MPSBase : banco de dados compreensivo de genes diferencialmente expressos em mucopolissacaridoses

As Mucopolissacaridoses (MPSs) são doenças de acúmulo lisossomal causadas pela deficiência de enzimas necessárias para o metabolismo de glicosaminoglicanos (GAGs). Essas e outras doenças raras têm ganhado uma significativa melhoria nos processos de pesquisa e diagnóstico com o advento das tecnologias de análise de DNA em larga escala. Para doenças monogênicas, como as MPSs, diferentes pesquisas têm focado na identificação de perfis de transcrição gênica em resposta à deficiência enzimática. Atualmente, há 13 estudos publicamente disponíveis avaliando os perfis de transcrição para 6 tipos diferentes de MPS. A análise de dados de transcriptoma requer conhecimentos em programação, o que pode dificultar a análise.Para mitigar essa barreira de acesso à informação, foi desenvolvido um banco de dados de interface amigável, abrangendo todos esses estudos e foi disponibilizado para acesso pela internet. Combinando diferentes grupos experimentais par-a-par, chegamos a mais de 50 comparações entre diferentes condições da doença, assim como grupos controle. As análises foram realizadas pelo R usando os pacotes limma, affy e oligo para análises de microarranjo e edgeR para RNA-Seq. Todos os dados de microarranjo foram normalizados com o método média robusta multi-arranjo (RMA) e o valor p foi corrigido pela taxa de falsas descobertas (FDR). Utilizando esses métodos, foram identificados os genes diferencialmente expressos (DEGs) e com esses foi feito o enriquecimento de vias e termos ontológicos com os pacotes enrichKEGG e ClusterProfiler. Com o intuito de melhorar a experiência do usuário, foram incluídas representações gráficas para ambos DEGs e vias e termos enriquecidos

Genetic Modulation of Cerebral Vasculopathy in Children with Sickle Cell Anemia

Palestras de difusão da cultura científica dirigidas aos colaboradores internos do INSA, podendo incluir participantes externos envolvidos nos estudos e colegas de investigação da área de trabalho em questão.Sickle cell anemia (SCA) arises from homozygosity for the mutation c.20A>T in the HBB gene which originates hemoglobin S (HbS). In hypoxic conditions, HbS polymerizes inside erythrocytes deforming them and ultimately leading to hemolysis and vaso-occlusion. SCA shows a multifactorial-like behaviour with a high heterogeneity of clinical features, with stroke being the most severe of them. This heterogeneity may arise from underlying genetic modifiers, namely those affecting vascular hemostasis. These include genes like the ones encoding VCAM-1 and its ligand integrin α4 (expressed in activated human endothelium and leucocytes/stress reticulocytes, respectively), but also eNOS (expressed in human endothelium and regulating vascular tone). The aim of this study was to identify putative genetic modulators of stroke risk by analyzing 70 pediatric SCA patients, grouped according to their degree of cerebral vasculopathy. Molecular analysis was performed using Next-Generation Sequencing (NGS) and Sanger Sequencing. R software was used for statistical analyses and association studies. In silico studies were performed using PHASE, TFbind, PROMO and Human Splicing Finder software tools. We identified six different VCAM1 promoter variants and seven haplotypes. The VCAM1 promoter rs1409419_T allele was associated with stroke events, while one VCAM1 promoter haplotype was found to be protective of stroke. In the ITGA4 gene, forty variants were found, six of them novel. All patients presented with at least one variant in this gene. We observed co-inheritance of specific sets of ITGA4 variants indicating the presence of haplotypes not previously described. Three NOS3 variants were analysed and seven haplotypes were identified. The NOS3 promoter rs2070744_C allele was associated with stroke events, while the intron 4 VNTR 27bp_4a allele was found to be in association with risk of stroke. Our results reinforce the role of endothelial molecules and blood cell interaction in SCA severity. The association between specific variants in VCAM1 and ITGA4, as well as in NOS3, with certain cerebral vasculopathy predictors further enhances their putative modulating effect on pediatric stroke severity and prognosis. These findings provide additional clues on the SCA pathophysiology and uncover features of both genes that may prove to be crucial as potential therapeutic targets.N/

Paediatric cerebral vasculopathy in sickle cell anaemia: contribution of genetic modifiers

