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

BMC Med Genet. 2019 Jun 27;20(1):115. doi: 10.1186/s12881-019-0819-6.


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.

Keywords: CALR mutations; JAK2 V617F; Myeloproliferative neoplasms.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adolescent
  • Adult
  • Aged
  • Aged, 80 and over
  • Alleles
  • Biomarkers, Tumor
  • Calreticulin / genetics*
  • Cost-Benefit Analysis
  • Female
  • Genotyping Techniques / methods
  • Humans
  • Janus Kinase 2 / genetics
  • Leukemia, Myeloid, Chronic, Atypical, BCR-ABL Negative
  • Male
  • Middle Aged
  • Mutation*
  • Myeloproliferative Disorders / diagnosis*
  • Myeloproliferative Disorders / genetics*
  • Phenotype
  • Polycythemia Vera
  • Primary Myelofibrosis
  • Real-Time Polymerase Chain Reaction* / economics
  • Real-Time Polymerase Chain Reaction* / methods
  • Sensitivity and Specificity
  • Thrombocythemia, Essential
  • Young Adult


  • Biomarkers, Tumor
  • Calreticulin
  • calreticulin, human
  • JAK2 protein, human
  • Janus Kinase 2