DPSPDNT - Posters/abstracts em congressos internacionais
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- 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.
- 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.
- To Correct or not to Correct (for treatment): Estimating Pre-treatment LDL-C Concentrations in Genetically Characterized Patients with Familial Hypercholesterolaemia on Lipid-lowering MedicationPublication . Stevens, C.A.T.; Elshorbagy, A.; Vallejo-Vaz, A.J.; Dharmayat, K.; Lyons, A.; Bourbon, M.; Chora, J.; Humphries, S.E.; Catapano, A.L.; Hovingh, G.; Mata, P.; Santos, R.; Soran, H.; Watts, G.F.; Raal, F.; Freiberger, T.; Ray, K.K.; on behalf of all the EAS FHSC CollaboratorsBackground and Aims: Pretreatment LDL-C measurements aid familial hypercholesterolaemia (FH) diagnosis, and are crucial in epidemiologic studies investigating FH, but are often unavailable because individuals are already on lipid-lowering medication (LLM). Several formulae have been reported to estimate pre-treatment LDL-C in people on LLM by ‘correcting’ their LDL-C concentrations for LLM type and dosage, based on observational or trial evidence of drug efficacy. We compared 4 published correction factors in estimating pre-treatment LDL-C in patients with FH. Methods: Cross-sectional analysis of adults with pathogenic/likely-pathogenic FH variants in the EAS-FH Studies Collaboration (FHSC) Registry. At the time of LDL-C measurement, N=3012 participants were not on LLM (Untreated group), and N=3226 were on LLM monotherapy, with information on LLM type and dosage allowing estimation of pre-treatment LDL-C (Corrected group) based on correction factors by Ruel 2018, Ellis 2016, Haralambos 2015 and Besseling 2014. We compared the groups for clinical characteristics and LDL-C by gene and variant. Results: The Corrected group was older than the Untreated group (median[IQR]: 50[39,63] vs. 38[28,50]y), with similar proportion of women (54.5% vs. 56.8%;p=0.14) but more comorbidities (all p<0.001). In the Corrected group, 3120 were on statins, 106 on ezetimibe, none on PCSK9-inhibitors. The Corrected group had higher LDL-C vs. Untreated group, with the difference greater at upper percentiles, regardless of correction factor. LDL-C was highest in those with LDLR>APOB>PCSK9 gene variants, but Corrected was still higher than Untreated LDL-C within each gene group. The difference in Corrected vs. Untreated LDL-C varied by variant, from +0.6 to +3.5mmol/L (20 commonest variants). The LDL-C differences persisted after adjusting for age, sex and comorbidities. Conclusions: Application of current LDL-C correction factors appears to overestimate pre-treatment LDL-C in epidemiologic settings, or the Untreated and Corrected groups might have inherently different LDL-C profiles. The accuracy of using LDL-C correction factors in FH therefore warrants further investigation.
- Social prescribing (SP) as a community health promoting tool: lessons from a country exchangePublication . Costa, Luciana; Costa, Alexandra; Papartyte, L.Issue: Social Prescribing (SP) describes evidence-based interventions designed to improve health and wellbeing by referring individuals with non-medical, health-related social needs to health-promoting community-based support and services. Differences in national health systems, sociopolitical infrastructures and terminology, has however created a heterogeneity on SP practice and limited its implementation. Description of the problem: On may 2022 the Portuguese National Institute of Health Dr Ricardo Jorge in partnership with EurohealthNet, organised a Country Exchange Visit to Lisbon. The purpose was to discuss SP and other health promoting primary care strategies in practice at European level and explore the political, financial and practical enablers that connect community and primary health care and social services, in order to build up knowledge-base to foster further SP policy and practice. Results: Participants from ten European health authorities visit on site several community-based initiatives and shared information about relevant activities in their countries. In particular, brainstorming sessions between participants and the twelve key SP partners from the voluntary and community sector, were continuously facilitated by organizers. A summary report was published, providing an overview of the knowledge exchange. Lessons: Overall, the importance to strengthen the collaboration between health and social sectors was recorrently highlighted. Co-funding between the governmental bodies and development of intersectorial partnership projets were pointed out as key innovative approaches to launch and scale up sustainable SP implementation. It was noted that despite the evidence showing the health benefits of a SP model, quality of individual interventions should be ensured and supported by common evaluation frameworks. The development of new training and competence modules adapted to contextual needs was considered particularly relevant to foster SP implementation. Key messages: - Social prescribing represents an innovative tool that enables health systems to meet in a better way the new societal challenges by a complete paradigm shift to a biopsychosocial model of care. - Implementation of social prescribing and its integration into national health systems is crucial to develop new models of care and health to face 21st century health problems.
