Silvia Maria Meira Magalhães

Polymorphisms of DNA repair genes are related to the pathogenesis of myelodysplastic syndrome

Some studies show that alterations in DNA repair genes polymorphisms are associated with the pathogenesis and susceptibility of Myelodysplastic Syndrome (MDS). We genotyped 60 MDS patients for six DNA repair gene polymorphisms: BRCA1 rs4793191, BRCA2 rs9567623, RAD51 rs1801320, XRCC5 rs3835, XRCC6 rs2267437 and LIG4 rs1805388. The G/C heterozygote genotype of rs1801320 polymorphism was associated with a decreased chance of developing MDS (p = 0.05). Additionally, the G/G homozygous genotype was associated with the presence of one cytopenia in whole blood. The genotype C/G and CG + GG of the rs2267437 polymorphism was associated with normal karyotype (p = 0.010) and bone marrow cellularity normocellular + hypercellular (p = 0.023). We found that the A/G heterozygous genotype of the rs3835 polymorphism is associated with decreased chance of developing MDS (p < 0.001). These results support the importance of RAD51, XRCC5 and XRCC6 genes polymorphisms in the maintenance of genomic stability promoting a better understanding of the genesis and etiology of MDS. Copyright

Proteins related to the spindle and checkpoint mitotic emphasize the different pathogenesis of hypoplastic MDS

Some studies show that alterations in expression of proteins related to mitotic spindle (AURORAS KINASE A and B) and mitotic checkpoint (CDC20 and MAD2L1) are involved in chromosomal instability and tumor progression in various solid and hematologic malignancies. This study aimed to evaluate these genes in MDS patients. The cytogenetics analysis was carried out by G-banding, AURKA and AURKB amplification was performed using FISH, and AURKA, AURKB, CDC20 and MAD2L1 gene expression was performed by qRT-PCR in 61 samples of bone marrow from MDS patients. AURKA gene amplification was observed in 10% of the cases, which also showed higher expression levels than the control group (p=0.038). Patients with normo/hypercellular BM presented significantly higher expression levels than hypocellular BM patients, but normo and hypercellular BM groups did not differ. After logistic regression analysis, our results showed that HIGH expression levels were associated with increased risk of developing normo/hypercellular MDS. It also indicated that age is associated with AURKA, CDC20 and MAD2L1 HIGH expression levels. The distinct expression of hypocellular patients emphasizes the prognostic importance of cellularity to MDS. The amplification/high expression of AURKA suggests that the increased expression of this gene may be related to the pathogenesis of disease.

Influence of functional polymorphisms in DNA repair genes of myelodysplastic syndrome

Myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic stem cell (HSC) malignances characterized by peripheral cytopenias and predisposition to acute myeloid leukemia transformation. Several studies show that the MDS pathogenesis is a complex and heterogeneous process that involves multiple steps through a sequence of genetic lesions in the DNA which lead to functional changes in the cell and the emergence and subsequent evolution of pre-malignant clone. Double strand breaks (DSB) lesions are the most severe type of DNA damage in HSCs, which, if not properly repaired, might contribute to the development of chromosomal abnormalities, which in turn may lead to leukemia development. We assessed the mRNA expression levels of ATM, BRCA1, BRCA2, RAD51, XRCC5, XRCC6 and LIG4 genes in bone marrow samples of 47 MDS patients in order to evaluate the association with functional polymorphisms rs228593, rs4793191, rs9567623, rs1801320, rs3835, rs2267437 and rs1805388, respectively, and try to detect clinical associations. We found that the rs228593, rs2267437 and rs1805388 functional polymorphisms probably alter the level of expression of the ATM, XRCC6 and LIG4 genes, respectively, being important in the maintenance of genomic instability in MDS.

HFE gene mutation and oxidative damage biomarkers in patients with myelodysplastic syndromes and its relation to transfusional iron overload: an observational cross-sectional study

Objective A relation between transfusional IOL (iron overload), HFE status and oxidative damage was evaluated. Design, setting and participants An observational cross-sectional study involving 87 healthy individuals and 78 patients with myelodysplastic syndromes (MDS) with and without IOL, seen at University Hospital of the Federal University of Ceará, Brazil, between May 2010 and September 2011. Methods IOL was defined using repeated measures of serum ferritin ≥1000 ng/mL. Variations in the HFE gene were investigated using PCR/restriction fragment length polymorphism (RFLP). The biomarkers of oxidative stress (plasmatic malonaldehyde (MDA), glutathione peroxidase (GPx) and superoxide dismutase (SOD)) were determined by spectrophotometry. Results The HFE gene variations were identified in 24 patients (30.77%) and 5 volunteers (5.74%). The H63D variant was observed in 35% and the C282Y variant as heterozygous in 5% of patients with MDS with IOL. One patient showed double heterozygous variant (C282Y/H63D) and serum ferritin of 11 649 ng/mL. In patients without IOL, the H63D variant was detected in 29.34%. Serum MDA levels were highest in patients with MDS with IOL, with a significant difference when compared with patients without IOL and healthy volunteers, pointing to the relationship between IOL and oxidative stress. The GPx and SOD were also significantly higher in these patients, indicating that lipid peroxidation increase was followed by an increase in antioxidant capacity. Higher ferritin levels were observed in patients with HFE gene variation. 95.7% of patients with MDS with the presence of HFE gene variations had received more of 20 transfusions. Conclusions We observed a significant increase in MDA levels in patients with MDS and IOL, suggesting an increased lipid peroxidation in these patients. The accumulation of MDA alters the organisation of membrane phospholipids, contributing to the process of cellular degeneration. Results show that excess iron intensifies the process of cell damage through oxidative stress. Trial registration number Local Ethics Committee (licence 150/2009).

