Genome-wide association study across European and African American ancestries identifies a SNP in DNMT3B contributing to nicotine dependence

Cigarette smoking is a leading cause of preventable mortality worldwide. Nicotine dependence, which reduces the likelihood of quitting smoking, is a heritable trait with firmly established associations with sequence variants in nicotine acetylcholine receptor genes and at other loci. To search for additional loci, we conducted a genome-wide association study (GWAS) meta-analysis of nicotine dependence, totaling 38,602 smokers (28,677 Europeans/European Americans and 9925 African Americans) across 15 studies. In this largest-ever GWAS meta-analysis for nicotine dependence and the largest-ever cross-ancestry GWAS meta-analysis for any smoking phenotype, we reconfirmed the well-known CHRNA5-CHRNA3-CHRNB4 genes and further yielded a novel association in the DNA methyltransferase gene DNMT3B. The intronic DNMT3B rs910083-C allele (frequency=44–77%) was associated with increased risk of nicotine dependence at P=3.7 × 10−8 (odds ratio (OR)=1.06 and 95% confidence interval (CI)=1.04–1.07 for severe vs mild dependence). The association was independently confirmed in the UK Biobank (N=48,931) using heavy vs never smoking as a proxy phenotype (P=3.6 × 10−4, OR=1.05, and 95% CI=1.02–1.08). Rs910083-C is also associated with increased risk of squamous cell lung carcinoma in the International Lung Cancer Consortium (N=60,586, meta-analysis P=0.0095, OR=1.05, and 95% CI=1.01–1.09). Moreover, rs910083-C was implicated as a cis-methylation quantitative trait locus (QTL) variant associated with higher DNMT3B methylation in fetal brain (N=166, P=2.3 × 10−26) and a cis-expression QTL variant associated with higher DNMT3B expression in adult cerebellum from the Genotype-Tissue Expression project (N=103, P=3.0 × 10−6) and the independent Brain eQTL Almanac (N=134, P=0.028). This novel DNMT3B cis-acting QTL variant highlights the importance of genetically influenced regulation in brain on the risks of nicotine dependence, heavy smoking and consequent lung cancer.

[1]  Scott F. Saccone,et al.  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. , 2012, Addiction.

[2]  George Bartzokis,et al.  Differences between smokers and nonsmokers in regional gray matter volumes and densities , 2004, Biological Psychiatry.

[3]  M. Brilliant,et al.  Regulatory Polymorphisms in Human DBH Affect Peripheral Gene Expression and Sympathetic Activity , 2014, Circulation research.

[4]  Sarah M. Scholl,et al.  Characteristics and smoking patterns of intermittent smokers. , 2012, Experimental and clinical psychopharmacology.

[5]  S. Wacholder,et al.  Time to smoke first morning cigarette and lung cancer in a case-control study. , 2014, Journal of the National Cancer Institute.

[6]  Luigi Ferrucci,et al.  Imputation of Variants from the 1000 Genomes Project Modestly Improves Known Associations and Can Identify Low-frequency Variant - Phenotype Associations Undetected by HapMap Based Imputation , 2013, PloS one.

[7]  J. Mill,et al.  Methylation quantitative trait loci in the developing brain and their enrichment in schizophrenia-associated genomic regions , 2015, Nature neuroscience.

[8]  S. Naidu,et al.  Abnormalities of the DNA Methylation Mark and Its Machinery: An Emerging Cause of Neurologic Dysfunction , 2014, Seminars in Neurology.

[9]  S. Belinsky,et al.  Global identification of genes targeted by DNMT3b for epigenetic silencing in lung cancer , 2014, Oncogene.

[10]  P. Lee,et al.  Systematic review with meta-analysis of the epidemiological evidence in the 1900s relating smoking to lung cancer , 2012, BMC Cancer.

[11]  Megan E. Piper,et al.  Time to first cigarette in the morning as an index of ability to quit smoking: implications for nicotine dependence. , 2007, Nicotine & tobacco research : official journal of the Society for Research on Nicotine and Tobacco.

[12]  Angela R Laird,et al.  Chronic cigarette smoking is linked with structural alterations in brain regions showing acute nicotinic drug-induced functional modulations , 2016, Behavioral and Brain Functions.

[13]  N. Breslau,et al.  Predicting smoking cessation and major depression in nicotine-dependent smokers. , 2000, American journal of public health.

[14]  Laura J Bierut,et al.  A multiancestry study identifies novel genetic associations with CHRNA5 methylation in human brain and risk of nicotine dependence. , 2015, Human molecular genetics.

[15]  William S. Bush,et al.  Large-scale association analysis identifies new lung cancer susceptibility loci and heterogeneity in genetic susceptibility across histological subtypes , 2017, Nature Genetics.

