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CHRNB3 Is More Strongly Associated with Fagerstrm Test for Cigarette Dependencebased Nicotine Dependence Than Cigarettes Per D

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CHRNB3 Is More Strongly Associated with Fagerstrm Test for Cigarette Dependencebased Nicotine Dependence Than Cigarettes Per D bs_bs_banner RESEARCH REPORT doi:10.1111/j.1360-0443.2012.03922.x CHRNB3 is more strongly associated with Fagerström Test for Cigarette Dependence-based nicotine dependence than cigarettes per day: phenotype definition changes genome-wide association studies results John P. Rice1, Sarah M. Hartz1, Arpana Agrawal1, Laura Almasy2, Siiri Bennett3, Naomi Breslau4, Kathleen K. Bucholz1, Kimberly F. Doheny5, Howard J. Edenberg6, Alison M. Goate1, Victor Hesselbrock7, William B. Howells1, Eric O. Johnson8, John Kramer9, Robert F. Krueger16, Samuel Kuperman10, Cathy Laurie3, Teri A. Manolio11, Rosalind J. Neuman1, John I. Nurnberger Jr12, Bernice Porjesz13, Elizabeth Pugh5, Erin M. Ramos11, Nancy Saccone14, Scott Saccone1, Marc Schuckit15 & Laura J. Bierut1, as part of the GENEVA Consortium Department of Psychiatry, Washington University School of Medicine, St Louis, MO, USA,1 Department of Genetics, Southwest Foundation for Biomedical Research, San Antonio, TX, USA,2 Department of Biostatistics, University of Washington, Seattle, WA, USA,3 Department of Epidemiology, Michigan State University, East Lansing, MI, USA,4 Center for Inherited Disease Research, Johns Hopkins School of Medicine, Baltimore, MD, USA,5 Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, USA,6 Department of Psychiatry, University of Connecticut, Farmington, CT, USA,7 Division of Health, Social and Economic Research, ResearchTriangle Institute International, ResearchTriangle Park, NC, USA,8 Department of Psychiatry, University of Iowa College of Medicine, Iowa City, IA, USA,9 Division of Child Psychiatry, University of Iowa Hospitals, Iowa City, IA, USA,10 National Human Genome Research Institute, Bethesda, MD, USA,11 Department of Psychiatry, Indiana University School of Medicine, Indianapolis, IN, USA,12 Department of Psychiatry and Behavioral Sciences, State University of New York, Brooklyn, NY, USA,13 Department of Genetics, Washington University School of Medicine, St Louis, MO, USA,14 Department of Psychiatry, University of California–San Diego, La Jolla, CA, USA15and Department of Psychology, University of Minnesota, Minneapolis, MN, USA16 ABSTRACT Aims Nicotine dependence is a highly heritable disorder associated with severe medical morbidity and mortality. Recent meta-analyses have found novel genetic loci associated with cigarettes per day (CPD), a proxy for nicotine dependence. The aim of this paper is to evaluate the importance of phenotype definition (i.e. CPD versus Fagerström Test for Cigarette Dependence (FTCD) score as a measure of nicotine dependence) on genome-wide association studies of nicotine dependence. Design Genome-wide association study. Setting Community sample. Participants A total of 3365 subjects who had smoked at least one cigarette were selected from the Study of Addiction: Genetics and Environment (SAGE). Of the participants, 2267 were European Americans, 999 were African Americans. Measurements Nicotine dependence defined by FTCD score Ն4, CPD. Findings The genetic locus most strongly associated with nicotine dependence was rs1451240 on chromosome 8 in the region of CHRNB3 [odds ratio (OR) = 0.65, P = 2.4 ¥ 10-8]. This association was further strengthened in a meta-analysis with a previously published data set (combined P = 6.7 ¥ 10-16, total n = 4200). When CPD was used as an alternate phenotype, the association no longer reached genome-wide significance (b=-0.08, P = 0.0004). Conclusions Daily cigarette consumption and the Fagerstrom Test for Cigarette Dependence show different associations with polymorphisms in genetic loci. Keywords CHRNB3, genome-wide association studies, meta-analysis, phenotype definitions. Correspondence to: John P. Rice, Department of Psychiatry, Washington University School of Medicine, St Louis, MO 63110, USA. E-mail: [email protected] Submitted 12 September 2011; initial review completed 7 November 2011; final version accepted 18 April 2012 © 2012 The Authors, Addiction © 2012 Society for the Study of Addiction Addiction, 107, 2019–2028 2020 John P. Rice et al. INTRODUCTION variants associated with nicotine dependence, especially in a multi-ethnic sample. Tobacco use is one of the leading causes of mortality To clarify the relationship between FTCD-based nico- world-wide. Because most regular smoking occurs in the tine dependence and CPD in the context of a GWAS, we context of nicotine dependence, nicotine dependence is defined FTCD-based nicotine-dependent cases and non- frequently the focus of studies on tobacco use [1]. Among nicotine-dependent controls from the Study of Addiction: current smokers, approximately 