A, MAO B Dopamine ß-hydroxylase SEROTONIN Tryptophan hydroxylase (TPH) Serotonin transporter (5-HTT) Nicotine Metabolism CYP2A6 enzyme CYP2D6 enzyme This diagram presents several mechanisms by which genetics might play a role in tobacco use and dependence by affecting nicotine pharmacodynamics and pharmacokinetics. Genes can alter nicotine pharmacodynamics through a variety of mechanisms; however, the two most extensively studied, to date, relate to nicotine’s effects on the neurotransmitters dopamine and serotonin. With respect to dopamine, the following functions are genetically modulated: Dopamine synthesis can be altered via changes in tyrosine hydroxylase, the starting point for biosynthesis of dopamine. Tyrosine enters cells and is converted to L-dopa by tyrosine hydroxylase. L-Dopa then is converted to dopamine, via aromatic amino acid decarboxylase. Dopamine receptor activation also can be affected by genes.There are five different dopamine receptor types (DRD1, DRD2, DRD3, DRD4, DRD5). In preliminary studies, DRD2 and DRD4 appear to be the most closely associated with tobacco use. Dopamine reuptake is affected by alterations in the genes that code for dopamine transporter (also referred to as SLC6A3). Altering the body’s ability to clear dopamine from the synapse will alter nicotine’s pharmacologic effects. Dopamine metabolism is mediated by catechol-O-methyltransferase, monoamine oxidase (MAO) A and B, and dopamine -hydroxylase. Genetic variations that alter the body’s production of these chemicals will affect nicotine pharmacodynamics. Serotonin synthesis and reuptake can be altered as a function of genetic variation in the tryptophan hydroxylase (TPH) and serotonin transporter (5-HTT) genes, respectively. The TPH gene codes for a primary enzyme in the biosynthesis of serotonin, and 5-HTT is responsible for the body’s ability to clear serotonin from the synapse. ♪ Note to instructor(s): This listing is not comprehensive. Other genes are being examined. With respect to nicotine pharmacokinetics, the rate of nicotine metabolism appears to be regulated, at least in part, by the genetic alterations that affect the function of the cytochrome P450 liver enzymes. Two key enzymes have been studied in this context: the CYP2A6 and CYP2D6 enzymes. Of these, the CYP2A6 polymorphisms appear to be more closely associated with tobacco use. The next three slides take a closer look at the effects of genetics on (1) the dopamine reward pathway and (2) the body’s ability to metabolize nicotine.

Dopamine Reward Pathway Genetic Effects on the Dopamine Reward Pathway This slide shows a synapse and the process of chemical neurotransmission. As an electrical impulse arrives at the terminal, it triggers vesicles containing dopamine to move toward the terminal membrane. The vesicles fuse with the terminal membrane to release their contents (in this case, dopamine). Once inside the synaptic cleft (the space between the two neurons), the dopamine can bind to specific proteins called dopamine receptors on the membrane of a neighboring neuron. This binding of dopamine to the dopamine receptors activates the dopamine reward pathway, providing the pleasurable, “rewarding” sensations that lead to nicotine dependence in smokers. Variations in the genes that affect the reward pathway function can alter an individual’s response to nicotine. For example, some persons, as a result of genetic variation in the D2 dopamine receptor gene, have an increased density of dopamine receptors. This likely will affect the rate at which one becomes nicotine dependent. Also, it is conceivable that persons who have impaired dopamine synthesis, rapid dopamine metabolism, or enhanced dopamine reuptake might not experience normal levels of nicotine-induced pleasure. This slide is made available to the public through the National Institute on Drug Abuse at www.nida.nih.gov/Teaching2/largegifs/slide7.gif.

