In Vitro and Ex Vivo Analysis of CHRNA3 and CHRNA5 Haplotype Expression

Genome-wide association studies implicate variations in CHRNA5 and CHRNA3 as being associated with nicotine addiction (NA). Multiple common haplotypes (“risk”, “mixed” and “protective”) exist in Europeans; however, high linkage disequilibrium between variations in CHRNA5 and CHRNA3 makes assigning causative allele(s) for NA difficult through genotyping experiments alone. We investigated whether CHRNA5 or CHRNA3 promoter haplotypes, associated previously with NA, might influence allelic expression levels. For in vitro analyses, promoter haplotypes were sub-cloned into a luciferase reporter vector. When assessed in BE(2)-C cells, luciferase expression was equivalent among CHRNA3 haplotypes, but the combination of deletion at rs3841324 and variation at rs503464 decreased CHRNA5 promoter-derived luciferase activity, possibly due to loss of an SP-1 and other site(s). Variation within the CHRNA5 5’UTR at rs55853698 and rs55781567 also altered luciferase expression in BE(2)-C cells. Allelic expression imbalance (AEI) from the “risk” or “protective” haplotypes was assessed in post-mortem brain tissue from individuals heterozygous at coding polymorphisms in CHRNA3 (rs1051730) or CHRNA5 (rs16969968). In most cases, equivalent allelic expression was observed; however, one individual showed CHRNA5 AEI that favored the “protective” allele and that was concordant with heterozygosity at polymorphisms ∼13.5 kb upstream of the CHRNA5 transcription start site. Putative enhancer activity from these distal promoter elements was assessed using heterologous promoter constructs. We observed no differences in promoter activity from the two distal promoter haplotypes examined, but found that the distal promoter region strongly repressed transcription. We conclude that CHRNA5 promoter variants may affect relative risk for NA in some heterozygous individuals.

[1]  C. D. Fowler,et al.  Habenular α5* nicotinic receptor signaling controls nicotine intake , 2011, Nature.

[2]  W. Berrettini,et al.  Acetylcholine Receptor (AChR) α5 Subunit Variant Associated with Risk for Nicotine Dependence and Lung Cancer Reduces (α4β2)2α5 AChR Function , 2011, Molecular Pharmacology.

[3]  U. Pastorino,et al.  Promoter polymorphisms and transcript levels of nicotinic receptor CHRNA5. , 2010, Journal of the National Cancer Institute.

[4]  Ming D. Li,et al.  Associations of Variants in CHRNA5/A3/B4 Gene Cluster with Smoking Behaviors in a Korean Population , 2010, PloS one.

[5]  William Wheeler,et al.  Multiple Independent Loci at Chromosome 15q25.1 Affect Smoking Quantity: a Meta-Analysis and Comparison with Lung Cancer and COPD , 2010, PLoS genetics.

[6]  H. Sasaki,et al.  CHRNA5 gene D398N polymorphism in Japanese lung adenocarcinoma. , 2010, The Journal of surgical research.

[7]  M. Gill,et al.  Evidence for cis-acting regulation of ANK3 and CACNA1C gene expression. , 2010, Bipolar disorders.

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

[9]  Tariq Ahmad,et al.  Meta-analysis and imputation refines the association of 15q25 with smoking quantity , 2010, Nature Genetics.

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

[11]  Z. Herceg,et al.  Aberrant DNA methylation links cancer susceptibility locus 15q25.1 to apoptotic regulation and lung cancer. , 2010, Cancer research.

[12]  G. Basile,et al.  Non smoking for successful aging: therapeutic perspectives. , 2010, Current pharmaceutical design.

[13]  Scott F. Saccone,et al.  The CHRNA5-CHRNA3-CHRNB4 nicotinic receptor subunit gene cluster affects risk for nicotine dependence in African-Americans and in European-Americans. , 2009, Cancer research.

[14]  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.

