CYP3A4 allelic variants with amino acid substitutions in exons 7 and 12:: Evidence for an allelic variant with altered catalytic activity

Objective: To determine the existence of mutant and variant C Y P3A4 alleles in three racial groups and to assess functions of the variant alleles by complementary deoxyribonucleic acid (cDNA) expression, Methods: A bacterial artificial chromosome that contains the complete C Y P3A4 gene: was isolated and the exons and surrounding introns were directly sequenced to develop primers to polymerase chain reaction (PCR) amplify and sequence the gene from lymphocyte DNA. DNA samples from Chinese, black, and white subjects were screened. Mutating the affected amino acid in the wild-type cDNA and expressing the variant enzyme with use of the baculovirus system was used to functionally evaluate the variant allele having a missense mutation, Results: To investigate the existence of mutant and variant C Y P3A4 alleles in humans, all 13 exons and the 5'-flanking region of the human C Y P3A4 gene in three racial groups were sequenced and four alleles were identified. An A-->G point mutation in the 5'-flanking region of the human C Y P3A4 gene, designated C Y P3A4*1B, was found in the three different racial groups, The frequency of this allele in a white population was 4.2%, whereas it was 66.7% in black subjects. The C Y P3A4*1B allele was not found in Chinese subjects. A second variant allele, designated C Y P3A4*2 having a Ser222Pro change, was found at a frequency of 2.7% in the white population and was absent in the black: subjects and Chinese subjects analyzed. Baculovirus-directed cDNA expression revealed that the C Y P3A4*2 P450 had a lower intrinsic clearance for the C Y P3A4 substrate nifedipine compared with the wild-type: enzyme but was not significantly different from the wild-type enzyme for testosterone 6 beta-hydroxylation. Another rare allele, designated C Y P3A4*3, was found in a single Chinese subject who had a Met445Thr change in the conserved heme-binding region of the P450. Conclusions: These are the fu st examples of potential function polymorphisms resulting from missense mutations in the C Y P3A4 gene, The C Y P3A4*2 allele was found to encode a P450 with substrate-dependent altered kinetics compared with the wild-type P450.