Open AccessImproved Diagnosis of the Transition to JAK2V617F Homozygosity: The Key Feature for Predicting the Evolution of Myeloproliferative Neoplasms
Author(s) -
Mariana Selena Gonzalez,
Carlos Daniel de Brasi,
Michele Bianchini,
Patricia Gargallo,
Carmen Stanganelli,
Ilana Zalcberg,
Irene Larripa
Publication year - 2014
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0086401
Subject(s) - essential thrombocythemia , biology , loss of heterozygosity , genomic dna , genetics , polymerase chain reaction , complementary dna , allele , digital polymerase chain reaction , population , variants of pcr , polycythemia vera , taqman , microbiology and biotechnology , medicine , dna , gene , immunology , environmental health
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.