Background: The genetic lesion underlying the pathogenesis of chronic myeloproliferative disorders (MPDs) has been identified in the Janus kinase 2 (JAK2) gene. A point mutation in codon 617 causes a valine to phenylalanine substitution (V617F) in the JH2 autoinhibitory region of the protein, resulting in constitutive activation of the tyrosine kinase. The high prevalence of this conserved mutation in MPD makes it an excellent candidate as a diagnostic molecular marker.
Methods and Results: We report here the development and validation of a single oligonucleotide probe-based PCR approach using fluorescence melting curve analysis for point mutation detection in DNA derived from unfractionated peripheral blood samples. Using this assay and serial dilutions of an erythroleukemia cell line harboring the homozygous JAK2 V617F mutation, we successfully detected the mutation within a background of wild type sequences at a sensitivity of 2.5%. Our novel fluorescence probe-based assay was compared with allele-specific PCR-gel assay and sequencing techniques. Using the single probe assay, we examined 70 cases with a presumptive diagnosis of MPD, of which 38 (54%) yielded positive results for the presence of the JAK2 V617F mutation, and 92 follicular lymphoma cases, which were negative for the JAK2 V617F mutation. Additionally, the probe-based assay detected a previously unreported T>C base substitution at nucleotide 2342 (JAK2, codon 616), which was not detected by an allele-specific PCR assay.
Conclusion: The single fluorescent probe-based assay described here is a rapid, homogeneous, and robust method for the detection of the JAK2 V617F mutation with favorable performance characteristics that make it advantageous for clinical diagnosis.