title: High resolution melting analysis: a rapid and accurate method to detect CALR mutations.
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.
authors: Hematology Department, Hospital Universitario de Gran Canaria Dr. Negrin, Las Palmas de Gran Canaria, Spain; Morfology Department, Universidad de Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Spain.
authors: Cristina
authors: C
authors: Bilbao-Sieyro
authors: Hematology Department, Hospital Universitario de Gran Canaria Dr. Negrin, Las Palmas de Gran Canaria, Spain.
authors: Guillermo
authors: G
authors: Santana
authors: Hematology Department, Hospital Universitario de Gran Canaria Dr. Negrin, Las Palmas de Gran Canaria, Spain.
authors: Melania
authors: M
authors: Moreno
authors: Hematology Department, Hospital Universitario de Gran Canaria Dr. Negrin, Las Palmas de Gran Canaria, Spain.
authors: Laura
authors: L
authors: Torres
authors: Preventive Medicine Department, Hospital Universitario de Gran Canaria Dr. Negrin, Las Palmas de Gran Canaria, Spain.
authors: Gonzalo
authors: G
authors: Santana-Lopez
authors: Hematology Department, Hospital Universitario de Gran Canaria Dr. Negrin, Las Palmas de Gran Canaria, Spain.
authors: Carlos
authors: C
authors: Rodriguez-Medina
authors: Hematology Department, Hospital Universitario de Gran Canaria Dr. Negrin, Las Palmas de Gran Canaria, Spain.
authors: María
authors: M
authors: Perera
authors: Laboratori de Biologia Molecular Servei de Patologia, Hospital del Mar, Barcelona, Spain.
authors: Beatriz
authors: B
authors: Bellosillo
authors: Hematology Department, Hospital Universitario de Gran Canaria Dr. Negrin, Las Palmas de Gran Canaria, Spain.
authors: Silvia
authors: S
authors: de la Iglesia
authors: Hematology Department, Hospital Universitario de Gran Canaria Dr. Negrin, Las Palmas de Gran Canaria, Spain; Medical Sciences Department, Universidad de Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Spain.
authors: Teresa
authors: T
authors: Molero
authors: Hematology Department, Hospital Universitario de Gran Canaria Dr. Negrin, Las Palmas de Gran Canaria, Spain.
authors: Maria Teresa
authors: MT
authors: Gomez-Casares
mesh_terms: D001483:Base Sequence; D037282:Calreticulin / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D005820:Genetic Testing / Q000379:methods; D005838:Genotype; D006801:Humans; D053614:Janus Kinase 2 / Q000235:genetics; D008875:Middle Aged; D009154:Mutation; D009691:Nucleic Acid Denaturation; D020641:Polymorphism, Single Nucleotide; D055728:Primary Myelofibrosis / Q000175:diagnosis / Q000235:genetics*; D053628:Receptors, Thrombopoietin / Q000235:genetics; D015203:Reproducibility of Results; D012189:Retrospective Studies; D012680:Sensitivity and Specificity; D013920:Thrombocythemia, Essential / Q000175:diagnosis / Q000235:genetics*; D044366:Transition Temperature
chemical_list: D037282:Calreticulin; D053628:Receptors, Thrombopoietin; C082218:MPL protein, human; C507924:JAK2 protein, human; D053614:Janus Kinase 2