Browsing by Issue Date, starting with "2017-11-16"
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- Regulation of the alternative splicing of tumor-related RAC1b by signal transduction pathwaysPublication . Gonçalves, Vânia; Matos, Paulo; Pereira, Joana; Henriques, Andreia; Jordan, PeterIntroduction In colon cancer distinct genetic subtypes have been described, one of which involves overexpression of RAC1b, a variant generated by alternative splicing. Aberrant splicing is known to occur in cancer and can be caused by mutation in a gene or splicing factor but also represents a dynamic response to oncogene-induced cellular signaling and in this case it may be pharmacologically targeted. Here we explore how signaling pathways are involved in the deregulation of alternative RAC1b splicing in colorectal tumor cells.
- Human biomonitoring in risk assessment: analysis of the current practice and 1st examples HBM in risk assessments of HBM4EU priority chemicalsPublication . Santonen, T.; Heinälä, M.; Bessems, J.; Buekers, J.; Cornelis, C.; Vermeire, T.; Woutersen, M.; van Engelen, J.; Borges, T.; Rousselle, C.; Ougier, E.; Louro, Henriqueta; Alvito, Paula; Martins, Carla; Assunção, Ricardo; Silva, Maria João Silva; Krul, L.; Pronk, A.; Schaddelee-Scholten, B.; Gonzalez, M.C.; de Alba, M.; Díaz, G.; Castaño, A.; Viegas, S.; Humar-Juric, T.; Kononenko, L.; Abraham, K.; Vinggaard, A.M.In chemicals risk assessment frameworks, the default approach is to assess external intake from different sources of exposure and via different routes of exposure. They are often assessed separately. This approach includes various uncertainties and often overestimates the real uptake since default, conservative estimates are used e.g. for the absorption of the chemical. At the same time, actual (real life) exposure may be underestimated by not taking into account that exposure to a chemical substance may occur from different sources, which may fall under separate legislative frameworks. Examples are triclosan that is used in biocidal products as well as in consumer products and importantly, most if not all chemicals that are produced by workers where at the same time these workers may be exposed as part of the general population. In some cases, other tools to assess exposure via all possible routes may be insufficient; an example is occupational exposure via hand-to-mount exposure, which has been shown to occur for example in the case of many metals, like lead, through contaminated hands. Without biomonitoring, exposure in these cases could become severely underestimated. Human Biomonitoring (HBM) is an important tool to survey the real life body burden – or internal exposure – of humans resulting from ‘total’ exposure to chemicals via different routes (lung, skin, digestive tract) and ‘via’ different legislative frameworks on chemicals. By providing more accurate data on actual body burdens (internal exposure), inclusion of HBM data could improve human health risk assessment for both the general population (exposure via air, consumer products, drinking water and food) as well as for workers (exposure via inhalation and/or skin) separately or as part of the population.
- IRES-dependent translation of shorter p53 isoforms is affected by mutations in p53Publication . Lacerda, Rafaela; Neves, Ana Rita; Maruo, M; Romão, Luísa; Matsuda, M; Candeias, MarcoFull-length p53 (FLp53) is a tumour suppressor protein that has been considered a master regulator of many cellular functions. Several isoforms have been described for p53 so far and some of the functions of shorter p53 isoforms have been elucidated and they are different from and complement FLp53 activity. p53 is the most commonly mutated gene in cancer and depending on its mutation status p53 may act as a tumour suppressor or a proto-oncogene. Recently, we have shown that the most common p53 cancer mutants express a larger number and higher levels of shorter p53 protein isoforms that are translated from the mutated FLp53 mRNA (Candeias et al. EMBO R. 2016). Also, we found that cells expressing these shorter p53 isoforms exhibit mutant p53 “gain-of-function” cancer phenotypes, such as enhanced cell survival, proliferation, invasion and adhesion, altered mammary tissue architecture and invasive cell structures. Here, we found that some of these mutations affect the function of an Internal Ribosome Entry Site (IRES) in p53 mRNA. We investigated which mutations influence — by altering IRES structure and function — IRES-dependent translation of shorter p53 isoforms and to what extent this may lead to the onset or progression of some types of tumours.
