Context: A specific mutation, JAK2(V617F), was recently recognized as having diagnostic value for myeloproliferative disorders. No practical assay is currently available for routine use in a clinical laboratory.
Objective: We report the development of a real-time polymerase chain reaction melting curve analysis assay that is appropriate for molecular diagnostics testing.
Design: Specific primers and fluorescence resonance energy transfer probes were designed, and patients with a previously diagnosed myeloproliferative disorder, de novo acute myeloid leukemia, or reactive condition were selected. The DNA was extracted from fresh and archived peripheral blood and bone marrow specimens, and real-time polymerase chain reaction melting curve analysis was performed on the LightCycler platform (Roche Applied Science, Indianapolis, Ind).
Results: The JAK2 region was successfully amplified, and wild-type amplicons were reproducibly discriminated from JAK2(V617F) amplicons. Titration studies using homozygous wild-type and mutant cell lines showed the relative areas under a melting curve were proportional to allele proportion, and the assay reliably detected one mutant in 20 total cells. JAK2(V617F) was identified in patients previously diagnosed with a myeloproliferative disorder or acute myeloid leukemia transformed from myeloproliferative disorder, whereas a wild-type genotype was identified in patients with reactive conditions or de novo acute myeloid leukemia.
Conclusions: These findings demonstrate the suitability of this assay for identifying JAK2(V617F) in a clinical laboratory setting. Furthermore, the semiquantitative detection of JAK2(V617F) in archived specimens provides a new tool for studying the prognostic significance of this mutation.