title: A novel molecular assay using hybridisation probes and melt curve analysis forCALRexon 9 mutation detection in myeloproliferative neoplasms.
abstract: OBJECTIVE
  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 1-5CALRmutations.
  
  
  METHODS
  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 ofCALRwild-type andCALRmutation 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 inCALRexon 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.
authors.affiliation: Research and Development Division, Advanced Molecular Systems, Galway, Ireland.
authors.forename: Thomas
authors.initials: T
authors.lastname: Keaney
authors.affiliation: Research and Development Division, Advanced Molecular Systems, Galway, Ireland.|Molecular Diagnostic Research Group, School of Natural Sciences, National University of Ireland, Galway, Ireland.
authors.forename: Louise
authors.initials: L
authors.lastname: O'Connor
authors.affiliation: Department of Haematology, National University of Ireland, Galway, Ireland.|Galway University Hospital, Galway, Ireland.
authors.forename: Janusz
authors.initials: J
authors.lastname: Krawczyk
authors.affiliation: Department of Haematology, National University of Ireland, Galway, Ireland.|Galway University Hospital, Galway, Ireland.
authors.forename: Moutaz A
authors.initials: MA
authors.lastname: Abdelrahman
authors.affiliation: Department of Haematology, National University of Ireland, Galway, Ireland.|Galway University Hospital, Galway, Ireland.
authors.forename: Amjad H
authors.initials: AH
authors.lastname: Hayat
authors.affiliation: Department of Haematology, National University of Ireland, Galway, Ireland.|Galway University Hospital, Galway, Ireland.
authors.forename: Margaret
authors.initials: M
authors.lastname: Murray
authors.affiliation: Department of Haematology, National University of Ireland, Galway, Ireland.|Galway University Hospital, Galway, Ireland.
authors.forename: Michael
authors.initials: M
authors.lastname: O'Dwyer
authors.affiliation: Department of Haematology, Belfast City Hospital, Belfast, UK.
authors.forename: Melanie
authors.initials: M
authors.lastname: Percy
authors.affiliation: Cancer Molecular Diagnostics, St. James's Hospital, Dublin, Ireland.
authors.forename: Stehpen
authors.initials: S
authors.lastname: Langabeer
authors.affiliation: Cancer Molecular Diagnostics, St. James's Hospital, Dublin, Ireland.
authors.forename: Karl
authors.initials: K
authors.lastname: Haslam
authors.affiliation: Research and Development Division, Advanced Molecular Systems, Galway, Ireland.
authors.forename: Barry
authors.initials: B
authors.lastname: Glynn
authors.affiliation: Research and Development Division, Advanced Molecular Systems, Galway, Ireland.
authors.forename: Ciara
authors.initials: C
authors.lastname: Mullen
authors.affiliation: Research and Development Division, Advanced Molecular Systems, Galway, Ireland.
authors.forename: Evelyn
authors.initials: E
authors.lastname: Keady
authors.affiliation: Research and Development Division, Advanced Molecular Systems, Galway, Ireland.
authors.forename: Sinéad
authors.initials: S
authors.lastname: Lahiff
authors.affiliation: Research and Development Division, Advanced Molecular Systems, Galway, Ireland.|Molecular Diagnostic Research Group, School of Natural Sciences, National University of Ireland, Galway, Ireland.
authors.forename: Terry J
authors.initials: TJ
authors.lastname: Smith
mesh_terms: D037282:Calreticulin / Q000235:genetics*; D015342:DNA Probes / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics; D005091:Exons / Q000235:genetics*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D009154:Mutation / Q000235:genetics*; D009196:Myeloproliferative Disorders / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
chemical_list: D037282:Calreticulin; D015342:DNA Probes; D004273:DNA, Neoplasm
keywords: MOLECULAR ONCOLOGY; MYELOPROLIFERATIVE DISEASE; PCR