Genetic Modifiers of Sickle Cell Anemia Phenotype in a Cohort of Angolan Children.

The aim of this study was to identify genetic markers in the HBB Cluster; HBS1L-MYB intergenic region; and BCL11A, KLF1, FOX3, and ZBTB7A genes associated with the heterogeneous phenotypes of Sickle Cell Anemia (SCA) using next-generation sequencing, as well as to assess their influence and prevalence in an Angolan population. Hematological, biochemical, and clinical data were considered to determine patients' severity phenotypes. Samples from 192 patients were sequenced, and 5,019,378 variants of high quality were registered. A catalog of candidate modifier genes that clustered in pathophysiological pathways important for SCA was generated, and candidate genes associated with increasing vaso-occlusive crises (VOC) and with lower fetal hemoglobin (HbF) were identified. These data support the polygenic view of the genetic architecture of SCA phenotypic variability. Two single nucleotide polymorphisms in the intronic region of 2q16.1, harboring the BCL11A gene, are genome-wide and significantly associated with decreasing HbF. A set of variants was identified to nominally be associated with increasing VOC and are potential genetic modifiers harboring phenotypic variation among patients. To the best of our knowledge, this is the first investigation of clinical variation in SCA in Angola using a well-customized and targeted sequencing approach.

Characterization of a cohort of Angolan children with sickle cell anemia treated with hydroxyurea.

BACKGROUND: 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 fix 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 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.

Differential expression of adhesion molecules in sickle cell anemia and gut microbiome effect.

Sickle cell anemia (SCA) causes a long-standing vascular inflammation state, leading to endothelial dysfunction and chronic overexpression of several adhesion molecules, which contributes to acute and constant vaso-occlusive (VOC) episodes. It has been demonstrated that hydroxyurea (HU) can reduce VOC events, organ damage, blood transfusions, and even the adhesion properties to endothelial cells of SCA subjects. Due to VOC episodes, these patients are also more susceptible to recurrent bacterial translocation and dysbiosis. Given this, our study aimed to uncover the interplay between adhesion molecules, gut microbiome, and hydroxyurea in a population of Angolan SCA children. Serum and fecal samples were obtained before and after HU treatment in 35 children. After HU, four of these adhesion molecules were significantly reduced: sE-selectin (p = 0.002), ADAMTS13 (p = 0.023), sICAM-1 (p = 0.003), and sVCAM-1 (p = 0.018). A positive correlation was observed between the number of neutrophils and sICAM-1, platelets, and sP-selectin, and also between leukocytes, sICAM-1, and sVCAM-1. Most taxa showing a significant correlation mainly belonged to the Clostridiales order. Specifically, from the Clostridium genera, the groups g19, g21, and g34 were all negatively correlated with HbF levels; g19, g21, and g24 positively correlated with leukocytes; g19 positively with neutrophils and sVCAM-1; and g34 positively with E- and P-selectin. Serratia, an opportunistic pathogen, was positively correlated with sE-selectin and sICAM-1 levels. Additionally, a negative correlation was observed between sP-selectin and Bifidobacterium. Research studies in this area could improve our understanding and contribute to finding new prognostic biomarkers to guarantee precise SCA patient stratification and predict severe complications.

Are Genetic Modifiers the Answer to Different Responses to Hydroxyurea Treatment?-A Pharmacogenetic Study in Sickle Cell Anemia Angolan Children.

Sickle 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.

DNA methyltransferase expression (DNMT1, DNMT3a and DNMT3b) as a potential biomarker for anti-VEGF diabetic macular edema response.

PURPOSE: DNA methylation is involved in Diabetic Retinopathy progression showing a metabolic memory mechanism. However, the association of DNA methyltransferase with diabetic macular edema is still unknown. We aimed to describe the differences in DNA methyltransferase gene expression in patients with different diabetic macular edema responses. METHODS: A total of 27 diabetic patients, aged 59-90 years, were prospectively enrolled in this cross-sectional study. The participants were classified into control group (CG, n = 11), diabetic macular edema responders (rDME, n = 9) and non-responder diabetic macular edema (nrDME, n = 7) after anti-vascular endothelial growth factor (anti-VEGF) treatment. Only cases with a complete ophthalmological examination, digital 133° color fundus, and SD-OCT assessments were used. After RNA extraction and first-strand cDNA synthesis, quantitative real-time PCR was performed with specific primers on the CFX Connect™ Real-Time PCR Detection System to assess differential transcriptional expression patterns. RESULTS: The DNMT1 gene showed a positive correlation (r = 0.617; p = 0.043) with Best Corrected Visual Acuity (BCVA) in CG, a positive correlation (r = 0.917; p = 0.010) with HbA1c in nrDME and a negative correlation (r = -0.659; p = 0.049) with GCL-IPL thickness in rDME. DNMT3A gene showed a positive correlation (r = -0.890; p = 0.001) with Sub-foveal Choroidal thickness in rDME whereas DNMT3b gene showed a negative correlation (r = -0.815; p = 0.007) with HbA1c and RNFL (r = -0.664; p = 0.026) in CG. CONCLUSIONS: Patients with similar metabolic profile risk factors showed associated DNA methyltransferase transcriptional expression patterns differences fitting with the anti-VEGF diabetic macular edema response. Further studies are needed to clarify if these results (1) reflect disease evolution, (2) translate the therapeutic impact, (3) or can help to predict the therapeutic resistance profile.

