Sara Teresinha Olalla Saad

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.

Prevalence of homozygosity for the deleted alleles of glutathione S-transferase mu (GSTM1) and theta (GSTT1) among distinct ethnic groups from Brazil: relevance to environmental carcinogenesis?

Arruda VR, Grignolli CE, Goncalves MS, Soares MC, Menezes R, Saad STO, Costa FF. Prevalence of homozygosity for the deleted alleles of glutathione S‐transferase mu (GSTMl) and theta (GSTTl) among distinct ethnic groups from Brazil: relevance to enviromental carcinogenesis? Clin Genet 1998: 54: 210–214. 0 Munksgaard, 1998

The release of nitric oxide and superoxide anion by neutrophils and mononuclear cells from patients with sickle cell anaemia

The aim of this work was to investigate the release of nitric oxide and superoxide by neutrophils and mononuclear cells from patients with sickle cell anaemia. Nitric oxide release was assayed by the ability of leucocytes to inhibit thrombin‐induced washed platelet aggregation. Superoxide release was assessed by a cytochrome c reduction assay. Neutrophils from sickle cell anaemia patients released nitric oxide in a similar manner to those from healthy controls, because inhibition of platelet aggregation by neutrophils from sickle cell anaemia or from healthy controls was blocked by the inhibitor of nitric oxide synthesis N ω‐nitro‐l‐arginine methyl ester (300 μm), but not by N ω‐nitro‐d‐arginine methyl ester (300 μm) and was reversed by l‐arginine (1 mm). Additionally, a similar number of neutrophils from sickle cell anaemia patients and from healthy controls was required to inhibit platelet aggregation. Mononuclear cells from sickle cell anaemia patients inhibited platelet aggregation only in the presence of superoxide dismutase (60 U ml−1). Phorbol 12‐myristate 13‐acetate (PMA, 30 nm)‐ or zymosan (100 particles/cell)‐induced release of superoxide by mononuclear cells from sickle cell anaemia patients was significantly higher than that observed in mononuclear cells from healthy controls (P < 0.001 and P < 0.01 respectively, Mann‐Whitney test). The levels of superoxide released by neutrophils from sickle cell anaemia patients were similar to those from healthy controls. We conclude that mononuclear cells from sickle cell anaemia patients release more superoxide than those from healthy controls, when stimulated with PMA or zymosan in vitro. Considering that superoxide inactivates nitric oxide, that nitric oxide is an important endogenous vasodilator, and that superoxide produces oxidant damage, this greater production of superoxide by mononuclear cells from sickle cell anaemia patients may represent an additional risk factor for the obstruction of the microcirculation and tissue damage in these patients.

Single nucleotide polymorphism array lesions, TET2, DNMT3A, ASXL1 and CBL mutations are present in systemic mastocytosis.

We hypothesized that analysis of single nucleotide polymorphism arrays (SNP-A) and new molecular defects may provide new insight in the pathogenesis of systemic mastocytosis (SM). SNP-A karyotyping was applied to identify recurrent areas of loss of heterozygosity and bidirectional sequencing was performed to evaluate the mutational status of TET2, DNMT3A, ASXL1, EZH2, IDH1/IDH2 and the CBL gene family. Overall survival (OS) was analyzed using the Kaplan-Meier method. We studied a total of 26 patients with SM. In 67% of SM patients, SNP-A karyotyping showed new chromosomal abnormalities including uniparental disomy of 4q and 2p spanning TET2/KIT and DNMT3A. Mutations in TET2, DNMT3A, ASXL1 and CBL were found in 23%, 12%, 12%, and 4% of SM patients, respectively. No mutations were observed in EZH2 and IDH1/IDH2. Significant differences in OS were observed for SM mutated patients grouped based on the presence of combined TET2/DNMT3A/ASXL1 mutations independent of KIT (P = 0.04) and sole TET2 mutations (P<0.001). In conclusion, TET2, DNMT3A and ASXL1 mutations are also present in mastocytosis and these mutations may affect prognosis, as demonstrated by worse OS in mutated patients.

Increased adhesive properties of neutrophils in sickle cell disease may be reversed by pharmacological nitric oxide donation

Adhesive interactions between blood cells and endothelium lead to vaso-occlusion in sickle cell disease. This study suggests that pharmacological nitric oxide donation may reduce neutrophil adhesion to vascular endothelium. Increased leukocyte adhesion to vascular endothelium contributes to vaso-occlusion in sickle cell disease. Since nitric oxide bioavailability is decreased in sickle cell disease and nitric oxide may inhibit leukocyte adhesion, we investigated whether stimulation of NO-signaling pathways can reduce the adhesive properties of neutrophils from sickle cell disease individuals (sickle cell diseaseneu). sickle cell diseaseneu presented greater adhesion in vitro to both fibronectin and ICAM-1 than control neutrophils. Co-incubation of sickle cell diseaseneu with the nitric oxide-donor agents, sodium nitroprusside and dietheylamine NONOate (DEANO), and the guanylate cyclase stimulator, BAY41-2272, all significantly reduced the increased adhesion to fibronectin/ICAM-1. Oxadiazolo[4,3-a]quinoxalin-1-one, a guanylate cyclase inhibitor, reversed sodium nitroprusside/DEANO-diminished adhesion to fibronectin, implicating cGMP-dependent signaling in this mechanism. Interestingly, intracellular cGMP was significantly higher in neutrophils from sickle cell disease individuals on hydroxyurea (sickle cell diseaseHUneu). Accordingly, sickle cell diseaseHUneu adhesion to fibronectin/ICAM-1 was significantly lower than that of sickle cell diseaseneu. Agents that stimulate the nitric oxide/cGMP-dependent pathway may have beneficial effects on leukocyte function if used in these subjects.

