Advantages and Versatility of Fluorescence-Based Methodology to Characterize the Functionality of LDLR and Class Mutation Assignment
Data(s) |
14/10/2015
14/10/2015
11/11/2014
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Resumo |
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 I-125-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 I-125-labeled lipoproteins. Flow cytometry results are in full agreement with the data obtained by the I-125 methodology. Additionally confocal microscopy allowed the assignment of different class mutation to the variants assayed. Use of fluorescence yielded similar results than I-125-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. |
Identificador |
PLOS ONE 9 (11) : (2014) // Article ID e112677 1932-6203 http://hdl.handle.net/10810/15864 10.1371/journal.pone.0112677 |
Idioma(s) |
eng |
Publicador |
Public Library Science |
Relação |
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0112677#abstract0 |
Direitos |
2014 Etxebarria et al. 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 credited. info:eu-repo/semantics/openAccess |
Palavras-Chave | #density lipoproteinr eceptor #heterozygous familial hypercholesterolemia #molecular-genetics #missense mutations #cytoplasmic domain #human fibroblasts #ligand-binding #cells #internalization #identification |
Tipo |
info:eu-repo/semantics/article |