Detection of the JAK2(V617F) mutation in myeloproliferative disorders by melting curve analysis using the LightCycler system.
BACKGROUND
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.


METHODS
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.
Olsen
Randall J
RJ
Department of Pathology, Baylor College of Medicine, Houston, Texas, USA.
Tang
Zhouwen
Z
Farkas
Daniel H
DH
Bernard
David W
DW
Zu
Youli
Y
Chang
Chung-Che
CC
D001854:Bone Marrow Cells / Q000737:chemistry / Q000473:pathology; D004252:DNA Mutational Analysis / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D053614:Janus Kinase 2; D007951:Leukemia, Myeloid / Q000097:blood / Q000235:genetics / Q000473:pathology; D025202:Molecular Diagnostic Techniques; D009154:Mutation; D009196:Myeloproliferative Disorders / Q000097:blood / Q000235:genetics* / Q000473:pathology; D011505:Protein-Tyrosine Kinases / Q000097:blood / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000097:blood / Q000235:genetics*; D012189:Retrospective Studies; D020133:Reverse Transcriptase Polymerase Chain Reaction; D044366:Transition Temperature
D011518:Proto-Oncogene Proteins; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; D053614:Janus Kinase 2