Splicing factor SF3B1 mutations and ring sideroblasts in myelodysplastic syndromes: a Brazilian cohort screening study.

Background Myelodysplastic syndromes (MDS) comprise a group of malignant clonal hematologic disorders characterized by ineffective hematopoiesis and propensity for progression to acute myeloid leukemia. Acquired mutations in the gene encoding RNA splicing factor 3B subunit 1 (SF3B1) are highly associated with the MDS subtypes presenting ring sideroblasts, and represent a specific nosological entity. The effects of these mutations on clinical outcomes are diverse and contrasting. Methods A cohort of 91 Brazilian MDS patients, including patients with ring sideroblasts in the bone marrow, were screened for mutations in the SF3B1 hotspots (exons 12–15) by direct Sanger sequencing. Results SF3B1 heterozygous mutations were identified in six patients (7%), all of them with ring sideroblasts, thus confirming the association between SF3B1 mutations and myelodysplastic syndrome subtypes bearing this morphologic feature (frequency of 6/13, p-value < 0.0001). Conclusion This is the first screening of SF3B1 mutations in a cohort of Brazilian myelodysplastic syndrome patients. Our findings confirm that mutations in this splicing gene correlate with bone marrow ringed sideroblasts.

Proteins of the mitotic checkpoint and spindle are related to chromosomal instability and unfavourable prognosis in patients with myelodysplastic syndrome

Aims To study the immunoexpression of proteins related to the mitotic checkpoint (cell division cycle 20 (CDC20), mitotic arrest deficient 2 (MAD2)) and the mitotic spindle (Aurora-B) in patients with myelodysplastic syndrome (MDS). Methods Protein expression was analysed in bone marrow tissue samples from 40 patients with MDS using immunohistochemistry. Prognostic markers (transfusion dependency, depth of cytopenias, chromosomal abnormalities and survival) were also studied. Results Higher MAD2 expression was observed among patients with platelets <50×109/L than among patients with platelets ≥50×109/L (42.6±22.8% vs 22.7±19.1%, respectively). Higher CDC20 expression was identified among patients with three dysplasias compared with patients who presented with one or two dysplasias (33.9±24.1% vs 10.5±5.7% vs 12.8±7.8%, respectively), among patients who exhibited a complex versus non-complex karyotype (50.0±30.2% vs 18.4±14%, respectively) and among patients with platelets <50×109/L vs platelets ≥50×109/L (38.2±26.2% vs 16.1±12.4%, respectively). Higher Aurora-B expression was found in patients with an abnormal versus normal karyotype (21.2±13.2% vs 7.5±5.0%, respectively). High expression of MAD2 and CDC20 (≥50%) was associated with severe thrombocytopenia. We also found statistically significant differences in the overall survival rate when comparing different degrees of CDC20, MAD2 and Aurora-B protein expression. Conclusions To the best of our knowledge, this is the first report to demonstrate that these proteins are associated with chromosomal abnormalities and poor prognosis in patients with MDS.

Daily and Weekly Iron Supplementations are Effective in Increasing Hemoglobin and Reducing Anemia in Infants.

OBJECTIVE The objective of this study was to evaluate the effect of daily and weekly iron supplementation compared with control on hemoglobin values and anemia prevalence in infants. METHODS In this cluster-randomized study, we evaluated infants aged 12-24 months (n = 210) from three public daycare centers, during 4 months. Intervention-group A was allocated 25 mg elemental iron once weekly; intervention-group B received 12.5 mg elemental iron once daily; control-group C received 0.5 ml of a natural color additive. Hemoglobin was assessed before and after intervention. RESULTS Baseline mean hemoglobin was 8.81 ± 0.89 g/dl (group A), 9.70 ± 1.56 g/dl (group B) and 10.96 ± 0.92 g/dl (group C); after intervention, mean hemoglobin was 10.03 ± 0.78 g/dl (p < 0.0001), 10.65 ± 0.97 g/dl (p < 0.0001) and 11.30 ± 0.80 g/dl (p = 0.0034) for groups A, B and C, respectively. Anemia prevalence was as follows: group A, 100% at baseline and 83.3% at end of study, p = 0.0001; group, B 75.0% and 41.7%, p = 0.0002; group C, 50.0% and 37.5%, p = 0.182. CONCLUSIONS Weekly and daily iron supplementation were effective in increasing hemoglobin levels and reducing anemia in infants.

