Departamento de Genética Humana
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- Addressing a challenging enzyme in vitro: proof of principle on the therapeutic potential of an antisense oligonucleotide approach for ML IIPublication . Matos, Liliana1. To confirm the specificity of selected ASOs to skip the GNPTAB exon 19, a scramble ASO (expected to have no effect on GNPTAB gene) was transfected in WT & ML II patient fibroblasts. RT-PCR results showed that this ASO did not changed exon 19 splicing pattern proving the specificity of GNPTAB ASOs (Fig1A). Also, the enzymatic activity of various hydrolases was analysed in the same transfected cells. Results showed that the scramble ASO seems to interfere, even if little, with hydrolases activities of treated WT & ML II fibroblasts as shown by the differences in activities observed (Fig1B). The experiments number need to be increased to confirm these results. 2. To analyse the effect of exon 19 skipping on the GlcNAc-PT, a WT construct with the full GNPTAB cDNA (pGNPTAB_WT) and a mutant without exon 19 (pGNPTAB_delEx19) were tested in HEK293T cells. RT-PCR results after transfection showed the expected transcript pattern in both constructs (Fig2A). Moreover, GNPTAB protein expression was analysed by Western Blot (WB) using an antibody (Ab) specific for a myc-His tag located in constructs downstream the GNPTAB insert. Both constructs expressed a band corresponding to the α/β precursor, with or without exon 19. However, regarding the cleaved β-subunit the results are not so clear since we observed 2 bands (~48KDa) for the WT construct and further analysis is needed to understand if any of them corresponds to the target (Fig2B). Both constructs were also transfected in ML II fibroblasts and cDNA analysis showed the expected transcript pattern. The activity of hydrolases will also be assessed to check if it increases with the delEx19 construct expression, suggesting a potential therapeutic effect. 3. We generated a novel Ab for the GlcNAc-PT β-subunit (rabbits). We expected to detect the protein in WT but not in ML II fibroblasts. However, WB results showed a band with the expected protein size in both cases (Fig3A). So, to test the Ab specificity different assays were done. The pre-bleed serum of the immunized rabbits was tested by WB and no target band was detected confirming that animals did not have Abs against the human protein (Fig3B). Also, the 2 synthetic β-subunit peptides used to immunize the animals were detected by WB confirming the specificity of the produced Abs (Fig3C). Protein sequencing (mass spectrometry) was also performed in extracts of WT & ML II fibroblasts after SDS-Page to search for the GNPTAB protein. As our Ab target has 45KDa, bands within 37-50KDa were analysed but the protein was not detected not even in the WT, suggesting that the protein amount used was insufficient for target protein detection. Finally, the Ab was tested by WB in HEK293T cells transfected with both constructs and the expected protein pattern at least for the α/β precursor was observed (Fig3D). This last experiment needs to be repeated, however results obtained in the various assays suggest that our Ab is specific for GNPTAB protein detection.
- Can an Antisense Oligonucleotide Exon Skipping Rewrite the Story of N-Acetylglucosamine-1-Phosphotransferase Deficiency?Publication . Gonçalves, M.; Moreira, L.; Encarnação, M.; Gaspar, P.; Duarte, A.J.; Santos, J.I.; Coutinho, M.F.; Prata, M.J.; Omidi, M.; Pohl, S.; Silva, F.; Oliveira, P.; Matos, L.; Alves, S.Mucolipidosis II (ML II) is a Lysosomal Storage Disorder caused by N-acetylglucosamine-1-phosphotransferase (GlcNAc-PT) deficiency, which impairs the trafficking of lysosomal hydrolases. Of all ML II mutations, c.3503_3504delTC in GNPTAB exon 19 is the most frequent, making it a good target for a personalized therapy. Here, we explored an innovative therapeutic strategy based on the use of antisense oligonucleotides (ASOs). Previously, in ML II patients’ fibroblasts, we tested ASOs to induce exon 19 skipping in pre-mRNA, successfully generating an in-frame mRNA (Matos et al., 2020). Now, our aim is to determine whether this in-frame transcript leads to increased GlcNAc-PT levels improving ML II cellular phenotype.