- Concordance between variants detected by clinical exome, gene panel and Sanger sequencingPublication . Mendonça, Joana; Silva, Catarina; Theisen, Patrícia; Gonçalves, João; Vieira, LuísIntroduction:Exome sequencing (ES) is becoming a preferred methodology for detecting DNA changes in genetic diseases with no known molecular cause or no definitive diagnosis. This results from the fact that next-generation sequencing technology allows a greater number of bases to be sequenced at an increasingly lower cost. However, sequencing a high number of genes requires an evaluation of the analytical performance of ES before it is used in the clinical setting. Methods: Fifteen genomic DNA samples were used to prepare sequencing libraries with the TruSight One Sequencing Panel (Illumina) consisting of 4813 disease-associated genes ('clinical exome'), according to the manufacturer's procedures. Libraries were sequenced on the MiSeq (Illumina) and the results were analyzed using the MiSeq Reporter and IGV. Variants identified in ES were compared with those validated previously in a subset of genes using the TruSight Cancer gene panel (Illumina) and Sanger sequencing. This study was conducted in 2 phases. In the first, the clinical exome of 9 samples was sequenced and the variants obtained were compared with known variants in 8 genes. In the second phase, 6 samples were sequenced and the variants in 8 genes were analyzed without prior knowledge of the results obtained in the other methods. Furthermore, it was not known that one of these samples had been sequenced in the first phase of the study. Results: In the first phase, ES identified all the exonic (n=41) and intronic flanking (n=15) variants validated in the MSH2, MLH1, APC, MUTYH, BRCA1, BRCA2, STK11 and TP53 genes, while no additional changes have been detected. In the second phase, ES detected a total of 50 variants in MSH2, MLH1, APC, BRCA1, BRCA2, TP53, CDH1 and ATM genes which were found to include each of the 46 variants previously validated and 4 additional changes located outside the genomic regions defined in the gene panel. The same 15 exonic variants were identified in the sample independently processed and sequenced in both phases. Taken together, 87 variants were independently identified using different sequencing approaches. Discussion: The results of this work showed a complete agreement between variants identified by clinical exome, gene panel and Sanger sequencing. Moreover, these results support the notion that the clinical exome panel can also be used as a set of sub-panels of genes applicable to different genetic diseases.
- Behind the curtain: unveiling DIS3L2 role in NMD and human cancerPublication . Costa, Paulo; Saramago, Margarida; Viegas, Sandra; Arraiano, Cecília; Romão, LuísaNonsense-mediated mRNA decay (NMD) is a surveillance mechanism that targets and degrades mRNAs carrying premature translation-termination codons (PTCs), preventing the production of truncated proteins potentially harmful for the cells. In addition to this, several studies have shown that NMD regulates the levels of many physiological mRNAs that encode full-length proteins. Nevertheless, NMD is inhibited in tumor microenvironment and (de)regulates oncogenes and tumor suppressors in several types of cancer. In humans, the mRNA degradation pathways involve exonuclease proteins, such as the DIS3-like protein family (DIS3, DIS3L1 and DIS3L2); however, it is not known whether these proteins are involved in NMD. In order to unveil the role of DIS3L2 in NMD, we performed its knockdown, by RNA interference, in HeLa cells and measured, by RT-qPCR, the mRNA levels and half-lives of various natural NMD targets. Our results show that some NMD targets are highly stabilized in DIS3L2-depleted cells. In addition, mRNA half-life analysis indicate that these NMD targets are in fact direct DIS3L2 substrates. By performing DIS3L2, TUT4 and TUT7 triple knockdown, we also observed that DIS3L2-mediated decay depends on the terminal uridylyl transferases (TUTases) Zcchc6/11 (TUT7/4) activity. Among the NMD targets regulated by DIS3L2, we highlight GADD45A. GADD45A is involved in cell cycle arrest, DNA damage response and apoptotic process. Furthermore, GADD45A deregulation is associated with several types of cancer, such as, esophageal, lung, bladder and pancreatic. Together, our findings establish the role of DIS3L2 and uridylation in NMD and in the regulation of oncogenes and tumor suppressor gene expression. These results might be highly relevant for the advance in diagnosis, prognosis and treatment of many human cancers.
