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Purpose To determine the various haptoglobin genotypes and their influence on the clinico-laboratory manifestations among young Nigerian sickle cell anemia (SCA) patients. Patients and methods A total of 101 SCA patients and 64 controls were studied. SCA was diagnosed by polymerase chain reaction (PCR). Haptoglobin genotype was determined by PCR followed by agarose gel electrophoresis. The patients' laboratory and clinical parameters were differentiated by haptoglobin genotypes. Results The Hp1 and Hp2 alleles frequencies were 0.62 and 0.38 in the patients and 0.73 and 0.27 in the controls, respectively, and these did not differ significantly (p>0.05). The haptoglobin genotype distribution among the patients and controls were Hp1-1, 43 (42.6%); Hp2-1, 40 (39.6%); Hp2-2, 18 (17.8%) and Hp1-1, 35 (54.7%); Hp2-1, 24 (37.5%); Hp2-2, 5 (7.8%), respectively, with no difference between the two groups (P>0.05). No significant difference was found in the clinical events and laboratory parameters of the patients when partitioned according to the various haptoglobin genotypes (P> 0.05). Conclusion This study found that haptoglobin gene polymorphism does not have a significant influence on the clinico-laboratory manifestations among SCA patients.

The impaired bioavailability of endogenous nitric oxide (NO) in sickle cell anemia (SCA) may be influenced by polymorphisms in the endothelial nitric oxide synthase gene (eNOS). We compared allelic/genotypic frequencies of the eNOS polymorphisms T-786C, VNTR4a/b and G894T between 89 adult SCA patients and 100 healthy controls, and investigated the relationship between these SNPs and markers of hemolysis [lactate dehydrogenase (LDH), indirect bilirubin (IB) and reticulocyte counts], inflammation [interleukins IL-1β, IL-6, IL-8, Tumor Necrosis Factor (TNF-α) and C-reactive protein (CRP)] and endothelial dysfunction (ED) [soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble intercellular adhesion molecule-1 (sICAM-1), soluble L-selectin (sL-selectin), von Willebrand Factor (vWF) antigen and D-dimers] in the patients. The frequencies of the mutant -786C allele and -786C/C genotype were significantly higher in patients (p = 0.02 and p = 0.04, respectively) but not significantly correlated with the markers. For VNTR4a/b and G894T, the allelic/genotypic frequencies did not statistically differ between patient and control groups. Patients carrying the 4a allele and those with the 894G/G genotype showed a significant decrease in IB (p = 0.02 and p = 0.04, respectively), and only patients with the 4a allele exhibited reduced IL-1β (p = 0.01). The correlation profiles between markers of inflammation and ED significantly differed between patients carrying the mutant alleles and those with wild-type genotypes. This appears to be the first report on the relationship between eNOS gene polymorphisms and markers of hemolysis, inflammation and ED in Brazilian SCA patients. Our results indicate that the SNPs analyzed may influence the phenotypic variability of these patients.

STAT3 is a cytokine-signaling transcription factor critical for gene regulation. Gain-of-function (GOF) mutations in STAT3 are associated with lymphoproliferation, autoimmune cytopenias, increased susceptibility to infection, early-onset solid-organ autoimmunity, short stature, and eczema. We studied the JAK/STAT signaling pathway gene expression and the cytokine profile in two families carrying STAT3-GOF variants to shed light on the STAT3-GOF-associated variable expressivity, including the identification of disease markers. Considering 92 target genes, KIT and IL2RA were downregulated only in patients with high clinical penetrance, while CXCL8 was markedly downregulated for all of them. Unlike previous studies, SOCS3-a STAT3 inhibitor-was not upregulated in patients. In addition, low levels of IL-2 and a reduced numbers of Tregs cells were strikingly prevalent in patients. This study shows a disruptive role of STAT3-GOF variants in the regulatory axis activities CXCL8/STAT3, KIT/STAT3, IL2/CD25/Treg, which, by slightly different mechanisms, underlie the broad clinical spectrum seen in the studied patients. In addition, we suggest the investigation of CXCL8 as a biomarker for identifying STAT3-GOF mutation.

Patients with homozygous beta thalassemia and the β⁰β⁰ genotype produce almost only fetal hemoglobin and a small proportion of hemoglobin A2. However, in the bone marrow of these patients there are descriptions of two different subpopulations of erythroblasts, one with high production of HbF (High HbF cells) and another with low production of HbF (Low HbF cells). The High F cells are probably those that survive through erythropoiesis and originate the viable red blood cells. The molecular mechanisms that are responsible for this high production of HbF are not clear. Therefore, the central aim of our work was to study, in cultures of CD34⁺ cells from β⁰β⁰ patients, differentiated to erythrocytes in vitro, the molecular characteristics of cells related to high production of HbF (High HbF), compared to those with low production of HbF (Low HbF). For this purpose, peripheral blood was collected from patients and CD34⁺ cells were identified, and further differentiated in vitro and then isolated by flow cytometry (FACS Aria). After separating the High HbF and Low HbF groups, the cells were morphologically evaluated by optical and confocal microscopy and image cytometry. Next RNA was extracted from the pool of isolated cells and the RNA sequencing assay (RNAseq) was performed, so that it was possible to analyze the transcripts that were differentially expressed between each of the groups. Finally, we validated the sequencing data by real-time PCR. The sorting was performed on the 10th day of cell culture, when the cells presented a double labeling profile for anti-transferrin (CD71) and anti-glycophorin (CD235) antibodies, the mean intensity and fluorescence (MIF) of the markers was 1058 and 1365 for CD71 in healthy control and β-thalassemia groups, respectively, and for CD235 was 430 in controls and 516 for patients. The cells were in an intermediate stage of maturation. After the isolation of sub-populations of High HbF and Low HbF cells, confocal microscopy indicated a clear difference in the intracellular HbF levels of the analyzed cells, confirming that the sorting by cytometry had occurred as desired. Imaging cytometry analysis revealed a MIF of 7.8 x 10⁵ for High HbF cells and 1.7 x 10⁵ for Low HbF cells, when we assessed the cell pools and when single cells were studied, these values ranged from 2.2 x 10⁵ for Low HbF cells to 1.9 x 10⁶ of mean intensity and fluorescence for High cells. After all data generated by RNAseq were filtered, we obtained a list of 16 enriched and differentially-expressed genes between High HbF and Low HbF cells. From these genes, it was possible to identify two that were apparently associated with the increased production of fetal hemoglobin, the genes ZBTB8B and LIN28B. ZBTB8B is a protein-coding gene with a zinc finger domain and may be involved in transcriptional regulation, it was included in our analyses since this gene is part of the same family as ZBTB7A, an important transcription factor that represses several genes involved in cell differentiation and proliferation and that has already been described as a regulator of HbF production. The LIN28B gene encodes an RNA-binding protein, which is described as a possible regulator of HbF levels during fetal development, by increasing the expression of gamma globin, culminating in the increase of intracellular levels of fetal hemoglobin through a direct action on the let-7 microRNA family and BCL11A gene.These results not only contribute to a better understanding of the mechanisms of gene regulation involved in the production of HbF, but indicate a potential new therapeutic approach to increase the production of HbF in hemoglobinopathies. Disclosures Costa: Novartis: Consultancy.

