Browsing by Author "Alves, S."
Now showing 1 - 10 of 18
Results Per Page
Sort Options
- Clinical, biochemical and molecular characterization of cystinuria in a cohort of 12 patients.Publication . Barbosa, M.; Lopes, A.; Mota, C.; Martins, E.; Oliveira, J.; Alves, S.; De Bonis, P.; Mota, M. do C.; Dias, Carlos Matias; Rodrigues-Santos, P.; Fortuna, A.M.; Quelhas, D.; Lacerda, L.; Bisceglia, L.; Cardoso, M.L.Cystinuria is a rare autosomal inherited disorder characterized by impaired transport of cystine and dibasic aminoacids in the proximal renal tubule. Classically, Cystinuria is classified as type I (silent heterozygotes) and non-type I (heterozygotes with urinary hyperexcretion of cystine). Molecularly, Cystinuria is classified as type A (mutations on SLC3A1 gene) and type B (mutations on SLC7A9 gene). The goal of this study is to provide a comprehensive clinical, biochemical and molecular characterization of a cohort of 12 Portuguese patients affected with Cystinuria in order to provide insight into genotype–phenotype correlations. We describe seven type I and five non-type I patients. Regarding the molecular classification, seven patients were type A and five were type B. In SLC3A1 gene, two large genomic rearrangements and 13 sequence variants, including four new variants c.611-2A>C; c.1136+44G>A; c.1597T (p.Y533N); c.*70A>G, were found. One large genomic rearrangement was found in SLC7A9 gene as well as 24 sequence variants including 3 novel variants: c.216C>T (p.C72C), c.1119G>A (p.S373S) and c.*82C>T. In our cohort the most frequent pathogenic mutations were: large rearrangements (33.3% of mutant alleles) and a missense mutation c.1400T>C ( p.M467T) (11.1%). This report expands the spectrum of SLC3A1 and SLC7A9 mutations and provides guidance in the clinical implementation of molecular assays in routine genetic counseling of Portuguese patients affected with Cystinuria.
- Development of RNA based approaches to exploit alternative therapies for Lysosomal Storage DiseasesPublication . Matos, L.; Santos, J.I.; Rocha, M.; Coutinho, M.F.; Gaspar, P.; Voltolini Velho, R.; Braulke, T.; Prata, M.J.; Alves, S.Treatment strategies such enzyme-replacement therapy and substrate reduction, among others, are available for some Lysosomal Storage Diseases, yet still with some limitations. In recent years, the RNA molecule became one of the most promising targets for therapeutic intervention and currently, a large number of RNA-based therapies are being investigated at the basic research level and in late-stage clinical trials. Actually, some of them are already approved for medical use (e.g. Spinal muscular atrophy and Duchenne muscular dystrophy). RNA-based approaches can act at pre-mRNA level (by splicing modulation/correction using antisense oligonucleotides or U1snRNA vectors), at mRNA level (inhibiting gene expression by siRNAs and antisense oligonucleotides) or at DNA level (by editing mutated sequences through the use of CRISPR/Cas). Currently, we are developing some of these therapeutic approaches for LSDs. Two main research lines are ongoing: one involves the use of antisense U1 snRNAs to overcome the effect of a splice site mutation causing Mucopolysaccharidosis type IIIC and the other is based on the use of splice switching oligonucleotides to induce the skipping and consequently circumvent the effects of the most common causal mutation in Mucolipidosis type II.
- Doenças Lisossomais de Sobrecarga – Novas metodologias de diagnósticoPublication . Alves, S.Enquadramento e objetivos: O “II Curso de Rastreio, Diagnostico das Doenças Hereditárias do Metabolismo (DHM) ” é organizado pela Unidade de Rastreio Neonatal (URN), Metabolismo e Genética, do Departamento de Genética Humana do Instituto Nacional de Saúde Doutor Ricardo Jorge, IP. O programa possui uma forte componente teórico-prática, assenta no conhecimento e experiência acumulada da URN e inclui a apresentação e discussão de dados laboratoriais dos doentes rastreados e diagnosticados na Unidade e o seu seguimento clínico. Os participantes irão adquirir conhecimentos básicos sobre a organização de rastreio e diagnóstico laboratorial de DHM e integração de dados clinico-laboratoriais. A iniciativa conta com a colaboração de clínicos, técnicos e investigadores especialistas nesta área do conhecimento.
- Ficha de Atividades Laboratoriais no âmbito das Doenças Lisossomais de SobrecargaPublication . Alves, S.Material utilizado em visitas de estudo ao INSA no âmbito Doenças Lisossomais de Sobrecarga.