Epigenetic and Drug Response Modulation of Epigalocaten-In-3-Gallate in Staphylococcus aureus with Divergent Resistance Phenotypes.

Healthcare-associated methicillin-resistant Staphylococcus aureus infections represent extremely high morbidity and mortality rates worldwide. We aimed to assess the antimicrobial potential and synergistic effect between Epigalocatenin-3-gallate (EGCG) and different antibiotics in S. aureus strains with divergent resistance phenotypes. EGCG exposure effects in epigenetic and drug resistance key modulators were also evaluated. S. aureus strains (n = 32) were isolated from infected patients in a Lisbon hospital. The identification of the S. aureus resistance phenotype was performed through automatized methods. The antibiotic synergistic assay was performed through disk diffusion according to EUCAST guidelines with co-exposure to EGCG (250, 100, 50 and 25 µg/mL). The bacteria's molecular profile was assessed through FTIR spectroscopy. The transcriptional expression of OrfX, SpdC and WalKR was performed by using qRT-PCR. FTIR-spectroscopy analysis enabled the clear discrimination of MRSA/MSSA strains and the EGCG exposure effect in the bacteria's molecular profiles. Divergent resistant phenotypes were associated with divergent transcriptional expression of the epigenetic modulator OrfX, particularly in MRSA strains, as well as the key drug response modulators SpdC and WalKR. These results clearly demonstrate that EGCG exposure alters the expression patterns of key epigenetic and drug response genes with associated divergent-resistant profiles, which supports its potential for antimicrobial treatment and/or therapeutic adjuvant against antibiotic-resistant microorganisms.

Efficacy and safety of pharmacological interventions for managing sickle cell disease complications in children and adolescents: Systematic review with network meta-analysis.

This study aimed to synthesize the evidence on the effects of disease-modifying agents for managing sickle cell disease (SCD) in children and adolescents by means of a systematic review with network meta-analyses, surface under the cumulative ranking curve (SUCRA) and stochastic multicriteria acceptability analyses (SMAA) (CRD42022328471). Eightteen randomized controlled trials (hydroxyurea [n = 7], l-arginine [n = 3], antiplatelets [n = 2], immunotherapy/monoclonal antibodies [n = 2], sulfates [n = 2], docosahexaenoic acid [n = 1], niprisan [n = 1]) were analyzed. SUCRA and SMAA demonstrated that hydroxyurea at higher doses (30 mg/kg/day) or at fixed doses (20 mg/kg/day) and immunotherapy/monoclonal antibodies are more effective for preventing vaso-occlusive crisis (i.e., lower probabilities of incidence of this event; 14, 25, and 30%, respectively), acute chest syndrome (probabilities ranging from 8 to 30%), and needing of transfusions (11-31%), while l-arginine (100-200 mg/kg) and placebo were more prone to these events. Therapies were overall considered safe; however, antiplatelets and sulfates may lead to more severe adverse events. Although the evidence was graded as insufficient and weak, hydroxyurea remains the standard of care for this population, especially if a maximum tolerated dose schedule is considered.

Efficacy and safety of pharmacological interventions for managing sickle cell disease in children and adolescents: protocol for a systematic review with network meta-analysis.

