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Introduction Mutations affecting genes involved in oxidative and signaling pathways may be associated with kidney disease in sickle cell anemia. We determined the allele and genotype frequencies of some polymorphisms in the promoter regions of the Heme Oxygenase-1 (HMOX1) [rs2071746 (A > T) and (GT)n repeats, short (S) and long (L) alleles] and Bone Morphogenetic Protein Receptor type-1B (BMPR1B) [rs17022863 (A > G), rs4331783 (A > G) and rs1470409 (A > G)] genes in 75 adult patients with sickle cell anemia and 160 healthy controls and investigated whether these polymorphisms may influence the estimated glomerular filtration rate for the patients. Methods The single nucleotide polymorphisms were genotyped using the TaqMan assays, the HMOX1(GT)n repeats were determined by polymerase chain reaction fragment size analysis and the estimated glomerular filtration rate was calculated by the Modification of Diet in Renal Disease formula. Results Regarding the HMOX1rs2071746, the estimated glomerular filtration rate median was significantly higher in TT patients (p = 0.019), including when TT was compared with AT + AA (p = 0.009); for the (GT)n repeats, the estimated glomerular filtration rate medians of SS, SL and LL significantly differed (p = 0.009), being the LL estimated glomerular filtration rate median significantly higher, when compared with the LS + SS (p = 0.005). These results suggest that both the homozygotes, TT for rs2071746 and LL for (GT)n repeats, lead to a higher risk of developing renal complications. Concerning the BMPR1B, the frequencies of GG for rs17022863 and AA for rs4331783 were significantly higher in patients than in controls (p = 0.002 and p = 0.008, respectively), however no association with estimated glomerular filtration rate was found. Conclusion These results contribute to a better understanding of the genetic factors related to the development of nephropathy in sickle cell anemia patients.

Hyperbilirubinemia in patients with sickle cell anemia (SCA) as a result of enhanced erythrocyte destruction, lead to cholelithiasis development in a subset of patients. Evidence suggests that hyperbilirubinemia may be related to genetic variations, such as the UGT1A1 gene promoter polymorphism, which causes Gilbert syndrome (GS). Here, we aimed to determine the frequencies of UGT1A1 promoter alleles, alpha thalassemia, and βS haplotypes and analyze their association with cholelithiasis and bilirubin levels. The UGT1A1 alleles, -3.7 kb alpha thalassemia deletion and βS haplotypes were determined using DNA sequencing and PCR-based assays in 913 patients with SCA. The mean of total and unconjugated bilirubin and the frequency of cholelithiasis in GS patients were higher when compared to those without this condition, regardless of age (P 0.05). However, not cholelithiasis but total and unconjugated bilirubin levels were associated with βS haplotype. These findings confirm in a large cohort that the UGT1A1 polymorphism influences cholelithiasis and hyperbilirubinemia in SCA. HbF and alpha thalassemia also appear as modulators for cholelithiasis risk.

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.