GENETIC EFFECTS on NICOTINE METABOLISM 4.4% 9.8% Nicotine-1'- N-oxide 0.4% Nicotine Nornicotine Nicotine glucuronide Nicotine 4.2% The functions of the enzymes that metabolize nicotine also are genetically modulated. When an individual consumes nicotine, approximately 80% of it is broken down into its metabolite cotinine by the cytochrome P450 oxidase enzymes in the liver. Benowitz NL, Jacob P, Fong I, Gupta S. (1994). Nicotine metabolic profile in man: Comparison of cigarette smoking and transdermal nicotine. J Pharmacol Exp Ther 268:296–303. Trans-3'- hydroxycotinine ~80% 33.6% Trans-3'- hydroxycotinine Cotinine Cotinine 13.0% Trans-3'- hydroxycotinine glucuronide Cotinine glucuronide 7.4% 12.6% Norcotinine Cotinine- N-oxide 2.0% 2.4% Reprinted with permission, Benowitz et al. (1994).

GENETIC EFFECTS on NICOTINE METABOLISM (cont’d) Specifically, the CYP2A6 enzyme appears to be primarily responsible for the metabolism of nicotine to cotinine. The gene that codes for this enzyme differs between people, such that some people have slower enzymatic activity than others. If a person rapidly metabolizes nicotine, he or she might be more likely to smoke larger numbers of cigarettes per day than slower metabolizers, in order to attain adequate nicotine blood levels. Thus rapid metabolizers would be exposed to substantially higher levels of the cancer-causing tars and the other compounds present in tobacco and tobacco smoke. In this way, it is possible that genes also could predict who, among tobacco users, might be most likely to develop lung cancer. Similarly, it is possible that slow metabolizers of nicotine might be less likely than faster metabolizers to become cigarette smokers, because slow metabolizers would have had higher blood levels and therefore experienced more nausea (and other negative reactions) when they smoked their first cigarette. This might make them less likely to try a second cigarette. Thus, it is not entirely clear whether the speed of metabolism increases, or decreases, one’s likelihood of establishing tobacco dependence. Ongoing genetic studies of smoking aim to test this, and other, genetic hypotheses. CYP2A6 Aldehyde oxidase Cotinine

GENES AND TOBACCO USE: SOCIETAL AND POLICY IMPLICATIONS If genetic tests become available, should society encourage genetic testing for tobacco dependence? Single gene or multiple gene? Prevalence of the gene(s) in the population? Is there an effective intervention to prevent smoking in those who are susceptible? Impact of positive tests, and negative tests? What if…some day we are able to identify persons who might be at risk for tobacco use and dependence? Should society encourage widespread genotyping for this disorder? This is an interesting conceptual question. A few key factors should be considered in this debate. First, genetic testing is most valuable when a single gene is highly predictive of the outcome and when effective interventions exist. For some diseases, a clear link exists between genes and disease occurrence. For smoking, the link is less clear. There are many predictors of tobacco use—some of which are environmental, such as whether one’s friends smoke or whether one is exposed to tobacco advertising. These are external factors. There also are internal factors that predict tobacco use, such as one’s personality and other psychological characteristics, the physiologic makeup of one’s body, and the way one’s body metabolizes nicotine. Some of these internal factors can be genetically mediated. In some cases, genes may interact with other factors, either internal or external (Swan et al., 2003). When multiple genes or multiple factors are independently or jointly responsible for an outcome, the genetic test becomes less valuable because it is just one piece in a larger puzzle that describes an individual’s risk. Second, although having a specific variant of a gene might increase one’s risk for the outcome, the overall risk might still be quite small. Generally, the more genes involved, the less useful is the genotype information. This becomes even more evident when multiple genes, each of low prevalence in the general population, must occur concurrently in order for an elevated risk to be realized (Hall et al., 2002). Third, if it became possible to effectively identify persons at risk for dependence, and it could be accomplished at a reasonable cost to society, how would those at risk be treated? In the case of smoking, it makes little sense to screen for dependence potential—if a person were not genetically susceptible for smoking, does this mean that he or she should be advised that it is okay to smoke? Although it could be argued that genetically susceptible persons might need to receive more intensive smoking prevention interventions than nonsusceptible persons, unless the test were highly accurate in its ability to predict smoking onset, and the intervention highly effective, the risks associated with labeling of persons likely are too high. Finally, knowledge of one’s genetic predisposition for smoking might adversely affect one’s likelihood of starting to smoke or reduce one’s likelihood of trying to quit. Hall W, Madden P, Lynskey M. (2002). The genetics of tobacco use: Methods, findings and policy implications. Tobacco Control 11:119–124. Swan GE, Hudmon KS, Khroyan TV. (2003). Tobacco dependence. In Handbook of Psychology, edited by Nezu AM, Nezu CM, Geller PA, Weiner IB. New York: Wiley.

