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- Portugal - Developments on the Safety of Manufactured Nanomaterials and Advanced MaterialsPublication . Louro, HenriquetaItem 4. Developments in Member countries relevant to the WPMN Programme of Work: Tour de Table (ENV/CBC/NANO(2025). Delegations will be invited to highlight past or planned activities at the national level that are most relevant for the discussion of the WPMN Programme of Work, and for the work of the various steering groups. An updated document will be prepared after the WPMN.
- Targetable Genetic Alterations in High-Risk Neuroblastoma Patients Enrolled in the SIOPEN HR-NBL1 StudyPublication . Anseri, Elnaz Saberi; Bellini, Angela; Pötschger, Ulrike; Taschner-Mandl, Sabine; Planchon, Julien Masliah; Attignon, Valéry; Auger, Nathalie; Beiske, Klaus; Goodmann, Angharad; Jeison, Marta; Mazzocco, Katia; Morini, Martina; Capasso, Mario; Mühlethaler-Mottet, Annick; Noguera, Rosa; Font de Mora, Jaime; Martinsson, Tommy; Van Roy, Nadine; Vicha, Ales; Marques, Barbara; Chesler, Louis; George, Sally; Tweddle, Deborah; Ladenstein, Ruth; Schleiermacher, GudrunBackground: In high-risk neuroblastoma (HR-NB), new treatment strategies, including targeted treatments, are required to improve outcomes. Aim: To determine the frequency of genetic alterations (SNVs/Indels) in genes considered to be targetable and/or to play a role in oncogenesis in HR-NB. Methods: Diagnostic tumor samples from 709 patients treated in the SIOPEN HR-NBL1 trial (INRG stage M: 636 patients; localized: 70 patients; Ms: 3 patients; 269 MYCN amplified) were analyzed. Targeted Sequencing (TrueSeq Custom Amplicon) of 85 genes involved in oncogenesis and therapy response was performed on (n=484 samples), along with other targeted sequencing approaches (n=377 samples), whole-exome sequencing (n=32 samples), and whole-genome sequencing (n=8 samples) across ten European centers. Final results were reported on the panel of 85 genes. Variant calling and copy number alterations were analyzed using Mutect2 and FACETS. Matched germline data were available for 54 patients.
- Ingested titanium dioxide nanomaterials: new approach to investigate intestinal genotoxicity and key cellular/molecular effectsPublication . Ventura, Célia; Rolo, Dora; Gramacho, Ana C.; Vieira, Adriana; Roque, Rossana; Valente, Ana; Vital, Nádia; Pinto, Fátima; Alvito, Paula; Assunção, Ricardo; Martins, Carla; Bettencourt, Ana; Gonçalves, Lídia; Pereira, Joana; Matos, Paulo; Jordan, Peter; Vieira, Luís; Silva, Catarina; Silva, Maria Joao; Louro, HenriquetaOral exposure to titanium dioxide nanomaterials (TiO2NMs) is due to their presence in food, food contact materials, medicines and cosmetics. The gastrointestinal tract(GIT) represents primary site of contact, that may result in systemic exposure, if biological barriers are surpassed. The INGESTnano project aimed to investigate nano-bio interactions at the cellular/molecular levels within the context of the intestinal tract and digestion processes, for understanding potential effects on human health. A group of three TiO₂NMs(NM-102, NM-103, NM-105) was selected as case study using a new approach methodology(NAM), incorporating the in vitro human digestion simulation prior to biological assays in Caco-2 and HT29-MTX-E12 intestinal cells. The endpoints included cyto- and genotoxicity, cell uptake, intestinal permeability, GIT transport and epigenomic modifications. The results showed a more pronounced cytotoxicity in HT29-MTX-E12 cells for digested NM-105, as compared to undigested, concomitantly with subtle changes in hydrodynamic-size. DNA-damage induction was more relevant for NM-105, and the micronucleus assay showed chromosomal damage in HT29-MTX-E12 cells for all TiO2NMs, especially after in vitro digestion.All NMs, digested or not, were internalized by intestinal cells, but did not affect transepithelial resistance, nor the epithelial markers in polarized enterocytes. NM-102 was retained in lysosomes, while NM-103 and NM-105 showed transcytosis, a potential gateway for systemic distribution. Using Reduced Representation Bisulfite Sequencing, several differentially methylated genes were identified for the TiO₂NMs, either digested or not. Pathway and Gene Ontology analyses showed that each TiO2NMs has a different functional impact on intestinal cells, probably linked to specific physicochemical properties, and digestion seems to reduce this impact. A trend towards CpG hypermethylation was observed upon NM-105 exposure, unlike for the other TiO2NMs. This integrated approach enabled the identification of key events and molecular pathways elicited by TiO2NMs, highlighting the importance of considering the digestion on the induction of adverse outcomes.
