Genetic polymorphisms and functional characterization of the 5'-flanking region of the human CYP2C9 gene: in vitro and in vivo studies.
OBJECTIVE
Genetic polymorphisms were identified in the 5'-flanking region of the human CYP2C9 gene, and their effects on the phenotype were evaluated on the basis of the luciferase reporter gene assay and the in vivo pharmacokinetics of phenytoin.


METHODS
Genetic polymorphisms were screened by polymerase chain reaction-single-strand conformational polymorphism analysis, following sequencing with DNA samples obtained from 50 healthy volunteers and 133 adult epileptic patients. HepG2 hepatoma cells were cotransfected with various sequence patterns of 5'-flanking region-luciferase reporter gene constructs. Pharmacokinetic parameters of phenytoin in relation to the corresponding sequence patterns were estimated by the Bayesian method, and the results were compared with in vitro activities.


RESULTS
Genetic analysis revealed the existence of 7 single nucleotide polymorphisms (SNPs). Allele frequencies of T-->C transition at position -1912 (T-1912C), C-1886G, C-1566T, G-1538A, C-1189T, G-982A, and A-162G were 0.019, 0.019, 0.077, 0.019, 0.579, 0.019, and 0.003, respectively. Some mutations occurred simultaneously, and a total of 6 sequence patterns (patterns 1-6) were observed. The luciferase reporter gene assay indicated that the presence of mutation(s) resulted in a reduction in luciferase activity of 41.4% (pattern 2) to 86.8% (pattern 5) compared with the activity of the wild-type construct. The calculated intrinsic clearance of phenytoin was also lower (up to a 40% reduction for pattern 2) when a mutation(s) was present.


CONCLUSIONS
In addition to the two major mutations in the coding region (CYP2C9*2 and CYP2C9*3 ), mutations in the 5'-flanking region of the human CYP2C9 gene appear to contribute to the large interindividual variability in drug metabolism activity.
Shintani
M
M
Department of Clinical Pharmacokinetics, Division of Pharmaceutical Sciences, Graduate School, Kyushu University, Japan.
Ieiri
I
I
Inoue
K
K
Mamiya
K
K
Ninomiya
H
H
Tashiro
N
N
Higuchi
S
S
Otsubo
K
K
D000328:Adult; D000927:Anticonvulsants / Q000493:pharmacokinetics*; D001189:Aryl Hydrocarbon Hydroxylases; D001499:Bayes Theorem; D065729:Cytochrome P-450 CYP2C9; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D004827:Epilepsy / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics*; D005260:Female; D017930:Genes, Reporter / Q000235:genetics; D014644:Genetic Variation; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008297:Male; D009154:Mutation; D010641:Phenotype; D010672:Phenytoin / Q000493:pharmacokinetics*; D010957:Plasmids; D011110:Polymorphism, Genetic; D018807:Polymorphism, Single-Stranded Conformational; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000235:genetics*
D000927:Anticonvulsants; D010672:Phenytoin; D003577:Cytochrome P-450 Enzyme System; D008156:Luciferases; D013250:Steroid Hydroxylases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; D039181:Steroid 16-alpha-Hydroxylase