DETERMINATION OF ALLELE BURDEN OF JAK2V617F MUTATION IN GENOMIC AND PLASMA CELL FREE DNA IN HEALTHY INDIVIDUALS

RIERA L; RAMOS JG; GAYDOU L,; BOSQUIAZZO VL,; CARDOZO MA, ; FOLLONIER AM,

Congreso; LXISociedad Argentina de Investigación Clínica (SAIC); 2016

Mutations in JAK2 gene are common in chronic BCR-ABL (-) negative myeloproliferative neoplasms. The most frequent mutation is observed in exon 14 G1849T resulting in the substitution of a valine for a phenylalanine in the 617 position (JAK2 V617F) leading to a protein with constitutive tyrosine kinase activity.Some studies have shown low percentages (1-3%) of the mutation in healthy individuals, suggesting that it may be present before the onset of the disease. It is necessary not only to know the cut-off value of the JAK2 mutation allele burden in healthy individuals but also to develop quantitative methods with robust responses with low limits of detection (0.1-1%). We designed an allele specific real time PCR assays (qPCR) for the determination of allele burden of JAK2V617F (percentage of alleles JAK2V617F / total alleles JAK2) insamples of genomic DNA (gDNA) and plasma cell free DNA (cfDNA).The calibration curve was made with a mixture of gDNA from healthy individuals and gDNA derived from a homozygous JAK2V617F-Human-erythroleukemia cell line.gDNA was obtained from peripheral blood of 100 healthy individuals using a modified Miller and Dykes technique, while cfDNA was obtained from the plasma of 32 healthy patients using the QIAmp DNA Blood Mini kit. In order to discard subclinic haematological diseases a blood count for each individual was performed.The JAK2V617F mutationwas detectedboth the gDNAandcfDNAof healthy people.Allele burden ingDNA was 0.066% [95% CI: 0.047-0.093%], while in cfDNA was 0.054% [95% CI: 0.034-0.087%]. Statisticaldifferences were not detected betweengenomic and plasmatic compartments (p=0.23). There was no correlation between the allele burden of JAK2V617F and the patient?s age, gender or the different hematimetric parameters. These results demonstrate that the developed methodology allowed the quantification of low allele burden in healthy individual samples.