Browsing by Author "Vargas, Sofia"
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- 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.
- Differential Endothelial VCAM1 Expression and Implications for Sickle Cell Anemia VasculopathyPublication . Silva, Marisa; Coelho, Andreia; Vargas, Sofia; Faustino, PaulaBackground: Vascular disease is systemic in sickle cell anemia (SCA), with profound effects in organs like the brain,where stroke is the most severe end of the cerebral vasculopathy spectrum. Endothelial dysfunction is an important pathobiological mechanism in SCA systemic vasculopathy, with upregulation of adhesion molecules (e.g., VCAM-1), lower nitric oxide bioavailability, and increased oxidative stress. In previous association studies, we found positive associations between the presence of three specific VCAM1 gene promoter haplotypes and i) high blood flow velocities in the median cerebral artery, and ii) a chronic hemolysis biochemical marker. Aims: The aims of our work were: a) to investigate the functional role of those VCAM1 promoter haplotypes in endothelial cell response following endothelial activation through TNF-α stimulation; b) to assess the modulation role of proinflammatory and/or pro-oxidative stimuli on endothelial VCAM1 expression; and, finally, to evaluate how hydroxyurea (HU) treatment would affect that expression. Methods: After molecular cloning of three VCAM1 promoter haplotype constructs, using pGL4 promoterless vectors, haplotype sequence was confirmed, by Sanger sequencing, prior to transfection. Transfection experiments for each construct were performed, with or without TNF-α stimulation, using EAhy926, and HBEC as macrovascular and microvascular endothelial cell models, respectively. Differences in promoter activity were assessed by luciferase reporter assay. RNA was extracted from non-transfected EAhy926 and HBEC cell cultures stimulated or not with TNF-a and/or hemin, and with or without HU treatment. RT-qPCR was performed to analyze VCAM1 expression. HMOX1 and NOS3 were also analyzed for comparison purposes. Results: Our results showed that two VCAM1 promoter haplotypes, previously associated with pediatric cerebral vasculopathy and hemolysis in SCA, increased promoter activity in transfected and TNF-α-stimulated EA.hy926 and HBEC cells, consistent with a higher VCAM1 expression in macro and microvascular settings. In non-transfected cells, we also observed TNF-a-induced VCAM1 overexpression as well as heme-induced overexpression of HMOX1 in both cell models. Heme did not affect VCAM1 nor NOS3 expression and the latter was also not affected by TNF-a stimulus. Hydroxyurea treatment lowered TNF-a-induced VCAM1 and NOS3 expression but did not affect heme-induced HMOX1 expression. Summary/Conclusion: These data further indicate that VCAM1 haplotypes we previously associated with pediatric cerebral vasculopathy and hemolysis in SCA, induce higher VCAM1 expression potentially affecting both cerebral and systemic vasculopathy risk. The differential endothelial expression of VCAM1, NOS3, and HMOX1 after proinflammatory and/or pro-oxidative stimuli also reinforces their genetic modulation role in SCA systemic vasculopathy.
- 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.
- Hemorheological alterations in sickle cell anemia and their clinical consequences – the role of genetic modulatorsPublication . Silva, Marisa; Vargas, Sofia; Coelho, Andreia; Dias, Alexandra; Ferreira, Teresa; Morais, Anabela; Maia, Raquel; Kjollerstrom, Paula; Lavinha, João; Faustino, PaulaSickle cell anemia (SCA) is an autosomal recessive disease caused by the HBB:c.20A>T mutation that leads to hemoglobin S synthesis. The disease presents with high clinical heterogeneity characterized by chronic hemolysis, recurrent episodes of vaso-oclusion and infection. This work aimed to characterize by in silico studies some genetic modulators of severe hemolysis and stroke risk in children with SCA, and understand their consequences at the hemorheological level. Association studies were performed between hemolysis biomarkers as well as the degree of cerebral vasculopathy and the inheritance of several polymorphic regions in genes related with vascular cell adhesion and vascular tonus in pediatric SCA patients. In silico tools (e.g. MatInspector) were applied to investigate the main variant consequences. Variants in vascular adhesionmolecule-1 (VCAM1) gene promoter and endothelial nitric oxide synthase (NOS3) gene were significantly associated with higher degree of hemolysis and stroke events. They potentially modify transcription factor binding sites (e.g. VCAM1 rs1409419 T allele may lead to an EVI1 gain) or disturb the corresponding protein structure/function. Our findings emphasize the relevance of genetic variation inmodulating the disease severity due to their effect on gene expression or modification of protein biological activities related with sickled erythrocyte/endothelial interactions and consequent hemorheological abnormalities.