Alpha-thalassemias are among the most common genetic diseases in the world. They are characterized by hypochromic and microcytic anemia and great clinical variability, ranging from a practically asymptomatic phenotype to severe anemia, which can lead to intrauterine or early neonatal death. Deletions affecting the α-globin genes, located on chromosome 16p13.3, are the main causes of α-thalassemia. Multiplex ligation-dependent probe amplification (MLPA) can be used to detect rearrangements that cause α-thalassemia, particularly large deletions involving the whole α cluster and/or deletions in the HS-40 region. Here, MLPA was used to investigate the molecular basis of α-thalassemia in five unrelated patients, three of whom had Hb H disease. In addition to the -α3.7 deletion identified in the patients with Hb H disease, four different α0 deletions removing 15 to 225 kb DNA segments were found: two of them remove both the α genes, one affects only the regulatory element (HS-40) region, and another one extends over the entire α cluster and the HS-40 region. These results illustrate the diversity of α-thalassemia deletions in the Brazilian population and highlight the importance of molecular investigation in cases that present with microcytosis and hypochromia without iron deficiency and normal or reduced Hb A2 levels..

Hb Bristol-Alesha [HBB: c.202G>A; β 67 Val>Met] is a rare structural variant of hemoglobin (Hb) resulting from a GTG>ATG substitution at codon 67 of the β-globin gene that leads to the replacement of valine by methionine in the corresponding position of the β-globin chain. The methionine residue is subsequently modified to aspartic acid [β67(E11)Val-Met→Asp], possibly by autoxidation mechanisms. This substitution prevents normal non-polar binding of Val67 to the heme group, resulting in molecular instability and severe hemolysis. We identified Hb Bristol-Alesha (in the heterozygous state), as the cause of severe congenital hemolytic anemia in an 11-month-old girl of mixed (native Indian and European) ethnic origin from the Midwestern region of Brazil, whose parents were clinically and hematologically normal. The mutation on the β-globin gene was found to have been coinherited with the α212 patchwork allele.

Aceruloplasminemia is a rare autosomal recessive genetic disease characterized by mild microcytic anemia, diabetes, retinopathy, liver disease, and progressive neurological symptoms due to iron accumulation in pancreas, retina, liver, and brain. The disease is caused by mutations in the Ceruloplasmin (CP) gene that produce a strong reduction or absence of ceruloplasmin ferroxidase activity, leading to an impairment of iron metabolism. Most patients described so far are from Japan. Prompt diagnosis and therapy are crucial to prevent neurological complications since, once established, they are usually irreversible. Here, we describe the largest series of non-Japanese patients with aceruloplasminemia published so far, including 13 individuals from 11 families carrying 13 mutations in the CP gene (7 missense, 3 frameshifts, and 3 splicing mutations), 10 of which are novel. All missense mutations were studied by computational modeling. Clinical manifestations were heterogeneous, but anemia, often but not necessarily microcytic, was frequently the earliest one. This study confirms the clinical and genetic heterogeneity of aceruloplasminemia, a disease expected to be increasingly diagnosed in the Next-Generation Sequencing (NGS) era. Unexplained anemia with low transferrin saturation and high ferritin levels without inflammation should prompt the suspicion of aceruloplasminemia, which can be easily confirmed by low serum ceruloplasmin levels. Collaborative joint efforts are needed to better understand the pathophysiology of this potentially disabling disease. info:eu-repo/semantics/publishedVersion

Sickle cell anemia (SCA) pathophysiology is characterized by the activation of sickle red blood cells, reticulocytes, leukocytes, platelets, and endothelial cells, and with the expression of several inflammatory molecules. Therefore, it is conceivable that variations in levels of proinflammatory cytokines may act as a signaling of differential clinical course in SCA. Here, we evaluated the clinical impact of proinflammatory cytokines interleukin 1-β (IL-1β), interleukin 6 (IL-6), and interleukin 8 (IL-8) in 79 patients with SCA, followed in a single reference center from northeastern Brazil. The main clinical/laboratory data were obtained from patient interview and medical records. The proinflammatory markers IL-1β, IL-6, and IL-8 were evaluated by using commercially available enzyme-linked immunosorbent assay kits. According to levels of the proinflammatory markers, we observed that patients who had a higher frequency of VOC per year (P = 0.0236), acute chest syndrome (P = 0.01), leg ulcers (P = 0.0001), osteonecrosis (P = 0.0006), stroke (P = 0.0486), and priapism (P = 0.0347) had higher IL-6 levels compared with patients without these clinical complications. Furthermore, increased levels of IL-8 were found in patients who presented leg ulcers (P = 0.0184). No significant difference was found for IL-1β levels (P > 0.05). In summary, the present study emphasizes the role of inflammation in SCA pathophysiology, reveals an association of IL-8 levels and leg ulcer occurrence, and indicates that IL-6 levels can be used as a useful predictor for poor outcomes in SCA.

Norovirus is a highly prevalent pathogen that can infect a wide range of host species. Thus far, there have only been two reports of norovirus infection in rats. Diagnostic assays for the detection of norovirus are well established, but a specific molecular assay for the diagnosis of norovirus infection in laboratory rats has not yet been reported. In this study, we describe the development of a sensitive, semi-nested RT-PCR assay for detection of norovirus in fecal samples from Rattus norvegicus, reared in animal facilities under different sanitary barrier conditions. Additionally, we describe the first report of the presence of norovirus in rat colonies from Brazilian animal facilities.

Pyrimidine-5'-nucleotidase type I (P5'NI) deficiency is an autosomal recessive condition that causes nonspherocytic hemolytic anemia, characterized by marked basophilic stippling and pyrimidine nucleotide accumulation in erythrocytes. We herein present two African descendant patients, father and daughter, with P5'N deficiency, both born from first cousins. Investigation of the promoter polymorphism of the uridine diphospho glucuronosyl transferase 1A (UGT1A) gene revealed that the father was homozygous for the allele (TA7) and the daughter heterozygous (TA6/TA7). P5'NI gene (NT5C3) gene sequencing revealed a further change in homozygosity at amino acid position 56 (p.R56G), located in a highly conserved region. Both patients developed gallstones; however the father, who had undergone surgery for the removal of stones, had extremely severe intrahepatic cholestasis and, liver biopsy revealed fibrosis and siderosis grade III, leading us to believe that the homozygosity of the UGT1A polymorphism was responsible for the more severe clinical features in the father. Moreover, our results show how the clinical expression of hemolytic anemia is influenced by epistatic factors and we describe a new mutation in the P5'N gene associated with enzyme deficiency, iron overload, and severe gallstone formation. To our knowledge, this is the first description of P5'N deficiency in South Americans.

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 HU's therapeutic effects may occur through novel vascular mechanisms.

