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- 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.
- A machine learning approach for the prioritization of disease-associated genetic variants from ngs datasetsPublication . Martiniano, Hugo; Vilela, Joana; Marques, Ana R.; Santos, João; Rasga, Célia; Vicente, AstridOur objective is to develop variant prioritization methods using machine learning approaches to predict genotype-phenotype associations, that can detect the most likely causal variants associated to a given phenotype.
- Induced Pluripotent Stem Cells derived cardiomyocytes from a patient with Fabry Disease: a work in process cell modelPublication . Duarte, Ana; Ribeiro, Diogo; Bragança, Jose; Amaral, OlgaFabry disease (FD) is one of the commonest Lysosomal Storage Disorders (LSDs) and is caused by mutations in the alpha-galactosidase A gene (GLA) from which results a deficient activity of the lysosomal hydrolase alphagalactosidase A (α-Gal A). This deficiency leads to progressive multisystemic accumulation of glycolipids, namely, globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3), in plasma and in a wide range of cells, particularly in the relevant cells affected by the disease like vascular endothelial cells, podocytes, cardiomyocytes, and arterial smooth muscle cells. Taking FD as a cardiac disorder, our aim was to differentiate induced pluripotent stem cells (iPSCs) reprogrammed from FD patients’ fibroblasts into cardiomyocytes, a cellular type usually targeted by this disease. Since only mature cardiomyocytes can fully recapitulate the same disease phenotypes present in vivo and serve as an accurate disease model, we have to surpass the immature state to achieve a complete disease model.
- Identification of neurotransmission and synaptic risk genes and disrupted biological processes in Autism Spectrum DisorderPublication . Vilela, Joana; Martiniano, Hugo; Marques, Ana Rita; Santos, João; Rasga, Célia; Oliveira, Guiomar; Vicente, Astrid M.The objective of this study is the identification of Single Nucleotide Variants (SNVs) in neurotransmission and synaptic genes (NS genes) that play a role in ASD etiology, and the biological processes and global networks affected.
- Interplay between glycemia and the genetics of ENOS and ACE genes for the susceptibility to the onset and development of hypertension on the Portuguese populationPublication . Aguiar, Laura; Ferreira, Joana; Matos, Andreia; Mascarenhas, Mário Rui; Menezes Falcão, Luiz; Faustino, Paula; Bicho, Manuel; Inácio, ÂngelaIntroduction: Hypertension is a multifactorial condition of anthropometric, physiologic, metabolic, genetic, and environmental nature. In Portugal, the mean prevalence of hypertension in the population is 45.5%. Objective: The aim of this study is to evaluate the contribution of anthropometric, physiological, and genetic factors (eNOS and ACE) to the development of hypertension in a Portuguese population. Methods: A case-control study was conducted in a sample of 377 individuals, 243 hypertensives, and 134 normotensives. The polymorphic analyses of intron 4 VNTR in the eNOS gene and the insertion/deletion (I/D) in ACE gene were performed by polymerase chain reaction (PCR). Results: High body mass index (BMI) values, high glycemia levels, and the 4a allele of the eNOS were associated with hypertension. Among the hypertensive group, the allele 4a (eNOS) was associated with high levels of HbA1c, and the D allele (ACE) with glycemia. Conclusion: Our results highlight the contribution of eNOS and ACE genes as important players for the onset and development of hypertension in the Portuguese population. We believe that a combinatory clinical approach including the traditional anthropomorphic and physiological parameters together with genetic studies can be more elucidative in establishing a susceptibility profile on multifactorial conditions as hypertension.
