Percorrer por autor "Kjollerstrom, Paula"
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- Alpha-thalassemia due to novel deletions and complex rearrangements in the subtelomeric region of chromosome 16pPublication . Ferrão, José; Silva, Marisa; Gonçalves, Lúcia; Gomes, Susana; Loureiro, Pedro; Coelho, Andreia; Miranda, Armandina; Seuanes, Filomena; Batalha Reis, Ana; Pina, Francisca; Maia, Raquel; Kjollerstrom, Paula; Monteiro, Estela; F. Lacerda, João; Lavinha, João; Gonçalves, João; Faustino, PaulaIntroduction: Inherited deletions removing the α-globin genes and/or their upstream regulatory elements (MCSs) give rise to alpha-thalassemia, one of the most common genetic recessive disorders worldwide. The pathology is characterized by microcytic hypochromic anemia due to reduction of the α-globin chain synthesis, which are essential for hemoglobin tetramerization. Material and Methods: In order to clarify the suggestive α-thalassemia phenotype in eleven patients, we performed Multiplex Ligation-dependent Probe Amplification with commercial and synthetic engineered probes, gap-PCR, and Sanger sequencing to search for deletions in the subtelomeric region of chromosome 16p. Results: We have identified five distinct large deletions, two of them novel, and one indel. The deletions range from approximately 3.3 to 323 kb, and i) remove the whole α-globin cluster; or ii) remove exclusively the upstream regulatory elements leaving the α-globin genes structurally intact. The indel consists in the loss of MCS-R2 (HS-40), which is the most important distal regulatory element for the α-globin gene expression, and the insertion of 39 bp, seemingly resulting from a complex rearrangement involving two DNA segments (probably from chromosome 3q) bridging the deletion breakpoints with a CC-bp orphan sequence in between. Finally, in one patient no α-globin deletion or point mutation were found. This patient revealed to be a very unusual case of acquired alpha-thalassemia associated with a myelodysplastic syndrome. Conclusions: Our study widens the spectrum of molecular lesions by which α-thalassemia may occur and emphasizes the importance of diagnosing large α-zero-deletions to provide patients with appropriate genetic counseling.
- Alpha-thalassemia due to novel deletions and complex rearrangements in the subtelomeric region of chromosome 16pPublication . Ferrão, José; Silva, Marisa; Gonçalves, Lúcia; Gomes, Susana; Loureiro, Pedro; Coelho, Andreia; Miranda, Armandina; Seuanes, Filomena; Batalha Reis, Ana; Valtonen-André, Camila; Sonesson, Annika; Pina, Francisca; Maia, Raquel; Kjollerstrom, Paula; Monteiro, Estela; F. Lacerda, João; Lavinha, João; Gonçalves, João; Faustino, PaulaIntroduction: Inherited deletions removing the α-globin genes and/or their upstream regulatory elements (MCSs) give rise to alpha-thalassemia, one of the most common genetic recessive disorders worldwide. The pathology is characterized by microcytic hypochromic anemia due to reduction of the α-globin chain synthesis, which are essential for hemoglobin tetramerization. Material and Methods: In order to clarify the suggestive α-thalassemia phenotype in eleven patients, we performed Multiplex Ligation-dependent Probe Amplification with commercial and synthetic probes, gap-PCR, and Sanger sequencing to search for deletions in the subtelomeric region of chromosome 16p. Results: We have identified six distinct large deletions, three of them novel, and one indel. The deletions range from approximately 3.3 to 323 kb, and i) remove the whole α-globin cluster; or ii) remove exclusively the upstream regulatory elements leaving the α-globin genes structurally intact. The indel consists in the loss of MCS-R2 (HS-40), which is the most important distal regulatory element for the α-globin gene expression, and the insertion of 39 nt, seemingly resulting from a complex rearrangement involving two DNA segments (probably from chromosome 3q), bridging the deletion breakpoints with a CC-bp orphan sequence in between. Finally, in one patient no α-globin deletion or point mutation were found. This patient revealed to have acquired alpha-thalassemia associated with a myelodysplastic syndrome. Conclusions: Our study widens the spectrum of molecular lesions by which α-thalassemia may occur and emphasizes the importance of diagnosing large α0-deletions to provide patients with appropriate genetic counseling.