Background/objective: The severity and outcome of COVID-19 are determined by the level of overstimulation of the immune response, age, and comorbidities in the patients infected by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). Lymphopenia is the most consistent finding that characterizes the hemogram in COVID-19 patients. We evaluated the hemogram and compared the lymphocyte count (LC),neutrophil-lymphocyte ratio (NLR), and platelet-lymphocyte ratio (PLR) at diagnosis in COVID-19 patients hospitalized at the Clinical Hospital of the State University of Campinas (UNICAMP), Campinas, São Paulo, Brazil. Methods: In this retrospective study, we reviewed the medical notes of 320 adult hospitalized patients with PCR-confirmed COVID-19 at the Clinical Hospital of UNICAMP, Campinas, from March 2020 to March 2021. The hemogram (performed using automated counter-XN 9000™, Sysmex, Japan) at COVID-19 diagnosis was analyzed, and NLR and PLR were calculated. The primary outcomes were discharge (n = 257 patients who recovered from the disease and were discharged from the hospital), and death (n = 63 those who died during treatment). Statistical analyses were performed using SPSS (version 22). Unpaired data of deceased and discharged COVID-19 patients were compared using Mann-Whitney tests. All results were significant if p < 0.05 or except otherwise stated. Results: Compared to the 257 discharged patients, the 63 deceased patients were older 56.0 vs 64.7 ys respectively, p = 0.000), the males are more in each group and the duration of hospitalization was not different (18.6 vs 19.7 days respectively, p = 0.12). The leukocyte (8.89 ± 4.50 vs 10.37 ± 7.03, p = 0.289) and platelet counts (227.00 ± 91.15 vs 197.79 ± 97.47, p = 0.119) were not significantly different in the two groups, the hematocrit was higher in the discharged than in the deceased patients (38.84 ± 6.86 vs 35.89 ± 8.57, p = 0.021). The LC was lower in the deceased (0.81 ± 0.59 × 103 vs 1.09 ± 0.80x103/μL, p = 0.002), and negatively correlated with the age of the patients(r = -0.145, p=0.009 at a significant level of 0.01). The deceased group had a higher NLR (17.52 ± 19.20 vs 10.06 ± 12.31, p < 0.001) and PLR (366.32 ± 275.03 vs 319.23 ± 331.54, p = 0.047) higher than the discharged group, and both parameters were strongly correlated (r = 0.734, p < 0.001 significant level of 0.01). One hundred and thirty-eight (53.7%) of the discharged patients and 45 (71.4%) of the deceased had LC of < 1.0 × 103/μL. The LC is associated with the disease outcome (χ2 = 6.498, df = 1, p = 0.011), and the odds for a deceased to have a lymphopenia is 1.9 times that for the discharged patients [OR = 1.87 (95% CI = 1.135-3.085). Discussion: Though lymphopenia is consistent in COVID-19, the cause is unclear. Acute recruitment of lymphocytes to the site of infection (mainly the lung) may explain this, thus the lymphopenia may worsen and the LRs will be elevated with the increasing severity of COVID-19. The negative correlation of LC with age and higher odds of lymphopenia in the deceased patients suggest that LC and the LRs at diagnosis could be easily accessible and useful predictors of severity and mortality in these patients. Conclusion: Our study supports that lymphopenia is negatively associated with mortality in COVID-19 patients and that the deceased patients have elevated NLR and PLR at diagnosis. These parameters are easily derived from the hemogram and could be utilized as affordable and accessible predictors of outcomes in patients with COVID-19.

A anemia falciforme é uma doença hereditária recessiva causada por uma mutação em um único nucleotídeo no gene da beta globina. Esta mutação provoca uma alteração estrutural na qual o ácido glutâmico é substituído por uma valina acarretando na falcização das hemácias quando desoxigenadas. Alguns fármacos com ação hipometilante são capazes de induzir a expressão de gama globina através da inibição da metilação no promotor desse gene. Porém, pouco se sabe a respeito desta regulação e sobre os genes envolvidos neste processo. Visando compreender esta regulação, células CD34+, de indivíduos controle, foram tratadas com 1μM de Decitabina no 9°dia de cultura celular para indução da produção de Hemoglobina Fetal (HbF). Os níveis de expressão dos fatores de transcrição e modificadores da cromatina foram avaliados por meio da plataforma PCR Array (Qiagen™ Germany). Nesse ensaio, foram evidenciados 24 fatores de transcrição diferencialmente expressos, dentre eles, o HNF4α (Hepatocyte Nuclear Factor 4 Alpha) regulado positivamente em 26,1 vezes, HNF1α (Hepatocyte Nuclear Factor 1 Homeobox A) 20,0 vezes e o TCF7L2 (Transcription Factor 7 Like 2) 5,9 vezes, em células tratadas com Decitabina comparadas ao controle. Selecionamos o gene HNF4α como um candidato para silenciamento utilizando a ferramenta CRISPR/Cas9 em células HUDEP-2 (immortalized human erythroid progenitor) por ser o fator de transcriação com maior valor diferencial de expressão entre os listados. O HNF4α é descrito como um fator de transcrição que regula a expressão de vários genes hepáticos, podendo atuar no desenvolvimento do fígado, rim e intestinos e é expresso em tecido hematopoiético. As células HUDEP-2 foram tratadas com Decitabina 50 nM e coletadas após 72 horas. Os níveis de HbF foram mensurados por citometria de fluxo em triplicas biológicas. A porcentagem de células positivas para HbF em HUDEP-2, tratada com Decitabina, foi de 12,27 ± 0,7% n = 3, enquanto nas células controle 1,0 ± 0,06% n = 3, p < 0,0001. Estes resultados corroboram o aumento da expressão observado nas células CD34+. As células HUDEP-2 foram nucleofectedas com Cas9 high fidelity (104 pmol), complexo crRNA: tracrRNA (120 pmol) e 1uM de gRNA HNF4α usando o kit de células humanas CD34+ no dispositivo AMAXA Nucleofector 4D (Lonza). 48 horas após a nucleofecção, as células HUDEP-2 editadas foram submetidas à seleção de clones e expandidas por aproximadamente 28 dias. O DNA genômico dos clones foi sequenciado, e quatro clones -HNF4α-HUDEP-2 foram encontrados com indels no quarto éxon do gene HNF4α. Esses clones foram expandidos em cultura com um controle, e os níveis de HbF foram quantificados com anticorpo anti-HbF por citometria de fluxo. Os níveis de HbF destes quatro clones foram de 8,2 ± 2,4%, o nível do controle foi de 0,9 ± 0,02% p < 0,0258. Os clones editados, embora não homogeneamente, expressaram significativamente mais HbF do que o controle. Nossos resultados sugerem que o HNF4α pode desempenhar um papel importante na ativação do gene da gama globina em células HUDEP-2. Adicionalmente, o aumento na expressão dos genes TCF7L2 e HNF1α são sugestivos de um possível papel na regulação de HbF. Esses dados são importantes, pois indicam novos possíveis alvos para terapia gênica em hemoglobinopatias.

