Improved Diagnosis of the Transition to JAK2V617F Homozygosity: The Key Feature for Predicting the Evolution of Myeloproliferative Neoplasms
- Autores
- Gonzalez, Mariana Selena; de Brasi, Carlos Daniel; Bianchini, Michele; Gargallo, Patricia Martha; Stanganelli, Carmen Graciela; Zalcberg, Ilana; Larripa, Irene Beatriz
- Año de publicación
- 2013
- Idioma
- inglés
- Tipo de recurso
- artículo
- Estado
- versión publicada
- Descripción
- 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 52.5364.2% and 51.4664.21%, 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.
Fil: Gonzalez, Mariana Selena. Consejo Nacional de Invest.cientif.y Tecnicas. Instituto de Medicina Experimental;
Fil: de Brasi, Carlos Daniel. Consejo Nacional de Invest.cientif.y Tecnicas. Instituto de Medicina Experimental;
Fil: Bianchini, Michele. DTO. DE GENETICA;
Fil: Gargallo, Patricia Martha. INST. DE INVEST. HEMATOLOGICAS;
Fil: Stanganelli, Carmen Graciela. Academia Nacional de Medicina de Buenos Aires. Instituto de Invest. Hematologicas "mariano R. Castex";
Fil: Zalcberg, Ilana. Molecular Biology, Laboratory, Instituto Nacional do Caˆncer, Rio de Janeiro, Brazil;
Fil: Larripa, Irene Beatriz. Consejo Nacional de Invest.cientif.y Tecnicas. Instituto de Medicina Experimental; - Materia
-
JAK2 V617F
qPCR
gDNA
cDNA - Nivel de accesibilidad
- acceso abierto
- Condiciones de uso
- https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
- Repositorio
- Institución
- Consejo Nacional de Investigaciones Científicas y Técnicas
- OAI Identificador
- oai:ri.conicet.gov.ar:11336/507
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Improved Diagnosis of the Transition to JAK2V617F Homozygosity: The Key Feature for Predicting the Evolution of Myeloproliferative NeoplasmsGonzalez, Mariana Selenade Brasi, Carlos DanielBianchini, MicheleGargallo, Patricia MarthaStanganelli, Carmen GracielaZalcberg, IlanaLarripa, Irene BeatrizJAK2 V617FqPCRgDNAcDNAhttps://purl.org/becyt/ford/3https://purl.org/becyt/ford/3.4Most 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 52.5364.2% and 51.4664.21%, 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.Fil: Gonzalez, Mariana Selena. Consejo Nacional de Invest.cientif.y Tecnicas. Instituto de Medicina Experimental;Fil: de Brasi, Carlos Daniel. Consejo Nacional de Invest.cientif.y Tecnicas. Instituto de Medicina Experimental;Fil: Bianchini, Michele. DTO. DE GENETICA;Fil: Gargallo, Patricia Martha. INST. DE INVEST. HEMATOLOGICAS;Fil: Stanganelli, Carmen Graciela. Academia Nacional de Medicina de Buenos Aires. Instituto de Invest. Hematologicas "mariano R. Castex";Fil: Zalcberg, Ilana. Molecular Biology, Laboratory, Instituto Nacional do Caˆncer, Rio de Janeiro, Brazil;Fil: Larripa, Irene Beatriz. Consejo Nacional de Invest.cientif.y Tecnicas. Instituto de Medicina Experimental;Public Library Science2013-12-27info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/507Gonzalez, Mariana Selena; de Brasi, Carlos Daniel; Bianchini, Michele; Gargallo, Patricia Martha; Stanganelli, Carmen Graciela; Zalcberg, Ilana; Larripa, Irene Beatriz; Improved Diagnosis of the Transition to JAK2V617F Homozygosity: The Key Feature for Predicting the Evolution of Myeloproliferative Neoplasms; Public Library Science; Plos One; 9; 1; 27-12-2013; 1-8;1932-6203enginfo:eu-repo/semantics/altIdentifier/doi/10.1371/journal.pone.0086401info:eu-repo/semantics/altIdentifier/url/http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0086401info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:08:50Zoai:ri.conicet.gov.ar:11336/507instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-29 10:08:51.087CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse |
dc.title.none.fl_str_mv |
Improved Diagnosis of the Transition to JAK2V617F Homozygosity: The Key Feature for Predicting the Evolution of Myeloproliferative Neoplasms |
title |
Improved Diagnosis of the Transition to JAK2V617F Homozygosity: The Key Feature for Predicting the Evolution of Myeloproliferative Neoplasms |
spellingShingle |
Improved Diagnosis of the Transition to JAK2V617F Homozygosity: The Key Feature for Predicting the Evolution of Myeloproliferative Neoplasms Gonzalez, Mariana Selena JAK2 V617F qPCR gDNA cDNA |
title_short |
Improved Diagnosis of the Transition to JAK2V617F Homozygosity: The Key Feature for Predicting the Evolution of Myeloproliferative Neoplasms |
title_full |
Improved Diagnosis of the Transition to JAK2V617F Homozygosity: The Key Feature for Predicting the Evolution of Myeloproliferative Neoplasms |
title_fullStr |
Improved Diagnosis of the Transition to JAK2V617F Homozygosity: The Key Feature for Predicting the Evolution of Myeloproliferative Neoplasms |
