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A novel molecular assay using hybridisation probes and melt curve analysis for CALR exon 9 mutation detection in myeloproliferative neoplasms
  1. Thomas Keaney1,
  2. Louise O'Connor1,2,
  3. Janusz Krawczyk3,4,
  4. Moutaz A Abdelrahman3,4,
  5. Amjad H Hayat3,4,
  6. Margaret Murray3,4,
  7. Michael O'Dwyer3,4,
  8. Melanie Percy5,
  9. Stehpen Langabeer6,
  10. Karl Haslam6,
  11. Barry Glynn1,
  12. Ciara Mullen1,
  13. Evelyn Keady1,
  14. Sinéad Lahiff1,
  15. Terry J Smith1,2
  1. 1Research and Development Division, Advanced Molecular Systems, Galway, Ireland
  2. 2Molecular Diagnostic Research Group, School of Natural Sciences, National University of Ireland, Galway, Ireland
  3. 3Department of Haematology, National University of Ireland, Galway, Ireland
  4. 4Galway University Hospital, Galway, Ireland
  5. 5Department of Haematology, Belfast City Hospital, Belfast, UK
  6. 6Cancer Molecular Diagnostics, St. James's Hospital, Dublin, Ireland
  1. Correspondence to Dr Louise O'Connor Molecular Diagnostics Research Group, Orbsen Building, National University of Ireland Galway, Galway, Ireland; louise.oconnor{at}


Aims Somatic insertions/deletions in exon 9 of the calreticulin gene have been identified in patients with essential thrombocythemia and primary myelofibrosis. Over 55 mutations have been discovered, 80% of which consist of either type 1 52-bp deletion or type 2 5-bp insertion. Other mutations (types 3–5) in conjunction with types 1 and 2 account for >87% of identified mutations. The aim of this study was development of a rapid PCR-based assay using LightCycler Hybridisation Probes for the detection of type 1–5 CALR mutations.

Method A real-time PCR assay using a novel HybProbe set was developed for use on the LightCycler 480 Instrument II. The acceptor probe was labelled with LC640 and Faststart DNA Master HybProbe kit was used for PCR reactions.

Results Assay limit of detection was determined to be seven target copies with a probability of 95%. The specificity of the assay was determined by using synthetic constructs of CALR wild-type and CALR mutation types 1–5 with no non-specific detection observed. Samples from 21 patients with essential thrombocythemia (ET) and 12 patients with primary myelofibrosis (PMF), together with 29 control samples from patients diagnosed with various conditions, were screened using the assay. Of these, 24 were found to have mutations in CALR exon 9, with the assay detecting 8 type 1 mutations, 12 type 2 mutations, 2 type 24 mutations, 1 type 20 mutation and 1 31-bp deletion.

Conclusions The novel assay described has potential for application as a rapid, sensitive, high-throughput screening method in the clinical diagnostics setting.

  • PCR

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  • Handling editor Mary Frances McMullin

  • Contributors TK and LOC contributed equally. TK performed the experimental work described in the manuscript. LOC is a member of the project management team. Contributed intellectually to the design of the study and prepared the manuscript. JK is a member of the project management team responsible for the study concept and design. Assisted with collection of clinical samples. AHH and MM assisted with collection of clinical samples and contributed to study concept and design. MOD is a member of the project management team responsible for the study concept and design. MP, SL and KH provided access to clinical samples and provided critical review of the manuscript. BG , CM , EK and SL assisted with technical aspects of assay development. TS is a member of the project management team responsible for the study concept and design, critical review of the experimental work. Responsible for manuscript preparation and critical review.

  • Funding Irish Research Council (EBPPG/2013/62).

  • Competing interests None declared.

  • Patient consent Obtained.

  • Ethics approval National University of Ireland Galway Research Ethics Committee and GUH REC.

  • Provenance and peer review Not commissioned; externally peer reviewed.