Sickle cell disease (SCD), one of the most common hemoglobinopathies worldwide, is characterized by a chronic inflammatory component, with systemic release of inflammatory cytokines, due to hemolysis and vaso-occlusive processes. Patients with SCD demonstrate dysfunctional T and B lymphocyte responses, and they are more susceptible to infection. Although dendritic cells (DCs) are the main component responsible for activating and polarizing lymphocytic function, and are able to produce pro-inflammatory cytokines found in the serum of patients with SCD, minimal studies have thus far been devoted to these cells. In the present study, we identified the subpopulations of circulating DCs in patients with SCD, and found that the bloodstream of the patients showed higher numbers and percentages of DCs than that of healthy individuals. Among all the main DCs subsets, inflammatory DCs (CD14+ DCs) were responsible for this rise and correlated with higher reticulocyte count. The patients had more activated monocyte-derived DCs (mo-DCs), which produced MCP-1, IL-6, and IL-8 in culture. We found that a CD14+ mo-DC subset present in culture from some of the patients was the more activated subset and was mainly responsible for cytokine production, and this subset was also responsible for IL-17 production in co-culture with T lymphocytes. Finally, we suggest an involvement of heme oxygenase in the upregulation of CD14 in mo-DCs from the patients, indicating a potential mechanism for inducing inflammatory DC differentiation from circulating monocytes in the patients, which correlated with inflammatory cytokine production, T lymphocyte response skewing, and reticulocyte count.

A new calorimetric technique is described which allows the measurement of metabolic heat rates in biological materials which are triggered by changes in the oxygen tension. It uses the high permeability for oxygen of a thin-walled Teflon flow channel of a chip calorimeter in order to adjust variable but defined oxygen concentrations in the samples. Using this technique, for the first time, the heat production rate of sickle cell erythrocytes (SS-RBCs) in sickled and non-sickled state could be compared. To measure heat rate changes in real time when sickled SS-RBCs return to the non-sickled state, the heat dissipation was measured during the re-oxygenation of the cell samples which had been treated before under anoxic conditions. We found higher heat production rate of the blood cells in non-sickled state, which is in agreement with the reported increased glucose uptake.

