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Leuk Res. 2015 Jan;39(1):82-7. doi: 10.1016/j.leukres.2014.11.019. Epub 2014 Nov 29.

Evaluation of methods to detect CALR mutations in myeloproliferative neoplasms.

Author information

1
Faculty of Medicine, University of Southampton, Southampton, UK; Wessex Regional Genetics Laboratory, Salisbury NHS Foundation Trust, Salisbury, UK.
2
Wessex Regional Genetics Laboratory, Salisbury NHS Foundation Trust, Salisbury, UK.
3
Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany.
4
Cambridge Institute for Medical Research and Wellcome Trust/MRC Stem Cell Institute, University of Cambridge, Cambridge, UK.
5
Faculty of Medicine, University of Southampton, Southampton, UK; Wessex Regional Genetics Laboratory, Salisbury NHS Foundation Trust, Salisbury, UK. Electronic address: ncpc@soton.ac.uk.

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.

KEYWORDS:

CALR; MARIMO; MPN

PMID:
25499808
DOI:
10.1016/j.leukres.2014.11.019
[Indexed for MEDLINE]

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