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

Abstract

CYP2C9 is a polymorphic gene for which there are four known allelic variants; CYP2C9*1, CYP2C9*2, CYP2C9*3, and CYP2C9*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. The CYP2C9*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 K(m) value 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 omega-1 hydroxylation of lauric acid. V(max) values differed less than K(m) values between the CYP2C9*1 and CYP2C9*5 proteins. In vitro intrinsic clearances for CYP2C9*5, calculated as the ratio of V(max)/K(m), 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.

Influence of CYP2C9 Genotype on Warfarin Dose Among African–Americans and European–Americans

“…To our knowledge, the 226 African Americans who comprise 50% of the POAT cohort represent the largest population of African Americans genotyped for CYP2C9. Inclusion of the recently discovered variants [26,31,32] in the genotyping provides a robust estimate of the CYP2C9 allele frequencies in this racially underrepresented group.…”

Pharmacogenetics of CYP2C9 and interindividual variability in anticoagulant response to warfarin

“…The allelic frequency of this variant should be very low in Japanese and no information is available in Caucasian and African-American populations. CYP2C9*5 was detected in five of 110 African-American 17 and three of 183 Tanzanians, 18 but none in Caucasian or Asians. Although no information is available for the in vivo enzyme activity of CYP2C9*5 at present, recombinant proteins of CYP2C9*5 and CYP2C9*4 exhibited markedly reduced enzyme activities as compared with that of the wild-type CYP2C9 in vitro.…”

“…Population differences in CYP2C9 activity and allelic frequencies of CYP2C9 variants There are substantial differences in the allelic frequencies of CYP2C9 variants, at least for CYP2C9*2 and CYP2C9*3 alleles, across different ethnic populations: the respective allelic frequencies of CYP2C9*2 and CYP2C9*3 in African-Americans (1-3.6% and 0.5-1.5%, respectively) and Asians (0% and 1.7-5%, respectively) tended to be lower than the corresponding values of Caucasians (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19).1% and 5.3-10%, respectively). 8,9 As the enzyme activities of CYP2C9 variant proteins are reduced as compared with the wild-type protein 42 at least in in vitro experiments, there is a possibility that the population differences in the allelic frequencies of distinct CYP2C9 variants may emerge as those in certain phenotypic traits of CYP2C9 (eg, CL po,u ).…”

“…Although no information is available for the in vivo enzyme activity of CYP2C9*5 at present, recombinant proteins of CYP2C9*5 and CYP2C9*4 exhibited markedly reduced enzyme activities as compared with that of the wild-type CYP2C9 in vitro. 17,19 While the allelic frequency of CYP2C9*6, 20 a null allele of CYP2C9, has been shown to be 0.6% in African-American, none of the Caucasians or Asians have been shown to possess this variant allele. The finding that a homozygote of CYP2C9*6 developed severe phenytoin toxicity by an oral phenytoin dosing of 300 mg/day indicates that this allele would be associated with a substantially reduced in vivo enzyme activity.…”

Integrating Field Analyses with Laboratory Exposures to Assess Ecosystems Health

“…Alleles CYP2C9*2, *3, *4 and *5 lead to amino acid replacements, and these mutations have been shown to decrease catalytic activities in vitro (Rettie et al, 1994;Haining et al, 1996;Sullivan-Klose et al, 1996;Dickmann et al, 2001;Kidd et al, 2001;Takahashi et al, 2004). CYP2C9*3 is reported to lead to lower activities in vivo (Sullivan-Klose et al, 1996;Shintani et al, 2001;Takahashi et al, 2004).…”