Browsing by Author "Benito-Vicente, 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.
- 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.
- Further evidence of novel APOB mutations as a cause of Familial HypercholesterolemiaPublication . Alves, A.C.; 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. In the last 2 years functional mutations in other fragments of exon 26 and 29 as well as in exon 3 and 22 have been reported in FH patients. However with Next Generation Sequencing techniques others alterations in fragments not studied in routine diagnosis are being found and need to be functional characterized. The main aim of this project was to characterize 2 novel alterations in APOB, exon 19 and 26, in order to identify the genetic cause of the hypercholesterolemia in these patients.
- 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.
- Structural analysis of APOB variants, p.(Arg3527Gln), p.(Arg1164Thr) and p.(Gln4494del), causing Familial Hypercholesterolaemia provides novel insights into variant pathogenicityPublication . Fernández-Higuero, J.A.; Etxebarria, A.; Benito-Vicente, A.; Alves, A.C.; Arrondo, J.L.; Ostolaza, H.; Bourbon, M.; Martin, C.Familial hypercholesterolaemia (FH) is an inherited autosomal dominant disorder resulting from defects in the low-density lipoprotein receptor (LDLR), in the apolipoprotein B (APOB) or in the proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. In the majority of the cases FH is caused by mutations occurring within LDLR, while only few mutations in APOB and PCSK9 have been proved to cause disease. p.(Arg3527Gln) was the first mutation in APOB being identified and characterized. Recently two novel pathogenic APOB variants have been described: p.(Arg1164Thr) and p.(Gln4494del) showing impaired LDLR binding capacity, and diminished LDL uptake. The objective of this work was to analyse the structure of p.(Arg1164Thr) and p.(Gln4494del) variants to gain insight into their pathogenicity. Secondary structure of the human ApoB100 has been investigated by infrared spectroscopy (IR) and LDL particle size both by dynamic light scattering (DLS) and electron microscopy. The results show differences in secondary structure and/or in particle size of p.(Arg1164Thr) and p.(Gln4494del) variants compared with wild type. We conclude that these changes underlie the defective binding and uptake of p.(Arg1164Thr) and p.(Gln4494del) variants. Our study reveals that structural studies on pathogenic variants of APOB may provide very useful information to understand their role in FH disease.
- The importance of an integrated analysis of clinical, molecular, and functional data for the genetic diagnosis of familial hypercholesterolemiaPublication . Benito-Vicente, A.; Alves, A.C; Etxebarria, A.; Medeiros, A.M.; Martin, C.; Bourbon, M.Purpose: Familial hypercholesterolemia (FH) is one of the most common monogenic disorders, and the high concentrations of low-density lipoprotein (LDL) cholesterol presented since birth confers on these patients an increased cardiovascular risk. More than 1,600 alterations have been described in the LDL receptor gene (LDLR), but a large number need to be validated as mutations causing disease to establish a diagnosis of FH. This study aims to characterize, both at the phenotypic and genotypic levels, families with a clinical diagnosis of FH and present evidence for the importance of the integration of clinical, molecular, and functional data for the correct diagnosis of patients with FH.Methods:A detailed analysis of the phenotype and genotype presented by 55 families with 13 different alterations in the LDLR was conducted. For eight of these, an extensive functional characterization was performed by flow cytometry, confocal microscopy, and reverse transcriptase polymerase chain reaction.Results:Carriers of neutral alterations presented a significantly lower incidence of premature cardiovascular disease, lower levels of atherogenic lipoproteins and a large number of these individuals had LDL-cholesterol values below the 75th percentile. presented a significantly lower incidence of premature cardiovascular disease, lower levels of atherogenic lipoproteins and a large number of these individuals had LDL-cholesterol values below the 75th percentile However, the functional study was essential to determine the pathogenicity of variants.Conclusion:The data collected illustrate the importance of this integrated analysis for the correct assessment of patients with FH who can otherwise be misdiagnosed.