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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 homozygo...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3903535/ https://www.ncbi.nlm.nih.gov/pubmed/24475114 http://dx.doi.org/10.1371/journal.pone.0086401 |
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author | Gonzalez, Mariana Selena De Brasi, Carlos Daniel Bianchini, Michele Gargallo, Patricia Stanganelli, Carmen Zalcberg, Ilana Larripa, Irene Beatriz |
author_facet | Gonzalez, Mariana Selena De Brasi, Carlos Daniel Bianchini, Michele Gargallo, Patricia Stanganelli, Carmen Zalcberg, Ilana Larripa, Irene Beatriz |
author_sort | Gonzalez, Mariana Selena |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-3903535 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-39035352014-01-28 Improved Diagnosis of the Transition to JAK2 (V617F) Homozygosity: The Key Feature for Predicting the Evolution of Myeloproliferative Neoplasms Gonzalez, Mariana Selena De Brasi, Carlos Daniel Bianchini, Michele Gargallo, Patricia Stanganelli, Carmen Zalcberg, Ilana Larripa, Irene Beatriz PLoS One Research Article 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. Public Library of Science 2014-01-27 /pmc/articles/PMC3903535/ /pubmed/24475114 http://dx.doi.org/10.1371/journal.pone.0086401 Text en © 2014 Gonzalez et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Gonzalez, Mariana Selena De Brasi, Carlos Daniel Bianchini, Michele Gargallo, Patricia Stanganelli, Carmen Zalcberg, Ilana Larripa, Irene Beatriz Improved Diagnosis of the Transition to JAK2 (V617F) Homozygosity: The Key Feature for Predicting the Evolution of Myeloproliferative Neoplasms |
title | Improved Diagnosis of the Transition to JAK2
(V617F) Homozygosity: The Key Feature for Predicting the Evolution of Myeloproliferative Neoplasms |
title_full | Improved Diagnosis of the Transition to JAK2
(V617F) Homozygosity: The Key Feature for Predicting the Evolution of Myeloproliferative Neoplasms |
title_fullStr | Improved Diagnosis of the Transition to JAK2
(V617F) Homozygosity: The Key Feature for Predicting the Evolution of Myeloproliferative Neoplasms |
title_full_unstemmed | Improved Diagnosis of the Transition to JAK2
(V617F) Homozygosity: The Key Feature for Predicting the Evolution of Myeloproliferative Neoplasms |
title_short | Improved Diagnosis of the Transition to JAK2
(V617F) Homozygosity: The Key Feature for Predicting the Evolution of Myeloproliferative Neoplasms |
title_sort | improved diagnosis of the transition to jak2
(v617f) homozygosity: the key feature for predicting the evolution of myeloproliferative neoplasms |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3903535/ https://www.ncbi.nlm.nih.gov/pubmed/24475114 http://dx.doi.org/10.1371/journal.pone.0086401 |
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