Browsing by Author "Duarte, Ana Joana"
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- Allelic frequency of CHIT1 variants in the Portuguese populationPublication . Duarte, Ana Joana; Ribeiro, Diogo; Amaral, OlgaChitotriosidase (EC.3.2.1.14) is an enzyme secreted by activated macrophages. This chitinase is useful as a biochemical marker in several lysosomal and nonlysosomal diseases due to its increased activity in such conditions (MIM#600031). In Gaucher Disease type 1 (GD, MIM#230800) patients, the chitotriosidase activity is increased in about 600-fold compared with normal controls. Chitotriosidase is not only useful as a disease severity marker but also measures the effectiveness of the therapy in GD. However, chitotriosidase gene (CHIT1, 1q31-q32) mutations modify the plasma chitotriosidase levels and therefore introduce a degree of variability that can be misleading. The most well known cause of chitotriosidase deficiency is the common 24 bp duplication. Nonetheless, other mutations that occur in this gene also lead to altered catalytic properties. However, as suggested by Bussink and collaborators, slight modifications in assay conditions may help avoiding such problems. In the present work we determined the frequency of two mutations which had appeared in a preliminary screening.
- Can Cell-type specific variability be involved in a rare variant of Unverricht-Lundborg? Investigation with iPSC generated modelsPublication . Duarte, Ana Joana; Ribeiro, Diogo; Moreira, Luciana; Amaral, OlgaHomozygosity for a private synonymous mutation in the cystatin-B gene (CSTB, MIM:601145; c.66G>A; p.Q22Q) was detected in a Portuguese patient with a rare, atypical form of Unverricht-Lundborg disease (ULD, MIM #254800). This apparently silent mutation leads to mis-splicing of CSTB pre-mRNA where a normal and an abnormal transcript were detected. Using iPSCs as a source of different cell types, we intend to clarify if the observed abnormal RNA splicing is cell-type specific, and to characterise the subsequent protein mislocalization. In conclusion, we hope to be able to contribute to the understanding of cell-type specific implications in the pathogenesis of ULD.
- Cellular characterization of normal and mutant cystatin BPublication . Duarte, Ana Joana; Ribeiro, Diogo; Chaves, João; Amaral, OlgaUnverricht- Lundborg disease (ULD or EPM1, MIM 254800), is a myoclonic epilepsy, caused by mutations in the cystatin B gene (CSTB gene) which lead to impaired function of cystatin B compromising its function. The normal protein is an endogenous inhibitor of cysteine proteinases and has several cellular localizations, it is found in the nucleus, cytosol and lysosome. Identification of a rare molecular mechanism causal of Unverricht Lundborg disease in a unique Portuguese patient triggered research in this field. Skin fibroblasts were obtained with informed consent from a homozygous patient with a rare genotype and from a normal control (anonymized). Fibroblast cell cultures were expanded using standard methods. Western Blotting (WB) and immunofluorescence (IF) experiments were carried out following previous described methods (Pinto et al, 2012). For cell fractionation analysis, cells were collected by scraping and suspending in ice-cold phosphate buffer saline (PBS) and cell fractions where obtained using the methods described elsewhere (Suzuki et al, 2010). Immunofluorescence experimental results revealed consistency with the western blot analysis. Although with a decrease of about 4 fold, in relation to normal, cystatin B is clearly present in the cells’ total fraction and in the nuclear fraction. However, in the cytoplasm the decrease seems to be higher which could suggest a subsequent lower protective anti-protease function compromising cellular and lysosomal integrity. Discussion and future perspectives In patient’s fibroblasts the protein quantity is diminished. Although the nuclear location seems to be preserved in the patient´s cells, the cytoplasmic/lysosomal fraction is clearly decreased.
- Characterization of a rare Unverricht-Lundborg disease mutationPublication . Duarte, Ana Joana; Ribeiro, Diogo; Chaves, João; Amaral, OlgaCystatin B (CSTB) gene mutations cause Unverricht–Lundborg disease (ULD), a rare form of myoclonic epilepsy. The previous identification of a Portuguese patient, homozygous for a unique splicing defect (c.66GNA; p.Q22Q), provided awareness regarding the existence of variant forms of ULD. In this work we aimed at the characterization of thismutation at the population level and at the cellular level. The cellular fractionation studies here carried out showed mislocalization of the protein and add to the knowledge on this disease.
