High Resolution Melting Analysis: A Rapid and Accurate Method to Detect CALR Mutations

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.

A novel molecular assay using hybridisation probes and melt curve analysis forCALRexon 9 mutation detection in myeloproliferative neoplasms

“…Other techniques are available for detecting CALR mutations, including HRM19 and Sanger sequencing 20. Both methods have been reported to give false positive and negative results.…”

“…The HRM technique has been used in many studies and a range of mutation types emerged from these studies. For example, three different mutation types were detected in one study19 and six different mutations detected in a second study 20. HRM technology is similar to the HybProbe technology described in this study, but without the inclusion of a specific set of detection probes.…”

Molecular diagnostics of myeloproliferative neoplasms

“…Given the multitude and complexity of CALR exon 9 mutations, methodologies that can accommodate this variability will provide most utility in a diagnostic setting. Whilst initial reports employed PCR of CALR exon 9 followed by fragment analysis or Sanger sequencing to determine CALR mutation status , subsequent studies have explored the utility of alternative HRM techniques . A comparison of the limits of detection for HRM, next‐generation sequencing (NGS), and fragment analysis techniques to detect CALR mutations found a bespoke NGS approach capable of detecting mutations when present at a level of 1.25%, an improvement in sensitivity over HRM and fragment analysis .…”

High absolute basophil count is a powerful independent predictor of inferior overall survival in patients with primary myelofibrosis

“…For mutation testing, DNA was isolated from full blood by the QIAamp DNA Blood Mini Kit (Qiagen, ID 51104). JAK2 V617F was assessed by the allele-specific polymerase chain reaction (PCR), as described previously [22], CALR1 and MPL exon 10 mutations were screened by the high-resolution melting dye assays [23,24] and any sample sequence that deviated from normal was Sanger-sequenced.…”