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- Are genetic modifiers the answer to different responses to hydroxyurea treatment? A pharmacogenetic study in sickle cell anemia Angolan childrenPublication . Ginete, Catarina; Delgadinho, Mariana; Santos, Brígida; Pinto, Vera; Silva, Carina; Miranda, Armandina; Brito, MiguelSickle cell anemia (SCA) is an inherited disease affecting the hemoglobin that is particularly common in sub-Saharan Africa. Although monogenic, phenotypes are markedly heterogeneous in terms of severity and life span. Hydroxyurea is still the most common treatment for these patients, and the response to treatment is highly variable and seems to be an inherited trait. Therefore, identifying the variants that might predict hydroxyurea response is important for identifying patients who will have a poorer or non-response to treatment, and the ones that are more prone to suffer from severe side effects. In the present pharmacogenetic study, we analyzed the exons of 77 genes described in the literature as potentially associated with hydroxyurea metabolism in Angolan children treated with hydroxyurea and evaluated the drug response considering fetal hemoglobin levels, other hematological and biochemical parameters, hemolysis, number of vaso-occlusive crises and hospitalizations. Thirty variants were identified in 18 of those genes as possibly associated with drug response, five of them in gene DCHS2. Other polymorphisms in this gene were also associated with hematological, biochemical, and clinical parameters. Further research examining the maximum tolerated dose and fixed-dose with a larger sample size is necessary to corroborate these findings.
- Genotypic diversity among Angolan children with sickle cell anemiaPublication . Delgadinho, Mariana; Ginete, Catarina; Santos, Brígida; Miranda, Armandina; Brito, MiguelBackground. Sickle cell anemia (SCA) is an inherited blood disorder that affects over 300,000 newborns worldwide every year, being particularly prevalent in Sub-Saharan Africa. Despite being a monogenic disease, SCA shows a remarkably high clinical heterogeneity. Several studies have already demonstrated the existence of some polymorphisms that can provide major clinical benefits, producing a mild phenotype. Moreover, the existence of distinct haplotypes can also influence the phenotype patterns of certain populations, leading to different clinical manifestations. Our aim was to assess the association between polymorphisms in genes previously related to SCA disease severity in an Angolan pediatric population. Methods. This study analyzed clinical and biological data collected from 192 Angolan children. Using NGS data, we classified the HBB haplotypes based on four previously described SNPs (rs3834466, rs28440105, rs10128556, and rs968857) and the genotype for the SNPs in HBG2 (rs7482144), BCL11A (rs4671393, rs11886868, rs1427407, rs7557939), HBS1L-MYB (rs66650371) and BGLT3 (rs7924684) genes. Results. The CAR haplotype was undoubtedly the most common HBB haplotype in our population. The HbF values and the ratio of gamma chains were statistically significant for almost all of the variants studied. We reported for the first time an association between rs7924684 in the BGLT3 gene and gamma chains ratio. Conclusions. The current findings emphasize the importance personalized medicine would have if applied to SCA patient care since some of the variants studied might predict the phenotype and the overall response to treatment
- How hydroxyurea alters the gut microbiome: a longitudinal study involving Angolan children with sickle cell anemiaPublication . Delgadinho, Mariana; Ginete, Catarina; Santos, Brígida; Fernandes, Carolina; Silva, Carina; Miranda, Armandina; Vasconcelos, Jocelyne Neto de; Brito, MiguelSickle cell anemia (SCA) is an inherited hematological disorder and a serious global health problem, especially in Sub-Saharan Africa. Although hydroxyurea (HU) is the leading treatment for patients with SCA, its effects on the gut microbiome have not yet been explored. In this context, the aim of this study was to investigate this association by characterizing the gut microbiome of an Angolan SCA pediatric population before and after 6 months of HU treatment. A total of 66 stool samples were obtained and sequenced for the 16S rRNA gene (V3-V4 regions). Significant associations were observed in alpha and beta-diversity, with higher values of species richness for the children naïve for HU. We also noticed that children after HU had higher proportions of several beneficial bacteria, mostly short-chain fatty acids (SCFAs) producing species, such as Blautia luti, Roseburia inulinivorans, Eubacterium halli, Faecalibacterium, Ruminococcus, Lactobacillus rogosae, among others. In addition, before HU there was a higher abundance of Clostridium_g24, which includes C. bolteae and C. clostridioforme, both considered pathogenic. This study provides the first evidence of the HU effect on the gut microbiome and unravels several microorganisms that could be considered candidate biomarkers for disease severity and HU efficacy.
- Gut microbiota impact on Angolan children with sickle cell diseasePublication . Brito, Miguel; Delgadinho, Mariana; Ginete, Catarina; Mendes, Joana; Vasconcelos, J.; Santos, BrígidaIntroduction: Clinical manifestations of Sickle cell disease (SCD) are very heterogeneous, and the intestinal microbiome appears to be crucial in the modulation of inflammation, cell adhesion, and induction of aged neutrophils, which are the main interveners of recurrent vaso-occlusive crisis. Enterocyte injury, increased permeability, altered microbial composition, and bacterial overgrowth have all been documented as microbial and pathophysiologic changes in the gut microbiome of SCD patients in recent research studies. Microbiota analysis in SCD populations will be essential to demonstrate the importance of specific bacteria and their function in this disease and provide new insights for attenuating symptoms and new drug targets. Purpose: Given this, our aim is to sequence by NGS bacterial 16S RNA gene in order to characterize the gut microbiome of SCD children and healthy siblings, as a control.