- Hemorheological alterations in sickle cell anemia and their clinical consequences: the role of genetic modulatorsPublication . Silva, Marisa; Vargas, Sofia; Coelho, Andreia; Dias, Alexandra; Ferreira, Teresa; Morais, Anabela; Maia, Raquel; Kjollerstrom, Paula; Lavinha, João; Faustino, PaulaSickle cell anemia (SCA) is an autosomal recessive chronic hemolytic anemia, caused by homozygosity for the HBB:c.20A>T mutation. The disease presents with high clinical heterogeneity, stroke being the most devastating manifestation. This study aimed to identify genetic modulators of severe hemolysis and stroke risk in children with SCA, as well as understand their consequences at the hemorheological level. Sixty-six children with SCA were categorised according to their degree of cerebral vasculopathy (Stroke/Risk/Control). Relevant data were collected from patients’ medical records. Several polymorphic regions in genes related to vascular cell adhesion and tonus were characterized by molecular methodologies. Data analyses were performed using R software. Several in silico tools (e.g. TFBind, MatInspector) were applied to investigate the main variant consequences. Some genetic variants in vascular adhesion molecule-1 gene promoter and endothelial nitric oxide synthase gene were associated with higher levels of hemolysis and stroke events. They modify important transcription factor binding sites or disturb the corresponding protein structure/function. Our findings emphasize the relevance of the genetic variants in modulating the degree of hemolysis and development of cerebral vasculopathy due to their effect on gene expression, modification of protein biological activities related with erythrocyte/endothelial interactions and consequent hemorheological abnormalities in SCA.
- Macro- and microvascular endothelial dysfunction modulation by VCAM1 haplotypes of pediatric sickle cell anemia patientsPublication . Silva, Marisa; Vargas, Sofia; Coelho, Andreia; Faustino, PaulaSickle cell anemia (SCA) is a multifactorial-like monogenic disease that results from homozygosity for the HBB:c.20A>T mutation. Children with SCA usually present a systemic vascular disease with profound effects in organs like the brain, with stroke being the most severe end of the cerebral vasculopathy spectrum. Endothelial dysfunction plays a major role in vasculopathy and several adhesion molecules, such as vascular cell adhesion molecule 1 (VCAM-1), are produced by a cytokine-activated endothelium. In previous genotype/phenotype association studies, we found positive associations of specific VCAM1 gene promoter variants and haplotypes to high blood flow velocities in the median cerebral artery, and to chronic hemolysis biochemical markers. Our aims in this study were to: (i) assess the role of those variants, together with imaging, serological and hematological parameters as potential biomarkers of cerebral vasculopathy in SCA children, and (ii) evaluate the functional effects of the VCAM1 promoter haplotypes on endothelial cell response following endothelial activation by TNF-alpha stimulation. We investigated seventy children with SCA of sub-Saharan ancestry, with focussing on cerebral vasculopathy, as well as on stroke risk (as measured by transcranial Doppler ultrasound). PCR and Sanger sequencing were used for genotyping VCAM-1 gene. Statistical analyses were performed using SPSS (v.25.0) software. When statistical significance was identified for specific haplotypes, plasmid constructs were created by molecular cloning using a promoterless pGL4.10[luc2] vector. Haplotype sequence of each construct was confirmed by Sanger sequencing prior to transfection to EAhy926 and HBEC (macrovascular and microvascular endothelial cell models, respectively) with and without TNF-alpha stimulation. Differences in promoter activity were then assessed by luciferase reporter assays. We analysed 6 VCAM1 promoter variants and 7 haplotypes with potential modulating effect. The rs1409419_T allele and haplotype 7 (Hap7) were positively associated with stroke, stroke risk, and high levels of LDH. On the other hand, haplotype 1 (Hap1) was negatively associated with stroke. Luciferase reporter assays showed differences in promoter activity, in both endothelial cell models, as a result of Hap1 and Hap7 transfection. Hap1 endothelial cell transfection led to a decrease, while Hap7 transfection led to an increase in promoter activity. These results are consistent with: (i) lower VCAM1 expression, hence a protective effect, due to Hap1 and (ii) higher expression due to Hap7 and consequently, increased vasculopathy risk, in a pro-inflammatory milieu. The association between specific haplotypes and endothelial cell response further enhances the modifier effect of VCAM1 not only on macro- and microvascular endothelial dysfunction but also on systemic SCA vasculopathy. Furthermore, we suggest that those haplotypes, together with the imaging, biochemical and hematological parameters, may be used to design a sensitive and specific biomarker panel for SCA vasculopathy risk, severity and prognosis.