- Genetic modifiers of the intermediate phenotypes in sickle cell anemiaPublication . Aguiar, Laura; Matos, Ângela; Gil, Ângela; Ferreira, Joana; Braga, Lígia; Almeida, Salomé; Kjollerstrom, Paula; Faustino, Paula; Bicho, Manuel; Inácio, ÂngelaSickle cell anemia (SCA) is an inherited blood disorder characterized by the presence of hemoglobin S (HbS). This disease is caused by a single point mutation in the beta-globin gene with a corresponding amino acid substitution at the sixth position of the beta-globin chain. Vaso-occlusion and hemolytic anemia are the major features of this disease, however, SCA patients present clinical and hematologic variability that cannot be only explained by the single mutation. Others genetic modifiers and environmental factors are important for the clinical phenotype. We studied the association between several hematological and biochemical parameters and a set of genetic variants in 26 pediatric SCA patients. Myeloperoxidase (MPO) and placental growth factor (PlGF) were determined by ELISA (R&D Systems Inc.). Amplification of DNA samples for the rs1050829 characterization, in the glucose-6-phosphate dehydrogenase (G6PD) gene, was performed by PCR followed by restriction fragment length analysis. A multiplex PCR assay was used for simultaneous amplification of glutathione S-transferases mu (GSTM1) and theta (GSTT1). All statistical tests were performed with SPSS 24.0 software. Our results show higher levels of MPO (p<0.001) and PlGF (p=0.048) in SCA patients, compared with healthy adult controls. Moreover, in these patients we found associations between: 1) lower levels of total hemoglobin and the GSTM1 null genotype (p=0.044); 2) higher levels of HbS with the rs1050829_G genotype (hemizygous males) in the G6PD gene (p=0.026). We suggest that the mentioned polymorphisms in GSTM1 and G6PD genes may act as genetic modifiers in SCA, which could be useful for the prediction of increased susceptibility to complications. Furthermore, our results reinforce the importance to study biochemical parameters for a better understanding of the clinical outcome of this disease.
- Integrative network approach to identify new players involved in NMD or its regulationPublication . Nogueira, Gonçalo; Pinto, Francisco; Romão, LuísaNonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature translation-termination codons (PTCs). The physiological importance of NMD is manifested by the fact that about one third of all genetic diseases and some forms of cancer are caused by nonsense or frameshift mutations that introduce PTCs, and NMD can modulate the clinical phenotype of these diseases. Noteworthy, in total, genetic diseases attributable to PTCs affect millions of patients worldwide. Recent studies have shown that NMD also targets mRNAs transcribed from a large subset of wild-type genes, shaping their levels. NMD is a complex process where several proteins interact with each other and cooperate to induce degradation of a given transcript. Although this pathway has been extensively studied, the interactions and connectivity among these components is only partly elucidated. Aiming to expand the knowledge about the NMD pathway, we are combining bioinformatics, network analysis and experimental work to identify new proteins involved in NMD or its regulation. Our work, begins with a network analysis approach that integrates publicly available data regarding different types of interactions: 1) protein-protein, 2) kinase-target, 3) phosphatase-target, 4) miRNA-target, 5) transcription factor-target, 6) gene co-expression and 7) ubiquitination-target. Additionally, our network include data regarding known NMD-targets and NMD-triggering features. The generated network will be used to find novel NMD-associated proteins, prioritizing candidates with simultaneous interactions with different mRNA processing pathways (mRNA splicing, mRNA transport, mRNA translation and mRNA decay). Following data integration, we will develop a scoring algorithm to select the most central proteins in the generated network, which can be essential to further understand NMD and its regulation. The predicted candidates will be experimentally validated and their role in NMD will be tested. Due to the diversity of regulatory links integrated in this workflow, we propose it can be applied to find molecular bridges between related biological processes and generate novel hypotheses about the molecular mechanisms co-regulating these phenomena.
- Caracterização de regiões polimórficas em genes ligados à hipertensão - NOS3, G6PD e HBA numa população Moçambicana e numa população portuguesaPublication . Semente, Ildegário; Matos, Ângela; Faustino, Paula; Bicho, Manuel; Inácio, ÂngelaIntrodução: A hipertensão arterial é uma doença multifatorial, de elevada prevalência em Moçambique e Portugal (respectivamente 33,1%[1] e 42,2%[2]). Uma vez que a sintase do óxido nítrico endotelial (NOS3), a glucose-6-fosfato-desidrogenase (G6PD) e a alfa-globina (HBA) têm sido propostas como potenciais moduladoras da hipertensão arterial, pretendem-se neste estudo caraterizar as variantes genéticas mais comuns em duas populações, uma Moçambicana e outra Portuguesa. Material e Métodos: Foram analisadas 22 amostras de DNA provenientes do Hospital Central de Maputo e 87 provenientes do Hospital de Santa Maria. Para a pesquisa do número de repetições em tandem (VNTR) no intrão 4 do gene NOS3 foi usada a PCR, para a pesquisa do SNP rs1050829 no gene G6PD foi usado a PCR-RFLP e para a pesquisa da deleção alfa-talassémica de -3,7kb no agrupamento génico da alfa-globina foi usada uma metodologia de Gap-PCR. As frequências alélicas e genotípicas foram calculadas e analisadas com recurso ao programa estatístico SPSS 22.0 Resultados: Os resultados mostram que em relação ao gene HBA, a população Moçambicana analisada apresenta a frequência de 59% para o alelo mutado e de 41% para o alelo normal, em contraste com o observado para a população Portuguesa onde foi detetada a frequência de 1% para o alelo mutado e 99% para o alelo normal. No gene G6PD, observou-se na população Moçambicana a frequência de 76% para o alelo mutado e 24% para o alelo normal e para a população Portuguesa analisada observou-se que o alelo mutado apresenta a frequência de 1% e o alelo normal a frequência de 99%. Para o VNTR em NOS3, na população Moçambicana os alelos 4a e 4b apresentam respetivamente a frequência de 32% e 68%, enquanto na população Portuguesa os mesmos alelos apresentam respetivamente a frequência de 12% e 88%. Discussão: Estes resultados preliminares mostram a caracterização da frequência de regiões polimórficas em três genes potencialmente influentes no desenvolvimento da hipertensão em duas populações distintas. Encontra-se em estudo um outro conjunto de indivíduos controlos, não hipertensos, que permitirão através de estudos de associação avaliar a contribuição dos referidos polimorfismos para esta patologia. [1] Damasceno A et al. Hypertension 2009; 54:77. [2] Polonia J et al. Journal of Hypertension 2014; 32:1211.
