title: Novel multiplex bead-based assay with LNA-modified probes for detection of MPL exon 10 mutations.
abstract: MPL exon 10 mutations were the second class of mutations shown to be associated with the pathogenesis of some Philadelphia chromosome - negative myeloproliferative neoplasms (MPNs). Recently, their identification gained wide recognition in the diagnostic work-up for suspected cases of JAK2 V617F negative MPNs. Various molecular approaches have been applied, yet universally accepted method is still lacking. We aimed at development and validation of a novel bead-based liquid assay using Locked nucleic acids (LNA)-modified oligonucleotide probes for multiplexed detection of the following MPL mutations: W515L/K/A/R. Testing on both artificial plasmid constructs and on clinical samples revealed that the method was comparable in terms of specificity to direct sequencing and had a much higher sensitivity of 1% mutant alleles. This method could be successfully implemented in the diagnostic work-up for MPNs. Furthermore, this system allows further multiplexing for single-tube identification of different mutations associated with MPNs.
authors.affiliation: Department of Clinical Immunology, Alexandrovska University Hospital, Medical University, Sofia, Bulgaria.
authors.forename: Milena I
authors.initials: MI
authors.lastname: Ivanova
authors.forename: Velizar S
authors.initials: VS
authors.lastname: Shivarov
authors.forename: Evgueniy A
authors.initials: EA
authors.lastname: Hadjiev
authors.forename: Elissaveta J
authors.initials: EJ
authors.lastname: Naumova
mesh_terms: D019411:Clinical Laboratory Techniques / Q000379:methods*; D005091:Exons / Q000235:genetics*; D006801:Humans; D009154:Mutation / Q000235:genetics*; D009196:Myeloproliferative Disorders / Q000175:diagnosis* / Q000235:genetics*; D009841:Oligonucleotides; D010677:Philadelphia Chromosome; D053628:Receptors, Thrombopoietin / Q000235:genetics*
chemical_list: D009841:Oligonucleotides; D053628:Receptors, Thrombopoietin; C477371:locked nucleic acid; C082218:MPL protein, human