Identification and Functional Characterization of a New CYP2C9 Variant (CYP2C9*5) Expressed among African Americans

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Abstract
CYP2C9 is a polymorphic gene for which there are four known allelic variants; CYP2C9*1,CYP2C9*2, CYP2C9*3, andCYP2C9*4. In the present study, DNA from 140 European Americans and 120 African Americans was examined by single-strand conformational polymorphism and restriction fragment length polymorphism analyses, resulting in the identification of a new CYP2C9 variant, CYP2C9*5. This variant is derived from a C1080G transversion in exon 7 of CYP2C9 that leads to an Asp360Glu substitution in the encoded protein. TheCYP2C9*5 variant was found to be expressed only in African Americans, such that approximately 3% of this population carries the CYP2C9*5 allele. The variant was expressed in, and purified from, insect cells infected with a recombinant baculovirus. Comparative kinetic studies using the purified wild-type protein CYP2C9*1; the Ile359Leu variant, CYP2C9*3; and the Asp360Glu variant, CYP2C9*5 were carried out using (S)-warfarin, diclofenac, and lauric acid as substrates. The major effect of the Asp360Glu mutation was to increase the Kmvalue relative to that of CYP2C9*1 for all three substrates: 12-fold higher for (S)-warfarin 7-hydroxylation, 5-fold higher for the 4′-hydroxylation of diclofenac, and 3-fold higher for the ω-1 hydroxylation of lauric acid. Vmax values differed less than Km values between the CYP2C9*1 and CYP2C9*5 proteins. In vitro intrinsic clearances for CYP2C9*5, calculated as the ratio ofVmax/Km, ranged from 8 to 18% of CYP2C9*1 values. The corresponding ratio for CYP2C9*3 was 4 to 13%. Accordingly, the in vitro data suggest that carriers of the CYP2C9*5 allele would eliminate CYP2C9 substrates at slower rates relative to persons expressing the wild-type protein.