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- Genetic Modulators of Hemolytic Anemia in Angolan Children with Sickle Cell AnemiaPublication . Germano, Isabel; Santos, Brígida; Delgadinho, Mariana; Lopes, Pedro; Arez, Ana Paula; Brito, Miguel; Faustino, PaulaSickle Cell Anemia (SCA) is a recessive genetic disease caused by the c.20A>T variant in HBB gene. It is characterised by sickled erythrocytes, chronic hemolytic anemia and vaso-occlusive events. However, these manifestations are heterogeneous due to environmental and genetic modifying factors. The aim of this study was to investigate genetic modifiers of hemolytic anemia in pediatric SCA patients living in Africa, where the disease is a severe public health problem. The study was conducted on 200 Angolan SCA 3-12 year-old children. Thirteen polymorphic regions in genes previously associated with vascular cell adhesion (VCAM1 and CD36), vascular tonus (NOS3) or erythrocyte hemoglobinisation (HBA), were genotyped using PCR, RFLP, Gap-PCR and Sanger sequencing. Hematological and biochemical phenotypes were obtained at steady state and clinical adverse events were collected from patients’ medical records. Results revealed a high level (67.5%) of α-thalassemia co-inheritance (del. 3.7kb in HBA), which improve patients’ health by delaying the onset of the disease, decreasing anemia and the number of blood transfusions. Two SNPs in CD36 (rs1984112 and rs1413661) showed impact on anemia severity. Particularly, genotypes containing the rs1413661_allele C revealed to be risk factors for severe anemia, as they were associated with lower hemoglobin levels, increased number of hospitalizations and transfusions. This is the first report associating this SNP with SCA pathology. Moreover, the rs1041163_allele C in VCAM1 was associated with lower LDH levels, inversely the rs2070744_allele C in NOS3 was associated with higher LDH levels and a higher number of hospitalizations, being a possible risk factor for increased hemolytic rate. This study contributed to the understanding of SCA complex pathophysiology. It confirmed the positive role of α-thal., both in SCA related anemia and in its clinical manifestations. In addition, it reinforced the importance of vascular cell adhesion in hemolytic anemia variability. In this context, we propose the SNP rs1413661 in CD36 as an important novel genetic modulator of SCA in Africa.
- 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.
- Biomarcadores e moduladores genéticos da Anemia Falciforme numa coorte de crianças Angolanas com Anemia FalciformePublication . Santos, Brígida; Germano, Isabel; Delgadinho, Mariana; Ginete, Catarina; Arez, Ana Paula; Faustino, Paula; Brito, MiguelA anemia falciforme é uma doença monogénica autossómica recessiva com grande variabilidade fenotípica e é influenciada por factores ambientais e genéticos. A doença é caracterizada por episódios vaso-oclusivos recorrentes, hemólise crónica e susceptibilidade a infecções. Objectivos: Avaliar a correlação clínica e laboratorial com polimorfismos genéticos em crianças angolanas com anemia falciforme. Métodos: Foram seleccionadas 200 crianças com anemia falciforme seguidas no Hospital Pediátrico David Bernardino (Luanda) e no Hospital Geral do Bengo. A cada criança foi feita a caracterização clínica e laboratorial (hemograma, contagem de reticulócitos, desidrogenase láctea, bilirrubinas e doseamento da hemoglobina fetal). A análise genética incluiu a identificação de Haplótipos cuja classificação foi baseada em quatro SNPs descritas anteriormente (rs3834466, rs28440105, rs10128556 e rs968857), polimorfismos nos genes HBG2 (rs7482144), BCL11A (rs4671393), HBS1L-MYB (rs28384513, rs4895441) e correlacionados com a quantificação de HbF e à variabilidade fenotípica, Os polimorfismos nos genes VCAM1, NOS3 e CD36 foram correlacionados com os biomarcadores de hemólise. A análise estatistica foi feita pelo teste ANOVA (valor de P<0,05) e teste Qui-quadrado. Resultados: O haplotipo mais prevalente foi o CAR/CAR identificado em 91,7% seguido pelo CAR/BEN em 5,7%. Houve significância estatística entre haplotipos e a hemoglobina fetal e os parâmetros hematológicos e associação entre os polimorfismos rs3783599, rs1984112, rs3917023 e os biomarcadores de hemólise e consequentemente maior número de transfusões sanguíneas. Conclusões: Os dados obtidos contribuem para o conhecimento genético populacional de crianças angolanas com anemia falciforme e destacam o papel dos moduladores genéticos na variabilidade fenotípica da doença.
