Variants in nicotinic receptors and risk for nicotine dependence

Abstract

Objective: A recent study provisionally identified numerous genetic variants as risk factors for the transition from smoking to the development of nicotine dependence, including an amino acid change in the alpha5 nicotinic cholinergic receptor (CHRNA5). The purpose of this study was to replicate these findings in an independent data set and more thoroughly investigate the role of genetic variation in the cluster of physically linked nicotinic receptors, CHRNA5-CHRNA3-CHRNB4, and the risk of smoking.

Method: Individuals from 219 European American families (N=2,284) were genotyped across this gene cluster to test the genetic association with smoking. The frequency of the amino acid variant (rs16969968) was studied in 995 individuals from diverse ethnic populations. In vitro studies were performed to directly test whether the amino acid variant in the CHRNA5 influences receptor function.

Results: A genetic variant marking an amino acid change showed association with the smoking phenotype (p=0.007). This variant is within a highly conserved region across nonhuman species, but its frequency varied across human populations (0% in African populations to 37% in European populations). Furthermore, functional studies demonstrated that the risk allele decreased response to a nicotine agonist. A second independent finding was seen at rs578776 (p=0.003), and the functional significance of this association remains unknown.

Conclusions: This study confirms that at least two independent variants in this nicotinic receptor gene cluster contribute to the development of habitual smoking in some populations, and it underscores the importance of multiple genetic variants contributing to the development of common diseases in various populations.

Figure 1

Comparative sequence analysis surrounding aspartate D 398 of the α5 nicotinic receptor. The amino acid change to asparagine in the variant receptor (rs16969968).

Figure 2

Allele frequency distribution (%) of cSNP rs16969968 in different ethnic populations (A allele is white segment). Populations were grouped together based on their genetic structures reported by Rosenberg et al (23).

Figure 3

The CHRNA5 D398N polymorphism affects nAChR function but not expression of α4β2α5 nAChRs. A. Concentration-response curves for epibatidine-evoked changes in intracellular calcium were measured from HEK293T cells transfected with plasmids containing a calcium-sensing aequorin cDNA, α4 and β2 cDNAs and either α5D398 or α5N398 cDNA. Two-way ANOVA indicated that the response curves for each nAChR variant were significantly different (n = 12 per variant, p < 0.0001). However, EC₅₀ values were not altered by the variant forms of α5 (n = 12, p = 0.098). B. nAChRs possessing the D398 variant of α5 also exhibited a significantly greater maximal response to epibatidine as compared to nAChRs containing the N398 variant (n = 12 per variant, p < 0.0001). C. [¹²⁵I] epibatidine binding on membranes prepared from cells used for the aequorin assay indicate that there is no difference in the expression of the receptors when either the D398 or N398 variant of α5 is co-transfected with α4 and β2 (n = 12, p = 0.375). D. Western analysis demonstrates that the variant forms of α5 do not differ in expression (n = 4 per variant, p = 0.78). Inset: sample western from α4β2α5D398 (D398) α4β2α5N398 (N398) and α4β2 (-) transfected cells. Data shown for all graphs are mean ± SEM.