- Genetics as part of health science education: reflecting on a changing approachPublication . Amaral, Olga; Ferreira, FilipaGenetics goes beyond the human species and this should be the paramount power of genetics education. The current wave of interest in Science should be the force towards a change in the way information reaches the public. The prior underestimation of the understanding capabilities of the student and non-student populations resulted in a loss of engagement in science and might lead to misinformation. In the past decades, INSA has had an approach to communication in Health Science that depends on strong views and areas of expertise, various programmed activities and, as in research, it depends on expectations. Along the line of community involvement, GENEtic.COMunication (GENE.COM) is a project submitted to a competitive call launched by Ciencia Viva. With that project, the focus is on a niche of knowledge with great impact on Health, in society, as well as on personal and public budgets: the area of the rare genetic metabolic diseases. The project intended to contribute to the dissemination of information about Human Genetics, particularly in the area of rare genetic diseases. The aim was to communicate knowledge in an easily understandable way; inform health professionals, patients, students, teachers, as well as patient associations. This contact could provide the establishment of a dialogue between professionals working in the field and different publics. Furthermore, the switch to virtual tools relies on easier access to the information while implying diminished associated costs, such as transportation, time and space limitations. The problem may be how to make passive learning more engaging. As we know, assessment drives learning and there are simple and quick ways to assess the efficacy of the “take home message”, which may improve engagement and provide the needed feedback. Attempts to contribute to a more informed society should be disclosed and supported as they can lead to more positive attitudes and empowered choices. By providing digital contents in a way that they can be followed by various target audiences we also aim at reaching audiences that would not typically seek us.
- Mutational Spectrum and Geographic Distribution of Alpha-thalassemia in an Adult Microcytic and/or Hypochromic Population Living in Portugal: Results from the First National Health Examination Survey (INSEF 2015)Publication . Santos, Daniela; Kislaya, Irina; Lopes, Pedro; Matias-Dias, Carlos; Barreto, Marta; Faustino, PaulaAlpha-thalassemia (α-thal) is one of the most common monogenic disorders in the world. Its clinical severity varies from almost asymptomatic, mild microcytic hypochromic anemia, to a lethal hemolytic condition, depending on the number of affected α-globin genes (1 to 4). The disease is most commonly originated by deletions on 16p13.3. The aim of this study was to identify the molecular basis, geographic distribution and prevalence of mild forms of α-thal in Portugal. This is a cross-sectional population-based study, based on the first Portuguese National Health Examination Survey (INSEF), which included individuals living in Portugal for more than 12 months, aged between 25 and 74 years old. For this INSEF sub-study, we analysed 4812 participants from whom a Complete Blood Count was performed and selected the 204 participants presenting red blood cell microcytosis (Mean Corpuscular Volume, MCV <80fL) and/or hypochromia (Mean Corpuscular Hemoglobin, MCH <27pg). DNA from these samples was used to search for deletions in HBA cluster by Gap-PCR and Multiplex Ligation-dependent Probe Amplification. We found 52 individuals heterozygous for the -α3.7kb deletion, one homozygous for this deletion and one heterozygous for the -α4.2kb deletion. Two cases presented triplicated α-globin genes (αααanti 3.7kb). Thus, α-thal was observed in 54 individuals (26.5 %) of the analysed population. Carriers of the –α3.7kb deletion have hypochromic red blood cells (MCH mean 26.0 ± 0.9 pg) but normal or borderline volume (MCV mean 81.4 ± 2.7 fL). The geographic distribution of affected participants showed two regions with highest prevalence of α-thal: LVT and RA Madeira. Although the mild forms of α-thal themselves are of no clinical significance, their major importance is the modifying effect that they have on various severe forms of β-thalassemia and sickle cell disease. Furthermore, α-thal trait can be confused with iron deficiency anemia as the hematological parameters are quite similar. Therefore, iron status should be properly assessed to distinguish between the two conditions and α-thal confirmation at DNA level is necessary for a definitive diagnosis.
- Translational Regulation of the Human PERK by Upstream Open Reading FramesPublication . Fernandes, Rafael; Lopes, Pedro; Romão, LuísaUpstream open reading frames (uORFs) are cis-acting elements located within the 5’ leader sequence (5’UTR) of transcripts, which can regulate translation of the correspondent main open reading frame (mORF). During endoplasmic reticulum (ER) stress, the accumulation of unfolded proteins activates the ER-resident PKR-like ER kinase (PERK), which results in phosphorylation of eIF2α to inhibit global mRNA translation, while allowing the selective uORF-mediated translation of downstream effectors responsible for stress resolution or, ultimately, cell death. The dual role of PERK in regulating cell fate was implicated in human diseases, like diabetes, neurodegenerative disorders and cancer. Moreover, mutations in the EIF2AK3 gene (encoding PERK) were associated to the rare genetic disease, Wolcott-Rallison Syndrome (WRS). In this work, we aimed to study the translational regulatory role of 5 AUG- and 3 non-AUG-uORFs identified in the PERK 5’UTR and assess its biological relevance. While uORF2 and the non-AUG-uORFs 5, 6 and 7 (numbered according to their distance to the 5’ end of the mRNA) do not seem to have a regulatory role, uORF1, uORF3, uORF4 and uORF8 together present a strong repressive effect over mORF translation in basal conditions. Curiously, we found that when PERK is overexpressed, it leads to the spontaneous activation of a portion of PERK in the absence of any stress stimulus, possibly highlighting the biological relevance of its uORF-mediated translational regulation. Conversely, during ER stress, increased bypass of uORF1 results in a modest degree of translational de-repression, which may help to counterbalance the increased rate of PERK protein turnover observed in these conditions. We also observed that alteration of the PERK uORFs by mutations found in WRS patients modify mORF expression, providing a possible link to the disease. Altogether, we highlight the importance of including 5’UTRs in the screening of disease-related mutations and the necessity of functional studies to assess their role in pathogenesis.
