Percorrer por autor "Cardoso, Maria Teresa"
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- Assessing Lysosomal Disorders in the NGS Era: Identification of Novel Rare VariantsPublication . Encarnação, Marisa; Coutinho, Maria Francisca; Silva, Lisbeth; Ribeiro, Diogo; Ouesleti, Souad; Campos, Teresa; Santos, Helena; Martins, Esmeralda; Cardoso, Maria Teresa; Vilarinho, Laura; Alves, SandraLysosomal storage diseases (LSDs) are a heterogeneous group of genetic disorders with variable degrees of severity and a broad phenotypic spectrum, which may overlap with a number of other conditions. While individually rare, as a group LSDs affect a significant number of patients, placing an important burden on affected individuals and their families but also on national health care systems worldwide. Here, we present our results on the use of an in-house customized next-generation sequencing (NGS) panel of genes related to lysosome function as a first-line molecular test for the diagnosis of LSDs. Ultimately, our goal is to provide a fast and effective tool to screen for virtually all LSDs in a single run, thus contributing to decrease the diagnostic odyssey, accelerating the time to diagnosis. Our study enrolled a group of 23 patients with variable degrees of clinical and/or biochemical suspicion of LSD. Briefly, NGS analysis data workflow, followed by segregation analysis allowed the characterization of approximately 41% of the analyzed patients and the identification of 10 different pathogenic variants, underlying nine LSDs. Importantly, four of those variants were novel, and, when applicable, their effect over protein structure was evaluated through in silico analysis. One of the novel pathogenic variants was identified in the GM2A gene, which is associated with an ultra-rare (or misdiagnosed) LSD, the AB variant of GM2 Gangliosidosis. Overall, this case series highlights not only the major advantages of NGS-based diagnostic approaches but also, to some extent, its limitations ultimately promoting a reflection on the role of targeted panels as a primary tool for the prompt characterization of LSD patients.
- Assessing Niemann-Pick Type C (NP-C) through a multi-omics approach genomic and transcriptomic profile of challenging casesPublication . Encarnação, Marisa; Coutinho, Maria Francisca; Cho, Soo-Min; Cardoso, Maria Teresa; Chaves, Paulo; Ribeiro, Isaura; Santos, Juliana Inês; Gaspar, Paulo; Quelhas, Dulce; Lacerda, Lúcia; Leão-Teles, Elisa; Futerman, Anthony H.; Vilarinho, Laura; Alves, SandraNiemann-Pick type C (NP-C) is a neurodegenerative Inherited Metabolic Disease with a heterogeneous clinical presentation, due to mutations in either the NPC1 or NPC2 genes. We studied patients with clinical diagnosis of NP-C but presenting inconclusive results regarding biomarkers testing and molecular analysis. Using NGS- targeted DNA sequencing we have identified some novel putative mutations and subsequent cDNA analysis allowed us to stablish the functional effect of a silent mutation (previously reported as a polymorphism) in the NPC1 splicing process. We demonstrated that this mutation leads to exon skipping, frameshift and premature stop codon and identified it in two NP-C patients from two unrelated Portuguese families. This mutation most likelly leads to a very unstable transcript that was overlooked. Also, to better characterize the pathomechanisms related to specific disease-causing mutations in NP-C patients, we analysed gene expression profiles in cultured skin fibroblasts and compared them to control individuals using Massively Parallel RNA Single-Cell Sequencing (MARS-Seq). The most prominent hits from this transcriptomics analysis were validated by qRT-PCR. The MARS-Seq analysis showed that a number of genes were upregulated and a significant number of the highly enriched genes are related to the unfold protein response (UPR) and Endoplasmic Reticulum (ER) stress, in a specific patient, which deserves further studies. ER stress is a hallmark of many neurodegenerative diseases, including LSD and can be due to misfolded/unfolded proteins as result of a specific missense mutation. Our preliminary results suggest that UPR activation is variable among NP-C patients, and this is likely to depend on the mutation type. Several other factors may contribute to this though, which could explain the heterogeneous presentation of this pathology. Additionally, we have investigated the same patients studied in MARS-Seq at the protein and celular levels. Interestingly, and according to recently published work, we observed that, for the analyzed mutations a significant part of the mutated NPC1 protein was retained/ delayed in the ER.
- 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. (...)
