Detection of CALR mutations in peripheral blood of myeloproliferative neoplasm patients with high resolution melting curve analysis

Abstract

Objective To establish a rapid, accurate and low-cost screening method for the detection of calreticulin (CALR) mutations in myeloproliferative neoplasms (MPN). Methods Seventy cases diagnosed with MPN were collected from 2012 to 2016. PCR combined with high resolution melting (HRM) analysis were used to screen the CALR mutations, and Sanger sequencing and T-A sequencing were applied to verify the HRM positive samples. CALR wild type DNA, type 1 and type 2 mutant DNA samples were selected and analyzed 4 times/day for 5 days to detected the CVs of Tm (melting temperature) respectively. JAK2 mutations were also analyzed in MPN patients to compare the association between JAK2 and CALR mutations. Results PCR-HRM analysis showed 7 cases (26.9%) and 5 cases (20.8%) patients with CALR mutations were screened out from 26 essential thrombocythaemia (ET) cases and 24 primary myelofibrosis (PMF) cases, but no CALR mutations were found in cases with polycythaemia vera (PV). All mutations were confirmed by direct sequencing or cloning sequencing. The CVs for HRM analysis of CALR wild type DNA, type 1 and type 2 mutant DNA samples were 1.91%, 1.59% and 1.43%, respectively.There were 47 cases with JAK2 V617F and 1 case with exon12 mutation. No coexistence of JAK2 mutation and CALR mutations were found in a single sample. Conclusion PCR-HRM can be used for rapid screening of CALR mutation. Subsequent sequencing can be applied for rapid diagnosis of MPN patients in clinical practice.(Chin J Lab Med, 2017, 40: 456-459) Key words: Myeloproliferative disorders; Hematologic neoplasms; Calreticulin; Mutation; High resolution melting