Advantages and Versatility of Fluorescence-Based Methodology to Characterize the Functionality of LDLR and Class Mutation Assignment
Main Author: | |
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Publication Date: | 2014 |
Other Authors: | , , , , |
Format: | Article |
Language: | eng |
Source: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Download full: | http://hdl.handle.net/10400.18/2467 |
Summary: | Familial hypercholesterolemia (FH) is a common autosomal codominant disease with a frequency of 1∶500 individuals in its heterozygous form. The genetic basis of FH is most commonly mutations within the LDLR gene. Assessing the pathogenicity of LDLR variants is particularly important to give a patient a definitive diagnosis of FH. Current studies of LDLR activity ex vivo are based on the analysis of 125I-labeled lipoproteins (reference method) or fluorescent-labelled LDL. The main purpose of this study was to compare the effectiveness of these two methods to assess LDLR functionality in order to validate a functional assay to analyse LDLR mutations. LDLR activity of different variants has been studied by flow cytometry using FITC-labelled LDL and compared with studies performed previously with 125I-labeled lipoproteins. Flow cytometry results are in full agreement with the data obtained by the 125I methodology. Additionally confocal microscopy allowed the assignment of different class mutation to the variants assayed. Use of fluorescence yielded similar results than 125I-labeled lipoproteins concerning LDLR activity determination, and also allows class mutation classification. The use of FITC-labelled LDL is easier in handling and disposal, cheaper than radioactivity and can be routinely performed by any group doing LDLR functional validations. |
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Advantages and Versatility of Fluorescence-Based Methodology to Characterize the Functionality of LDLR and Class Mutation AssignmentDoenças Cardio e Cérebro-vascularesFamilial HypercholesterolemiaSaúde PúblicaPortugalFamilial hypercholesterolemia (FH) is a common autosomal codominant disease with a frequency of 1∶500 individuals in its heterozygous form. The genetic basis of FH is most commonly mutations within the LDLR gene. Assessing the pathogenicity of LDLR variants is particularly important to give a patient a definitive diagnosis of FH. Current studies of LDLR activity ex vivo are based on the analysis of 125I-labeled lipoproteins (reference method) or fluorescent-labelled LDL. The main purpose of this study was to compare the effectiveness of these two methods to assess LDLR functionality in order to validate a functional assay to analyse LDLR mutations. LDLR activity of different variants has been studied by flow cytometry using FITC-labelled LDL and compared with studies performed previously with 125I-labeled lipoproteins. Flow cytometry results are in full agreement with the data obtained by the 125I methodology. Additionally confocal microscopy allowed the assignment of different class mutation to the variants assayed. Use of fluorescence yielded similar results than 125I-labeled lipoproteins concerning LDLR activity determination, and also allows class mutation classification. The use of FITC-labelled LDL is easier in handling and disposal, cheaper than radioactivity and can be routinely performed by any group doing LDLR functional validations.Public Library of ScienceRepositório Científico do Instituto Nacional de SaúdeEtxebarria, A.Benito-Vicente, A.Alves, A.C.Ostolaza, H.Bourbon, M.Martin, C.2014-11-26T17:24:07Z2014-11-112014-11-11T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10400.18/2467engPLoS One. 2014 Nov 11;9(11):e112677. doi: 10.1371/journal.pone.0112677. eCollection 2014.1932-620310.1371/journal.pone.0112677info:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-07-20T15:39:18Zoai:repositorio.insa.pt:10400.18/2467Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T18:37:29.418623Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Advantages and Versatility of Fluorescence-Based Methodology to Characterize the Functionality of LDLR and Class Mutation Assignment |
title |
Advantages and Versatility of Fluorescence-Based Methodology to Characterize the Functionality of LDLR and Class Mutation Assignment |
spellingShingle |
Advantages and Versatility of Fluorescence-Based Methodology to Characterize the Functionality of LDLR and Class Mutation Assignment Etxebarria, A. Doenças Cardio e Cérebro-vasculares Familial Hypercholesterolemia Saúde Pública Portugal |
title_short |
Advantages and Versatility of Fluorescence-Based Methodology to Characterize the Functionality of LDLR and Class Mutation Assignment |
title_full |
Advantages and Versatility of Fluorescence-Based Methodology to Characterize the Functionality of LDLR and Class Mutation Assignment |
title_fullStr |
Advantages and Versatility of Fluorescence-Based Methodology to Characterize the Functionality of LDLR and Class Mutation Assignment |
title_full_unstemmed |
Advantages and Versatility of Fluorescence-Based Methodology to Characterize the Functionality of LDLR and Class Mutation Assignment |
title_sort |
Advantages and Versatility of Fluorescence-Based Methodology to Characterize the Functionality of LDLR and Class Mutation Assignment |
author |
Etxebarria, A. |
author_facet |
Etxebarria, A. Benito-Vicente, A. Alves, A.C. Ostolaza, H. Bourbon, M. Martin, C. |
author_role |
author |
author2 |
Benito-Vicente, A. Alves, A.C. Ostolaza, H. Bourbon, M. Martin, C. |
author2_role |
author author author author author |
dc.contributor.none.fl_str_mv |
Repositório Científico do Instituto Nacional de Saúde |
dc.contributor.author.fl_str_mv |
Etxebarria, A. Benito-Vicente, A. Alves, A.C. Ostolaza, H. Bourbon, M. Martin, C. |
dc.subject.por.fl_str_mv |
Doenças Cardio e Cérebro-vasculares Familial Hypercholesterolemia Saúde Pública Portugal |
topic |
Doenças Cardio e Cérebro-vasculares Familial Hypercholesterolemia Saúde Pública Portugal |
description |
Familial hypercholesterolemia (FH) is a common autosomal codominant disease with a frequency of 1∶500 individuals in its heterozygous form. The genetic basis of FH is most commonly mutations within the LDLR gene. Assessing the pathogenicity of LDLR variants is particularly important to give a patient a definitive diagnosis of FH. Current studies of LDLR activity ex vivo are based on the analysis of 125I-labeled lipoproteins (reference method) or fluorescent-labelled LDL. The main purpose of this study was to compare the effectiveness of these two methods to assess LDLR functionality in order to validate a functional assay to analyse LDLR mutations. LDLR activity of different variants has been studied by flow cytometry using FITC-labelled LDL and compared with studies performed previously with 125I-labeled lipoproteins. Flow cytometry results are in full agreement with the data obtained by the 125I methodology. Additionally confocal microscopy allowed the assignment of different class mutation to the variants assayed. Use of fluorescence yielded similar results than 125I-labeled lipoproteins concerning LDLR activity determination, and also allows class mutation classification. The use of FITC-labelled LDL is easier in handling and disposal, cheaper than radioactivity and can be routinely performed by any group doing LDLR functional validations. |
publishDate |
2014 |
dc.date.none.fl_str_mv |
2014-11-26T17:24:07Z 2014-11-11 2014-11-11T00:00:00Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10400.18/2467 |
url |
http://hdl.handle.net/10400.18/2467 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
PLoS One. 2014 Nov 11;9(11):e112677. doi: 10.1371/journal.pone.0112677. eCollection 2014. 1932-6203 10.1371/journal.pone.0112677 |
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info:eu-repo/semantics/openAccess |
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openAccess |
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Public Library of Science |
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Public Library of Science |
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