INTRODUCTION: Sickle cell disease (SCD), an inherited haemoglobinopathy, has important impact on morbidity and mortality, especially in paediatrics. Previous systematic reviews are limited to adult patients or focused only on few therapies. We aim to synthesise the evidence on efficacy and safety of pharmacological interventions for managing SCD in children and adolescents. METHODS AND ANALYSIS: This systematic review protocol is available at Open Science Framework (doi:10.17605/OSF.IO/CWAE9). We will follow international recommendations on conduction and report of systematic reviews and meta-analyses. Searches will be conducted in PubMed, Scopus and Web of Science (no language nor time restrictions) (first pilot searches performed in May 2022). We will include randomised controlled trials comparing the effects of disease-modifying agents in patients with SCD under 18 years old. Outcomes of interest will include: vaso-occlusive crisis, haemoglobin levels, chest syndrome, stroke, overall survival and adverse events. We will provide a narrative synthesis of the findings, and whenever possible, results will be pooled by means of pairwise or Bayesian network meta-analyses with surface under the cumulative ranking curve analyses. Different statistical methods and models will be tested. Dichotomous outcomes will be reported as OR, risk ratio or HR, while continuous data will be reported as standard mean differences, both with 95% CI/credibility interval. The methodological quality of the trials will be evaluated using the Risk of Bias 2.0 tool, and the certainty of the evidence will be assessed with the Grading of Recommendations Assessment, Development and Evaluation approach. ETHICS AND DISSEMINATION: This study refers to a systematic review, so no ethics approval is necessary. We intent to publish our findings in international, peer-reviewed journal. Data will also be presented to peers in scientific events. Additionally, the results obtained in this study may contribute towards the update of therapeutic guidelines and for the development of health policies for SCD. PROSPERO REGISTRATION NUMBER: CRD42022328471.

Gut microbiota profile of COVID-19 patients: Prognosis and risk stratification (MicroCOVID-19 study).

Background: Gut microbiota is intrinsically associated with the immune system and can promote or suppress infectious diseases, especially viral infections. This study aims to characterize and compare the microbiota profile of infected patients with SARS-CoV-2 (milder or severe symptoms), non-infected people, and recovered patients. This is a national, transversal, observational, multicenter, and case-control study that analyzed the microbiota of COVID-19 patients with mild or severe symptoms at home, at the hospital, or in the intensive care unit, patients already recovered, and healthy volunteers cohabiting with COVID-19 patients. DNA was isolated from stool samples and sequenced in a NGS platform. A demographic questionnaire was also applied. Statistical analysis was performed in SPSS. Results: Firmicutes/Bacteroidetes ratios were found to be significantly lower in infected patients (1.61 and 2.57) compared to healthy volunteers (3.23) and recovered patients (3.89). Furthermore, the microbiota composition differed significantly between healthy volunteers, mild and severe COVID-19 patients, and recovered patients. Furthermore, Escherichia coli, Actinomyces naeslundii, and Dorea longicatena were shown to be more frequent in severe cases. The most common COVID-19 symptoms were linked to certain microbiome groups. Conclusion: We can conclude that microbiota composition is significantly affected by SARS-CoV-2 infection and may be used to predict COVID-19 clinical evolution. Therefore, it will be possible to better allocate healthcare resources and better tackle future pandemics.

Genetic modulation of anemia severity, hemolysis level, and hospitalization rate in Angolan children with Sickle Cell Anemia.

BACKGROUND: Sickle Cell Anemia (SCA) is a genetic disease caused by the c.20 A > T mutation in HBB gene, generally characterized by sickle erythrocytes, chronic hemolytic anemia, and vaso-occlusive events. This study aimed to investigate genetic modulators of anemia severity, chronic hemolytic rate, and clinical manifestations in pediatric SCA patients from Angola, where the disease is a severe public health problem. METHODS AND RESULTS: The study was conducted on 200 SCA children living in Luanda or Caxito province. Their clinical phenotype was collected from patients' hospital records. Hematological and biochemical phenotypes were characterized in steady state condition. Twelve polymorphic regions in VCAM1, CD36 and NOS3 genes were genotyped using PCR, RFLP, and Sanger sequencing. CD36 gene promoter variants showed a significant impact on anemia severity. Particularly, the rs1413661_C allele was associated with lower hemoglobin levels, and increased number of hospitalizations and transfusions. This is the first report associating this SNP with SCA phenotypic heterogeneity. Moreover, the rs1041163_C allele in VCAM1 was associated with lower LDH levels; inversely the rs2070744_C allele in NOS3 was related with higher LDH levels and number of hospitalizations, being a risk factor for increased hemolytic rate. CONCLUSION: This study highlights, for the first time in the Angolan population, the importance of the genetic modifiers of vascular cell adhesion and nitric oxide metabolism in SCA pediatric phenotypic variability.

Microbial gut evaluation in an angolan paediatric population with sickle cell disease.

