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- Functional Studies in LDLR to Improve Genetic Diagnosis in Familial HypercholesterolemiaPublication . Alves, Ana Catarina; Graça, Rafael; Ferreira, Maria Simões; Correia, Bernardo Amaro; Medeiros, Ana Margarida; Miranda, Beatriz; Chora, Joana Rita; Bourbon, MafaldaFamilial hypercholesterolemia (FH) is the most common inherited disorder of lipid metabolism, affecting approximately 1 in 300 individuals. FH is an autosomal semidominant disorder and was the first genetic lipid metabolism disorder to be molecularly characterized. Individuals with FH present elevated blood cholesterol levels from birth, leading to a high cumulative risk of developing cardiovascular disease. Pathogenic variants in the low-density lipoprotein receptor (LDLR) gene are the primary cause of FH, with more than 4,000 variants identified to date. However, only 15% of these have been functionally characterized in vitro, demonstrating their impact (or lack of) on LDL receptor function. This study highlights the role of functional studies in genetic diagnosis and personalized medicine, using the Portuguese Familial hypercholesterolemia Study (EPHF) as an example of how these studies have clinical impact. Functional studies of LDLR are crucial for understanding how this receptor activity regulates cholesterol clearance. In a cohort of 1088 individuals (1073 heterozygotes and 15 homozygotes) with a clinical diagnosis of FH, 164 different LDLR variants were identified. To date, 70 of these variants have been functionally studied within the EPHF and 38 have been characterized by other labs. This functional characterization contributed to obtain a definitive genetic diagnosis for 847 individuals, marking a significant step towards personalized medicine and the effective management of FH. These studies not only improve the classification of LDLR variants but also directly influence treatment decisions and patient outcomes. By advancing the functional characterization of these variants, we contribute to more specialized and precise diagnostics, leading to more tailored therapeutic approaches for cholesterol-related disorders.
- 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.
- Genetic Similarity Between nAutism Spectrum Disorder and Comorbid Brain DisordersPublication . Vilela, Joana; Martiniano, Hugo; Marques, Ana Rita; Santos, João Xavier; Rasga, Célia; Oliveira, Guiomar; Vicente, Astrid MouraBackground: A range of brain disorders are comorbid with Autism Spectrum Disorder (ASD), such as Epilepsy, Intellectual disability or Anxiety. Previous evidence suggested a shared genetic influence between ASD and several brain disorders. In this study, we sought to further substantiate the genetic similarity between ASD and a set of comorbid brain disorders. Methods: We constructed a network of 29 brain disorders based on their genetic similarity, estimated from the Jaccard coefficient between disease pairs. The Leiden algorithm was used to identify network disease communities. In 3,881 ASD cases from a whole-genome sequencing dataset, we further searched for de novo loss-of-function (LoF) Single Nucleotide Variants (SNVs) in genes shared by the disease communities. Results: Through network analysis we identified three disease communities, including a heterogeneous community that is genetically more similar to ASD, and that also includes Epilepsy, Bipolar Disorder, Attention-Deficit/Hyperactivity Disorder combined type, and some disorders in the Schizophrenia Spectrum. Several of the genes shared by diseases communities are strong ASD candidate genes, such as SHANK3, SCN2A, ASH1L or CHD2, and harbour the highest number of rare de novo LoF SNVs in ASD patients. Conclusion: This study provided further evidence for a shared genetic architecture between ASD and several other brain disorders, including some frequent comorbidities of ASD. It also showed that ASD patients have rare de novo LoF variants in genes associated with frequent comorbid disorders, and identified genes that overlap between brain disease communities.
- 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.
- 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.
- Prediction of Genes Associated With Autism Spectrum Disorder Using Sequence And Graph Embedding MethodsPublication . Inácio, João; Vilela, Joana; Marques, Ana Rita; Santos, João Xavier; Rasga, Célia; Vicente, Astrid; Martiniano, HugoIntroduction: Autism Spectrum Disorder (ASD) is a complex neurodevelopmental disorder, characterized by a strong genetic component, heterogeneous phenotypic presentation and complex genetic architecture. [1, 2] Identification of ASD risk genes and biological pathways is key to understand this disorder.[3, 4] Despite the large number of genes associated with ASD, its genetic etiology is still not fully understood. To address this challenge, we developed a machine-learning approach for ASD risk gene prediction. Methods: We developed a Machine Learning–based approach to identify and rank ASD-associated genes. Results and discussion: Prediction of ASD-associated Genes The best model was used to create a ranked list of ASD-associated genes. Using the top decile of the predicted genes, we performed a decile enrichment analysis using Phenotypes from the Human Phenotype Ontology (HPO) database and gene lists for neurodevelopmental disorders and psychiatric disorders unrelated to ASD. Conclusions: We developed a machine learning approach based on sequence and Graph embedding Methods to predict ASD-associated genes. Our approach compares favorably with state-of-the-art approaches in identifying genes targeted by loss-of-function (LOF) mutations in the MSSNG and Simons Simplex Collection cohorts and shows specificity towards ASD-related phenotypes.
