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- Is exon skipping the key to correct N-acetylglucosamine-1-phosphotransferase deficiency? An antisense oligonucleotide therapeutic approachPublication . Gonçalves, Mariana; Moreira, Luciana; Encarnação, Marisa; Gaspar, Paulo; Santos, Juliana Inês; Coutinho, Maria Francisca; Prata, Maria João; Omidi, Maryam; Pohl, Sandra; Silva, Frederico; Oliveira, Paula; Matos, Liliana; Alves, SandraIntroduction: 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. Aims: Now, our aim is to determine whether this in-frame transcript leads to increased GlcNAc-PT levels improving ML II cellular phenotype. Methodology: First, the GlcNAc-PT activity was measured in fibroblasts by a radioactive assay, but activity levels were similar in ML II and control fibroblasts (treated and 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 the exon 19) and a mutant (with the mutation c.3503_3504delTC) that were transfected in HEK293T cells. Then GlcNAc-PT expression was analyzed by Western Blot (WB). Additionally, we have measured the activity of several lysosomal hydrolases and evaluated the expression of α-galactosidase A (α-Gal) by WB after ASO treatment of control and patient cells. To further help in the validation of this therapy we are also generating a novel GlcNAc-PT antibody in rabbits. Results: Our results demonstrated 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, the c.3503_3504delTC construct showed no expression, with no detectable α/β precursor band. We also observed a slight increase in the activity of various lysosomal hydrolases in ML II fibroblasts treated with the ASO, particularly 24h and 48h post-treatment. However, only the values relatively to the α-Gal were statistically significant, but the WB analysis using an antibody against this enzyme did not detect any band in ASO-treated ML II fibroblasts. We also developed a novel antibody for GlcNAc-PT. Preliminary results revealed a band corresponding to the β-subunit in both control and ML II patient fibroblasts (unexpected), but in overexpression assays both WT and del_ex19 constructs presented α/β precursor bands. So, further assays are needed to assess their specificity. Conclusion: Our ASO-based approach effectively promotes the skipping of exon 19. 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.
- A Multiplex Biomarker panel: A powerful tool for LSDs DiagnosisPublication . Neiva, Raquel; da Silva Gaspar, Paulo Jorge Miranda; Sousa e Silva, Lisbeth Elena; Gonçalves, Isabel; Ferreira, Sara; Diogo, Luisa; Vilarinho, LauraIntrodução / Descrição do Caso: Lysosomal Storage Disorders (DLSs) are a set of rare, chronic and multisystemic pathologies with a variable mode of presentation and severity. Acid sphingomyelinase deficiency (ASMD), historically known as Niemann–Pick disease (NPD) types A, A/B, and B, is a rare, progressive, potentially fatal lysosomal storage disease caused by pathogenic variants in SMPD1 gene. The disease manifestations frequently involve hepatosplenomegaly with progressive organ dysfunction, interstitial lung disease, and bleeding. The cellular damage caused by sphingomyelin accumulation can be irreversible and can lead to life-threatening complications with reduced life expectancy. ASMD can be underestimate and the diagnostic odyssey arise from an overlap in symptomology with other diseases, including primary hepatic disease, Gaucher disease, NPC, and lysosomal acid lipase deficiency.
- Early Diagnosis of Mucopolysaccharidoses in PediatricsPublication . da Silva Gaspar, Paulo Jorge Miranda; Neiva, Raquel; Sousa e Silva, Lisbeth Elena; Diogo, Luisa; Ferreira, A.; Miranda, A.; Ribeiro, S.; Antunes, D.; Garcia, P.; Rodrigues, E.; Campos, T.; Janeiro, P.; Lopes, Altina; Pereira, Cristina; Nogueira, Célia; Sousa, S.; Ferreira, S.; Alves, Sandra; Leão Teles, Elisa; Vilarinho, LauraIntroduction: Mucopolysaccharidoses (MPSs) are a group of Lysosomal Storage Disorders with multisystem involvement, presenting different degrees of severity and evolution. At early disease stages and late onset forms, diagnosis can be postponed for years or even missed. The FIND PROJECT was designed to claim awareness to the redflags of MPSs at pediatric age and to provide a useful tool for physicians to diagnose these pathologies, since most of them are amenable to enzyme replacement therapy.
