免疫抑制剂代谢相关基因多态性检测芯片的研制及其在肝移植患者中的应用  

Development of a chip for detecting immunosuppressant metabolism associated genetic polymorphism and its application in liver transplantation patients

作　　者：姜楠[1,2] 李洋[1,2] 胥顺[3] 傅斌生[1,2] 汪国营[1,2] 李华[1,2] 汪根树[1,2] 张剑[1,2] 杨扬[1,2] 陈规划[1,2] 

机构地区：[1]中山大学附属第三医院肝移植中心 [2]中山大学器官移植研究所 广东省器官移植研究中心,广州510630 [3]中山大学达安基因股份有限公司

出　　处：《器官移植》2013年第3期127-135,共9页Organ Transplantation

基　　金：国家重点基础研究发展计划(973计划)(2009CB522404);广东省科技计划项目(2009B030801007);广州市科技计划项目(2009Z1-E211)

摘　　要：目的建立一种快速、准确的检测免疫抑制剂常见代谢相关基因多态性的微阵列芯片技术,明确肝移植供、受体细胞色素P450家族细胞色素3A5(cytochrome P4503A5,CYP3A5)及多药耐药1(multidrug resistance1,MDR1)基因多态性对术后患者他克莫司(FK506)浓度/剂量比(C/D)的影响。方法选取CYP3A5和MDR1基因外显子的8个常见变异基因位点(CYP3A5Exon1、Exon2、Exon3、Exon13和MDR1Exon1、Exon12、Exon21、Exon26)设计特异性野生型、突变型引物检测探针,通过AD3200微阵列芯片点样仪点样于醛基基片上,室温保湿过夜固定。采用该基因芯片对109份肝移植供、受者全血标本进行检测,每份样本重复检测3次,GenePix4100共聚焦扫描仪阅读芯片,GenePixPro410芯片图像分析软件分析结果,根据同一检测位点野生型探针和突变型探针在阴阳性状态的信号值比值来确定探针的切值(cut-off值)。引物探针和样本微阵列芯片检测结果与脱氧核糖核酸(DNA)测序作比对进行验证。进一步分析临床资料完整的32例肝移植患者,比较供、受体CYP3A5和MDR1基因多态性对患者FK506C/D值的影响。结果芯片样点分布均匀、清晰、规整度好、无漏点和连点,突变型探针和野生型探针的荧光信号强度区分明显;按照突变型探针和野生型探针的荧光强度比值设定cut-off值,结果易于判断,芯片检测结果与测序结果符合率高达99%。供体CYP3A5基因Exon3第6986位点A/G单核苷酸多态性(CYP3A5*3)与FK506C/D值有关,术后2、4、12周供体CYP3A5*1/*1和*1/*3型患者的FK506C/D值明显低于*3/*3型患者(均为P<0.05),而供、受体CYP3A5其它基因型及供受体MDR1各基因型组间FK506C/D值相比差异无统计学意义(均为P>0.05)。结论供体CYP3A5基因第6986位点携带*1等位基因的患者需更高剂量FK506才能达到目标血药浓度。CYP3A5和MDR1基因多态性的微阵列芯片技术的检测效果理想,可重复性强,为肝移植术后免疫抑�Objective To develop a rapid and accurate chip to detect the immunosuppressant metabolism-associated genetic polymorphism, and to study the effect of genetic polymorphism of cytochrome P450 3A5 （CYP3A.5） and multidrug resistance 1 （MDR1） in liver transplant donors and recipients on the concentration/dosage （C/D） of tacrolimus （FK506） after transplantation. Methods The specific wild-type and mutant probes were designed through selecting 8 common variant gene loci （CYP3A5 Exonl, Exon2, Exon3, Exonl3 and MDR1 Exonl, Exon12, Exon21, Exon26） in genetic exon of CYP3A5 and MDR1. The probes were immobilized onto the activated slides with aldehyde surface using AD3200 microarray arrayer, and were incubated overnight in a humid chamber at room temperature. The chips were used to detect 109 whole blood samples of liver transplant donors and recipients, and each sample was repeatedly measured for 3 times. After hybridization, GenePix 4100 confocal scanner and GenePix Pro 410 software were used to scan and analyze the chips. The cut-off values of clips were measured according to the ratio of negative/positive signal value of wild and mutant-type probes in the same detecting site. The detecting results of primer probe and microarray were compared and verified by sequencing of deoxyribonucleic acid （DNA）. Clinical data of 32 liver transplant patients were further analyzed. The influence of genetic polymorphism of CYP3A5 and MDR1 in donors and recipients on FK506 C/D value was compared. Results Uniform distributed, distinct and regular on the chip were observed without missing or continuous spots. The difference of fluorescence signal intensity was clear between the wild and mutant-type probes. The cut-off values were set up to judge easily according to the fluorescence intensity ratio of wild and mutant-type probes. The coincidence rate of chip and sequencing result was 99%. The A/G mononucleotide polymorphism of exon 3 of CYP3A5 （ CYP3A5 ＊ 3 ） at 6986 locus was correlated with the FK.506 C/D value

关 键 词：微阵列芯片技术 寡核苷酸序列分析 基因位点 他克莫司 细胞色素3A5 多药耐药基因 

分 类 号：R617[医药卫生—外科学]