60% are nicotine- Genetics and Environment (SAGE), a multi-ethnic, case– dependent based on the Fagerström Test for Cigarette control sample selected for alcohol dependence [21]. By Dependence (FTCD), a well-established scale for assessing including a diverse set of study participants, we have the nicotine dependence [2]. Evidence for genetic factors con- opportunity to extend our investigation beyond the pre- tributing to the risk of smoking behaviors and nicotine vious studies in European Americans, and specifically dependence is seen in the clustering of heavy smoking address the role that phenotype definition plays in and nicotine dependence in families and the similarity of genome-wide association studies. smoking behaviors in genetically identical twins [3,4]. Numerous studies have found an association between METHODS nicotine dependence and single nucleotide polymor- phisms (SNPs) in the a5 nicotinic receptor gene, Data CHRNA5 [5–12]. To maximize the power to detect genetic This analysis uses a subset of subjects who have ever variants associated with smoking quantity (as an alter- smoked from the SAGE, part of the Gene Environment native to nicotine dependence), three large research con- Association Studies (GENEVA) program of the National sortia performed genome-wide association meta-analyses Institutes of Health (NIH) Genes, Environment, and of cigarettes per day (CPD) in a combined sample of more Health Initiative [22]. For the overall SAGE project, unre- than 75 000 subjects [13–15]. The strongest association lated alcohol-dependent cases (n = 1897) and non- was the variant in CHRNA5, with a combined P-value alcohol-dependent control subjects (n = 1937) were less than 1 ¥ 10-70. However, several other variants were selected from three large, complementary data sets: the discovered with genome-wide significance: variants near Collaborative Genetic Study of Nicotine Dependence the nicotinic receptor subunit genes CHRNB3 and (COGEND), the Collaborative Study on the Genetics of CHRNA6 on chromosome 8 (rs6474412, P = 1.4 ¥ 10-8, Alcoholism (COGA) and the Family Study of Cocaine a region associated previously with other nicotine Dependence (FSCD). Characteristics of the individual phenotypes [12,16–19]), variants near the nicotine data sets are given in the supporting information metabolizing enzyme genes CYP2A6 and CYP2B6 on (Table S3). The Institutional Review Board at each con- chromosome 19 (rs4105144, P = 2.2 ¥ 10-12) and vari- tributing institution reviewed and approved the protocols ants in a non-coding region located on chromosome for genetic studies of substance dependence under which 10q23 (rs1329650, P = 5.7 ¥ 10-10). all subjects were recruited. Subjects provided informed Because genome-wide association studies (GWAS) consent for genetic studies and agreed to allow their have stringent P-value requirements, the issue of statis- genetic and phenotypic information to be shared with tical power is highly relevant. Although most attempts at qualified investigators through NIH repositories. For each maximizing power in GWAS focus on increasing sample of the three studies, we describe the sampling schemes size, as in the above meta-analyses, power can also be used to recruit subjects and select for genotyping. increased by reducing phenotypic variance by increasing either precision of measurement or phenotypic homoge- COGEND neity of the subjects. Although CPD is the most common phenotypic COGEND was designed as a community based case– measurement of smoking behavior, there is strong epi- control family study of nicotine dependence. The demiological evidence that the number of cigarettes COGEND ascertainment protocol identified current smoked per day varies across cultures and ethnicities. smokers with nicotine dependence defined by an FTCD For example, African Americans smoke fewer cigarettes score Ն 4 (maximum score of 10); non-nicotine- than do European Americans [20]. However, the FTCD dependent subjects who had smoked at least 100 ciga- score, ranging from 0 to 10 where CPD can account for rettes and had a life-time FTCD score of zero were also a maximum of four levels, defined as 1–10, 11–20, recruited. All subjects were ascertained from Detroit and 21–30 or 31 or more, appears to be an invariant St Louis. Approximately 53 000 subjects were screened measure of nicotine dependence across ethnicities [20]. by telephone, 2800 were interviewed personally and Therefore, we hypothesized that a GWAS with FTCD nearly 2700 donated blood samples for genetic studies. rather than CPD may have increased power to detect The COGEND study contributed 275 nicotine-dependent © 2012 The Authors, Addiction © 2012 Society for the Study of Addiction Addiction, 107, 2019–2028 CHRNB3 associated with nicotine dependence 2021 cases and 1082 non-nicotine-dependent smoking con- COGEND. For our case definition, we also included 100 trols to these nicotine
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