- Differential Diagnosis of Alpha-Mannosidosis in MPSsPublication . da Silva Gaspar, Paulo Jorge Miranda; Gonçalves, Diana; Neiva, Raquel; Sousa e Silva, Lisbeth Elena; Vilarinho, LauraIntroduction: Mucopolysaccharidosis (MPSs) and oligosaccharidosis, two subgroups of lysosomal storage disorders (LSDs), face diagnostic challenges due to their wide spectrum of clinical presentations and overlapping symptoms. One of the oligosaccharidose is α-mannosidosis, an extremely rare and often undiagnosed disorder. It is characterized by a deficiency in the enzymatic activity of α-mannosidase, which is responsible for cleaving mannose from N-linked oligosaccharides. This study aimed to investigate the activity of α-mannosidase in dried blood spot (DBS) samples that had undergone screening for MPSs.
- Deleção intersticial 7q33q34 em fetos de gravidez gemelar monocoriónica diamnióticaPublication . Simão, Laurentino; Marques, Bárbara; Ferreira, Cristina; Serafim, Sílvia; Alves, Cristina; Silva, Marisa; Viegas, Mónica; Peliano, Ricardo; Brito, Filomena; Bernardeco, Joana; Cruz, Jader; Pedro, Sónia; Martins, Ana; Tarelho, Ana; Carvalho, Inês; Cohen, Álvaro; Correia, HildebertoIntrodução: O acompanhamento de gestações gemelares pode revelar-se desafiante se houver alterações ecográficas e discrepâncias entre os fetos. Deleções intersticiais 7q, abrangendo diferentes regiões e apresentando tamanho variável, são raras, e encontram-se quase exclusivamente descritas em pós-natal. Objectivos: Apresentamos o caso de uma gestante, de 32 anos, com gravidez gemelar monocoriónica e diamniótica de 16 semanas, referenciada por bolsas jugulares bilaterais, crescimento no P10-P20 e discrepância no pico sistólico de velocidade da artéria cerebral média (PSV-ACM). Foi efetuada colheita de liquido amniótico para estudo por microarray cromossómico (CMA). Metodologia: Foi efeituado diagnóstico rápido de aneuploidias (DRA) por QF-PCR (Devyser®), ao que se seguiu CMA com array CytoScan 750K (Thermo Fischer®) e cariótipo. Resultados: O DRA revelou um resultado normal. O CMA permitiu a identificação de uma deleção intersticial com 9,0 Mb em 7q33q34 - arr[GRCh37] 7q33q34(133411316_142427027)x1. A alteração engloba 12 genes mórbidos. O cariotipo confirmou o resultado: 46,XX,del(7)(q32.3q34)dn. Após aconselhamento genético, o casal optou por interrupção da gestação. Conclusões: Deleções intersticiais na região 7q32 a 7q35 apresentam grande variabilidade fenotípica. As características mais comuns são: atraso de desenvolvimento e da linguagem, défice intelectual, dismorfias faciais e atraso de crescimento. Nos raros casos descritos com alterações cromossómicas parcialmente sobreponíveis ao caso em estudo, a CNV tem sido classificada como patogénica. No único caso com referência ao período pré-natal e com alteração quase totalmente sobreponível, descreve-se decréscimo de movimentos fetais, baixo peso à nascença, atraso de desenvolvimento, défice intelectual, dismorfias faciais e infeções múltiplas. A alteração cromossómica encontrada poderá explicar a relativa restrição de crescimento fetal, não tendo as bolsas jugulares, presentes em ambos os fetos e a discrepância PSV-ACM sido até agora descritos. Gestações com alterações ecográficas e CNVs, mas com reduzida bibliografia, são desafiantes na interpretação dos resultados a nível laboratorial e clínico. Só a descrição de mais casos permitirá um ganho de conhecimento em saúde.