- Challenges of the Application of In Vitro Digestion for Nanomaterials Safety AssessmentPublication . Vital, Nádia; Gramacho, Ana Catarina; Silva, Mafalda; Cardoso, Maria; Alvito, Paula; Kranendonk, Michel; Silva, Maria Joao; Louro, HenriquetaConsidering the increase in the production and use of nanomaterials (NM) in food/feed and food contact materials, novel strategies for efficient and sustainable hazard characterization, especially in the early stages of NM development, have been proposed. Some of these strategies encompass the utilization of in vitro simulated digestion prior to cytotoxic and genotoxic assessment. This entails exposing NM to fluids that replicate the three successive phases of digestion: oral, gastric, and intestinal. Subsequently, the resulting digestion products are added to models of intestinal cells to conduct toxicological assays, analyzing multiple endpoints. Nonetheless, exposure of intestinal cells to the digested products may induce cytotoxicity effects, thereby posing a challenge to this strategy. The aim of this work was to describe the challenges encountered with the in vitro digestion INFOGEST 2.0 protocol when using the digestion product in toxicological studies of NM, and the adjustments implemented to enable its use in subsequent in vitro biological assays with intestinal cell models. The adaptation of the digestion fluids, in particular the reduction of the final bile concentration, resulted in a reduced toxic impact of digestion products.
- Classification of microcytic anemias using machine learning methodsPublication . Neves Leitão, Beatriz; Vinga, Susana; Faustino, PaulaA prevalência mundial da anemia é estimada em 24,8% e de entre as suas possíveis causas sobressaem a carência nutricional em ferro (anemia ferropénica) e algumas doenças genéticas (hemoglobinopatias como, por exemplo, beta-talassémia e alfa-talassémia). O diagnóstico da etiologia das anemias microcíticas requer métodos laboratoriais caros e morosos, mas é fundamental para a decisão clínica referente ao tratamento e, quando apropriado, para o aconselhamento genético. Neste trabalho aplicaram-se algoritmos de aprendizagem automática (machine learning) para diferenciação das referidas anemias microcíticas usando apenas as informações obtidas no hemograma, um dos exames laboratoriais mais comuns em medicina. Os resultados destacaram o excelente desempenho dos classificadores desenvolvidos com o algoritmo de florestas aleatórias (random forests), tanto na classificação binária quanto na multiclasse, demonstrando o potencial da inteligência artificial na identificação da etiologia dessas anemias.
- An engineered U1 snRNA-based therapeutic approach can efficiently rescue a 5’ splice site mutation causing Mucolipidosis type IIIPublication . Peretto, L.; Gonçalves, M.; Santos, J.I.; Duarte, A.J.; Moreira, L.; Encarnação, M; Coutinho, M.F.; Pinotti, M.; Balestra, D.; Alves, S.; Matos. L.A significant number of splicing mutations have been identified in Lysosomal Storage Disorders (LSDs). Mucolipidosis III (ML III) is a LSD caused by GlcNAc-1-phosphotransferase deficiency, which impairs the trafficking of lysosomal hydrolases. 10% of the genetic defects in ML III are splicing mutations, and around 45% affect 5' splice-sites (ss) thus constituting a good target for mutation specific therapies. The use of engineered U1 snRNA (either modified U1 snRNAs or exon-specific U1s - ExSpeU1s) has been applied as a potential therapeutic strategy to correct 5’ss defects. Here we used engineered U1 snRNAs to correct the GNPTAB exon 17 skipping caused by the 5’ss mutation (c.3335+6T>G) found in a ML III patient. First, we performed transfection of exon-trapping minigenes expressing exon 17 surrounded by a portion of introns - pGNPTAB_WT and pGNPTAB_+6, in HEK293T cells to analyze if they reproduce the WT and mutant splicing patterns. Then, to evaluate the potential of 2 modified U1’s, 3 ExSpeU1s and 2 modified U6’s to restore mRNA splicing, these vectors were cotransfected into HEK293T cells along with the mutant +6 minigene as well as electroporated in patient’s fibroblasts. Then, cells were harvested, and RT-PCR analysis was performed. Both minigenes reproduced the control or ML III patient cDNA’s splicing patterns, thus, different concentrations of the modified U1’s and ExSpeU1s were tested together with the mutant minigene. The cDNA analysis showed almost 100% of exon 17 inclusion when one of the ExSpeU1s, was overexpressed in HEK293T cells. The combination of the 2 modified U6’s with the modified U1’s or the ExSpeU1s allowed exon 17 inclusion at some extent, but not as