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- Next-generation sequencing of LDLR and APOB genes in patients with a clinical diagnosis of Familial HypercholesterolaemiaPublication . Alves, A.C.; Bourbon, M.Familial hypercholesterolemia (FH) is a genetic condition characterized by a high cholesterol concentration in the blood. The most frequent causes of FH are inherited defects in the Low Density Lipoprotein Receptor gene (LDLR) but, in a small percentage of patients, mutations in the apolipoprotein B gene (APOB) and in the propotein convertase subtilisin/kexin type 9 gene (PCSK9) are also responsible for FH. These 3 genes are currently studied in the “Portuguese FH study”. From the 563 families with a clinical diagnosis of FH studied only 41% of these have a mutation in one of the 3 studied genes, so other gene defects must exist to explain the cause of hypercholesterolemia in the remaining families. The aim of this study was the exclusion of previously unidentified LDLR and APOB gene defects in 65 severely affected patients, as well as the exclusion of mutations in LDLRAP1 and CYP7A1 genes in patients with possible recessive hypercholesterolemia. The whole sequencing of LDLR and APOB genes of the 65 index patients, without mutations in LDLR or PCSK9 genes or in fragments of exon 26 and 29 of APOB gene was performed. A pool of the 65 DNAs was sequenced by pyrosequecing with custom design primers and a total of 227688 nucleotide reads were obtained, corresponding to a mean coverage of 35x/fragment/individual. CYP7A1 and LDLRAP1 genes were analysed by PCR and direct sequencing. A total of 87 alterations were detected by pyrosequencing. More than half were previously described SNPs and 32 were novel possible pathogenic variants. The majority of these variants (25) were in exons 26 and 29. From the 32 novel variations identified by pyrosequencing only 4 were found by Sanger sequencing. Three alterations (2 novel and 1 described) were found by Sanger sequencing and were not detected by pyrosequencing. Three SNP’s were also studied do to their low alleles estimated. After family studies of these 10 variants, 3 alterations did not co-segregate in the family, 4 alterations were not possible to verify co-segregation and 3 of the alterations found are possibly mutations causing disease, but functional studies are required to prove pathogenicity. No mutations were found in LDLRAP1 and CYP7A1 genes in 10 patients with possible recessive hypercholesterolemia. Patients, in whom it was not possible to find the genetic cause of the hypercholesterolaemia, will require further studies, since all show a severe clinical phenotype of FH.
- Biobancos e Investigação Genética: Orientações ÉticasPublication . Ventura, CéliaNas últimas décadas, muitas instituições de saúde ou académicas foram estabelecendo colecções organizadas de amostras biológicas associadas a dados pessoais. Estes biobancos têm suscitado o interesse da comunidade científica pela sua pertinência relativamente à investigação sobre doenças comuns com impacto em saúde pública. Este interesse levou, inclusivamente, ao aparecimento de vários biobancos privados com fins comerciais. A tendência actual é a construção de biobancos com milhares de amostras para estudar a associação entre variantes genéticos e doenças complexas, a sua interacção com factores ambientais e também com os fármacos, no último caso para garantir uma medicação mais dirigida e eficaz. O sucesso da implementação de um biobanco depende da participação colectiva da sociedade, sendo esta fortemente influenciada por factores socioculturais, tais como a confiança nas instituições e nos seus profissionais. Estes têm a responsabilidade moral de salvaguardar a autonomia e integridade dos participantes em investigação. Uma das formas de respeitar esta autonomia é através do consentimento informado. Contudo, o consentimento informado do participante relaciona-se frequentemente com a utilização primária da amostra, enquanto o seu uso a longo prazo não é mencionado. Assim, é necessário definir princípios para a utilização de amostras antigas em novos estudos, quando um novo consentimento do participante se torna difícil ou impossível de obter. Por outro lado, a obtenção de amostras para biobancos, com o intuito de as utilizar em múltiplos estudos indefinidos à data da colheita, levou a formas alternativas de consentimento informado, consideradas por alguns autores como distorções do conceito original. Neste trabalho é proposto um modelo base de consentimento informado genérico para ser aplicado em associação com medidas adicionais de controlo dos biobancos, como a anonimização reversível ou codificação das amostras, associada a um sistema honest broker, bem como à vigilância por comissões de ética. É também proposto um modelo para a administração de biobancos, assim como critérios para a divulgação e integração responsável dos resultados de investigação na área clínica. A comercialização dos produtos biológicos é outro tema de discussão, pois o seu direito de propriedade e a partilha de benefícios estão ainda por definir na maioria dos países. Muitas organizações nacionais e internacionais elaboraram códigos de conduta, mas as discrepâncias são imensas. O debate social, com a intervenção de eticistas, legisladores e cientistas é necessário para atingir o equilíbrio entre os interesses individuais e colectivos. In the last decades many health services or academic institutions have established organized collections of biological samples associated with personal data. These biobanks have captured the interest of the scientific community for