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

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

Trung NT1,2,3, Quyen DT2,3, Hoan NX2,4, Giang DP2,3, Trang TTH1,2,3, Velavan TP2,4, Bang MH2,5, Song LH6,7.

Author information

1
Centre for Genetic Consultation and Cancer Screening, 108 Institute of Clinical Medical and Pharmaceutical Sciences, Hanoi, Vietnam.
2
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.
3
Department of Molecular Biology, 108 Institute of Clinical Medical and Pharmaceutical Sciences, Hanoi, Vietnam.
4
Institute of Tropical Medicine, Universitätsklinikum Tübingen, Tübingen, Germany.
5
Faculty of Gastroenterology, 108 Institute of Clinical Medical and Pharmaceutical Sciences, Hanoi, Vietnam.
6
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. lehuusong@108-icid.com.
7
Faculty of Tropical and Infectious Diseases, 108 Institute of Clinical Medical and Pharmaceutical Sciences, Hanoi, Vietnam. lehuusong@108-icid.com.

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.

KEYWORDS:

CALR mutations; JAK2 V617F; Myeloproliferative neoplasms

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