Sickle cell disease (SCD) is one of the most common genetic conditions worldwide. It can contribute up to 90% of under-5 mortality in sub-Saharan Africa. Clinical manifestations are very heterogeneous, and the intestinal microbiome appears to be crucial in the modulation of inflammation, cell adhesion and induction of aged neutrophils, 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 studies. Our aim was to sequence the bacterial 16S rRNA gene in order to characterize the gut microbiome of Angolan children with SCA and healthy siblings as a control. A total of 72 stool samples were obtained from children between 3 and 14 years old. Our data showed that the two groups exhibit some notable differences in microbiota relative abundance at different classification levels. Children with SCA have a higher number of the phylum Actinobacteria. As for the genus level, Clostridium cluster XI bacteria was more prevalent in the SCA children, whereas the siblings had a higher abundance of Blautia, Aestuariispira, Campylobacter, Helicobacter, Polaribacter and Anaerorhabdus. In this study, we have presented the first microbiota analysis in an Angolan paediatric population with SCD and provided a detailed view of the microbial differences between patients and healthy controls. There is still much to learn before fully relying on the therapeutic approaches for gut modulation, which is why more research in this field is crucial to making this a reality.

How Hydroxyurea Alters the Gut Microbiome: A Longitudinal Study Involving Angolan Children with Sickle Cell Anemia.

Sickle 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.

Drug Resistance and Epigenetic Modulatory Potential of Epigallocatechin-3-Gallate Against Staphylococcus aureus.

Antimicrobial resistance of human pathogens, such as methicillin-resistant Staphylococcus aureus, is described by the World Health Organization as a health global challenge and efforts must be made for the discovery of new effective and safe compounds. This work aims to evaluate epigallocatechin-3-gallate (EGCG) epigenetic and modulatory drug potential against S. aureus in vitro and in vivo. S. aureus strains were isolated from commensal flora of healthy volunteers. Antibiotic susceptibility and synergistic assay were assessed through disk diffusion accordingly to EUCAST guidelines with and without co-exposure to EGCG at final concentrations of 250 µg/ml, 100 µg/ml, 50 µg/ml, and 25 µg/ml. Transcriptional expression of orfx, spdC, and WalKR was performed through qRT-PCR. A 90-day interventional study was performed with daily consumption of 225 mg of EGCG. Obtained data revealed a high prevalence of S. aureus colonization in healthcare workers and clearly demonstrated the antimicrobial and synergistic potential of EGCG as well as divergent resistant phenotypes associated with altered transcriptional expression of epigenetic and drug response modulators genes. Here, we demonstrate the potential of EGCG for antimicrobial treatment and/or therapeutic adjuvant against antibiotic-resistant microorganisms and report divergent patterns of epigenetic modulators expression associated with phenotypic resistance profiles.

Genotypic Diversity among Angolan Children with Sickle Cell Anemia.

BACKGROUND: 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.

Co-Inheritance of alpha-thalassemia and sickle cell disease in a cohort of Angolan pediatric patients.

The 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 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.

Environmentally Relevant Concentrations of Bisphenol A Interact with Doxorubicin Transcriptional Effects in Human Cell Lines.

The worldwide production of synthetic chemicals, including endocrine disruptor chemicals (EDCs), such as Bisphenol A (BPA) has increased significantly in the last two decades. Human exposure to BPA, particularly through ingestion, is continuous and ubiquitous. Although, considered a weak environmental estrogen, BPA can induce divergent biological responses through several signaling pathways, including carcinogenesis in hormone-responsive organs. However, and despite the continuous increase of tumor cell-resistance to therapeutic drugs, such as doxorubicin (DOX), information regarding BPA drug interactions is still scarce, although its potential role in chemo-resistance has been suggested. This study aims to assess the potential interactions between environmentally relevant levels of BPA and DOX at a therapeutic dosage on Hep-2 and MRC-5 cell lines transciptome. Transcriptional effects in key-player genes for cancer biology, namely c-fos, p21, and bcl-xl, were evaluated through qRT-PCR. The cellular response was analyzed after exposure to BPA, DOX, or co-exposure to both chemicals. Transcriptional analysis showed that BPA exposure induces upregulation of bcl-xl and endorses an antagonistic non-monotonic response on DOX transcriptional effects. Moreover, the BPA interaction with DOX on c-fos and p21 expression emphasize its cellular specificity and divergent effects. Overall, Hep-2 was more susceptible to BPA effects in a dose-dependent manner while MRC-5 transcriptional levels endorsed a non-monotonic response. Our data indicate that BPA environmental exposure may influence chemotherapy outcomes, which emphasize the urgency for a better understanding of BPA interactions with chemotherapeutic agents, in the context of risk assessment.