- The tumor suppressor p53 acquires oncogenic functions due to a translational switch during integrated stress responsePublication . Lacerda, Rafaela; Fonseca Costa, Inês; López-Iniesta, M.; Romão, Luísa; Candeias, Marco M.To cope with the stress stimuli to which they are often exposed, eukaryotic cells have developed adaptive pathways that restore cellular homeostasis. Under stress conditions there is an overall decrease of protein synthesis, and a concomitant induction of alternative mechanisms of mRNA translation initiation. The tumour suppressor protein p53 has been considered the guardian of the genome and a master regulator of many cellular functions. However, apart from the full-length p53 (FLp53), several p53 isoforms have been described so far. Some functions of shorter p53 isoforms have already been elucidated and they are different from and complement FLp53 activity, the most mutated gene in cancer. Here we show that the integrated stress response (ISR) leads to the specific induction of Δ160p53 isoform. Using bicistronic constructs we confirmed the presence of an Internal Ribosome Entry Site (IRES) in p53 mRNA that controls Δ160p53 isoform translation. Subjecting cells to endoplasmic reticulum stress showed that eIF2α phosphorylation is a key event leading to cap-independent expression of Δ160p53 during ISR. Additionally, cancer-specific mutations in p53 also enhanced cap-independent translation of Δ160p53 via Δ160p53IRES. An antisense morpholino oligo targeting Δ160IRES efficiently reduced Δ160p53 protein levels and significantly impaired oncogenic functions of Δ160p53. Our data support a model in which an IRES structure in the coding region of p53 is activated under stress conditions, leading to the expression of the oncogenic shorter Δ160p53 isoform, whose structure is affected by cancer-specific mutations in the p53 gene. A better understanding of Δ160p53IRES structure and function may be advantageous for a more efficient therapeutic targeting of p53.
- Regulation of IRES-mediated translation in p53Publication . Fonseca Costa, Inês; Lacerda, Rafaela; López-Iniesta, M.; Romão, Luísa; Candeias, Marco M.The tumor microenvironment is characterized by several stresses impairing canonical translation. However, specific mRNAs harbouring internal ribosome entry sites (IRES), such as several tumour suppressors and oncogenes, can overcome this impairment. The tumor suppressor TP53 gene, an important transcription factor that ensures cellular homeostasis, is frequently mutated in human cancers. Over the years, several p53 isoforms have been identified, which in some cases result from alternative initiation of translation regulated by an IRES. Recently, we have associated mutant p53 “gain-of-function” cancer phenotype, such as enhanced cell survival, invasion, proliferation, and adhesion, with the expression of higher levels of shorter p53 isoforms, such as Δ160p53 isoform.1 Here, we used a bicistronic system containing two reporter luciferases (renilla luciferase and firefly luciferase) to assess IRES-mediated translation. Several p53 mRNA elements were tested in this system and, interestingly, we have found an inhibitory element of IRES-mediated translation. Overall, IRES-regulated translation in malignant cells is used to translate specific proteins that promote cancer progression. Thus, inhibiting translation of oncogenes via IRES could prevent the formation of tumor cells and their adaptation to unfavourable conditions in the tumor microenvironment. 1. Candeias, M. M., Hagiwara, M. & Matsuda, M. Cancer‐specific mutations in p53 induce the translation of Δ160p53 promoting tumorigenesis. EMBO Rep. 17, 1542–1551 (2016).