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.