This large study of nicotine dependent smokers and non-dependent smokers tested the hypothesis that variants in nicotine metabolizing genes are associated with nicotine dependence. This hypothesis is based on the theory that nicotine metabolism, which varies between individuals, can influence smoking behaviors and alter the risk of developing nicotine dependence. We studied polymorphisms in genes that potentially metabolize nicotine: CYP2A6, CYP2B6, the FMO gene family, and the UGT gene family.
A number of polymorphisms in FMO gene family members showed association with nicotine dependence in our discovery sample. We followed 5 of these polymorphisms into a replication sample of heavy and light smokers and a significant association remained with 2 of these SNPs. Two highly correlated SNPs in FMO1 (rs7877, rs10912675) show significant association in the replication sample after Bonferroni correction (nominal p-values 0.0192 and 0.0067, respectively; corrected p-values 0.0384 and 0.0134, respectively). In both samples, these SNPs have similar risks of developing nicotine dependence or heavy smoking (OR of 0.77 and 0.80 for nicotine dependence, respectively; OR of 0.87 and 0.85 for heavy smoking, respectively). These two SNPs are located in the 3′ UTR and 5′ UTR of FMO1, respectively, suggesting a potential role for regulation of gene expression. These SNPs are not known to be in high LD with any exonic variants and their exact function is unclear. Had we used association tests on the CPD measure in the initial sample (more directly comparable to the phenotype in the replication sample), we would have followed up many of the same SNPs and would have tagged the bin that shows replication.
Because human FMO1 was not known to metabolize nicotine, we determined the catalytic efficiency of nicotine N
-oxidation by this enzyme. In vitro
experiments established that human FMO1, like pig FMO1 [14
] catalyzes the non-stereospecific N-oxidation of nicotine. Contrary to conventional wisdom, FMO1, an extrahepatic enzyme in humans, is a better catalyst of nicotine N
-oxidation than the hepatic enzyme, FMO3. Although the KM
of FMO1 is relatively high, it is lower than that of FMO3. We also note that the KM
for CYP2A6 is 140 μM, also well above the plasma concentration of nicotine [15
In adult humans, FMO1 is an extrahepatic enzyme with relatively high levels of expression in the kidney and shows moderate inter-individual variability in protein levels [16
]. FMO1 is likely to be a major contributor to renal metabolism and clearance of therapeutic drugs [16
]. However, we report here that FMO1-catalyzed nicotine metabolism results in the formation of approximately equal amounts of cis
-oxide, whereas only trans
-oxide has been detected in the urine of smokers [14
]. This suggests that renal FMO1 does not contribute significantly to the formation of nicotine N
-oxide excreted by smokers. However, FMO1 may play a role in nicotine metabolism in other extrahepatic tissues. FMOs are expressed in the human brain [17
] and may contribute to the level of nicotine present in this organ. One of the two or more FMOs present in the human brain has been purified and partially characterized [18
]. This brain FMO, based on substrate specificity, is likely FMO1 because it catalyzes the N
-oxidation of imipramine, an FMO1 mediated reaction [19
]. If FMO1 activity catalyzed the N
-oxidation of nicotine in the brains of smokers, the nicotine N
-oxide formed could serve as a substrate pool available for reduction back to nicotine. The reduction of the N
-oxide of tertiary amines has been suggested to play a role in the pharmacology of both imipramine and tamoxifen [20
There are some limitations to this study that need to be noted. While our work highlights the potential role of FMO1
in the development of nicotine dependence, heavy smoking, and nicotine metabolism, we have not shown an association of the polymorphisms in FMO1
with nicotine metabolism. Nor have we demonstrated a biological mechanism by which these polymorphisms contribute to nicotine metabolism. Secondly, although the SNPs associated with nicotine dependence lie in FMO1,
highly correlated SNPs span the entire cluster of FMO
genes, indicating that causal variant(s) may be in one of the other FMO
genes. In addition, this study was undertaken in subjects of European descent and these findings may not generalize to other populations. This is important because it is known that nicotine metabolism varies between populations [22
]. Finally, we note that enzyme was not highlighted in three recent meta-analyses of smoking behavior [24
]. Our finding may therefore be a replicated false positive. However, this discrepancy could also be due to heterogeneous sample selection, different assessment such as current cigarettes per day, coverage from different platforms, and a relatively small effect size for the variants in FMO1.
In this study, we did not find common variants in CYP2A6 associated with nicotine dependence. However, our coverage of CYP2A6 is minimal, including only two SNPs, neither of which are known to alter CYP2A6 activity. Our negative association does not rule out the importance of CYP2A6 in the development of nicotine metabolism. Furthermore, although the other nicotine metabolizing genes received better coverage, most rare and some common variants were not tagged.
In summary, the association of polymorphisms in FMO1 with nicotine dependence and heavy smoking along with the demonstration that this enzyme is a catalyst of nicotine N-oxidation suggest that polymorphisms in FMO1 may be significant risk factors in the development of nicotine dependence. The mechanism by which this enzyme plays a role in nicotine dependence is unclear; however the role of brain metabolism in the pharmacology of nicotine warrants further study.