- Constructing a cardiac cell model from a patient with Fabry diseasePublication . Duarte, Ana Joana; Ribeiro, Diogo; Amaral, Olga• We successfully achieve an iPSC line from a patient with Fabry Disease • Our line has the characteristics of stem cells and has the hability to differentiate into the 3 germ layers • After induction with specific cardiac effectors we achieved beating iPSC-CMs
- Correction of a Splicing Mutation Affecting an Unverricht-Lundborg Disease Patient by Antisense TherapyPublication . Matos, Liliana; Duarte, Ana Joana; Ribeiro, Diogo; Chaves, João; Amaral, Olga; Alves, SandraUnverricht-Lundborg disease (ULD) is a common form of progressive myoclonic epilepsy caused by mutations in the cystatin B gene (CSTB) that encodes an inhibitor of several lysosomal cathepsins. Presently, only pharmacological treatment and psychosocial support are available for ULD patients. To overcome the pathogenic effect of the ULD splicing mutation c.66G>A (exon 1), we investigated whether an antisense oligonucleotide therapeutic strategy could correct the defect in patient cells. A specific locked nucleic acid (LNA) antisense oligonucleotide was designed to block a cryptic 5′ss in intron 1. Overall, this approach allowed the restoration of the normal splicing pattern. Furthermore, the recovery was both sequence and dose-specific. In general, this work provides a proof of principle on the correction of a CSTB gene defect causing ULD through a mutation-specific antisense therapy. It adds evidence to the feasibility of this approach, joining the many studies that are paving the way for translating antisense technology into the clinical practice. The insights detailed herein make mutation-based therapy a clear candidate for personalized treatment of ULD patients, encouraging similar investigations into other genetic diseases.
- CRISPR/Cas in iPSCs from sphingolipidoses patientsPublication . Moreira, Luciana; Duarte, Ana Joana; Ribeiro, Diogo; Amaral, OlgaBackground: Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR) were found as an immune adaptive mechanism in bacteria and quickly were applied to various fields as a promising tool for gene editing. Ultimately, the use of CRISPR/Cas-mediated gene editing can provide specific cellular models of disease, correct causal mutations in LSDs and create mutants for functional studies. Aim: In this work, experimental conditions were tested to validate a CRISPR/Cas9 approach to generate a KO. Conclusion: The cells were amenable to edition by CRISPR/Cas9 and the results obtained proved that editing was achieved. All results contribute to the improvement of our knowledge regarding this technology, broadening the validation of CRISPR application and making it an accessible option.
- CSTB: from cell to population and backPublication . Amaral, Olga; Duarte, Ana JoanaCystatin B (CSTB) gene mutations cause Unverricht–Lundborg disease (ULD, EPM1, OMIM #254800), a rare form of myoclonic epilepsy. In this work, we aimed at investigating this rare disease by first analyzing the patient results, then examining the population through a population screening and, finally, exploring the cellular phenotype in order to assess the involvement of lysosomes.
- Doenças lisossomais de sobrecarga em Portugal: 10 anos de experiência em estudos moleculares no INSA (2006-2016)Publication . Coutinho, Maria Francisca; Duarte, Ana Joana; Matos, Liliana; Santos, Juliana Inês; Amaral, Olga; Alves, SandraAs Doenças Lisososomais de Sobrecarga (DLS) são um grupo de mais de 50 doenças hereditárias do metabolismo, sendo a maioria causada por defeitos em enzimas lisossomais específicas. A característica distintiva das DLS é a acumulação lisossomal do(s) substrato(s) não degradado(s), bem como a acumulação de outro material secundariamente à disfunção lisossomal. A apresentação clínica destas patologias é bastante heterogénea, variando desde formas pré-natais, até apresentações infantis ou na idade adulta, sendo frequente a presença de atraso psicomotor e neurodegeneração progressiva. Neste artigo são apresentados os resultados de vários estudos de caracterização molecular efetuados ao longo da última década (2006-2016) em doentes portugueses com as seguintes DLS: Mucopolissacaridose II, Mucopolissacaridose IIIA, Mucopolissacaridose IIIB, Mucopolissacaridose IIIC, Sialidose, Galactosialidose, Gangliosidose GM1, Mucolipidose II alfa/beta, Mucolipidose III alfa/beta, Mucolipidose III gama e Doença de Unverricht-Lundborg. De um modo geral, estes trabalhos permitiram conhecer as variações genéticas associadas a estas DLS, analisar a sua distribuição na população portuguesa e compreender o seu papel na forma de apresentação clínica destas patologias.
- Doenças lisossomais de sobrecarga: da epidemiologia genética ao desenvolvimento de modelos de doençaPublication . Moreira, Luciana; Coutinho, Maria Francisca; Moutinho, Maria Eduarda; Almeida, Matilde Barbosa; Gonçalves, Francisca; Carvalho, Sofia; Amaral, Olga; Duarte, Ana Joana; Encarnação, Marisa; Gaspar, Paulo; Gonçalves, Mariana; Matos, Liliana; Ribeiro, Diogo; Rocha, Hugo; Santos, Juliana Inês; Alves, Sandra; .