Browsing by Author "Morais, Anabela"
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- 2-DIGE of Red Blood Cells from Sickle-Cell Disease Patients with Severe Vaso-Occlusion.Publication . Vaz, Fátima; Charro, Nuno; Morais, Anabela; Lavinha, João; Penque, Deborah
- Aplicação de scores de gravidade à anemia das células falciformes: um problema ainda por resolverPublication . Coelho, Andreia; Dias, Alexandra; Morais, Anabela; Nunes, Baltazar; Faustino, Paula; Lavinha, João
- Biomarkers and genetic modulators of cerebral vasculopathy in sub-Saharan ancestry children with sickle cell anemiaPublication . Silva, Marisa; Vargas, Sofia; Coelho, Andreia; Ferreira, Emanuel; Mendonça, Joana; Vieira, Luís; Maia, Raquel; Dias, Alexandra; Ferreira, Teresa; Morais, Anabela; Mota Soares, Isabel; Lavinha, João; Silva, Rita; Kjollerstrom, Paula; Faustino, PaulaWe investigated biomarkers and genetic modulators of the cerebral vasculopathy (CV) subphenotype in pediatric sickle cell anemia (SCA) patients of sub-Saharan African ancestry. We found that one VCAM1 promoter haplotype (haplotype 7) and VCAM1 single nucleotide variant rs1409419_T were associated with stroke events, stroke risk, as measured by time-averaged mean of maximum velocity in the middle cerebral artery, and with high serum levels of the hemolysis biomarker lactate dehydrogenase. Furthermore, VCAM-1 ligand coding gene ITGA4 variants rs113276800_A and rs3770138_T showed a positive association with stroke events. An additional positive relationship between a genetic variant and stroke risk was observed for ENPP1 rs1044498_A. Conversely, NOS3 variants were negatively associated with silent cerebral infarct events (VNTR 4b_allele and haplotype V) and CV globally (haplotype VII). The -alpha3.7kb–thal deletion did not show association with CV.However, it was associated with higher red blood cell and neutrophil counts, and lower mean corpuscular volume, mean corpuscular hemoglobin and red cell distribution width. Our results underline the importance of genetic modulators of the CV sub-phenotype and their potential as SCA therapeutic targets. We also propose that a biomarker panel comprising biochemical, hematological, imaging and genetic data would be instrumental for CV prediction, and prevention.
- Early modification of sickle cell disease clinical course by UDP-glucuronosyltransferase 1A1 gene promoter polymorphismPublication . Martins, Rute; Morais, Anabela; Dias, Alexandra; Soares, Isabel; Rolão, Cristiana; Ducla-Soares, JL; Braga, Lígia; Seixas, Teresa; Nunes, Baltazar; Olim, Gabriel; Romão, Luísa; Lavinha, João; Faustino, PaulaElevated erythrocyte destruction in sickle cell disease (SCD) results in chronic hyperbilirubinaemia and, in a subset of patients, cholelithiasis occurs. We investigated whether the (TA)n promoter polymorphism in the UDP-glucuronosyltransferase 1A1 gene (UGT1A1) may modify bilirubin metabolism, influencing bilirubinaemia, predisposition to cholelithiasis and subsequent cholecystectomy, in a group of 153 young SCD patients (mean age 12.0 +/- 9.0 years) predominantly of Bantu beta S haplotype. The concomitant effect of alpha thalassaemia was also analysed. Among the several UGT1A1 genotypes found, the most frequent were the (TA)6/(TA)6 (n = 37), (TA)6/(TA)7 (n = 60) and (TA)7/(TA)7 (n = 29). These groups of patients did not significantly differ in age, gender ratio and haemoglobin, foetal haemoglobin and reticulocyte levels. On the other hand, total bilirubin levels were significantly different between groups, with an increased (TA) repeat number being associated with higher bilirubinaemia. Furthermore, both cholelithiasis and cholecystectomy were more frequent in groups with higher (TA) repeat number, although the former association was not statistically significant. None of the mentioned parameters is statistically different within UGT1A1 groups with the presence of alpha thalassaemia. Thus, the UGT1A1 promoter polymorphism may represent an important nonglobin genetic modifier of Bantu SCD patients' clinical manifestations, even at a young age.
- Endothelial factors and stroke risk in pediatric sickle cell anemia patients: insights from VCAM1 and ITGA4 variantsPublication . Silva, Marisa; Vargas, Sofia; Coelho, Andreia; Mendonça, Joana; Vieira, Luís; Kjollerstrom, Paula; Maia, Raquel; Silva, Rute; Dias, Alexandra; Ferreira, Teresa; Morais, Anabela; Mota Soares, Isabel; Lavinha, João; Faustino, PaulaSickle 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 adhesion/endothelial dysfunction. These include genes encoding the VCAM-1 molecule and its ligand VLA-4 (ITGA4 or integrin α4), increasingly studied due to their expression in activated human endothelium and leucocytes/stress reticulocytes, respectively. 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 (p=0.008; O.R.= 4.33; C.I.95% =1.391-14.257), while one VCAM1 promoter haplotype was found to be protective of stroke (p=0.011; O.R.=0.22; C.I.95% =0.048-0.784). On 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. Additionally the presence of specific variants seems to result in a predisposition for either high reticulocyte count, elevated lactate dehydrogenase, raised bilirubin levels or increased transcranial Doppler velocity values. Our results reinforce the role of endothelial molecules and blood cell interaction in SCA severity. The association between specific VCAM1, as well as ITGA4, variants 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.
