Anderson F. Cunha

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.

Partitioning of molecular variation at local spatial scales in the vulnerable neotropical freshwater turtle, Hydromedusa maximiliani (Testudines, Chelidae): implications for the conservation of aquatic organisms in natural hierarchical systems

Abstract Hydromedusa maximiliani is a vulnerable freshwater turtle endemic to mountainous regions of the Atlantic rainforest in southeastern Brazil. Random amplified polymorphic DNA (RAPD) were surveyed with the purpose of assessing the genetic structure and determining the partitioning of molecular variation in H. maximiliani across the natural spatial hierarchical scale of its habitat. The goal of the study was to integrate ecological data with estimates of molecular genetics diversity to develop strategies for the conservation of this freshwater turtle. Specimens were sampled from rivers and streams across three drainages. Nine of the 80 primers used generated 27 scoreable bands of which 10 (37%) were polymorphic and produced 16 RAPD phenotypes. Significant heterogeneity was found in the distribution of RAPD molecular phenotypes across the three drainages. Analysis of molecular variance for molecular phenotypes showed that the heterogeneity had a spatial structure since a significant amount (22%) of the total variance was attributable to variation among rivers and streams. Since the genetic variation of this turtle seems to be structured according to the natural hierarchical system of rivers and streams within drainages, it is suggested that local populations should be considered as separate management units.

Preliminary Phylogeographic Analysis of the Neotropical Freshwater Turtle Hydromedusa maximiliani (Chelidae)

Abstract Phylogeography of Hydromedusa maximiliani was assessed by sequencing a 416 bp fragment from the mtDNA control region. Based on this fragment, 18 haplotypes were detected among individuals from four populations throughout southeastern Brazil. Two major geographical groups could be distinguished, a western population represented by individuals from São Paulo state, and an eastern population, represented by individuals from Rio de Janeiro and Minas Gerais states. Divergence dates of 8–16 million years ago for eastern and western H. maximiliani populations fit orographical activities resulting in Serra do Mar and the Serra da Mantiqueira mountain uplifts during Pliocene and Miocene.

New mutations detected by denaturing high performance liquid chromatography during screening of exon 6 bcr-abl mutations in patients with chronic myeloid leukemia treated with tyrosine kinase inhibitors

Point mutations within the ABL kinase domain are the most frequent mechanism for reactivation of kinase activity of the BCR-ABL gene and have been associated with clinical resistance to tyrosine kinase (TK) inhibitors in patients with CML, conferring a poor prognosis. T315I (Treonine→Isoleucine) is a mutation in the exon 6 of BCR-ABL gene that makes the protein resistant to kinase inhibitors currently used for treating CML. Denaturing High-performance liquid chromatography (D-HPLC) allows for high throughput mutation screening. In this study, we screened mutations in exon 6 of the BCR-ABL gene in patients presenting failure or sub optimal response according to Leukemia Net criteria and correlated the presence of mutations with clinical outcome. Genomic DNA was extracted from peripheral blood samples from 93 patients with CML (5 intolerant and 88 resistant). The PCR product was analysed by D-HPLC, and the patients samples with abnormal D-HLPC profiles were submitted to automated sequencing, using specific primers. Overall survival (OS) was calculated from the date of mutation analysis, for the whole group and for both groups (mutation versus no mutation). We screened mutations in exon 6 of the BCR-ABL gene in 93 CML TKI - resistant patients. Twenty-three out of 93 samples (25%) showed an abnormal elution profile. Automated sequencing confirmed the presence of a nucleotide change in 19 out of 23 cases: one polymorphism, T315T, seven known point mutations: T315I, F317L, V339L, M351T, E355G and F359V and three novel mutations: C305R, D325D and I360S. OS for the whole group was 80% in a median observation time of 30 months. OS for patients without the mutation was 87% and with the mutation was 56%, in a median observation time of 37 and 10 months, respectively (p < 0.0001, RR = 68). D-HPLC is a practical and sensitive method for routine clinical monitoring for emergence of kinase domain mutations and may be useful for optimising therapy in CML. The screening of mutations in exon 6 is clinically relevant, once the presence of mutations confers a poor outcome. Early detection of emerging mutant clones may help in decision-making for alternative treatment.

Gene expression profiles of erythroid precursors characterise several mechanisms of the action of hydroxycarbamide in sickle cell anaemia

Hydroxycarbamide (HC) (or hydroxyurea) has been reported to increase fetal haemoglobin levels and improve clinical symptoms in sickle cell anaemia (SCA) patients. However, the complete pathway by which HC acts remains unclear. To study the mechanisms involved in the action of HC, global gene expression profiles were obtained from the bone marrow cells of a SCA patient before and after HC treatment using serial analysis of gene expression. In the comparison of both profiles, 147 differentially expressed transcripts were identified. The functional classification of these transcripts revealed a group of gene categories associated with transcriptional and translational regulation, e.g. EGR‐1, CENTB1, ARHGAP4 and RIN3, suggesting a possible role for these pathways in the improvement of clinical symptoms of SCA patients. The genes involved in these mechanisms may represent potential tools for the identification of new targets for SCA therapy.

IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES INDUCED BY HYDROXYUREA IN RETICULOCYTES FROM SICKLE CELL ANAEMIA PATIENTS

1 The major effect associated with hydroxyurea (HU) treatment of sickle cell anaemia (SCA) patients is an increase in fetal haemoglobin (HbF) synthesis, which inhibits the polymerization of haemoglobin S. 2 Hydroxyurea improves clinical symptoms by reducing the frequency of pain and vaso‐occlusive crises, acute chest syndrome, transfusion requirements and hospitalization. 3 The molecular mechanisms responsible for HU‐mediated induction of fetal globin transcription are not completely understood. Therefore, the aim of the present study was to identify differentially expressed genes participating in these mechanisms. 4 We established two suppression subtractive hybridization (SSH) libraries from reticulocytes obtained from SCA patients either not on or on HU treatment. The gene expression of some of the genes identified was subsequently evaluated by real‐time polymerase chain reaction (PCR). 5 Genes identified with altered expression included SUDS3, FZD5 and PHC3, which may be associated with the regulation of globin expression. 6 This is the first demonstration of an association between HU treatment and the expression of genes identified in erythroid cells.

JAK2 V617F prevalence in Brazilian patients with polycythemia vera, idiopathic myelofibrosis and essential thrombocythemia

Polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF) are myeloproliferative disorders (MPD) that arise from the clonal proliferation of a pluripotent hematopoietic progenitor, leading to the overproduction of one or more myeloid lineages. Recently, a specific mutation in the JAK2 gene, which encodes a tyrosine kinase, has been shown to be associated with the myeloproliferative phenotype observed in PV, ET and IMF. In this study of Brazilian patients, the JAK2 V617F mutation [c.1887G > T) was detected in four out of 49 patients with PV (96%), 14 out of 25 patients with IMF (56%), and in eight out of 29 patients with ET, which is in accordance with previous screenings of this mutation in other populations.

Analyses of the three 1-Cys Peroxiredoxins from Aspergillus fumigatus reveal that cytosolic Prx1 is central to H2O2 metabolism and virulence

Standing among the front defense strategies against pathogens, host phagocytic cells release various oxidants. Therefore, pathogens have to cope with stressful conditions at the site of infection. Peroxiredoxins (Prx) are highly reactive and abundant peroxidases that can support virulence and persistence of pathogens in distinct hosts. Here, we revealed that the opportunistic human pathogen A. fumigatus presents three 1-Cys Prx (Prx6 subfamily), which is unprecedented. We showed that PrxB and PrxC were in mitochondria, while Prx1 was in cytosol. As observed for other Prxs, recombinant Prx1 and PrxC decomposed H2O2 at elevated velocities (rate constants in the 107 M−1s−1 range). Deletion mutants for each Prx displayed higher sensitivity to oxidative challenge in comparison with the wild-type strain. Additionally, cytosolic Prx1 was important for A. fumigatus survival upon electron transport dysfunction. Expression of Prxs was dependent on the SakAHOG1 MAP kinase and the Yap1YAP1 transcription factor, a global regulator of the oxidative stress response in fungi. Finally, cytosolic Prx1 played a major role in pathogenicity, since it is required for full virulence, using a neutropenic mouse infection model. Our data indicate that the three 1-Cys Prxs act together to maintain the redox balance of A. fumigatus.

Correlations with Point Mutations and Severity of Hemolitic Anemias: The Example of Hereditary Persistence of Fetal Hemoglobin with Sickle Cell Anemia and Beta Thalassemia

Hemolytic anemias are a group of diseases characterized by a reduction in red blood cells (RBC) life span mainly caused by a deregulation in the hemoglobin formation. Among these diseases, Sickle Cell Disease (SCD) and Beta Thalassemia (Thal) are the most common disorders involved in the premature destruction of RBC. Understanding the molecular mechanisms involved in the outcome of these diseases as well as the metabolic pathways surrounding its onset constitutes a very useful approach to target treatment strategies for such diseases. In this chapter we will discuss the state-of-the-art aspects about this theme highlighting the importance of several point mutations in hemolytic anemia using  thalassemia and sickle cell disease as examples. In addition, the molecular aspects involved in the Hereditary Persistence of Fetal Hemoglobin (HPFH), also an important disorder caused by point mutations and deletions, and its association with the severity of Thal and SCD will be also discussed. In this context lies the manifestation of better prognostic to patients having an increase in fetal hemoglobin and SCD or Thal concomitantly. Therefore a parallel discussion towards the advances currently described in the literature and associations of gene expression and different drugs that increase the production of fetal hemoglobin (HbF) are pointed out as a mechanism to improve the quality of life of SCD and Thal patients.