Dai Da-peng, Li Chuan-bao, Wang Shuang-hu, Geng Pei-wu, Hu Guo-xin, Cai Jian-ping
Key Laboratory of Geriatrics, Beijing Hospital & Beijing Institute of Geriatrics, Ministry of Health, Beijing 100730, China.
Department of Clinical Laboratory, Beijing Hospital, Ministry of Health, Beijing 100730, China.
Zhonghua Yi Xue Za Zhi. 2013 Nov 26;93(44):3537-41.
To perform a pharmacogenetic study of a patient with warfarin hypersensitivity and determine the biological function of a novel CYP2C9 mutation.
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.
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 wild-type 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.
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.
对一名对华法林过敏的患者进行药物遗传学研究，并确定一种新型CYP2C9突变的生物学功能。
从对华法林过敏的患者中提取基因组DNA，用于通过直接测序对CYP2C9、VKORC1和CYP4F2进行基因筛查。将获得的序列与公共核苷酸数据库进行比对和比对，以阐明这3个基因中是否存在位点突变。通过聚合酶链反应（PCR）定点诱变获得CYP2C9*2、*3、*13和1400T>C的全长cDNA片段，并用于昆虫表达载体的构建。根据制造商的说明，使用Bac-to-Bac杆状病毒表达系统获得表达5种CYP2C9变体的昆虫细胞微粒体。然后通过蛋白质印迹法定量每种变体的CYP2C9蛋白表达水平，并使用甲苯磺丁脲作为探测底物来评估每种CYP2C9变体的体外代谢特征。计算Vmax和Km值，并用于评估新型1400T>C突变体的生物学影响。
在受试患者的DNA中检测到一种新型核苷酸突变1400T>C。该位点突变导致CYP2C9蛋白第467位氨基酸发生变化（L467P）。发现其他两个候选基因VKORC1和CYP4F2为野生型等位基因。当在昆虫细胞微粒体中表达时，CYP2C9*2、*3、*13和L467P变体对甲苯磺丁脲的相对清除率分别为野生型蛋白的91.58%、13.55%、0.11%和1.59%。
CYP2C9基因的1400T>C突变大大降低了CYP2C9的体外酶活性。在服用由CYP2C9代谢的药物时，携带这种突变等位基因的患者应更加谨慎，因为他们的代谢率比野生型携带者慢。体内随后的药物蓄积可能会引起潜在的严重不良反应。
