多重连接依赖性单核苷酸多态性复合分析技术的建立及其在乳腺癌化疗药物相关基因检测中的应用  

Establishment of a multiplex ligation-dependent SNP genotyping method and its application in the detection of genes related to chemotherapeutic drugs in breast cancer

作　　者：唐金海[1] 赵建华[4] 吴建中[2] 陆建伟[3] 潘立群[5] 许芝银[6] 

机构地区：[1]江苏省肿瘤医院普外科,南京210009 [2]江苏省肿瘤医院科研部,南京210009 [3]江苏省肿瘤医院内科,南京210009 [4]江苏省临床检验中心 [5]南京中医药大学第三附属医院中医外科 [6]南京中医药大学第一附属医院中医外科

出　　处：《中华肿瘤杂志》2009年第2期108-113,共6页Chinese Journal of Oncology

基　　金：江苏省社会发展科技计划项目（BS2007077）;江苏省科技发展计划重点招标项目（BS2006006）

摘　　要：目的探讨由耐热连接酶介导的、基于等位基因特异性寡核苷酸探针连接技术的多基因单核苷酸多态性（SNP）分型检测方法，在分析与乳腺癌化疗药物相关的代谢酶基因多态性中的应用价值。方法选取10个与紫杉类、蒽环类和环磷酰胺代谢相关的酶基因SNP位点，利用SNP分型检测方法对126例乳腺癌患者的血液标本进行分型柃测，观察其多态性分布，并分析SNP与乳腺癌化疗疗效的关系。结果所建方法的检测下限为6．25ng基因组DNA，连接产物在测序仪上各荧光检测峰的峰位精确，与预期大小基本一致（偏差范围0．08～0．69bp；x^-=0．31bp，s=0．18bp）；随机抽取8份样本进行重复检测并测序验证，结果均完全一致。10个SNP位点在乳腺癌患者中均存在不同频率的多态性，其中GSTP1基因型和GSTM1基因型的组合与以蒽环类为基础的化疗疗效相关（P=0．037），低GSTs活性组（GSTP1携带突变型＋GSTM1缺失型）患者的疗效最好，有效率为85．7％；GSTM1基因型及其与GSTP1和（或）CYP3A5＊3基冈型的组合与以紫杉醇为基础的化疗疗效相关（均P〈0．05），低GSTs活性组和药物慢代谢组（低GSTs活性组＋CYP3A5＊3携带野生型）均具有更好的疗效，有效率均为100．0％。结论所建方法特异性和重复性好，可用于多基因多SNP位点的复合检测。SNP组合对于预测化疗疗效具有更好的临床应用价值。Objective To establish a method for SNP genotyping of multi-genes by allele-specific oligonucleotide probe ligation mediated by a thermostable ligase, and to explore the genetic polymorphisms of drug-metabolizing enzymes in breast cancer patients and their association with chemotherapeutic responses. Methods 10 SNP loci of enzyme genes related to chemotherapeutic drugs such as taxanes,anthracyclines and cyclophosphamide were selected, and were genotyped for blood samples from 126 breast cancer patients by the established method. Their correlations with therapeutic responses were retrospectively evaluated. Results The lower detection limit of genomic DNA by this developed method was 6.25 ng. The fluorescent peak locations of ligation products on ABI PRISM 377 DNA sequencer were accurate and consistent with prospective sizes in bases （ Bias range 0.08-0.69 bp, ^-x = 0.31 bp, s = 0.18 bp）. Same genotyping results were obtained for repeat tests of 8 random samples, which were further confirmed by sequencing analysis. The 10 SNP loci were polymorphic of different frequency in the breast cancer patients. The combinations with GSTP1 genotypes and GSTMI genotypes were related to anthracycline-based chemotherapy efficacy （P = 0.037）, and the low GSTs activity group （GSTP1 variant allele ＋ GSTM1 null） showed the best effects （85.7%）. GSTM1 genotypes and their combinations with GSTP1 and/or CYP3A5 ＊ 3 genotypes were related to taxane-based therapy efficacy （ P 〈 0.05 for all ） , and both the low GSTs activity group and the drug slow-metabolising group （low GSTs activity group ＋ CYP3A5 ＊ 3 wild allele） showed better effects （ 100% ）. Conclusion The established method is reliable and applicable in multiplex SNPs genotyping of multi-genes. SNPs combination may have a better clinical application value for prediction of chemotherapeutic responses.

关 键 词：多态现象 代谢酶基因 乳腺肿瘤 

分 类 号：R686[医药卫生—骨科学]