- 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 the HBB gene. It is characterized 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 a steady state and clinical adverse events were collected from the patient's medical records. Results revealed a high level (67.5%) of α-thalassemia co-inheritance (del. 3.7kb in HBA), which improves 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 an impact on anemia severity. Particularly, genotypes containing the rs1413661_allele C were 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.
- Haplotype distribution and genotypic diversity among Angolan children with sickle cell diseasePublication . Delgadinho, Mariana; Santos, Brígida; Brito, MiguelSickle cell disease (SCD) is an inherited blood disorder that affects over 300,000 newborns worldwide every year. Despite being a monogenic disease, SCD shows a remarkably high clinical heterogeneity, and analysis of the HBB gene cluster has revealed five distinct haplotypes: Senegal (SEN), Benin (BEN), Central African Republic (CAR), Cameroon (CAM), and Arab-Indian (ARAB). The aim of this study was to assess the frequency of HBB haplotypes, as well as to correlate other genetic predictors that could have an impact on SCD phenotype in an Angolan pediatric population.
- Co-inheritance of alpha-thalassemia and sickle cell disease in a cohort of Angolan pediatric patientsPublication . Santos, Brígida; Delgadinho, Mariana; Ferreira, Joana; Germano, Isabel; Miranda, Armandina; Arez, Ana Paula; Faustino, Paula; Brito, MiguelThe aim of this study was to explore the association between alpha-thalassemia, fetal hemoglobin, hematological indices, and clinical adverse events in Angolan sickle cell disease pediatric patients. A total of 200 sickle cell disease (SCD) children were sampled in Luanda and Caxito. A venous blood sample was collected and used for hematological analyses, fetal hemoglobin quantification, and genotyping of 3.7 kb alpha-thalassemia deletion by GAP-PCR. The frequency of the 3.7 kb alpha-thalassemia deletion in homozygosity was 12.5% and in heterozygosity was 55.0%. An increase in alpha-thalassemia frequency was observed in children older than 5 years old (11.7% vs. 13.00%). Furthermore, 3.7 kb alpha-thalassemia deletion homozygotes had a significantly higher age of the first manifestation, lower number of blood transfusions by year, higher hemoglobin, lower mean corpuscular volume, mean corpuscular hemoglobin, and lower hemolytic rate observed by a lower number of reticulocytes count. There were no differences in fetal hemoglobin between the three genotypes. Moreover, the number of stroke events, osteomyelitis, splenomegaly, splenectomy, and hepatomegaly were lower when alpha-thalassemia was co-inherited. For the first time in the Angolan population, the effect of alpha-thalassemia deletion in sickle cell disease was analyzed and results reinforce that this trait influences the hematological and clinical aspects and produces a milder phenotype.
- Preliminary findings from the follow-up of pregnant sickle cell disease patients in Luanda, AngolaPublication . Brito, Miguel; Ginete, Catarina; Ferreira, J.; Delgadinho, Mariana; Sebastião, Cruz; Mateus, A.; Mendes, M.; Quinto, F.; Simão, F.; Fernandes, F.; Vasconcelos, J.Background: In Angola, the prevalence of Sickle Cell Disease (SCD) is almost 2%, and the carriers reach 21% of the population. Although its presentation tends to be very heterogeneous, chronic hemolytic anemia, frequent painful crises, and extensive organ damage are common features of these patients. Pregnancy in SCD patients is associated with an increase in severe outcomes, namely, a high risk of eclampsia and pre-eclampsia, stroke, and even death. Therefore, it is crucial to maintain continuous medical surveillance during pregnancy, especially in women with previous strokes. Moreover, health services in low- and middle-income countries are generally not prepared to follow these patients. Aims: We aim to present the preliminary findings from the cohort study conducted at the Lucrecia Paim Maternity Hospital (Luanda, Angola), which aims to determine pregnancy complications in SCD women, especially those responsible for maternal death, and, by supporting the obstetric consultations in this hospital, contribute to the reduction of mortality and morbidity rates. Methods: Pregnancy monitoring includes analysis of clinical history and incidents (number of hospitalizations, blood transfusions, strokes, and other clinical complications), hematological and biochemical analysis, transcranial Doppler to assess cerebral hemodynamics and genetic analysis (confirmation of the diagnosis, genotyping of four SNPs in the β-cluster to assess the haplotype, and evaluation of the presence of the 3.7kb deletion of the α-globin gene). Results: To date, 61 women are being followed in the obstetric consultations, with ages from 18 to 40 years old (mean 26.1±5.4). There are no records of previous strokes, although 83.9% of the patients have been previously transfused (47 out of 56), 98.2% have been hospitalized (55 out of 56) due to SCD complications and 19.6% (10 out of 54) had at least one miscarriage. At the first appointment, total hemoglobin values ranged from 4.70 to 10.40 g/dL (n=52, mean 7.18±1.30), erythrocytes from 1.46 to 5.42 x1012/L (n=52, mean 2.46±0,72), white blood cells count from 1.67 to 61.88 x109/L (n=51, mean 12.20±8.69), platelets from 24.2 to 710.0 x109/L (n=52, mean 272.2±155.9), and lactate dehydrogenase (LDH) from 263.3 to 2836.7 (n=50, mean 708.1±450.46). The CAR/CAR haplotype, which is usually associated with a more severe prognosis, is the most prevalent in this population (57.7%, 30 out of 52), followed by the CAR/SEN haplotype (25.0%, 13 out of 52). In this population, 17.3% (9 out of 52) are homozygous for the 3.7kb α-thalassemia deletion and 44.2% (23 out of 52) are carriers. This deletion influences hematological and clinical aspects of sickle cell disease patients, resulting in less severe phenotypes. TCD time-averaged mean of the maximum velocity (TAMMx) at the middle cerebral arteries ranged between 41 to 132 cm/s (n=61, mean 84cm/s), and peak systolic velocity (PSV) from 61 to 180 cm/s (mean 129 cm/s). At the basilar artery level, TAMMx obtained were between 29 to 102 cm/s (n=60, mean 52 cm/s) and PSV ranged from 43 to 141 cm/s (mean 78 cm/s). Summary - Conclusion: The main goal of this project is to study pregnancy-related complications and outcomes by giving support to an Angolan cohort of SCD pregnant women. We also intend to obtain TCD reference values of cerebral blood flow velocities in pregnant women with SCD as a risk predictor of vascular events as there are no values in the literature for this specific population.
- Sickle cell disease and gut health: the influence of intestinal parasites and the microbiome on Angolan childrenPublication . Delgadinho, Mariana; Ginete, Catarina; Santos, Brígida; Vasconcelos, Jocelyne Neto; Arez, Ana Paula; Brito, MiguelParasitic infections are a common problem in developing countries and can intensify morbidity in patients with sickle cell disease (SCD), increasing the severity of anemia and the need for transfusions. It has been demonstrated that both helminths and protozoa can affect gut microbiome composition. On the other hand, the presence of specific bacterial communities can also influence parasite establishment. Considering this, our aim was to associate the presence of intestinal parasites with the results of hematological analyses and microbiome composition evaluations in a population of Angolan children with and without SCD. A total of 113 stool samples were collected, and gut microbiome analysis was performed using 16S sequencing and real-time PCR to detect eight different intestinal parasites. In our population, more than half of children (55%) had at least one parasitic infection, and of these, 43% were co-infected. Giardia intestinalis and Ascaris lumbricoides were more frequently found in children from the rural area of Bengo. Moreover, SCD children with ascariasis exhibited higher values of leukocytes and neutrophils, whereas the total hemoglobin levels were lower. In regards to the gut microbiome, the presence of intestinal parasites lowered the prevalence of some beneficial bacteria, namely: Lactobacillus, Bifidobacterium, Cuneatibacter, Bacteroides uniformis, Roseburia, and Shuttleworthia. This study presents the prevalence of several intestinal parasites in a high-risk transmission area with scarce information and opens new perspectives for understanding the interaction between parasites, the microbiome, and SCD.
- Characterization of a cohort of Angolan children with sickle cell anemia treated with hydroxyureaPublication . Santos, Brígida; Ginete, Catarina; Gonçalves, Elisângela; Delgadinho, Mariana; Miranda, Armandina; Faustino, Paula; Arez, Ana Paula; Brito, MiguelBackground: Sickle Cell Anemia (SCA) is a monogenic disease, although its severity and response to treatment are very heterogeneous. Objectives: This study aims to characterize a cohort of Angolan children with SCA and evaluate their response to hydroxyurea (HU) treatment and the potential side effects and toxicity. Methods: The study enrolled 215 patients between 3 and 12 years old before and after the administration of HU, at a fixed dose of 20 mg/kg/day for 12 months. Results: A total of 157 patients started HU medication and 141 of them completed the 12-month treatment. After initiating HU treatment, the frequency of clinical events decreased (transfusions 53.4%, hospitalizations 47.1%). The response to HU medication varied among patients, with some experiencing an increase in fetal hemoglobin (HbF) of <5%. The mean increase in HbF was 11.9%, ranging from 1.8% to 31%. Responders to HU treatment were 57%, inadequate responders 38.7%, and non-adherent 4.2%. No clinical side effects related to HU were reported. Hematological toxicities were transient and reversible. Children naïve to HU and with lower HbF reported a higher number of hospitalizations caused by malaria infection. During HU treatment, the frequency of malaria episodes did not appear to be affected by HbF levels. Conclusions: the present study provided a valuable contribution to the understanding of the clinical and laboratory profiles of Angolan children with SCA. These findings support the evidence that the implementation of prophylactic measures and treatment with HU is associated with increased survival in children with SCA.