DGH - Apresentações orais em encontros internacionais
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- Molecular characterization of the Portuguese patients with defects in GlcNAc-phosphotransferase: a key enzyme in the M6-P dependent lysosomal traffickingPublication . Coutinho, Maria Francisca; Encarnação, Marisa; Gomes, Rui; Prata, Maria João; Lacerda, Lúcia; Bargal, Ruth; Filocammo, Mirella; Raas-Rothschild; Tappino, Barbara; Laprise, Cathrine; Sirois-Gagnon, D.; Costa, Roberto; Ribeiro, Helena; Lopes, Lurdes; Alves, SandraIntroduction: GlcNAc-phosphotransferase is one of the enzymes responsible for the formation of M6P residues and plays a key role in lysosomal trafficking, since most soluble acid hydrolases reach these organelles through the M6P pathway. It is composed of six subunits (α2β2γ2), products of two genes recently cloned: GNPTAB (mutated in mucolipidosis II/IIIA patients) and GNPTG (mutated in MLIIIC patients). Methods: Using both gDNA and cDNA extracted from patient’s fibroblasts, we performed a molecular study of both genes in 13 MLII/III patients (10Portuguese, 1Finnish, 1Spanish of Arab origin and 1Indian). Expression studies were performed by quantitative real-time PCR. Results: We identified 11 different mutations, 8 of them novel: 6 in the GNPTAB gene (c.121delG;c.440delC;c.2249_50insA;W81L;I403T and E667) and 2 in the GNPTG gene (c.610-1G.T and c.639delT). Interestingly, although the MLII-causing mutations have been mostly found to be private or rare, there is one (c.3503_3504delTC) that shows a broad distribution having been detected among different populations. This same mutation was also the most frequent one in our patients. Such distribution pattern prompted us to perform a haplotypic study. We analysed 37 patients (23Italians, 8Arab Muslims, 1Turkish and 5Portuguese) for 3 intragenic polymorphisms and 2 microsatellite markers flanking the GNPTAB gene, identifying a common haplotype. Regarding the mRNA expression studies, real-time results suggest the existence of feedback regulation mechanisms between α/β and the γ subunits. Discussion/Conclusion: This work enabled the establishment of a strong genotype-phenotype correlation, which is of crucial importance to an improved genetic counselling for ML families. The sharing of an ancestral haplotype by patients carrying the deletion implies a common origin of this mutation, while the higher level of diversity observed at the most distant locus indicates that it is a relatively ancient one. The developed strategies constitute valuable tools that allow carrier detection and prenatal- molecular diagnostics of these diseases.
- Developmental follow-up of 90 adolescents and adult PKU patients: results and challenges of a multidisciplinary approachPublication . Carmona, CarlaIntroduction: Severe neurological disability caused by Phenylketonuria (PKU) can largely be prevented by a strict reduced phenylalanine (Phe) diet started in the neonatal period. However, the deficits observed in specific domains of school performance and a specific profile in intellectual quotient tests (IQ) seemed to suggest a degree of neurobiological impairment. In fact, early-treated PKU patients are, in some cases, referred as having a slight decrease in IQ coupled with impairments in specific aspects of cognition, including specific deficits in executive function. The main purposes of this study was to characterize our adolescent and adult PKU patients and understand the way they adapt to this chronic condition at different ages and in the different contexts of life. Methods: We studied 90 patients aged 12 – 31 years. We considered the quality of dietetic control (the annual medians of Phe) as independent variables. The treatment outcome was evaluated considering IQ as a global value, the subtest profile in IQ tests, the level of school education attained and their professional career. Results: Although their global IQ levels are in the normal range, we found a profile of cognitive and behavioural difficulties. These difficulties condition the level of studies attained and their professional careers, suggesting the need special multidisciplinary supervision through life.
