Browsing by Author "Ferreira, Maria Simões"
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- ClinVar Analysis of APOB Variants Associated with Familial HypercholesterolemiaPublication . Ferreira, Maria Simões; Alves, Ana Catarina; Bourbon, MafaldaFamilial hypercholesterolemia (FH) is an autosomal semi dominant disorder of lipid metabolism characterized by elevated LDL cholesterol levels associated with an increased cardiovascular risk. FH can be caused by variants in LDLR, APOB, and PCSK9 genes. Although most of the cases are due to variants in the LDLR gene, the APOB gene is responsible for 5-10% of the cases. The number of variants in this gene has been increasing due to Next Generation Sequencing; however, for most of these variants, their effect on the LDLR cycle is not known. The aim of this work was to evaluate the APOB variants reported in ClinVar associated with familial hypercholesterolemia. The ClinVar repository was consulted on May 2023 for this analysis, and all APOB variants submitted associated with FH were extracted. Variants submitted with no associated condition (“not specified”, “not provided”) or associated with other conditions (“Hypobetalipoproteinemia”, “Familial hypobetalipoproteinemia”, “Inborn genetic diseases”, “Cardiovascular phenotype”) were excluded. Information regarding classification, review status, and functional characterization was considered for this analysis A total of 2586 APOB variants were identified associated with FH, most of which were reported simultaneously with another condition. A total of 844 variants were submitted only associated to FH condition. From these, the majority were missense variants (592), followed by synonymous alterations (169); 38 frameshifts and nonsense variants were found. In terms of ClinVar classification, 18 were classified as pathogenic, 8 as likely pathogenic, and 234 as likely benign. The remaining were considered variants of uncertain significance (VUS) (566) or presented conflicting interpretations of pathogenicity (18). Most APOB variants (708) presented a review status of clinical significance corresponding to one star (one submitter or multiple submitters with conflicting interpretations of evidence), with only 121 variants presenting two stars (multiple submitters with the same interpretation of the evidence); 15 variants did not present any star (submission did not include evidence). The great majority of these variants lack functional studies proving their effect. The lack of evidence and knowledge about APOB variants compromises their correct classification and the diagnosis of FH, being crucial to share information regarding the variants between countries. Functional studies are very important to understand the effect of the variants in protein function and can contribute to changing a classification of VUS. It would be important develop specification for classification of APOB variants to for an accurate classification.
- Functional characterization of two APOB variants from exon 29 found in individuals with clinical diagnosis of Familial HypercholesterolemiaPublication . Ferreira, Maria Simões; Alves, Ana Catarina; Larrea-Sebal, Asier; Martín, César; Bourbon, MafaldaFamilial hypercholesterolemia (FH) is an inherited lipid disorder characterized by increased levels of LDL cholesterol. About 5-10% of FH cases occur due to variants in the APOB gene, but these alterations can be a more common cause of FH than expected since most of APOB variants identified is still unknown their effect on the metabolism. The majority of the variants are missense but there are a few nonsense variants and small indels in exon 29 identified in individuals with hypercholesterolemia phenotype that can cause FH. The aim of this project was to functional characterize APOB variants from exon 29 identified in individuals referred to the Portuguese FH Study to assess if these are the genetic cause of disease. LDL from index cases and relatives was isolated through sequential ultracentrifugation. ED-LDLR was purified from HEK293 cells transfected with the pcDNA3.1-EC-LDLR-His plasmid by affinity chromatography. Purified ED-LDLR fragments were coated onto 96-well plates and incubated with the different APOB variants. Antibodies were used for ligand detection, and absorbance was determined at 405 nm. CHO-ldlA7 cells were transfected with wt LDLR plasmid and incubated with FITC-labeled LDL to determine LDL binding and uptake by flow cytometry. p.(Gln4316*) and p.(Glu4387Asnfs*7) alterations from exon 29 showed reduced affinity for the LDL receptor. Uptake and binding assays results were similar, so these variants may affect the binding of apoB to the LDL receptor. The alterations studied were not present in a normolipidemic panel. APOB variants studied in this work produce truncated forms of apoB, but they are unlikely to lead to nonsense-mediated decay processes due to their location near the end of the gene. Functional studies can provide important evidence for variant pathogenicity assessment being these essential to provide an accurate diagnosis. These assays can confirm the clinical diagnosis by highlighting the cause of disease, and contribute to a personalized treatment and stratify patient associated cardiovascular risk.