title_full_unstemmed |
Improved Diagnosis of the Transition to JAK2V617F Homozygosity: The Key Feature for Predicting the Evolution of Myeloproliferative Neoplasms |
title_sort |
Improved Diagnosis of the Transition to JAK2V617F Homozygosity: The Key Feature for Predicting the Evolution of Myeloproliferative Neoplasms |
dc.creator.none.fl_str_mv |
Gonzalez, Mariana Selena de Brasi, Carlos Daniel Bianchini, Michele Gargallo, Patricia Martha Stanganelli, Carmen Graciela Zalcberg, Ilana Larripa, Irene Beatriz |
author |
Gonzalez, Mariana Selena |
author_facet |
Gonzalez, Mariana Selena de Brasi, Carlos Daniel Bianchini, Michele Gargallo, Patricia Martha Stanganelli, Carmen Graciela Zalcberg, Ilana Larripa, Irene Beatriz |
author_role |
author |
author2 |
de Brasi, Carlos Daniel Bianchini, Michele Gargallo, Patricia Martha Stanganelli, Carmen Graciela Zalcberg, Ilana Larripa, Irene Beatriz |
author2_role |
author author author author author author |
dc.subject.none.fl_str_mv |
JAK2 V617F qPCR gDNA cDNA |
topic |
JAK2 V617F qPCR gDNA cDNA |
purl_subject.fl_str_mv |
https://purl.org/becyt/ford/3 https://purl.org/becyt/ford/3.4 |
dc.description.none.fl_txt_mv |
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 52.5364.2% and 51.4664.21%, 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. Fil: Gonzalez, Mariana Selena. Consejo Nacional de Invest.cientif.y Tecnicas. Instituto de Medicina Experimental; Fil: de Brasi, Carlos Daniel. Consejo Nacional de Invest.cientif.y Tecnicas. Instituto de Medicina Experimental; Fil: Bianchini, Michele. DTO. DE GENETICA; Fil: Gargallo, Patricia Martha. INST. DE INVEST. HEMATOLOGICAS; Fil: Stanganelli, Carmen Graciela. Academia Nacional de Medicina de Buenos Aires. Instituto de Invest. Hematologicas "mariano R. Castex"; Fil: Zalcberg, Ilana. Molecular Biology, Laboratory, Instituto Nacional do Caˆncer, Rio de Janeiro, Brazil; Fil: Larripa, Irene Beatriz. Consejo Nacional de Invest.cientif.y Tecnicas. Instituto de Medicina Experimental; |
description |
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 52.5364.2% and 51.4664.21%, 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. |
publishDate |
2013 |
dc.date.none.fl_str_mv |
2013-12-27 |
dc.type.none.fl_str_mv |
info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion http://purl.org/coar/resource_type/c_6501 info:ar-repo/semantics/articulo |
format |
article |
status_str |
publishedVersion |
dc.identifier.none.fl_str_mv |
http://hdl.handle.net/11336/507 Gonzalez, Mariana Selena; de Brasi, Carlos Daniel; Bianchini, Michele; Gargallo, Patricia Martha; Stanganelli, Carmen Graciela; Zalcberg, Ilana; Larripa, Irene Beatriz; Improved Diagnosis of the Transition to JAK2V617F Homozygosity: The Key Feature for Predicting the Evolution of Myeloproliferative Neoplasms; Public Library Science; Plos One; 9; 1; 27-12-2013; 1-8; 1932-6203 |
url |
http://hdl.handle.net/11336/507 |
identifier_str_mv |
Gonzalez, Mariana Selena; de Brasi, Carlos Daniel; Bianchini, Michele; Gargallo, Patricia Martha; Stanganelli, Carmen Graciela; Zalcberg, Ilana; Larripa, Irene Beatriz; Improved Diagnosis of the Transition to JAK2V617F Homozygosity: The Key Feature for Predicting the Evolution of Myeloproliferative Neoplasms; Public Library Science; Plos One; 9; 1; 27-12-2013; 1-8; 1932-6203 |
dc.language.none.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
info:eu-repo/semantics/altIdentifier/doi/10.1371/journal.pone.0086401 info:eu-repo/semantics/altIdentifier/url/http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0086401 |
dc.rights.none.fl_str_mv |
info:eu-repo/semantics/openAccess https://creativecommons.org/licenses/by-nc-sa/2.5/ar/ |
eu_rights_str_mv |
openAccess |
rights_invalid_str_mv |
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/ |
dc.format.none.fl_str_mv |
application/pdf application/pdf |
dc.publisher.none.fl_str_mv |
Public Library Science |
publisher.none.fl_str_mv |
Public Library Science |
dc.source.none.fl_str_mv |
reponame:CONICET Digital (CONICET) instname:Consejo Nacional de Investigaciones Científicas y Técnicas |
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CONICET Digital (CONICET) |
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CONICET Digital (CONICET) |
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Consejo Nacional de Investigaciones Científicas y Técnicas |
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CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas |
repository.mail.fl_str_mv |
dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar |
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13.070432 |