- Detection of copy number variants in the human genome: Is long-read sequencing an alternative to genomic microarrays?Publication . Silva, Catarina; Ferrão, José; Marques, Barbara; Pedro, Sónia; Correia, Hildeberto; Rodrigues, António Sebastião; Vieira, LuísIntroduction: Copy number variations (CNVs) represent ~13% of the human genome and can harbour important genes and regulatory elements. High-resolution whole genome microarray (MA) analysis is the gold standard tool for detection of CNVs associated with genetic disorders. While short-read sequencing (SRS) can address SV detection, the use of long-read sequencing as proven to overcome SRS mapping inaccuracy in highly repetitive DNA regions and improve genome contiguity. We applied whole genome nanopore sequencing (NS) to call CNVs and compared the results with those obtained by microarray. Methodology: Genomic DNA from 2 cell lines (EOL-1 and 697) were processed using the CytoSan HD Array (Affymetrix) and ChAS software (ThermoFisher). A minimum CNV calling size threshold of 35 Kb was used. DNA was also sequenced on the MinION device (Oxford Nanopore Technologies) following a rapid library preparation method. Sequencing data were basecalled using Guppy, mapped with LRA, and SVs called using both CuteSV and Sniffles2. Sanger sequencing was performed to demonstrate breakpoint positions for 3 CNVs. R packages were used to perform comparisons between MA and NS data. Results: A total of 49 CNVs were confirmed after curated MA analysis in both cell lines, ranging in size from 35 Kb to 79 Mb. From those, 43 CNVs (87.7%) were called in nanopore data by either one (4 CNVs) or both (39 CNVs) callers with a mean whole genome coverage of ~12X. Six of 43 CNVs were called as inversions instead. In 3 CNVs the size of the variant was found to be smaller (ranging from ~5 to 22 Kb) than the threshold of MA analysis. The correlation between CNV sizes obtained with MA and NS was of 0.71 with Sniffles2 and 0.74 with CuteSV, whereas the correlation between callers was of 0.99. The breakpoint precision obtained for NS was much higher (ranging for CuteSV from 2 to 42 bp; and for Sniffles2 from 0 to 87 bp) than the one obtained for MA (ranging from 774 to 7618 bp). Conclusions: NS technology proved to be technically effective in the detection of CNVs of different types and sizes and thus posing itself as an alternative to MA in the detection of pathogenic SVs associated with genetic diseases. However, NS data analysis requires fine-tuning of the analysis conditions as well as the use of different methods, for greater reliability of results in a clinical context.
- The disease modelling value of a folklore FAIRYtale: SHEDing light over a special group of genetic disordersPublication . Carvalho, S.; Santos, J.I.; Moreira, L.; Gaspar, P.; Gonçalves, M.; Encarnação, M.; Ribeiro, D.; Duarte, A.; Prata, M.J.; Coutinho, M.F.; Alves, SandraThe problem we are addressing: Despite extensive research, the links between accumulation of glycosaminoglycans (GAGs) and the clinical features seen in patients suffering from various forms of Mucopolysaccharidoses (MPSs) have yet to be further elucidated. These Lysosomal Storage Diseases (LSDs) present symptoms, which may (or may not) include critical musculoskeletal and cardiovascular alterations, respiratory problems, and serious neurological dysfunctions. The skeletal and brain systems are the hardest ones to access and, consequently, those in greatest need of additional knowledge and novel therapeutic solutions.
