Detection and characterization of novel polymorphisms in the CYP2E1 gene

: To investigate whether interindividual variation in CYP2E1 levels can be explained by genetic polymorphism, we analysed DNA samples from 40 healthy individuals by single-strand conformational polymorphism analysis for polymorphisms in the CYP2E1 coding sequence and promoter region. DNA sequencing of samples showing mobility shifts on single-strand conformational polymorphism detected polymorphisms at positions -316 (A to G), -297 (T to A), -35 (G to T), 1107 (G to C; intron 1), 4804 (G to A Val179Ile; exon 4) and 10157 (C to T; exon 8). All individuals positive for either A(-316)G, G(-35)T, G(4804)A or the previously described RsaI polymorphism at -1019 were also positive for T(-297)A, which had the highest allele frequency of the observed polymorphisms (0.20). A(-316)G, G(-35)T and G(4804)A were detected at allele frequencies of 0.022, 0.052 and 0.013, respectively. The functional significance of the upstream polymorphisms was examined by preparing constructs of positions -549 to +3 of CYP2E1 containing the observed combinations of the polymorphisms fused to luciferase reporter genes and transfecting HepG2 cells. For the G(-35)T/T(-297)A construct, a 1.8-fold increase in luciferase activity compared with the wild-type sequence (P = 0.06) and 2.5-fold compared with T(-297)A only (P = 0.025) was observed. No significant difference in activity was observed between the other constructs. The significance of the predicted Val179Ile base change from G(4804)A was determined by expression of the wild-type and mutated full length cDNAs in lymphoblastoid cells. No significant difference in kinetic constants for chlorzoxazone hydroxylation between mutant and wild-type was observed. In summary, this study demonstrated six novel CYP2E1 polymorphisms, including three upstream of the promoter, but with the possible exception of G(-35)T, none appeared to be of functional significance.