their relevance to the study of common disorders with public health impact. This interest as also led to the emergence of private for-profit biobanks. The trend is to build biobanks with thousands of samples in order to investigate an association of genetic variants with complex diseases and their interaction with environmental factors, as well as with drugs, in the last case to achieve a more direct and efficient medication. The success of biobanks depends on the society’s participation, which is influenced by sociocultural factors like trust in the institutions and in their professionals. These have the moral responsibility of safeguarding the autonomy and integrity of the study participants. One way of respecting this autonomy is through informed consent. However, most often the participant informed consent in genetic research refers to the primary use of the sample whereas its long term use is not addressed. Therefore, it is necessary to define principles for using previously collected samples when a new consent is difficult or even impossible to obtain. Moreover, collection of samples for biobanks with the purpose of using those in several unforeseen studies has also led to alternative forms of informed consent, which are considered by some as distortions of the original concept. A model of a general informed consent is here presented, which is associated with additional forms of biobank control including sample reversible anonymization or codification associated with an honest broker system, as well as surveillance by research ethics committees. A model for biobank administration is also proposed as well as criteria for the responsible disclosing and integration of research results in the clinical setting. Commercialization of biological products is another issue of discussion because in most countries sample property rights and benefit sharing are poorly defined. Guidelines have been proposed by many governmental or non-governmental organizations, although the lack of harmonization is enormous. A societal debate, including ethicists, legislators and scientists is needed to achieve a balance between individual and collective interests.
- A genome-wide association study using a DNA pooling strategy identifies BBS9 and GLIS3 as novel loci influencing patient’s outcome after strokePublication . Manso, H.; Paulos-Pinheiro, S.; Krug, T.; Sobral, J.; Albergaria, I.; Gaspar, G.; Ferro, J.M.; Oliveira, S.A.; Vicente, A.M.Stroke is a major cause of morbidity in developed countries and therefore finding adequate treatments to promote patient’s recovery is a priority task, requiring the elucidation of the molecular pathways influencing brain recovery. Few studies, however, have assessed the role of genes in stroke outcome. This study describes a pilot genome-wide association study (GWAS) to identify genetic factors contributing to patient’s outcome, using a DNA pooling design. Methods: Patient’s outcome was assessed using the modified Rankin Scale (mRS) three months after stroke. Using the 250K Affymetrix GeneChip Mapping Assay® – Nsp I, we compared SNP allele frequencies in a pool of non-disabled stroke patients (N=87, mRS=0), with a pool of severely disabled or deceased patients (N=100, mRS>=3). The 100 most interesting SNPs were selected for validation by individual genotyping. Results: 36 SNPs were validated, showing significant differences between patients with extremely good and extremely poor outcome at three months (1.7x10-4 ).
- Onset of Ciguatera Fish Poisoning and associated dinoflagellates in temperate regionsPublication . Alverca, ElsaCiguatera Fish Poisoning (CFP) is a form of human poisoning, quite common and widespread in tropical and subtropical regions. It results from the ingestion of fish contaminated with toxins produced by benthic dinoflagellates belonging to the genus Gambierdiscus. These toxins, namely the lipid-soluble ciguatoxins (CTXs), are first bioaccumulated in herbivorous fish and transferred to larger carnivorous fish through the marine food web until reaching humans. Although Gambierdiscus dinoflagellates are the only known CTXs producers, in ciguatera-endemic regions they co-occur with other toxic dinoflagellate species of the genera Ostreopsis, Prorocentrum, Amphidinium and Coolia, forming a characteristic benthic/epiphytic dinoflagellate assemblage. From this group, species of Ostreopsis are the ones that raise a major concern as they can produce palytoxin analogues, one of the most potent marine biotoxins. Until recently, the CFP was considered a tropical/subtropical endemic problem. However, a growing number of ciguatera poisoning cases are being reported in Europe and the presence of Gambierdiscus sp. both in the Mediterranean Sea and in Canary Islands has also been detected, suggesting that this problem is already affecting regions in temperate latitudes. Additionally, during the last 15 years, reports of toxic episodes involving benthic dinoflagellates belonging to the ciguatera community, in particular Ostreopsis, have had a marked increase in warm-temperate areas particularly the Mediterranean Sea, affecting several countries of this region like Spain, Italy, France, Greece and more recently Portugal. Although, these phenomena are already recognized as an emergent public health problem in temperate regions, and specific monitoring plans are now being implemented in the mostly affected Mediterranean countries, effective and uniform programs for risk assessment are still lacking. Within this talk, this issue, as well as the challenges posed in the management of the Harmful Benthic Algal Blooms will be addressed.
