CAJ | 학술논문

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检测,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6.25 ng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0.08～0.69 bp;x=0.31 bp,s=0.18 bp);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基因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.
목적 탐토유내열련접매개도적、기우등위기인특이성과핵감산탐침련접기술적다기인단핵감산다태성(SNP)분형검측방법,재분석여유선암화료약물상관적대사매기인다태성중적응용개치.방법 선취10개여자삼류、은배류화배린선알대사상관적매기인SNP위점,이용SNP분형검측방법대126례유선암환자적혈액표본진행분형검측,관찰기다태성분포,병분석SNP여유선암화료료효적관계.결과 소건방법적검측하한위6.25 ng기인조DNA,련접산물재측서의상각형광검측봉적봉위정학,여예기대소기본일치(편차범위0.08～0.69 bp;x=0.31 bp,s=0.18 bp);수궤추취8빈양본진행중복검측병측서험증,결과균완전일치.10개SNP위점재유선암환자중균존재불동빈솔적다태성,기중GSTP1기인형화GSTM1기인형적조합여이은배류위기출적화료료효상관(P=0.037),저GSTs활성조(GSTP1휴대돌변형+GSTM1결실형)환자적료효최호,유효솔위85.7%;GSTM1기인형급기여GSTP1화(혹)CYP3A5*3기인형적조합여이자삼순위기출적화료료효상관(균P<0.05),저GSTs활성조화약물만대사조(저GSTs활성조+CYP3A5*3휴대야생형)균구유경호적료효,유효솔균위100.0%.결론 소건방법특이성화중복성호,가용우다기인다SNP위점적복합검측.SNP조합대우예측화료료효구유경호적림상응용개치.
Objective To establish a method for SNP genotyping of multi-genes by allele-specific oligenucleotide 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 eyclophosphamide were selected, and were gcnotyped 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 multl-genes. SNPs combination may have a better clinical application value for prediction of chemotherapeutic responses.