Functional characterization of SNPs in CHRNA3/B4 intergenic region associated with drug behaviors

Abstract

The cluster of human neuronal nicotinic receptor genes (CHRNA5/A3/B4) (15q25.1) has been associated with a variety of smoking and drug-related behaviors, as well as risk for lung cancer. CHRNA3/B4 intergenic single nucleotide polymorphisms (SNPs) rs1948 and rs8023462 have been associated with early initiation of alcohol and tobacco use, and rs6495309 has been associated with nicotine dependence and risk for lung cancer. An in vitro luciferase expression assay was used to determine whether these SNPs and surrounding sequences contribute to differences in gene expression using cell lines either expressing proteins characteristic of neuronal tissue or derived from lung cancers. Electrophoretic mobility shift assays (EMSAs) were performed to investigate whether nuclear proteins from these cell lines bind SNP alleles differentially. Results from expression assays were dependent on cell culture type and haplotype. EMSAs indicated that rs8023462 and rs6495309 bind nuclear proteins in an allele-specific way. Additionally, GATA transcription factors appeared to bind rs8023462 only when the minor/risk allele was present. Much work has been done to describe the rat Chrnb4/a3 intergenic region, but few studies have examined the human intergenic region effects on expression; therefore, these studies greatly aid human genetic research as it relates to observed nicotine phenotypes, lung cancer risk and potential underlying genetic mechanisms. Data from these experiments support the hypothesis that SNPs associated with human addiction-related phenotypes and lung cancer risk can affect gene expression, and are potential therapeutic targets. Additionally, this is the first evidence that rs8023462 interacts with GATA transcription factors to influence gene expression.

Pleiotropic effects of Chr15q25 nicotinic gene cluster and the relationship between smoking, cognition and ADHD

“…The C allele of this same SNP has been previously associated with externalizing and delinquent behaviors (Stephens et al, 2012). Remarkably, experimental studies demonstrated that the GATA transcription factors, involved in fine receptor regulation during development, are only capable of binding when the C allele is present (Flora et al, 2013).…”

“…Flora and collaborators suggest that this transcription factor might play a role in controlling nicotinic receptor subunit expression in specific cell lines and developmental stages, which could be negatively affected by tobacco exposure (Flora et al, 2013).…”

Evidence for Association Between Low Frequency Variants in CHRNA6/CHRNB3 and Antisocial Drug Dependence

“…The four nonsynonymous variants classified as probably damaging by Polyphen-2 may be of interest for further analysis. We found twice as many variants in CHRNA6 introns and UTRs in cases compared to control, which may lead to subtle differences in transcription (Ehringer et al, 2010; Flora et al, 2013; Gallego et al, 2013; Kamens et al, 2015). …”

“…Non-coding regions in the Chrn genes in rodents have been shown to play an important role in gene regulation (Bruschweiler-Li et al, 2010; McDonough and Deneris, 1997; McDonough et al, 2000; Scofield et al, 2010; Xu et al, 2006). Moreover, in vitro gene expression studies using cell culture models to examine human genetic variants in the non-coding regions of CHRN genes have demonstrated differential functional effects for SNP alleles (Ehringer et al, 2010; Flora et al, 2013; Gallego et al, 2013; Kamens et al, 2015). Because expression of individual nicotinic receptor subunits is tightly regulated, both temporally and spatially (Albuquerque et al, 2009; Gotti et al, 2006, 2009), the importance of genetic variants in non-coding regions must also be explored.…”

Scientific overview: 2013 BBC plenary symposium on tobacco addiction

“…The α5 risk variant, rs16969968 G/A, causes an Asp398Asn amino acid substitution, and the risk allele (Asn398) produces hypofunctional α5-containing nAChRs with reduced Ca 2+ permeability and faster desensitization rates than non-risk alleles (Bierut et al, 2008; Kuryatow et al, 2011). Other polymorphisms are associated with different levels of α5 or α3 mRNA (Wang, 2009), and the functional significance of several other gene variants in the CHRNA5/A3/B4 gene cluster is currently being investigated (Flora et al, 2013). …”