Gorbenko A S, Stolyar M A, Olkhovskiy I A, Abdullaev A O, Sudarikov A B, Dunaeva E A, Mironov K O, Shipulin G A
Krasnoyarsk branch of the "National Research Center for Hematology", 660036, Krasnoyarsk, Russia.
Federal Research Center "Krasnoyarsk Scientific Center of the Siberian Branch of the Russian Academy of Sciences", 660036, Krasnoyarsk, Russia.
Klin Lab Diagn. 2018;63(9):588-592. doi: 10.18821/0869-2084-2018-63-8-588-592.
The detection of somatic mutations in the 9 exon of the calreticulin gene (CALR) is regulated by the clinical recommendations as a diagnostic criterion for chronic Ph-negative myeloproliferative neoplasms (MPN). Some methods of nucleic acids testing are used to identify CALR gene mutations with different requirements for special skills of personnel and expensive equipment. The purpose of this work is to compare the results of the detection of CALR gene mutations in venous blood samples by allele-specific RT-PCR with subsequent electrophoresis, fragment analysis and Sanger- or pyro- sequencing. We used 1284 blood samples of patients with suspected MPN and 20 blood donor samples. Mutations in the CALR gene of the I and II type were identified using PCR-RT with the original primers and TaqMan probes. Also, all samples were tested for mutations in the CALR gene by electrophoretic detection of PCR results in an agarose gel. The use of allele-specific RT-PCR followed by electrophoretic detection made it possible to determine clinically significant mutations in the CALR gene in 81 venous blood samples of JAK2- and MPL-negative patients, including 42 cases of type I mutation, 33 cases of type II mutation and 8 rare CALR mutations. Mutations in the 9 exon of the CALR gene were not detected in any of the 20 blood donor samples or in 121 blood samples of patients with polycythemia vera. In randomly selected 20 negative samples, CALR gene mutations were also not detected using Sanger sequencing. All positive samples were confirmed by fragment analysis, as well as with Sanger- sequencing and pyro- sequencing. The described combined approach to detect mutations of the CALR gene in peripheral blood samples can be used in clinical diagnostic laboratories that have a standard set of equipment for electrophoresis of nucleic acids and a PCR-RT. We also propose a confirmatory test based on the pyrosequencing of DNA using the system of genetic analysis "PyroMark Q24".
钙网蛋白基因（CALR）第9外显子体细胞突变的检测受临床推荐规范的调控，作为慢性Ph阴性骨髓增殖性肿瘤（MPN）的诊断标准。一些核酸检测方法用于鉴定CALR基因突变，这些方法对人员的特殊技能和昂贵设备有不同要求。本研究的目的是比较采用等位基因特异性逆转录聚合酶链反应（RT-PCR）并随后进行电泳、片段分析以及桑格测序或焦磷酸测序来检测静脉血样本中CALR基因突变的结果。我们使用了1284份疑似MPN患者的血样和20份献血者血样。使用原始引物和TaqMan探针通过PCR-RT鉴定I型和II型CALR基因的突变。此外，通过在琼脂糖凝胶中电泳检测PCR结果，对所有样本进行CALR基因突变检测。采用等位基因特异性RT-PCR并随后进行电泳检测，使得在81份JAK2和MPL阴性患者的静脉血样本中确定了具有临床意义的CALR基因突变，其中包括42例I型突变、33例II型突变和8例罕见的CALR突变。在20份献血者血样或121份真性红细胞增多症患者血样中均未检测到CALR基因第9外显子的突变。在随机选择的20份阴性样本中，使用桑格测序也未检测到CALR基因突变。所有阳性样本均通过片段分析以及桑格测序和焦磷酸测序得到确认。所描述的用于检测外周血样本中CALR基因突变的联合方法可用于具备核酸电泳和PCR-RT标准设备的临床诊断实验室。我们还提出了一种基于使用“PyroMark Q24”基因分析系统对DNA进行焦磷酸测序的验证试验。
