Received for publication November 7, 2006.
Revised February 24, 2007.
Accepted for publication February 26, 2007.

CHARACTERIZATION OF THE NOVEL DEFECTIVE CYP2C9*24 ALLELE

Abstract

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 anticoagulant warfarin, have a narrow therapeutic index. The human CYP2C9 gene is highly polymorphic. We found a new sequence variation in exon 7 of 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 in HEK293 cell system. CO 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 HEK293 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, while 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 cosistent with our findings in vivo. In conclusion, we have identified a novel defective CYP2C9 variant allele of potential importance for drug metabolism in vivo.

Key words: CYP cloning, CYP expression, CYP2C, polymorphisms