GENES AND TOBACCO USE: SOCIETAL AND POLICY IMPLICATIONS (cont’d) Improved treatment for tobacco dependence? Perhaps the most promising benefit of genetic research on smoking Improved understanding of tobacco dependence might result in development of more effective medications Pharmacotherapy treatment matching It has been suggested that the possibility of improved treatment for dependence, and therefore reduced smoking prevalence, will be the most likely benefit resulting from genetic research on smoking (Hall et al., 2002). Certainly if genetic research leads to new knowledge regarding the mechanisms underlying the development and maintenance of dependence, then new, more effective medications might be created. In addition, through pharmacogenomics research we might have improved knowledge as to which patients, based on their genetic profiles, would be best treated with which medications. Researchers are beginning to examine how DNA variants affect outcome with pharmacologic treatments, with a goal of determining which genetic profiles respond most favorably to specific pharmacologic aids for cessation (e.g., Berrettini & Lerman, 2005; Lerman et al., 2005; Lerman & Niaura, 2002; Munafo et al., 2005; Swan et al., 2005). Berrettini WH, Lerman CE. (2005). Pharmacotherapy and pharmacogenetics of nicotine dependence. Am J Psychiatry 152:1441–1451. Hall W, Madden P, Lynskey M. (2002). The genetics of tobacco use: Methods, findings and policy implications. Tobacco Control 11:119–124. Lerman C, Jepson C, Wileyto EP, et al. (2005). Role of functional genetic variation in the dopamine D2 receptor (DRD2) in response to bupropion and nicotine replacement therapy for tobacco dependence: Results of two randomized clinical trials. Neuropsychopharmacology [online], August 10. Lerman C, Niaura R. (2002). Applying genetic approaches to the treatment of nicotine dependence. Oncogene 21:7412–7420. Munafo MR, Lerman C, Niaura R, Shields AE, Swan GE. (2005). Smoking cessation treatment: Pharmacogenetic assessment. Curr Opin Mol Ther 7:202–208. Swan GE, Valdes AM, Ring HZ, et al. (2005). Dopamine receptor DRD2 genotype and smoking cessation outcome following treatment with bupropion SR. Pharmacogenomics J 5:21–29.

GENES and TOBACCO USE: SUMMARY Research in the area of genetics and smoking is in its infancy; however, there appears to be a genetic component to tobacco use. Tobacco use is a complex behavior, with many determinants. More research is needed. Genetic research and testing must proceed with caution because the societal stakes are high. Although research in the area of genetics and smoking is in its infancy, compelling evidence demonstrates a genetic component to tobacco use. However, tobacco use is a complex behavior that is affected by multiple factors, both internal and external. Genetics is only one piece of a much larger puzzle. More research is needed (Caron et al., 2005; Sullivan & Kendler, 1999). Although widespread testing for dependence is not likely to become the norm, benefits can be gained by advancing scientific knowledge of genetic determinants of tobacco use and dependence and pharmacotherapy matching for cessation. However, as with all genetic research and testing, results must be proceed with caution because the societal stakes are high (Caron et al., 2005). Caron L, Karkazis K, Raffin TA, Swan G, Koenig BA. (2005). Nicotine addiction through a neurogenomic prism: Ethics, public health, and smoking. Nicotine Tob Res 7:181–197. Sullivan PF, Kendler KS. (1999). The genetic epidemiology of smoking. Nicotine Tob Res 1:S51–S57.