- Generation of Cellular Models for Fabry Disease: Unlocking the Potential of iPSCs and Gene EditingPublication . Duarte, Ana Joana; Moreira, Luciana; Ribeiro, Diogo; Alves, Sandra; Gaspar, Paulo; Bragança, José; Amaral, OlgaIntroduction: Fabry Disease (FD) is a lysosomal storage disorder caused by mutations in the GLA gene, resulting in a defective α-GAL A enzyme. This deficiency leads to the accumulation of Gb3 and lyso-Gb3 within lysosomes, resulting in a multisystem disease. Through reprogramming, we obtained induced pluripotent stem cells (iPSCs) derived from fibroblasts of a patient with FD2 and from a wild-type (WT) control. We used CRISPR/Cas9 to correct the c.860G>A mutation present in the patient’s cells, as well as to generate a WT GLA knockout (KO). The resulting cells were then differentiated into cardiomyocytes, a cell type affected by this disease. Methods: We reprogrammed the fibroblasts into iPSCs using episomal vectors or Sendai virus. For gene editing, single-guide RNAs (sgRNAs) and Cas9 were nucleofected, and the editing was confirmed by Sanger sequencing. Following colony selection, isogenic cell lines were established. The FD iPSCs, the corrected FD iPSCs, and the WT iPSCs were then differentiated into iPSC-derived cardiomyocytes (iPSC-CMs). Results: Seven new cell models were generated. Functional studies of the FD iPSCs showed the maintenance of the molecular and biochemical characteristics and a normal karyotype. The KO cell line recapitulated the biological features observed in FD patient cells, with reduced GLA expression, lower α-Galactosidase A (α-Gal A) activity (1.5 nmol/h/mg protein), and Gb3 accumulation. The corrected cell line was generated with 75.8% efficiency and 69.6% on-target efficacy. Enzyme activity increased to 579 nmol/h/mg protein (vs. 0.78 nmol/h/mg protein in FD iPSCs), accompanied by a marked reduction in Gb3 levels. We successfully generated iPSC-CM lines, which were validated by qRT-PCR and immunofluorescence. Discussion: Cell modelling is essential for studying the pathophysiology of disease mechanisms. By retaining the characteristics of the original cells, iPSCs are a valuable biological resource for generating specific differentiated cell types affected by the disease, which would otherwise be difficult to access. This study also explored the therapeutic potential of gene editing as a promising approach to altering the course of rare diseases.
- Tracking Mycotoxin Exposure in Portugal: New Insights and Key DeterminantsPublication . Namorado, Sónia; Maris, Elias; Chen, A.; Pero-Gascon, Roger; de Boevre, Marthe; De Saeger, Sarah; Silva, Maria João; Alvito, Paula; .Human biomonitoring (HBM) is a vital tool for assessing exposure to environmental chemicals. Mycotoxins have been associated with various adverse health effects, including estrogenic, immunotoxic, nephrotoxic, and teratogenic outcomes. In this presentation an overview of two recent HBM studies on multiple mycotoxins exposure biomarkers conducted in Portugal will be given. The first study analyzed 37 mycotoxin biomarkers in urine samples from 94 adult participants (48.4 ± 15.2 years), as part of the National Food, Nutrition, and Physical Activity Survey (2015–2016). Six different mycotoxins -DON, ZEN, AOH, OTA, FB1, and CIT- was confirmed through the quantification of 12 urinary biomarkers in paired 24 h and first-morning urine samples. DON and its metabolites were among the most frequently detected biomarkers and AOH was identified for the first time in urine samples from a European population. Associations between urinary mycotoxin biomarkers and consumption of specific food items were also observed. More recently, a subset of 295 first-morning urine samples from adults (28–39 years) was collected between 2019-2020, as part of a cross-sectional study embedded within the Portuguese National Health Examination Survey (INSEF). These samples were analyzed using a newly optimized and validated LC-MS/MS method capable of detecting 40 mycotoxins and/or their metabolites in urine. DON and tenuazonic acid were the most frequently detected, with detection rates of 85% and 96%, respectively. Further investigations into key exposure determinants—including dietary habits, demographic factors, and geographical variations—are ongoing and will be presented. Altogether, these findings highlight the importance of continued surveillance and the integration of HBM into national food safety and public health strategies.
