Minimal doses of hydroxyurea for sickle cell disease

The use of hydroxyurea (HU) can improve the clinical course of sickle cell disease. However, several features of HU treatment remain unclear, including the predictability of drug response and determination of adequate doses, considering positive responses and minimal side effects. In order to identify adequate doses of HU for treatment of sickle cell disease, 10 patients, 8 with sickle cell anemia and 2 with Sß thalassemia (8SS, 2Sß), were studied for a period of 6 to 19 months in an open label dose escalation trial (10 to 20 mg kg-1 day-1). Hemoglobin (Hb), fetal hemoglobin (Hb F) and mean corpuscular volume (MCV) values and reticulocyte, neutrophil and platelet counts were performed every two weeks during the increase of the HU dose and every 4 weeks when the maximum HU dose was established. Reduction in the number of vasoocclusive episodes was also considered in order to evaluate the efficiency of the treatment. The final Hb and Hb F concentrations, and MCV values were significantly higher than the initial values, while the final reticulocyte and neutrophil counts were significantly lower. There was an improvement in the concentration of Hb (range: 0.7-2.0 g/dl) at 15 mg HU kg-1 day-1, but this concentration did not increase significantly when the HU dose was raised to 20 mg kg-1 day-1. The concentration of Hb F increased significantly (range: 1.0-18.1%) when 15 mg HU was used, and continued to increase when the dose was raised to 20 mg kg-1 day-1. The final MCV values increased 11-28 fl (femtoliters). However, reticulocyte (range: 51-205 x 109/l) and neutrophil counts (range: 9.5-1.3 x 109/l) obtained at this dose were significantly lower than those obtained with 15 mg kg-1 day-1. All patients reported a decrease in frequency or severity of vasoocclusive episodes. These results suggest that a hydroxyurea dose of 15 mg kg-1 day-1 seems to be adequate for treatment of sickle cell disease in view of the minimal side effects observed and the improvement in laboratory and clinical parameters.

Interleukin 8 as a vaso-occlusive marker in Brazilian patients with sickle cell disease

Sickle cell disease has a worldwide distribution and is a public health problem in Brazil. Although vaso-occlusive crisis (VOC) is one of the most important clinical features of the disease, there are still several steps of its pathogenesis which are unknown. The increase of the chemotactic factor interleukin 8 (IL-8) has been reported to be involved in sickle cell disease crisis, but this has not been demonstrated conclusively. In the present study we analyzed serum IL-8 levels by ELISA and hematological parameters and hemoglobin patterns by standard techniques in 23 (21 SS and 2 SC) Brazilian patients with sickle cell syndromes during VOC caused by different inducing factors, 22 (21 SS and 1 SC) sickle cell patients out of crisis, and 11 healthy controls. Increased IL-8 levels were observed in 19 of 23 VOC patients (79.2%), 3 of them with more than 1,000 pg/ml. Seventeen of 22 (77.3%) non-crisis patients showed low IL-8 levels (less than 15 pg/ml). Healthy controls had low IL-8 levels. A significant difference in serum IL-8 levels was observed between crisis and non-crisis sickle cell patients (P<0.0001). There was no correlation between IL-8 levels and hematological data or hemoglobin patterns. High serum IL-8 levels were observed in VOC patients independently of the crisis-inducing factor. We conclude that in the studied population, IL-8 concentration may be a useful VOC marker, although the mechanism of the pathogenic process of sickle cell VOC syndromes remains unclear.

Effect of hydroxyurea on G gamma chain fetal hemoglobin synthesis by sickle-cell disease patients