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 (r(s) = 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.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T19:03:49Z No. of bitstreams: 1 S0004-27302013000100005.pdf: 348533 bytes, checksum: c8345ad912e0be6c0f2e4b2f2c995bd5 (MD5) Made available in DSpace on 2013-08-22T19:03:49Z (GMT). No. of bitstreams: 1 S0004-27302013000100005.pdf: 348533 bytes, checksum: c8345ad912e0be6c0f2e4b2f2c995bd5 (MD5) Previous issue date: 2013-02-01 Made available in DSpace on 2013-09-30T20:06:09Z (GMT). No. of bitstreams: 2 S0004-27302013000100005.pdf: 348533 bytes, checksum: c8345ad912e0be6c0f2e4b2f2c995bd5 (MD5) S0004-27302013000100005.pdf.txt: 52872 bytes, checksum: 314d6833f1fc326e92a35d96062e2c40 (MD5) Previous issue date: 2013-02-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:56:46Z No. of bitstreams: 2 S0004-27302013000100005.pdf: 348533 bytes, checksum: c8345ad912e0be6c0f2e4b2f2c995bd5 (MD5) S0004-27302013000100005.pdf.txt: 52872 bytes, checksum: 314d6833f1fc326e92a35d96062e2c40 (MD5) Made available in DSpace on 2014-05-20T13:56:46Z (GMT). No. of bitstreams: 2 S0004-27302013000100005.pdf: 348533 bytes, checksum: c8345ad912e0be6c0f2e4b2f2c995bd5 (MD5) S0004-27302013000100005.pdf.txt: 52872 bytes, checksum: 314d6833f1fc326e92a35d96062e2c40 (MD5) Previous issue date: 2013-02-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) OBJETIVO: Analisar a influência da associação dos polimorfismos do gene da sintase do óxido nítrico endotelial (NOS3) para as posições -786T>C, Glu298Asp e íntron 4b/a e a aptidão cardiorrespiratória sobre as concentrações de nitrito/nitrato, pressão arterial, perfil lipídico e prevalência de doenças cardiometabólicas em adultos. SUJEITOS E MÉTODOS: Noventa e duas pessoas foram divididas de acordo com o genótipo: não polimórficas (NP) e polimórficas (P). Posteriormente, foram subdivididas pela aptidão cardiorrespiratória associada ao genótipo: alta (ANP e AP) ou baixa (BNP e BP). RESULTADOS: Os indivíduos que apresentavam polimorfismo para as posições Glu298Asp+Íntron 4b/a e Glu298Asp+-786T>C e baixa aptidão cardiorrespiratória apresentaram maiores valores de colesterol total e maior prevalência de dislipidemia. CONCLUSÃO: Nossos dados demonstram que os polimorfismos do gene da NOS3 para essas duas associações influenciam os níveis de colesterol plasmático, e essa associação foi mais claramente observada quando os indivíduos apresentavam menor nível de aptidão cardiorrespiratória. OBJECTIVE: To evaluate the influence of the interaction between endothelial nitric oxide synthase gene (NOS3) polymorphisms at positions -786T>C, Glu298Asp and intron 4b/a, and cardiorespiratory fitness on plasma nitrite/nitrate levels, blood pressure, lipid profile, and prevalence of cardiometabolic disorders. SUBJECTS and METHODS: Ninety-two volunteers were genotyped for NOS3 polymorphisms at positions (-786T>C and Glu298Asp) and (intron 4b/a) and divided according to the genotype: non-polymorphic (NP) and polymorphic (P). After that, they were subdivided according to the cardiorespiratory fitness associated with genotype: high (HNP and HP) and low (LNP and LP). RESULTS: The subjects with polymorphism for the interactions at positions Glu298Asp + intron 4b/a, and Glu298Asp+-786T>C showed the highest values in total cholesterol, as well as dyslipidemia. CONCLUSION: Our findings show that NOS3 gene polymorphisms at positions -786T>C, Glu298Asp, and intron 4b/a exert negative effects on the lipid profile compared with those who do not carry polymorphisms. Universidade Estadual Paulista (Unesp) Instituto de Biociências Laboratório de Fisiologia Cardiovascular e Atividade Física Universidade Estadual de Campinas (FCM-Unicamp) Faculdade de Ciências Médicas Centro de Hematologia e Hemoterapia Universidade Estadual Paulista (Unesp) Instituto de Biociências Laboratório de Evolução Molecular Universidade Estadual Paulista (Unesp) Instituto de Biociências Laboratório de Fisiologia Cardiovascular e Atividade Física Universidade Estadual Paulista (Unesp) Instituto de Biociências Laboratório de Evolução Molecular

Hereditary persistence of fetal hemoglobin deletion type-2 (HPFH-2) and Sicilian-δβ-thalassemia are conditions described as large deletions of the human β-like globin cluster, with absent β-globin chains and a compensatory variable increase in γ-globin. HPFH, in general, may be distinguished from DB-Thalassemia by higher fetal hemoglobin (HbF) levels, absence of anemia and hypochromic and microcytic erythrocytes. MicroRNAs (miRNAs) regulate a range of cellular processes including erythropoiesis and regulation of transcription factors such as the BCL11A and SOX6 genes, which are related to the regulation of γ-globin expression. In this report, a possible association among the overexpression of miRNAs and the expression of the γ-globin gene was analyzed in these two conditions. Forty-nine differentially expressed miRNAs were identified by microarrays in CD34+-derived erythroid cells of two subjects heterozygous for Sicilian-δβ-thalassemia, 2 for HPFH-2 and 3 for controls after 13 days of culture. Some of these miRNAs may participate in γ-globin gene regulation and red blood cell function. The BCL11A gene was found to be potentially targeted by 12 miRNAs that were up-regulated in HPFH-2 or in DB-Thal. A down-regulation of BCL11A gene expression in HPFH-2 was verified by quantitative polymerase chain reaction. These data suggest an important action for miRNA that may partially explain the phenotypic differences between HPFH-2 and Sicilian δβ-thalassemia and the increased expression of γ-globin in these conditions.

Sickle cell disease (SCD) is a complex disease that is characterized by the polymerization of deoxyhemoglobin S, altered red blood cell membrane biology, endothelial activation, hemolysis, a procoagulant state, acute and chronic inflammation, and vaso-occlusion. Among the physiological changes that occur during pregnancy, oxygen is consumed by fetal growth, and pregnant women with SCD are more frequently exposed to low oxygen levels. This might lead to red blood cells sickling, and, consequently, to vaso-occlusion. The mechanisms by which SCD affects placental physiology are largely unknown, and chronic inflammation might be involved in this process. This study aimed to evaluate the gene expression profile of inflammatory response mediators in the placentas of pregnant women with sickle cell cell anemia (HbSS) and hemoglobinopathy SC (HbSC). Our results show differences in a number of these genes. For the HbSS group, when compared to the control group, the following genes showed differential expression: IL1RAP (2.76-fold), BCL6 (4.49-fold), CXCL10 (−2.12-fold), CXCR1 (−3.66-fold), and C3 (−2.0-fold). On the other hand, the HbSC group presented differential expressions of the following genes, when compared to the control group: IL1RAP (4.33-fold), CXCL1 (3.05-fold), BCL6 (4.13-fold), CXCL10 (−3.32-fold), C3 (−2.0-fold), and TLR3 (2.38-fold). Taken together, these data strongly suggest a differential expression of several inflammatory genes in both SCD (HbSS and HbSC), indicating that the placenta might become an environment with hypoxia, and increased inflammation, which could lead to improper placental development.

We describe here a new frameshift mutation of β-thalassemia in a Uruguayan family with Italian ancestry [β48 (-T); HBB:c.146delT]. This frameshift results in formation of premature stop codon (TGA) 40 bp downstream and in a short unstable product that is degraded in the cell.

Phosphatidylinositol phosphate kinases (PIPKs) are enzymes that participate in diverse intracellular signaling pathways. They are classified into 3 functionally distinct subfamilies - PIPKI (α, β, γ), PIPKII (α, β, γ), and PIPKIII - located in various subcellular compartments. Recently, the PIPKIIα and β-globin genes were found to be overexpressed in reticulocytes from 2 siblings with hemoglobin H disease, suggesting a possible relationship between PIPKIIa and the production of globins. The main aim of this study was to determine the expression profiles of PIPK genes in healthy individuals during in vitro erythropoiesis using quantitative real-time polymerase chain reaction and to compare these profiles with profiles of globin genes. Our results showed that expression of all PIPKs increases as the cells differentiate, coinciding with the expression profiles of globins. Analysis of the effects of globins on PIPK genes revealed that they varied significantly between the globins, the most noticeable being the effect of α-globin on PIPKIIα (P < 0.0001) and γ-globin on PIPKIIγ (P < 0.0001). The relationship between the expression of PIPKs and globin genes was statistically significant, particularly between PIPKIIα and α-globin (P = 0.0002) and PIPKIIγ and β-globin (P < 0.0001). Linear correlation analysis revealed a strong relationship between PIPKIIα and α-globin genes. This study is the first to establish the expression profiles of PIPK genes during in vitro erythropoiesis in healthy individuals and suggests a parallel between the expression of PIPK and globin genes, reinforcing the hypothesis that they may be related.

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.

