Shintani M, Ieiri I, Inoue K, Mamiya K, Ninomiya H, Tashiro N, Higuchi S, Otsubo K
Department of Clinical Pharmacokinetics, Division of Pharmaceutical Sciences, Graduate School, Kyushu University, Japan.
Clin Pharmacol Ther. 2001 Aug;70(2):175-82. doi: 10.1067/mcp.2001.117367.
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.
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.
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.
In addition to the two major mutations in the coding region (CYP2C92 and CYP2C93 ), mutations in the 5'-flanking region of the human CYP2C9 gene appear to contribute to the large interindividual variability in drug metabolism activity.
鉴定人类CYP2C9基因5'-侧翼区的基因多态性，并基于荧光素酶报告基因检测和苯妥英钠的体内药代动力学评估其对表型的影响。
采用聚合酶链反应-单链构象多态性分析筛选基因多态性，随后对50名健康志愿者和133名成年癫痫患者的DNA样本进行测序。将HepG2肝癌细胞与5'-侧翼区-荧光素酶报告基因构建体的各种序列模式共转染。用贝叶斯方法估计苯妥英钠与相应序列模式相关的药代动力学参数，并将结果与体外活性进行比较。
基因分析揭示了7个单核苷酸多态性（SNP）的存在。-1912位（T-1912C）的T→C转换、C-1886G、C-1566T、G-1538A、C-1189T、G-982A和A-162G的等位基因频率分别为0.019、0.019、0.077、0.019、0.579、0.019和0.003。一些突变同时发生，共观察到6种序列模式（模式1-6）。荧光素酶报告基因检测表明，与野生型构建体的活性相比，突变的存在导致荧光素酶活性降低41.4%（模式2）至86.8%（模式5）。当存在突变时，计算得到的苯妥英钠内在清除率也较低（模式2降低高达40%）。
除了编码区的两个主要突变（CYP2C92和CYP2C93）外，人类CYP2C9基因5'-侧翼区的突变似乎也导致了药物代谢活性的个体间巨大差异。
