RAPID LOW-COST DETECTION OF TYPE 2CALR MUTATION BY ALLELE-SPECIFIC RT-PCR FOR DIAGNOSIS OF MYELOPROLIFERATIVE NEOPLASMS

Abstract

Background: 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. Aim: 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. Materials and Methods: 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. Results: 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. Conclusion: 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.