Sickle cell anaemia (SCA) arises from homozygosity for the mutation c.20A>T in the HBB gene. However, it shows a multifactorial-like behaviour with high heterogeneity of clinical features. Cerebral vasculopathy (CVA), namely paediatric ischemic stroke, is one of its most devastating consequences. The risk of CVA development, specifically stroke or silent cerebral infarction, may be modulated by underlying genetic modifiers, for example those affecting vascular homeostasis. In this study, we aimed to investigate the impact of variants in genes related with endothelial adhesion (VCAM1 and ITGA4) and nitric oxide metabolism (NOS3) on CVA in a group of 70 SCA children well characterized according to their CVA degree. In addition, the effect of the same genetic variants on biochemical/haematological biomarkers of chronic haemolysis was also analysed. Moreover, we also evaluated the putative additional modulating role of variants previously identified as stroke risk factors by genome-wide associated studies: GOLGB1 Y1212C, ENPP1 K173Q and PON1 Q192R. Molecular analysis was performed using PCR, PCR-RFLP, next-generation sequencing and Sanger sequencing. SPSS software was used for statistical analyses and association studies. One of the seven VCAM1 promoter haplotypes found and the VCAM1 promoter rs1409419_T showed association with moderate to high time-averaged mean of maximum velocity in the middle cerebral artery. The same association was observed for the variant ENPP1 K173Q. On the other hand, we observed that ITGA4 variants rs113276800_A and rs3770138_T were associated with stroke events. As for NOS3, one of the six haplotypes and the intron 4 VNTR_4b allele (5 repeats) were associated with lower risk of silent cerebral infarction. Chronic haemolysis biomarker levels also seemed to be influenced by genetic variants. LDH levels was higher in the presence of VCAM1 promoter rs1409419_T and one VCAM1 haplotype but lower in patients with one of the two ITGA4 haplotypes found. Genetic modulation also occurred in total bilirubin levels, which were higher in association with VCAM1 rs3783613_C allele. Our results, namely the association between specific variants with certain cerebral vasculopathy predictors further enhances their putative modulating effect on SCA paediatric stroke risk, severity and prognosis. These findings provide additional clues on the SCA pathophysiology and uncover features in these genes that may prove to be crucial as potential therapeutic targets.INSA_202DGH720 and ISAMBinfo:eu-repo/semantics/publishedVersio

Genetic modulation of stroke in children with sickle cell anaemia

Sickle cell anaemia (SCA) is an autosomal recessive genetic disease that leads to the synthesis of haemoglobin S (HbS). The pathophysiology of the disease is centred on HbS polymerization inside the red blood cells, which become sickle-shaped (SSRBCs), rigid, viscous and adherent-prone to the vascular endothelium, favouring the occurrence of chronic haemolysis and vaso-occlusion. The main vascular problems of SCA arise from several pathways including endothelial dysfunction and nitric oxide (NO) metabolism. Children with SCA have a much higher risk (11% by age 20 years) of developing stroke or silent cerebral infarcts (up to 37%) than the general paediatric population. Abnormal interactions between SSRBCs and the cerebral arterial endothelium lead to endothelial injury, vaso-occlusion and tissue ischemia and result in cerebral vasculopathy (CVA) through a yet unknown pathophysiological mechanism. Current risk screening strategies rely mainly on imaging techniques (transcranial Doppler ultrasonography and magnetic resonance imaging) and children with altered results undergo regular blood transfusion and/or hydroxyurea therapy to reduce stroke risk/recurrence. However, we need more specific/sensitive biomarkers for stroke prediction/prognosis. Genetic modulators may be paramount in SCA pathophysiology and in CVA severity. They include variants in VCAM1 (endothelial dysfunction), ITGA4 (cell-cell adhesion), and NOS3 (nitric oxide metabolism. The main goals of this work are: a) improve the knowledge on the genetic architecture of paediatric cerebral vasculopathy in SCA; b) assessing the consequences of those genetic variants on gene expression/protein function; c) identify genotypic/phenotypic markers of SCA sub-phenotypes; and d) analyse their potential as genetic modulators of disease severity. This would be crucial in assessing potential pharmacological targets specifically aimed to the vascular system and instrumental for the design of novel preventive, prophylactic or therapeutic strategies.INSA_202DGH72 and ISAMBN/

Phenolic, dietetic fibre and sensorial analyses of apples from regional varieties produced in conventional and biological mode

