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O trabalho foi desenvolvido no DGH do INSA em colaboração com participantes do DGH do Porto e de Lisboa ao abrigo do projeto PTDC/BIM-MEC/4762/2014. Fabry Disease (FD) is a multisystemic X-linked disorder that belongs to the group of lysosomal storage disorders (LSDs). Causal mutations on alpha-galactosidase A (α-Gal A) commonly lead to abnormal protein and consequently to FD. Since it is an X-linked disease, males are primarily affected. This work describes the generation of induced Pluripotent Stem Cells (iPSCs) from skin fibroblasts from a FD patient, using non-integrative episomal vectors. Differentiation of iPSCs can be applied to generate a variety of cell types with high degree of genetic complexity that would otherwise be difficult to obtain. Portuguese Foundation of Science and Technology (FCT) project PTDC/BIM-MEC/4762/2014 (PI-O.A.) and UIDB/00211/2020. R.S. was the recipient of an FCT Grant from project PTDC/BIM-MEC/4762/2014 (MCTES). info:eu-repo/semantics/publishedVersion

Free PMC Article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6275066/ Note: Where it reads BBB-BMD/6301, should be PTDC/BIM-MEC/4762/2014. Erro de edição, tendo sido solicitado correção. In order to delineate a better approach to functional studies, we have selected 23 missense mutations distributed in different domains of two lysosomal enzymes, to be studied by in silico analysis. In silico analysis of mutations relies on computational modeling to predict their effects. Various computational platforms are currently available to check the probable causality of mutations encountered in patients at the protein and at the RNA levels. In this work we used four different platforms freely available online (Protein Variation Effect Analyzer- PROVEAN, PolyPhen-2, Swiss-model Expert Protein Analysis System-ExPASy, and SNAP2) to check amino acid substitutions and their effect at the protein level. The existence of functional studies, regarding the amino acid substitutions, led to the selection of the distinct protein mutants. Functional data were used to compare the results obtained with different bioinformatics tools. With the advent of next-generation sequencing, it is not feasible to carry out functional tests in all the variants detected. In silico analysis seems to be useful for the delineation of which mutants are worth studying through functional studies. Therefore, prediction of the mutation impact at the protein level, applying computational analysis, confers the means to rapidly provide a prognosis value to genotyping results, making it potentially valuable for patient care as well as research purposes. The present work points to the need to carry out functional studies in mutations that might look neutral. Moreover, it should be noted that single nucleotide polymorphisms (SNPs), occurring in coding and non-coding regions, may lead to RNA alterations and should be systematically verified. Functional studies can gain from a preliminary multi-step approach, such as the one proposed here. Financial support was received from the Portuguese Foundation of Science and Technology (FCT), project PTDC/BIM-MEC/4762/2014 (PI-O.A.) Diogo Ribeiro and Luciana Moreira were grant recipients from FCT under the same project. Ana Duarte was a PhD student with grant a FCTSFRH/BD/118009/2016. info:eu-repo/semantics/publishedVersion

Brief Report Unverricht-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. This work was funded by FCT (Fundação para a Ciência e a Tecnologia—MCTES, Portugal) project PTDC/BBB-BMD/6301/2014. Liliana Matos was grant recipient from FCT under the same project info:eu-repo/semantics/publishedVersion

15th Annual WORLDSymposium 2019 Scientific Meeting - Lysosomal Disease Research, 4-8 Feb 2019, Orlando, Florida, USA. 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. Lysosomal storage diseases (LSDs) are a group of metabolic disorders caused by defects in lysosomal proteins leading to accumulation of undigested macromolecules within the cells. The lack of good in vitro models hinders research of the pathophysiologic mechanisms and the development of new therapies. Induced pluripotent stem cells (iPSCs) are patient-specific and can be differentiated in any cell type. The advantage of iPSCs is to enable targeted studies in cells with the patient’s own background leading to more straightforward results than other models. Combining CRISPR and iPSCs is, therefore, a promising strategy. We aim to use CRISPR/Cas-mediated gene editing to provide more specific cellular models of disease, to correct causal mutations in LSDs and to create mutants for functional studies. In this work, we generated and characterized iPSCs from human fibroblasts obtained from Gaucher and Fabry patients (through Gaslini Institute) and will edit them with a CRISP/Cas9 approach. Because both gene editing and iPSCs generation require manipulating the cell’s genome, we envisage multiple check points along the workflow. It will be useful to compare the “native” mutated cells with the corrected cells that modulate the “disease in a dish”. Gene editing is still recent and the methods require improvement, namely increasing transfection rates and mutagenesis efficiency with less off-targets. Nevertheless, CRISPR/Cas is a promising alternative to other therapies, and every result contributes to the enhancement of this technology, broadening the validation of CRISPR application and making it an accessible option. FCT Funding: PTDC/BIM-MEC/4762/2014 info:eu-repo/semantics/publishedVersion

Objectives. Development of a simple mutation directed method in order to allow lowering the cost of mutation testing using an easily obtainable biological material. Assessment of the feasibility of such method was tested using a GC-rich amplicon. Design and Methods. A method of denaturing high-performance liquid chromatography (dHPLC) was improved and implemented as a technique for the detection of variants in exon 9 of the IDUA gene. The optimized method was tested in 500 genomic DNA samples obtained from dried blood spots (DBS). Results. With this dHPLC approach it was possible to detect different variants, including the common p.Trp402Ter mutation in the IDUA gene. The high GC content did not interfere with the resolution and reliability of this technique, and discrimination of G-C transversions was also achieved. Conclusion. This PCR-based dHPLC method is proved to be a rapid, a sensitive, and an excellent option for screening numerous samples obtained from DBS. Furthermore, it resulted in the consistent detection of clearly distinguishable profiles of the common p.Trp402Ter IDUA mutation with an advantageous balance of cost and technical requirements.

This study investigated whether endothelin (ET)-1-induced increase in myocardial distensibility is preserved in heart failure (HF) and whether it is modulated by nitric oxide (NO) and prostaglandins. New Zealand white rabbits were treated with doxorubicin (1 mg/kg, intravenously twice a week for 8 weeks, DOX-HF group) or saline (control group). Effects of ET-1 (0.1, 1, 10 nM) were tested in papillary muscles from the DOX-HF group and a control group in the presence of: i) intact endocardial endothelium (EE); ii) damaged EE; iii) NG -nitro-L-arginine (L-NNA; NO synthase inhibitor), and iv) indomethacin (INDO; cyclooxygenase inhibitor). In the presence of an intact EE, ET-1 promoted concentration-dependent positive inotropic and lusitropic effects that were maintained after damaging the EE, in the presence of L-NNA or INDO and in the DOX-HF Group. ET-1 reduced resting tension at the end of the isometric twitch (increased diastolic distensibility) by 3.2±1.3 %, 6.0±1.6 % and 8.8±2.7 % (at 0.1, 1 and 10 nM, respectively), in muscles with intact EE, effect that was completely abolished after damaging EE, in the presence of L-NNA or INDO or in the DOX-HF Group. This study demonstrated that the increase in myocardial distensibility induced by ET-1 is absent in HF and is dependent of NO and prostaglandin release.

Publicado em: Molecular Genetics and Metabolism 105 (2012) S15–S69. Disponivel em: http://www.sciencedirect.com/science/article/pii/S1096719211004343