Conference Contribution Details
Mandatory Fields
Keaney , T., O’Connor, T., Krawczyk, J., O’Dwyer, M., Murray, M., Hayat, A., Abdelrahman, M., Glynn, B., Mullen, C?., Lahiff, S., Smith, T.J.
Haematology Association of Ireland Annual Conference
Development of novel molecular assay using hybridisation probes and melt curve analysis for CALR mutations in myeloproliferative neoplasms
Sligo, Ireland
Conference Organising Committee Chairperson
Optional Fields
BackgroundSomatic insertions/deletions in exon 9 of the Calreticulin (CALR) gene have recently been identified in patients with Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) who are negative for both JAK2 and MPL mutations. To date, over forty mutations in exon 9 have been discovered, over 80% of which consist of either type-1 52-bp deletion (p.L367fs*46) or type-2 5-bp insertion (p.K385fs*47), with a further three mutations (types 3 – 5) in conjunction with types 1 and 2 accounting for approximately 88% of identified CALR exon 9 mutations(1, 2). This study aimed to develop a rapid, user friendly assay using LightCycler™ Hybridization Probes (HybProbes) and melt curve analysis for the detection of type1-type 5 CALR mutations.    MaterialsA real-time PCR assay using HybProbes and melt curve analysis was developed for the detection of type 1-type 5 mutations in CALR for use on the LightCycler® 480. Limit of detection and specificity of the assay was established using synthetic constructs. DNA was extracted from PBMCs from twenty five patients with various myeloid malignancies including two patients with ET and tested with the assay. ResultsThe limit of detection of the assay was determined to be 10 copies of target, while the ability of the assay to detect CALR mutations of types 1-5 was confirmed using synthetic constructs. Twenty five samples from patients with various haematological disorders were analysed.One sample was found to be positive for type 2 5-bp TTGTC insertion. This patient had been diagnosed with ET and was JAK2 and MPL negative. The result was confirmed by Sanger sequencing. ConclusionsMelt curve analysis provides a sensitive, specific and a rapid method for the detection of CALR mutations.  CALR gene mutation detection can aid in the specific diagnosis of a myeloproliferative neoplasm, and help distinguish this clonal disease from a benign reactive process. CALR Diagnosis has been proposed to become part of the world health organization diagnostic criteria for ET and PMF in the same manner as Jak 2 did in 2008(3) and the assay developed as part of this study could provide a rapid, reliable, sensitive, high throughput screening method which could be easily be incorporated in a clinical setting.References 1.  Klampfl T, Gisslinger H, Harutyunyan AS, Nivarthi H, Rumi E, Milosevic JD, et al. Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med Dec 19;369(25):2379-90.2.  Nangalia J, Massie CE, Baxter EJ, Nice FL, Gundem G, Wedge DC, et al. Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2. N Engl J Med Dec 19;369(25):2391-405.   
Advanced Molecular Systems Ltd. and Irish Research Council Employment-based Post-graduate Programme Fellowship
Publication Themes
Biomedical Science and Engineering