基质辅助激光解吸电离飞行时间质谱技术检测华法林代谢酶基因多态性  

Development and application of MALDI-TOF-MS for detection of genetic polymorphism of warfarin metabolizing enzymes

作　　者：郭俊[1] 李小燕[1] 蔡伦[1] 张魁[2] 董然[2] 王绿娅[1] 杜杰[1] 

机构地区：[1]首都医科大学附属北京安贞医院,北京市心肺血管疾病研究所,心血管重塑相关疾病教育部重点实验室 [3]首都医科大学附属北京安贞医院,北京市心肺血管疾病研究所心脏外科,北京100029

出　　处：《临床检验杂志》2014年第6期410-413,共4页Chinese Journal of Clinical Laboratory Science

基　　金：科研基地建设-心血管重大疾病防治协同创新中心(2011协同创新中心)(PXM2013_014226_07_000088)

摘　　要：目的用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)技术建立检测人群中华法林代谢酶基因多态性的方法。方法选取2011年1月至2013年1月北京安贞医院心脏外科心脏机械瓣膜置换术后接受华法林抗凝治疗的患者10例,采集外周血,提取全血基因组DNA,用MALDI-TOF-MS技术检测10例患者华法林代谢酶基因CYP2C9*2(rs1799853)、CYP2C9*3(rs1057910)、CYP2C9*6(rs9332131)、VKORC1 C>T(rs9923231)(-1639G>A)、GGCX G>C(rs11676382)的单核苷酸多态性(SNP)位点,并用Sanger测序法进行验证。结果用MALDI-TOF-MS技术可以同时完成10份标本3个华法林代谢酶基因5个SNP位点的检测。10名服华法林的心脏机械瓣膜置换术患者中,发现VKORC1-1639G>A AG型2例,AA型8例,A等位基因频率为90%。未发现CYP2C9*2(rs1799853)、CYP2C9*3(rs1057910)、CYP2C9*6(rs9332131)和GGCX G>C(rs11676382)突变。经与Sanger测序法比较,符合率为100%。结论成功建立MALDI-TOF-MS技术检测华法林代谢酶基因多态性的平台,该法具有高通量、快速、准确的特点,在个性化医疗上有较大的推广应用价值。Objective To develop a high throughput method for detecting genetic polymorphism of warfarin metabolism-related genes by matrix assisted laser desorption ionisation-time of flight mass spectrometry（MALDI-TOF-MS） method. Methods Ten Chinese Han patients who were undergoing mechanical heart valve replacement（MHVR） and receiving stabilized warfarin therapy from January 2011 to January 2013 were recruited in our study. Genomic DNA was extracted from peripheral blood of the patients. Single nucleotide polymorphisms（SNPs） of warfarin metabolism-related genes CYP2C9 ＊ 2 （rs1799853）, CYP2C9 ＊ 3 （rs1057910）, CYP2C9 ＊ 6 （rs9332131）, VKORC1 C 〉 T（ rs9923231 ）（-1639G 〉 A） and GGCX G 〉 C （rs11676382） were detected by MALDI-TOF-MS and identified by Sanger sequencing. Results The results displayed that five loci of SNPs from the three warfarin metabolism-related genes could be simultaneously detected by MALDI-TOF-MS, and 10 samples could be carried out on one chip. Among the 10 samples, 2 of AG and 8 of AA for VKORC1-1639G 〉 A were found and the A allele frequency was 90%. No G 〉 C mutation of CYP2C9 ＊ 2 （ rs1799853 ） , CYP2C9 ＊ 3 （ rs1057910 ）, CYP2C9 ＊ 6（ rs9332131 ） and GGCX G 〉 C （ rs11676382 ） was found. Reliability test indica- ted that the agreement between the MALDI-TOF-MS and the Sanger sequencing methods was 100%. Conclusion A method of MAL- DI-TOF-MS for detection of genetic polymorphism was successfully developed, which should be high throughput, fast and accurate for the detection of genetic polymorphism of the warfarin metabolism-related enzyme genes and available in personalized medical treatment.

关 键 词：心脏机械瓣膜置换术 华法林 基质辅助激光解吸电离飞行时间质谱技术 单核苷酸多态性 

分 类 号：R446[医药卫生—诊断学]