effectively as with the best ExSpeU1 alone. The electroporation of the 2 modified U1’s and of the 3 ExSpeU1s was done, and the cDNA analysis of patient’s fibroblasts treated with 2 ExSpeU1s (ExSpeU1 int17-1 or int17-2) showed around 35% and 15% of exon 17-including transcripts, respectively. To confirm these results, given that the lentiviral transduction is a more efficient delivery technique than electroporation, the gene cassettes of the 2 most promising ExSpeU1s were cloned in a lentivirus vector and after obtaining the viral mediums, their transduction in patient’s fibroblasts is being optimized. The cDNA analysis of preliminary experiments is still ongoing. In conclusion, we have developed an RNA therapy based on engineered U1 snRNAs for a ML III 5’ss mutation. We showed that an ExSpeU1 (binding downstream of the mutated 5´ss) can restore proper exon 17 definition in vitro, opening the opportunity for a personalized therapeutic intervention.
- An engineered U1 snRNA-based therapeutic approach can efficiently rescue a 5’ splice site mutation causing Mucolipidosis type IIIPublication . Peretto, L.; Gonçalves, M.; Santos, J.I.; Duarte, A.J.; Moreira, L.; Encarnação, M.; Coutinho, M.F.; Pinotti, M.; Balestra, D.; Alves, S.; Matos, L.A significant number of splicing mutations have been identified in Lysosomal Storage Disorders (LSDs). Mucolipidosis III (ML III) is a LSD caused by GlcNAc-1-phosphotransferase deficiency, which impairs the trafficking of lysosomal hydrolases. 10% of the genetic defects in ML III are splicing mutations, and around 45% affect 5' splice-sites (ss) thus constituting a good target for mutation specific therapies. The use of engineered U1 snRNA (either modified U1 snRNAs or exon-specific U1s - ExSpeU1s) has been applied as a potential therapeutic strategy to correct 5’ss defects. Here we used engineered U1 snRNAs to correct the GNPTAB exon 17 skipping caused by the 5’ss mutation (c.3335+6T>G) found in a ML III patient.
- Exposure to mycotoxins in the Portuguese adult populationPublication . Maris, Elias; Namorado, Sónia; Chen, A.; Pero-Gason, Roger; De Boevre, Marthe; De Saeger, Sarah; Silva, Maria João; Alvito, PaulaMycotoxins are toxic fungal metabolites commonly found in food, posing health risks such as immunosuppression, carcinogenicity, and endocrine disruption. Despite regulatory limits, chronic low-level exposure remains a concern. Understanding real-life exposure in populations is essential for effective risk assessment. This study aims to investigate mycotoxin exposure among young adults in Portugal, contributing to evidence-based public health interventions. This study leveraged data and biospecimens from the INSEF-ExpoQuim survey, a cross-sectional study nested within thPortuguese National Health Examination Survey (INSEF). Data was collected via REDCap-assisted telephone interviews, covering sociodemographic and exposure-relevant variables. A subset of 295 first morning urine samples was collected from adults aged 28–39 years between May 2019 and March 2020. Urine samples were analyzed by a newly optimized and validated LC-MS/MS method targeting 40 mycotoxins and/or their corresponding metabolites in urine. Urinary creatinine was measured using a validated colorimetric method to allow adjustment and standardization of mycotoxin concentrations, ensuring accurate exposure assessment and comparability. This methodological approach enabled a robust characterization of mycotoxin exposure in a representative Portuguese population cohort.The study included 58% females and 42% males. Most participants had medium to high education, and urbanization was nearly evenly split between towns/suburbs (36.9%) and rural areas (35.9%), with fewer living in cities (27.1%). The majority were employed, and sampling was primarily conducted in summer and autumn. The number of mycotoxin co-exposures in the Portuguese population ranged from 0 to 5, with two simultaneous exposures being most common (n = 160). Among the 40 mycotoxins analysed, deoxynivalenol and tenuazonic acid were most frequently detected, with frequency of detection of 85% and 96%, respectively. This study offers robust biomonitoring data on mycotoxin exposure in Portuguese young adults using a validated LC-MS/MS method. The high prevalence of deoxynivalenol and tenuazonic acid suggests low level dietary contamination. These findings support the need for continued monitoring and the integration ofhuman biomonitoring into national food safety strategies. Detailed sociodemographic analyses are planned to further clarify exposure patterns and enable targeted public health interventions.
