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Updated: Apr 20, 2026
Genetic Variant Detection in the CALR gene using High Resolution Melting Analysis
Published on: August 26, 2020
Rapid and sensitive detection of CALR exon 9 mutations using high-resolution melting analysis
Ken-Hong Lim1, Huan-Chau Lin2, Caleb Gon-Shen Chen3
1Graduate Institute of Oncology, National Taiwan University College of Medicine, Taipei, Taiwan; Division of Hematology and Oncology, Department of Internal Medicine, Mackay Memorial Hospital, Taipei, Taiwan; Laboratory of Good Clinical Research Center, Department of Medical Research, Mackay Memorial Hospital, Tamsui District, New Taipei City, Taiwan; Department of Medicine, Mackay Medical College, New Taipei City, Taiwan.
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.
