Advantages and versatility of fluorescence-based methodology to characterize the functionality of LDLR and class mutation assignment.
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.
Unidad de Biofísica (CSIC, UPV/EHU) and Departamento de Bioquímica y Biología Molecular, Universidad del País Vasco, Apdo. 644, 48080 Bilbao, Spain.
Aitor
A
Etxebarria
Unidad de Biofísica (CSIC, UPV/EHU) and Departamento de Bioquímica y Biología Molecular, Universidad del País Vasco, Apdo. 644, 48080 Bilbao, Spain.
Asier
A
Benito-Vicente
Grupo de Investigação Cardiovascular, Unidade I&D, Departamento de Promoção da Saúde e Prevenção de Doenças Não Transmissíveis, Instituto Nacional de Saúde Dr. Ricardo Jorge, Lisboa 1649-040, Portugal; Centre for Biodiversity, Functional and Integrative Genomics, Faculty of Sciences, University of Lisboa, 1749-016 Lisboa, Portugal.
Ana C
AC
Alves
Unidad de Biofísica (CSIC, UPV/EHU) and Departamento de Bioquímica y Biología Molecular, Universidad del País Vasco, Apdo. 644, 48080 Bilbao, Spain.
Helena
H
Ostolaza
Grupo de Investigação Cardiovascular, Unidade I&D, Departamento de Promoção da Saúde e Prevenção de Doenças Não Transmissíveis, Instituto Nacional de Saúde Dr. Ricardo Jorge, Lisboa 1649-040, Portugal; Centre for Biodiversity, Functional and Integrative Genomics, Faculty of Sciences, University of Lisboa, 1749-016 Lisboa, Portugal.
Mafalda
M
Bourbon
Unidad de Biofísica (CSIC, UPV/EHU) and Departamento de Bioquímica y Biología Molecular, Universidad del País Vasco, Apdo. 644, 48080 Bilbao, Spain.
Cesar
C
Martin
D000818:Animals; D016466:CHO Cells; D003198:Computer Simulation; D003412:Cricetulus; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism / Q000493:pharmacokinetics; D005456:Fluorescent Dyes / Q000378:metabolism / Q000493:pharmacokinetics; D006801:Humans; D006938:Hyperlipoproteinemia Type II / Q000235:genetics*; D008077:Lipoproteins, LDL / Q000737:chemistry / Q000493:pharmacokinetics; D018613:Microscopy, Confocal; D009154:Mutation; D011973:Receptors, LDL / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection
D005456:Fluorescent Dyes; C573412:LDLR protein, human; D008077:Lipoproteins, LDL; D011973:Receptors, LDL; D016650:Fluorescein-5-isothiocyanate