Browsing by Author "Quelhas, Dulce"
Now showing 1 - 8 of 8
Results Per Page
Sort Options
- 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.
- Challenges in the Definitive Diagnosis of Niemann–Pick Type C—Leaky Variants and Alternative TranscriptsPublication . Encarnação, Marisa; Ribeiro, Isaura; David, Hugo; Coutinho, Maria Francisca; Quelhas, Dulce; Alves, SandraNiemann-Pick type C (NPC, ORPHA: 646) is a neuro-visceral, psychiatric disease caused predominantly by pathogenic variants in the NPC1 gene or seldom in NPC2. The rarity of the disease, and its wide range of clinical phenotypes and ages of onset, turn the diagnosis into a significant challenge. Other than the detailed clinical history, the typical diagnostic work-up for NPC includes the quantification of pathognomonic metabolites. However, the molecular basis diagnosis is still of utmost importance to fully characterize the disorder. Here, the authors provide an overview of splicing variants in the NPC1 and NPC2 genes and propose a new workflow for NPC diagnosis. Splicing variants cover a significant part of the disease-causing variants in NPC. The authors used cDNA analysis to study the impact of such variants, including the collection of data to classify them as leaky or non-leaky pathogenic variants. However, the presence of naturally occurring spliced transcripts can misdiagnose or mask a pathogenic variant and make the analysis even more difficult. Analysis of the NPC1 cDNA in NPC patients in parallel with controls is vital to assess and detect alternatively spliced forms. Moreover, nonsense-mediated mRNA decay (NMD) analysis plays an essential role in evaluating the naturally occurring transcripts during cDNA analysis and distinguishing them from other pathogenic variants' associated transcripts.
- De Barsy syndrome and ATP6V0A2-CDGPublication . Leao-Teles, Elisa; Quelhas, Dulce; Vilarinho, Laura
- 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.
- 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).