[15]  John P. Rice,et al.  Multiple distinct risk loci for nicotine dependence identified by dense coverage of the complete family of nicotinic receptor subunit (CHRN) genes , 2009, American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics.

[16]  K. Shianna,et al.  A Genome-Wide Association Study in Chronic Obstructive Pulmonary Disease (COPD): Identification of Two Major Susceptibility Loci , 2009, PLoS genetics.

[17]  William Wheeler,et al.  Genome-Wide and Candidate Gene Association Study of Cigarette Smoking Behaviors , 2009, PloS one.

[18]  M. Spitz,et al.  The CHRNA5-A3 region on chromosome 15q24-25.1 is a risk factor both for nicotine dependence and for lung cancer. , 2008, Journal of the National Cancer Institute.

[19]  Andrew D. Johnson,et al.  Polymorphisms affecting gene transcription and mRNA processing in pharmacogenetic candidate genes: detection through allelic expression imbalance in human target tissues , 2008, Pharmacogenetics and genomics.

[20]  Tatiana Foroud,et al.  Variants in nicotinic receptors and risk for nicotine dependence. , 2008, The American journal of psychiatry.

[21]  H. Wit Faculty Opinions recommendation of Alpha-5/alpha-3 nicotinic receptor subunit alleles increase risk for heavy smoking. , 2008 .

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

[23]  P. Muglia,et al.  α-5/α-3 nicotinic receptor subunit alleles increase risk for heavy smoking , 2008, Molecular Psychiatry.

[24]  Andrew D. Johnson,et al.  Allelic Expression Imbalance of Human mu Opioid Receptor (OPRM1) Caused by Variant A118G* , 2005, Journal of Biological Chemistry.

[25]  Michael C O'Donovan,et al.  Strong bias in the location of functional promoter polymorphisms , 2005, Human mutation.

[26]  Michael C O'Donovan,et al.  Haplotypes at the dystrobrevin binding protein 1 (DTNBP1) gene locus mediate risk for schizophrenia through reduced DTNBP1 expression. , 2005, Human molecular genetics.

[27]  Mark Daly,et al.  Haploview: analysis and visualization of LD and haplotype maps , 2005, Bioinform..

[28]  M. Owen,et al.  Allelic expression of APOE in human brain: effects of epsilon status and promoter haplotypes. , 2004, Human molecular genetics.

[29]  A. C. Collins,et al.  Nicotine Activation of α4* Receptors: Sufficient for Reward, Tolerance, and Sensitization , 2004, Science.

[30]  S. Gabriel,et al.  The Structure of Haplotype Blocks in the Human Genome , 2002, Science.

[31]  C. Evans,et al.  Surgeon general's report. , 2001, Journal of the American Dental Association.

[32]  S. Terzano,et al.  Transcriptional regulation of the human alpha5 nicotinic receptor subunit gene in neuronal and non-neuronal tissues. , 2000, European journal of pharmacology.

[33]  Qun Lu,et al.  Habenular a5 nicotinic receptor subunit signalling controls nicotine intake , 2011 .

[34]  Ryan M. Smith,et al.  Nicotinic α5 receptor subunit mRNA expression is associated with distant 5′ upstream polymorphisms , 2011, European Journal of Human Genetics.

[35]  C. Gieger,et al.  Sequence variants at CHRNB 3 – CHRNA 6 and CYP 2 A 6 affect smoking behavior , 2010 .

[36]  Y. Mineur,et al.  Genetics of nicotinic acetylcholine receptors: Relevance to nicotine addiction. , 2008, Biochemical pharmacology.

[37]  Nicholas G Martin,et al.  Cholinergic nicotinic receptor genes implicated in a nicotine dependence association study targeting 348 candidate genes with 3713 SNPs. , 2007, Human molecular genetics.

[38]  Office on Smoking The Health Consequences of Smoking: A Report of the Surgeon General , 2004 .