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Clin Chim Acta. 2015 Feb 2;440:133-9. doi: 10.1016/j.cca.2014.11.011. Epub 2014 Nov 15.

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

Author information

1
Graduate 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. Electronic address: khlim@mmh.org.tw.
2
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.
3
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; Institute of Molecular and Cellular Biology, National Tsing-Hua University, Hsinchu, Taiwan.
4
Laboratory of Good Clinical Research Center, Department of Medical Research, Mackay Memorial Hospital, Tamsui District, New Taipei City, Taiwan.
5
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.
6
Division of Hematology and Oncology, Department of Internal Medicine, Mackay Memorial Hospital, Taipei, Taiwan.
7
Graduate Institute of Oncology, National Taiwan University College of Medicine, Taipei, Taiwan. Electronic address: yykuo@ntu.edu.tw.
8
Division of Hematology, Department of Internal Medicine, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan; Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan.

Abstract

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.

KEYWORDS:

Calreticulin (CALR); Essential thrombocythemia; High-resolution melting analysis; Mutation; Myeloproliferative neoplasm

PMID:
25447704
DOI:
10.1016/j.cca.2014.11.011
[Indexed for MEDLINE]

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