Improved diagnosis of the transition to JAK2 (V⁶¹⁷F) homozygosity: the key feature for predicting the evolution of myeloproliferative neoplasms.
Most cases of BCR-ABL1-negative myeloproliferative neoplasms (MPNs), essential thrombocythemia, polycythemia vera and primary myelofibrosis are associated with JAK2 (V617F) mutations. The outcomes of these cases are critically influenced by the transition from JAK2 (V617F) heterozygosity to homozygosity. Therefore, a technique providing an unbiased assessment of the critical allele burden, 50% JAK2 (V617F), is highly desirable. In this study, we present an approach to assess the JAK2 (V617F) burden from genomic DNA (gDNA) and complementary DNA (cDNA) using one-plus-one template references for allele-specific quantitative-real-time-PCR (qPCR). Plasmidic gDNA and cDNA constructs encompassing one PCR template for JAK2 (V617F) spaced from one template for JAK2(Wild Type) were constructed by multiple fusion PCR amplifications. Repeated assessments of the 50% JAK2(V617F) burden within the dynamic range of serial dilutions of gDNA and cDNA constructs resulted in 52.53 ± 4.2% and 51.46 ± 4.21%, respectively. The mutation-positive cutoff was estimated to be 3.65% (mean +2 standard deviation) using 20 samples from a healthy population. This qPCR approach was compared with the qualitative ARMS-PCR technique and with two standard methods based on qPCR, and highly significant correlations were obtained in all cases. qPCR assays were performed on paired gDNA/cDNA samples from 20 MPN patients, and the JAK2 (V617F) expression showed a significant correlation with the allele burden. Our data demonstrate that the qPCR method using one-plus-one template references provides an improved assessment of the clinically relevant transition of JAK2 (V617F) from heterozygosity to homozygosity.
Instituto de Medicina Experimental (IMEX), National Research Council (CONICET)-Academia Nacional de Medicina (ANM), Buenos Aires, Argentina.
Mariana Selena
MS
Gonzalez
Instituto de Medicina Experimental (IMEX), National Research Council (CONICET)-Academia Nacional de Medicina (ANM), Buenos Aires, Argentina ; Instituto de Investigaciones Hematológicas (IIHEMA), ANM, Buenos Aires, Argentina.
Carlos Daniel
CD
De Brasi
Centro de Investigaciones Oncológicas-Fundación Cáncer (CIO-FUCA), Instituto Alexander Fleming, Buenos Aires, Argentina.
Michele
M
Bianchini
Instituto de Investigaciones Hematológicas (IIHEMA), ANM, Buenos Aires, Argentina.
Patricia
P
Gargallo
Instituto de Investigaciones Hematológicas (IIHEMA), ANM, Buenos Aires, Argentina.
Carmen
C
Stanganelli
Molecular Biology, Laboratory, Instituto Nacional do Câncer, Rio de Janeiro, Brazil.
Ilana
I
Zalcberg
Instituto de Medicina Experimental (IMEX), National Research Council (CONICET)-Academia Nacional de Medicina (ANM), Buenos Aires, Argentina ; Instituto de Investigaciones Hematológicas (IIHEMA), ANM, Buenos Aires, Argentina.
Irene Beatriz
IB
Larripa
D017931:DNA Primers / Q000235:genetics; D005787:Gene Frequency; D006801:Humans; D053614:Janus Kinase 2 / Q000235:genetics*; D054438:Leukemia, Myeloid, Chronic, Atypical, BCR-ABL Negative / Q000175:diagnosis* / Q000503:physiopathology*; D019656:Loss of Heterozygosity; D020125:Mutation, Missense / Q000235:genetics*; D009196:Myeloproliferative Disorders / Q000235:genetics* / Q000503:physiopathology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods
D017931:DNA Primers; C507924:JAK2 protein, human; D053614:Janus Kinase 2