- Regulatory RNAs targeted by Copy Number Variation in Autism Spectrum DisorderPublication . Marques, Ana Rita; Martiniano, H.; Santos, J.X.; Asif, M.S.; Oliveira, G.; Romão, Luísa; Vicente, AstridIntroduction: Autism Spectrum Disorder (ASD) is a highly heterogeneous neurodevelopmental disorder with an unclear etiology. Genetic factors are estimated to account for ~50-80% of the familial ASD risk but most of the genetic determinants are still not known. Several copy number variants (CNVs) targeting ASD candidate genes explain some ASD cases. Still, further exploration of noncoding RNAs targeted by CNVs is necessary. MicroRNA (miRNA) and long noncoding RNA (lncRNA) are regulatory molecules, abundantly expressed in the brain, that play an important role during early stages of neural development. Thus, they are strong candidates for ASD. The goal of this work is to identify miRNA and lncRNA genes targeted by CNVs in a cohort of ASD patients and examine their target genes and biological pathways. Methods: We compared the frequency of miRNA and lncRNA genes targeted by CNVs in a cohort of 2446 ASD subjects and 9649 ancestry-matched control subjects. Genetic data from ASD patients was obtained from the Autism Genome Project and the control group from the Database of Genomic Variant (DGV). Both cases and controls were quantified using the same detection method. AGP data was transformed to hg19 annotation followed by functional annotation using the most recent dataset from MIRBASE. Statistical analysis was performed using Fisher’s exact test followed by Bonferroni correction (p-value<0.05). Results: We found 9 miRNAs exclusively targeted by CNVs in ASD subjects and 7 miRNAs more frequently targeted by CNVs in ASD subjects, when compared to controls. From these, only 2 were already known to be associated with ASD. Interestingly, we identified 4 novel miRNAs associated with ASD that were previously described to be associated with Schizophrenia, a disorder that presents some phenotypic overlap with ASD. Putative targets of these 16 miRNAs were enriched for ASD risk genes described in SFARI database. Gene enrichment analysis indicates that these genes are involved in neurodevelopmental processes, which is consistent with literature. In addition, we also found 102 novel lncRNAs more frequently targeted by CNVs in ASD. Discussion: These results support our hypothesis that genetic variants targeting noncoding regulatory RNAs are involved in ASD pathophysiology. This innovative approach will allow the identification of novel biomarkers and drug targets in ASD, which can contribute to a better diagnosis and treatment.
- Shorter p53 isoform expression through na Internal Ribosome Entry Site (IRES) in p53 mRNAPublication . Neves, Ana Rita; Lacerda, Rafaela; Marques-Ramos, Ana; Romão, Luísa; Matsuda, M; Candeias, MarcoThe tumour suppressor p53 gene is one of the most studied cancer-related genes. So far, many p53 isoforms have been identified either resulting from alternative splicing, alternative translation or alternative promoter usage. It is known that cap-dependent translation is repressed under stress conditions to preserve energy. Therefore, other translational mechanisms are required to keep the synthesis of stress-response proteins. Internal Ribosome Entry Sites (IRESes) were first discovered in viruses, and then observed in eukaryotes, as secondary structures present in RNA that were capable of recruiting ribosomes to the vicinity of an initiation codon inserted in an optimal environment allowing cap-independent translation of mRNAs. Translation of Δ40p53, a p53 isoform, is one example of this non-canonical mechanism due to the presence of an IRES near an alternative initiation codon (AUG40). Here, we will present a new IRES in p53 mRNA, including details on the localization and regulation of this IRES under normal and stress conditions.