一种同时检测急性髓系白血病FLT3-ITD和NPM1基因突变的快速和简便方法  

Establishment of a rapid and easy method for simultaneous detection of FLT3-ITD and NPM1 gene mutations in acute myeloid leukemia

作　　者：陆滢[1] 汪琼[2] 牧启田[1,3] 余梦霞[2] 黄勤 金洁[3] 

机构地区：[1]浙江省宁波市第一医院血液科,315000 [2]宁波大学医学院 [3]浙江大学附属第一医院血液科浙江大学血液病研究所 [4]Department of Pathology, City of Hope National Medical Center, Duarte 91010,CA,USA

出　　处：《中华医学遗传学杂志》2012年第2期163-166,共4页Chinese Journal of Medical Genetics

基　　金：基金项目：国家高技术研究发展计划（863计划）重大课题（2006AA02A405）

摘　　要：目的建立用聚丙烯酰胺凝胶电泳法（polyacrylamidegelelectrophoresis，PAGE）同时快速检测正常核型急性髓细胞白血病（cytogeneticallynormalacutemyeloidleukemia，CN—AML）患者FLT3-ITD和NPM1基因突变的方法。方法在多重PCR基础上，用毛细管电泳法（capillaryelectrophoresis，CE）和PAGE凝胶电泳法同时检测117例初发CN-AML患者的FLT3-ITD和NPM1基因突变。结果突变型双链DNA分子，如突变型FLT3和NPM1的长度均比野生型长（FLT3-mut：420bp〉FLT3-wt：327～332bp，NPM1-mut：172bp〉NPMI—wt：168bp），因此，在PAGE凝胶电泳中突变双链DNA比未突变的DNA移动得慢，从而可将突变检出。117例CN—AML患者均通过CE检测得到验证，其结果与PAGE凝胶电泳结果完全一致（FLT3-ITD＋／NPM1-患者18例，占15．4％；FLT3-ITD＋／NPM1＋患者19例，占16．2％；FLT3-ITD-／NPM1＋患者25例，占21．4％；FLT3-ITD-／NPM1-患者55例，占47．0％）。结论两种电泳法均可快速、简便地同时检测CN—AML患者FLT3-ITD和NPM1基因突变。CE检测敏感，图像清晰；PAGE凝胶电泳法则操作简单，成本低，结果可靠，更适于在基层医院开展初步筛查。Objective To establish a stable, rapid multiplex PCR assay combined with PAGE gel electrophoresis for simultaneously detecting FLT3 -ITD and NPM1 mutations in acute myeloid leukemia （AML）. Methods Capillary electrophoresis （CE） and PAGE gel electrophoresis were simultaneously used to analyze FLT3 -ITD and NPM1 mutations in 117 de novo AML patients with normal cytogenetic findings. Results For certain mutations, the length of mutated double-stranded DNA is longer than wild-type DNA. Since FLT3 -mut （420 bp） is longer than FLT3 -wt （327-332 bp）, and NPM1 -mut （172 bp） is longer than NPM1-wt （168 bp）, heteroduplex will move more slowly during PAGE gel electrophoresis than homoduplex. Therefore the mutations may be detected. A total of 117 CN-AML patients were analyzed with CE and PAGE gel electrophoresis, and the results were identical, which included 18 （15.4%） patients with FLT3-ITD＋/NPM1 -, 19 （16. 2%） patients with FLT3-ITD＋/NPM1 ＋, 25 （21. 4%） patients with FLT3-ITD-/NPM1＋, and 55 （47.0%） patients with FLT3-ITD--/NPM1--. Conclusion Both types of electrophoresis assays may provide a rapid and handy assay for simultaneous detection of FLT3-ITD and NPM1 mutations. CE is relatively sensitive, stable; while PAGE electrophoresis is relatively simple, cheap, and reliable, which may be suitable for primary hospitals and preliminary screening.

关 键 词：聚丙烯酰胺凝胶电泳 毛细管电泳 急性髓系白血病 FLT3基因 NPM1基因 

分 类 号：R733.71[医药卫生—肿瘤]