- 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.
- Deciphering the genetic modifiers of sickle cell anemia in children: the role of CYB5R3 genePublication . Nascimento, Djanira; Lopes, Pedro; Kjollerstrom, Paula; Maia, Raquel; Faria, Teresa; Faustino, PaulaIntroduction: Sickle cell anemia (SCA) is an autosomal recessive disorder caused by the c.20A>T alteration in the beta-globin gene (HBB), leading the synthesis of abnormal hemoglobin S (HbS). Under hypoxic conditions, HbS polymerizes within red blood cells (RBCs), causing them to adopt the characteristic sickle shape. This results in a clinically heterogenous disease, characterized by chronic hemolytic anemia, vaso-occlusive crises, and multi-organ damage. The CYB5R3 gene encodes the enzyme NADH-cytochrome b5 reductase 3, which plays a crucial role in protecting hemoglobin (Hb) from oxidative damage to unfunctional methemoglobin. Patients with SCA may develop methemoglobinemia, particularly under conditions of oxidative stress. This study aimed to evaluate the potential modulatory effects of a CYB5R3 variant, along with other well-established genetic modifiers within the globin genes, on the phenotypic variability of SCA in pediatric age. Methodology: Eighty-one children with SCA were followed by pediatricians at two hospitals in the Greater Lisbon area, who characterized their clinical, hematological, and biochemical phenotypes. For this specific study, CYB5R3, HBG2, HBA1, and HBA2 genes were genotyped using PCR, Sanger sequencing, and Gap-PCR. Association analyses were performed using SPSS. Results and Discussion: Co-inheritance of α-thalassemia with SCA was observed in 43.2% of the children and proved to be beneficial, as it was associated with higher RBC counts, milder anemia, and a significant reduction in hemolysis biomarkers (bilirubin and reticulocyte counts). Similarly, elevated fetal Hb levels (HbF ≥10%) were also beneficial, leading to less severe hemolytic anemia. In the HBG2 gene, the rs7482144_T allele had a frequency of 15% and was associated with higher HbF, reduced hemolytic parameters, lower HbS level, milder anemia, and a lower frequency of clinical comorbidities, except for heart disease. In the CYB5R3 gene, the rs1800457_G allele showed a very high allelic frequency of 35% and appears to exert a deleterious effect because patients carrying this allele presented with more severe anemia, elevated hemolysis biomarkers, and a greater tendency toward hepatomegaly and cardiac comorbidities. This study contributes to understanding how genetic modifiers influence SCA severity, complication risk and eventually treatment response. Identifying these factors supports personalized medicine and may help uncover new therapeutic targets.
- 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.
