白血病NPM1基因突变检测方法的临床适用性比较  

Comparison of the cfinical application of different methods for detection of NPM1 gene mutations in leukemia

作　　者：邹积艳[1] 朱平[1] 刘红星[2] 张英[1] 王赫[2] 蔡鹏[2] 卜定方[1] 

机构地区：[1]北京大学第一医院血液科,100034 [2]北京市道培医院特检中心

出　　处：《中华检验医学杂志》2009年第1期35-39,共5页Chinese Journal of Laboratory Medicine

基　　金：科技部国际科技合作重大项目（2006DFB31430）;国家自然科学基金资助项目（30470939）

摘　　要：目的分析急性髓细胞白血病（AML）的NPM1（nucleophosmin）基因第12外显子突变，对比3种常用检测方法的临床适用性。方法随机选择54份AML患者的冻存骨髓细胞标本，提取DNA后PCR扩增NPM1基因第12外显子，分别进行PCR-毛细管电泳、变性高效液相色谱（DHPLC）和直接测序检测。FLT3内部串联重复（ITD）突变的检测采用FLT3PCR产物分别进行琼脂糖凝胶电泳和PCR-毛细管电泳检测。结果7例AML患者发现NPM1基因突变，其中5例为常见的A型突变，即960bp处插入TCTG4个碱基；1例为D型突变，即960bp处插入CCTG4个碱基；另1例为新发现的1种突变，在958bp处丢失TGGCAGTG8个碱基，插入GCCCGCGGTTTA 12个碱基。3种基因突变的检测方法检出率均为100％。毛细管电泳检测NPM1基因突变更快速可靠，且可同时检测FLT3-ITD突变。DHPLC的分辨率受实验因素的影响较多。直接测序步骤相对繁琐，而且有杂合子基因序列误读的可能性。结论AML存在一种NPM1基因的958bp位点12个碱基置换的基因突变；AML的NPM1基因突变临床检测采用PCR-毛细管电泳法更方便。Objective To analyze nucleophosmin （NPM1） gene mutations in exon 12 in patients with acute myeloid leukemia （AML） and evaluate the clinical appliance of three methods which are frequently used for detecting gene mutation. Methods Genomic DNA from bone marrow of 54 AML patients was detected by PCR for NPM1 exon 12 and screened by PCR-capillary electrophoresis, denature highperformance liquid chromatography （DHPLC） and direct sequencing separately. FLT3-ITD （FMS-like tyrosine kinease internal tandem duplication） was detected by agarose gel electrophoresis and PCR-capillary electrophoresis. Results Seven AML sample harbored NPM1 gene mutations. Five of them were the most common mutation, known as type A （an insertion of a TCTG tetranucleotide at position 960 bp）. One of them was type D （ an insertion of a CCTG tetranucleotide at position 960 bp）. The new variant was a deletion of a TGGCAGTG sequence at 958 bp and insertion of a GCCCGCGG3TFA sequence instead. The detection ratio of the three methods was all 100% and capillary electrophoresis was more rapid, reliable and easier than the other two methods. Moreover it could detect FLT3-ITD simultaneously. The resolving power of DHPLC was affected by many factors. The direct sequencing method was tedious and the heterozygous sequence might be misread. Conclusions There is a new mutation at position 958 bp with a 12-nucleotide insertion and substitution. PCR-capillary electrophoresis is convenient to screen NPM1 mutations of AML in clinical practice.

关 键 词：白血病 髓样 急性 核蛋白质类 突变 电泳 毛细管 色谱法 高压液相 聚合酶链反应 

分 类 号：R686[医药卫生—骨科学]