Fernando Ferreira Costa

DNA polymorphisms at the BCL11A, HBS1L-MYB, and β-globin loci associate with fetal hemoglobin levels and pain crises in sickle cell disease

Sickle cell disease (SCD) is a debilitating monogenic blood disorder with a highly variable phenotype characterized by severe pain crises, acute clinical events, and early mortality. Interindividual variation in fetal hemoglobin (HbF) expression is a known and potentially heritable modifier of SCD severity. High HbF levels are correlated with reduced morbidity and mortality. Common single nucleotide polymorphisms (SNPs) at the BCL11A and HBS1L-MYB loci have been implicated previously in HbF level variation in nonanemic European populations. We recently demonstrated an association between a BCL11A SNP and HbF levels in one SCD cohort [Uda M, et al. (2008) Proc Natl Acad Sci USA 105:1620–1625]. Here, we genotyped additional BCL11A SNPs, HBS1L-MYB SNPs, and an SNP upstream of Gγ-globin (HBG2; the XmnI polymorphism), in two independent SCD cohorts: the African American Cooperative Study of Sickle Cell Disease (CSSCD) and an SCD cohort from Brazil. We studied the effect of these SNPs on HbF levels and on a measure of SCD-related morbidity (pain crisis rate). We strongly replicated the association between these SNPs and HbF level variation (in the CSSCD, P values range from 0.04 to 2 × 10−42). Together, common SNPs at the BCL11A, HBS1L-MYB, and β-globin (HBB) loci account for >20% of the variation in HbF levels in SCD patients. We also have shown that HbF-associated SNPs associate with pain crisis rate in SCD patients. These results provide a clear example of inherited common sequence variants modifying the severity of a monogenic disease.

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.

The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome

Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription–PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning.

Newer Aspects of the Pathophysiology of Sickle Cell Disease Vaso-Occlusion

Sickle cell disease is an inherited disorder of hemoglobin (Hb) synthesis, caused by a single nucleotide substitution (GTG>GAG) at the sixth codon of the β-globin gene, leading to the production of a defective form of Hb, Hb S. When deoxygenated, Hb S polymerizes, damaging the sickle erythrocyte and it is this polymerization that is the primary indispensable event in the molecular pathogenesis of sickle cell disease. Hb S polymerization results in a series of cellular alterations in red cell morphology and function that shorten the red cell life span and leads to vascular occlusion. Sickle cell disease vaso-occlusion is now known to constitute a complex multifactorial process characterized by recurrent vaso-occlusion, ischemia-reperfusion injury, and oxidative stress with consequent vascular endothelial cell activation that induces a chronic inflammatory state in sickle cell disease individual and is propagated by elevated levels of circulating inflammatory cytokines. Activation of the endothelium results in the induction of endothelial adhesion molecule expression that mediates red and white cell adhesion to the vessel wall and the formation of heterocellular aggregates, followed by secondary red cell trapping, all of which contribute to reduced blood flow and eventually obstruction of the micro-circulation. Reduced nitric oxide bioavailability, caused principally by its consumption by cell-free Hb, liberated during intravascular hemolysis, contributes to this process by facilitating vasoconstriction and adhesion molecule activity.

Prevalence of the mutation C677 → T in the methylene tetrahydrofolate reductase gene among distinct ethnic groups in Brazil

Vascular disease is a serious public health problem in the industrialized world, and is a frequent cause of death among the adult population of Brazil. Mild hyperhomocysteinemia has been identified as a risk factor for arterial disease, venous thrombosis, and neural tube defects. Individuals homozygous for the thermolabile variant of methylenetetrahydrofolate reductase (MTHFR-T) are found in 5-15% of the general population and have significantly elevated plasma homocysteine levels which represent one of the genetic risk factors for vascular diseases. We have analyzed the prevalence of individuals homozygous for the MTHFR-T in 327 subjects representing the three distinct ethnic groups in Brazil. The prevalence of homozygotes for the mutated allele MTHFR-T was high among persons of Caucasian descent (10%) and considerably lower among Black (1.45%) and Indians persons populations (1.2%). These data suggest that screening for the MTHFR-T allele should help in identifying individuals with a high risk of vascular disease among populations with a heterogeneous background.

Increased risk for acute myeloid leukaemia in individuals with glutathione S-transferase mu 1 (GSTM1) and theta 1 (GSTT1) gene defects

Abstract: Objectives: Glutathione S‐transferases (GST) modulate the effects of exposure to various cytotoxic and genotoxic agents, including those associated with increased risks of the myelodysplastic syndrome (MDS), acute myeloid leukaemia (AML) and aplastic anemia (AA). Both the GST mu 1 (GSTM1) and GST theta 1 (GSTT1) genes have a null variant allele in which the entire gene is absent. In this study, we tested whether null genotypes for the GSTM1 and GSTT1 genes altered the risks for MDS, AML and AA. Methods: Genomic DNA from 49 MDS, 38 AML and 37 AA patients and 276 controls was analysed using the polymerase chain reaction (PCR). Results: The frequencies of GSTM1 (73.6%) and GSTT1 (34.2%) null genotypes were significantly higher in AML patients than in the controls (36.9 and 18.1%, respectively). A higher frequency of the combined null genotype for both genes was also observed in patients with AML (26.3% compared with 5.0% in the controls). In contrast, no differences in the frequencies of the null genotypes were found among MDS patients, AA patients and the controls. Conclusion: Our observation of a 4.7‐fold (95% CI: 2.1–11.0) and 2.3‐fold (95% CI: 1.0–5.2) increased risk associated with the GSTM1 and GSTT1 null genotypes, respectively, and a 6.6‐fold (95% CI: 2.4–7.9) increased risk associated with the combined null genotype presents preliminary evidence that the inherited absence of this carcinogen detoxification pathway may be an important determinant of AML.

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.

DNA array analysis for red blood cell antigens facilitates the transfusion support with antigen-matched blood in patients with sickle cell disease.

Background  Blood samples from patients with sickle cell disease (SCD) present to transfusion service with numerous antibodies, making the searching for compatible red blood cells (RBC) a challenge. To overcome this problem we developed an effective strategy to meet needs of supplying RBC‐compatible units to SCD patients using DNA arrays.

Sickle Cell Disease in a Brazilian Population from Sao Paulo: A Study of the βs Haplotypes

In this study we have determined the frequency of βs haplotypes in a Brazilian sickle cell disease population from Sao Paulo, Brazil, by analyzing sequence variations in the immediate 5’ fl

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.