Functional characterization of novel allelic variants of CYP2C9 recently discovered in southeast Asians.
CYP2C9 was recently resequenced in 150 Asian subjects from Singapore. Several new coding variants were reported, and these variants are now named CYP2C9*14 (R125H), CYP2C9*15 (S162X), CYP2C9*16 (T299A), CYP2C9*17 (P382S), CYP2C9*18 (D397A), and CYP2C9*19 (Q454H). The CYP2C9*18 variant also contained an I359L change previously associated with the CYP2C9*3 allele. In this study, we assessed the functional consequences of the new coding changes. cDNAs containing each of the new coding changes were constructed by site-directed mutagenesis and expressed in a bacterial cDNA expression system, the allelic proteins were partially purified, and their ability to hydroxylate a prototype CYP2C9 substrate was assayed. Expression of cDNAs in Escherichia coli containing either the D397A change or the S162X (premature stop codon) could not be detected either spectrally or at the apoprotein level. CYP2C9.14 and CYP2C9.16 exhibited 80 to 90% lower catalytic activity toward tolbutamide at two substrate concentrations compared with wild-type CYP2C9.1. Kinetic analysis confirmed that CYP2C9.14 and CYP2C9.16 have a higher Km and a >90% lower intrinsic clearance of tolbutamide compared with wild-type CYP2C9.1. Both CYP2C9.17 and CYP2C9.19 proteins exhibited modest 30 to 40% decreases in catalytic activity toward tolbutamide. Thus, CYP2C9*15 and CYP2C9*18 may represent null alleles, whereas CYP2C9*14 and CYP2C9*16 allelic variants produce proteins that are clearly catalytically defective in vitro, indicating the existence of new defective putative alleles of CYP2C9 in Asians.
DeLozier
Tracy C
TC
Human Metabolism Section, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC 27709, USA.
Lee
Soo-Chin
SC
Coulter
Sherry J
SJ
Goh
Boon Cher
BC
Goldstein
Joyce A
JA
D000483:Alleles; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D002681:China / Q000208:ethnology; D065729:Cytochrome P-450 CYP2C9; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D014644:Genetic Variation; D006801:Humans; D006900:Hydroxylation; D007194:India / Q000208:ethnology; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D020641:Polymorphism, Single Nucleotide; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012846:Singapore; D014044:Tolbutamide / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology
D018076:DNA, Complementary; D011994:Recombinant Proteins; D014044:Tolbutamide; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases