Rapid, low cost and sensitive detection of Calreticulin mutations by a PCR based amplicon length differentiation assay for diagnosis of myeloproliferative neoplasms

Abstract

Background Calreticulin ( CALR ) gene mutations are currently recommended as biomarkers in diagnosis of patients with myeloproliferative neoplasms (MPN) with Jak2 V617F negative phenotype. Our aim was to establish a rapid, low cost and sensitive assay for identification of CALR gene mutations and to validate the diagnostic performance of the established assay in a patient cohort with different clinical MPN phenotypes. Methods One hundred five Philadelphia-negative MPN patients, including polycythemia vera (PV), essential thrombocythaemia (ET), and primary myelofibrosis (PMF) were initially screened for JAK2 mutations by amplification-refractory mutation system (ARMS-PCR) methodology and were further subjected to detection of CALR gene mutations by our in-house assay, a PCR based amplicon length differentiation assay (PCR-ALDA). The PCR-ALDA methodology was compared with real time PCR and Sanger sequencing methods. Furthermore, the analytical sensitivity of the assay was established. Results PCR - ALDA approach was able to detect and discriminate the pseudo-positive samples containing more than 1% CALR mutant alleles. CALR mutations were not detected in 63 Jak2 V617F positive cases in all three methods. In contrast, amongst 42 Jak2 V617F negative cases, both PCR-ALDA and Sanger sequencing coherently identified 12 CALR mutants compared to 10 CALR mutants detected by real-time PCR method. Conclusion PCR-ALDA can be utilized as an easy-to-use, rapid, low cost and sensitive tool in the detection of CALR mutations in Philadelphia-negative MPN patients. Electronic supplementary material The online version of this article (10.1186/s12881-019-0819-6) contains supplementary material, which is available to authorized users.

Calreticulin Mutations in Myeloproliferative Neoplasms Patients Diagnosed in UKM Medical Centre

“…Since first discovered in 2013 by Nangalia et al (8) and Klampfl et al (9), after performing whole exome sequencing on myeloproliferative neoplasms patients who were JAK2 negative, more than 50 different types of mutations were documented in the CALR gene (10). Rapid advances in molecular techniques through PCR have since allowed these mutations to be detected through various methods such as high resolution melting (HRM) curve analysis (11), amplicon length based analysis (ALDA) (6,12) and recently an antibody based immunohistochemical method has been evaluated for use which showed good correlation with PCR based method (13). The method by Jeong JH et al was chosen primarily due to its simplicity of using only 3 primers with affordable, widely available agarose gel for detecting the mutations.…”

Production and Characterization of Peptide Antibodies to the C-Terminal of Frameshifted Calreticulin Associated with Myeloproliferative Diseases

“…In general, the treatment of MPNs using disease modifiers is further complicated, as the specific treatment depends on the individual mutation [ 16 ]. Diagnosis of MPNs is aided by the detection of the various associated mutations by polymerase chain reaction, sequencing, or other DNA-based methods [ 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 ], but the detection of disease-associated mutations may be facilitated or aided by mutation-specific immunohistochemistry (IHC) [ 31 , 32 , 33 ], methods which supplement each other. Moreover, antibodies such as these are invaluable reagents for studying the properties of CRT in relation to MPN.…”