Dulcineia M. Albuquerque

An inherited mutation leading to production of only the short isoform of GATA-1 is associated with impaired erythropoiesis

Acquired somatic mutations in exon 2 of the hematopoietic transcription factor GATA-1 have been found in individuals with Down syndrome with both transient myeloproliferative disorder and acute megakaryoblastic leukemia. These mutations prevent the synthesis of the full-length protein but allow the synthesis of its short isoform, GATA-1s. Experiments in mice suggest that GATA-1s supports normal adult megakaryopoiesis, platelet formation and erythropoiesis. Here we report a mutation, 332G → C, in exon 2 of GATA1, leading to the synthesis of only the short isoform in seven affected males from two generations of a family. Hematological profiles of affected males demonstrate macrocytic anemia, normal platelet counts and neutropenia in most cases. Altogether, data suggest that GATA-1s alone, produced in low or normal levels, is not sufficient to support normal erythropoiesis. Moreover, this is the first study to indicate that a germline splicing mutation does not lead to leukemia in the absence of other cooperating events, such as Down syndrome.

Use of a nested polymerase chain reaction (N-PCR) to detect Trypanosoma cruzi in blood samples from chronic chagasic patients and patients with doubtful serologies

Chagas disease, caused by Trypanosoma cruzi, is an important endemic illness in Latin America. Serologic tests for T. cruzi detection in blood are sensitive, but their specificity is unsatisfactory. Direct detection of parasites in blood, either by xenodiagnosis or hemoculture, is highly specific but of low sensitivity. Molecular assays such as the Polymerase chain reaction (PCR), which amplifies certain repetitive sequences of nuclear DNA has been used as a good alternative tool for T. cruzi detection in human blood. The present study aimed to test PCR diagnosis in chagasic chronic patients and doubtful serologic patients attended in GEDOCH (Chagas Disease Study Group/UNICAMP, Brazil). A 149 bp fragment originated from nuclear DNA was specifically detected in chronic chagasic patients. The results of these tests were compared with serologic diagnosis performed using standard techniques and xenodiagnosis. We found that 43 out of 50 patients previously serodiagnosed as chagasic were positive using the N-PCR method. Thirteen of 30 patients with doubtful serologic results were confirmed as positive by N-PCR. Our results suggest that the N-PCR may be a complementary tool to serology in the diagnosis of Chagas disease, and that it is usefull for parasite detection in patients with chronic disease and patients with doubtful serologic results.

Therapy with hydroxyurea is associated with reduced adhesion molecule gene and protein expression in sickle red cells with a concomitant reduction in adhesive properties.

Propagation of the vaso‐occlusive process in sickle cell anaemia (SCA) is a complex process involving the adhesion of steady–state SCA patients red cells and reticulocytes to the vascular endothelium. The effect of hydroxyurea therapy (HUT) on the adhesive properties of sickle cells and the expression of adhesion molecule genes by erythroid cells of SCA individuals is not yet fully understood. The expressions of the CD36 gene and the VLA‐4‐integrin subunit genes, CD49d (α‐subunit) and CD29 (β‐subunit), were compared in the reticulocytes of steady–state SCA patients and patients on HUT using real‐time PCR. Basal adhesion of red cells from these subjects was also compared using static adhesion assays, as was surface protein expression, using flow cytometry. Basal sickle red cell adhesion to fibronectin was significantly greater than that of normal cells (P < 0.01); in contrast, HUT was associated with significantly lower levels (P < 0.01) of red cell adhesion that were similar to those of control cells; this decrease could not be justified solely by altered reticulocyte numbers in this population. Accordingly, flow cytometry demonstrated that reticulocytes from patients on HUT had significantly lower CD36 and CD49d surface expressions (P < 0.01) and, importantly, significantly lower expressions of the CD36, CD49d and CD29 genes (P < 0.05) than reticulocytes of SCA patients not on HUT. Taken together, data support the hypothesis that HUT reduces the adhesive properties of sickle cells and that this decrease appears to be mediated, at least in part, by a decrease in the gene and, consequently, surface protein expression of adhesion molecules such as VLA‐4 and CD36.

