Rapid and sensitive detection of CALR exon 9 mutations using high-resolution melting analysis.


BACKGROUND Somatic CALR exon 9 mutations have recently been identified in patients with JAK2/MPL-unmutated myeloproliferative neoplasm, and have become an important clonal marker for the diagnosis of essential thrombocythemia (ET) and primary myelofibrosis. In the present study, we sought to use high-resolution melting analysis (HRMA) as a screening method for the detection of CALR mutations. METHODS 32 JAK2/MPL-unmutated ET patients were retrospectively enrolled and 8 healthy adults were used as wild-type control. CALR exon 9 mutation was independently screened by HRMA with the CFX Connect real-time system and Sanger sequencing. TA-cloning was used to detect CALR exon 9 mutations in patients suspected to have low mutant allele burden. RESULTS The maximal sensitivity of HRMA in identifying both CALR type 1 and type 2 mutants from patients' genomic DNA was 2.5%. Twenty-two samples were found to have distinct melting curves from wild-type. The presence of CALR mutations in 16 of these 22 samples was confirmed by Sanger sequencing, while the other 6 samples were wild-type by sequencing. After TA-cloning, CALR mutations were detected in 5 of 6 patients from 1 (6%) of 16 clones to 1 (2%) of 50 clones. Therefore, HRMA identified CALR mutations in 21 (65.6%) of 32 ET patients compared to 16 (50%) patients by Sanger sequencing, with a false positive rate of 3% and no false negative. CONCLUSION The HRMA developed in our system is a rapid and sensitive technique for the detection of CALR exon 9 mutations.

DOI: 10.1016/j.cca.2014.11.011
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@article{Lim2015RapidAS, title={Rapid and sensitive detection of CALR exon 9 mutations using high-resolution melting analysis.}, author={Ken-Hong Lim and Huan-Chau Lin and Caleb Gon-Shen Chen and Wei-Ting Wang and Yu-Cheng Chang and Yi-Hao Chiang and Ching-Sung Lin and Nai-Wen Su and Ying-Wen Su and Johnson Lin and Yi-Fang Chang and Ming-Chih Chang and R F Hsieh and Yuan-Yeh Kuo and W H Chou}, journal={Clinica chimica acta; international journal of clinical chemistry}, year={2015}, volume={440}, pages={133-9} }