Sickle cell disease (SCD) is a serious condition, chronic and undoubtedly represents a public health problem worldwide. SCD is caused by a point mutation in codon 6 of the β globin gene resulting in the production of a structurally abnormal hemoglobin, hemoglobin S. Although the cause of the disease has been known for more than fifty years, therapeutic options are still quite limited. High levels of fetal hemoglobin (HbF) in the blood are associated with a better clinical outcome in SCD patients. In some individuals, the expression of γ-globin gene persists into adulthood in elevated levels, which is called hereditary persistence of fetal hemoglobin (HPFH). A single nucleotide mutation from C to G at position -195 of the HBG1 gene promoter, called non deletional HPFH Brazilian type (nd-HPFH-B), augments the levels of HbF in patients in 7%- 30%. Nd-HPFH-B has been described by our group, but the mechanism and how this single mutation rises HbF levels differently in red blood cells is still unknown. Genome editing using CRISPR/Cas9 in HUDEP-2 cell, an erythroid precursor line, has been developed through homologous direct repair from a small single DNA strand containing the guanine in -195 position at HBG1 gene promotor. All the other genes, including the second HBG1 allele were unaltered. This point mutation has been carried out by CRISPR/Cas9 high fidelity system, capable of performing a specific break in the DNA target sequence, that improves homologous recombination rate of the donor sequence containing the -195 C For the first time, we generated a HUDEP-2 cell line with the -195 C>T mutation in HBG1 promoter using CRISPR/Cas9 genome editing. The HUDEP-2 cells were nucleofected with Cas9 high fidelity ribonucleoprotein (104 pmol), crRNA:tracrRNA (120 pmol) complex and 1uM ssODN -195, using CD34+ human cell kit and program E-001 in AMAXA Nucleofector 4D- device (Lonza). Seven days after nucleofection, the transformed cells were submitted to clonal selection for 25 days. The genomic DNA from 48 clones were submitted by Sanger Sequencing. The sequencing analysis showed highest Crispr/Cas9 efficiency in genomic DNA cut (77.08%; 37/48) and satisfactory ssODN -195 homologous recombination (10.4%; 5/48). Five nd-HPFH-B HUDEP-2 clones and three other clones without the mutation, but with indels after Cas9 DNA cut (controls), were expanded in culture and the HbF levels were measure with anti-HbF antibody by flow cytometry in two biological replicates. HbF levels in nd-HPFH-B HUDEP-2 clones were 6.02%±1.4, 8.25% ± 0.28, 10.18% ± 3.71, 11.95% ± 0.49, 26,3% ± 4,6 while in controls were 1.69% ± 0.26, 1.66% ± 0.26, 0.59% ± 0.06. Two nd-HPFH-B clones were differentiated into erythrocyte in vitro, and fetal hemoglobin levels persisted at high levels seen previously. In addition, α-globin, β-globin and γ-globin mRNA levels were evaluated in three nd-HPFH-B HUDEP-2 clones and two control clones. The mRNA HBG1/HBG1+HBB percentage in nd-HPFH-B were 96.16% ± 4.10 against 22.63% ± 9.64 in controls. The monoallelic single nucleotide mutation -195 C>G is capable to increase the fetal hemoglobin levels up to 30% in nd-HPFH-B HUDEP-2, and our results shows that this is a potential experimental in vitro model to be used in future studies. Disclosures No relevant conflicts of interest to declare.

Ceruloplasmin (CP) is a multicopper ferroxidase that oxidizes ferrous iron promoting the binding of ferric iron to transferrin. The secreted form of CP (sCP) produced mainly by the liver is essentially absent in patients with aceruloplasminemia, a rare type of hereditary iron overload with brain, liver and pancreatic siderosis. Alternative RNA splicing generates a form of CP that is anchored by glycosylphosphatidylinositol (GPI-CP) to the membrane of astrocytes and immune cells. GPI-CP has been reported to help stabilize ferroportin, the only known iron exporter in mammal cells, so we aimed to investigate whether ferroportin expression is abnormal in circulating blood cells in aceruloplasminemia and in paroxysmal nocturnal hemoglobinuria (PNH), a naturally-occurring human model of acquired deficiency of GPI-anchored proteins. Peripheral blood samples were collected from two patients with aceruloplasminemia with different mutations on the CP gene: CP c.2879-1 G>T (splice site mutation) and CP c.2756 T>C (missense mutation), both with undetectable levels of sCP (T were similar to those seen in the control. Nevertheless, monocytic expression of GPI-CP and ferroportin were significantly reduced in CP c.2756 T>C and PNH, when compared to the control. These data confirm previous observations that B lymphocytes and monocytes express GPI-CP and ferroportin, and concomitant reduction of both expressions in PNH and in CP c.2756 T>C support that GPI-CP fosters ferroportin stability on the cell membrane. We also show that, while germline mutations of the CP gene generally cause undetectable sCP, there is heterogeneity in GPI-CP expression, which may remain preserved, as observed in CP c.2879-1 G>T. Further studies are necessary to clarify why this splice site mutation would still allow GPI-anchoring, while the CP c.2756 T>C point mutation abrogates the ability to anchor GPI-CP. While the preservation of lymphocytic GPI-CP was not surprising in an essentially myeloid PNH clone, normal GPI-CP in B lymphocytes in aceruloplasminemia suggests there are lineage-specific differences in physiological expression of ceruloplasmin forms between B lymphocytes and monocytes, with possible implications to the importance of iron metabolism in immune responses. We also noticed that the CP c.2756 T>C patient with monocytic reduction ferroportin presented with slightly more intense anemia and microcytosis. This could result from lower expression of ferroportin in bone marrow macrophages, with impaired iron delivery to erythroblasts, in analogy to monocytes. Finally, acquired ferroportin deficiency in PNH monocytes implies that loss of GPI-anchored protein not only exposes these cells to lysis by complement, but also to intracellular iron retention, generation of reactive oxygen species and may be involved in the pathophysiology of PNH. In summary, our data show that heterogeneity in GPI-CP expression in B lymphocytes and monocytes results in differential expression of ferroportin in aceruloplasminemia and PNH, and future studies should aim at investigating the implications of dysregulated iron metabolism in immune cells. Disclosures Fertrin: Apopharma Inc.: Honoraria; Alexion Pharmaceuticals Brasil: Speakers Bureau.

We are reporting here the results of differential gene expression experiments comparing two siblings, a 21-yr-old male and a 19-yr-old female, with the same alpha-thalassemia genotype (-alpha(3.7)/(--SEA)) and quite different levels of Hb H in the peripheral blood (18.7 and 5%, respectively). By using mRNA differential-display reverse-transcription-PCR and suppression subtractive hybridization, two main transcripts were selected in both procedures and validated by qRT-PCR, one corresponding to the phosphatidylinositol phosphate 4-kinase type II-alpha (PIP4KIIA) gene and the other to the beta-globin gene, both over expressed in the patient with the higher percentage of Hb H. Type II PIP kinases produce phosphatidylinositol 4,5 biphosphate, a critical and pleiotropic regulatory molecule involved in diverse cellular activities, including gene expression. Our results suggest that PIP4KIIA may be one of the factors related to the regulation of the beta-globin gene expression and the different levels of Hb H in alpha-thalassemic patients.

The JAK2 V617F mutation, present in the majority of polycythemia vera (PV) patients, causes constitutive activation of JAK2 and seems to be responsible for the PV phenotype. However, the transcriptional changes triggered by the mutation have not yet been totally characterized. In this study, we performed a large-scale gene expression study using serial analysis of gene expression in bone marrow cells of a newly diagnosed PV patient harboring the JAK2 V617F mutation and in normal bone marrow cells of healthy donors. JUNB was one of the genes upregulated in PV, and we confirmed, by quantitative real-time PCR, an overexpression of JUNB in hematopoietic cells of other JAK2 V617F PV patients. Using Ba/F3-EPOR cell lines and primary human erythroblast cultures, we found that JUNB was transcriptionally induced after erythropoietin addition and that JAK2 V617F constitutively induced JunB protein expression. Furthermore, JUNB knockdown reduced not only the growth of Ba/F3 cells by inducing apoptosis, but also the clonogenic and proliferative potential of human erythroid progenitors. These results establish a role for JunB in normal erythropoiesis and indicate that JunB may play a major role in the development of JAK2 V617F myeloproliferative disorders.

