Percorrer por autor "Sandiares, Ana Catarina"
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- ARPE-19-A Stable Cell Line Expressing a Variant of Unknown Significance in the NPC1 GenePublication . Monteiro, Beatriz; Peixoto, Maria Inês; Ortigoza-Escobar, Juan Darío; Alves, Mariana; Sandiares, Ana Catarina; Gonçalves, Mariana; Vaz Moreira, Luciana; Coutinho, Maria Francisca; Matos, Liliana; Alves, Sandra; Encarnação, MarisaBackground: Niemann-Pick type C is a lysosomal storage disorder that results from pathogenic variants in the NPC1 gene or in some cases from NPC2 pathogenic alterations. The disease presents a remarkable clinical variability that in some cases resembles common diseases, often resulting in a diagnostic odyssey or at least delaying proper diagnosis. In addition, the NPC1 gene is highly polymorphic, and consequently, when missense variants are identified after gene sequencing, accurate classification of their pathogenicity is essential to ensure appropriate access to available therapies and to provide reliable genetic counseling. Objectives: To get insights into the pathogenicity of a novel variant in NPC1, p.Cys800Ser, we created stable cell lines expressing this variant, in parallel with cell lines expressing the NPC1 wild-type and NPC1 pathogenic variants. Methods: We leveraged an isogenic cell line in which the NPC1 gene was knocked down and subsequently infected it with retroviruses carrying NPC1-WT and NPC1 variants C-terminally fused with an mNeonGreen tag. Three different NPC1 variants were included in this study: two known pathogenic variants, p.Ala1035Val and p.Pro1007Ala, and the novel p.Cys800Ser, whose significance was unknown. Results: We observed in the stable cell line expressing NPC1 p.Cys800Ser that the mutated NPC1 protein is transported to the lysosome similarly to the p.Pro1007Ala variant and affects lysosomal distribution. Conclusions: Using this approach, we could analyze the pathogenicity of each variant separately and these cell lines could be used for personalized medicine-based approaches and multi-omic 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.