- Genomics and transcriptomics approach - diagnosis of a challenging case of Niemann-Pick type C (NP-C)Publication . Encarnação, Marisa; Coutinho, Maria Francisca; Cho, Soo-Min; Cardoso, Maria Teresa; Chaves, Paulo; Gaspar, Paulo; Santos, Juliana Inês; Ribeiro, Isaura; Quelhas, Dulce; Lacerda, Lúcia; Leão-Teles, Elisa; Futerman, Anthony H.; Vilarinho, Laura; Alves, SandraNP-C is a neurodegenerative Inherited Metabolic Disease with a heterogeneous clinical presentation, and due to mutations in either the NPC1 or NPC2 genes.We have studied a patient with clinical diagnosis of NP-C but presenting inconclusive results regarding molecular analysis.To better characterize this patient, we have performed NGS-based technologies (targeted DNA sequencing and single cell-RNA sequencing).For the molecular diagnosis we used a NGS gene panel followed by the analysis of cDNA.Latter, we used massively parallel single cell RNA-seq (MARS-Seq) to address gene profiling changes and characterize the pathomechanisms related to specific disease-causing mutations. Using our targeted NGS panel we identified two novel mutations in NPC1 gene.Next, through cDNA analysis of one of the patient parents we were able to understand the impact of the silent mutation.This mutation leads to exon skipping giving origin to an out-of-frame transcript and eliciting the nonsense-mediated decay pathway.Thus, we were not able to easily detect the mutant transcript which turned the molecular diagnosis more challenging. Apparently the presence of the other mutation (a missense mutation) impairs the NPC protein folding leading to its ER retention.The MARS-Seq analysis of this patient showed that a number of upregulated genes are related to the unfold protein response (UPR) and ER stress, which deserves further studies.
- Investigating p.Ala1035Val in NPC1: New Cellular Models for Niemann–Pick Type C DiseasePublication . David, Hugo; Monfregola, Jlenia; Ribeiro, Isaura; Cardoso, Maria Teresa; Sandiares, Ana Catarina; Moreira, Luciana; Coutinho, Maria Francisca; Quelhas, Dulce; Ballabio, Andrea; Alves, Sandra; Encarnação, MarisaNiemann-Pick type C (NPC) is a lysosomal storage disorder (LSD) caused by pathogenic variants in either the NPC1 or NPC2 genes, which encode proteins involved in the lysosomal export of unesterified cholesterol. In patients of Western European descent, the p.Ile1061Thr variant in NPC1 is especially prevalent. However, mounting evidence has positioned p.Ala1035Val as the most common variant in Portugal and the second most prevalent variant worldwide. By analyzing 10 Portuguese NPC patients homozygous for p.Ala1035Val, we found an SNP in cis on position 858 (p.Ile858Val), which we hypothesize could have a disease-modifying effect. To address this query, we created variant-specific in vitro models of NPC by stably transducing NPC1-/- ARPE-19 cells with constructs encoding different fluorescently-tagged variants of NPC1, which we used, alongside patient-derived skin fibroblasts, to investigate lysosomal positioning and the trafficking routes elicited by p.Ile1061Thr and p.Ala1035Val (with and without the p.Ile858Val SNP in cis). Our results corroborate the previously described decrease in p.Ile1061Thr-NPC1 trafficking to the lysosome and suggest a similar, if not worse, scenario for the p.Ala1035Val variant, especially when in cis with p.Ile858Val. This is the first reported functional study addressing the impact of the p.Ala1035Val variant at the cellular level, paving the way for novel therapeutic options.
- Leukocyte Imbalances in Mucopolysaccharidoses PatientsPublication . Lopes, Nuno; Maia, Maria L.; Pereira, Cátia S.; Mondragão-Rodrigues, Inês; Martins, Esmeralda; Ribeiro, Rosa; Gaspar, Ana; Aguiar, Patrício; Garcia, Paula; Cardoso, Maria Teresa; Rodrigues, Esmeralda; Leão-Teles, Elisa; Giugliani, Roberto; Coutinho, Maria F.; Alves, Sandra; Macedo, M. FátimaMucopolysaccharidoses (MPSs) are rare inherited lysosomal storage diseases (LSDs) caused by deficient activity in one of the enzymes responsible for glycosaminoglycans lysosomal degradation. MPS II is caused by pathogenic mutations in the IDS gene, leading to deficient activity of the enzyme iduronate-2-sulfatase, which causes dermatan and heparan sulfate storage in the lysosomes. In MPS VI, there is dermatan sulfate lysosomal accumulation due to pathogenic mutations in the ARSB gene, leading to arylsulfatase B deficiency. Alterations in the immune system of MPS mouse models have already been described, but data concerning MPSs patients is still scarce. Herein, we study different leukocyte populations in MPS II and VI disease patients. MPS VI, but not MPS II patients, have a decrease percentage of natural killer (NK) cells and monocytes when compared with controls. No alterations were identified in the percentage of T, invariant NKT, and B cells in both groups of MPS disease patients. However, we discovered alterations in the naïve versus memory status of both helper and cytotoxic T cells in MPS VI disease patients compared to control group. Indeed, MPS VI disease patients have a higher frequency of naïve T cells and, consequently, lower memory T cell frequency than control subjects. Altogether, these results reveal MPS VI disease-specific alterations in some leukocyte populations, suggesting that the type of substrate accumulated and/or enzyme deficiency in the lysosome may have a particular effect on the normal cellular composition of the immune system.