[16]  P. Sullivan,et al.  The genetic epidemiology of smoking. , 1999, Nicotine & tobacco research : official journal of the Society for Research on Nicotine and Tobacco.

[17]  Ming D. Li,et al.  The Contribution of Rare and Common Variants in 30 Genes to Risk Nicotine Dependence , 2014, Molecular Psychiatry.

[18]  A. Singleton,et al.  Genetic variability in the regulation of gene expression in ten regions of the human brain , 2014, Nature Neuroscience.

[19]  M. Daly,et al.  Estimation of the multiple testing burden for genomewide association studies of nearly all common variants , 2008, Genetic epidemiology.

[20]  Anders Albrechtsen,et al.  Weighting sequence variants based on their annotation increases power of whole-genome association studies , 2016, Nature Genetics.

[21]  Scott F. Saccone,et al.  Risk for nicotine dependence and lung cancer is conferred by mRNA expression levels and amino acid change in CHRNA5. , 2009, Human molecular genetics.

[22]  L. Kozlowski,et al.  The Fagerström Test for Nicotine Dependence: a revision of the Fagerström Tolerance Questionnaire. , 1991, British journal of addiction.

[23]  V. Salomaa,et al.  Association of the DBH Polymorphism rs3025343 With Smoking Cessation in a Large Population-Based Sample , 2017, Nicotine & tobacco research : official journal of the Society for Research on Nicotine and Tobacco.

[24]  Andrew A. Rooney,et al.  Forest Plot Viewer: a new graphing tool. , 2011, Epidemiology.

[25]  Jie Huang,et al.  Comparison of HapMap and 1000 Genomes Reference Panels in a Large-Scale Genome-Wide Association Study , 2017, PloS one.

[26]  S. Flis,et al.  DNA methyltransferase inhibitors and their emerging role in epigenetic therapy of cancer. , 2013, Anticancer research.

[27]  Luigi Ferrucci,et al.  Abundant Quantitative Trait Loci Exist for DNA Methylation and Gene Expression in Human Brain , 2010, PLoS genetics.

[28]  Ming D. Li,et al.  Genome-wide meta-analyses identify multiple loci associated with smoking behavior , 2010, Nature Genetics.

[29]  M. Miquel,et al.  Have we been ignoring the elephant in the room? Seven arguments for considering the cerebellum as part of addiction circuitry , 2016, Neuroscience & Biobehavioral Reviews.

[30]  L. Wain,et al.  Novel insights into the genetics of smoking behaviour, lung function, and chronic obstructive pulmonary disease (UK BiLEVE): a genetic association study in UK Biobank , 2015, The Lancet. Respiratory medicine.

[31]  S. Shiffman,et al.  DSM criteria for tobacco use disorder and tobacco withdrawal: a critique and proposed revisions for DSM-5. , 2012, Addiction.

[32]  Hongyu Zhao,et al.  Genome-Wide Association Study of Nicotine Dependence in American Populations: Identification of Novel Risk Loci in Both African-Americans and European-Americans , 2015, Biological Psychiatry.

[33]  S. Steinberg,et al.  A rare missense mutation in CHRNA4 associates with smoking behavior and its consequences , 2016, Molecular Psychiatry.

[34]  M. Kanai,et al.  Empirical estimation of genome-wide significance thresholds based on the 1000 Genomes Project data set , 2016, Journal of Human Genetics.

[35]  B. Lushniak,et al.  The Health consequences of smoking—50 years of progress : a report of the Surgeon General , 2014 .

[36]  Gonneke Willemsen,et al.  Heritability of Smoking Initiation and Nicotine Dependence , 2005, Behavior genetics.

[37]  D. Haber,et al.  DNA Methyltransferases Dnmt3a and Dnmt3b Are Essential for De Novo Methylation and Mammalian Development , 1999, Cell.

[38]  N. Martin,et al.  Genome-wide association study on detailed profiles of smoking behavior and nicotine dependence in a twin sample , 2013, Molecular Psychiatry.

[39]  S. Wacholder,et al.  Time to First Morning Cigarette and Risk of Chronic Obstructive Pulmonary Disease: Smokers in the PLCO Cancer Screening Trial , 2015, PloS one.

[40]  K. Fagerström,et al.  Measuring degree of physical dependence to tobacco smoking with reference to individualization of treatment. , 1978, Addictive behaviors.

[41]  P. Lee,et al.  Indirectly estimated absolute lung cancer mortality rates by smoking status and histological type based on a systematic review , 2013, BMC Cancer.

[42]  L. Bierut,et al.  Cis-Regulatory Variants Affect CHRNA5 mRNA Expression in Populations of African and European Ancestry , 2013, PloS one.

[43]  W. Loh,et al.  Are tobacco dependence and withdrawal related amongst heavy smokers? Relevance to conceptualizations of dependence. , 2012, Journal of abnormal psychology.

