title: A sensitive detection method for MPLW515L or MPLW515K mutation in chronic myeloproliferative disorders with locked nucleic acid-modified probes and real-time polymerase chain reaction.
abstract: Acquired mutations in the juxtamembrane region of MPL (W515K or W515L), the receptor for thrombopoietin, have been described in patients with primary myelofibrosis or essential thrombocythemia, which are chronic myeloproliferative disorders. We have developed a real-time polymerase chain reaction assay for the detection and quantification of MPL mutations that is based on locked nucleic acid fluorescent probes. Mutational analysis was performed using DNA from granulocytes. Reference curves were obtained using cloned fragments of MPL containing either the wild-type or mutated sequence; the predicted sensitivity level was at least 0.1% mutant allele in a wild-type background. None of the 60 control subjects presented with a MPLW515L/K mutation. Of 217 patients with myelofibrosis, 19 (8.7%) harbored the MPLW515 mutation, 10 (52.6%) with the W515L allele. In one case, both the W515L and W515K alleles were detected by real-time polymerase chain reaction. By comparing results obtained with conventional sequencing, no erroneous genotype attribution using real-time polymerase chain reaction was found, whereas one patient considered wild type according to sequence analysis actually harbored a low W515L allele burden. This is a simple, sensitive, and cost-effective procedure for large-scale screening of the MPLW515L/K mutation in patients suspected to have a myeloproliferative disorder. It can also provide a quantitative estimate of mutant allele burden that might be useful for both patient prognosis and monitoring response to therapy.
authors.affiliation: UF di Ematologia, University of Florence, Viale Morgagni 85, 50134 Florence, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Pancrazzi
authors.forename: Paola
authors.initials: P
authors.lastname: Guglielmelli
authors.forename: Vanessa
authors.initials: V
authors.lastname: Ponziani
authors.forename: Gaetano
authors.initials: G
authors.lastname: Bergamaschi
authors.forename: Alberto
authors.initials: A
authors.lastname: Bosi
authors.forename: Giovanni
authors.initials: G
authors.lastname: Barosi
authors.forename: Alessandro M
authors.initials: AM
authors.lastname: Vannucchi
mesh_terms: D000483:Alleles; D016022:Case-Control Studies; D002908:Chronic Disease; D015342:DNA Probes / Q000737:chemistry*; D005820:Genetic Testing / Q000191:economics; D006801:Humans; D053614:Janus Kinase 2 / Q000235:genetics; D009154:Mutation; D009196:Myeloproliferative Disorders / Q000235:genetics*; D009841:Oligonucleotides / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D053628:Receptors, Thrombopoietin / Q000235:genetics*; D012680:Sensitivity and Specificity
chemical_list: D015342:DNA Probes; D009841:Oligonucleotides; D053628:Receptors, Thrombopoietin; C477371:locked nucleic acid; C082218:MPL protein, human; D053614:Janus Kinase 2