title: Rapid and sensitive detection of CALR exon 9 mutations using high-resolution melting analysis.
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.
  
  
  CONCLUSIONS
  The HRMA developed in our system is a rapid and sensitive technique for the detection of CALR exon 9 mutations.
authors.affiliation: 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.
authors.forename: Ken-Hong
authors.initials: KH
authors.lastname: Lim
authors.affiliation: 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.
authors.forename: Huan-Chau
authors.initials: HC
authors.lastname: Lin
authors.affiliation: 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.
authors.forename: Caleb Gon-Shen
authors.initials: CG
authors.lastname: Chen
authors.affiliation: Laboratory of Good Clinical Research Center, Department of Medical Research, Mackay Memorial Hospital, Tamsui District, New Taipei City, Taiwan.
authors.forename: Wei-Ting
authors.initials: WT
authors.lastname: Wang
authors.affiliation: 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.
authors.forename: Yu-Cheng
authors.initials: YC
authors.lastname: Chang
authors.affiliation: 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.
authors.forename: Yi-Hao
authors.initials: YH
authors.lastname: Chiang
authors.affiliation: Laboratory of Good Clinical Research Center, Department of Medical Research, Mackay Memorial Hospital, Tamsui District, New Taipei City, Taiwan.
authors.forename: Ching-Sung
authors.initials: CS
authors.lastname: Lin
authors.affiliation: 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.
authors.forename: Nai-Wen
authors.initials: NW
authors.lastname: Su
authors.affiliation: 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.
authors.forename: Ying-Wen
authors.initials: YW
authors.lastname: Su
authors.affiliation: Division of Hematology and Oncology, Department of Internal Medicine, Mackay Memorial Hospital, Taipei, Taiwan.
authors.forename: Johnson
authors.initials: J
authors.lastname: Lin
authors.affiliation: 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.
authors.forename: Yi-Fang
authors.initials: YF
authors.lastname: Chang
authors.affiliation: 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.
authors.forename: Ming-Chih
authors.initials: MC
authors.lastname: Chang
authors.affiliation: 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.
authors.forename: Ruey-Kuen
authors.initials: RK
authors.lastname: Hsieh
authors.affiliation: Graduate Institute of Oncology, National Taiwan University College of Medicine, Taipei, Taiwan. Electronic address: yykuo@ntu.edu.tw.
authors.forename: Yuan-Yeh
authors.initials: YY
authors.lastname: Kuo
authors.affiliation: 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.
authors.forename: Wen-Chien
authors.initials: WC
authors.lastname: Chou
mesh_terms: D000328:Adult; D037282:Calreticulin / Q000235:genetics*; D016022:Case-Control Studies; D015331:Cohort Studies; D004252:DNA Mutational Analysis / Q000379:methods*; D005091:Exons; D006801:Humans; D053614:Janus Kinase 2 / Q000235:genetics; D009154:Mutation; D012680:Sensitivity and Specificity; D013920:Thrombocythemia, Essential / Q000235:genetics*
chemical_list: C576483:CALR protein, human; D037282:Calreticulin; C507924:JAK2 protein, human; D053614:Janus Kinase 2
keywords: Calreticulin (CALR); Essential thrombocythemia; High-resolution melting analysis; Mutation; Myeloproliferative neoplasm