Increased soluble guanylate cyclase activity in the red blood cells of sickle cell patients

Activation of soluble guanylate cyclase (sGC) has been reported to up‐regulate γ‐globin gene transcription in erythroid cell lines and primary erythroblasts. sGC is activated by nitric oxide (NO), subsequently catalysing the conversion of guanosine triphosphate to cyclic guanosine monophosphate (cGMP), which mediates various physiological responses. To study the importance of this mechanism in the erythroid cells of sickle cell patients, cGMP levels were measured in the red blood cells (RBC) of normal individuals, steady‐state sickle cell patients (SS) and SS patients on hydroxyurea (HU) therapy (SS + HU). cGMP levels were found to be significantly higher in RBC of SS patients (SS RBC) than in RBC of normal individuals, and were further increased in RBC of SS + HU patients. cGMP levels correlated with fetal haemoglobin (HbF) levels in SS/SS + HU patients, but not with reticulocyte count. Furthermore, NO‐stimulated sGC activity, following incubation of cells with a NO donor, was significantly greater in SS RBC than in normal RBC. These results demonstrate, for the first time, an increased metabolism of NO mediated by sGC in the SS RBC, which is further increased by hydroxyurea. Augmentation of cGMP levels by NO in erythroid cells may constitute a mechanism for induction of HbF and other erythrocyte functions and represent a possible therapeutic target for treatment of sickle cell disease.

SUCCESSFUL USE OF HYDROXYUREA IN BETA-THALASSEMIA MAJOR

To the Editor: Hydroxyurea has been used in the treatment of sickle cell anemia to elevate hemoglobin levels and reduce clinical complications.1 The potential usefulness of hydroxyurea in the thala...

Effect of Cytokines and Chemokines on Sickle Neutrophil Adhesion to Fibronectin

A role for leukocytes in sickle cell vaso-occlusive crisis is becoming increasingly recognized. Neutrophil counts are higher in sickle cell patients and neutrophils from these patients demonstrate increased adhesion to endothelial monolayers under certain circumstances. The effects of selected cytokines on the adhesion mechanisms of normal neutrophils and neutrophils from sickle cell anaemia patients (SCA neutrophils) were investigated. Neutrophils were separated from the blood of homozygous (HbSS) SCA patients and healthy controls. Following pre-incubation (25 min, 37°C) of the cells with cytokines, the adhesion of the cells to fibronectin (FN)-coated plates (20 µg/ml) was determined (60 min, 37°C, 5% CO2). Basal adhesion of normal and SCA neutrophils to FN was not statistically different. Pretreatment of normal neutrophils with either IL-6 (10–100 pg/ml), GCSF (1– 10 ng/ml) or IL-8 (1–100 ng/ml) had no significant effect upon their adhesion to FN. In contrast, SCA neutrophil adhesion to FN was increased significantly following pre-incubation with IL-6, G-CSF and IL-8 (p < 0.01). RANTES (1–100 ng/ml) had no significant effect on either normal or SCA neutrophil adhesion to FN. Flow-cytometric analyses demonstrated that IL-8 (10 ng/ml) significantly augments CD11b (Mac-1 integrin subunit) expression on SCA neutrophils, but not normal neutrophils. IL-6 and G-CSF (10 pg/ml and 10 ng/ml, respectively), however, had no effect on SCA neutrophil adhesion molecule expression. In conclusion, SCA neutrophil adhesion mechanisms may increase in the presence of certain cytokines, in vivo, and this activation may contribute to the physiopathology of sickle cell disease.

Clinical, hematological, and molecular characterization of sickle cell anemia pediatric patients from two different cities in Brazil.

This study focused on clinical, hematological, and molecular aspects of sickle cell anemia pediatric patients from two different cites in Brazil. Seventy-one patients from São Paulo and Salvador, aged 3 to 18 years, were evaluated. Hematological analyses, betaS globin gene haplotypes, and alpha2 3.7kb-thalassemia were performed. Numbers of hospitalizations due to vaso-occlusive crises, infections, stroke, and cholelithiasis were investigated. São Paulo had more hospitalizations from vaso-occlusion, cholelithiasis, and stroke than Salvador. The Ben/CAR genotype predominated in both cities. Alpha2 3.7kb-thalassemia had a frequency of 28.2% in Salvador, mostly with Ben/CAR genotype (45.0%), while São Paulo had 22.5% with similar frequencies of the Ben/ CAR and CAR/CAR genotypes. Sickle cell anemia patients from São Paulo also had more episodes of stroke, which was observed among CAR/CAR, atypical, and BEN/CAR haplotypes. In Salvador stroke was only observed in the Ben/CAR genotype. Cholelithiasis had similar frequencies in the two cities. These data suggest a milder phenotype among patients in Salvador, possibly due to genetic, environmental, and socioeconomic factors. Further studies are needed to elucidate modulating factors and phenotype association.

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.