- Truncating APOB Variants Impair LDL Metabolism: Functional Evidence From Binding StudiesPublication . Ferreira, Maria Simões; Larrea-Sebal, Asier; Martín, César; Apellaniz-Ruiz, Maria; Ernaga-Lorea, Ander; Bourbon, Mafalda; Alves, Ana CatarinaFamilial hypercholesterolaemia ( is a condition caused by pathogenic variants in LDLR APOB or PCSK 9 genes, characterised by high levels of LDL cholesterol and premature cardiovascular disease ( APOB variants account for 5 10 of FH cases, most being due to missense variants however, this can be higher than initially estimated Although truncating variants are typically associated with hypocholesterolaemia phenotype, some have been reported in clinical FH patients and can be the cause of diseaseThis study is part of the PerMedFH project and aimed to functionally characterise four nonsense APOB variants in exon 29 identified in patients with a clinical diagnosis of FH, emphasising on their molecular consequences and possible contribution to the FH phenotype. This study is part of the PerMedFH project and aimed to functionally characterise four nonsense APOB variants in exon 29 identified in patients with a clinical diagnosis of FH, emphasising on their molecular consequences and possible contribution to the FH phenotype
- Pharmacogenomic Risk Profiling of Statin Response in Portuguese Familial Hypercholesterolemia and General Population CohortsPublication . Chora, Joana Rita; Grade, M. M.; Antunes, M.; Bourbon, M.Background/Aims: Genetic variants in pharmacogenes involved in statin transport, metabolism, and excretion can increase the risk of adverse effects, particularly statin-associated musculoskeletal symptoms (SAMS). Loss-of-function haplotypes in SLCO1B1 are reliably linked to elevated SAMS risk. Familial hypercholesterolemia (FH), a high cardiovascular disease risk condition, requires lifelong lipid-lowering therapy, often from a young age. However, studies of statin pharmacogenomics in FH patients remain scarce. The Portuguese population, shaped by millennia of admixture, displays a complex genetic background with potential implications for pharmacogenomic diversity. This study aimed to assess the prevalence of high-risk alleles in genes related to statin metabolism and transport, including variants relevant to therapeutic efficacy, in both the general Portuguese population and FH patients. Methods: We analysed 738 adults from the nationally representative e_Cor cohort and 489 clinical FH patients. Genotyping of 13 statin-related single-nucleotide variants (SNV) was conducted using both OpenArrayTM technology and NGS target sequencing. Statin-medicated individuals from both cohorts (N=903) were further analysed for pharmacogenomic risk. Results: Compared to public databases, the frequency of the APOE rs429358 risk allele was significantly lower in the general Portuguese cohort, but significantly higher in statin-medicated individuals and FH patients. Genotype distributions of SLCO1B1, ABCB1, HMGCR, and MTHFR SNVs differed significantly between medicated individuals from the general population and FH patients. Among all participants, 2% had SLCO1B1 poor-function and 22% decreased-function haplotypes; these frequencies were slightly higher in the medicated group (3% and 23%, respectively). Notably, 40% of poor-function and 32% of decreased-function carriers were prescribed a statin dose/type considered high-risk for SAMS. Differences between FH-positive and negative individuals were only observed for APOE rs429358. Conclusion: This study presents a comprehensive overview of statin-related pharmacogenetic variation in the Portuguese population and FH patients. The high prevalence of actionable variants underlines the importance of pharmacogenomic-informed prescribing in high-risk groups. Integrating genetic information into clinical decision-making can optimise statin therapy, mitigate adverse effects, and enhance the effectiveness of personalised lipid-lowering strategies in Portugal.