Hydroxyurea is used for sickle-cell disease patients in order to increase fetal hemoglobin synthesis and consequently decrease the severity of pain episodes. Fetal hemoglobin, which is formed by gamma-globin chains A and G, is present in a constant composition throughout fetal development: about 75% of Ggamma and 25% of Agamma. In contrast, adult red cells contain about 40% of Ggamma and 60% of Agamma. In the present study, we analyzed the effect of hydroxyurea induction on the gamma chain composition of fetal hemoglobin in 31 sickle-cell disease patients treated with hydroxyurea. The control group was composed of 30 sickle-cell disease patients not treated with hydroxyurea in clinical steady state. The patients were older than 13 years and were not matched for age. All patients were seen at Hemocentro/UNICAMP and Boldrini Infantile Center, Campinas, SP, Brazil. The levels of total hemoglobin were significantly higher in patients treated with hydroxyurea (mean ± SD, 9.6 ± 2.16 g/dl) than in untreated patients (8.07 ± 0.91 g/dl). Fetal hemoglobin levels were also higher in treated patients (14.16 ± 8.31%) than in untreated patients (8.8 ± 4.09%), as was the Ggamma/Agamma ratio (1.45 ± 0.78 vs 0.98 ± 0.4, P < 0.005). The increase in the Ggamma/Agamma ratio in patients treated with hydroxyurea suggests the prevalence of a pattern of fetal hemoglobin synthesis, whereas patients not treated with hydroxyurea maintain the adult pattern of fetal hemoglobin synthesis. Because no correlation was observed between the Ggamma/Agamma ratio and total hemoglobin or fetal hemoglobin levels, the increase in Ggamma chain synthesis may not imply a higher production of hemoglobin.

Risk factors for alloimmunization by patients with sickle cell disease

Blood transfusion in patients with sickle cell disease (SCD) is limited by the development of alloantibodies to erythrocytes. In the present study, the frequency and risk factors for alloimmunization were determined. Transfusion records and medical charts of 828 SCD patients who had been transfused and followed at the Belo Horizonte Blood Center, Belo Horizonte, MG, Brazil, were retrospectively reviewed. Alloimmunization frequency was 9.9% (95% CI: 7.9 to 11.9%) and 125 alloantibodies were detected, 79% of which belonged to the Rhesus and Kell systems. Female patients developed alloimmunization more frequently (P = 0.03). The median age of the alloimmunized group was 23.3 years, compared to 14.6 years for the non-alloimmunized group (P < 0.0001). Multivariate analyses were applied to the data for 608 hemoglobin (Hb) SS or SC patients whose number of transfusions was recorded accurately. Number of transfusions (P = 0.00006), older age (P = 0.056) and Hb SC (P = 0.02) showed independent statistical associations with alloimmunization. Hb SC patients older than 14 years faced a 2.8-fold higher (95% CI: 1.3 to 6.0) risk of alloimmunization than Hb SS patients. Female Hb SC patients had the highest risk of developing alloantibodies. In patients younger than 14 years, only the number of transfusions was significant. We conclude that an increased risk of alloimmunization was associated with older patients with Hb SC, specially females, even after adjustments were made for the number of transfusions received, the most significant variable.

The clinical impact of MTHFR polymorphism on the vascular complications of sickle cell disease

Sickle cell disease (SCD) is one of the most common inherited diseases in the world and the patients present notorious clinical heterogeneity. It is known that patients with SCD present activation of the blood coagulation and fibrinolytic systems, especially during vaso-occlusive crises, but also during the steady state of the disease. We determined if the presence of the factor V gene G1691A mutation (factor V Leiden), the prothrombin gene G20210A variant, and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism may be risk factors for vascular complications in individuals with SCD. We studied 53 patients with SCD (60% being women), 29 with SS (sickle cell anemia; 28 years, range: 13-52 years) and 24 with SC (sickle-hemoglobin C disease; 38.5 years, range: 17-72 years) hemoglobinopathy. Factor V Leiden, MTHFR C677T polymorphism, and prothrombin G20210A variant were identified by PCR followed by further digestion of the PCR product with specific endonucleases. The following vascular complications were recorded: stroke, retinopathy, acute thoracic syndrome, and X-ray-documented avascular necrosis. Only one patient was heterozygous for factor V Leiden (1.8%) and there was no prothrombin G20210A variant. MTHFR 677TT polymorphism was detected in 1 patient (1.8%) and the heterozygous form 677TC was observed in 18 patients (34%, 9 with SS and 9 with SC disease), a prevalence similar to that reported by others. No association was detected between the presence of the MTHFR 677T allele and other genetic modulation factors, such as alpha-thalassemia, ß-globin gene haplotype and fetal hemoglobin. The presence of the MTHFR 677T allele was associated with the occurrence of vascular complications in SCD, although this association was not significant when each complication was considered separately. In conclusion, MTHFR C677T polymorphism might be a risk factor for vascular complications in SCD.

