The role of the CYP2C9-Leu³⁵⁹ allelic variant in the tolbutamide polymorphism

Tolbutamide undergoes hydroxylation in humans via a cytochrome P450-mediated pathway. The primary P450 isozyme responsible for this metabolism is thought to be CYP2C9. Population studies have indicated the existence of slow metabolizers of tolbutamide (~1 in 500) suggesting a rare polymorphism associated with 2C9. Several allelic variants of 2C9 have been identified; however, the effect of these allelic variations on metabolism in vivo is not established. In the present study, the coding regions, intron-exon junctions, and upstream region of CYP2C9 were amplified by PCR and sequenced in two slow metabolizers. One individual was homozygous for Leu³⁵⁹/Leu³⁵⁹ and the other individual was heterozygous for Arg¹⁴⁴/ Cys¹⁴⁴ and for Ile³⁵⁹/Leu³⁵⁹. No other genetic variations in 269 were detected in these individuals, PCR-RFLP tests showed that Arg¹⁴⁴Tyr³⁵⁸Ile³⁵⁹6Gly⁴¹⁷ is the principle CYP2C9 allele. Frequencies of the rarer Leu³⁵⁹ and Cys¹⁴⁴ alleles were 0.06 and 0.08, respectively, in a Caucasian-American population and 0.005 and 0.01 respectively in African-Americans. The frequency of the Leu³⁵⁹ allele was 0.026 in Chinese-Taiwanese, but the Cys¹⁴⁴ allele was not detected in this population, Studies in a recombinant yeast expression system showed that the Leu³⁵⁹ variant had the highest K(m) and the lowest V(mac) for hydroxylation of tolbutamide of all the CYP2C9 allelic variants. This allelic variant also had the highest K, for the 7-hydroxylation of S-warfarin. The present data suggest that the incidence of the Leu³⁵⁹ allelic variant of CYP2C9 may account for the occurrence of poor metabolizers of tolbutamide.