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- Upstream of precise disease models for better downstream decision makingPublication . Duarte, Ana; Moreira, Luciana; Ribeiro, Diogo; Bragança, José; Amaral, OlgaInborn errors of metabolism are a common cause of inherited diseases. Diseases of carbohydrate metabolism pathway include lysosomal storage diseases (LSDs), which are a significant subgroup with approximately 70 LSDs. Grouped according to their defective lysosomal proteins and pathways they are usually characterized by intralysosomal accumulation of substrate. Accumulation may occur at different levels in diverse types of cells, some of which are of difficult access. Patient, molecular, cell and tissue heterogeneity hinders the development of further therapeutic approaches. We are currently establishing human Induced Pluripotent Stem Cells (iPSCs) from fibroblasts of LSDs patients and controls. The use of disease-specific cell models, mimicking the cell-target of the specific disease, may help to appropriately study the pathogenesis as well as the therapeutics. Integrating new techniques in the work pipeline for the establishment of models may lead to more accurate models while ensuring the safeguard of the patient’s background. Advanced technologies like microarray and NGS profiling add to the traditional techniques such as Immunofluorescence, directed sequencing and conventional cytogenetics. As in the diagnosis process, we may better understand the prognosis, and contribute to cost avoidance, by combining genomic and protein profiling checkpoints in the cell-model establishment pipeline. The investment in the upstream checkpoints might prove to be helpful in ensuring the integrity of the cell models.
- 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.
- Future perspectives using Lysosomal Storage Disease iPSCs models and gene editing therapyPublication . Ribeiro, Diogo; Duarte, Ana Joana; Amaral, OlgaLysosomal storage diseases (LSDs) are characterized by accumulation of macromolecules in the late endocytic system. Their collective frequency of 1/5000 live births and are caused by inherited defects in genes that mainly encode lysosomal proteins (1). In the Portuguese population, lysosomal storage disorders (LSDs) have a prevalence of 1/4000 live births. Tay Sachs disease (TSD, MIM#272800) variant B1 is one of the most prevalent in the Portuguese population (2). The TSD variant B1 is caused by mutations on the HEXA gene (MIM#606869.0006), leading to hexosaminidase A malfunction. The mutation subject of this study, p.R178H (rs28941770), is frequent in specific populations. In the Portuguese it has a carrier frequency of 1:340, and in the North of Portugal it was estimated to be 1:119. Fabry disease ((FD, MIM#301500)) is one of the most frequent LSDs, it is caused by mutations on the GLA gene (MIM#300644), such as the mutation p.W287X (rs104894839), leading to alpha-galactosidase A impairment. Gaucher disease (GD) is also a frequent LSD and it is, usually, due to deficient activity of lysosomal acid beta glucosidase (GBA1, MIM#606463). It has several phenotypic forms (GD1, 230800; GD2, 230900; and GD3; 231000) of which the most severe are neurodegenerative and elude common therapies. In our group, we are attempting to use gene editing through CRISPR/Cas9 as a therapeutic tool to correct specific mutations involved in the abovementioned diseases. Our aim is first to obtain induced pluripotent stem cells (iPSCs) derived from these cell lines and then to correct the mutational defects.
- Tay Sachs disease variant B1: iPSC and NGS as the basis for a cellular modelPublication . Ribeiro, Diogo; Duarte, Ana J.; Moreira, Luciana; Santos, Renato; Encarnaçao, Marisa; Silva, Lisbeth; Coutinho, M. Francisca; Alves, Sandra; Amaral, OlgaTay Sachs disease variant B1 (TSD B1; OMIM 272800) is a neurodegenerative lysosomal storage disease (LSD) which, although rare, is the most frequent form of TSD in Portugal. The mutation p.R178H (c.533G>A; rs28941770), associated with TSD B1, leads to a mutant HexA protein with altered kinetics and reduced residual activity. The availability of disease-relevant cell types derived from induced pluripotent stem cells (iPSCs) provides a model for studying the pathogenic mechanisms and, eventually, test therapeutic approaches for TSD B1 patients. The main objectives of this project is, by using iPSCs, to generate a neuronal TSD B1 specific cellular model and to implement the genetic profiling by Next Generation Sequencing (NGS) to examine potential changes in the manipulated cells. In the present work, the iPSC reprogramming and differentiation into neural progenitor cells (NPCs) is presented, as well as the NGS results from the donor cells. As a first step, iPSCs from a control fibroblast cell line and from TSD B1 fibroblasts were obtained by using a non-integrative approach with episomal vectors, the control was further differentiated into NPCs. When generating iPSCs it is important to have multiple characterisation steps. The iPSCs have to mimic the donor background at genetic and protein levels. Furthermore, iPSCs or iPSC-derived lines need to be free of the reprogramming and differentiation markers while presenting specific cell-type markers. The frailty of these cells and adverse conditions (first lockdowns and later accidental deprivation of liquid nitrogen) led to the interruption of the iPSC work. In the meantime, we did a lysosomal-related gene profiling of the DNA from the original cell lines. By using an in-house customized NGS panel, we obtained results of the cells in a “naïve” state to later compare with TSD B1 iPSCs and NPCs obtained from control iPSCs. The results presented consist of the preliminary work to obtain a cell model of TS-B1.
- 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.