- Genetic background of individuals with clinical diagnosis of FH from the Portuguese FH Study cohortPublication . Medeiros, Ana Margarida; Alves, Ana Catarina; Chora, Joana Rita; Miranda, Beatriz Raposo; Bourbon, MafaldaAim: Familial Hypercholesterolemia (FH) is a common genetic disorder of lipid metabolism associated to increased CAD risk. Three genes are associated with FH (LDLR, APOB, PCSK9). Variants in FH phenocopies genes (LDLRAP1, APOE, LIPA, ABCG5, ABCG8), LDL-C polygenic risk score (PRS) and hyper-Lp(a) can mimic the FH phenotype. In the present work we intend to unravel the genetic background in individuals with clinical diagnosis of FH. Methods: A biochemical and genetic study was performed to 1005 patients with clinical diagnosis of FH referred to the Portuguese FH Study until December 2021. Since 2017, genetic diagnosis is performed by an NGS panel with 8 genes and 6-SNPs to determine PRS. Results: FH was genetically confirmed in 41% of the cases. In the FH-negative cohort (N=590), 30% (N=177) present Lp(a)>50mg/dl, 16% (N=95) have high PRS, 1% (N=7) have other monogenic cause and 1% (N=7) have one pathogenic variant in ABCG5/ABCG8. Additionally, 11% (N=61) carry heterozygous VUS in either LDLR, APOB or PCSK9 and 5% (N=29) carry heterozygous variants of unknown significance (VUS) in FH phenocopies genes. No identifiable cause of dyslipidemia was found in the remaining 36% patients. Conclusions: Overall, FH was confirmed genetically in 41% of the cohort. In 50% of the FH negatives the FH phenotype can be caused by Hyper-Lp(a) or high PRS. A small part of patients has pathogenic variants in ABCG5/8 in heterozygosity and this can be the cause of hypercholesterolemia and should be further investigated. This extended NGS panel is important to identify FH/FH-phenocopies and therefore personalize each patient’s treatment
- LDLR variant classification by the ClinGen Familial Hypercholesterolemia Variant Curation Expert Panel - first 10 rounds of curationPublication . Chora, Joana Rita; Lacocca, Michael A.; Elnagheeb, Marwa; Kullo, Iftikhar J.; Bourbon, Mafalda; on behalf of the ClinGen FH VCEPFamilial hypercholesterolemia (FH) is the most common monogenic disorder of lipid metabolism associated with increased cardiovascular risk. FH is caused by pathogenic variants in three main FH genes, of which LDLR is the most frequent. Genetic testing can confirm the clinical diagnosis, but there are currently over 3700 different LDLR variants deposited in ClinVar. In 2022 the Clinical Genome Resource FH Variant Curation Expert Panel (VCEP) recommendations to classify LDLR variants were published. Here, we present the results of the first ten rounds of LDLR variant classification by the FH VCEP. The FH VCEP is currently composed of 12 reviewers, 19 curators, and 10 associated laboratories. Training of biocurators started in August/2021, sustained curation in November/2021, and the most recent round (10th) will end in June/2023. Variants with conflicting classifications in ClinVar and other variants in the same codon (required to properly classify variants) are being prioritized. The FH VCEP preparation team is composed of one alternating curator that uploads de-identified case data sent from associated labs, and one dedicated curator that screens and uploads published papers with functional studies into ClinGen’s Variant Curation Interface (VCI) prior to the curation round. Each variant is assessed using the VCI independently by two curators or one senior curator and approved by three reviewers before being officially published to the Evidence Repository and ClinVar. Once we complete our first ten curation rounds, we will have evaluated 451 LDLR variants: 25 with previous ClinVar review status of no stars (criteria not provided), 333 with one-star (conflicting and single submitter) and 93 with two-stars (multiple submitters, no conflicts). Prior ClinVar classifications were 246 conflicting, 148 Pathogenic/Likely pathogenic (P/LP), 45 Uncertain Significance (VUS), 10 Benign/Likely benign (B/LB) and 2 not provided, totalling only 35% of variants with definite classifications. Currently, 231 variants are published in ClinVar at three-star status, 85 are approved and in the process of being published, 64 are nearly approved and 71 are under evaluation. Among the 374 variants already with an FH VCEP classification, 157 were classified as P/LP, 186 as VUS, 24 as B/LB and only 7 remained conflicting, improving the number of definite classifications to 48% and decreasing the number of conflicting classifications by ~35-fold. Accurate genetic diagnosis relies on correct variant interpretation. FH VCEP guidelines significantly decrease conflicting classifications and provide expert-level consensus data that will facilitate genetic diagnosis, ultimately improving patient management and prognosis.