- Hepatitis C Virus infection, iron metabolism, liver fibrosis and response to Direct Acting Antivirals (DAAs)Publication . Ferreira, Joana; Faustino, Paula; Bicho, Manuel; Serejo, FátimaIntroduction: Chronic Hepatitis C (CHC) is characterized by hepatic and extra-hepatic manifestation. Among hepatic manifestations, liver fibrosis is probably the most significant as it can lead to cirrhosis, hepatocellular carcinoma and death. Iron overload is one of the extra-hepatic manifestations induced by Hepatitis C Virus (HCV) infection and can induce fibrosis-promoting signals in the parenchymal and non-parenchymal cells, which accelerate disease progression and exacerbate liver pathology. Aims: Evaluate the changes in iron metabolism induced by HCV elimination, the association of liver fibrosis with biochemical and genetic parameters of iron metabolism before and after DAAs treatment and baseline parameters that could predict the improvement of liver fibrosis after treatment. Materials and Methods: 329 patients with CHC (age: 49.93 [45.57-50.28] years; 124 female and 205 male). 134 treated with DAAs with sustained response (age: 53.42 [51.47-55.36]; 58 female and 56 male). Liver fibrosis stage was assessed by transient elastography using a Fibroscan and patients were divided in two groups (F1/2_mild and moderate fibrosis and F3/4_severe fibrosis and cirrhosis), biochemical parameters of iron metabolism (Total Iron_Fe, Total Iron Binding Capacity_TIBC, Transferrin Saturation_TS, Ferritin_FT, Haptoglobin_Hp) were evaluated by standard methods and genetic polymorphisms within Transferrin_TF HFE, Bone Morphogenetic Protein 2_BMP2, Hepcidin_HAMP and Ferroportin_SLC40A1 genes were analyzed by PCR, sequencing or NGS. Statistical analysis was performed using SPSS 26.0 for windows. Results: Patients showed lower values of Fe, TS and FT and higher values of Hp after treatment.
- Contributo dos genes NOS e ECA para a suscetibilidade de elevar a glicemia em hipertensosPublication . Aguiar, Laura; Semente, Ildegário; Ferreira, Joana; Matos, Andreia; Mascarenhas, Mário Rui; Menezes Falcão, Luíz; Faustino, Paula; Bicho, Manuel; Inácio, ÂngelaIntrodução: A Hipertensão Arterial (HTA) é um fator de risco cardiovascular muito prevalente em Portugal. Esta patologia é multifatorial, envolvendo fatores genéticos e ambientais. A resistência à insulina e a hipertensão são componentes do síndroma metabólico e frequentemente coexistem. Níveis aumentados de glicemia associados a hipertensão aumentam significativamente o risco de doença cardiovascular. Objetivo: Este estudo teve como objetivo investigar a potencial implicação de polimorfismos genéticos nos genes da sintase do óxido nítrico endotelial (eNOS) e da enzima conversora da angiotensina (ECA) no desenvolvimento da HTA e hiperglicemia, na população portuguesa. Material e métodos: Foi realizado um estudo de caso-controlo para uma amostra de 377 indivíduos portugueses, dos quais 243 hipertensos e 134 normotensos. As análises polimórficas do número variável de repetições em tandem (VNTR) no intrão 4 (repetição em tandem de 27 pb) do gene eNOS e do polimorfismo ECA I/D (inserção/ deleção) foram realizadas por reação em cadeia da polimerase (PCR). Todas as análises estatísticas foram realizadas recorrendo ao software SPSS, versão 24.0, sendo o nível de significância estatística estabelecido para p <0,05. Resultados: Encontrou-se uma associação entre o alelo 4a do gene eNOS com a hipertensão (p =0,001), verificando-se ainda que na hipertensão, a presença do alelo 4a está associada a valores superiores de HbA1c (p=0,031). Em relação ao gene ECA, não se encontram diferenças estatisticamente significativas entre doentes e controlos, contudo verificou-se que a presença do alelo D em hipertensos está associada a níveis mais elevados de glicemia (p=0,017). Conclusão: Os nossos resultados mostram uma associação entre os genes eNOS e ECA com fenótipos clínicos associados a hiperglicemia, na população portuguesa. Indivíduos com níveis elevados de glicemia têm maior risco de desenvolver hipertensão. A identificação de polimorfismos genéticos que possam influenciar o desenvolvimento e gravidade da HTA, pode permitir um diagnóstico mais precoce e específico, que pode proporcionar melhores estratégias terapêuticas e de prevenção, para esta doença tão prevalente em Portugal.
