Browsing by Author "Ribeiro, Isaura"
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- Arylsulfatase B mutations in Portuguese MPS VI patientsPublication . Amaral, Olga; Dias, Aureliano; Pinto, Eugénia; Ribeiro, Isaura; Sá Miranda, M.C.Mucopolysaccharidosis type VI (MPS VI, OMIM 253200) is a rare autosomal recessive disorder characterized by the deficient activity of arylsulfatase B (ARSB, EC 3.1.6.12). In Portugal, the birth prevalence of the rare MPS VI is 0.42/100000. With the emerging availability of promising enzyme replacement therapy for this disease, mutation analysis becomes an important tool not only for the genetic counselling of individuals at risk, but also in the prognosis of the disease and identification of cases which might benefit of an early therapeutic intervention. In this work we present the preliminary results obtained through mutation analysis of 12 Portuguese patients. This study involved PCR amplification and direct automated sequencing analysis of exons and intron boundaries. The identification of seven mutations is reported: two recently described deletions, three missense mutations (two of them new), one novel nonsense mutation and also one new splicing mutation. Additionally, two previously described point mutations were detected in the form of a complex allele. Seven patients were homozygous for various mutations, while the remaining five were compound heterozygotes. Interestingly, three mutations seem to have an increased frequency in the Portuguese sample studied jointly c.1533del23 (Petry et al.,2003), c.427delG (Karageorgos et al.,2004) and R315Q (Villani et al.,1999) represent 58% of the patients alleles. In some of the cases Western blot analysis was also carried out. The impact of the various mutations at the protein level and the resulting phenotypic implications are discussed.
- 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.
- 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.
- Molecular profile and peripheral markers of neurodegeneration in patients with Niemann-Pick type C: Decrease in Plasminogen Activator Inhibitor type 1 and Platelet-Derived Growth Factor type AAPublication . Hammerschmidt, Tatiane Grazieli; Encarnação, Marisa; Lamberty Faverzani, Jéssica; de Fátima Lopes, Franciele; Oliveira, Fabiano Poswar de; Fischinger Moura de Sousa, Carolina; Ribeiro, Isaura; Alves, Sandra; Giugliani, Roberto; Regla Vargas, CarmenNiemann-Pick type C1 (NPC1) is a fatal inherited disease, caused by pathogenic variants in NPC1 gene, which leads to intracellular accumulation of non-esterified cholesterol and glycosphingolipids. This accumulation leads to a wide range of clinical manifestations, including neurological and cognitive impairment as well as psychiatric disorders. The pathophysiology of cerebral damage involves loss of Purkinje cells, synaptic disturbance, and demyelination. Miglustat, a reversible inhibitor of glucosylceramide synthase, is an approved treatment for NPC1 and can slow neurological damage. The aim of this study was to assess the levels of peripheric neurodegeneration biomarkers of NPC1 patients, namely brain-derived neurotrophic factor (BDNF), platelet-derived growth factors (PDGF-AA and PDGF-AB/BB), neural cell adhesion molecule (NCAM), PAI-1 Total and Cathepsin-D, as well as the levels of cholestane-3β,5α,6β-triol (3β,5α,6β-triol), a biomarker for NPC1. Molecular analysis of the NPC1 patients under study was performed by next generation sequencing (NGS) in cultured fibroblasts. We observed that NPC1 patients treated with miglustat have a significant decrease in PAI-1 total and PDGF-AA concentrations, and no alteration in BDNF, NCAM, PDGF-AB/BB and Cathepsin D. We also found that NPC1 patients treated with miglustat have normalized levels of 3β,5α,6β-triol. The molecular analysis showed four described mutations, and for two patients was not possible to identify the second mutated allele. Our results indicate that the decrease of PAI-1 and PDGF-AA in NPC1 patients could be involved in the pathophysiology of this disease. This is the first work to analyze those plasmatic markers of neurodegenerative processes in NPC1 patients.
