Abstract
BackgroundThe 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.ResultsNineteen 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%.ConclusionsThis study establishes a sensitive, reliable and rapid HRM method to screen for the presence of CALR mutations.
Highlights
Philadelphia-negative myeloproliferative neoplasms include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) [1]
Exome sequencing techniques have allowed the identification of somatic mutations at exon 9 of calreticulin gene (CALR) in 56%–88% of JAK2/MPL-unmutated ET and PMF [3,4]
Calreticulin is a highly conserved endoplasmic reticulum (ER) Ca+2 binding chaperone that contributes to glycoprotein folding, it is found in the nucleus, cell membranes and extracellular matrix, and has been related to calcium homeostasis, cell adhesion and immune response [5]
Summary
Philadelphia-negative myeloproliferative neoplasms include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) [1]. Exome sequencing techniques have allowed the identification of somatic mutations at exon 9 of calreticulin gene (CALR) in 56%–88% of JAK2/MPL-unmutated ET and PMF [3,4]. Most of the mutants correspond to a 52 bp deletion (L367fs*46) and a 5 bp TTGTC insertion (K385fs*47) [3,4,6,7] These frameshift mutations instead of generating a premature protein termination, produce a significantly altered C-terminal domain with still unpredicted consequences [4]. 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
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