- Migration solvents for free isocyanates in food contact materials using experimental designPublication . André, Catarina; Curvacho, Carina; Matos, Ana Sofia; Castanheira, IsabelAgglomerated cork stoppers are currently used for still wines, semi-sparkle and gaseous wines, beer and cider. Methylene diphenyl diisocyanate (MDI) is presently the adhesive in use due to its lowest toxicity comparing with toluene diisocyanate (TDI) previously employed. However, free monomeric MDI can migrate from agglomerated cork stoppers to food stuff and this is worrying subject according to food contact materials. The objective of this study is to determine which solvent is better to extract isocyanates from agglomerated cork stoppers, essentially MDI to quantify its free monomer. A long term migration study was performed in order to compare with soxhlet extraction. Firstly a global migration test was performed in order to select the best solvents for soxhlet extraction. A Design of Experiments (DOE) with two factors, solvent and wax (to spike adhesive film), at six and two levels, respectively, was done. Six different solvents were put in contact with adhesive film during ten days at a temperature of forty degrees. Experiments were replicated and repeated three times. Through a TWO-WAY ANOVA the significance of solvents was evaluated and the three better solvents selected. Afterwards, soxhlet extraction was performed with the three better solvents, ethanol, acetonitrile and n-heptane, regarding ANOVA results. The other control factors were cycles’ number (20, 55 and 90) and sample type (natural, agglomerated cork stopper and adhesive film). In a fractional factorial DOE, with three factors at three levels, nine experiments with three replicates were performed. The significance of the factors/interactions was evaluated and the best level’s factors were selected. The Design of Experiments reveals to be a suitable statistical tool to determine the best conditions to measure the migration of free isocyanates from agglomerated cork stoppers to real foodstuff. The best solvent to monitor the migration from cork to wine by soxhlet extraction was ethanol although in long term migration the best solvent was acetonitrile.
- O RNA mensageiro como alvo terapêutico nas distrofias muscularesPublication . Santos, Rosário
- Genetic variation in CHIT 1Publication . Duarte, Ana; Ribeiro, Diogo; Amaral, OlgaChitotriosidase (EC.3.2.1.14) is an enzyme secreted by activated macrophages. This chitinase is useful as a biochemical marker in several lysosomal and nonlysosomal diseases due to its increased activity in such conditions (MIM#600031. CHIT1 variants may account for additional biochemical variability and should be considered when using chitotriosidase activity as a secondary evaluation marker in diagnosis and disease prognosis.
- Novel and rare large deletions in the globin gene clusters causing different types of thalassemiaPublication . Coelho, Andreia; Fernandes, Emília; Batalha-Reis, Ana; Sonesson, Annika; Picanço, Isabel; Miranda, Armandina; Faustino, PaulaThe major component of the red blood cells is hemoglobin A which consists of 2α- and 2β-globin chains encoded by α- and β-globin genes located in two different gene clusters (16p13.3 and 11p.15.5, respectively). Molecular defects (usually point mutation or short deletion) that give rise to a quantitative reduction of the corresponding globin chain, result in a hereditary hypochromic and microcytic anemia called thalassemia. However, rarely, the molecular basis of the pathology could be a large deletion affecting several globin genes and/or their distal regulatory sequence. Four patients with hematological phenotypes suggestive of thalassemia, in whom no globinic molecular abnormalities had been found by standard diagnostic procedures, were screened for deletions in the telomeric region of chromosome 16 and 11, by Multiplex Ligation-dependent Probe Amplification (MLPA) assay. To further characterize the breakpoints of the deletions found, we employed synthetic MLPA probemixes designed in our laboratory, as well as PCR and DNA sequencing. We identified two cases of α-thalassemia caused by two distinct large deletions which remove all α-like structural genes and their distal regulatory sites: both are telomeric, one presents at least 271.14 kb of length and the other, at least, 231 kb. Concerning β-globin cluster screening, two deletions were found: one has at least 186 kb, encloses the entire cluster and its locus control region, and gives rise to a εγδβ0-thalassemia. The other presents at least 3 kb, has its 5’ breakpoint located within the second intron of the β-globin gene and its 3’ end within the L1 repetitive region of the cluster. Both α- and β-cluster larger deletions are novel and were named --CMB/αα and PORTUGUESE εγδβ0-Thal, respectively. The other two smaller deletions, given the uncertainty regarding their breakpoints, might be similar to others already published. In all patients, genotypes are well correlated with the different thalassemic phenotypes presented. MLPA proves to be a useful technique to identify known and unknown large deletions affecting globin gene clusters.