- Hazard characterization of the mycotoxins enniatins and beauvericin to identify data gaps and improve risk assessment for human healthPublication . Behr, Anne-Cathrin; Fæste, Christiane Kruse; Azqueta, Amaya; Tavares, Ana M.; Spyropoulou, Anastasia; Solhaug, Anita; Olsen, Ann-Karin; Vettorazzi, Ariane; Mertens, Birgit; Zegura, Bojana; Streel, Camille; Ndiaye, Dieynaba; Spilioti, Eliana; Dubreil, Estelle; Buratti, Franca Maria; Crudo, Francesco; Eriksen, Gunnar Sundstøl; Snapkow, Igor; Teixeira, João Paulo; Rasinger, Josef D.; Sanders, Julie; Machera, Kyriaki; Ivanova, Lada; Gaté, Laurent; Le Hegarat, Ludovic; Novak, Matjaz; Smith, Nicola M.; Tait, Sabrina; Fraga, Sónia; Hager, Sonja; Marko, Doris; Braeuning, Albert; Louro, Henriqueta; Silva, Maria João; Dirven, Hubert; Dietrich, JessicaEnniatins (ENNs) and beauvericin (BEA) are cyclic hexadepsipeptide fungal metabolites which have demonstrated antibiotic, antimycotic, and insecticidal activities. The substantial toxic potentials of these mycotoxins are associated with their ionophoric molecular properties and relatively high lipophilicities. ENNs occur extensively in grain and grain-derived products and are considered a food safety issue by the European Food Safety Authority (EFSA). The tolerable daily intake and maximum levels for ENNs in humans and animals remain unestablished due to key toxicological and toxicokinetic data gaps, preventing full risk assessment. Aiming to find critical data gaps impeding hazard characterization and risk evaluation, this review presents a comprehensive summary of the existing information from in vitro and in vivo studies on toxicokinetic characteristics and cytotoxic, genotoxic, immunotoxic, endocrine, reproductive and developmental effects of the most prevalent ENN analogues (ENN A, A1, B, B1) and BEA. The missing information identified showed that additional studies on ENNs and BEA have to be performed before sufficient data for an in-depth hazard characterisation of these mycotoxins become available.
