Improved Diagnosis of the Transition to JAK2V617F 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 JAK2V617F mutations. The outcomes of these cases are critically influenced by the transition from JAK2V617F heterozygosity to homozygosity....

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Published in:PloS one 2014-01, Vol.9 (1), p.e86401
Main Authors: Gonzalez, Mariana Selena, De Brasi, Carlos Daniel, Bianchini, Michele, Gargallo, Patricia, Stanganelli, Carmen, Zalcberg, Ilana, Larripa, Irene Beatriz
Format: Article
Language:English
Subjects:
Alleles
Complementary DNA
Correlation
Councils
Deoxyribonucleic acid
Dilution
DNA
Heterozygosity
Homozygosity
Kinases
Leukemia
Medical diagnosis
Mutation
Myelofibrosis
Neoplasms
Patients
Polycythemia
Polycythemia vera
Polymerase chain reaction
Thrombosis
Tumors
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Summary:Most cases of BCR-ABL1-negative myeloproliferative neoplasms (MPNs), essential thrombocythemia, polycythemia vera and primary myelofibrosis are associated with JAK2V617F mutations. The outcomes of these cases are critically influenced by the transition from JAK2V617F heterozygosity to homozygosity. Therefore, a technique providing an unbiased assessment of the critical allele burden, 50% JAK2V617F, is highly desirable. In this study, we present an approach to assess the JAK2V617F 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 JAK2V617F spaced from one template for JAK2Wild Type were constructed by multiple fusion PCR amplifications. Repeated assessments of the 50% JAK2V617F 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 JAK2V617F 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 JAK2V617F from heterozygosity to homozygosity.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0086401