- Desenvolvimento de um ensaio para genotipagem do vírus da Hepatite C numa população de infectados em ambiente prisionalPublication . Avó, Ana Patricia; Pádua, Elizabeth; Crespo, Ana[PT] O vírus da hepatite C (VHC) é caracterizado por uma elevada variabilidade genética traduzida na sua classificação em 6 diferentes genótipos, e em mais de 80 subtipos com distintos padrões de distribuição epidemiológica no mundo. Os vários genótipos estão associados a uma diferente evolução da infecção e progressão da doença, sendo por isso a classificação do VHC a principal ferramenta do clínico para a determinação da dosagem e duração do tratamento dos doentes. O presente estudo pretendeu desenvolver um método alternativo de genotipagem e subtipagem do VHC, no contexto de um laboratório de referência, que permitisse por um lado, reduzir os custos, e por outro lado, obter uma melhoria na classificação do vírus. No presente trabalho foram analisadas amostras de referência e amostras clínicas de 72 reclusos infectados por VHC, com resultados previamente conhecidos dos níveis de RNA e de genotipagem obtidos pelo método comercial LiPA. As amostras foram sujeitas a extracção de RNA e síntese de cDNA, amplificação por heminested PCR e sequenciação, e ainda, a análise filogenética de sequências nucleotídicas das regiões C/E1 e NS5B do VHC. Os resultados foram comparados com os previamente conhecidos obtidos pelo método LiPA. Independentemente das regiões genómicas, a subtipagem do VHC pelo método desenvolvido foi conseguida em 95,8% (69/72) dos casos, uma proporção bastante superior comparativamente à obtida no método LiPA (47,2%, 34/72), mostrando assim um melhor desempenho em identificar ou discriminar o subtipo VHC. Informação molecular conjunta para as duas regiões genómicas foi obtida em 88,9% (64/72) dos casos com identificação de um recombinante intergenótipo 1b/2k e um recombinante intragenótipo 2q/2k para a região C/E1 e 2k/2a para a região NS5B. Adicionalmente, 2 casos apresentaram uma classificação de VHC discordante, 1b/4a e 3a/4a, respectivamente para as regiões C/E1 e NS5B analisadas. Contudo, nestes casos não foi possível descartar a hipótese de possíveis infecções mistas. O elevado desempenho do método desenvolvido na detecção e classificação do VHC pode possibilitar de um modo mais preciso uma correlação clínica com os subtipos virais do VHC, e deste modo viabilizar um melhor entendimento do papel da variabilidade genómica na história natural e progressão da infecção do VHC.
- Influence of LPL, APOAIV, APOAV, APOCIII and USF1 polymorphisms in a Portuguese population with clinical diagnosis of Familial Combined HyperlipidaemiaPublication . Santos, T.; Rato, Q.; Gaspar, I.M.; Bourbon, M.Familial Combined Hyperlipidaemia (FCHL) is a genetic disorder characterized by highly atherogenic profile with presence of sdLDL, hyperlipidaemia (hypertriglyceridaemia and/or hypercholesterolaemia), different lipid profiles in members of the same family and high apoB levels. Some polymorphisms in several genes (LPL -93T>G/D9N, APOAIV Q360H and V13M, APOAV -1131T>C and S19W, APOCIII 3238C>G, USF1s1 and USF1s2) have been associated with higher triglycerides (TG) levels or FCHL. Hypertriglyceridaemia has been suggested by some authors as an independent risk factor for cardiovascular diseases (CVD). The purpose of the present study was to verify if these associations are valid in a Portuguese FCHL population and if the above polymorphisms also affect sdLDL concentration since these particles are formed from triglyceride-rich VLDL (VLDL1). The molecular study of the above polymorphisms was performed in 45 FCHL index patients and 116 relatives by PCR amplification and direct sequencing. Total cholesterol (TC), TG, sdLDL and apoB values were measured in automated analysers. The results were analyzed with SPSS software using t-test. It was found at least one of the described polymorphism in 69 individuals (P1) but not in 92 (P2). We verified that individuals with at least one of these alterations had not only higher TG levels, as we were expecting, but also higher levels of sdLDL (TG: P1=186,1±11,0mg/dL, P2=136,7±7,3mg/dL, p<0,001; sdLDL: P1=33,2±2,3mg/dL, P2:22,6±2,2mg/dL, p=0,002). We didn’t found any significant relation between these polymorphisms and TC (P1=233,9±9,0mg/dL, P2=218,3±6,4, p=0,311). In P1 we also found an association between TG levels and CVD (with CVD: TG=227,5±23,4mg/dL, without CVD: TG=176,5±12,2mg/dL, p=0,036) that was not present in P2 (with CVD: TG=131,4±19,2mg/dL, without CVD: TG=137,3±7,9mg/dL, p=0,984). Our results not only reinforce the idea that hypertriglyceridaemia is a risk factor for CVD as suggested by some authors but also suggest that this condition is responsible for the increase of sdLDL particles in FCHL Portuguese patients.