 Guiné R P F1, Sousa R2, Alves A2, Teixeira L2, Figueiredo C2, Fonseca S2, Soares S2, Sousa I2, Almeida P2, Correia A C2, Jordão A M1, Lopes A D3, Ferreira D1 (1. CI & DETS, Instituto Politécnico de Viseu, ESAV, Portugal;2. Instituto Politécnico de Viseu, ESAV, Portugal; 3. Direcção Regional de Agricultura e Pescas do Centro, DSAP, DPAP, Viseu, Portugal) Abstract: In the present work apples from eleven regional varieties originating from Portugal were studied in terms of their content in phenolic compounds and dietetic fibres (soluble and insoluble).  In some cases, apples from two different production modes were analysed: conventional and biological.  Some of the most commercialized apples in Portugal were also studied for comparison purposes.  In addition, a sensorial evaluation of some of the varieties was performed.  From the present work it was possible to conclude that the regional varieties studied contain higher amounts of dietetic fibre than those of commercial varieties and other fruits.  With respect to the content in total phenolic compounds, the apples of regional varieties showed values clearly higher than those quantified in commercial varieties, and being concentrated mainly on the peel, then on the seeds, and finally on the pulp.  From the sensorial analyses was possible to infer that the regional varieties which were more appreciated were Bravo de Esmolfe and Camoesa de Alcongosta. Keywords: apple, conventional production, biological production, soluble fibre, insoluble fibre, polyphenols, sensorial analysis, portugal Citation: Guiné R P F, Sousa R, Alves A, Teixeira L, Figueiredo C, Fonseca S, Soares S, Sousa I, Almeida P, Correia A C, Jordão A M, Lopes A D, Ferreira D.  Phenolic, dietetic fibre and sensorial analyses of apples from regional vareties produced in conventional and biological mode.  Agric Eng Int: CIGR Journal, 2010, 12(2): 70-78.   &nbsp

Fetal hemoglobin level and stroke risk in children with sickle cell anemia

21ª Reunião da Sociedade Portuguesa de Genética Humana, 16-18 nov 2017Sickle Cell Anemia (SCA) is a hereditary anemia caused by a missense mutation in HBB and it is characterized by chronic hemolysis, recurrent episodes of vaso-occlusion and infection. Cerebral vasculopathy is one of the most devastating complications of the disease and even young children with SCA have a high risk of stroke. It is known that both environmental and genetic determinants are able to modulate the onset, course and outcome of the disease. Among those, the level of fetal hemoglobin (HbF) has been proposed as the most significant disease modulator. Thus, in this work, we aimed to investigate if the level of HbF in SCA children is related with the risk of stroke and if it is modulated by variants in genes, such as HBG2, BCL11A, HBS1L-MYB, and KLF1. Sixty-seven children (3 years of age) with SCA were enrolled in this study. Hematological and imaging data were retrospectively obtained from patients’ medical records at Greater Lisbon area hospitals. Patients were grouped according to their degree of cerebral vasculopathy evaluated by transcranial Doppler velocities and magnetic resonance imaging. Molecular analyses were performed using Next-Generation Sequencing, Sanger sequencing and PCR-RFLP. In silico studies and statistical analyses were done using the PolyPhen-2 and SPSS softwares, respectively. The association studies revealed that low HbF levels were associated with stroke events in SCA children (p=0.005). At the molecular level, it was observed that patients with the rarest genotypes in HBG2 (rs7482144_TT+TC) presented higher levels of HbF (p=0.031). Additionally, the rs11886868_C and the rs4671393_A alleles in BCL11A also seemed to predispose to higher HbF levels. Moreover, eleven distinct variants in KLF1 were detected (one of them novel, the p.Q342H) with 83% of the patients having at least one variant in this gene. The group of patients who have co-inherited the above mentioned variants in HBG2 and BCL11A together with at least one KLF1 variant presented the highest HbF levels (p=0.021). Our results corroborate previous studies suggesting that a low level of HbF in SCA patients is a risk factor for stroke. Furthermore, we report for the first time the importance of KLF1 variants in combination with other genetic modifiers to the final phenotypic expression of HbF in SCA children with different degrees of cerebral vasculopathy. Consequently, this study allowed the delineation of a genetic pattern with prognostic value for SCA.info:eu-repo/semantics/publishedVersio

Avaliação das medidas lineares e angulares de cavalos de vaquejada em Araguaína, Tocantins

Objetivou-se caracterizar morfologicamente através da metodologia das proporções corporais e angulares para cavalos de vaquejada na região de Araguaína, Tocantins. Foram utilizados 63 animais, de ambos os sexos, sendo mensuradas 17 medidas lineares e 8 angulares, utilizando-se de um hipômetro, fita métrica e um artrogoniômetro. Os resultados observados no estudo, demonstraram uma grande variação na estatura, peso, perímetro torácico, peso vivo, garupa e largura do peito, e houve diferença entre os sexos, principalmente para estatura, peso, perímetro torácico e garupa dos animais, caracterizando-os em animais de impulso, explosão e velocidade, como também de animais de trabalho e de carga (tração). Possivelmente essa diferença possa está associada a maior pressão de seleção das fêmeas, uma vez que a intensidade de seleção nas mesmas é maior que nos machos. Não foi observada uma associação favorável e alta entre medidas dos ângulos dos membros traseiros e dianteiros, o que de certa forma poderia favorecer melhor marcha e andamento, bem como comodidade. Assim, recomenda-se que a seleção de animais seja também direcionada para comodidade e andamento dos animais