- A Global Survey Of Genetic Testing Methods For Familial Hypercholesterolemia. A Study From The EAS FHSC RegistryPublication . Karungi, Irene, On behalf of the EAS FHSC Investigators; Chora, Joana Rita; Elshorbagy, A.; Stevens, C.A.T.; Vallejo-Vaz, A. J.; Ray, K. K.; Raal, F. J.; Humphries, S. E.; Freiberger, T.; Bourbon, M.Contexto: A hipercolesterolemia familiar (HF) é causada principalmente por variantes patogénicas nos genes LDLR, APOB ou PCSK9, levando a um LDL-C elevado ao longo da vida e a um aumento do risco de doenças cardiovasculares. Os testes genéticos oferecem um diagnóstico definitivo da HF, no entanto, falta uma abordagem padronizada para os métodos de teste genético da FH a nível global. Métodos: Foi enviado um inquérito estruturado a investigadores nacionais de 68 países ativos no registo da Colaboração de Estudos EAS-FH. Os domínios do inquérito incluíram critérios para encaminhamento para testes genéticos, técnicas de teste, triagem genética e interpretação da patogenicidade variante. Os dados foram analisados descritivamente, e foram feitas comparações entre casos índice e não-índice e países de rendimento elevado versus não elevado. Resultados: Dos 68 países convidados, 55 (81%) responderam. Entre os países com critérios clínicos estabelecidos para adultos (85%) e crianças (76%), a rede holandesa de clínicas de lípidos foi o critério mais utilizado para o encaminhamento de adultos e crianças (72% e 57%, respetivamente) para testes genéticos. Os critérios de Simon-Broome e MedPed foram usados com menor frequência, reportados apenas por países de rendimento elevado para adultos (7% e 2%, respetivamente) e crianças (12% e 2%, respetivamente). Para testes de casos índice, a maioria dos países utilizou painel génico de sequenciação de próxima geração (NGS) (62%), enquanto para casos não indexados, a maioria baseou-se em variantes específicas por sequenciação Sanger (71%). Este padrão era semelhante tanto em países de rendimento elevado como não elevado; no entanto, alguns países usaram a mesma abordagem de teste tanto para casos índice como não indexados (Figura). As variantes de número de cópia (CNVs) foram avaliadas em 65% dos países, dos quais 86% incorporaram análise CNV em plataformas NGS, enquanto 14% utilizaram amplificação de sonda dependente de ligação multiplex (MLPA) e testes de microarray. Todos os países testados para LDLR, 97% para APOB, e 95% para PCSK9. Em 96% dos países, os relatórios de testes incluíram interpretação de patogenicidade variante (com a maioria a seguir as diretrizes do American College of Medical Genetics and Genomics). Conclusões: A variabilidade generalizada nas práticas de testes genéticos destaca a necessidade crítica de padronização para garantir um diagnóstico de FH eficaz, consistente e comparável a nível global.
- HbA1c laboratory quality performance of National External Quality Assessment Program (PNAEQ) in the EurA1c project: 2017–2024Publication . Correia, Helena; das Neves Pereira da Silva, Susana; Miranda, Armandina; Faria, Ana; Weykamp, Cas; Siebelder, CarlaIntroduction: The determination of glycated hemoglobin (HbA1c) is used in patients with diabetes mellitus as a key to monitoring the long-term blood glucose control (1). The Portuguese National External Quality Assessment Program (PNAEQ), organizes since 2003 the Hemoglobin glycated (HbA1c) program for External Quality Assessment Laboratory and integrated the IFCC EurA1c project in 2017. External Quality Assessment (EQA) is a powerful education tool to monitor quality that, by identifying poor performing laboratories and test systems, can be used as a tool to improve quality (2). Objective: Monitor the analytical performance of HbA1c determination, performed by PNAEQ participants, based on results of their participation in EurA1c project (bias (%) and coefficient of variation (CV%). Methods: From 2017 to 2024, PNAEQ laboratory participants (mean=41), from public and private sectors of ambulatory and hospital care, analysed in first round, two control samples per year, (n=16), with different concentrations (samples below and above 48mmol/mol). The samples sent from EurA1c project were lyophilized hemolysate specimens manufactured from the same pool. Participants HbA1c results, in IFCC units, were evaluated by comparison with target values assessed by EurA1c project. Usually, values were assigned by five laboratories using the IFCC Reference Measurement Procedure. The statistical analysis followed the EurA1c project´s methodology. Outliers- defined as results differing by more than 25% from the target value- were excluded prior to calculating the mean, CV% and bias%. Performance comparisons between different concentration samples were performed with Wilcoxon test considering a significance of 5%. Results: During the study period, an average of 41 laboratories participated (range: 26–49). A total of 17 different types of equipment and 9 distinct methodologies were used. From 2022 onwards, a noticeable shift in equipment and/or methodology was observed among several participants. Notably, five laboratories consistently submitted results for all samples throughout the study. There was no significant difference in the mean CV% between pathological samples (CV% = 5.1) and non-pathological samples (CV% = 5.7), with a p-value of 0.151. Similarly, no significant difference was observed in Bias% between pathological samples (Bias% = 1.0) and non-pathological samples (Bias% = 1.6), with a p-value of 0.069. Before pandemic period (2017-2019), the mean bias% was 0.02, in the pandemic period (2020-2021) was 3.18, and after pandemic 1.43. Based on the minimum EFLM quality specifications for HbA1c bias (2,7%), the mean bias of participants meets the established criteria in 13 samples. Regarding the desirable specification (1.8%), 10 samples and regarding the optimal specification (0.9%), 5 samples were compliant. During the pandemic period, 3 samples did not meet the minimum specifications. Conclusion: During the study period, the laboratories changed equipment without showing performance improvements. There is clear sign of a decline in performance in the pandemic period (2020-2022) with no improvement to the level of pre pandemic period. Most samples met the minimum quality specifications for HbA1c bias established by the EFLM, with a substantial proportion also meeting desirable and optimal targets. These findings highlight the effectiveness of the EurA1c external quality assessment framework in supporting continuous quality improvement in HbA1c testing across Portuguese laboratories.