- Mucopolysaccharidoses in Tunisia: a molecular portrait of allelic heterogeneity and consanguinityPublication . Coutinho, Maria Francisca; Ouesleti, Souad; Ribeiro, Isaura; Miled, A.; Mosbahi, D.S.; Alves, SandraThere are 11 different enzymes involved in the stepwise degradation of glycosaminoglycans (GAGs). Deficiencies in each of those enzymes result in eight different Mucopolysaccharidoses (MPSs), all sharing a series of clinical features, though in variable degrees. Typical symptoms include organomegally, dysostosys multiplex and coarse facies. CNS, hearing, vision and cardiovascular function may also be compromised. Traditionally, MPSs are recognized through analysis of urinary GAGs. Still, initial screenings of urinary GAGs allow discrimination between broad classes of MPSs but cannot distinguish subgroups. In fact, a definitive diagnosis may only be accessed through a combination of enzymatic assays and molecular analyses. Currently, there are countless laboratories in Europe where those biochemical and genetic tests are carried out. Nevertheless, developing countries often lack the necessary resources/expertise for proper diagnosis of rare genetic diseases. Being one of the labs where molecular genetic testing for virtually all MPSs is available for research purposes, we receive several samples from other countries, whose clinicians and/or centers struggle to get a molecular characterization of affected individuals. Here we present our results on the molecular characterization of MPS patients we have been receiving from Tunisia. Nine families suffering from five different diseases were studied so far: 3 MPS II; 2 MPS IIIA; 2 MPS IIIB; 1 MPS IIIC and 1 MPS VI. We have identified 9 different mutations, 5 of which were novel: 1 in the IDS gene (c.1333delC); another in the SGSH gene (p.D477N); 2 in the NAGLU gene (p.L550P and p.E153X) and yet another in the ARSB gene (p.L82P). All detected mutations were further analyzed with the most suitable approaches. Special attention was paid to the novel alterations, particularly to the missense ones, whose impact on protein structure and function was evaluated in silico. In general, there was a strong correlation between the observed clinical phenotype and the genotype assessed through molecular analysis. Also noteworthy is the astonishing level of homozygosity in our sample (100%), with each family presenting its own molecular defect, a pattern consistent with the occurrence of consanguineous matings in Tunisia, where such marriages are thought to provide social, economic and cultural benefits. Altogether, our results provide a preliminary overview of the molecular basis, enzymatic defects and clinical manifestations of MPSs in Tunisia, further supporting previous reports on the high impact of inbreeding and regional endogamy on the occurrence of autosomal recessive disorders in that country. Hopefully, these results will not only contribute to improve genetic counseling for affected families, but also to highlight the need for reinforced and continuous information of general public and health professionals on the potential negative medical impact of intra-family marriages, particularly in Northern Africa, Middle East and South Asia.
- Mucopolysaccharidosis type III in PortugalPublication . Caseiro, Carla; Rocha, Sónia; Ferreira, Célia; Ribeiro, Helena; Pinto, Eugénia; Pinto, Fernanda; Sousa, Domingos; Pinto, Eugénia; Ribeiro, Isaura; Laranjeira, Francisco; Coutinho, Maria Francisca; Alves, Sandra; Lacerda, LúciaIntroduction: Mucopolysaccharidosis type III (MPS III, Sanfilippo syndrome) is a rare autosomal recessively inherited disorder composed at least by four different subtypes: type A (OMIM # 252900), type B (OMIM # 252920), type C (OMIM # 252930) and type D (OMIM # 252940). Each subtype is caused by a deficiency in a different enzyme involved in the catabolic pathway for heparan sulfate. From the clinical point of view, type III patients can be classified within the MPS neurodegenerative phenotype, given visceromegaly, coarse facies, joint disease and bone dysplasia are relatively mild and progresses steadily. Behavioural disturbances and hyperactivity are often reported in the early stages of the disease. Objectives: The aim of this study is to draw the attention to the several subtypes which may be diagnosed within type III MPS, pointing out for mucopolysacharidosis-like patients with unclear etiology. Methods: MPS type III patients are diagnosed by the quantification of GAG heparan sulfate in urine, identification of the enzymatic deficiency in peripheral blood and gene sequencing for causal mutations. Results: From 34 MPS type III families, 4 patients were found to be IIIA, 24 IIIB and 6 IIIC. Type IIIB is the most common, accounting for 70% of all Portuguese cases with MPS III. Conclusions: Biochemical and molecular characterization allows carrier identification and informed family planning decisions. A negative diagnosis for subtype A to D does not rule out the disease and those patients with a high clinical suspicion are currently further tested for a recently proposed subtype, MPS IIIE.
- New splicing mutation in the cystatin B genePublication . Amaral, Olga; Freitas, Joel; Pinto, Eugénia; Duarte, Ana Joana; Ribeiro, Isaura; Ribeiro, Diogo; Chaves, João