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Title: High resolution melting analysis: A rapid and accurate method to detect CALR mutations
Authors: Bilbao-Sieyro, Cristina 
Santana, Guillermo
Moreno, Melania
Torres, Laura
Santana-Lopez, Gonzalo
Rodriguez-Medina, Carlos
Perera, María
Bellosillo, Beatriz
De La Iglesia, Silvia
Molero Labarta, María Teresa 
Gómez Casares, María Teresa 
UNESCO Clasification: 32 Ciencias médicas
3205 Medicina interna
Keywords: Calreticulin
CALR mutations
High Resolutions Melting Analysis
Issue Date: 2014
Journal: PLoS ONE 
Abstract: Background: The recent discovery of CALR mutations in essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients without JAK2/MPL mutations has emerged as a relevant finding for the molecular diagnosis of these myeloproliferative neoplasms (MPN). We tested the feasibility of high-resolution melting (HRM) as a screening method for rapid detection of CALR mutations.Methods: CALR was studied in wild-type JAK2/MPL patients including 34 ET, 21 persistent thrombocytosis suggestive of MPN and 98 suspected secondary thrombocytosis. CALR mutation analysis was performed through HRM and Sanger sequencing. We compared clinical features of CALR-mutated versus 45 JAK2/MPL-mutated subjects in ET.Results: Nineteen samples showed distinct HRM patterns from wild-type. Of them, 18 were mutations and one a polymorphism as confirmed by direct sequencing. CALR mutations were present in 44% of ET (15/34), 14% of persistent thrombocytosis suggestive of MPN (3/21) and none of the secondary thrombocytosis (0/98). Of the 18 mutants, 9 were 52 bp deletions, 8 were 5 bp insertions and other was a complex mutation with insertion/deletion. No mutations were found after sequencing analysis of 45 samples displaying wild-type HRM curves. HRM technique was reproducible, no false positive or negative were detected and the limit of detection was of 3%.Conclusions: This study establishes a sensitive, reliable and rapid HRM method to screen for the presence of CALR mutations.
ISSN: 1932-6203
DOI: 10.1371/journal.pone.0103511
Source: Plos One[ISSN 1932-6203],v. 9 (7)
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