Abstract
BackgroundCalreticulin (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.MethodsOne 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.ResultsPCR - 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.ConclusionPCR-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.
Highlights
Calreticulin (CALR) gene mutations are currently recommended as biomarkers in diagnosis of patients with myeloproliferative neoplasms (MPN) with Janus Kinase 2 (Jak2) V617F negative phenotype
Baseline characteristics of MPN patients Recruited patients were diagnosed based on clinical manifestations and haematological tests and subsequently classified into three groups: polycythemia vera (PV), essential thrombocythemia (ET) and Primary myelofibrosis (PM) based on the revised World Health Organization (WHO) diagnostic criteria [11]
PLT levels were significantly higher in ET patients than PV and primary myelofibrosis (PMF) groups (P < 0.0001)
Summary
Calreticulin (CALR) gene mutations are currently recommended as biomarkers in diagnosis of patients with myeloproliferative neoplasms (MPN) with Jak 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. Three distinct phenotypes of PV, ET and PMF mainly constitute to Philadelphia-negative myeloproliferative syndromes [2]. Genetic alterations in both Janus Kinase 2 (Jak2) and thrombopoietin receptor myeloproliferative leukemic (MPL) virus oncogenes serve as molecular targets in the diagnosis of MPN [3]. Trung et al BMC Medical Genetics (2019) 20:115 other Jak variants in exon 10 and 12 [4, 5] and non-synonymous MPL variant in exon 10 (W515 L and W515K) [5, 6] were reported to occur among 5% of patients with MPN
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