title: Identification of the JAK2 V617F mutation in chronic myeloproliferative disorders using FRET probes and melting curve analysis.
abstract: We developed and validated a real-time polymerase chain reaction assay using fluorescent hybridization probes and melting curve analysis to identify the JAK2 V617F mutation, which is implicated in a substantial proportion of chronic myeloproliferative disorders (CMPDs). DNA from 161 samples was isolated from peripheral blood granulocytes and formalin-fixed bone marrow clot sections in patients with CMPDs and without myeloproliferative disorders previously genotyped for the JAK2 V617F (G-->T) mutation, which included 114 wild types (GG) and 47 mutants (GT and TT). Melting curve analysis of these samples yielded 114 wild types, 42 heterozygotes, and 5 homozygotes showing 100% concordance. Analytic sensitivity of the assay for mutant DNA was 5% for the LightTyper (Roche Applied Sciences, Indianapolis, IN) and 10% for the LightCycler (Roche Applied Sciences). Consistent with earlier reports, 78% of the non-chronic myelogenous leukemia CMPD patients and 8% of non-CMPD patients displayed this mutation. This study demonstrates that clinical genotyping of the JAK2 V617F mutation can be performed by melting analysis using both freshly isolated and formalin-fixed tissues.
authors.affiliation: Department of Clinical Pathology, Cleveland Clinic Foundation and the Cleveland Clinic, Lerner College of Medicine, Cleveland, OH 44195, USA.
authors.forename: Gurunathan
authors.initials: G
authors.lastname: Murugesan
authors.forename: Samer
authors.initials: S
authors.lastname: Aboudola
authors.forename: Hadrian
authors.initials: H
authors.lastname: Szpurka
authors.forename: Mary Ann
authors.initials: MA
authors.lastname: Verbic
authors.forename: Jaroslaw P
authors.initials: JP
authors.lastname: Maciejewski
authors.forename: Raymond R
authors.initials: RR
authors.lastname: Tubbs
authors.forename: Eric D
authors.initials: ED
authors.lastname: Hsi
mesh_terms: D001854:Bone Marrow Cells / Q000473:pathology; D004252:DNA Mutational Analysis / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer; D005557:Formaldehyde; D005838:Genotype; D006098:Granulocytes / Q000473:pathology; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D053614:Janus Kinase 2; D009154:Mutation; D009196:Myeloproliferative Disorders / Q000235:genetics*; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016707:Tissue Fixation; D044366:Transition Temperature
chemical_list: D011518:Proto-Oncogene Proteins; D005557:Formaldehyde; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; D053614:Janus Kinase 2