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
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/507

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repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling 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
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv 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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