Dybkov M V, Zavelevich M P, Gluzman D F, Telegeev G D
Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kyiv 03143, Ukraine.
RE Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, National Academy of Sciences of Ukraine, Kyiv 03022, Ukraine.
Exp Oncol. 2022 May;44(1):83-86. doi: 10.32471/exp-oncology.2312-8852.vol-44-no-1.17329.
Approximately 15% to 24% of essential thrombocythemia (ET) and 25-35% of primary myelofibrosis cases carry a mutation in the calreticulin (CALR) gene. Sanger sequencing, qPCR, high resolution melt or targeted next generation sequencing usually used to detect these mutations are expensive and require costly equipment. Nevertheless, type 1 CALR mutations are detectable by using polymerase chain reaction (PCR) and agarose gel electrophoresis.
To offer the use of the allele-specific reverse transcription (RT) PCR for rapid low-cost detection of the type 2 mutation in the CALR gene.
Allele-specific primers designed for detecting type 2 mutation (5-bp insertion; c.1154_1155 ins TTGTC) of the CALR gene were used for allele-specific RT-PCR analysis of cDNA of the patient with JAK2-, MPL-negative ET, whose mutation in CALR gene has been identified by Sanger sequencing. RT-PCR samples were analyzed by agarose gel electrophoresis.
The type 2 mutation (K385fs*47 ins5) in CALR gene was detected by Sanger sequencing in JAK2- and MPL-negative ET patient. The cDNA obtained was then re-analyzed by using allele-specific RT-PCR with newly designed primers. Normal and type 2 mutation alleles of the CALR gene were detected by gel electrophoresis. The results of allele-specific RT-PCR were consistent with the data of Sanger sequencing.
Allele-specific RT-PCR analysis may be used for the fast low-cost detection of the major type 2 mutation (ins 5) of the CALR gene in patients with MPNs.
大约15%至24%的原发性血小板增多症（ET）以及25%至35%的原发性骨髓纤维化病例携带钙网蛋白（CALR）基因突变。通常用于检测这些突变的桑格测序、定量聚合酶链反应（qPCR）、高分辨率熔解曲线分析或靶向新一代测序成本高昂且需要昂贵设备。然而，通过聚合酶链反应（PCR）和琼脂糖凝胶电泳可检测到1型CALR突变。
提供一种等位基因特异性逆转录（RT）PCR方法，用于快速低成本检测CALR基因的2型突变。
设计用于检测CALR基因2型突变（5碱基插入；c.1154_1155 ins TTGTC）的等位基因特异性引物，用于对JAK2、MPL阴性的ET患者的cDNA进行等位基因特异性RT-PCR分析，该患者的CALR基因突变已通过桑格测序鉴定。RT-PCR样本通过琼脂糖凝胶电泳进行分析。
通过桑格测序在JAK2和MPL阴性的ET患者中检测到CALR基因的2型突变（K385fs*47 ins5）。然后使用新设计的引物通过等位基因特异性RT-PCR对获得的cDNA进行重新分析。通过凝胶电泳检测到CALR基因的正常和2型突变等位基因。等位基因特异性RT-PCR的结果与桑格测序数据一致。
等位基因特异性RT-PCR分析可用于快速低成本检测骨髓增殖性肿瘤（MPN）患者中CALR基因的主要2型突变（插入5）。
