This study validates the importance of genetic variants within the α5-α3-β4 nicotinic acetylcholine receptor gene cluster that contribute to the risk of a light smoker transitioning to heavy smoking and sheds light on a potential biological mechanism. There are two distinct genetic associations – one marked by rs16969968 which results in an amino acid change in the α5 nicotinic cholinergic receptor (CHRNA5)
and a second marked by a SNP rs578776 in the 3’ untranslated region of the α3 nicotinic cholinergic receptor (CHRNA3). Because the r2
between these two variants is low (0.15), the statistical significance at both cannot be explained solely by the linkage disequilibrium between them. Therefore, these data imply that there are two independent loci in this region that alter the risk for nicotine dependence and habitual smoking. These findings confirm our previous results in a case-control series that examined nicotine dependence (6
) and the recent work by Berrettini and colleagues that studied heavy smoking (36
In these three genetic studies that demonstrated the associations, there are important differences in the definition of phenotypes, recruitment procedures, and analytic methods. The COGA sample was recruited as a high-risk population for alcoholism, and a broader smoking phenotype was used: habitual smoking, defined as smoking 20 cigarettes a day for 6 months or more, and light smoking, defined as smoking 10 cigarettes or fewer per day. A family based analytic design for association was used which is less likely to be biased by population stratification. The previous genetic study sample by our group was recruited from the community, the phenotypic status was defined by the Fagerström Test for Nicotine Dependence, and a case control design of unrelated individuals was used (5
). Similar SNPs were analyzed in these two studies. The recent publication by Berrettini was based on genetic studies of heart disease and other common illnesses in a population based sample of 15,000 people (36
). Smoking status was collected and a quantitative phenotype defined by cigarettes per day was studied in a secondary genetic analysis. Though different genetic variants were tested in the Berrettini sample, the associated SNPs are highly correlated (in linkage disequilibrium) with the variants in our studies. The consistency of results across three different studies shows that these genetic findings are robust across populations, phenotypic classification systems, and analytic methods.
Three additional research groups recently published strong evidence of genetic association of the α5-α3-β4 nicotinic acetylcholine receptor gene cluster on chromosome 15 with lung cancer(37
). The findings highlighted in their work are highly correlated with the genetic variant that results in the amino acid change in the α5 nicotinic acetylcholine receptor gene. The groups differed in their interpretation of whether this genetic association with lung cancer acts through the indirect effect of smoking or whether this variant also directly increases the vulnerability to lung cancer.
When a genetic association is found, it represents not only association with the tested variants, but all genetic variants (tested and untested) that are highly correlated. Identifying the variant that causes functional changes requires biological investigations. We have focused on the amino acid change in the α5 nicotinic cholinergic receptor for further studies.
The aspartic acid at position 398 in CHRNA5
in humans occurs at a residue that is otherwise invariant across vertebrate species. Frogs, chickens, rodents, cattle and non-human primates all possess an aspartic acid residue at this location. In humans, the amino acid may be either an aspartic acid, the predominant residue at this position, or asparagine. The α5 nicotinic subunit is not involved in receptor binding in vivo
) , and this variant is located in the cytoplasmic loop between transmembrane domains.
Evidence that the amino acid change is functionally relevant is supported by the fact that in vitro
, α4β2α5nicotinic receptors with the aspartic acid variant (D398) exhibited a greater maximal response to a nicotinic agonist than did α4β2α5 nicotinic receptors with the asparagine amino acid substitution (N398). Because the allele that codes for asparagine is associated with increased risk for developing nicotine dependence, and nicotinic receptors containing the α5 subunit with this amino acid (N398) exhibit reduced function in vitro
, reduced function of α4β2α5 nicotinic receptors may lead to an elevated risk for developing nicotine dependence. The observation that decreased nAChR function is associated with increased risk for nicotine dependence is consistent with the observation that individuals who are extensive metabolizers of nicotine (reduced receptor activation per cigarette) are at increased risk for nicotine dependence (40
). We believe that this combined evidence of high conservation across species and biological change in receptor function supports the amino acid variant in the α5 nicotinic receptor as a causative biological factor that alters the risk of nicotine dependence, though we cannot definitively rule out the other correlated SNPs across the three gene cluster.
The α4β2α5-containing nicotinic receptors are expressed on dopaminergic neurons in the striatum (16
) where they modulate nicotine-stimulated dopamine release (15
). In addition, α4β2α5 nicotinic receptors also are found on GABAergic neurons in the striatum and ventral tegmental area (42
).This region of the brain is associated with the reward pathway, and the neurotransmitter dopamine plays a crucial role in the development of dependence. Individuals with reduced α4β2α5 cholinergic receptor activity may require greater amounts of nicotine to achieve the same activation of the dopaminergic pathway. Alternatively, reduced activity of the receptor complex on GABAergic neurons may lead to increased dopaminergic activity in response to nicotine. How the altered receptor activity caused by the CHRNA5
amino acid change modifies liability to nicotine dependence via the reward system in response to nicotine requires further study.
The “at risk” allele differs dramatically across human populations. It is predominantly seen in populations of European and Middle Eastern descent and is uncommon or non-existent in populations of African, Asian, or American origin. Interestingly, African Americans have a lower prevalence of nicotine dependence than European Americans (43
), and this may be explained in part by the low prevalence of this risk allele in the populations of African descent.
There is less information available regarding the second independent finding marked by the genetic variant rs578776 in this gene cluster. This SNP, rs578776, is located in the 3’ untranslated region of the CHRNA3 gene. The 3’ untranslated regions contain regulatory sequences, and we can speculate that this SNP is a putative functional variant. It is important to also note that there are correlated SNPs with rs578776 in CHRNA5 and CHRNA3 and the localization of the functional alleles may be in either gene. Further experiments are needed to identify the potential functional variants and the biologic mechanisms.
In summary, there are at least two independent genetic variants in the CHRNA5-CHRNA3-CHRNB4 gene cluster on chromosome 15 which are highly associated with smoking behaviors, and we have extended our work to identify a potential biological mechanism for one of the findings. This study provides strong evidence that an amino acid change in the α5 nicotinic receptor, which is highly conserved across species, results in a functional change that increases a smoker’s risk of transitioning from non-dependence (light smoking) to dependence (habitual smoking) on nicotine. This variant is common in populations of European and Middle Eastern descent and increases the risk of developing nicotine dependence, but rare in populations of African, American and Asian descent. Intriguingly, three recent papers demonstrated that this genetic locus also contributes to the risk of developing lung cancer. A second independent finding in this gene cluster is also seen, though further study is needed to localize the potential functional allele and to determine whether the variant affects function of the CHRNA5 or CHRNA3 gene. These converging genetic associations and biological data support the importance of CHRNA5 and potentially CHRNA3 in the development of nicotine dependence and highlight the pharmacogenetic response to nicotine which increases the susceptibility to dependence. Importantly, this finding may help predict response to pharmacologic therapies, such as varenicline and nicotine replacement, for those smokers who attempt to quit, and may shed important light onto the biological mechanisms that contribute to lung cancer.