- A novel large alpha zero-thalassemia deletion found in a Portuguese 32 years old womanPublication . Reis, Ana; Coelho, Andreia; Faustino, Paula; Reichert, Alice; Meireles, Flora; Brito, Ana; Miranda, Armandina; Matos, Susana; Júnior, Esmeraldina
- Genotoxicidade da MCLR em vários modelos biológicos: linhas celulares, eritrócitos de murganho e linfócitos humanosPublication . Dias, Elsa; Louro, Henriqueta; Pereira, Paulo; Silva, Maria JoãoO conhecimento sobre as propriedades toxicológicas das microcistinas é ainda bastante limitado, em particular no que respeita à sua actividade cancerígena. É globalmente aceite que as microcistinas são promotores tumorais, mas é ainda ambígua a sua eventual genotoxicidade. Alguns autores defendem que as microcistinas são genotóxicas e que induzem quebras no ADN, outros defendem que as microcistinas não são genotóxicas. Alguns destes resultados são dificilmente comparáveis porque foram obtidos através de metodologias distintas, nomeadamente no que respeita ao tipo de toxina usado, ao modelo celular testado e ao método para avaliação de efeito genotóxico aplicado. A nossa equipa tem vindo a estudar as propriedades genotóxicas da MCLR em diversos modelos biológicos: linha celular Vero-E6 (rim de macaco), linha celular HepG2 (hepatoma humano) e eritrócitos de murganho. Para tal temos usado dois tipos de abordagem metodológica: o Teste do Cometa, que avalia danos ao nível da cadeia de ADN, e o Ensaio do Micronúcleo, que avalia a actividade clastogénica (quebra cromossómica) ou aneugénica (perda de cromossomas). Naqueles modelos demonstrámos que a MCLR não induz danos no ADN pelo teste do Cometa mas induz o aumento da frequência de micronúcleos in vitro e in vivo. Nesta apresentação iremos discutir os nossos resultados mais recentes acerca do efeito da MCLR na indução de micronúcleos em linfócitos humanos e comparar com os resultados publicados por outros autores. Iremos também discutir o mecanismo de acção genotóxica da MCLR e a sua importância para a avaliação do risco de exposição humana a água contaminada com cianobactérias produtoras de microcistinas.
- A multiplex assay for X-linked intellectual disability assessmentPublication . Jorge, Paula; Marques, Isabel; Oliveira, Bárbara; Santos, RosárioX-linked intellectual disability (XLID) represents a common cause of monogenic mental retardation, where X-linked conditions are easily identified in affected males, who inevitably manifest a phenotype when harboring a mutant allele, due to their hemizygozity (Chiurazzi et al., 2008). Among the genetic causes involved in XLID, mutations in the Fragile Mental Retardation 1 (FMR1), AF4/FMR2 family member 2 (FMR2) and Aristaless Related Homeobox (ARX) genes emerge as important causes. FMR1 (FRAXA, locus A) and FMR2 (AFF2, FRAXE, locus E) contain polymorphic repetitive regions susceptible to suffer dynamic mutations, a process that may give rise to pathogenic expansions. The expansion to over 200 [CGG] triplets in the FMR1 gene, known as full mutation, is associated with the Fragile X Syndrome [FXS; MIM#300624], the most common form of familial severe intellectual disability (ID). In the case of the ARX gene, different mutational phenomena occur that include insertions, deletions, duplications, missense, nonsense and splice mutations. This makes it difficult to establish genotype/phenotype correlations and concomitantly clinical and molecular guidelines for the molecular analysis of ARX gene (Shoubridge et al., 2010). With the aim to perform pre-screening in ID populations, a multiplex molecular test was developed based on basic PCR methods. The method was applied in intellectually-disabled individuals after exclusion of FMR1 full mutation, focusing on mutational hotspots within the FMR1, FMR2 and ARX genes.
- Nanosized Titanium Dioxide Particles: Evaluation of Genotoxic Effects in Human LymphocytesPublication . Tavares, Ana; Antunes, Susana; Louro, Henriqueta; Lavinha, João; Silva, Maria JoãoThe number of consumer products containing nanomaterials (NM) in the European market showed a 6-fold increase in 2010 (RIVM report, 2010), reflecting the growing relevance of nanotechnology and the broad field of applications of NM across consumer, medical and industrial products. Although the use of NM may offer enormous benefits, it may also pose risks to human health, especially to workers who may face higher exposure, and to environment. Several studies have reported that the greater surface area per mass renders NM more reactive than larger-sized particles of similar chemistry. Size, surface properties, agglomeration state, biopersistence and dose are also likely to modify cell responses to NM, presenting a challenge to the assessment of their potential hazards to human health. Titanium dioxide (TiO2) and zinc oxide (ZnO) nanoparticles (NP) are frequently used in sunscreens and cosmetics. Although cytotoxic and genotoxic properties of TiO2-NP and ZnO-NP have been investigated, conflicting results have been reported. Differences inherent to cell lines, NPs characterization, dispersion protocols, exposure times and assays, together with the lack of positive NM controls have lead to difficulties in the toxicity assessment of those NM. As a part of a larger project (www.nanogenotox.eu), aimed at establishing a robust methodology to evaluate the potential genotoxicity of manufactured NM, the objective of the present work was to characterize the potential genotoxic effects of TiO2-NP (anatase, hydrophilic rutile, hydrophobic rutile and rutile/anatase) in primary cultures of human lymphocytes. The cytokinesis-block micronucleus (CBMN) assay was carried out according to OECD guidelines. Dispersions of each NP were freshly prepared and cultures were exposed to NP concentrations (5-250 μg/mL), during 30h. Concurrent control cultures were processed: vehicle control, positive control (mytomicin C, MMC) and a reference NP (ZnO-NP). The results show that none of the four TiO2-NP tested induced a dose-related increase in MN frequency in lymphocytes. Likewise, the cytokinesis-block proliferation index (CBPI) was not significantly affected by TiO2-NP treatments, indicating no influence on cell cycle progression. As to the positive control, MMC induced a significant increase in the frequency of MN and a concomitant decrease in the CBPI, whereas ZnO-NP caused a decrease in the CBPI without affecting the frequency of MN. In conclusion, the results suggest that the tested TiO2-NP are not clastogenic or aneugenic in human lymphocytes, under the selected test conditions. Further data using different cell types and other endpoints, together with the study of in vivo genotoxic and toxicokinetics parameters, are expected to clarify if these TiO2-NP can be considered as non-genotoxic to humans.