- Occupational studies developed in Portugal in the scope of HBM4EU – Main results and way forwardPublication . Martins, Carla; Ribeiro, Edna; Ladeira, Carina; Pinhal, Herminia; Nogueira, Ana; Reis, Silvia; Louro, Henriqueta; Silva, Maria João; Viegas, SusanaIntroduction: Within the EU human biomonitoring initiative (HBM4EU), targeted national studies on occupational settings, where exposure to chemicals could occur, were performed. These studies aimed to provide information on occupational exposure to chemicals using biomonitoring approaches to support the implementation of new risk management measures (RMM) and policy actions at national and European levels. Methods: Cross-sectional studies were performed in two occupational settings: i) a setting with plating, welding and painting activities, targeting exposure to chromium VI [Cr(VI)]; and ii) a setting dedicated to processing of electronic waste (e-waste), targeting exposure to metals. Both studies included a control group obtained from the Portuguese general population, employed in different companies with no known exposure to Cr(VI) or involvement on tasks related with recycling of e-waste. Studies enrolled 50 workers and 27 controls (chromates study) and 26 workers and 12 controls (e-waste study). Spot urine samples were collected in the beginning and the end of the work week, and concentrations of metals were determined. Protocols were submitted to Ethics Committees with the approvals being granted before recruiting the studies’ participants (companies and workers). Descriptive statistical analysis was performed as well as inferential analysis (Wilcoxon test, Mann–Whitney test, and Kruskal–Wallis test). Results: Regarding chromates study, median levels obtained for workers were 0.33 µg/g crea for Cr, in the post-shift samples. Results showed that painters presented higher levels of Cr in post-shift urine samples than the control group (p<0.05), although not presenting differences between pre-shift and post-shift urine samples (p>0.05). Platers showed higher levels of Cr in post-shift urine samples when compared to pre-shift samples (p<0.05). Welders did not present statistically significant differences between pre- and post-shift samples and in relation to control group (p>0.05). Regarding RMM, the use of respiratory protection equipment by painters was identified as a promotor of lower exposures (p<0.05). Regarding e-waste study, median levels obtained for workers were 0.21, 0.43 and 12.70 µg/g crea for cadmium (Cd), mercury (Hg) and lead (Pb), respectively, in the post-shift samples. Statistically significant differences between workers and control group were found for urinary Pb levels (p<0.05), and no significant differences were found between pre- and post-shift urine samples for Cd, Hg and Pb. Conclusions: The results obtained showed that workers are exposed to metals in these occupational settings, thus, RMM in place need to be improved. Based on the results obtained, the implementation and enforcement of new regulatory actions should be considered. The results also claim attention for the need of updating the occupational limit values as it is being discussed at European level for Cr(VI), Pb and Cd due to the related health effects [3]. The differentiated data provided by biomonitoring tools demonstrated that these should be used more often to perform exposure assessment.
- Hepatitis C Virus infection, iron metabolism, liver fibrosis and response to Direct Acting Antivirals (DAAs)Publication . Ferreira, Joana; Faustino, Paula; Bicho, Manuel; Serejo, FátimaIntroduction: Chronic Hepatitis C (CHC) is characterized by hepatic and extra-hepatic manifestation. Among hepatic manifestations, liver fibrosis is probably the most significant as it can lead to cirrhosis, hepatocellular carcinoma and death. Iron overload is one of the extra-hepatic manifestations induced by Hepatitis C Virus (HCV) infection and can induce fibrosis-promoting signals in the parenchymal and non-parenchymal cells, which accelerate disease progression and exacerbate liver pathology. Aims: Evaluate the changes in iron metabolism induced by HCV elimination, the association of liver fibrosis with biochemical and genetic parameters of iron metabolism before and after DAAs treatment and baseline parameters that could predict the improvement of liver fibrosis after treatment. Materials and Methods: 329 patients with CHC (age: 49.93 [45.57-50.28] years; 124 female and 205 male). 134 treated with DAAs with sustained response (age: 53.42 [51.47-55.36]; 58 female and 56 male). Liver fibrosis stage was assessed by transient elastography using a Fibroscan and patients were divided in two groups (F1/2_mild and moderate fibrosis and F3/4_severe fibrosis and cirrhosis), biochemical parameters of iron metabolism (Total Iron_Fe, Total Iron Binding Capacity_TIBC, Transferrin Saturation_TS, Ferritin_FT, Haptoglobin_Hp) were evaluated by standard methods and genetic polymorphisms within Transferrin_TF HFE, Bone Morphogenetic Protein 2_BMP2, Hepcidin_HAMP and Ferroportin_SLC40A1 genes were analyzed by PCR, sequencing or NGS. Statistical analysis was performed using SPSS 26.0 for windows. Results: Patients showed lower values of Fe, TS and FT and higher values of Hp after treatment.
- Exosomal microRNAs as possible biomarkers for a rare disease affecting lipidsPublication . Encarnação, Marisa; David, Hugo; Ribeiro, Isaura; Vieira, Luís; Carneiro Silva, Catarina; Martins, Esmeralda; Cardoso, Maria Teresa; Futerman, Anthony H.; Quelhas, Dulce; Alves, SandraExosomes mediate the communication between cells and the characterization of their content can provide important insights into health and disease. Their cargo includes proteins, lipids and nucleic acids (including microRNAs (miRNAs)). miRNAs regulate many cellular processes, including metabolism. (...)