Increased parameters of oxidative stress and its relation to transfusion iron overload in patients with myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are group of stem cell disorders characterised by peripheral cytopaenias and a variable risk of progression to acute myeloid leukaemia. Most patients have anaemia and many develop transfusion dependence and iron overload (IOL), considered to be a negative independent prognostic factor associated with a higher risk of leukemic transformation and shorter survival.1 Iron pool is regarded as an important regulator of the production of reactive oxygen species (ROS). The excessive production of ROS and reactive nitrogen species causes lipid peroxidation, which can suppress self-renewal, the number of haematopoietic stem cells and directly induce DNA damage and genomic instability.2 However, the role of iron-mediated oxidative stress in the physiopathology of MDS remains uncertain. The present study aimed to evaluate plasma nitrite (NO2 −) and plasma malonaldehyde, secondary product of lipid peroxidation, in patients with MDS and correlate them with IOL due to transfusion dependence in MDS patients. Consecutive adult patients with MDS with and without IOL, followed at the University Hospital of the Federal University of Ceara, Brazil, were enrolled. The study was approved by the local ethics committee (licence 150/2009). They were classified according to the presence or absence of IOL defined as serum ferritin of 1000 ng/ml or …

Tissue doppler echocardiography detects preclinical markers of cardiac lesion in MDS patients

Myelodysplastic syndrome (MDS) is a clonal hematopoietic stem cell disorder of elderly people. Cardiac dysfunction is a marker of grim prognosis in MDS. We evaluated cardiac dysfunction of MDS patients with or without transfusion dependency by tissue doppler echocardiography. We found the average values of ventricular end-systolic and end-diastolic volumes in transfusion dependency MDS group higher than others. These results were strongly correlated to hemoglobin levels. Tissue Doppler Echocardiography should be routinely performed in MDS patients to detect preclinical cardiac alterations and prevent more heart insults in this group of chronic anemic aged patients.

Avaliação das concentrações de malonaldeído e nitrito em pacientes com anemia falciforme em tratamento ou não com hidroxiureia

Objective: To determine the serum levels of malondialdehyde and nitrite in patients with sickle cell anemia treated or not with hydroxyurea in outpatient?s setting. Methods: Of the 65 patients with sickle cell anemia selected for the study, 51 of them were not treated with hydroxyurea (Group 1), 14 made chronic use of hydroxyurea (Group 2) and 20 individuals had no hemoglobinopathies (Control Group). Results: The Control Group had a lower and more homogeneous concentration of malondialdehyde levels as compared to the other groups. The results of Groups 1 and 2 showed increased values of malondialdehyde levels when compared to the Control Group. Considering the values of Groups 1 and 2, there were no significant changes in the malondialdehyde levels. There was no significant difference in the serum levels of nitrite between the groups. Group 2 presented a statistically significant correlation between serum malondialdehyde levels and the clinical variables investigated. In turn, Group 1 showed correlation only with occurrence of three or more vaso-occlusive crises. There was no correlation between nitrite levels and the clinical variables. Conclusion: The results revealed that during the pathogenesis of sickle cell anemia, an increase in lipid peroxidation was observed. On the other hand, no changes in oxidative parameters were detected during treatment with hydroxyurea, probably due to the short period of treatment of the patients studied. Objetivo: Determinar os niveis sericos de malonaldeido e de nitrito em pacientes com anemia falciforme em tratamento ou nao com hidroxiureia e em acompanhamento ambulatorial. Metodos: Dos 65 pacientes com diagnostico de anemia falciforme selecionados para o estudo, 51 nao fizeram tratamento com hidroxiureia (Grupo 1) e 14 fizeram uso cronico de hidroxiureia (Grupo 2), sendo que 20 individuos nao tinham hemoglobinopatias (Grupo Controle). Resultados: O Grupo Controle possuia menor e mais homogenea concentracao dos niveis de malonaldeido em relacao aos outros grupos. Os resultados do Grupo 1 e do Grupo 2 mostraram valores aumentados dos niveis de malonaldeido quando comparados ao Grupo Controle. Quando comparados os valores dos Grupos 1 e 2, nao foram observadas alteracoes significativas nos niveis de malonaldeido. Nao houve diferenca significativa nos niveis sericos de nitrito entre os grupos. Verificou-se que, no Grupo 2, houve uma correlacao estatisticamente significativa dos niveis sericos de malonaldeido com as variaveis clinicas investigadas. Por sua vez, o Grupo 1 mostrou correlacao somente com a ocorrencia de tres ou mais crises vaso-oclusivas. Nao se verificou nenhuma correlacao nos niveis de nitrito com as variaveis clinicas. Conclusao: Os resultados revelaram que, durante o estabelecimento da patogenese da anemia falciforme, pode ser observado um aumento na peroxidacao lipidica. Por outro lado, durante o tratamento com a hidroxureia, nao foi detectada nenhuma alteracao nos parâmetros oxidativos, provavelmente devido ao curto periodo de tratamento dos pacientes em estudo.