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

Tran Thi Huyen Trang¹  Ngo Tat Trung¹  Nghiem Xuan Hoan²  Dao Thanh Quyen²  Mai Hong Bang²  Le Huu Song²  Thirumalaisamy P. Velavan²  Dao Phuong Giang² 

[1] Centre for Genetic Consultation and Cancer Screening, 108 Institute of Clinical Medical and Pharmaceutical Sciences;Vietnamese - German Center for Medical Research, 108 Institute of Clinical Medical and Pharmaceutical Sciences;

关键词: Myeloproliferative neoplasms;    JAK2 V617F;    CALR mutations;   

DOI  :  10.1186/s12881-019-0819-6

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown