Identification and functional characterization of a new CYP2C9 variant (CYP2C9*5) expressed among African Americans.

CYP2C9 is a polymorphic gene for which there are four known allelic variants; CYP2C91, CYP2C92, CYP2C93, and CYP2C94. 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, CYP2C95. This variant is derived from a C1080G transversion in exon 7 of CYP2C9 that leads to an Asp360Glu substitution in the encoded protein. The CYP2C95 variant was found to be expressed only in African Americans, such that approximately 3% of this population carries the CYP2C95 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 CYP2C91; the Ile359Leu variant, CYP2C93; and the Asp360Glu variant, CYP2C95 were carried out using (S)-warfarin, diclofenac, and lauric acid as substrates. The major effect of the Asp360Glu mutation was to increase the K(m) value relative to that of CYP2C91 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 omega-1 hydroxylation of lauric acid. V(max) values differed less than K(m) values between the CYP2C91 and CYP2C95 proteins. In vitro intrinsic clearances for CYP2C95, calculated as the ratio of V(max)/K(m), ranged from 8 to 18% of CYP2C91 values. The corresponding ratio for CYP2C93 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.