[44]  S. Steinberg,et al.  Genome-wide meta-analysis reveals common splice site acceptor variant in CHRNA4 associated with nicotine dependence , 2015, Translational Psychiatry.

[45]  Michael Boehnke,et al.  LocusZoom: regional visualization of genome-wide association scan results , 2010, Bioinform..

[46]  Madison,et al.  Quitting Smoking Among Adults — United States, 2001–2010 , 2012 .

[47]  C. Gieger,et al.  Sequence variants at CHRNB3–CHRNA6 and CYP2A6 affect smoking behavior , 2010, Nature Genetics.

[48]  Lung-Ji Chang,et al.  De novo DNA methyltransferases Dnmt3a and Dnmt3b primarily mediate the cytotoxic effect of 5-aza-2′-deoxycytidine , 2005, Oncogene.

[49]  David C. Wilson,et al.  Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease , 2012, Nature.

[50]  Andre Franke,et al.  1000 Genomes-based imputation identifies novel and refined associations for the Wellcome Trust Case Control Consortium phase 1 Data , 2012, European Journal of Human Genetics.

[51]  Jun S. Liu,et al.  The Genotype-Tissue Expression (GTEx) pilot analysis: Multitissue gene regulation in humans , 2015, Science.

[52]  Yun Li,et al.  METAL: fast and efficient meta-analysis of genomewide association scans , 2010, Bioinform..

[53]  E F Domino,et al.  Nicotine effects on regional cerebral blood flow in awake, resting tobacco smokers , 2000, Synapse.

[54]  Manolis Kellis,et al.  HaploReg: a resource for exploring chromatin states, conservation, and regulatory motif alterations within sets of genetically linked variants , 2011, Nucleic Acids Res..

[55]  Scott F. Saccone,et al.  Novel genes identified in a high-density genome wide association study for nicotine dependence. , 2007, Human molecular genetics.

[56]  Frank Seifert,et al.  Smoking and structural brain deficits: a volumetric MR investigation , 2006, The European journal of neuroscience.

[57]  P. Strick,et al.  Cerebellum and nonmotor function. , 2009, Annual review of neuroscience.

[58]  G. Mills,et al.  Genome-wide association scan of tag SNPs identifies a susceptibility locus for lung cancer at 15q25.1 , 2008, Nature Genetics.

[59]  K. Cosgrove,et al.  Rare Nonsynonymous Variants in Alpha-4 Nicotinic Acetylcholine Receptor Gene Protect Against Nicotine Dependence , 2011, Biological Psychiatry.

[60]  Robert A Koeppe,et al.  Regional cerebral blood flow responses to smoking in tobacco smokers after overnight abstinence. , 2005, The American journal of psychiatry.

[61]  Daniel F. Gudbjartsson,et al.  A variant associated with nicotine dependence, lung cancer and peripheral arterial disease , 2008, Nature.

[62]  L. Tanoue.  Quitting Smoking Among Adults — United States, 2001–2010 , 2012 .

[63]  Qian Tao,et al.  DNA methyltransferase 3B (DNMT3B) mutations in ICF syndrome lead to altered epigenetic modifications and aberrant expression of genes regulating development, neurogenesis and immune function. , 2008, Human molecular genetics.

[64]  A. Peña,et al.  Environmental risk factors in inflammatory bowel disease. , 1996, Hepato-gastroenterology.

[65]  Brain grey matter deficits in smokers: focus on the cerebellum , 2012, Brain Structure and Function.

[66]  David Borsook,et al.  The cerebellum and addiction: insights gained from neuroimaging research , 2014, Addiction biology.

[67]  T. Baker,et al.  Measures of affect and nicotine dependence predict differential response to smoking cessation treatments. , 1992, Journal of consulting and clinical psychology.

[68]  Gary K. Chen,et al.  Genome-wide meta-analyses of smoking behaviors in African Americans , 2012, Translational Psychiatry.

[69]  Inês Barroso,et al.  Meta-analysis and imputation refines the association of 15q25 with smoking quantity , 2010, Nature Genetics.

[70]  J. Hokanson,et al.  Genome-wide association study of smoking behaviours in patients with COPD , 2012 .

[71]  N. Volkow,et al.  Dysfunction of the prefrontal cortex in addiction: neuroimaging findings and clinical implications , 2011, Nature Reviews Neuroscience.

[72]  Leif Groop,et al.  The (in)famous GWAS P-value threshold revisited and updated for low-frequency variants , 2016, European Journal of Human Genetics.

[73]  S. Dube,et al.  Vital signs: Current cigarette smoking among adults aged >=18 years --- United States, 2009 , 2010 .

[74]  Tania B. Huedo-Medina,et al.  Assessing heterogeneity in meta-analysis: Q statistic or I2 index? , 2006, Psychological methods.

[75]  E. Li,et al.  Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting , 2004, Nature.