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.

Objective α-Hemoglobin stabilizing protein (AHSP) binds α-hemoglobin (Hb), avoiding its precipitation and its pro-oxidant activity. In the presence of βHb, the αHb-AHSP complex is dismembered and βHb displaces AHSP to generate the quaternary structure of Hb. The relationship between Hb formation and alterations in AHSP expression, which may affect human erythropoiesis, has not yet been described in human cells. Hence, in this study, we examined the effects of AHSP knockdown in hemin-induced K562 and erythropoietin-induced CD34 + cells with particular reference to cellular aspects and gene expression. Materials and Methods Short-hairpin RNA expression vectors aimed at the AHSP mRNA target sequence were cloned and transfected into K562 and CD34 + cells. K562 and CD34 + cells were stimulated to erythroid differentiation. Cells were examined in terms of gene expression using quantitative real-time polymerase chain reaction; reactive oxygen species (ROS) production, apoptosis, and Hb production through flow cytometry assays; and immunofluorescence assays for globin chains. Results RNA interference–mediated knockdown of AHSP expression resulted in considerable αHb precipitation, as well as in a significant decrease in HbF formation. AHSP-knockdown cells demonstrated an increased ROS production and increased rate of apoptosis. Conclusion These findings strengthen the hypothesis that AHSP stabilizes the αHb chain, avoiding its precipitation and its ability to generate ROS, which implicate in cell death. Moreover, data indicate that AHSP may be highly significant for human hemoglobin formation and suggest that AHSP is a key chaperone protein during human erythropoiesis.

Three novel α-globin variants were found during a screening program for hemoglobinopathies in blood donors at the UNICAMP Hematology and Hemotherapy Center, Campinas, State of Sao Paulo, Southeastern Brazil. They were named for the town of origin of the carrier as Hb Itapira [α30(B11)Glu→Val], Hb Bom Jesus da Lapa [α30(B11)Glu→Ala] and Hb Boa Esperanca [α16(A14)Lys→Thr]. Hb Itapira, like Hb Bom Jesus da Lapa, shows an electrophoretic mobility similar to that of Hb S [β6(A3)Glu→Val, GAG→GTG] at alkaline pH; it is associated with a triplicate α-globin allele (αααanti 3.7) and corresponds to only 5.5% of the total hemoglobin (Hb). Hb Boa Esperanca, found in two different individuals, moves faster than Hb A and exhibits an abnormal functional performance.

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.

Background Preeclampsia is one of the leading causes of maternal and neonatal morbidity and mortality in the world, but its appearance is still unpredictable and its pathophysiology has not been entirely elucidated. Genetic studies have associated single nucleotide polymorphisms in genes encoding nitric oxide synthase and matrix metalloproteases with preeclampsia, but the results are largely inconclusive across different populations. Objectives To investigate the association of single nucleotide polymorphisms (SNPs) in NOS3 (G894T, T-786C, and a variable number of tandem repetitions VNTR in intron 4), MMP2 (C-1306T), and MMP9 (C-1562T) genes with preeclampsia in patients from Southeastern Brazil. Methods This prospective case-control study enrolled 77 women with preeclampsia and 266 control pregnant women. Clinical data were collected to assess risk factors and the presence of severe complications, such as eclampsia and HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome. Results We found a significant association between the single nucleotide polymorphism NOS3 T-786C and preeclampsia, independently from age, height, weight, or the other SNPs studied, and no association was found with the other polymorphisms. Age and history of preeclampsia were also identified as risk factors. The presence of at least one polymorphic allele for NOS3 T-786C was also associated with the occurrence of eclampsia or HELLP syndrome among preeclamptic women. Conclusions Our data support that the NOS3 T-786C SNP is associated with preeclampsia and the severity of its complications.

Point mutations affecting codons 12, 13 (exon 1) and 61 (exon 2) of the N-RAS gene and codons 12 and 13 (exon 1) of the K-RAS gene are identified in approximately 30.0% and 10.0%, respectively, of multiple myeloma (MM) patients living in the northern hemisphere. To date, there are no reports about the prevalence of RAS gene mutations in MM Brazilian patients, and this comprised the aim of the present study. DNA from bone marrow aspirates of 252 patients with MM (139 males and 113 females; aged 59.33 +/- 11.95 years) were investigated for whole exons 1 and 2 of the N-RAS gene and whole exon 1 of the K-RAS gene by direct sequencing of DNA amplified in vitro by the polymerase chain reaction. Fifty-three out of 252 (21.03%) MM patients presented RAS mutations. Heterozygous mutations at codons 4, 10 (exon 1), 61 and 65 (exon 2) of the N-RAS gene were identified in seven out of 252 (2.78%) patients. K-RAS heterozygous mutations at codons 7, 12, 13 (exon 1) were seen in 46 out of 252 (18.25%) patients. To the best of our knowledge, the mutation at codon 7 of K-RAS gene is reported for the first time in MM. Taken together, these results suggest that Brazilian MM patients are characterized by: (i) a low prevalence of RAS mutation and (ii) RAS mutations located at distinct regions of the critical codons of the N-RAS and K-RAS genes.

Analysis of DNA polymorphic sites is an important tool for the detection of gene flow in human evolutionary studies and to study the genetic background for gene mutations. The β-globin locus contains several single-base restriction fragment length polymorphism (RFLP) sites throughout chromosome 11. In addition to these polymorphic sequence repeats, others are being studied in order to expand our knowledge concerning the role between haplotype-genotype and phenotype associations. Far downstream of the expressed β-globin genes, there is a hypersensitive site (HS) whose function remains obscure. We sequenced this region in 27 thalassemia patients and found a new pattern in the micro-satellite-like AT-rich region of this site: a new TAA insertion in addition to the one previously described in sickle cell patients with a concomitant polymorphism (G→A). This new variation was found to be linked to the IVS-I-6 (T→C) mutation. This polymorphism may be useful for studies concerning genotype and phenotype associations.

Genetic variation in the glycoprotein B (gB) gene may play a role in human cytomegaloviruses (HCMVs) pathogenesis. Using restriction analysis of the gB gene product (PCR-RFLP), amplified by the nested polymerase chain reaction, the HCMV strains can be compared and classified into at least four HCMV groups. PCR single-strand conformation polymorphism (PCR-SSCP) is one of the techniques used to identify a mutant sequence or a polymorphism in a known gene. SSCP analysis has the advantage over RFLP analysis on detection of DNA polymorphisms and point mutations at a variety of positions in DNA fragments. However, the original SSCP protocols using the incorporation of radioactive label and polyacrylamide gel electrophoresis for detection are labour intensive and time-consuming. A simplified SSCP protocol is described to identify HCMV strains and the gB genotype, allowing the detection of sequence variations not residing in the endonuclease recognition sites.