- Resolving conflicting LDLR variants in ClinVar - Progress of the ClinGen familial hypercholesterolemia variant curation expert panelPublication . Chora, J.; Iacocca, M.; Elnagheeb, M.; Kullo, I.; Bourbon, M.; on behalf of the ClinGen FH VCEPFamilial hypercholesterolemia (FH) is the most common monogenic disorder of lipid metabolism. Genetic testing can confirm the clinical diagnosis, but there are currently over 3300 different variants in LDLR deposited in ClinVar and ~400 had conflicting classifications of pathogenicity. Here, we present the progress of LDLR variant classification by the FH Variant Curation Expert Panel (VCEP), composed of 13 reviewers, 17 curators, and 12 associated labs, with our LDLR consensus variant classification guidelines. Variants with conflicting classifications and other variants in the same codon (required to properly classify conflicting variants) are prioritized. Associated labs send internal variant case-level data, which is uploaded into the Variant Curation Interface (VCI) and supplemented by literature evidence. Each variant is assessed by one (very experienced) or two curators and approved by three reviewers before being officially published to ClinVar. As of December 2022, we have completed classification of 316 LDLR variants. Of those with prior conflicting classifications (n=165), 33% were classified as Pathogenic/Likely pathogenic (P/LP), 9% as Benign/Likely benign (B/LB), 55% as Variant of Uncertain Significance (VUS) by insufficient evidence and only 3% remained conflicting. Of the remaining 135 variants, 53% were classified as P/LP, 2% as B/LB and 45% as VUS. Until May 2023, we will evaluate 451 LDLR variants, 247 of them with prior conflicting classifications. Ultimately, efforts of the FH VCEP are aimed at improving FH genetic diagnosis, which relies on accurate LDLR variant classification. FH VCEP’s guidelines significantly decrease conflicting classifications, which will be especially helpful to the FH community.
- Implementation Gaps of the Portuguese Nacional Health Plan 2012 2021: Evidence Mapping AnalysisPublication . Costa, Alexandra; Costa, Luciana; Rosa, Arminda; Mexia, Ricardo; Matias-Dias, CarlosThe National Institute of Health Doutor Ricardo Jorge (INSA) is responsible for carrying out the evaluation of the Portuguese National Health Plan (PNS) 2012-2016, extended to 2020 (and later to 2021). As part of PNS evaluation, this study aims to identify gaps in the implementation of the PNS 2012-2021, and to identify the degree of alignment and coherence between the strategic axis, goals, and monitoring framework issued by the PNS at different levels of implementation (national, regional, local and municipal). Methods: A mix study, employing both qualitative and quantitative approaches was performed based upon a modified scoping review. Portuguese healthcare institutional websites and municipality websites were identified as the main data sources and secondary data were gathered. Evidence (plans, programs, or strategies) with publication date between 2015 and 2020 were included. A data collection matrix was validated by a group of experts from major stakeholders from academia and public health services. The matrix included 3 main categories following the PNS intervention logic (5 strategic axis, 4 goals and 37 monitoring indicators). Results: A total of 204 documents met the inclusion criteria. Evidence from national level (55%) and local level (35%) represented about 90% of the sample. Overall, three implementation gaps were identified: 1) absence of two Regional Health Plans (in a total of 5 mainland health regions); 2) lack of about 1/3 of the expected Local Health Plans (16 out of 53) and, 3) low transposition (or approximation) of the PNS monitoring framework into the hospital and primary care contract-programs, one of the main management tools for planning. Conclusions: The identification and analysis of implementation gaps contribute to the final evaluation of the PNS. Moreover, the results can guide the next planning cycle pointing out sensitive areas of implementation which need more attention from various stakeholders.
- 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.