Trypanosoma cruzi: parasite persistence in tissues in chronic chagasic Brazilian patients

Chagas disease in the chronic phase may develop into cardiac and/or digestive forms. The pathogenesis of the disease is not yet clear and studies have been carried out to elucidate the role of parasite persistence in affected organs. The aim of this study was to detect and quantify Trypanosoma cruzi in paraffin-embedded tissue samples from chronic patients using NPCR (nested polymerase chain reaction) and QPCR (quantitative polymerase chain reaction) methods. These results were correlated to anatomopathological alterations in the heart and gastrointestinal tract (GIT). Of the 23 patients studied, 18 presented the cardiac form and five presented the cardiodigestive form of Chagas disease. DNA samples were randomly isolated from formalin-fixed paraffin-embedded sections of heart and GIT tissue of 23 necropsies and were analyzed through NPCR amplification. T. cruzi DNA was detected by NPCR in 48/56 (85.7%) heart and 35/42 (83.3%) GIT samples from patients with the cardiac form. For patients with the cardiodigestive form, NPCR was positive in 12/14 (85.7%) heart and in 14/14 (100%) GIT samples. QPCR, with an efficiency of 97.6%, was performed in 13 samples (11 from cardiac and 2 from cardiodigestive form) identified previously as positive by NPCR. The number of T. cruzi copies was compared to heart weight and no statistical significance was observed. Additionally, we compared the number of copies in different tissues (both heart and GIT) in six samples from the cardiac form and two samples from the cardiodigestive form. The parasite load observed was proportionally higher in heart tissues from patients with the cardiac form. These results show that the presence of the parasite in tissues is essential to Chagas disease pathogenesis.

Surveillance of active human cytomegalovirus infection in hematopoietic stem cell transplantation (HLA sibling identical donor): search for optimal cutoff value by real-time PCR

BackgroundHuman cytomegalovirus (CMV) infection still causes significant morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Therefore, it is extremely important to diagnosis and monitor active CMV infection in HSCT patients, defining the CMV DNA levels of virus replication that warrant intervention with antiviral agents in order to accurately prevent CMV disease and further related complications.MethodsDuring the first 150 days after allogeneic HSTC, thirty patients were monitored weekly for active CMV infection by pp65 antigenemia, nested-PCR and real-time PCR assays. Receiver operating characteristic (ROC) plot analysis was performed to determine a threshold value of the CMV DNA load by real-time PCR.ResultsUsing ROC curves, the optimal cutoff value by real-time PCR was 418.4 copies/104 PBL (sensitivity, 71.4%; specificity, 89.7%). Twenty seven (90%) of the 30 analyzed patients had active CMV infection and two (6.7%) developed CMV disease. Eleven (40.7%) of these 27 patients had acute GVHD, 18 (66.7%) had opportunistic infection, 5 (18.5%) had chronic rejection and 11 (40.7%) died - one died of CMV disease associated with GVHD and bacterial infection.ConclusionsThe low incidence of CMV disease in HSCT recipients in our study attests to the efficacy of CMV surveillance based on clinical routine assay. The quantification of CMV DNA load using real-time PCR appears to be applicable to the clinical practice and an optimal cutoff value for guiding timely preemptive therapy should be clinically validated in future studies.