- Fatores genéticos moduladores dos níveis elevados de hemoglobina fetal na Anemia de Células FalciformesPublication . Almeida, Teresa; Mateus, Íris; Braga, Lígia; Freitas, Orquídia; Kjollerstrom, Paula; Faustino, PaulaA Drepanocitose ou Anemia das Células Falciformes (ACF) é uma anemia hereditária de transmissão autossómica recessiva caraterizada pela presença de hemoglobina S (HbS) devido à homozigotia para a mutação A>T no codão 6 do gene da beta-globina (HBB:c.20A>T). Embora se trate de uma doença monogénica, o seu fenótipo clínico é bastante heterogéneo. Este fato deve-se à influência modificadora tanto de fatores ambientais como de fatores genéticos. De entre estes últimos, salienta-se o nível elevado de hemoglobina fetal (HbF). O nível de HbF é também variável entre indivíduos drepanocíticos sendo condicionado por diversos fatores genéticos polimórficos existente no agrupamento génico da beta-globina e, por outros não associados a este agrupamento (exemplo dos fatores transcricionais BCL11A e KLF1) . Este trabalho teve como objetivos identificar molecularmente os fatores genéticos associados a níveis elevados de HbF num grupo de doentes com ACF seguidos no HDE. O grupo analisado foi constituído por 14 doentes drepanocíticos, com idades compreendidas entre os 3 e os 17 anos, todos com HbF elevada (média 15,8% ± 3,7). Após extração de DNA de alíquotas de sangue periférico foram caracterizados, recorrendo a metodologias de PCR-RFLP ou a sequenciação direta pelo método de Sanger, a mutação drepanocítica, quatro polimorfismos (SNPs) no promotor do gene HGB2, os haplotipos de restrição no agrupamento génico da beta-globina, um SNP no gene BCL11A e foi sequenciado o gene KLF1. O tratamento estatístico dos resultados foi efetuado usando o software SPSS vs 20. Após confrontação dos resultados obtidos com os encontrados anteriormente numa população semelhante, também de doentes drepanocíticos mas com HbF baixa (HbF <8%), foi possível concluir que o alelo T do SNP rs7482144 (local Xmn I a -158 de HBG2), o haplotipo Senegal, assim como o alelo C do SNP rs11886868 no gene BCL11A se encontram significativamente associados a níveis elevados de HbF neste grupo de doentes. Por outro lado, não foram encontradas alterações moleculares no gene KLF1 que pudessem ser associadas aos níveis de HbF. Em conclusão, neste estudo identificámos polimorfismos globínicos, um SNP (HGB2_rs7482144_alelo T) e um haplotipo (Senegal), assim como um polimorfismo não globínico (BCL11A_rs11886868_alelo C) que contribuem para os níveis elevados de HbF nestes doentes e, provavelmente, estarão também associados, conjuntamente com outros fatores, a fenótipos clínicos mais ligeiros.
- 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 modifiers of the intermediate phenotypes in sickle cell anemiaPublication . Aguiar, Laura; Matos, Ângela; Gil, Ângela; Ferreira, Joana; Braga, Lígia; Almeida, Salomé; Kjollerstrom, Paula; Faustino, Paula; Bicho, Manuel; Inácio, ÂngelaSickle cell anemia (SCA) is an inherited blood disorder characterized by the presence of hemoglobin S (HbS). This disease is caused by a single point mutation in the beta-globin gene with a corresponding amino acid substitution at the sixth position of the beta-globin chain. Vaso-occlusion and hemolytic anemia are the major features of this disease, however, SCA patients present clinical and hematologic variability that cannot be only explained by the single mutation. Others genetic modifiers and environmental factors are important for the clinical phenotype. We studied the association between several hematological and biochemical parameters and a set of genetic variants in 26 pediatric SCA patients. Myeloperoxidase (MPO) and placental growth factor (PlGF) were determined by ELISA (R&D Systems Inc.). Amplification of DNA samples for the rs1050829 characterization, in the glucose-6-phosphate dehydrogenase (G6PD) gene, was performed by PCR followed by restriction fragment length analysis. A multiplex PCR assay was used for simultaneous amplification of glutathione S-transferases mu (GSTM1) and theta (GSTT1). All statistical tests were performed with SPSS 24.0 software. Our results show higher levels of MPO (p<0.001) and PlGF (p=0.048) in SCA patients, compared with healthy adult controls. Moreover, in these patients we found associations between: 1) lower levels of total hemoglobin and the GSTM1 null genotype (p=0.044); 2) higher levels of HbS with the rs1050829_G genotype (hemizygous males) in the G6PD gene (p=0.026). We suggest that the mentioned polymorphisms in GSTM1 and G6PD genes may act as genetic modifiers in SCA, which could be useful for the prediction of increased susceptibility to complications. Furthermore, our results reinforce the importance to study biochemical parameters for a better understanding of the clinical outcome of this disease.
- 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.
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