- From bedside to cell biology: a century of history on lysosomal dysfunctionPublication . Coutinho, M.F.; Matos, L.S.; Alves, S.Lysosomal storage disorders (LSDs) are a group of rare genetic diseases, generally caused by a deficiency of specific lysosomal enzymes, which results in abnormal accumulation of undegraded substrates. The first clinical reports describing what were later shown to be LSDs were published more than a hundred years ago. In general, the history and pathophysiology of LSDs has impacted on our current knowledge of lysosomal biology. Classically, depending on the nature of the substrates, LSDs can be divided into different subgroups. The mucopolysaccharidoses (MPSs) are those caused by impaired degradation of glycosaminoglycans (GAGs). Amongst LSDs, the MPSs are a major group of pathologies with crucial historical relevance, since their study has revealed important biological pathways and highlighted interconnecting pathological cascades which are still being unveiled nowadays. Here we review the major historical discoveries in the field of LSDs and their impact on basic cellular knowledge and practical applications. Attention will be focused on the MPSs, with occasional references to other LSDs. We will show as studies on the metabolic basis of this group of diseases have increased our knowledge of the complex degradative pathways associated with the lysosome and established the basis to the development of specific therapeutic approaches aiming at correcting or, at least ameliorating their associated phenotypes.
- Genetically Modulated Substrate Reduction Therapy for Sanfilippo syndrome – proof of principlePublication . Santos, J.I.; Coutinho, Maria Francisca; Gaspar, P.; Alves, S.Introduction: Mucopolysaccharidosis type III (MPS III) refers to a group of five autosomal recessive neurodegenerative lysosomal storage disorders caused by the incomplete lysosomal degradation of the heparan sulphate (HS) that accumulates in patient cells and triggers disease. The main characteristic of this disease is the degeneration of the central nervous system, resulting in mental retardation and hyperactivity. Currently, there is no effective therapy available, with treatment limited to clinical management of neurological symptoms. Methods: Taking advantage of the RNA interference (RNAi) technology potential, we have designed and assayed a specific siRNA targeting an early stage of the HS biosynthetic cascade (XYLT1) in order to promote an effective reduction of the accumulating substrate. Fibroblasts from MPS III patients were transfected with the designed siRNA. Total RNA was extracted and target mRNA levels evaluated through real-time PCR. The effect on GAGs accumulation was quantified over time using a modified 1,9-dimethylmethylene blue assay. Results: Proof of principle on the effect of siRNA targeting XYLT1 was achieved for two independent control cell lines, with 8-12 fold decreases on the target mRNA levels, after 24h of incubation with concentrations of 20nM of each siRNA. Subsequent analysis on the effect of the same siRNA on MPS III cell lines resulted in significant lower expression of XYLT1 in types A, B and C, after 24-48h of siRNA incubation. Studies on type B are also ongoing. For types C and D, we have already assessed the treatment effect on storage and observed a significant reduction (50-70%) on the total GAGs levels. Conclusions: The effect of siRNA targeting XYLT1 was achieved, resulting in significant lower levels of XYLT1 mRNA. Studies on MPS IIIB are ongoing. Moreover, a significant reduction on GAGs’ accumulation was observed, and we are currently addressing this storage in the remaining MPS III cell lines.
- Molecular and computational analyses of genes involved in mannose 6-phosphate independent traffickingPublication . Coutinho, M.F.; Lacerda, L.; Pinto, E.; Ribeiro, H.; Macedo-Ribeiro, S.; Castro, L.; Prata, M.J.; Alves, S.The newly-synthesized lysosomal enzymes travel to the trans-Golgi network (TGN) and are then driven to the acidic organelle. While the best-known pathway for TGN-to-endosome transport is the delivery of soluble hydrolases by the M6P receptors (MPRs), additional pathways do exist, as showed by the identification of two alternative receptors: LIMP-2, implicated in the delivery of β-glucocerebrosidase; and sortilin, involved in the transport of the sphingolipid activator proteins prosaposin and GM2AP, acid sphingomyelinase and cathepsins D and H. Disruption of the intracellular transport and delivery pathways to the lysosomes may result in lysosomal dysfunction, predictably leading to a range of clinical manifestations of lysosomal storage diseases. However, for a great percentage of patients presenting such manifestations, no condition is successfully diagnosed. To analyse if, in this group, phenotypes could be determined by impairments in the known M6P-independent receptors, we screened the genes that encode for LIMP-2 and sortilin. No pathogenic mutations were identified. Other approaches will be needed to clarify whether sortilin dysfunction may cause disease.
- Molecular Characterization of a Novel Mucopolysaccharidosis (MPS) type VI-causing Mutation – Indirect Proof of Principle on its PathogenicityPublication . Coutinho, Maria Francisca; Encarnação, M.; Santos, J.I.; Alves, S.Introduction: With its unprecedented throughput, scalability and speed, next-generation sequencing (NGS) is revolutionizing clinical research. Targeted sequencing in particular is now available in many labs. Still, whenever a novel variant is detected, its pathogenicity must be carefully assessed and every now and again, a case pops up to highlight how tricky and delicate this process can be. Here we present a case of a molecular diagnosis of a patient with a clinical suspicion of MPS type VI, where even though the causal mutation was easy to detect by both Sanger and NGS, only through indirect studies could we present proof of principle on its pathogenicity.
- Mucolipidosis Type II and Type III: Nine cases from one Indian centrePublication . Bhat, M.; Sanjeeva, G.N.; Maganti, M.; Devaiah, S.; Silji, G.; Undamatla, J.; Coutinho, M.F.; Alves, S.