Evaluation of renal function in sickle cell disease patients in Brazil

The objective of this study was to investigate renal function in a cohort of 98 patients with sickle cell disease (SCD) followed up at a tertiary hospital in Brazil. Clinical and laboratory characteristics at the time of the most recent medical examination were analyzed. Renal function was evaluated by the estimation of glomerular filtration rate (GFR) by the criteria of the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI). We compared patients with normal GFR to patients with decreased GFR (<60 mL·min-1·(1.73 m²)-1) and hyperfiltration (>120 mL·min-1·(1.73 m²)-1). Comparison between patients according to the use of hydroxyurea and comparison of clinical and laboratory parameters according to GFR were also carried out. Average patient age was 33.8 ± 13.3 years (range 19-67 years), and 57 (58.1%) patients were females. The comparison of patients according to GFR showed that patients with decreased GFR (<60 mL·min-1·(1.73 m²)-1) were older, had lower levels of hematocrit, hemoglobin and platelets and higher levels of urea and creatinine. Independent risk factors for decreased GFR were advanced age (OR = 21.6, P < 0.0001) and anemia (OR = 39.6, P < 0.0001). Patients with glomerular hyperfiltration tended to be younger, had higher levels of hematocrit, hemoglobin and platelets and lower levels of urea and creatinine, with less frequent urinary abnormalities. Hydroxyurea, at the dosage of 500-1000 mg/day, was being administered to 28.5% of the patients, and there was no significant difference regarding renal function between the two groups. Further studies are required to establish the best therapeutic approach to renal abnormalities in SCD.

Development of cellular and gene therapies for b[beta]-Thalassemia and sickle cell disease

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Genomic architecture of sickle cell disease clinical variation in children from West Africa : a case-control study design

Contexte : L’anémie falciforme ou drépanocytose est un problème de santé important, particulièrement pour les patients d’origine africaine. La variation phénotypique de l’anémie falciforme est problématique pour le suivi et le traitement des patients. L’architecture génomique responsable de cette variabilité est peu connue. Principe : Mieux saisir la contribution génétique de la variation clinique de cette maladie facilitera l’identification des patients à risque de développer des phénotypes sévères, ainsi que l’adaptation des soins. Objectifs : L’objectif général de cette thèse est de combler les lacunes relatives aux connaissances sur l’épidémiologie génomique de l’anémie falciforme à l’aide d’une cohorte issue au Bénin. Les objectifs spécifiques sont les suivants : 1) caractériser les profils d’expressions génomiques associés à la sévérité de l’anémie falciforme ; 2) identifier des biomarqueurs de la sévérité de l’anémie falciforme ; 3) identifier la régulation génétique des variations transcriptionelles ; 4) identifier des interactions statistiques entre le génotype et le niveau de sévérité associé à l’expression ; 5) identifier des cibles de médicaments pour améliorer l’état des patients atteints d’anémie falciforme. Méthode : Une étude cas-témoins de 250 patients et 61 frères et soeurs non-atteints a été menée au Centre de Prise en charge Médical Intégré du Nourrisson et de la Femme Enceinte atteints de Drépanocytose, au Bénin entre février et décembre 2010. Résultats : Notre analyse a montré que des profils d’expressions sont associés avec la sévérité de l’anémie falciforme. Ces profils sont enrichis de génes des voies biologiques qui contribuent à la progression de la maladie : l’activation plaquettaire, les lymphocytes B, le stress, l’inflammation et la prolifération cellulaire. Des biomarqueurs transcriptionnels ont permis de distinguer les patients ayant des niveaux de sévérité clinique différents. La régulation génétique de la variation de l’expression des gènes a été démontrée et des interactions ont été identifiées. Sur la base de ces résultats génétiques, des cibles de médicaments sont proposées. Conclusion: Ce travail de thèse permet de mieux comprendre l’impact de la génomique sur la sévérité de l’anémie falciforme et ouvre des perspectives de développement de traitements ciblés pour améliorer les soins offerts aux patients.