Background. Previous studies on the role of inflammation in the pathophysiology of sickle cell disease (SCD) suggested that theCCR5Δ32allele, which is responsible for the production of truncated C-C chemokine receptor type 5 (CCR5), could confer a selective advantage on patients with SCD because it leads to a less efficient Th1 response. We determined the frequency of theCCR5Δ32polymorphism in 795 Afro-Brazilian SCD patients followed up at the Pernambuco Hematology and Hemotherapy Center, in Northeastern Brazil, divided into a pediatric group (3 months–17 years,n=483) and an adult group (18–70 years,n=312). The adult patients were also compared to a healthy control group (blood donors, 18–61 years,n=247).Methods. TheCCR5/CCR5Δ32polymorphism was determined by allele-specific PCR.Results. No homozygous patient for theCCR5Δ32allele was detected. The frequency of heterozygotes in the study population (patients and controls) was 5.8%, in the total SCD patients 5.1%, in the children 5.4%, in the adults with SCD 4.8%, and in the adult controls 8.1%. These differences did not reach statistical significance.Conclusions. Our findings failed to demonstrate an important role of theCCR5Δ32allele in the population sample studied here.

Aims Sickle cell disease (SCD) pathogenesis leads to recurrent vaso-occlusive and hemolytic processes, causing numerous clinical complications including renal damage. As vasoconstrictive mechanisms may be enhanced in SCD, due to endothelial dysfunction and vasoactive protein production, we aimed to determine whether the expression of proteins of the renin–angiotensin system (RAS) may be altered in an animal model of SCD. Main methods Plasma angiotensin II (Ang II) was measured in C57BL/6 (WT) mice and mice with SCD by ELISA, while quantitative PCR was used to compare the expressions of the genes encoding the angiotensin-II-receptors 1 and 2 ( AT1R and AT2R ) and the angiotensin-converting enzymes ( ACE1 and ACE2 ) in the kidneys, hearts, livers and brains of mice. The effects of hydroxyurea (HU; 50–75 mg/kg/day, 4 weeks) treatment on these parameters were also determined. Key findings Plasma Ang II was significantly diminished in SCD mice, compared with WT mice, in association with decreased AT1R and ACE1 expressions in SCD mice kidneys. Treatment of SCD mice with HU reduced leukocyte and platelet counts and increased plasma Ang II to levels similar to those of WT mice. HU also increased AT1R and ACE2 gene expression in the kidney and heart. Significance Results indicate an imbalanced RAS in an SCD mouse model; HU therapy may be able to restore some RAS parameters in these mice. Further investigations regarding Ang II production and the RAS in human SCD may be warranted, as such changes may reflect or contribute to renal damage and alterations in blood pressure.

We report the clinical and laboratory findings concerning three unrelated Brazilian patients investigated for polycythemia, whose definitive diagnosis could only be established after the presence of Hb Coimbra (b99 Asp ® Glu) was demonstrated. This illustrates the importance of properly investigating hereditary hemoglobinopathies in cases of erythrocytosis because in some populations variants with high oxygen affinity may be more frequent than expected but go undetected when conventional electrophoresis is used as the sole detection procedure.

We report here a new frameshift mutation in exon 3 of the β-globin gene, a single nucleotide deletion (-C) in between codons 140/141 (GCC/CTG→GCC/TG), found in an 8-year-old Argentinean girl with clinical picture of thalassemia intermedia. It leads to a β-chain that is elongated to 156 amino acids [(141)Trp-Pro-Thr-Ser-Ile-Thr-Lys-Leu-Ala-Phe-Leu-Leu-Ser-Asn-Phe-(156)Tyr-COOH]. The resulting hemoglobin, which we named Hb Florida, was not detected in peripheral blood; however, erythroid hyperplasia and dyserythropoiesis with large inclusion bodies on methyl violet staining were observed in bone marrow, suggesting that this is a hyperunstable variant producing a dominant β-thalassemia phenotype, since the other β-allele was completely normal. Am. J. Hematol. 81:358–360, 2006. © 2006 Wiley-Liss, Inc.

We report the coexistence of Hb Camperdown [beta104 (G6) Arg -> Ser] and beta°-thalassemia [beta39 (Gln -> stop codon)] in a nine-month-old Brazilian boy. He had a relatively more severe hypochromic and microcytic anemia in comparison to his mother's beta-thalassemia trait. His Hb Camperdown heterozygous father was clinically and hematologically normal. To our knowledge, this is the first description of an association of beta°-thalassemia with Hb Camperdown.

Stroke is a catastrophic complication of sickle cell anaemia (SCA) and is one of the leading causes of death in both adults and children with SCA. Evidence suggests that some genetic polymorphisms could be related to stroke development, but their association remains controversial. Here, we performed genotyping of five published single nucleotide polymorphisms, the α-thalassemia genotype, the G6PD A (-) variant deficiency, and the β(S) haplotype in a large series of SCA patients with well-defined stroke phenotypes. Of 261 unrelated SCA patients included in the study, 67 (9.5 %) presented a documented, primary stroke event. Markers of haemolysis (red blood cell (RBC) counts, p = 0.023; reticulocyte counts, p = 0.003; haemoglobin (Hb) levels, p 0.001; indirect bilirubin levels, p = 0.006; lactate dehydrogenase (LDH) levels, p = 0.001) were associated with stroke susceptibility. Genetically, only the β(S) haplotype (odds ratio (OR) 2.9, 95 % confidence interval (CI) 1.56 to 4.31; p = 0.003) and the α(3.7kb)-thalassemia genotype (OR 0.31, 95 % CI 0.11 to 0. 83; p = 0.02) were associated with increased and decreased stroke risk, respectively. In multivariate analysis, the β(S) haplotype was independently associated with stroke development (OR 2.26, 95 % CI 1.16 to 4.4; p = 0.016). Our findings suggest that only the β(S) haplotypes and the α(3.7kb)-thalassemia genotype modulate the prevalence of stroke in our SCA population. Genetic heterogeneity among different populations may account for the irreproducibility amongst different studies.

Background Based 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. Methods The 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. Results Sixty-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). Conclusions One 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.

Vaso-occlusion is a determinant for most signs and symptoms of sickle-cell anemia (SCA). The mechanisms involved in the pathogenesis of vascular complications in SCA remain unclear. It is known that genetic polymorphisms associated with thrombophilia may be potential modifiers of clinical features of SCA. The genetic polymorphisms C677T and A1298C relating to the enzyme methylenetetrahydrofolate reductase (MTHFR), a clotting Factor V Leiden mutation (1691G→A substitution of Factor V Leiden), and the mutant prothrombin 20210A allele were analyzed in this study. The aim was to find possible correlations with vascular complications and thrombophilia markers in a group of SCA patients in Pernambuco, Brazil. The study included 277 SCA patients, divided into two groups: one consisting of 177 nonconsanguineous SCA patients who presented vascular manifestations of stroke, avascular necrosis, leg ulcers, priapism, and acute chest syndrome (group 1); and the other consisting of 100 SCA patients without any reported vascular complication (group 2). Molecular tests were done using either polymerase chain reaction (PCR) restriction fragment length polymorphism or allele-specific PCR techniques. Comparisons between the groups were made using the χ(2) test. The 677 CT and TT genotypes showed a significant risk of vascular complications (p=0.015). No significant associations between the groups were found when samples were analyzed for the MTHFR A1298C allele (p=0.913), Factor V G1691 (p=0.555), or prothrombin G20210A mutation (p=1.000). The polymorphism MTHFR C677T seemed to be possibly predictive for the development of some vascular complications in SCA patients among this population.

