Characterization of the novel defective CYP2C9*24 allele.
CYP2C9 is one of the major drug-metabolizing enzymes, and it is involved in the oxidative metabolism of approximately 10% of clinically important drugs, among which some, such as the anticoagulant warfarin, have a narrow therapeutic index. The human CYP2C9 gene is highly polymorphic. We found a new sequence variation in exon 7 of the CYP2C9 gene (1060G>A) resulting in a substitution of acidic amino acid glutamate to basic lysine (E354K) when translated. The allele, designated CYP2C9(*)24, was present in heterozygous state in one warfarin-treated patient. To characterize the CYP2C9(*)24 allele, we expressed the wild-type and CYP2C9.24 protein in a recombinant yeast expression system and a human embryonic kidney (HEK)-293 cell system. Carbon monoxide difference spectra were recorded on dithionite-reduced microsomes, and protein was determined by Western blotting. Transfection with CYP2C9.1 cDNA resulted in detectable CYP2C9 protein in yeast or HEK-293 cells, whereas only small amounts of the protein were detected in yeast transfected with CYP2C9.24 cDNA. A strong differential absorption peak at 450 nm was observed with microsomes of yeast transfected with CYP2C9.1 cDNA, whereas no peak was detected with microsomes of yeast transfected with CYP2C9.24 cDNA or empty pYeDP60 plasmid. These results suggest that CYP2C9.24 may be improperly folded, both in yeast and mammalian cells, resulting in improper heme incorporation and rapid intracellular degradation. The data obtained in the expression systems are consistent with our findings in vivo. In conclusion, we have identified a novel defective CYP2C9 variant allele of potential importance for drug metabolism in vivo.
Herman
Darja
D
Institute of Biochemistry, Faculty of Medicine, Vrazov trg 2, SI-1000 Ljubljana, Slovenia.
Dolzan
Vita
V
Ingelman-Sundberg
Magnus
M
D000483:Alleles; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D065729:Cytochrome P-450 CYP2C9; D018698:Glutamic Acid / Q000235:genetics; D006801:Humans; D008239:Lysine / Q000235:genetics; D008861:Microsomes / Q000378:metabolism; D009154:Mutation; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
D018698:Glutamic Acid; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; D008239:Lysine