- HBM4EU chromates study: the Portuguese integrated and harmonized study on exposure to hexavalent chromium and related early effects.Publication . Viegas, Susana; Martins, Carla; Ribeiro, Edna; Ladeira, Carina; Pinhal, Hermínia; Nogueira, Ana; Santos, Sílvia; Tavares, Ana; Gomes, Bruno Costa; Afonso, Catarina Maia; Louro, Henriqueta; Silva, Maria JoaoIn the scope of the European Union (EU) human biomonitoring initiative, a multicentric study on different occupational settings from several European countries was performed, to provide information on occupational exposure to hexavalent chromium [Cr(VI)], a known lung carcinogen. Biomonitoring approaches were used to obtain exposure data to support the implementation of new risk management measures and policy actions at the national and European levels. This work describes the Portuguese contribution to the study, which aimed to assess workers' exposure to Cr, by using exposure biomarkers (urinary chromium [U-Cr]), and industrial hygiene samples (air and hand wipes) and to link exposure to potential long-term health effects by using effect biomarkers. Exposure determinants influencing exposure were explored from the contextual information and human biomonitoring data. The ultimate goal of the study was to appraise the risk management measures contributing to minimize exposure and protect workers' health. Several occupational settings and activities were considered, including plating, welding, and painting. A control group from the Portuguese general population was also included. Data on age, sex, and smoking habits from both groups were considered in the statistical analysis. Information on the risk management measures available for workers was collected and used to identify the ones that mainly contributed to reduce exposure. Environmental monitoring and human biomonitoring revealed that painters were the highest exposed group. The use of respiratory protection equipment showed an influence on total U-Cr levels for workers involved in painting activities. Concerning early health effects, the painters presented also a significantly higher level of DNA and chromosomal damage in peripheral blood cells, as compared to the control group, suggesting a plausible association between exposure to Cr(VI) and early genotoxic effects. The results showed that workers are exposed to Cr(VI) in those occupational settings. These findings point to the need to improve the prevention and risk management measures and the implementation and enforcement of new regulatory actions at the national level.
- Is Antisense Oligonucleotide-Mediated Exon Skipping a Potential Therapeutic Approach for Mucolipidosis II?Publication . Gonçalves, Mariana; Moreira, Luciana; Encarnação, Marisa; Duarte, Ana Joana; Gaspar, Paulo; Santos, Juliana Inês; Coutinho Maria Francisca; Prata, Maria João; Omidi, Maryam; Pohl, Sandra; Silva, Frederico; Oliveira, Paula; Matos, Liliana; Alves, SandraIntridution: Mucolipidosis II (ML II) is a Lysosomal Storage Disorder caused by N-acetylglucosamine-1-phosphotransferase (GlcNAc-PT) deficiency, which impairs lysosomal hydrolases trafficking. Here, we explored an innovative therapeutic strategy based on the use of antisense oligonucleotides (ASOs) to promote targeted skipping of GNPTAB exon 19, which harbors c.3503_3504del, the most frequent disease-causing variant. Previously, in ML II patients’ fibroblasts, we tested ASOs to induce exon 19 skipping, successfully generating an in-frame mRNA1. Now, our aim is to determine if this in-frame transcript leads to increased GlcNAc-PT levels. Methodology: First, the GlcNAc-PT activity was measured in fibroblasts, but activity levels were similar in ML II and control fibroblasts (treated/non-treated) showing that the assay is not proper to measure endogenous levels. To overcome this, we designed 3 constructs: a WT (full GNPTAB cDNA), a del_ex19 (without exon 19) and a mutant (with the mutation c.3503_3504del) that were transfected in HEK293T cells. Then GlcNAc-PT expression was analyzed by Western Blot (WB). Also, we measured the activity of several hydrolases and evaluated the expression of α-galactosidase A (α-Gal) by WB after ASO treatment. To further validate this therapy we also generated a novel GlcNAc-PT antibody in rabbits. Results: Our results showed that HEK293T cells were able to express all the constructs. The WB of both WT and del_ex19 constructs showed bands corresponding to the α/β precursor. However, only the WT construct expressed the β-subunit, suggesting that there is no GlcNAc-PT activity in the absence of exon 19. As expected, in the delTC construct WB no α/β precursor band was detected. We also observed a slight increase in the activity of various lysosomal hydrolases in ML II fibroblasts after treatment. However, only the α-Gal values were statistically significant, but the WB analysis for this enzyme did not reveal any band in ASO-treated ML II cells. We also developed a novel antibody for GlcNAc-PT. Preliminary results showed a β-subunit band both in control and patient fibroblasts (unexpected), but in overexpression both WT and del_ex19 constructs presented α/β precursor bands. So, further assays are needed to assess its specificity. Conclusion: Our ASO-based approach effectively promotes exon 19 skipping. However, this strategy, as far as we have been able to prove, is not able to restore any GlcNAc-PT enzymatic activity. Further validation, including co-localization studies are planned to clarify these findings.