Background Acute promyelocytic leukemia is a cytogenetically well defined entity. Nevertheless, some features observed at diagnosis are related to a worse outcome of the patients. Methods In a prospective study, we analyzed peripheral (PB) leukocyte count, immunophenotype, methylation status of CDKN2B, CDKN2A and TP73; FLT3 and NPM1 mutations besides nuclear chromatin texture characteristics of the leukemic cells. We also examined the relation of these features with patient’s outcome. Results Among 19 cases, 4 had a microgranular morphology, 7 presented PB leukocytes >10x109/l, 2 had FLT3-ITD and 3 had FLT3-TKD (all three presenting a methylated CDKN2B). NPM1 mutation was not observed. PB leukocyte count showed an inverse relation with standard deviation of gray levels, contrast, cluster prominence, and chromatin fractal dimension (FD). Cases with FLT3-ITD presented a microgranular morphology, PB leukocytosis and expression of HLA-DR, CD34 and CD11b. Concerning nuclear chromatin texture variables, these cases had a lower entropy, contrast, cluster prominence and FD, but higher local homogeneity, and R245, in keeping with more homogeneously distributed chromatin. In the univariate Cox analysis, a higher leukocyte count, FLT3-ITD mutation, microgranular morphology, methylation of CDKN2B, besides a higher local homogeneity of nuclear chromatin, a lower chromatin entropy and FD were associated to a worse outcome. All these features lost significance when the cases were stratified for FLT3-ITD mutation. Methylation status of CDNK2A and TP73 showed no relation to patient’s survival. Conclusion in APL, patients with FLT3-ITD mutation show different clinical characteristics and have blasts with a more homogeneous chromatin texture. Texture analysis demonstrated that FLTD-ITD was accompanied not only by different cytoplasmic features, but also by a change in chromatin structure in routine cytologic preparations. Yet we were not able to detect chromatin changes by nuclear texture analysis of patients with the FTLD-TKD or methylation of specific genes.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T18:52:52Z No. of bitstreams: 1 S0100-879X2011000900005.pdf: 549015 bytes, checksum: 8233b39a1c5a92d725053f42bc7cbac7 (MD5) Made available in DSpace on 2013-08-22T18:52:52Z (GMT). No. of bitstreams: 1 S0100-879X2011000900005.pdf: 549015 bytes, checksum: 8233b39a1c5a92d725053f42bc7cbac7 (MD5) Previous issue date: 2011-09-01 Made available in DSpace on 2013-09-30T19:44:21Z (GMT). No. of bitstreams: 2 S0100-879X2011000900005.pdf: 549015 bytes, checksum: 8233b39a1c5a92d725053f42bc7cbac7 (MD5) S0100-879X2011000900005.pdf.txt: 40286 bytes, checksum: 80b173c89c8a4d4e73483661405b03a8 (MD5) Previous issue date: 2011-09-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:58:15Z No. of bitstreams: 2 S0100-879X2011000900005.pdf: 549015 bytes, checksum: 8233b39a1c5a92d725053f42bc7cbac7 (MD5) S0100-879X2011000900005.pdf.txt: 40286 bytes, checksum: 80b173c89c8a4d4e73483661405b03a8 (MD5) Made available in DSpace on 2014-05-20T13:58:15Z (GMT). No. of bitstreams: 2 S0100-879X2011000900005.pdf: 549015 bytes, checksum: 8233b39a1c5a92d725053f42bc7cbac7 (MD5) S0100-879X2011000900005.pdf.txt: 40286 bytes, checksum: 80b173c89c8a4d4e73483661405b03a8 (MD5) Previous issue date: 2011-09-01 The health-promoting effects of exercise training (ET) are related to nitric oxide (NO) production and/or its bioavailability. The objective of this study was to determine whether single nucleotide polymorphism of the endothelial NO synthase (eNOS) gene at positions -786T>C, G894T (Glu298Asp) and at the variable number of tandem repeat (VNTR) Intron 4b/a would interfere with the cardiometabolic responses of postmenopausal women submitted to physical training. Forty-nine postmenopausal women were trained in sessions of 30-40 min, 3 days a week for 8 weeks. Genotypes, oxidative stress status and cardiometabolic parameters were then evaluated in a double-blind design. Both systolic and diastolic blood pressure values were significantly reduced after ET, which was genotype-independent. However, women without eNOS gene polymorphism at position -786T>C (TT genotype) and Intron 4b/a (bb genotype) presented a better reduction of total cholesterol levels (-786T>C: before = 213 ± 12.1, after = 159.8 ± 14.4, Δ = -24.9% and Intron 4b/a: before = 211.8 ± 7.4, after = 180.12 ± 6.4 mg/dL, Δ = -15%), and LDL cholesterol (-786T>C: before = 146.1 ± 13.3, after = 82.8 ± 9.2, Δ = -43.3% and Intron 4b/a: before = 143.2 ± 8, after = 102.7 ± 5.8 mg/dL, Δ = -28.3%) in response to ET compared to those who carried the mutant allele. Superoxide dismutase activity was significantly increased in trained women whereas no changes were observed in malondialdehyde levels. Women without eNOS gene polymorphism at position -786T>C and Intron 4b/a showed a greater reduction of plasma cholesterol levels in response to ET. Furthermore, no genotype influence was observed on arterial blood pressure or oxidative stress status in this population. Universidade Estadual Paulista Laboratório de Pesquisas em Fisiologia Cardiovascular e Atividade Física Universidade Estadual Paulista Instituto de Biociências Laboratório de Evolução Molecular Universidade Estadual de Campinas Faculdade de Ciências Médicas Centro de Hematologia e Hemoterapia Universidade Estadual Paulista Laboratório de Pesquisas em Fisiologia Cardiovascular e Atividade Física Universidade Estadual Paulista Instituto de Biociências Laboratório de Evolução Molecular

Background Human 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. Methods During 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. Results Using 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. Conclusions The 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.

Background and Objectives The human neutrophil antigen-2 (HNA-2) is expressed on a subpopulation of neutrophils as most subjects present a negative plus a positive HNA-2 population of neutrophils. The number of neutrophils expressing HNA-2 is variable and may increase in pregnancy, infections, myeloproliferative disorders and after G-CSF. This study investigated the presence of polymorphisms in the gene encoding HNA-2 (CD177) in individuals presenting different patterns of antigen expression and determined the association of single nucleotide polymorphisms (SNPs) with the heterogeneous HNA-2 expression. Materials and Methods Flow cytometry was employed to analyse the HNA-2 expression on neutrophils from 135 healthy subjects using two monoclonal antibodies (TAG4, 7D8). Sequencing reactions were performed on subjects whose antigen expression was low (≤ 50%), high (≥ 80%) or atypical (a nonreactive population plus two distinct positive cell populations). Results Five SNPs were detected, two of them (A793C, G1084A) were related to a low expression of HNA-2 (P = 0·031 and P = 0·004). Atypical antigen expression was observed in 5·9% (8/135) of the individuals, three nonpregnant women and five men. In these cases, the cDNA sequences revealed three SNPs (A134T, G156A and G1333A) strongly related to this atypical HNA-2 expression (P = 0·004, P = 0·006 and P

We describe here two new unstable beta-globin variants, Hb Caruaru and Hb Olinda, found in northeastern Brazil, both associated with chronic haemolytic anaemia. Haemoglobin Caruaru is caused by a single base substitution at codon 122 (TTC-->TCC), possibly originating from the germ line cells of the patient's grandmother. Haemoglobin Olinda is also a de novo mutation, caused by a 12 bp deletion leading to the removal of the 22nd to the 25th residues of the normal beta-globin chain.

HIV-1 infection has increased among women in recent years. The HIV-1 env gene (structural gene) has the greatest variation in all the HIV gene regions. In this study, 58 samples from infants infected with HIV-1 via perinatal transmission were analyzed. All the 58 samples were submitted to Nested-polymerase chain reaction of the env gene region for posterior viral genotyping using EN 70 and EN 85 (first polymerase chain reaction) and EN 80 and EN 95 (second polymerase chain reaction) primers, with the product of the 682 base pair amplification. After Nested-polymerase chain reaction for genotyping, purification of the product, and direct sequencing in a MegaBace 1000 automatic sequencer, 56 genotypes were found in the 58 HIV-1-positive children of the study, where 47 (83.93%) were HIV-1 subtype B infected and 9 (16.07%) were HIV-1 subtype F1 infected. The results demonstrate the predominance of subtype B followed by subtype F in Southeast Brazil.

