骨髓增生性疾病JAK2~(V617F)突变检测方法的比较研究

目的研究较好的检测骨髓增生性疾病(MPD)患者中JAK2~(V617F)点突变的方法。方法分别应用聚合酶链式反应-限制性片段长度多态性技术(PCR-RFLP)、等位基因特异性聚合酶链式反应(AS-PCR)检测16例慢性粒细胞白血病(CML)、22例真性红细胞增多症(PV)、26例原发性血小板增多症(ET)、12例慢性特发性骨髓纤维化(IMF)外周血单个核细胞基因组DNAJAK2~(V617F)点突变,结果经测序鉴定。结果测序证实经AS-PCR检测PV的阳性率为72.7%,比PCR-RFLP的阳性率(50%)检测提高22.7%;ET的阳性率为42.3%,比PCR-RFLP的阳性率(34.6%)检测提高7.7%。其中经PCR-RFLP检测的阳性样本全部包含在AS-PCR检测的阳性样本之中,二者结果均与测序结果相符。结论 AS-PCR与PCR-RFLP相比检测敏感性高,且操作简便、快捷,更具有临床大规模检测的应用价值。

The comparison of JAK2~(V617F) mutation detection methods in myeloproliferative disorders

Objective To investigate the better method to detect the occurrence of JAK2~(V617F) mutation in patients with my-eloproliferative disorders(MPD). Methods Genomic DNA was extracted from peripheral blood cell samples of 16 case of chronic myelogenous leukemia(CML), 22 cases of polycythacmia vera(PV), 26 cases of essential thrombocythaemia(ET) and 12 cases of idi-opathic myelofibrosis(IMF). Polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) and Allele-specific PCR(AS-PCR)were conducted to identify JAK2~(V617F) mutation individually. The sensitivity of two methods was compared success-fully and the results were confirmed by sequence analysis consequently. Results The occurrence of JAK2~(V617F) mutation in PV pa-tients detected by AS-PCR(72.7 %)was higher than that of PCR-RFLP(50 %). The occurrence of J AK2~(V617F) mutation in ET pa-tients detected by AS-PCR(42.3%)was higher than that of PCR-RFLP(34.6 %). All of the positive samples detected by PCR-RFLP were included in the positive samples of AS-PCR. The results of two methods were all coincided with sequence analysis. Conclusion AS-PCR is more sensitive,simple and convenient than PCR-RFLR. Consequently, it may have clinical significance in large scale detection of JAK2~V617F mutation.