CHRNB2 promoter region: association with subjective effects to nicotine and gene expression differences

Abstract

Smoking behavior is a complex, which includes multiple stages in the progression from experimentation to continued use and dependence. The experience of subjective effects, such as dizziness, euphoria, heart pounding, nausea and high, have been associated with varying degrees of persistence and subsequent abuse/dependence of marijuana, cocaine, tobacco and alcohol (Grant et al. 2005, Wagner & Anthony 2002). Previous studies have reported associations between neuronal nicotinic receptor (CHRN) genes and subjective effects to nicotine. We sought to replicate and expand this work by examining eight single nucleotide polymorphisms (SNPs) in a sample of adult smokers (n = 316) who reported subjective effects following cigarette smoking in a controlled laboratory environment. Two SNPs each in the CHRNB2, CHRNB3, CHRNA6 and CHRNA4 genes were examined. A significant association was found between two SNPs and physical effects reported after smoking the first experimental cigarette. SNP rs2072658 is upstream of CHRNB2 (P-value = 0.0046) and rs2229959 is a synonymous change in exon 5 of CHRNA4 (P value = 0.0051). We also examined possible functional relevance of SNP rs2072658 using an in vitro gene expression assay. These studies provided evidence that the minor allele of rs2072658 may lead to decreased gene expression, using two separate cell lines, P19 and SH-SY5Y (18% P < 0.001 and 26% P < 0.001 respectively). The human genetic study and functional assays suggest that variation in the promoter region of CHRNB2 gene may be important in mediating levels of expression of the β2 nicotinic receptor subunit, which may be associated with variation in subjective response to nicotine.

Figure 1

Mean physical effects score by rs2072658 genotype. Individuals with the AG genotype have significantly higher physical effects scores than individuals with the GG (common) genotype after smoking the first experimental cigarette (n=17 mean=21.3 and n=282 mean=11.6 respectively). There were no individuals in this study with the rare AA genotype. Error bars represent a 95% confidence interval on the mean, * indicates p<0.05.

Figure 2

Diagram of the human CHRNB2 promoter region and known SNPs from the UCSC Genome Browser March 2006 update (http://genome.ucsc.edu/), cross-referenced with dbSNP (http://www.ncbi.nlm.nih.gov/SNP/) and Ensembl (http://www.ensembl.org/).

Figure 3

Relative luciferase activity across cell types for three concentrations of nicotine. No significant differences were observed in relative activity between 0 μM, 1 μM and 10 μM chronic nicotine exposure. Error bars represent a 95% confidence interval on the mean.

Figure 4

Relative luciferase activity of four constructs in four cell types. The target polymorphism rs2072658 (minor allele A shaded grey) shows a significant reduction in expression in SH-SY5Y, P19, and differentiated SH-SY5Y cells, indicated by an asterisk. This reduction in relative luciferase activity is independent of other local SNP variation (major vs minor). Error bars represent a 95% confidence interval on the mean, * indicates p<0.05.