title: In vitro and ex vivo analysis of CHRNA3 and CHRNA5 haplotype expression.
abstract: 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.
authors.affiliation: Center for Neurobiology and Behavior, Department of Psychiatry, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America. gadoyle@mail.med.upenn.edu
authors.forename: Glenn A
authors.initials: GA
authors.lastname: Doyle
authors.forename: Min-Jung
authors.initials: MJ
authors.lastname: Wang
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Chou
authors.forename: John U
authors.initials: JU
authors.lastname: Oleynick
authors.forename: Steven E
authors.initials: SE
authors.lastname: Arnold
authors.forename: Russell J
authors.initials: RJ
authors.lastname: Buono
authors.forename: Thomas N
authors.initials: TN
authors.lastname: Ferraro
authors.forename: Wade H
authors.initials: WH
authors.lastname: Berrettini
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D001344:Autopsy; D001665:Binding Sites / Q000235:genetics; D001921:Brain / Q000378:metabolism; D045744:Cell Line, Tumor; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D005787:Gene Frequency; D020022:Genetic Predisposition to Disease / Q000235:genetics; D005838:Genotype; D006239:Haplotypes / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D011978:Receptors, Nicotinic / Q000235:genetics*; D012307:Risk Factors; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014029:Tobacco Use Disorder / Q000235:genetics; D044465:White People / Q000235:genetics
chemical_list: D020121:5' Untranslated Regions; D008156:Luciferases; D009419:Nerve Tissue Proteins; D011978:Receptors, Nicotinic; D016329:Sp1 Transcription Factor; C447158:CHRNA5 protein, human