高通量测序在FLT3-ITD阳性急性髓细胞白血病患者中的应用  

Application of High-throughput Sequencing in Acute Myeloid Leukemia Patients with Positive FLT3-ITD

作　　者：马亮[1] 姜永玮[1] 王少婷[1] 刘倩[1] 丛笑[1] 沈军[1] 曹永跃[1] 曹永彤[1] 

机构地区：[1]中日友好医院检验科,北京100029

出　　处：《中国实验血液学杂志》2016年第2期381-387,共7页Journal of Experimental Hematology

基　　金：中日友好医院青年课题(QN-2013-11)

摘　　要：目的:探讨高通量测序在FLT3基因ITD突变含量较低及存在多条ITD突变的初诊急性髓细胞白血病(AML)患者ITD序列分析中的应用,并分析ITD突变特点。方法:采用聚合酶链反应(PCR)扩增23例FLT3-ITD阳性AML患者FLT3基因,应用毛细管电泳法分析FLT3基因ITD突变,然后用带有不同序列标签的PCR引物再次扩增上述患者FLT3基因,应用illumina Miseq二代测序仪分析扩增产物,并将测序结果与UCSC数据库比对。结果:23例AML患者毛细管电泳检测显示,17例患者为1条ITD插入突变,3例患者为2条ITD插入突变,3例患者为3条ITD插入突变。高通量测序共检测到的33条ITD中17条ITD插入序列为FLT3野生型完全重复,16条ITD为野生型部分重复。1例伴多条ITD患者同一插入长度存在两种ITD序列,另外1例伴有2条ITD为同一ITD插入位点。ITD插入序列发生在p.Y572位至p.L602位之间,所有患者ITD均覆盖p.V592-p.E598之间的一个或多个氨基酸。结论:Illumina Miseq二代测序仪可灵敏、准确地分析FLT3-ITD序列,FLT3-ITD序列特征变化较大但突变热点区域较为集中。Objectice:To evaluate the application of high-throughput sequencing to sequence the FMS4 ike Tyrosine Kinase 3 internal tandem duplication(FLT3-ITD) in de novo acute myeloid leukemia(AML) patients with lower allelic ratio FLT3-ITD mutation or more than one ITD,and to analyze the feature of ITD.Methods:The genomic DNA of 23 AML patients with positive FLT3-ITD was amplified by PCR,capillary electrophoresis was used to detect the ITD mutation.Then,the FLT3 gene was amplified using primer with different barcode,and the product was analyzed by illumina Miseq,and the results were compared with UCSC database.Results:Out of 23 AML patients,17 had a single ITD,and 3 had 2 ITDs,and the remaining 3 had 3 ITD detected by capillary electrophoresis.The high-throughput sequencing showed that 17 ITD were the complate duplications of wild-type FLT3,and the remaining 16 ITD were partial duplications in the all 33 ITDs.The same length ITD mutation contained 2 different ITD sequences in one patient with more than one ITD,and the other patient with 2 ITD had the same ITD insertion position.The ITD occurred in the regions from p.Y572 to p.L602 of the FLT3 protein,and all the patient ITD covered one or more amino acid between p.V592 and p.E598.Conclusion:Illumina Miseq can analyze the sequence of ITDs precisely and accurately.ITD mutation varies widely,but the hotspots are concentrated.

关 键 词：高通量测序 FLT3基因 急性髓细胞白血病 毛细管电泳 ITD突变 

分 类 号：R440[医药卫生—诊断学] R733.71[医药卫生—临床医学]