Journal
PLOS ONE
Volume 9, Issue 11, Pages -Publisher
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0112677
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Funding
- Spanish Ministry of Economy and Competitiveness, Programa INNPACTO [IPT-2011-0817-010000]
- Spanish Ministerio de Ciencia y Tecnologia [BFU 2012-36241]
- Basque Government (Grupos Consolidados) [IT849-13]
- Basque Government (ETORTEK Program)
- Portuguese Science and Technology Foundation [SFRH/BD/27990/2006, PEst-OE/BIA/UI4046/2011]
- Fundação para a Ciência e a Tecnologia [SFRH/BD/27990/2006] Funding Source: FCT
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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.
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