title: Evaluation of methods to detect CALR mutations in myeloproliferative neoplasms.
abstract: The recent discovery of somatically acquired CALR mutations in a substantial proportion of patients with myeloproliferative neoplasms has provided a new marker of clonal disease, advancing both diagnosis and prognosis in these previously difficult to characterise disorders. The mutations, which can be challenging to detect on a routine basis, are heterogeneous insertions/deletions (indels) in exon 9 with mutant allele burden that vary substantially between patients. We evaluated four genetic screening methods for their ability to detect a series of different CALR mutations; Sanger sequencing, fragment analysis PCR, high resolution melt (HRM) and targeted next generation sequencing (NGS). The limit of detection (LoD) of each assay was tested using serial dilution series made with DNA from CALR positive sample DNA and a cell line, MARIMO, found to carry a heterozygous 61 nucleotide CALR deletion. All methods were capable of detecting each mutation; HRM and fragment analysis PCR were better at detecting low mutation levels compared to Sanger sequencing but targeted NGS had the lowest LoD at a 1% mutation burden.
authors.affiliation: Faculty of Medicine, University of Southampton, Southampton, UK; Wessex Regional Genetics Laboratory, Salisbury NHS Foundation Trust, Salisbury, UK.
authors.forename: Amy V
authors.initials: AV
authors.lastname: Jones
authors.affiliation: Wessex Regional Genetics Laboratory, Salisbury NHS Foundation Trust, Salisbury, UK.
authors.forename: Daniel
authors.initials: D
authors.lastname: Ward
authors.affiliation: Wessex Regional Genetics Laboratory, Salisbury NHS Foundation Trust, Salisbury, UK.
authors.forename: Matthew
authors.initials: M
authors.lastname: Lyon
authors.affiliation: Faculty of Medicine, University of Southampton, Southampton, UK; Wessex Regional Genetics Laboratory, Salisbury NHS Foundation Trust, Salisbury, UK.
authors.forename: William
authors.initials: W
authors.lastname: Leung
authors.affiliation: Wessex Regional Genetics Laboratory, Salisbury NHS Foundation Trust, Salisbury, UK.
authors.forename: Alison
authors.initials: A
authors.lastname: Callaway
authors.affiliation: Faculty of Medicine, University of Southampton, Southampton, UK; Wessex Regional Genetics Laboratory, Salisbury NHS Foundation Trust, Salisbury, UK.
authors.forename: Andrew
authors.initials: A
authors.lastname: Chase
authors.affiliation: Wessex Regional Genetics Laboratory, Salisbury NHS Foundation Trust, Salisbury, UK.
authors.forename: Carolyn L
authors.initials: CL
authors.lastname: Dent
authors.affiliation: Faculty of Medicine, University of Southampton, Southampton, UK; Wessex Regional Genetics Laboratory, Salisbury NHS Foundation Trust, Salisbury, UK.
authors.forename: Helen E
authors.initials: HE
authors.lastname: White
authors.affiliation: Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany.
authors.forename: Hans G
authors.initials: HG
authors.lastname: Drexler
authors.affiliation: Cambridge Institute for Medical Research and Wellcome Trust/MRC Stem Cell Institute, University of Cambridge, Cambridge, UK.
authors.forename: Jyoti
authors.initials: J
authors.lastname: Nangalia
authors.affiliation: Faculty of Medicine, University of Southampton, Southampton, UK; Wessex Regional Genetics Laboratory, Salisbury NHS Foundation Trust, Salisbury, UK.
authors.forename: Chris
authors.initials: C
authors.lastname: Mattocks
authors.affiliation: Faculty of Medicine, University of Southampton, Southampton, UK; Wessex Regional Genetics Laboratory, Salisbury NHS Foundation Trust, Salisbury, UK. Electronic address: ncpc@soton.ac.uk.
authors.forename: Nicholas C P
authors.initials: NC
authors.lastname: Cross
mesh_terms: D000483:Alleles; D037282:Calreticulin / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods; D004273:DNA, Neoplasm / Q000235:genetics; D005260:Female; D019337:Hematologic Neoplasms / Q000235:genetics*; D006801:Humans; D008126:Lod Score; D008297:Male; D009154:Mutation; D009196:Myeloproliferative Disorders / Q000235:genetics*; D009363:Neoplasm Proteins / Q000235:genetics*
chemical_list: C576483:CALR protein, human; D037282:Calreticulin; D004273:DNA, Neoplasm; D009363:Neoplasm Proteins
keywords: CALR; MARIMO; MPN