ALLELIC AND FUNCTIONAL VARIABILITY OF CYTOCHROME P4502C9

Single nucleotide substitutions are known to result in a different ami no acid at one of four sites in cytochrome P4502C9 (CYP2C9) namely: re sidue 144: Arg/Cys; residue 358: Tyr/Cys; residue 359: Ile/Leu and res idue 417: Gly/Asp. Polymerase chain reaction (PCR) - based amplificati on of the nucleotide fragments encompassing the four residues (144, 35 8-359 and 417) in 18 samples of human genomic DNA from a liver bank an d one sample of DNA extracted from the blood of a known poor metaboliz er of tolbutamide has been carried out. The products of PCR amplificat ion were analysed by either allele-specific restriction endonucleases or probed with radioactively labelled allele-specific oligonucleotides in dot blot hybridizations. Fourteen individuals were homozygous for Arg144 and four were heterozygous Arg/Cys144. All individuals analysed were homozygous for Tyr358 (n = 17) and for Gly417 (n = 18). With the exception of one heterozygote the other 17 subjects were homozygous f or Ile359. The genotype of the known poor metabolizer of tolbutamide w as homozygous for Arg144, Leu359 and Gly417. The relative levels of ex pression of the Cys and Arg144 alleles was studied in the heterozygote s. A relative 5- to 10-fold greater expression of the Cys- over the Ar g144 allele was noted in two heterozygotes. There was no apparent corr elation of genotype to the hydroxylation of the known CYP2C9 substrate s phenytoin, tolbutamide, torasemide and diclofenac. Apparent K-m valu es for the cDNA-expressed Arg144/Ile359, Cys144/Ile359 and Arg144/Leu3 59 variants towards tolbutamide were 91 mu M, 62 mu M and 229 mu M, re spectively. It is likely that functional changes occurring as a result of the Ile359Leu transition are responsible for the tolbutamide poor metabolizer phenotype.