Percorrer por data de Publicação, começado por "2025-05-04"
A mostrar 1 - 6 de 6
Resultados por página
Opções de ordenação
- 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.
- Regulatory Variants In LDLR And PCSK9 Promoters And 5'UTRs: Investigating The impact In Familial HypercholesterolaemiaPublication . Graça, R.; Menezes, J.; Fernandes, R.; Alves, A.C.; Romão, L.; Bourbon, M.Background and Aims: Familial Hypercholesterolaemia (FH) is a genetic disorder of lipid metabolism caused by pathogenic variants in LDLR, APOB, and PCSK9. While diagnostic efforts traditionally focus on coding variants, non-coding regions, such as promoters and 5'UTRs, remain understudied despite their importance. This work aims to characterise 100 variants in the promotor/5'UTR of LDLR and PCSK9. Methods: The promotor/5'UTR sequences of LDLR and PCSK9 were cloned by SOEing PCR upstream of the Firefly luciferase coding region in the pGL4.10. For LDLR, sequence from c.-319 to the initiation codon was retrieved from literature, while for PCSK9, sequence from c.-650 to the initiation codon was confirmed using a 5'-RACE strategy. The resulting constructs (LDLR_pGL4-WT and PCSK9_pGL4-WT) were subsequently modified through site-directed mutagenesis. LDLR and PCSK9 variants were transfected into CHO-ldlA7 and Huh7 cells, respectively. Cells were cultured in different cholesterol depletion states, and luciferase activity measured using a Dual-Luciferase Reporter Assay System. Results: Compared to their respective wild-type constructs, LDLR and PCSK9 variants displayed a diverse range of phenotypic effects, with statistically significant increases or decreases in promoter activity. These variations can differently impact the FH phenotype and hold significant implications for disease management and therapeutic strategies, as increases or decreases in promoter activity in the two genes have distinctly opposing effects on LDL-C levels. Moreover, as far as we know, this is the first experimental work defining the PCSK9 5’ UTR region. Conclusions: This study provides novel insights into the functional impact of LDLR and PCSK9 promoter/5'UTR variants on gene expression and their potential contributions to the FH phenotype. Importantly, these findings underscore the critical role of functional studies in variant classification, particularly for non-coding regions, which remain underrepresented in genetic diagnostics. By elucidating how these variants influence LDL-C levels through altered promoter activity, this work highlights their relevance in refining FH diagnosis and tailoring patient management strategies.
- Insights Into Homozygous Familial Hypercholesterolemia In PortugalPublication . Medeiros, Ana Margarida; Alves, Ana Catarina; Miranda, Beatriz; Chora, Joana Rita; Aguiar, Patrício; Amaro, Mário; Ferreira, Sofia; Gaspar, Ana; Gonçalves, Filipa Sousa; Lobarinhas, Goreti; Lourenço, Guilherme; Martins, Paula; Antunes, Sofia Moura; Palma, Isabel; Rato, Quitéria; Torres, Diogo; Rico, Miguel Toscano; Travessa, André; Bourbon, MafaldaBackground and Aims: Homozygous Familial Hypercholesterolemia (HoFH) is a rare, biallelic semidominant condition caused by pathogenic/likely pathogenic (P/LP) variants in LDLR, APOB, and/or PCSK9 genes. HoFH is characterized by a severe phenotype with LDL-C >400 mg/dL, xanthomas, and early-onset atherosclerotic cardiovascular disease (ASCVD). This work presents the clinical/genetic and follow-up data on individuals genetically identified with HoFH. Methods: A total of 1291 index-cases, with clinical diagnosis of FH, were referred to the Portuguese FH Study. Genetic diagnosis was performed using Sanger sequencing or NGS FH panel. Results: Fifteen individuals were identified as HoFH: 5 with identical biallelic LDLR variants, 8 with different biallelic variants (7 in LDLR, 1 in PCSK9), and 2 with biallelic variants in LDLR and APOB (digenic). Most variants are classified as P/LP; 3 are variants of unknown significance (VUS), but exhibited defects in LDL receptor activity. The cohort included mostly adults (73%) and females (87%), with a median age of 29.9±15.3years at referral (adults: 36.5±12.5years, children/adolescents: 12.0±1.9years). Clinical manifestations included tendon xanthomas (13%) and ASCVD (36%). At referral, all individuals were on statins therapy, with 50% using statin ezetimibe combination, 13% with PCSK9 inhibitors (PCSK9i), and 13% performed LDL apheresis. Five individuals carry at least one null allele (<10% activity), 9 carry defective alleles (10-70% activity), and one has null/null alleles. The latter presented the most severe phenotype (LDL-C=702mg/dL) despite intensive treatment (rosuvastatin, ezetimibe, LDL apheresis, and PCSK9i). Follow-up data were collected for 4 individuals: 2 with defective/defective alleles are now using PCSK9i, while 2 with null/defective and null/null are now using ANGPTL3 inhibitors. Conclusions: In general, Portuguese HoFH individuals are diagnosed late and do not reach the recommended target LDL-C levels. Genetic diagnosis enables precise identification of allele type, allowing more personalized therapeutic approaches, especially for null/null allele carriers who present reduced treatment responsiveness and require therapies independent of LDL receptor function.
