CAJ | 학술논문

目的明确1例华法林高敏感患者细胞色素P450 2C9酶(CYP2 C9)的新突变及其生物学功能.方法提取患者基因组DNA,PCR产物利用直接测序法对CYP2C9、维生素K环氧化物还原酶复合物1(VKORC1)及细胞色素P450 4F2酶(CYP4F2)基因进行序列测定,与国际数据库比对分析确定是否存在碱基突变.通过定点诱变方法获得3种典型缺陷型CYP2C9*2、* 3、*13及新突变型CYP2C9的cDNA,利用Bac-to-Bac Baculovirus Expression System试剂盒,获得高表达各型CYP2C9变异体的昆虫微粒体.Western印迹法进行CYP2C9蛋白定量,以甲苯磺丁脲为探针底物药,体外测定不同药物浓度下各型CYP2C9变异体的最大反应速率(Vmax)和米氏常数(Km),评价新突变类型对CYP2C9体外代谢活性的影响.结果患者CYP2C9基因中发现全新的突变类型:1400T＞C,可导致CYP2C9第467位氨基酸发生变异(L467P),VKORC1及CYP4F2基因无碱基突变.体外代谢实验结果显示,突变体CYP2C9*2、*3、*13和L467P的体外酶学活性分别为野生型的91.58％、13.55％、0.11％和1.59％.结论 1400T＞C突变可大大降低CYP2C9的体外酶学活性,提示携带此突变型的患者在使用经CYP2C9代谢的药物时,药物代谢速度会明显减慢,容易造成体内药物蓄积,发生潜在的药物不良反应.
목적 명학1례화법림고민감환자세포색소P450 2C9매(CYP2 C9)적신돌변급기생물학공능.방법 제취환자기인조DNA,PCR산물이용직접측서법대CYP2C9、유생소K배양화물환원매복합물1(VKORC1)급세포색소P450 4F2매(CYP4F2)기인진행서렬측정,여국제수거고비대분석학정시부존재감기돌변.통과정점유변방법획득3충전형결함형CYP2C9*2、* 3、*13급신돌변형CYP2C9적cDNA,이용Bac-to-Bac Baculovirus Expression System시제합,획득고표체각형CYP2C9변이체적곤충미립체.Western인적법진행CYP2C9단백정량,이갑분광정뇨위탐침저물약,체외측정불동약물농도하각형CYP2C9변이체적최대반응속솔(Vmax)화미씨상수(Km),평개신돌변류형대CYP2C9체외대사활성적영향.결과 환자CYP2C9기인중발현전신적돌변류형:1400T＞C,가도치CYP2C9제467위안기산발생변이(L467P),VKORC1급CYP4F2기인무감기돌변.체외대사실험결과현시,돌변체CYP2C9*2、*3、*13화L467P적체외매학활성분별위야생형적91.58％、13.55％、0.11％화1.59％.결론 1400T＞C돌변가대대강저CYP2C9적체외매학활성,제시휴대차돌변형적환자재사용경CYP2C9대사적약물시,약물대사속도회명현감만,용역조성체내약물축적,발생잠재적약물불량반응.
Objective To perform a pharmacogenetic study of a patient with warfarin hypersensitivity and determine the biological function of a novel CYP2C9 mutation.Methods Genomic DNAs were extracted from the warfarin hypersensitive patient and used for genetic screening of CYP2C9,VKORC1 and CYP4F2 by direct sequencing.Acquired sequences were aligned and blasted with public nucleotide database to clarify whether site mutations were present in these 3 genes.Full-length cDNA fragments of CYP2C9 * 2,* 3,* 13 and 1400T ＞ C were obtained by polymerase chain reaction (PCR)site-directed mutagenesis and used for the insect expression vector construction.According to the manufacturer's instruction,insect cell microsomes expressing 5 CYP2C9 variants were obtained with Bac-to-Bac Baculovirus Expression System.Then CYP2C9 protein expression level for each variant was quantified by Western blot and tolbutamide used as a probing substrate for assessing the in vitro metabolic characteristics of each CYP2C9 variant.Vmax and Km values were calculated and used for evaluating the biological impact of novel 1400T ＞ C mutant.Results One novel nucleotide mutation 1400T ＞ C was detected in DNAs of tested patient.And this site mutation resulted in one amino acid change at position 467 of CYP2C9 protein (L467P).Other two candidate genes VKORC1 and CYP4F2 were found to be the wildtype alleles.When expressed in insect cell microsomes,the relative clearance values of CYP2C9 * 2,* 3,* 13 and L467P variants to tolbutamide were 91.58％,13.55％,0.11％ and 1.59％ to that of wild-type protein respectively.Conclusions 1400T ＞ C mutation of CYP2C9 gene greatly reduces the in vitro enzymatic activity of CYP2C9.While taking the drugs metabolized by CYP2C9,the patients carrying this mutated allele should take more cautions because their metabolic rates are slower than those of wild-type carriers.Ensuing drug accumulation in vivo may cause potentially serious adverse reactions.