Identification of β thalassemia mutations in South Brazilians

We have evaluated the mutation profile in a sample of 127 unrelated β-thalassemia (β thal) individuals, diagnosed through A2 and fetal hemoglobin quantification by high-performance liquid chromatography (HPLC) from the Brazilian southernmost state, where a flow of Italian immigrants had occurred in the late 19th century, mainly from Northern Italy. The molecular analysis was performed by DNA sequencing of the most common mutations found in the Mediterranean region. The β0 codon 39 nonsense mutation was the most frequent alteration (50.9%), followed by β+ IVSI 110 G>A (18.1%), β0 IVSI 1 G>A (12.9%), β+ IVSI 6 T>C (9.5%), and other rare mutations (8.6%). The chosen gene sequence was able to identify 91% β-thal mutations in the population studied, showing some similarity with allele frequencies of the mainly colonizing countries of Rio Grande do Sul state. The comparison of our results to other Brazilian studies has shown significant differences. Therefore, we can conclude that the genotypic profile of β-thal shows great variability. Hence, it would be arbitrary to infer regional study results as being representative of the Brazilian whole population. Brazilian researchers of different regions should identify their most frequent genotypes to provide better understanding on this disease and state adequate public health policies.

Erythropoiesis-driven regulation of hepcidin in human red cell disorders is better reflected through concentrations of soluble transferrin receptor rather than growth differentiation factor 15

Growth differentiation factor 15 (GDF‐15) is a bone marrow‐derived cytokine whose ability to suppress iron regulator hepcidin in vitro and increased concentrations found in patients with ineffective erythropoiesis (IE) suggest that hepcidin deficiency mediated by GDF‐15 may be the pathophysiological explanation for nontransfusional iron overload. We aimed to compare GDF‐15 production in anemic states with different types of erythropoietic dysfunction. Complete blood counts, biochemical markers of iron status, plasma hepcidin, GDF‐15, and known hepcidin regulators [interleukin‐6 and erythropoietin (EPO)] were measured in 87 patients with red cell disorders comprising IE and hemolytic states: thalassemia, sickle cell anemia, and cobalamin deficiency. Healthy volunteers were also evaluated for comparison. Neither overall increased EPO, nor variable GDF‐15 concentrations correlated with circulating hepcidin concentrations (P = 0.265 and P = 0.872). Relative hepcidin deficiency was found in disorders presenting with concurrent elevation of GDF‐15 and soluble transferrin receptor (sTfR), a biomarker of erythropoiesis, and sTfR had the strongest correlation with hepcidin (rS = −0.584, P < 0.0001). Our data show that high concentrations of GDF‐15 in vivo are not necessarily associated with pathological hepcidin reduction, and hepcidin deficiency was only found when associated with sTfR overproduction. sTfR elevation may be a necessary common denominator of erythropoiesis‐driven mechanisms to favor iron absorption in anemic states and appears a suitable target for investigative approaches to iron disorders. Am. J. Hematol. 89:385–390, 2014.

In vitro and in vivo anti-angiogenic effects of hydroxyurea

Hydroxyurea (HU), or hydroxycarbamide, is used for the treatment of some myeloproliferative and neoplastic diseases, and is currently the only drug approved by the FDA for use in sickle cell disease (SCD). Despite the relative success of HU therapy for SCD, a genetic disorder of the hemoglobin β chain that results in red-cell sickling, hemolysis, vascular inflammation and recurrent vasoocclusion, the exact mechanisms by which HU actuates remain unclear. We hypothesized that HU may modulate endothelial angiogenic processes, with important consequences for vascular inflammation. The effects of HU (50-200 μM; 17-24 h) on endothelial cell functions associated with key steps of angiogenesis were evaluated using human umbilical vein endothelial cell (HUVEC) cultures. Expression profiles of the HIF1A gene and the miRNAs 221 and 222, involved in endothelial function, were also determined in HUVECs following HU administration and the direct in vivo antiangiogenic effects of HU were assessed using a mouse Matrigel-plug neovascularization assay. Following incubation with HU, HUVECs exhibited high cell viability, but displayed a significant 75% inhibition in the rate of capillary-like-structure formation, and significant decreases in proliferative and invasive capacities. Furthermore, HU significantly decreased HIF1A expression, and induced the expression of miRNA 221, while downregulating miRNA 222. In vivo, HU reduced vascular endothelial growth factor (VEGF)-induced vascular development in Matrigel implants over 7 days. Findings indicate that HU is able to inhibit vessel assembly, a crucial angiogenic process, both in vitro and in vivo, and suggest that some of HUs therapeutic effects may occur through novel vascular mechanisms.