- Osteoporosis: gene interaction between haptoglobin and HFE polymorphismsPublication . Aguiar, Laura; Ferreira, Joana; Laires, Maria José; Mascarenhas, Mário Rui; Inácio, Ângela; Faustino, Paula; Bicho, ManuelOsteoporosis is a common metabolic bone disease characterized by reduced bone mass and increased risk of fragility fractures. The pathogenesis of this disease is complex and influenced by multiple risk factors, where genetic factors play an important role. Osteoporosis and iron metabolism have an important relationship. Iron overload suppresses osteoblast formation and also stimulate osteoclast resorption of bone, suggesting that polymorphisms in genes affecting iron homeostasis can increase the susceptibility for the development of osteoporosis. In the present study, we aimed to analyse the epistatic relationship between two iron metabolism related genes - haptoglobin (Hp) and HFE – in osteoporosis. To achieve this, 313 patients with osteoporosis and 450 controls with normal bone mineral density were enrolled. Haptoglobin phenotype was determined by polyacrylamide gel electrophoresis (PAGE) and HFE polymorphisms (H63D and C282Y) were evaluated by PCR-RFLP. All statistical tests were performed with SPSS 24.0 software. Results showed that, no significant differences were found between the two populations (patients vs controls) concerning Hp phenotypes or HFE (H63D and C282Y) genotypes. However, individuals that have co-inherited the Hp 2.2 and the HFE_H63D HH have an increased risk for developing osteoporosis [p=0.049; OR (95% CI) = 2.509 (1.003-6.279)] (adjusted for age and body mass index). In summary, a significant epistatic interaction was detected between haptoglobin and HFE and osteoporosis, where Hp 2.2 in combination with HFE_H63D HH genotype appear to increase the risk for developing osteoporosis. Since these genes are related to iron metabolism, the results of this study reinforce an important action of this metabolism in the development of osteoporosis.
- Ancestry of the major long-range regulatory site of the α-globin genes in the Portuguese population with the common 3.7kb α-thalassemia deletionPublication . Pena, Rita; Lopes, Pedro; Gaspar, Gisela; Miranda, Armandina; Faustino, PaulaThe α-major regulatory element (known as HS-40) has a crucial role in the long-range regulation of the α-globin gene expression. This element is genetically polymorphic and six haplotypes (A to F) have been identified in different populations, with haplotype D almost exclusively found in African populations. This study aimed to identify the HS-40 haplotype associated with the common 3.7kb α-thalassemia deletion (-α3.7del) in the Portuguese population, and investigate its ancestry. We searched for the -α3.7del in 111 selected Portuguese individuals by Gap-PCR. In addition, a DNA fragment containing the HS-40 was amplified by PCR and Sanger sequenced. Statistical analysis was performed using R software. Fifty individuals have the wild-type α-globin genotype (group I), 34 are heterozygous for the -α3.7del (group II) and 27 are homozygous (group III). Regarding the HS-40, four haplotypes were found (A to D). The distribution of HS-40 haplotypes and genotypes are significantly different between groups with and without the -α3.7del (p<0.001), being haplotype D and genotype AD the most prevalent in group III. Furthermore, multiple correspondence analysis revealed that individuals without the -α3.7del are grouped with other European populations, while samples with the -α3.7del are split from these and found more related to the African population. This study revealed for the first time an association of a specific HS-40 haplotype with the -α3.7del in the Portuguese population, and its likely African ancestry. These results may have a clinical importance as in vitro analysis of haplotype D showed a descrease in its enhancer activity on α-globin genes.
