A quantitative assay for JAK2(V617F) mutation in myeloproliferative disorders by ARMS-PCR and capillary electrophoresis.
A point mutation in the Janus tyrosine kinase 2 (JAK2) gene has been described in patients with chronic myeloproliferative disorders (MPD), but the clinical significance of JAK2(V617F), which may be harbored in either the heterozygote or homozyote status, is still largely undefined. There are indirect suggestions that clinical phenotype and also some biological characteristics are dependent on the mutated allele levels. We have designed and validated in 179 MPD patients an amplification-refractory mutation sequencing PCR assay that allows the relative quantitation of mutated and normal JAK2 mRNAs using dye-labelled mutation-specific primers and capillary electrophoresis. Direct sequencing confirmed the specificity of the assay, which has a detection limit congruent with1% and allowed to identify 9% more JAK2-mutated patients as compared to conventional allele-specific PCR. The mutated mRNA ratio ranged from 5 to 51% in the JAK2(V617F) heterozygote and from 45 to 100% in the homozygote patients. Expression levels of both PRV-1 and NF-E2 gene, previously found to be overexpressed in MPD patients, were significantly correlated to the amount of mutated JAK2 mRNA. We propose that this method might complement current technologies based on genomic DNA analysis, and lead prospectively to a better clinically oriented assessment of the impact of JAK2(V617F) mutation in MPD.
Vannucchi
A M
AM
Department of Hematology, Azienda Ospedaliera-Universitaria Careggi, University of Florence, Florence, Italy. amvannucchi@unifi.it
Pancrazzi
A
A
Bogani
C
C
Antonioli
E
E
Guglielmelli
P
P
D019943:Amino Acid Substitution; D002908:Chronic Disease; D004252:DNA Mutational Analysis / Q000379:methods; D019075:Electrophoresis, Capillary / Q000379:methods; D058851:GPI-Linked Proteins; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D007519:Isoantigens / Q000235:genetics; D053614:Janus Kinase 2; D008562:Membrane Glycoproteins / Q000235:genetics; D009196:Myeloproliferative Disorders / Q000175:diagnosis* / Q000235:genetics*; D050984:NF-E2 Transcription Factor, p45 Subunit / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D011956:Receptors, Cell Surface / Q000235:genetics; D012680:Sensitivity and Specificity
C407242:CD177 protein, human; D058851:GPI-Linked Proteins; D007519:Isoantigens; D008562:Membrane Glycoproteins; D050984:NF-E2 Transcription Factor, p45 Subunit; C494692:NFE2 protein, human; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; D053614:Janus Kinase 2