Cytomegalovirus (CMV) genotype in allogeneic hematopoietic stem cell transplantation

BackgroundBased on sequence variation in the UL55 gene that encodes glycoprotein B (gB), human cytomegalovirus (CMV) can be classified into four gB genotypes. Previous studies have suggested an association between CMV gB genotype and clinical outcome in patients who underwent an allogeneic hematopoietic stem cell transplant (HSCT). The goals of this study were identify patients with active infection caused by CMV in recipients of HSCT; determine the prevalence of CMV genotypes in the study group; correlate genotype with CMV disease, acute GVHD and overall survival.MethodsThe diagnosis of active CMV infection after allogeneic HSCT was detected by antigenemia (AGM) and/or nested-PCR (N-PCR). Positive samples from patients with active CMV infection were submitted to genotyping using N-PCR to amplify a region of UL55, followed by restriction analysis based on HinfI and RsaI digestion. Real-time PCR (qPCR) was used to determine the viral load during active CMV infection and antiviral treatment.ResultsSixty-three allogeneic HSCT recipients were prospectively evaluated; 49/63 (78%) patients were infected with CMV genotypes – gB1 19/49 (39%), gB2 17/49 (35%), gB3 3/49 (6%), gB4 7/49 (14%) – and 3 (6%) had mixed CMV genotypes (gB1 + gB3, gB1 + gB4 and gB2 + gB4). Characterized by gastrointestinal disease, CMV disease occurred in 3/49 (6.1%) patients, who had CMV gB3 genotype. These gB3 genotype patients presented an increasing AGM number, mean 125 (± 250) (P = 0.70), and qPCR copies/ml, mean 37938 (SD ± 50542) (P = 0.03), during antiviral treatment, when compared with other CMV genotypes. According to CMV genotypes, stratified overall survival was 55% for gB1, 43% for gB2; 0% for gB3 and 57% for gB4 (P = 0.03).ConclusionsOne of the restrictions of the presented study was the low number of CMV gB sub-cohorts). However, we demonstrated that the frequency of active CMV infection in this HSCT population was high, and the most prevalent genotype in these patients with active CMV infection was gB1 and gB2 genotype (74%). In Brazil, HSCT recipients seem to carry mainly gB1 and gB2 CMV genotype.

Clinical and genetic risk factors for moderate hyperbilirubinemia in Brazilian newborn infants.

Objective:To identify clinical and genetic risk factors for moderate hyperbilirubinemia during the first week of life.Study Design:Using univariate and multivariate multiple regression analyses, the RR for clinical factors, the African variant of glucose-6-phosphate dehydrogenase (G6PD) deficiency (G202A/A376G), and (TA)n UGT1A1 polymorphisms were established in a cohort of 608 Brazilian newborn infants. Hyperbilirubinemia was monitored until 134.5±49.8 h of life (IQR, 111.0 to 156.7). The dependent variable was total bilirubinemia (TB)⩾12.9 mg per 100 ml estimated by transcutaneous or plasma bilirubin measurements.Result:The African variant of G6PD deficiency and (TA)7/(TA)7 and (TA)7/(TA)8 polymorphisms present in 6.1 and 12.0% of newborns, respectively, were not risk factors for moderate hyperbilirubinemia. Coexpression of G6DP deficiency and UGT1A1 polymorphisms occurred in 0.49% of the subjects. Independent clinical predictors for TB⩾12.9 mg per 100 ml were gestational age <38 weeks and reference curve percentiles >P40th.Conclusion:In this study, G6PD deficiency and UGT1A1 gene promoter polymorphisms were not risk factors for moderate hyperbilirubinemia. Genetic factors may vary considerably in importance among different populations.