- Fetal hemoglobin level and stroke risk in children with sickle cell anemiaPublication . Nicolau, Marta; Vargas, Sofia; Coelho, Andreia; Silva, Marisa; Mendonça, Joana; Vieira, Luís; Kjollerstrom, Paula; Maia, Raquel; Silva, Rute; Dias, Alexandra; Ferreira, Teresa; Morais, Anabela; Mota Soares, Isabel; Lavinha, João; Faustino, PaulaSickle 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.
- Genetic Modulation of Cerebral Vasculopathy in Children with Sickle Cell AnemiaPublication . Silva, Marisa; Vargas, Sofia; Coelho, Andreia; Mendonça, Joana; Vieira, Luís; Kjollerstrom, Paula; Maia, Raquel; Silva, Rita; Dias, Alexandra; Ferreira, Teresa; Morais, Anabela; Mota Soares, Isabel; Lavinha, João; Faustino, PaulaSickle 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.
- Genetic modulation of stroke in children with sickle cell anaemiaPublication . Silva, Marisa; Vargas, Sofia; Coelho, Andreia; Mendonça, Joana; Vieira, Luís; Kjollerstrom, Paula; Maia, Raquel; Silva, Rita; Dias, Alexandra; Ferreira, Teresa; Morais, Anabela; Mota Soares, Isabel; Lavinha, João; Faustino, PaulaSickle 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.
- Genetic modulators of fetal hemoglobin expression and ischemic stroke occurrence in African descendant children with sickle cell anemiaPublication . Nicolau, Marta; Vargas, Sofia; Silva, Marisa; Coelho, Andreia; Ferreira, Emanuel; Mendonça, Joana; Vieira, Luís; Kjollerstrom, Paula; Maia, Raquel; Silva, Rita; Dias, Alexandra; Ferreira, Teresa; Morais, Anabela; Mota Soares, Isabel; Lavinha, João; Faustino, PaulaSickle cell anemia (SCA) is an autosomal recessive monogenic disease with significant clinical variability. Cerebrovascular disease, particularly ischemic stroke, is one of the most severe complications of SCA in children. This study aimed to investigate the influence of genetic variants on the levels of fetal hemoglobin (Hb F) and biochemical parameters related with chronic hemolysis, as well as on ischemic stroke risk, in ninety-one unrelated SCA patients, children of sub-Saharan progenitors. Our results show that a higher Hb F level has an inverse relationship with the occurrence of stroke, since the group of patients who suffered stroke presents a significantly lower mean Hb F level (5.34 ± 4.57% versus 9.36 ± 6.48%; p = 0.024). Furthermore, the co-inheritance of alpha-thalassemia improves the chronic hemolytic pattern, evidenced by a decreased reticulocyte count (8.61 ± 3.58% versus 12.85 ± 4.71%; p < 0.001). In addition, our findings have confirmed the importance of HBG2 and BCL11A loci in the regulation of Hb F expression in sub-Saharan African SCA patients, as rs7482144_A, rs11886868_C, and rs4671393_A alleles are significantly associated with a considerable increase in Hb F levels (p = 0.019, p = 0.026, and p = 0.028, respectively). Concerning KLF1, twelve different variants were identified, two of them novel. Seventy-three patients (80.2%) presented at least one variant in this gene. However, no correlation was observed between the presence of these variants and Hb F level, severity of hemolysis, or stroke occurrence, which is consistent with their in silico-predicted minor functional consequences. Thus, we conclude that the prevalence of functional KLF1 variants in a sub-Saharan African background does not seem to be relevant to SCA clinical modulation.
- Genetic variation in CD36, HBA, NOS3 and VCAM1 is associated with chronic haemolysis level in sickle cell anaemia: a longitudinal studyPublication . Coelho, Andreia; Dias, Alexandra; Morais, Anabela; Nunes, Baltazar; Ferreira, Emanuel; Picanço, Isabel; Faustino, Paula; Lavinha, JoãoChronic haemolysis stands out as one of the hallmarks of sickle cell anaemia, a clinically heterogeneous autosomal recessive monogenic anaemia. However, the genetic architecture of this sub-phenotype is still poorly understood. Here, we report the results of an association study between haemolysis biomarkers (serum LDH, total bilirubin and reticulocyte count) and the inheritance of 41 genetic variants of ten candidate genes in a series of 99 paediatric SS patients (median current age of 9.9 yr) followed up in two general hospitals in Greater Lisboa area (median follow-up per patient of 5.0 yr). Although in a large number of tests a seemingly significant (i.e. P < 0.05) association was observed, the following ones were confirmed upon correction for multiple comparisons: (i) an increased serum LDH level was associated with haplotype 7 within VCAM1 gene; (ii) a lower total bilirubin was associated with the 3.7-kb deletion at HBA gene, rs2070744_T allele at NOS3 gene, and haplotype 9 within VCAM1 promoter; and (iii) a diminished reticulocyte count was associated with the 3.7-kb deletion at HBA, whereas an increased count was associated with rs1984112_G allele at CD36 gene. On the whole, our findings suggest a complex genetic architecture for the sickle cell anaemia haemolysis process involving multiple pathways, namely control of vascular cell adhesion, NO synthesis and erythrocyte volume and haemoglobinisation.
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