- Caracterização molecular de hemoglobinopatias na população portuguesa: um subestudo do projeto INSEFPublication . Santos, Daniela; Barreto, Marta; Kislaya, Irina; Lopes, Pedro; Mendonça, Joana; Machado, Miguel P.; Vieira, Luís; Dias, Carlos; Faustino, PaulaA anemia é um problema de saúde pública a nível mundial que ocorre quando o número de glóbulos vermelhos em circulação ou a sua capacidade de transporte de oxigénio é insuficiente para atender às necessidades do organismo. A anemia pode ter uma origem ambiental, por exemplo devido a carência nutricional em ferro, ou uma origem genética, onde se enquadram as anemias hereditárias originadas por alterações nos genes codificantes das cadeias globínicas da hemoglobina. Estas anemias hereditárias são denominadas por hemoglobinopatias, têm transmissão autossómica recessiva e incluem as talassémias e as variantes da hemoglobina. As talassémias, no estado de portador, apresentam-se geralmente associadas a um fenótipo hematológico de microcitose e/ou hipocromia e, eventualmente, anemia. Tendo em conta os movimentos migratórios recentes, sobretudo dos continentes africano e sul americano para a Europa, o padrão de distribuição destas patologias nalguns países da Europa tem-se alterado. Os últimos estudos de prevalência de hemoglobinopatias efetuados em Portugal foram realizados há mais de duas décadas. Assim, este trabalho teve como principal objetivo contribuir para o conhecimento mais recente das hemoglobinopatias em Portugal, sobretudo das talassémias. Para concretizar este objetivo foram analisados os participantes no estudo epidemiológico Inquérito Nacional de Saúde com Exame Físico (INSEF)* que constituem uma amostragem da população residente em Portugal, continental e ilhas, em 2015. Em 4808 participantes no INSEF foi caracterizado o fenótipo hematológico e naqueles que apresentaram hipocromia e/ou microcitose (n = 204) foram analisados os genes da β-globina (HBB) e da α-globina (HBA2 e HBA1). A pesquisa de deleções/inserções no agrupamento génico da α-globina foi efetuada por Gap-PCR e Multiplex Ligation-Dependent Probe Amplification (MLPA). O gene HBB foi estudado através de PCR-longo seguido de Next-Generation Sequencing (NGS). As variantes detetadas por NGS foram avaliadas in silico através das ferramentas bioinformáticas PolyPhen-2, Sorting Intolerant From Tolerant (SIFT) e varSEAK, de modo a analisar o seu impacto ao nível da estrutura e função da respetiva proteína e ao nível do splicing. As variantes consideradas patogénicas ou possivelmente patogénicas foram confirmadas por sequenciação de Sanger. Identificámos a presença da deleção α-talassémica de 3,7 kb em 53 indivíduos, da deleção de 4,2 kb em 1 indivíduo e ainda, em 2 indivíduos, a presença dos genes da α-globina triplicados. Relativamente ao estudo do gene HBB, foram identificadas 10 variantes genéticas diferentes, patogénicas ou possivelmente patogénicas, em 22 indivíduos, sendo que 7 tipos diferentes de mutações são responsáveis por β-talassémia [vulgarmente conhecidas por Cd39 (C>T), IVS-I-6 (T>C), IVS-I-110 (G>A), IVS-I-1 (G>A), Cd15 (G>A), Cd6(-A) e Cd41/42 (-CTTT)] e 3 por variantes de hemoglobina (Hb S, Hb C e Hb D-Portugal). Obtivemos neste subgrupo da população INSEF com fenótipo hematológico anómalo (n = 204) uma frequência de 26,6% para a α-talassémia, 10,8% para a β-talassémia e 1,5% para variantes de hemoglobina. Assim, os nossos resultados revelaram que 37,7% destes casos sintomáticos têm uma origem genética – uma hemoglobinopatia. Uma vez que, salvo raríssimas exceções, os portadores de β-talassémia apresentam o fenótipo de microcitose e hipocromia, os nossos resultados permitem sugerir uma prevalência geral de 0,5% de portadores de β-talassémia para a população residente em Portugal continental e ilhas em 2015. Este valor é semelhante ao anteriormente descrito para Portugal continental (Martins et al, J Med Genet, 1993, 30:235). A mesma extrapolação não poderá ser realizada para determinar a prevalência de α-talassémia uma vez que sabemos que uma proporção significativa destes portadores não manifesta qualquer alteração hematológica. Quanto às variantes de hemoglobina, uma vez que na grande maioria não afetam os índices hematimétricos, os 3 casos detetado justificam-se pela co-herança com α-talassémia ou da co-existência de anemia ferropénica. Assim, a determinação da prevalência de variantes de hemoglobina bastante importantes, como seja a Drepanocitose (presença de hemoglobina S), requerem outro tipo de abordagem metodológica. Observámos, ainda, que dos 204 indivíduos com microcitose e/ou hipocromia, 76 também apresentavam anemia. Contudo, no inquérito INSEF de autorreporte apenas 2 desses indivíduos referiram ter conhecimento desta condição. Para além disso, verificámos que grande maioria dos portadores de hemoglobinopatia detetados perceciona a sua saúde como sendo normal e, eventualmente, desconhece (não declarou ter conhecimento) de que é portador de uma doença genética. Este estudo permitiu-nos concluir que as α- e as β-talassémias, no seu estado de heterozigotia, são responsáveis por uma fração considerável dos fenótipos de microcitose e/ou hipocromia, acompanhados ou não de anemia, na população residente no nosso país. Para além disso, os nossos resultados apontam para a necessidade de sensibilização dos médicos de Medicina Geral e Familiar para estas patologias e para o reforço das estratégias de deteção, de informação e de acompanhamento dos portadores de uma hemoglobinopatia em Portugal.
