原发性血小板增多症及原发性骨髓纤维化患者MPL515突变的筛查  

Screening MPL515 mutations from 286 cases of essential thrombocythemia and primary myelofibrosis

作　　者：许笑[1] 张心菊[1] 吴之源[2] 许小平[3] 陈波斌[3] 胡婷婷[2] 陈宇明[2] 关明[1] 

机构地区：[1]复旦大学附属华山医院中心实验室,上海200040 [2]复旦大学附属华山医院中心检验科,上海200040 [3]复旦大学附属华山医院中心血液科,上海200040

出　　处：《中华检验医学杂志》2012年第7期634-638,共5页Chinese Journal of Laboratory Medicine

基　　金：国家临床重点专科资助课题（2010）;上海市科委重点项目资助课题（10411950200）;复旦大学新教师科研启动基金资助项目（JJF151002）

摘　　要：目的建立一种能够简便、灵敏地检测原发性血小板增多症（ET）及原发性骨髓纤维化（PMF）患者外周血MPLS15突变的方法，探讨我国ET和PMF患者的MPLS15突变频率及JAK2V617F突变情况。方法选取2008至2010年复旦大学附属华山医院ET患者261例和PMF患者25例，提取患者外周血DNA。使用SYBRGreenI荧光定量PCR检测标本的JAK2V617F突变。分别采用针对MPLS15wt、MPLW515L和MPLW515K这3种基因型设计的Taqman探针，使用荧光定量PCR法检测MPLS15突变，并通过T—A克隆挑取纯克隆测序确认。结果在261例ET患者中，检测到JAK2V617F突变119例（45．6％），MPLS15突变7例（2．7％），包括5例MPLW515L突变、1例MPLW515K突变和1例MPLW515L＋K突变。25例PMF患者中，JAK2V617F突变为10例（40．0％），MPL515突变为3例（12．0％），包括1例MPLW515L突变和2例MPLW515L＋K突变。1例MPLW515K突变的ET患者同时伴有JAK2V617F突变。结论JAK2V617F是ET和PMF的分子标志物，而在JAK2V617F阴性的病例中，MPLS15突变则是一个重要、有益的补充。Objective To establish a simple and sensitive method to detect MPLS15 mutations in peripheral blood of ET and PMF patients, and investigate the frequencies of the MPL515 and JAK2V617F mutations in Chinese patients. Methods Totally 261 patients of ET and 25 PMF cases were collected from Huashan Hospital of Fudan University and DNA samples were isolated from peripheral blood of these cases. SYBR Green I real-time PCR was used to detect JAK2V617F mutation. Taqman probe was designed to be specific for the three types of mutations （MPLS15wt, MPLWS15L and MPLWS15K）. Real-time PCR was used to detect MPLS15 mutations. The results were confirmed by sequencing after T-A cloning. Results Among 261 ET patients, 119 cases （45.6%） were identified as JAK2V617F mutation carriers and 7 cases （2. 7% ） were detected to be MPLS15 mutation carriers, including 5 cases with MPLWS15L, 1 case with MPLWS15K and 1 case with MPLWS15L ＋ K. Additionally 10 cases with JAK2V617F（40. 0% ） and 3 cases with MPLS15 （12. 0% ） were screened out in 25 PMF patients, including 1 case with MPLWS15L and 2 cases with MPLWS15L ＋ K. One ET patient was found to harbor concurrent JAK2V617F and MPLS15 mutations. Conclusion JAK2V617F mutation is the major molecular marker of ET and PMF, meanwhile MPLS15 mutation is important and useful complement. （Chin J Lab Med,2012,35：634-638）

关 键 词：血小板增多 骨髓纤维化 受体 血小板生成素 JANUS激酶2 突变 聚合酶链反应 

分 类 号：R558[医药卫生—血液循环系统疾病]