CAJ | 학술논문

目的评估分析DNA测序法检测华法林用药剂量相关基因——细胞色素氧化酶P4502C9(CYP2C9)和维生素K环氧化物还原酶(VKORC1)多态性指导华法林临床用药的价值.方法选取2011年7月至2012年7月武汉大学人民医院肿瘤科使用华法林经影像学和病理学检查确诊为肺癌患者140例,按检测CYP2C9和VKORC1基因且使用华法林(试验组70例)和不检测2个基因采取经验用药(对照组70例)分为2组,根据已知CYP2C9和VKORC1基因序列,设计针对CYP2C9和VKORC1基因多态性位点的特异性引物,PCR扩增包含CYP2C9和VKORC1基因多态性位点的DNA片段,并用DNA测序法验证；同时用DNA测序法检测试验组70例肺癌患者不同CYP2C9和VKORC1基因型的分布频率,且与对照组70例肺癌患者比较,以评价两组患者在使用华法林2周和4周后国际标准化比率(INR)的差异.结果 PCR产物凝胶电泳和DNA测序检测结果显示,针对CYP2C9和VKORC1基因的特异性引物可鉴别不同的基因型(CYP2C9*1、CYP2C9*2和CYP2C9*3；VKORC1-1639GG、VKORC1-1639AG和VKORC1-1639AA),其检测的特异度和敏感度均为100％.除CYP2C9*1/*1患者INR为100％外,CYP2C9*1/*2、CYP2C9*1/*3、CYP2C9*2/*2、CYP2C9*3/*3和CYP2C9* 2/*3均为0％.VKORC1-1639AG、VKORC1-1639AA和VKORC1-1639GG患者的INR分别为10％、90％和0％.采用基因型检测指导用药患者的INR值85.7％在第2周达到目标治疗范围,且明显高于对照组(48.6％,x2=21.9,P＜0.01)；第4周试验组患者的INR值均达到目标治疗范围(100％),对照组65例患者INR值达到目标治疗范围(92.9％)；但两组患者均未发生出血和血栓形成事件.结论设计针对肺癌患者华法林相关基因多态性的特异性引物,通过PCR扩增及DNA测序分析能快速、准确有效检测CYP2C9和VKORC1基因多态性,可靠地指导华法林在临床安全有效的使用.
목적 평고분석DNA측서법검측화법림용약제량상관기인——세포색소양화매P4502C9(CYP2C9)화유생소K배양화물환원매(VKORC1)다태성지도화법림림상용약적개치.방법 선취2011년7월지2012년7월무한대학인민의원종류과사용화법림경영상학화병이학검사학진위폐암환자140례,안검측CYP2C9화VKORC1기인차사용화법림(시험조70례)화불검측2개기인채취경험용약(대조조70례)분위2조,근거이지CYP2C9화VKORC1기인서렬,설계침대CYP2C9화VKORC1기인다태성위점적특이성인물,PCR확증포함CYP2C9화VKORC1기인다태성위점적DNA편단,병용DNA측서법험증；동시용DNA측서법검측시험조70례폐암환자불동CYP2C9화VKORC1기인형적분포빈솔,차여대조조70례폐암환자비교,이평개량조환자재사용화법림2주화4주후국제표준화비솔(INR)적차이.결과 PCR산물응효전영화DNA측서검측결과현시,침대CYP2C9화VKORC1기인적특이성인물가감별불동적기인형(CYP2C9*1、CYP2C9*2화CYP2C9*3；VKORC1-1639GG、VKORC1-1639AG화VKORC1-1639AA),기검측적특이도화민감도균위100％.제CYP2C9*1/*1환자INR위100％외,CYP2C9*1/*2、CYP2C9*1/*3、CYP2C9*2/*2、CYP2C9*3/*3화CYP2C9* 2/*3균위0％.VKORC1-1639AG、VKORC1-1639AA화VKORC1-1639GG환자적INR분별위10％、90％화0％.채용기인형검측지도용약환자적INR치85.7％재제2주체도목표치료범위,차명현고우대조조(48.6％,x2=21.9,P＜0.01)；제4주시험조환자적INR치균체도목표치료범위(100％),대조조65례환자INR치체도목표치료범위(92.9％)；단량조환자균미발생출혈화혈전형성사건.결론 설계침대폐암환자화법림상관기인다태성적특이성인물,통과PCR확증급DNA측서분석능쾌속、준학유효검측CYP2C9화VKORC1기인다태성,가고지지도화법림재림상안전유효적사용.
Objective To evaluate and analyze the clinical application value of detection of Warfarin-related gene polymorphisms,cytochrome P450 2C9 (CYP2C9) and Vitamin K epoxide reductase complex subunit 1 (VKORC1) polymorphisms.Methods From July of 2011 to July of 2012,the blood samples were randomly collected from 140 lung cancer patients from Department of Oncology in Renmin Hospital of Wuhan University.These lung cancer patients were diagnosed through imaging examination and pathological examination.CYP2C9 and VKORC1 polymorphisms were detected in 70 patients (studied group) but not detected in the other 70 patients (control group) before they used warfarin.According to known gene sequences of CYP2C9 and VKORC1,specific primers were designed to genotype the CYP2C9 *2 and CYP2C9 * 3 alleles as well as the VKORC1-1639G ＞ A polymorphism through PCR amplification and DNA sequencing.Meanwhile,the distribution of these alleles in the studied group was analyzed.The clinical significance of detection of these polymorphisms was evaluated by comparing the proportion of patients within the therapeutic INR (International Normalized Ratio) range between control and genotype-guided dosing groups using Chi square test after 2 and 4 weeks of Warfarin therapy.Results Based on the results of agarose gel electrophoresis of PCR products and DNA sequencing,the primers for CYP2C9 and VKORC1 polymorphisms were indeed specific to these SNPs (CYP2C9 * 1,CYP2C9 * 2 and CYP2C9 * 3 ;VKORC1-1639GG,VKORC1-1639AG and VKORC1-1639AA) and both of the specificity and sensitivity of these primers are 100％,thus contributiug for genotyping these alleles.The distribution of CYP2C9 * 1/* 1 was 100％,CYP2C9 * 1/* 2,CYP2C * 1/* 3,CYP2C9 * 2/* 2,CYP2C9 * 3/* 3 and CYP2C9 * 2/* 3 were 0％.The distribution of VKORC1-1639AG,VKORC1-1639AA and VKORC1-1639 GG were 10％,90％ and 0％ respectively.2 weeks after the treatment of Warfarin,85.7％ patients in the genotype-guided dosing group reached the stable therapeutic INR range,which was significantly higher than that in the control group (48.6％,x2 =21.9,P ＜ 0.01); 4 weeks later,all patients (100％) were inside the stable therapeutic INR range whereas only 65 patients (92.9％) in the control group reached the therapeutic INR range.No haemorrhage or thromboembolic events occurred in both groups.Conclusions CYP2C9 and VKORC1 polymorphisms can be accurately detected by PCR reaction with the designed primers and the subsequent DNA sequencing in patients with lung cancer.This method is validated to be reliable.The genotyping of the Warfarin-related genes detective method can effectively guide Warfarin-dosing.