- Contribution of HFE and HPSE genes and methaemoglobin reductase activity to heart failurePublication . Gaspar, Marcos A.; Aguiar, Laura; Ferreira, Joana; Faustino, Paula; Mascarenhas, Mário Rui; Menezes Falcão, Luiz; Bicho, Manuel; Inácio, ÂngelaIntroduction: Heart failure can be defined as a syndrome caused by a structural anomaly and/or by a committed cardiac function, which leads to an inadequate cardiac output unable to meet the metabolic necessities of the organism. We aim to understand if HFE and HPSE genes as well as methaemoglobin reductase activity, may influence the development of heart failure. Methodology: It was performed a case-control study, in which 252 DNA samples from Portuguese individuals were analysed, 143 derived from subjects with heart failure, and 109 from healthy controls. For HPSE genotyping (rs4693608), we performed endpoint PCR analysis. A multiplex ARMS (Amplification-Refractory Mutation System) assay was used for the simultaneous detection of two HFE polymorphisms (C282Y and H63D). Reductase methaemoglobin activity was determined by spectrophotometric methods. All statistical tests were performed with IBM® SPSS® Statistics 26.0 software. Statistical significance was defined as a p-value < 0.05. Results: Regarding the H63D polymorphism, results show the CG genotype as a risk factor [OR (95% CI) = 2.889 (1.041-8.018); p=0.042]. In what concerns HPSE gene, the GG genotype was found to have a protective effect [OR (95% CI) = 0.435 (0.193-0.982); p=0.045] while the presence of the A allele is a risk factor [OR (95% CI) = 2.297 (1.018-5.179); p=0.045. Considering methaemoglobin reductase, its activity was lower in patients than in healthy controls (p=0.019). Discussion: Intravenous iron supplementation is sometimes considered in heart failure treatment, emphasizing the results presented in the present study. Considering the high prevalence of heart failure in Portugal (400.000 individuals, according to Sociedade Portuguesa de Cardiologia), it is important to identify iron-related markers, since it may allow an earlier and more expert approach, which may provide better prevention and therapeutic strategies for this pathology.
- VCAM1 modulation on endothelial cells – implications for vasculopathy in sickle cell anemiaPublication . Silva, Marisa; Vargas, Sofia; Coelho, Andreia; Lavinha, João; Faustino, PaulaSickle cell anemia (SCA) is a highly heterogeneous and multifactorial-like monogenic disease that arises from homozygosity for the c.20A>T mutation in the HBB gene. Vascular disease is systemic in SCA, with profound effects in organs like the brain, where stroke is the most severe end of the cerebral vasculopathy (CVA) spectrum. Endothelial dysfunction plays a major role in vasculopathy with several adhesion molecules, such as VCAM1, being produced by the endothelium altered as a response to inflammatory cytokine (e.g., TNF-α) signalling. In previous association studies, we found positive associations between the presence of four specific VCAM1 gene promoter haplotypes and i) high blood flow velocities in the median cerebral artery, and ii) a chronic hemolysis biochemical marker. In this study, we aimed to assess the functional role of those VCAM1 promoter haplotypes in endothelial cell response following endothelial activation through TNF-α stimulation. After molecular cloning of 3 haplotype constructs, using a pGL4 promoterless vector, haplotype sequence was confirmed, by Sanger sequencing, prior to transfection. We used EAhy926, HUVEC and HBEC as different endothelial cell models, and performed transfection experiments for each construct, with and without TNF-α stimulation. Differences in promoter activity were assessed by luciferase reporter assay. All haplotypes showed differences in promoter activity, after TNF-α stimulation, in all cell models. Haplotype 1 showed decreased promoter activity, while haplotypes 7 and 8 showed increased activity after TNF-α stimulation, in all cell models. These results are consistent with lower VCAM1 expression due to haplotype 1, and therefore a protective effect. Conversely, a higher expression due to haplotypes 7 and 8, suggests an increased vasculopathy risk, in a pro-inflammatory milieu. The association between specific haplotypes and endothelial cell response further enhances the modifier effect of VCAM1 on endothelial dysfunction and its impact in SCA pathophysiology, as well as its potential role as a biomarker of SCA vasculopathy risk, severity and prognosis.