Browsing by Author "Amaral, Olga"
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- Advances in sphingolipidoses: CRISPR-Cas9 editing as an option for modelling and therapyPublication . Amaral, Olga; Santos, RenatoSphingolipidoses are inherited genetic diseases characterized by the accumulation of glycosphingolipids. Sphingolipidoses (SP), which usually involve the loss of sphingolipid hydrolase function, are of lysosomal origin, and represent an important group of rare diseases among lysosomal storage disorders. Initial treatments consisted of enzyme replacement therapy, but, in recent decades, various therapeutic approaches have been developed. However, these commonly used treatments for SP fail to be fully effective and do not penetrate the blood-brain barrier. New approaches, such as genome editing, have great potential for both the treatment and study of sphingolipidoses. Here, we review the most recent advances in the treatment and modelling of SP through the application of CRISPR-Cas9 genome editing. CRISPR-Cas9 is currently the most widely used method for genome editing. This technique is versatile; it can be used for altering the regulation of genes involved in sphingolipid degradation and synthesis pathways, interrogating gene function, generating knock out models, or knocking in mutations. CRISPR-Cas9 genome editing is being used as an approach to disease treatment, but more frequently it is utilized to create models of disease. New CRISPR-Cas9-based tools of gene editing with diminished off-targeting effects are evolving and seem to be more promising for the correction of individual mutations. Emerging Prime results and CRISPR-Cas9 difficulties are also discussed.
- 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.
- An account of the portuguese experience: advances and challenges in diagnosis, therapy and research in lysosomal disordersPublication . Amaral, OlgaIntroductory background: Inborn errors of metabolism constitute an important group of rare hereditary diseases. Such diseases started to be studied in Portugal in the early-mid 1980s. Among them, Lysosomal Storage Disorders (LSDs) constitute a challenging group of disorders. In Portugal, Gaucher disease and Tay Sachs variant B1 were the first to be studied and were the subject of intricate laboratory diagnosis and careful research. Aim: Provide an account of the experience with lysosomal storage disorders in Portugal.
- Applications of iPSCs in Gaucher DiseasePublication . Amaral, Olga; duarte, ana; Ribeiro, Diogo; Santos, Renato; Bragança, JoséIn recent years, human induced pluripotent cell (hiPSC) models have slowly become a trend in experimental modelling of disease, following and complementing animal based models. Human iPSCs provide an innovative manner for modelling Gaucher Disease (GD). Since 2008 several groups have created iPSCs models from GD patients, with various genotypes, and differentiated iPSCs to neural precursors and macrophages among many other types of cells. hiPSC models have been developed from multiple GD donors, recapitulating the disease phenotypic hallmarks. These models have provided a new platform for pathophysiology studies and for the testing of small molecules with therapeutic goals.
- 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.
- 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.
- Cell lines for the study of Lysosomal Storage Diseases: conservation and identityPublication . Correia, Maria I.; Duarte, Ana J.; Ribeiro, Diogo; Amaral, OlgaThe use of cell lines has revolutionized research in the area of human genetics. The possibility of allowing, at a low cost and relative ease, practical access to biological material bearing in mind the benefit to the patient/family by obtaining samples with adequate informed consent, is a great asset. The accessibility of cell lines from biobanks allows access to samples with all the ethical and feasibility concerns observed. Cell lines are usually cryopreserved in liquid nitrogen and can be maintained in viable conditions for long periods of time. Cell lines allow studies of the causes of the disease, namely: the establishment of cell models, for a better understanding of the pathophysiology; the study of gene interactions; toxicity assays and drug tests; gene editing studies and other types of research. Using fibroblasts from patients with Lysosomal Storage Diseases (LSDs), it was already possible in this laboratory to revert cells to the stem cell state by creating induced pluripotent stem cells (iPSCs) to serve as a model in future studies. This clearly demonstrates the potential of cell lines for research. As with other cell lines, iPSCs can be cryopreserved which increases their potential for use. In order to guarantee the integrity and viability of cryopreserved cell lines, in laboratories, not exclusively dedicated to cell culture (as would be the case of a biobank), it would be advisable to periodically perform random thawing of samples in order to guarantee the identity of the preserved cells, genetic stability and absence of contaminants. There are several ways to do this however, in this work, we present some of the techniques used based on minimal procedures to ensure the cellular integrity of cryopreserved lines. It would be desirable, even in small laboratories, that procedures like these were adopted in a standardized and routine way, to facilitate the success of the subsequent use of cells in research.
- 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 comon IDUA in the Portuguese populationPublication . Ribeiro, Diogo; Duarte, Ana; Amaral, OlgaMucopolysaccharidosis type I (MPS I; OMIM #252800) is an autosomal recessive disorder, which results from the defective activity of the lysosomal enzyme α-L-iduronidase (IDUA, EC 3.2.1.76). In MPS I, this enzyme deficiency results in a progressive accumulation of the undegraded substrates within tissue lysosomes and fluids, leading to the clinical manifestations of the disease. W402X has been described as common in patients of European Caucasian origin. Moreover, this mutation has been considered to play an important role in terms of the pathophysiology of MPS I. The results of current functional experiments will be presented.
- 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.