- Functional studies of APOB variants the experience of the Portuguese Familial Hypercholesterolemia StudyPublication . Ferreira, Maria Simões; Chora, Joana Rita; Medeiros, Ana Margarida; Bourbon, Mafalda; Alves, Ana CatarinaFamilial hypercholesterolemia (FH) is clinically characterized by increased levels of circulating LDL cholesterol leading to premature coronary heart disease. It can be caused by variants in LDLR, APOB, and PCSK9 genes. APOB variants are responsible for 5-10% of the FH cases, p.(Arg3527Gln) being the most common. Only recently the whole gene has been sequenced due to Next Generation Sequencing, increasing the variant spectrum of APOB and with it the number of variants that need to be functionally assessed. We aimed to characterize novel APOB variants identified in patients included in the Portuguese FH Study to confirm if they are the genetic cause of hypercholesterolemia. To better analyze these variants, we also create a database with all APOB rare variants found up to date in the Portuguese FH Study. The functional study of 5 variants is ongoing. To access if these variants affect apoB:LDL receptor binding, LDL from index cases and relatives with the variants was separated using sequential ultracentrifugation, and proliferation assays were performed with U937 cells. These cells do not synthesize cholesterol and they depend on apoB:LDL receptor binding to grow.
- Unveiling the Role of APOB Variants in Familial Hypercholesterolemia: Functional InsightsPublication . Ferreira, Maria Simões; Ramos, Diana; Rato, Inês; Jannes, Cinthia E.; Larrea-Sebal, Asier; Martín, César; Bourbon, Mafalda; Alves, Ana CatarinaFamilial hypercholesterolemia (FH) is a condition characterized by increased LDL cholesterol levels with APOB variants accounting for about 5-10% of FH cases. However, variants in this gene may be more common than initially estimated since the entire APOB gene has only recently started to be sequenced. Although most of the alterations are missense, nonsense variants and small indels in exon 29 were also identified in individuals with FH phenotype and can be the cause of disease. This work aimed to characterize APOB variants identified in individuals with clinical diagnosis of FH. Moreover, we intended to do an overview of the APOB variants presenting functional studies. PubMed repository was consulted to collect publications regarding functional characterization of APOB variants. For variant characterization, LDL was isolated through sequential ultracentrifugation. ED-LDLR fragments purified from HEK293 cells were incubated with the different APOB variants and antibodies, to determine apoB affinity for LDLR by ELISA assay. CHO-ldlA7 cells were transfected with wt LDLR plasmid and incubated with FITC-labeled LDL to determine LDL binding and uptake by flow cytometry. In the literature there are 23 APOB variants with functional studies, six of which characterized by our group. Fourteen variants affecting apoB normal function; the remaining results presenting normal apoB function. Recently we characterized 8 more variants: p.(Ala1393Val), p.(Asp1456Asn), p.(Met2042Thr), p.(Asp2213del), p.(Ile3374Thr), p.(Val4295Leu) and p.(Arg4519Thr) that do not appear to impact apoB's binding to the LDLR; p.(Gln4316*) demonstrated reduced affinity for the LDL receptor. Functional studies play a critical role in assessing the pathogenicity of genetic variants and are among the key criteria for variant classification. These in-depth analyses confirm clinical diagnosis and provide essential insights for developing personalized treatment strategies. In the future, we aim to increase the number of studied variants, starting with 15 more variants from the Portuguese FH Study.