Percorrer por autor "Etxebarria, A."
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- Advantages and versatility of fluorescence-based methodology to characterize the functionality of LDLR and class mutation assignmentPublication . Benito-Vicente, A.; Etxebarria, A.; Alves, A.C.; Bourbon, M.; Martin, C.INTRODUCTION: Familial hypercholesterolemia (FH) is a common autosomal dominant disease with a frequency of 1:500 individuals in its heterozygous form. The genetic basis of FH is mostly 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 or fluorescent-labeled 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.
- Advantages and Versatility of Fluorescence-Based Methodology to Characterize the Functionality of LDLR and Class Mutation AssignmentPublication . Etxebarria, A.; Benito-Vicente, A.; Alves, A.C.; Ostolaza, H.; Bourbon, M.; Martin, C.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.
- APOB: old gene, new perspective for Familial HypercholesterolaemiaPublication . Alves, A.C.; Etxebarria, A.; Martin, C.; Bourbon, M.Familial hypercholesterolemia (FH) is one of the most common diseases of lipid metabolism, has an autosomal dominant inheritance and was the first genetic disorder of lipid metabolism to be characterized molecularly. FH usually results from inherited defects in the low density lipoprotein receptor (LDLR) gene and is characterised by increased circulating low density lipoprotein (LDL) cholesterol that leads to lipid accumulation in arteries and tendons (xanthomas), causing premature arteriosclerosis and coronary heart disease. Mutations in other genes as the apolipoprotein B (APOB) and proprotein convertase subtilisin/kexin type 9 gene (PCSK9) are rare causes of FH. Until now only a few mutations in exon 26 and 29 have been reported to cause FH, being the APOB3527 the most common. The main aim of this project was to identify and characterize the genetic cause of severe hypercholesterolaemia in individuals with clinical diagnosis of FH, without mutations in LDLR and PCSK9 or in fragments of exon 26 and 29 of APOB routinely screened.
- Differences in secondary structure of p.Arg1164Thr and p.Gln4494del, two novel ApoB-100 mutantsPublication . Etxebarria, A.; Fernández-Higuero, J.A.; Benito-Vicente, A.; Alves, A.C.; Bourbon, M.; Ostolaza, H.; Martin, C.
- Functional characterization of 2 news variants in the APOB genePublication . Alves, A.C.; Medeiros, A.M.; Etxebarria, A.; Benito-Vicente, A.; Martin, C.; Bourbon, MafaldaFamilial hypercholesterolaemia (FH) is an autosomal dominant disorder of cholesterol metabolism. Loss of function mutations in LDLR and APOB and also gain of function mutations in PCSK9 have been associated with FH, but mutations in LDLR are the most common cause of FH. Until 2012 only mutations in two small fragments of exon 26 and 29 were described as causing FH. However with Next Generation Sequencing techniques others alterations in fragments not studied in routine diagnosis are being found and need to be functional characterized. In the past years 5 new functional mutations have been described in APOB fragments not routinely studied and our group characterized 2/5 as causing FH
- Functionally characterization of the most common LDLR missense alterations found in Portuguese FH patientsPublication . Alves, A.C.; Azevedo, S.; Benito-Vicente, A.; Etxebarria, A.; Barros, P.; Medeiros, A.M.; Martín, C.; Bourbon, MafaldaAims: Mutations in the LDLR gene are the major cause of familial hypercholesterolaemia (FH), which results in defective catabolism of LDL leading to premature coronary heart disease. Presently, more than 1700 different mutations in the LDLR gene have been described as causing FH but the majority of them remain without functional characterization. In the Portuguese Familial Hypercholesterolemia Study (PFHS), 123 LDLR alterations were found in 243 index patients and their relatives up to date. Until now, 70 of these alterations already have a final classification of pathogenic and 15 have been proved by in vitro studies to be non-pathogenic. The aim of the present work is to functionally characterize 16 LDLR missense alterations found in Portuguese FH patients and worldwide.
- Functionally characterization of the most common LDLR missense alterations found in Portuguese FH patientsPublication . Alves, A.C.; Azevedo, S.; Benito-Vicente, A.; Etxebarria, A.; Barros, P.; Medeiros, A.M.; Martín, C.; Bourbon, MafaldaMutations in the LDLR gene are the major cause of familial hypercholesterolaemia (FH), which results in defective catabolism of LDL leading to premature coronary heart disease. Presently, more than 1700 different mutations in the LDLR gene have been described as causing FH but the majority of them remain without functional characterization. In the Portuguese Familial Hypercholesterolemia Study (PFHS), 123 LDLR alterations were found in 243 index patients and their relatives up to date. Until now, 70 of these alterations already have a final classification of pathogenic and 15 have been proved by in vitro studies to be non-pathogenic. The aim of the present work was to functionally characterize the 16 most common LDLR alterations in our cohort without functional characterization found in Portuguese patients and worldwide.
- Genetic Diagnosis of Familial Hypercholesterolaemia: The Importance of an Integrated Analysis of Clinical, Molecular and Functional DataPublication . Alves, A.C.; Benito-Vicente, A.; Etxebarria, A.; Medeiros, A.M.; Martin, C.; Bourbon, MafaldaFamilial Hypercholesterolaemia (FH) is one of the most common monogenic disorders, being caused mostly by mutations in LDL receptor (LDLR) gene. The high levels of LDL cholesterol presented since birth confers these patients an increased cardiovascular risk. Laboratory techniques have improved greatly recently and new variants are found every day that need to be validated as mutations causing disease for the correct diagnosis of FH. The aim of this study was to characterize both at the phenotypic and genotypic level, families with a clinical diagnosis of FH and discuss the importance of the integration of clinical, molecular and functional data for the correct diagnosis of these patients.
- Novel Functional APOB Mutations outside LDL-Binding Region Causing FamilialPublication . Alves, A.C.; Etxebarria, A.; Martin, C.; Bourbon, M.
- Pathogenicity assessment of LDLR variants in patients with Familial HypercholesterolemiaPublication . Medeiros, A.M.; Alves, A.C.; Etxebarria, A.; Martin, C.; Bourbon, M.Mutations in LDLR gene are the major cause of Familial Hypercholesterolemia (FH) but there are several variants described whose pathogenicity is still unknown. To date 88 different mutations in LDLR have been identified in the Portuguese population most of them, although being present in other populations, don’t have functional studies reported. A previous work recently reported by our group investigated the pathogenicity of five missense LDLR variants by in vitro functional assays and 3 of them severely impaired receptor function. Here we present a novel functional characterization of five LDLR variants (p.Gly76Trp, p.Ala431Thr, p.Gly478Arg, p.Cys698Phe, c.-13A>G) found in Portuguese FH patients, two of them (p.Ala431Thr, p.Gly478Arg) previously described as pathogenic and used as control.