The contribution of common CYP2A6 alleles to variation in nicotine metabolism among European-Americans

Abstract

Objective: To study the association between cytochrome P450 2A6 (CYP2A6) genotype and metabolism of nicotine to cotinine, identify functional polymorphisms, and develop a predictive genetic model of nicotine metabolism.

Methods: The conversion of deuterated (D2)-nicotine to D2-cotinine was quantified in 189 European-Americans and the contribution of CYP2A6 genotype to variability in first-pass nicotine metabolism was assessed. Specifically, (i) single time point measures of D2-cotinine/(D2-cotinine+D2-nicotine) after oral administration were used as a metric of CYP2A6 activity; (ii) the impact of CYP2A6 haplotype was treated as acting multiplicatively; (iii) parameter estimates were calculated for all haplotypes in the subject pool, defined by a set of polymorphisms previously reported to affect function, including gene copy number; and (iv) a minimum number of predictive polymorphisms were justified to be included in the model based on statistical evidence of differences between haplotypes.

Results: The final model includes seven polymorphisms and fits the phenotype, 30-min after D2-nicotine oral administration, with R=0.719. The predictive power of the model is robust: parameter estimates calculated in men (n=89) predict the phenotype in women (n=100) with R=0.758 and vice versa with R=0.617; estimates calculated in current smokers (n=102) predict the phenotype in former-smokers (n=86) with R=0.690 and vice versa with R=0.703. Comparisons of haplotypes also demonstrate that CYP2A6*12 is a loss-of-function allele indistinguishable from CYP2A6*4 and CYP2A6*2 and that the CYP2A6*1B 5'-untranslated region conversion has negligible impact on metabolism. After controlling for CYP2A6 genotype, modest associations were found between increased metabolism and both female sex (P=4.8×10) and current smoking (P=0.02).

Conclusion: Among European-Americans, seven polymorphisms in the CYP2A6 gene explain the majority of variability in the metabolism of nicotine to cotinine after oral administration. Parameters determined from this in-vivo experiment can be used to predict nicotine metabolism based on CYP2A6 genotype.

Figure 1

CYP2A6 diplotype/ phenotype association. Time 1 (t1) =30 min. The boxplot provides a summary of the data distribution. The box represents the interquartile range, which includes 50% of values. The line across the box indicates the median. The whisker lines extend to the highest and lowest values that are within 1.5x the interquartile range. Further outliers are marked with circles.

Figure 2

CYP2A6 diplotype/ phenotype association among normal metabolizer alleles, CYP2A6*1B,*1D,*1H, and *14. Time 1 (t1) =30 min. The boxplot provides a summary of the data distribution. The box represents the interquartile range, which includes 50% of values. The line across the box indicates the median. The whisker lines extend to the highest and lowest values that are within 1.5x the interquartile range. Further outliers are marked with circles. N = CYP2A6 normal metabolizer haplotypes (*1B,*1D,*1H,*14); *1A = CYP2A6*1A(51A);*9 = CYP2A6*9; L = CYP2A6 putative loss-of-function haplotypes (*2, *4, *12). The N/N (n=95) group is significantly greater than the N/*1A (n=33, p=0.0001), N/*9 (n=20, p=6.1×10⁻⁵), and *1A/*1A (n=8, p=0.008) groups. The L/N (n=17) group is significantly greater than the L/*1A group (n=4, p=0.0002).

Figure 3

The Distribution of log(D₂cotinine/D₂nicotine)(A) and D₂cotinine/(D₂cotinine+D₂nicotine) (B) measured at time1 (t1, 30 minutes) among 189 European American subjects displayed as a cumulative histogram color-coded by diplotype class. White = normal/normal, Yellow = normal/intermediate, Green = intermediate/intermediate, Blue = normal/null, Purple = intermediate/null, Red = null/null. Normal haplotypes are *1A(51G),*1B, *1D,*1H and *14. Intermediate haplotypes are *1A(51A) and *9. Null haplotypes are *1D-Y351H, *2, *4 and *12. Genotype is not further differentiated.

Figure 3

The Distribution of log(D₂cotinine/D₂nicotine)(A) and D₂cotinine/(D₂cotinine+D₂nicotine) (B) measured at time1 (t1, 30 minutes) among 189 European American subjects displayed as a cumulative histogram color-coded by diplotype class. White = normal/normal, Yellow = normal/intermediate, Green = intermediate/intermediate, Blue = normal/null, Purple = intermediate/null, Red = null/null. Normal haplotypes are *1A(51G),*1B, *1D,*1H and *14. Intermediate haplotypes are *1A(51A) and *9. Null haplotypes are *1D-Y351H, *2, *4 and *12. Genotype is not further differentiated.