- Gapmer Antisense Oligonucleotides as a New Class of Genetic Substrate Reduction Agents in Mucopolysaccharidosis IIICPublication . Santos, Juliana I.; Gonçalves, Mariana; Almeida, Matilde B.; Rocha, Hugo; Duarte, Ana J.; Matos, Liliana; Moreira, Luciana V.; Encarnação, Marisa; Gaspar, Paulo; Prata, Maria J.; Coutinho, Maria F.; Alves, SandraBackground: Lysosomal storage disorders (LSDs) include over 70 rare inherited metabolic diseases caused by defective lysosomal enzymes or associated proteins, leading to the accumulation of undegraded substrates and progressive cellular dysfunction. Among these, Mucopolysaccharidoses (MPS) are a group characterized by storage of undegraded glycosaminoglycans (GAGs), including heparan, dermatan, keratan, and chondroitin sulfates. MPS type III (as known as Sanfilippo syndrome) predominantly affects the central nervous system (CNS) and manifests as severe neurodegeneration, behavioral abnormalities and cognitive decline. The subtype IIIC results from deficient activity of acetyl-CoA:α-glucosaminide N-acetyltransferase (HGSNAT) enzyme. Currently, treatment options for MPS III are limited, increasing the need to find alternative therapies. RNA-based therapies have recently gained attention as powerful alternatives to traditional treatments, with several already approved for clinical use in other diseases. Among these, antisense oligonucleotides (ASOs) stand out as a highly promising class of molecules for personalized medicine. ASOs are short, chemically synthesized strands of nucleic acids designed to bind specifically to target RNA sequences, thereby influencing gene expression. In this work, we explored the application of gapmer ASOs as a genetic substrate reduction therapy (gSRT) for MPS IIIC, with the goal of decreasing the expression of a gene, XYLT1, involved in the synthesis of the accumulated substrate, heparan sulfate (HS).
- Translational activation of Δ160p53 is triggered during the Integrated Stress Response to promote survivalPublication . Ramalho, Ana Catarina; López-Iniesta, Maria José; Lacerda, Rafaela; Parkar, Shrutee N.; Romão, Luísa; Candeias, Marco M.The p53 gene was discovered 45 years ago, but to this day some aspects of its regulation and function remain a mystery. This gene encodes for a group of protein isoforms that display different functions, acting cooperatively in stress response. Full-length p53 (FLp53), the best studied isoform, is a transcription factor with targets across several pathways, promoting cellular adaption during stress. Classical roles of FLp53 are the induction of cell cycle arrest and, a as a last resort, apoptosis. The short isoform Δ160p53 is an oncoprotein commonly overexpressed in tumours that can be translated from the same mRNA as FLp531. The exact mechanisms and factors that regulate Δ160p53 translation and how it exerts its pro-survival functions remain unexplored. In this work, it was demonstrated that the translation of Δ160p53 is mediated by an Internal Ribosome Entry Site (IRES) and its expression is induced by the Integrated Stress Response (ISR). Additionally, a mass spectrometry analysis uncovered potential new RNA-binding proteins involved in the translation of Δ160p53, and it was verified that translation factors specifically associated with alternative modes of initiation during the ISR promoted the translation of Δ160p53. In parallel, Δ160p53 was found to interact with FLp53 and modulate its transcriptional activity to favour a pro-survival response to stress. While suppressing genes involved in cell cycle arrest (CDKN1A) and apoptosis (PUMA), Δ160p53 enhances the expression of genes involved in cellular metabolism and protection against ferroptosis: SESN1, SESN2 and SLC7A11. Furthermore, Δ160p53 was shown to suppress ferroptosis and to interact with the SLC7A11 promoter. This study has uncovered new modes of p53 regulation and hints at a physiological role for Δ160p53 in the context of the ISR. However, abnormal levels of Δ160p53 may contribute to an exacerbated pro-survival p53 target signature, demonstrating the importance of p53 isoform balance.