We have evaluated the mutation profile in a sample of 127 unrelated beta-thalassemia (beta 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 beta 0 codon 39 nonsense mutation was the most frequent alteration (50.9%), followed by beta+ IVSI 110 GA (18.1%), beta 0 IVSI 1 GA (12.9%), beta+ IVSI 6 TC (9.5%), and other rare mutations (8.6%). The chosen gene sequence was able to identify 91% beta-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 beta-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.

Introduction: Mouse models have allowed a wider investigation of the inflammatory process in Sickle cell disease (SCD). Previous studies have demonstrated that neutrophil recruitment inresponse to LPS is upregulated in Berkeley sickle mice and the development of acute LPS-induced airway inflammation is associated with enhanced expression of cytokines, chemokines and MMPs expression in lungs. MicroRNAs (miRNAs) belong to a regulatory class of RNAs with great importance in several biological processes, including the development and fate of immune cells, and in both innate and adaptive immune responses, whereby strong evidence links miRNAs expression to signalling pathways and receptors. Some miRNAs are positive regulators of inflammation and others are involved in the negative-feedback regulation of inflammatory processes. A growing amount of evidence indicates that these molecules are involved in various lung diseases. However, the expression profile of miRNAs in lung injury in SCD have not been investigated. Since pulmonary complications are an important cause of morbidity and mortality in SCD, the aim of this study was to determine whether miRNAs implicated in LPS-mediated inflammation are involved in neutrophil recruitment and inflammation in lung injury in SCD using the sickle mice model. Methods: Acute lung inflammation and injury was induced in wild- type (WT, C57BL/6, n=5) and Berkeley SCD mice (n=5) by intranasal administration of LPS (50 ml of 250 mg/ml). The vehicle group received a similar volume of sterile PBS. BAL was performed 4h after LPS challenge. qPCR analysis was used to examine gene expression and ELISA was used to determine protein production. For miRNAs expression analysis, extraction of lung RNA was performed using mirVanaTM miRNA Isolation kit and TaqManTM MicroRNA Reverse Transcription.The expression level of miRNAs was determined using the Taqman miRNA expression assay by qPCR with U6 snRNA as an endogenous control. Results: Levels of miR-15a-5p, miR-16-5p, miR-26a-5p and miR-98-5p in lung tissue increased almost twofold at 4h after LPS challenge in SS compared with SS PBS group (2.9, 3.7; 2.3; 2.9-fold change, respectively). The expressions of miR-106a-5p, miR-146a-5p, miR-146b-5p, and miR-181a-5p were not altered (2.0; 1.2; 1.55; 1.4-fold change, respectively). However, the expressions of miR-203a-3p and miR-223-3p were significantly up-regulated compared to animals from the PBS group (4.3 and 8.8 fold change, respectively, p Conclusion: We have shown that theLPS-induced inflammatory response in SCD transgenic mice is associated with increases in miR-203a-3p and miR-223-3p expression in the mouse lung; suggesting a role of the miRNAs in the regulation of inflammatory mediator production, since these augmentations are associated with the increases in cytokines and chemokines. Furthermore, miR-146a-5p, a negative regulator of inflammatory gene expression, is not upregulated in SS mice and could contribute to the exaggerated inflammation observed in these animals. These data indicate that alterations in the miRNA expression may be associated with the inflammatory state in SCD. Financial Support: FAPESP Disclosures No relevant conflicts of interest to declare.

Alpha thalassemias are the commonest group of hereditary hemoglobin disorders in the world, the most common cause being deletions involving the α globin genes on 16p13.3. We found a new α0 deletionassociated with the -α3.7 deletion in a 3-month-old brown-skinned female Brazilian patient with Hb H disease. The deletion was identified by Multiplex Ligation-dependent Probe Amplification (MLPA) using the SALSA MLPA P140 C1 HBA kit (MRC Holland, Amsterdam, Holland). An approximately 360 kb-region of DNA extending from the telomeric region of the short arm of chromosome 16 to the DECR2 gene was analyzed, and the fragments generated were compared with Coffalyser.Net. In addition to the α3.7 deletion, an extensive trans deletion encompassing 70 to 105 kb of DNA and involving the α2and α1 genes and the contiguous LUC7L and ITFG3 genes was detected (Figure 1). The 5' breakpoint of the deletion is located between the ψα1and the α2 gene, while the 3' breakpoint region is between the ITFG3 and RGS11 genes. Familial analysis revealed that while the patient's father was heterozygous for the -α3.7 deletion, her mother and oldest sister were heterozygous for the α0 deletion and her middle sister also had Hb H disease. A polymorphism (C > T) in the 220 probe binding region in the hybrid gene (α2α1) (-α3.7 deletion) was also detected in the patient and her father, causing this probe to have a different pattern from that expected in MLPA. To our knowledge, this deletion does not have any similarities in terms of its breakpoint regions with other deletions previously described in the literature. It was named --ATB because the family came from the town of Atibaia in the state of São Paulo in southeast Brazil. Molecular characterization of α-thalassemia deletions is essential both for correct diagnosis of carriers of these deletions and for genetic counselling so that new cases of Hb H disease or Hb Bart's Hydrops Fetalis can be prevented. The hematologic and molecular data for the family studied here are shown in Table 1. Financial Support: FAPESP - 2014/00984-3, CNPq and CAPES/Brazil. Table 1. Hematologic and molecular data for the family members. Family Proband (3 months) Mother Father Sister (2 years) Sister (3 years) RBC RV: M:4,5-6,1/F: 4,2-5,4 4.55 5.81 5.39 5.50 5.07 Hb RV: M: 14-18/F: 12-16 7.75 12.1 14.8 7.6 10.4 MCH RV: 27-32 17 20.8 27.4 13.8 20.6 MCV RV: 80-99 54.1 64.2 79.6 41.5 61.4 A2 (%) VR: 1,6-4 0.78 2.13 2.80 2.40 2.80 F (%) RV: < 2 23.70 0.70 0.90 4.70 1 Hb profile A2, F, A, Bart's + H (10%) A2, A A2, A A2, A, H A2, A Genotype -α3.7/--ATB αα/--ATB αα/-α3.7 -α3.7/--ATB αα/--ATB RV: Reference values RBC: red blood cells (106/mm3); Hb: hemoglobin (g/dL); MCH: mean corpuscular hemoglobin (pg); MCV: mean corpuscular volume (fl). Figure 1. Figure 1. (A) Graph generated by the Coffalyser.Net software with the result for the proband. The x-axis represents the probes, and the y-axis the ratio of the signal intensity of the probes for the proband to the mean signal intensity for the normal controls. A ratio of 1 indicates the presence of both alleles, a ratio of 0.5 a loss of 1 allele and 0 the loss of that region in both alleles. (B) Schematic representation of 16p13.3. The oval represents the telomeric region, the arrows the locations of the probes and the boxes the genes. The blue line corresponds to the deleted fragment, the dotted lines denote the first and last deleted probes and the blank regions show where the breakpoints may be (adapted from MRC-Holland, 2014). Disclosures No relevant conflicts of interest to declare.

Background and Objectives B subgroups are rare and the genetic analysis reported to date has been limited. Materials and Methods Serological and molecular investigations were performed in blood from a B-subgroup donor. Results Red cells did not react with anti-B and anti-AB reagents. However, cells absorbed anti-B. Red cells presented positive reactions with anti-H, and saliva secreted H substance. The molecular study demonstrated a B allele with the substitutions 467C>T, 646T>A, 681G>A, 771C>T, 796C>A, 803G>C, 829G>A and an O allele with the sequence of O02. Conclusions It is probable that the presence in exon 7 of some of the O02 substitutions could have weakened the enzymatic activity of the encoded B transferase.