- Familial and Multifactorial Chylomicronemia Syndrome: Insights from Clinically Diagnosed Cases in PortugalPublication . Alves, Ana Catarina; Ferreira, Maria; Ferreira, Ana Cristina; Padeira, Gonçalo; Gaspar, Ana; Duarte, João Sequeira; Rato, Quitéria; Gonçalves, Filipa Sousa; Aguiar, Patrício; Cruz, Diogo; Bourbon, MafaldaFamilial Chylomicronemia Syndrome (FCS) is a rare autosomal recessive disorder caused by biallelic pathogenic variants in one of five canonical genes, LPL, APOC2, GPIHBP1, APOA5, and LMF1. These variants result in impaired hydrolysis of triglyceride-rich lipoproteins, leading to clinical features such as xanthomas, abdominal pain, hepatomegaly, hepatosplenomegaly, lipemia retinalis, and recurrent pancreatitis. In contrast, Multifactorial Chylomicronemia Syndrome (MCS) often involves monoallelic variants in these genes and/or a high polygenic risk score, contributing to the severe hypertriglyceridemia phenotype. Clinically, FCS and MCS have a similar presentation, requiring genetic analysis for differentiation. This study aimed to clinically and molecularly characterize 42 individuals with severe hypertriglyceridemia in Portugal. Biochemical lipid profile and molecular analysis of the five canonical genes were performed. Moulin's score was applied to 14 cases; for the remaining cases, all data could not be obtained. The average pre-treatment triglyceride level was 2570 mg/dL. Fourteen individuals had pancreatitis, four had hepatomegaly, and three presented with both conditions. Eight cases have biallelic variants: five in LPL (three with identical variants, two with different variants), one in APOC2, one frameshift variant in LMF1 and one total exon 4 deletion in GPIHBP1 (all with identical variants). For these cases, the Moulin score obtained was FCS very likely. Twenty cases have heterozygous variants in LPL, APOA5, LMF1, and GPIHBP1 and were classified as MCS. For one of these cases, the Moulin score was FCS very likely. Ten patients have a negative genetic study, 5 of which had a score of unlikely FCS. Four are still under study. Early identification of FCS is critical to prevent or mitigate its severe complications. A confirmed molecular diagnosis enables accurate differentiation between FCS and MCS, leading to improved clinical management and prognosis. This study underscores the importance of integrating genetic analysis into the diagnostic workup of severe hypertriglyceridemia.