- Alternative splicing of tumour-related Rac1b is regulated by upstream signalling pathwaysPublication . Gonçalves, Vânia; Matos, Paulo; Jordan, PeterThe small GTPase Rac1 regulates signalling pathways controlling actin-dependent cell motility as well as gene transcription. An alternative splicing variant Rac1b is overexpressed in a subset of colorectal tumours and cooperates with mutant B-Raf to sustain tumour cell viability. The alternative splicing mechanism regulating Rac1b expression involves two antagonistic splicing factors, ASF/SF2 and SRp20. Using a Rac1 minigene approach and siRNA-mediated depletion, we identified ASF/SF2 as an enhancer of endogenous Rac1b splicing whereas SRp20 acts as a silencer. Inhibition of the PI3-kinase pathway increased protein levels of ASF/SF2 and promoted Rac1b generation. By contrast, depletion of endogenous protein kinase SRPK1 led to decreased Rac1b expression. Together, these data indicate that altered upstream signaling pathways in colorectal cancer cells will target splicing factors that regulate alternative splicing of the small GTPase Rac1.
- Disclosing effects of tobacco smoke on occupationally exposed workers at Lisbon restaurantsPublication . Pacheco, Solange; Simoes, Tania; Torres, Vukosawa; Lopes, Carlos; Almeida, Bugalho; Penque, DeborahThis study represents a global assessment of IAQ in Lisbon smoking entertainment places and health effects on exposed workers. Most importantly, it may contribute to the better understanding of pathogenesis mechanisms due to SHS exposure. In addition it may lead to the discovery of specific biomarkers for occupational SHS-exposure that might precede respiratory diseases on their employees and promote more effective therapies.
- Analysis of the divergences in the nonsense-mediated mRNA decay mechanism among two globin transcripts highly conservedPublication . Pereira, Francisco JC; Romão, LuísaNonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature termination codons (PTCs, or nonsense codons), which has been highly conserved during evolution. Knowing that human a- and b-globin genes descend from a common ancestral, the aim of this work was to understand if both encoded mRNAs carrying a nonsense mutation, share parallel NMD profiles. We have previously shown that human b-globin mRNAs carrying a PTC located in close proximity to the translation initiation AUG codon escape NMD. This was called the “AUG-proximity effect”. The present work illustrates that the extension of the AUG-proximity effect, i.e. to what position in the open reading frame (ORF) an AUG-proximal PTC does not trigger NMD, is different between human a- and b-globin mRNAs. Remarkably, our data also demonstrate that, contrary to what occurs in the b-globin transcripts, a-globin mRNAs carrying an AUG-proximal PTC allow for efficient translation re-initiation, although it only partially explains their NMD resistance. In addition, our results reveal that in the a- and b-globin transcripts, the extension of the AUG-proximity effect is determined by the ORF sequence. Furthermore, we show how the mRNA secondary structure, which is affected by the ORF sequence, determines the AUG-proximity effect extension. Our data point out that the time taken to translate the short ORF, affected by its sequence and stability, besides being involved in modulating translation re-initiation, also plays an important role in establishing the extension of the AUG-proximity effect. Thus, although both mRNAs are evolutionarily related they show different features in their NMD response.
- A Rare Disease Patient ManagerPublication . Lopes, Pedro; Mendonça, Rafael; Rocha, Hugo; Oliveira, Jorge; Vilarinho, Laura; Santos, Rosário; Oliveira, JoséThe personal health implications behind rare diseases are seldom considered in widespread medical care. The low incidence rate and complex treatment process makes rare disease research an underrated field in the life sciences. However, it is in these particular conditions that the strongest relations between genotypes and phenotypes are identified. The rare disease patient manager, detailed in this manuscript, presents an innovative perspective for a patient-centric portal integrating genetic and medical data. With this strategy, patient’s digital records are transparently integrated and connected to wet-lab genetics research in a seamless working environment. The resulting knowledge base offers multiple data views, geared towards medical staff, with patient treatment and monitoring data; genetics researchers, through a custom locus-specific database; and patients, who for once play an active role in their treatment and rare diseases research.