基质辅助激光解吸电离飞行时间质谱技术检测药物代谢酶基因多态性平台的建立

目的 建立基于基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)技术检测患者体内药物代谢酶基因多态性的平台。方法 选取2013年10月～2014年6月在北京协和医院门诊就诊患者53例EDTA抗凝外周血,提取全血基因组DNA,用MALDI-TOF-MS技术检测53例患者药物代谢酶基因CYP2C9*2(rs1799853),CYP2C9*3(rs1057910),CYP2C19*2(rs4244285),CYP2C19*3(rs4986893),CYP2C19*4(rs28399504),CYP2C19*5(rs56337013)和CYP2C19*17(rs12248560)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53份样本,2个药物代谢酶基因,7个SNP位点的检测。53例患者中,发现CYP2C19*2(rs4244285)AG型25例,AA型6例,GG型22例,A等位基因频率为34.9%。CYP2C19*3(rs4986893)AG型4例,GG型49例,A等位基因频率为3.8%。CYP2C9*3(rs1057910)CA型5例,AA型48例,C等位基因频率为4.7%。未发现 CYP2C9*2(rs1799853),CYP2C19*4(rs28399504),CYP2C19*5(rs56337013)和CYP2C19*17(rs12248560)位点突变。经与Sanger测序法比较,两种检测方法结果的符合率为100%。结论 成功建立MALDI-TOF-MS技术检测药物代谢酶基因多态性的平台,该平台具有高通量、准确的特点,对个性化用药治疗具有重要的临床应用价值。

Establishment of MALDI TOF-MS Technique Platform for Detecting Cytochrome P450 Gene Polymorphism

Objective To establish the MALDI-TOF-MS technique platform for detecting Cytochrome P450 gene polymorphismfrom of patients.Methods Collected 53 EDTA anticoagulation peripheral blood samples from October 2013 to June 2014 in Peking Union Medical College Hospital.The whole genomic DNA was extracted from patients' peripheral blood.Used MALDI-TOF-MS to identify the SNP polymorphism of CYP2C9*2(rs1799853),CYP2C9*3(rs1057910),CYP2C19*2(rs4244285),CYP2C19*3(rs4986893),CYP2C19*4(rs28399504),CYP2C19*5(rs56337013)and CYP2C19*17(rs12248560).To verify the above SNP polymorphism by Sanger sequencing method.Results The MALDI-TOF-MS could perform 53 samples on two cytochrome gene and 7 SNP locus simultaneously.In all the 53 patients,25 AG,6 AA and 22 GG genotypes were identified in CYP2C19*2(rs4244285),the allele frequency of A genotype was 34.9%.4 AG and 49 GG genotypes were identified in CYP2C19*3(rs4986893),the allele frequency of A genotype was 3.8%.5 CA and 48 AA genotypes were identified in CYP2C9*3(rs1057910),the allele frequency of C genotype was 4.7%.No mutation loci were identified in CYP2C9*2(rs1799853),CYP2C19*4(rs28399504),CYP2C19*5(rs56337013)and CYP2C19*17(rs12248560).All the identification data were confirmed by Sanger sequencing.The coincidence rate was 100%.Conclusion The MALDI-TOF-MS technique platform for the cytochrome enzyme SNP was established.This platform has high throughput and accurate characteristics.It has important clinical application value for the treatment of personalized medicate.