Addiction, Function, and the ENCODE Project
Finding genomic function for genetic associations in nicotine addiction research: The ENCODE project’s role in future pharmacogenomic analysis
The ENCODE Project: ENCODE (Encyclopedia of DNA Elements) is a compilation of searchable data on several types of biochemical functions or “marks” across the genome; tool used in the prediction of SNPs roles in altering genomic function leading to explanations about how changes in DNA sequences are associated with behavioral phenotypes
The role of genetics in tobacco-related traits (smoking and nicotine addiction) has gained attention due to recent associations between these traits and SNPs in 3 of the nicotinic acetylcholine receptor subunit genes (CHRNA5, CHRNA3, and CHRNB4) on chromosome 15q25 as well as with the CYP2A6 gene on chromosome 19, and a new potential function of SNP, rs4105144.
Benefits of the findings in this review:
1. Replication in multiple studies
Liu et al. (2010), Furgberg Tobacco and Genetics Consortium (2010), and Thorgeirrson et al. (2010) found highly signification associatons between two SNPs (rs16969968 in CHRNA5 and rs1051730 in CHRNA3) and cigarettes smoked per day; these two SNPs are in high linkage disequilibrium indicating that their results are not fully independent, making specific associations with smoking behavior difficult
Smokers at highest genetic risk based on these two SNPs exposes themselves to over 700 additional cigarettes over the course of one year.
Possession of one of the risk alleles is associated with a 31% increase in lung cancer risk and possession of both alleles is associated with a 62% increase in lung cancer risk (Munafo et al.).
Benowitz and Jacob (1994), Nakajima et al. (1996), Messina et al. reviewed by Hukkanen et al. (2005), and Koudsi et al. (2010) show that the CYP2A6 gene encodes an enzyme that is the major metabolizer of nicotine, and that functional allelic variation in this gene contribute to differences in nicotine metabolism and nicotine addiction.
Studies on CYP2A6 revealed allelic risk for the chromosome 19 region on cigarettes smoked per day and nicotine dependence (Wassenaar et al. 2011).
Genetic variants that prevent metabolism of nicotine by the CYP2A6 enzyme is shown through the drug methoxsalen, which mimics these variants by blocking the enzyme, diminishing the desire to smoke by modulating nicotine metabolism in the liver (Sellers et al.).
Mutations in CYP2A6 cause lower rates of nicotine metabolism and less smoking (Sellers et al., 2000).
2. Findings are consistent with animal studies that demonstrate some of the brain regions in which these receptor subunit genes are expressed play a role in behavioral responses to nicotine
Nicotinic receptors containing the 3 protein subunits are primarily expressed in the medial habenula (MHb) and its primary output nucleus, the interpeduncular nucleus (IPN).
Gallego et al. (2012) shows the relationship between expression on the A5-A3-B4 cluster genes, the presence of nicotinic receptors containing the subunits, and nicotine withdrawal in mice that over-expressed the cluster. Mice that were over-expressed with the cluster genes shows higher sensitivity to nicotine and higher expression in the MHb region of the brain compared to wild-type mice.
Bierut et al. (2008) shows in mice that the SNP rs16969968 found within the CHRNA5 gene alters function of receptors contacting the alpha-5 subunit, and is associated with smoking-related behaviors in humans.
3. Some of the SNPs are likely to alter the function of the genes in which they occur
Frahm et al. (2011) shows that this SNP is also known as D398N because the mutant allele replaced an aspartate (D) with an asparagine (N) in the alpha-5 subunit. Over-expression of the alpha-5 subunit along with already over-expressed beta-4 resulted in reduced aversion to nicotine in comparison to control that did not receive alpha-5 subunit gene manipulation.
Wang et al. (2009, 2013) showed that several SNPs located near CHRNA5 are related to expression of the gene and are associated with risk of nicotine dependence.
4. The ENCODE project as an aid to predicting functions for tobacco behavior-related SNPs
The major areas on function rested in the ENCODE publication answers the following questions at near single-base resolution:
Is the nucleotide transcribed?
Is the nucleotide part of a transcription factor binding site (TFBS)?
Is the nucleotide part of a DNase I Hypersensitive Site (DHS)?
Is the nucleotide part of a region with altered chromatin marks (histone modifications or DNA methylation)?
Does the nucleotide physically interact with DNA at great distance from it on the chromo- some?
Thorgeiresson et al. (2010) shows that SNP rs41050144 is associated with the number of cigarettes smoked per day based on this SNPs ENCODE data. This SNP is approximately 2700 nucleotides upstream of the CYP2A6 gene, a candidate gene strongly linked to nicotine metabolism and smoking behaviors. The proximity of the SNP to the gene suggested genetic association due to linkage disequilibrium between them. Using ENCODE to search for information related to the SNP, we see that within a DNase I hypersensitive site (light gray bar) that the SNP is possibly located in a region of altered DNA structure, and that the alleles of this SNP might alter expression of a nearby gene/genes.DHS are frequently found in and around genes that are expressed. The dark gray bar for the CIP-seq track shows that one or more transcription factor binds to the region of DNA.
Clicking on the gray box a second time opens a new page called, “ENCODE Transcription Factor ChIP-seq (GR)” (not shown). The page indicates that the transcription factor binding to the DNA around rs4105144 was GR, or glucocorticoid receptor (gene name NR3C1), with supporting evidence that the CYP2A6 gene is regulated by the glucocorticoid receptor (Onica et al., 2008). The hypothesis that alleles at this SNP alter binding of the glucocorticoid receptor and provide a functional explanation for the SNP’s association with a smoking-related trait would not have been contemplated without the ENCODE data.
This review article was interesting in how it incorporated all of these GWAS about a common health problem in order to understand the effects of nicotine and and its effects on behavior. Being able to analyze ENCODE data to determine function on SNPs in genetic studies will allow for greater advancements in pharmacology as well as understanding these addictive behaviors associates with SNPs.