Human genetic studies have identified the cluster of nicotinic receptor genes (CHRNA5/A3/B4) on chromosome 15 as strong candidates for association with drug abuse-related behaviors, including smoking, alcohol phenotypes, and cocaine dependence. These genes have also been associated with lung cancer, but it is remains unclear what aspects of this association may be mediated through smoking behavior. In addition, we have found evidence that the intergenic region between the CHRNA3 and CHRNB4 genes is associated with more general measures of disinhibitory behavior, including conduct disorder, which is a known risk factor for early initiation of drugs and later problems. There is evidence from molecular, pharmacological, and animal studies that transcriptional regulation of these genes is likely to be co-regulated and complex, and that the 13 and 24 subunits might be good targets for development of smoking cessation drugs. We have initiated studies to assess the putative functional differences of alleles for SNPs that have been implicated in our human genetic studies. Our results provide evidence that at least two SNPs in this region may lead to differences in gene expression, using luciferase-gene assays in immortalized cell culture lines and in primary mouse neuronal embryonic cell cultures. The goals of this application are to extend these findings in several ways. First, we will use these assays to evaluate how the DNA sequence surrounding these SNPs may affect their ability to differentially drive gene expression ("promoter bashing"). Second, we will examine the effects of these SNPs in different cell types, including a variety of immortalized neuronal cells, lung cancer cells, and primary mouse neuronal embryonic cells (gene x cell type interaction). Third, we will challenge the cells with nicotine to assess whether this drug modifies gene expression patterns (gene x environment interaction). Fourth, we will determine whether allele-specific changes in gene expression using the luciferase assays lead to corresponding differences in protein subunit and receptor expression. Finally, we will assess function of nicotinic receptors whose subunit constituents were generated using our in vitro system containing different alleles for individual SNPs and haplotypes of SNPs. We expect the methods developed and applied to these genes to be easily applied to future studies of other nicotinic receptor genes that may also be associated with drug abuse-related behaviors. PUBLIC HEALTH RELEVANCE: Results from this project will facilitate a better understanding of how naturally occurring variations in the CHRNA3 and CHRNB4 genes might contribute to the underlying molecular mechanisms responsible for individual differences in behaviors relevant to drug use and abuse. Such knowledge should lead to the development of improved prevention and treatment of individuals who suffer from these disorders.

R21DA026901

2009-09-30

2013-08-31