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

BMC Medical Genetics

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[1] Centre for Genetic Consultation and Cancer Screening, 108 Institute of Clinical Medical and Pharmaceutical Sciences, Hanoi, Vietnam;Vietnamese - German Center for Medical Research, 108 Institute of Clinical Medical and Pharmaceutical Sciences, No 1, Tran Hung Dao Street, Hai Ba Trung District, Hanoi, Vietnam;Department of Molecular Biology, 108 Institute of Clinical Medical and Pharmaceutical Sciences, Hanoi, Vietnam;Vietnamese - German Center for Medical Research, 108 Institute of Clinical Medical and Pharmaceutical Sciences, No 1, Tran Hung Dao Street, Hai Ba Trung District, Hanoi, Vietnam;0000 0001 0196 8249, grid.411544.1, Institute of Tropical Medicine, Universitätsklinikum Tübingen, Tübingen, Germany;Vietnamese - German Center for Medical Research, 108 Institute of Clinical Medical and Pharmaceutical Sciences, No 1, Tran Hung Dao Street, Hai Ba Trung District, Hanoi, Vietnam;Department of Molecular Biology, 108 Institute of Clinical Medical and Pharmaceutical Sciences, Hanoi, Vietnam;Vietnamese - German Center for Medical Research, 108 Institute of Clinical Medical and Pharmaceutical Sciences, No 1, Tran Hung Dao Street, Hai Ba Trung District, Hanoi, Vietnam;Faculty of Gastroenterology, 108 Institute of Clinical Medical and Pharmaceutical Sciences, Hanoi, Vietnam;Vietnamese - German Center for Medical Research, 108 Institute of Clinical Medical and Pharmaceutical Sciences, No 1, Tran Hung Dao Street, Hai Ba Trung District, Hanoi, Vietnam;Faculty of Tropical and Infectious Diseases, 108 Institute of Clinical Medical and Pharmaceutical Sciences, Hanoi, Vietnam;

关键词: Myeloproliferative neoplasms;    JAK2;    CALR;   

DOI  :  10.1186/s12881-019-0819-6

来源: publisher

【 摘 要 】

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.

【 授权许可】

CC BY