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 -31.6 (A to G), -297 (T to A), -35 (G to T), 1107 (G to C; intron 1), 4804 (G to A Val(179)Ile; 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 Val(179)Ile 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,,T, none appeared to be of functional significance. Pharmacogenetics 8:543-552. (C) 1998 Lippincott Williams & Wilkins.