- Influência da genética do metabolismo do ferro nos fenótipos bioquímicos e hematológicos da insuficiência cardíacaPublication . Bicho, Manuel; Aguiar, Laura; Matias, Ana; Santos, Mafalda; Barbosa, Mário; Melício, Ana; Menezes Falcão, Luiz; Faustino, Paula; Inácio, ÂngelaIntrodução: A insuficiência cardíaca (IC) é uma síndrome que se caracteriza pela incapacidade do coração bombear sangue de forma eficiente para atender às necessidades metabólicas do organismo humano. Esta patologia multifatorial, frequentemente associada a deficiência de ferro e anemia, pode ser influenciada por diversos moduladores, entre estes, genes responsáveis pelo equilíbrio do metabolismo do ferro. Objetivos: O presente estudo teve como objetivo investigar a contribuição da variação genética em genes relacionados com o metabolismo do ferro, nomeadamente HFE, SLC40A1 e TMPRSS6, para os fenótipos bioquímicos e hematológicos relacionados com a cinética do ferro na IC no geral, na IC com fração de ejeção preservada (ICFEp) e na IC com fração de ejeção não preservada (ICFEnp), considerando as diferenças entre os sexos. Material e Métodos: O estudo englobou uma amostra populacional de 192 indivíduos com IC, com idades compreendidas entre os 35 e os 99 anos (mediana de 84 anos). A análise das variantes em estudo do gene HFE foi realizada através da técnica PCR-ARMS Multiplex. As análises dos restantes polimorfismos foram realizadas através da técnica Endpoint-Genotyping. Os parâmetros bioquímicos e hematológicos estudados (ferro sérico, ferritina, saturação da transferrina, hemoglobina, volume globular médio, Red Cell Distribution Width e capacidade total de fixação do ferro) foram recolhidos dos registos hospitalares dos doentes. A análise estatística foi realizada recorrendo ao software SPSS 28, sendo considerado o nível de significância estatística de p<0,05. Resultados: Neste estudo, a população com IC apresentou anemia ferropénica com baixos níveis de saturação de transferrina. Nos homens, o ferro sérico e o RDW também se apresentaram alterados. Diferenças significativas na presença do genótipo ou alelo variante foram encontradas para os parâmetros bioquímicos ou hematológicos estudados. Para os valores de ferro sérico foram encontradas diferenças significativas associadas a polimorfismos localizados nos três genes estudados. Quanto à ferritina, foram observadas diferenças significativas no HFE_H63D em mulheres e em homens e no SLC40A1_rs2304704 em mulheres, mais especificamente em mulheres com ICFEp. Para a saturação de transferrina, diferenças significativas foram encontradas no polimorfismo HFE_H63D em mulheres e no TMPRSS6_rs855791 em mulheres, particularmente em mulheres com ICFEnp e em homens com ICFEp. Em relação à hemoglobina, foram observadas diferenças significativas para o SLC40A1_rs1439816 em homens. Analisando o VGM, diferenças estatisticamente significativas foram observadas no SLC40A1_rs1439816 em homens com ICFEp e no SLC40A1_rs2304704 em mulheres, e em particular mulheres com ICFEp. Em relação ao RDW, diferenças significativas foram encontradas no polimorfismo C282Y em mulheres com ICFEnp e no polimorfismo H63D em mulheres com ICFEnp e em homens com ICFEp. Para a CTFF, diferenças significativas foram encontradas no rs855791 em mulheres com ICFEp. Conclusão: Este estudo confirmou a presença de indicadores de anemia ferropénica nesta população de indivíduos com IC, destacando a contribuição dos três genes analisados na modulação de fenótipos bioquímicos e hematológicos relacionados com o metabolismo do ferro. Observou-se que estas variantes genéticas têm impacto significativo tanto em homens quanto em mulheres, observando-se também diferenças nos subgrupos da IC com fração de ejeção preservada e não preservada.
- Hemocromatose - causa genética ou adquirida?Publication . Faustino, PaulaNesta comunicação oral apresentam-se e discutem-se as possíveis causas responsáveis pela a hemocromatose adquirida e genética e os seus diversos tipos. Explicam-se os principias mecanismos de absorção e reciclagem do ferro no Homem e a atuação da principal hormona responsável pela regulação dos níveis de ferro no organismo. Apresentam-se os biomarcadores bioquímicos usados no diagnóstico da doença e os métodos moleculares usados para caracterizar os genótipos de risco. Debate-se a penetrância e elevada variabilidade da doença e os seus fatores modificadores. Apresenta-se trabalho de investigação realizado no Departamento de Genética Humana do INSA no âmbito destas patologias.
- Hemoglobinopatias - InvestigaçãoPublication . Faustino, PaulaNesta comunicação oral apresentam-se as recomendações da OMS para combater a drepanocitose nos países Africanos e o papel da investigação para compreender os mecanismos da doença e desenvolver estratégias terapêuticas para a combater. Apresentam-se os fatores genéticos modificadores da doença, nomeadamente o nível de hemoglobina fetal e como esta pode ser manipulada farmacologicamente para benefício do doente. Mostram-se e discutem-se os resultados dos trabalhos de investigação de âmbito colaborativo desenvolvidos entre o INSA e algumas Instituições de Saúde Angolanas na área dos fatores genéticos modificadores das manifestações da drepanocitose. Discutem-se os tratamentos sintomáticos e os curativos, incluindo a terapia génica, e os obstáculos às suas utilizações em África.