- A first step to open the neuronal box of Gaucher CellsPublication . Ribeiro, Diogo; Duarte, Ana; Santos, Renato; Amaral, OlgaThis work focuses on the differentiation and gene expression characterization of neural progenitor cells obtained from human induced pluripotent cells (hiPSCs) reprogrammed from type 3 GD (GD3) fibroblasts. GD3 patient fibroblasts (from an international cell bank) were cultured and reprogramed as previously described (https://doi.org/10.1016/j.scr.2019.101595). The resulting hiPSCs were differentiated into pre-neuronal cells and, at this stage, they were examined. The gene expression behavior of all neurogenesis genes (NES, MAP2 and OTX2) was similar but higher expression was observed in GD3 hiPSCs than in GD3 neural progenitor cells. With this work, we can conclude that, when working with hiPSCs in the process of creating disease-specific cell models it is most important to carry out a general gene expression characterization of the different cell lines involved in all stages.
- Editorial: Somatic Cell Gene Editing for Treating DiseasesPublication . Wong, Raymond Ching-Bong; Huang, Junjiu; Li, Dali; Amaral, OlgaEditorial on the Special Issue with the Research Topic: Somatic Cell Gene Editing for Treating Diseases
- Engagement in science: virtual interaction as a flexible toolPublication . Amaral, OlgaThe pandemic lockdowns forced a rapid switch to digital platforms. Lockdowns closed schools and laboratories pushing scientist and educators to online activities. Digital platforms for communication are widely available for free use. These platforms, although different, are intuitive and user friendly, therefore facilitating this sudden imperative shift from in-person to virtual communication. In an effort to bring services or science information to people new solutions were adopted. Although in-person activities were interrupted, alternative solutions were set up in order to engage wide participation. Such examples can be found among various types of initiatives regarding human genetic disorders which successfully switched to online versions.
- The Biology of Lysosomes: From Order to DisorderPublication . Amaral, Olga; Martins, Mariana; Oliveira, Ana Rita; Duarte, Ana Joana; Mondragão-Rodrigues, Inês; Macedo, M. FatimaSince its discovery in 1955, the understanding of the lysosome has continuously increased. Once considered a mere waste removal system, the lysosome is now recognised as a highly crucial cellular component for signalling and energy metabolism. This notable evolution raises the need for a summarized review of the lysosome’s biology. As such, throughout this article, we will be compiling the current knowledge regarding the lysosome’s biogenesis and functions. The comprehension of this organelle’s inner mechanisms is crucial to perceive how its impairment can give rise to lysosomal disease (LD). In this review, we highlight some examples of LD fine-tuned mechanisms that are already established, as well as others, which are still under investigation. Even though the understanding of the lysosome and its pathologies has expanded through the years, some of its intrinsic molecular aspects remain unknown. In order to illustrate the complexity of the lysosomal diseases we provide a few examples that have challenged the established single gene—single genetic disorder model. As such, we believe there is a strong need for further investigation of the exact abnormalities in the pathological pathways in lysosomal disease.
- Segmental and total uniparental isodisomy (UPiD) as a disease mechanism in autosomal recessive lysosomal disorders: evidence from SNP arraysPublication . Labrijn-Marks, Ineke; Somers-Bolman, Galhana M.; In ’t Groen, Stijn L. M.; Hoogeveen-Westerveld, Marianne; Kroos, Marian A.; Ala-Mello, Sirpa; Amaral, Olga; Miranda, Clara sa; Mavridou, Irene; Michelakakis, Helen; Naess, Karin; Verheijen, Frans W.; Hoefsloot, Lies H.; Dijkhuizen, Trijnie; Benjamins, Marloes; van den Hout, Hannerieke J.M.; van der Ploeg, Ans T.; Pijnappel, W. W. M. Pim; Saris, Jasper J.; Halley, Dicky J.Analyses in our diagnostic DNA laboratory include genes involved in autosomal recessive (AR) lysosomal storage disorders such as glycogenosis type II (Pompe disease) and mucopolysaccharidosis type I (MPSI, Hurler disease). We encountered 4 cases with apparent homozygosity for a disease-causing sequence variant that could be traced to one parent only. In addition, in a young child with cardiomyopathy, in the absence of other symptoms, a diagnosis of Pompe disease was considered. Remarkably, he presented with different enzymatic and genotypic features between leukocytes and skin fibroblasts. All cases were examined with microsatellite markers and SNP genotyping arrays. We identified one case of total uniparental disomy (UPD) of chromosome 17 leading to Pompe disease and three cases of segmental uniparental isodisomy (UPiD) causing Hurler-(4p) or Pompe disease (17q). One Pompe patient with unusual combinations of features was shown to have a mosaic segmental UPiD of chromosome 17q. The chromosome 17 UPD cases amount to 11% of our diagnostic cohort of homozygous Pompe patients (plus one case of pseudoheterozygosity) where segregation analysis was possible. We conclude that inclusion of parental DNA is mandatory for reliable DNA diagnostics. Mild or unusual phenotypes of AR diseases should alert physicians to the possibility of mosaic segmental UPiD. SNP genotyping arrays are used in diagnostic workup of patients with developmental delay. Our results show that even small Regions of Homozygosity that include telomeric areas are worth reporting, regardless of the imprinting status of the chromosome, as they might indicate segmental UPiD.
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