- Development of Gene-Specific ACMG/AMP Guidelines for the Interpretation of APOB and PCSK9 Variants in Familial HypercholesterolemiaPublication . Chora, Joana Rita; Hooper, Amanda; Gutierrez-Ford, Christina; Kullo, Iftikhar; Bourbon, Mafalda; on behalf of the ClinGen Familial Hypercholesterolemia Variant Curation Expert PanelBackground and Aims: The general ACMG/AMP guidelines for standardized variant interpretation provide a critical framework for determining pathogenicity but require adaptation to specific genes and diseases. While LDLR-specific guidelines for familial hypercholesterolemia (FH) have been in use since 2020, similar adaptations for APOB and PCSK9 are needed to address the unique characteristics of these genes in FH diagnosis. Methods: The Clinical Genome Resource (ClinGen) consortium’s FH variant curation expert panel (FH VCEP) expanded its efforts to develop tailored guidelines for APOB and PCSK9. A panel of international FH experts proposed and voted on specifications for these genes, based on current evidence. These adaptations were compared to LDLR-specific criteria for consistency and refined based on gene-specific attributes. Results: The proposed guidelines address the unique features of APOB and PCSK9 variants. For both genes, adaptations included adjustment of thresholds for population frequency criteria (PM2/BA1/BS1), specification of appropriate functional studies (PS3/BS3) to reflect each gene’s role in the LDLR cycle, and documented critical protein regions (PM1). Several criteria were considered to be applicable in the same manner as for LDLR: phenotype specificity (PP4), frequency of cases (PS4), co-segregation data (PP1), and cases with more than onevariant (PM3/BP2). As null variants in APOB and loss-of-function variants in PCSK9 are not mechanisms for FH, the evidence code for truncating variants (PVS1) was deemed not applicable for both genes. Lack of segregation (BS4) in APOB was also deemed not applicable due to documented incomplete penetrance of APOB variants. Conclusions: With the increasing detection of variants through advances in sequencing and genotyping technologies, these guidelines represent a significant step toward standardizing APOB and PCSK9 variant interpretation in FH diagnosis. Following ClinGen approval, these gene-specific recommendations will enable high-confidence classification of APOB and PCSK9 variants in ClinVar, enhancing the accuracy of FH diagnosis and supporting personalized management strategies for patients worldwide.
- DLCN-PED – A New Proposal For Clinical Diagnosis Of Children And Young People With FHPublication . Miranda, Beatriz; Kafol, Jan; Humphries, Steve E.; Freiberger, Tomas; Medeiros, Ana Margarida; Sikonja, Jaka; Alves, Ana Catarina; Groselj, Urh; Bourbon, MafaldaFamilial hypercholesterolemia (FH) is a genetic disorder characterized by high cholesterol levels, and premature cardiovascular disease (CVD). The most used clinical diagnostic criteria to identify FH are the Dutch Lipid Clinic Network-(DLCN) and Simon Broome-(SB), being SB the only with specific criteria for children. This work aims to propose an adaptation of DLCN criteria, for children and young people (DLCN-PED) and assess the performance of this adaptation in the Portuguese and Slovenian FH registries. DLCN-PED includes data on clinical examinations, lipid profile, familial history of CVD and hypercholesterolemia. We propose that those with a DLCN-PED score3 should be referred for genetic testing. Index cases included were studied with an NGS panel including at least the three FH-causing genes prior to evaluation with DLCN-PED This work includes 1696 patients (Portugal=340, Slovenia=1356): 29% FH-positives (presenting likely pathogenic/pathogenic variants in FH genes), 71% FH-negatives (no detection of pathogenic variants). Individuals with variants of uncertain significance were not included. Scores had a range between 0-17, 58% presenting a score between 2-4. Across DLCN-PED scores, we observe a distribution overlap for both FH groups: FH-negatives from 0-8 and FH-positives from 0-17. Overall, DLCN-PED3 presents higher sensitivity compared to SB (93% vs 81%) and slightly lower specificity (55% vs 79%). DLCN-PED6 presents lower sensitivity compared to SB (67% vs 81%) but an enhanced specificity (91% vs 79%). The different analysis suggests that score cutoffs should be adapted according to the screening approach intended (sensitivity and specificity trade-off). Compared to DLCN, the DLCN-PED shows an improved performance (p-value=0.024). Given the crucial role of clinical diagnosis in FH identification, we have shown that the proposed DLCN-PED performs better than the general DLCN and is similar to SB with the advantage that is possible to personalize the cut offs and so it could improve FH assessment worldwide.