Genetic determinants of clinical heterogeneity in sickle cell disease

L’anémie falciforme est une maladie monogénique causée par une mutation dans le locus de la β-globine. Malgré le fait que l’anémie falciforme soit une maladie monogénique, cette maladie présente une grande hétérogénéité clinique. On présume que des facteurs environnementaux et génétiques contribuent à cette hétérogénéité. Il a été observé qu’un haut taux d’hémoglobine fœtale (HbF) diminuait la sévérité et la mortalité des patients atteints de l’anémie falciforme. Le but de mon projet était d’identifier des variations génétiques modifiant la sévérité clinique de l’anémie falciforme. Dans un premier temps, nous avons effectué la cartographie-fine de trois régions précédemment associées avec le taux d’hémoglobine fœtale. Nous avons ensuite effectué des études d’association pan-génomiques avec deux complications cliniques de l’anémie falciforme ainsi qu’avec le taux d’hémoglobine fœtale. Hormis les régions déjà identifiées comme étant associées au taux d’hémoglobine fœtale, aucun locus n’a atteint le niveau significatif de la puce de génotypage. Pour identifier des groupes de gènes modérément associés au taux d’hémoglobine fœtale qui seraient impliqués dans de mêmes voies biologiques, nous avons effectué une étude des processus biologiques. Finalement, nous avons effectué l’analyse de 19 exomes de patients Jamaïcains ayant des complications cliniques mineures de l’anémie falciforme. Compte tenu de la taille des cohortes de réplication disponibles, nous n’avons pas les moyens de valider statistiquement les variations identifiées par notre étude. Cependant, nos résultats fournissent de bons gènes candidats pour des études fonctionnelles et pour les réplications futures. Nos résultats suggèrent aussi que le β-hydroxybutyrate en concentration endogène pourraient influencer le taux d’hémoglobine fœtale. De plus, nous montrons que la cartographie-fine des régions associées par des études pan-génomiques peut identifier des signaux d’association additionnels et augmenter la variation héritable expliquée par cette région.

Up-regulation of NADPH oxidase components and increased production of interferon-gamma by leukocytes from sickle cell disease patients

We have previously demonstrated that mononuclear leukocytes from patients with sickle cell disease (SCD) release higher amounts of superoxide compared with normal controls. The aim of this study was to further study the NADPH oxidase system in these patients by investigating gene expression of NADPH oxidase components, phosphorylation of p47(phox) component, and the release of cytokines related to NADPH oxidase activation in mononuclear leukocytes from patients with SCD. gp91(phox) gene expression was significantly higher in monocytes from SCD patients compared with normal controls (P = 0.036). Monocytes from SCD patients showed higher levels of p47 phox phosphorylation compared with normal controls. INF-gamma release by lymphocytes from SCD patients was significantly higher compared with normal controls, after 48 h culture with phytohemagglutinin (P = 0.02). The release of TNF-alpha by monocytes from SCD patients and normal controls was similar after 24 and 48 h culture with lipopolysaccharide (P > 0.05). We conclude that monocytes from SCD patients show higher levels of gp91(phox) gene expression and p47(phox) phosphorylation, along with increased IFN-gamma release by SCD lymphocytes. These findings help to explain our previous observation showing the increased respiratory burst activity of mononuclear leukocytes from SCD patients and may contribute to inflammation and tissue damage in these patients.

Assessment of the In Vivo Genotoxicity of New Lead Compounds to Treat Sickle Cell Disease

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Mutagenicity of New Lead Compounds to Treat Sickle Cell Disease Symptoms in a Salmonella/Microsome Assay

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Design, Synthesis, and Pharmacological Evaluation of Novel Hybrid Compounds to Treat Sickle Cell Disease Symptoms. Part II: Furoxan Derivatives

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)