Detection and characterization of novel polymorphisms in the CYP2E1 gene

To investigate whether interindividual variation in CYP2E1 levels can be ex plained by genetic polymorphism, we analysed DNA samples from 40 healthy in dividuals by single-strand conformational polymorphism analysis for polymor phisms in the CYP2E1 coding sequence and promoter region. DNA sequencing of samples showing mobility shifts on single-strand conformational polymorphi sm 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 101 57 (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 observ ed polymorphisms (0.20). A(-316)G, G(-35)T and G(4804)A were detected at al lele frequencies of 0.022, 0.052 and 0.013, respectively. The functional si gnificance of the upstream polymorphisms was examined by preparing construc ts of positions -549 to +3 of CYP2E1 containing the observed combinations o f the polymorphisms fused to luciferase reporter genes and transfecting Hep G2 cells. For the G(-35)T/T(-297)A construct, a 1.8-fold increase in lucife rase 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 differ ence in activity was observed between the other constructs. The significanc e 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 hy droxylation between mutant and wild-type was observed. In summary, this Stu dy 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 Lippincot t Williams & Wilkins.