- NPC1 silent variant induces skipping of exon 11 (p.V562V) and unfolded protein response was found in a specific Niemann-Pick type C patientPublication . Encarnação, Marisa; Coutinho, Maria Francisca; Cho, Soo Min; Cardoso, Maria Teresa; Ribeiro, Isaura; Chaves, Paulo; Santos, Juliana Inês; Quelhas, Dulce; Lacerda, Lúcia; Leão Teles, Elisa; Futerman, Anthony H.; Vilarinho, Laura; Alves, SandraBackground: Niemann-Pick type C (NPC, MIM #257220) is a neuro-visceral disease, caused predominantly by pathogenic variants in the NPC1 gene. Here we studied patients with clinical diagnosis of NPC but inconclusive results regarding the molecular analysis. Methods: We used a Next-Generation Sequencing (NGS)-panel followed by cDNA analysis. Latter, we used massively parallel single-cell RNA-seq (MARS-Seq) to address gene profiling changes and finally the effect of different variants on the protein and cellular levels. Results: We identified novel variants and cDNA analysis allowed us to establish the functional effect of a silent variant, previously reported as a polymorphism. We demonstrated that this variant induces the skipping of exon 11 leading to a premature stop codon and identified it in NPC patients from two unrelated families. MARS-Seq analysis showed that a number of upregulated genes were related to the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress in one specific patient. Also, for all analyzed variants, the NPC1 protein was partially retained in the ER. Conclusion: We showed that the NPC1 silent polymorphism (p.V562V) is a disease-causing variant in NPC and that the UPR is upregulated in an NPC patient.
- Transcriptomics profiling of Niemann-Pick type C patients: activation of the unfold protein response in a specific casePublication . Encarnação, Marisa; Coutinho, Maria Francisca; Cho, Soo-Min; Cardoso, Maria Teresa; Chaves, Paulo; Gaspar, Paulo; Santos, Juliana Inês; Ribeiro, Isaura; Quelhas, Dulce; Lacerda, Lúcia; Leão-Teles, Elisa; Futerman, Anthony H.; Vilarinho, Laura; Alves, SandraBackground: Niemann-Pick type C (NP-C) is a neurodegenerative Lysosomal Storage Disease (LSD) with a heterogeneous clinical presentation secondary to abnormal intracellular accumulation of cholesterol. We have studied a patient with clinical diagnosis of NP-C but presenting inconclusive results regarding biomarkers testing and molecular analysis. To better characterize this patient, we have performed NGS-based technologies (targeted DNA sequencing and single cell-RNA sequencing). Methods: For the molecular diagnosis we used a NGS gene panel followed by the analysis of cDNA (in the patient and both parents). Latter, we have used massively parallel single cell RNA-seq (MARS-Seq) to address gene profiling changes and better characterize the pathomechanisms related to specific disease-causing mutations in this patient as well as in two NPC patients. The most prominent hits from this transcriptomics analysis were validated by qRT-PCR. Results and Discussion: Using our targeted NGS panel we identified two novel mutations in NPC1 gene (p.V505G; p.V562V). Next, through cDNA analysis of one of the patient parents we were able to understand the impact of the V562V silent mutation located in the middle of the exon 11. This mutation leads to exon 11 skipping giving origin to an out-of-frame transcript and eliciting the nonsense-mediated decay pathway. This mechanism contributed to the almost absence of the mutant transcript in the patient. Thus, we were not able to easily detect it in the sequencing electropherogram of the patient which turned the molecular diagnosis more challenging. By its turns, apparently the presence of the other mutation (the missense V505G) impairs the proper NPC protein folding leading to its ER retention. In fact, the MARS-Seq analysis of this patient showed that a number of genes were upregulated and a significant number of the highly enriched genes are related to the unfold protein response (UPR).