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

Ngo Tat Trung†,
Dao Thanh Quyen,
Nghiem Xuan Hoan,
Dao Phuong Giang,
Tran Thi Huyen Trang,
Thirumalaisamy P. Velavan,
Mai Hong Bang† and
Le Huu Song†Email author View ORCID ID profile

†Contributed equally

BMC Medical Genetics201920:115

https://doi.org/10.1186/s12881-019-0819-6

Received: 26 February 2019
Accepted: 3 May 2019
Published: 27 June 2019

Open Peer Review reports

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.