Background: Brazil became the South American epicenter for coronavirus disease (COVID-19) soon after the first case was diagnosed in February 2020 with the highest infection rate occurring in the state of Sao Paulo. COVID-19 is characterized by marked thrombo-inflammation mechanisms, and neutrophil-lymphocyte ratio (NLR) among many clinical and laboratory data, is becoming an inflammatory marker of severity and mortality of COVID-19. We evaluated the serial weekly lymphocyte ratios, which are easily derivable from the routine blood counts, in the survivors and non-survivors of COVID-19 at the Clinical Hospital of University of Campinas (UNICAMP), Campinas, Sao Paulo, Brazil, from time of diagnosis to the 3 rd week of care. This hospital is one of the referral centers for COVID-19 patients in this state. Methods: In this retrospective study, we reviewed the medical notes of 320 adults hospitalized patients with PCR-confirmed COVID-19 at the Clinical Hospital of UNICAMP, from March 2020 to March 2021. The serial weekly hematological parameters (analyzed using automated counter - XN 9000™, Sysmex, Japan) from the time of diagnosis were analyzed and lymphocytes ratios (neutrophil-lymphocyte, NLR, platelet-lymphocyte PLR, and monocyte-lymphocyte MLR) were calculated. The survivors (n=257) were those who recovered from the disease and were discharged from the hospital, while the non-survivors (n=63) were those who died in the course of treatment. Statistical analyses were performed using SPSS (version 22). Unpaired data of Survivors and Non-survivors with COVID-19 were compared using Mann-Whitney tests. Repeated measures were compared within and between groups using univariate and multivariate tests in general linear models. All results were considered significant if p Results: Of the 320 patients, 257 (80.3%) were survivors and had lower mean age than the non-survivors (57.73 vs 64.65 years, p We used general linear models for repeated measures and corrected for the patients who did not stay long enough to have a complete series of blood counts, Figure 1 A-G. Multivariate tests between the survivor and non-survivor groups showed significant variations with serial weekly lymphocyte count (p Conclusions: The non-survivors were older and their NLR and MLR tend to increase from the time of diagnosis while their PLR tend to decrease after the 2 nd week post-COVID-19 diagnosis and treatment. On the other hand, all three ratios significantly decrease in the survivors. While neutrophilia and lymphopenia improved in the survivor, they worsen in non-survivors. These cells may have contributed towards the recovery by ameliorating the inflammatory response in survivors, and death by worsening the response in non-survivors of COVID-19. This study shows that serial lymphocyte count, neutrophil count, NLR, PLR, and MLR could serve as good and easily accessible markers of outcomes in patients with COVID-19 and could be used for monitoring of response to treatment. Figure 1 Figure 1. Disclosures Costa: Novartis: Consultancy.

Stress erythropoiesis (SE) is characterized by an increase in erythropoietic activity in the bone marrow and in extramedullary sites. The central macrophage present in the erythroblastic island (EBI) plays a key role in regulating SE through the expression of molecules mediating cell adhesion, iron metabolism, and those capable of identifying and engulfing damaged and senescent erythrocytes. Those receptors are also expressed in monocytes (MC), suggesting that their monocytic expression could be involved in erythropoiesis and erythrophagocytosis. CD14 +CD16 + intermediate MC (I-MC) express markers that are typically found in EBI macrophages and, together with CD14 +CD16 - classic MC (C-MC), are able to remove circulating iron. Polycythemia vera (PV) is characterized by autonomous overproduction of red blood cells (RBCs) with extramedullary hematopoiesis most often caused by an acquired JAK2 V617F mutation, resulting in a state of chronic SE. The depletion of macrophages from EBIs in animal model of PV reverses splenomegaly and erythrocytosis indicating that they are essential for the development of chronic SE. It is unknown if MC play the same role in humans or if they have different expressions of those key molecules, which could contribute to the severity of the disease. We aimed to investigate the role of MCs in SE present in PV by characterizing the expression of molecules relevant to RBC adhesion, anti-inflammation, erythrophagocytosis, and iron metabolism in MCs from PV patients and from healthy controls (HC). Peripheral blood MCs were isolated from HC (n=21) and PV patients (n=17) and phenotyped by flow cytometry (FC) for sialoadhesin (CD169), VCAM1 (CD106), the receptor for the hemoglobin-haptoglobin complex (CD163), mannose receptor (CD206), SIRPα (CD172), ferroportin (Fpn), and separated into subtypes according to expression of CD14 and CD16. We also evaluated MC erythrophagocytosis by determining positivity for intracellular glycophorin (CD235a) (nHC=13 and nPV=17). In PV, we observed significantly higher expression of CD169 and CD106, and lower expression of CD172 in C-MC (1,167±216.6 vs 1,834±241.9, p=0.009; 1,427±217.1 vs 2,849±182.3, p=0.0004; 104,707±9,546 vs 77,070±8,756, p=0.0428, respectively); higher CD169 and CD106 in I-MC (2,221±322.4 vs 3,150±321.8, p=0.0371; 2,186±201.7 vs 2,721±153.5, p=0.0238, respectively); and higher CD206, CD163, CD172, and CD106 in CD14 lowCD16 + non-classical MCs (NC-MC) (181.2±8.5 vs 268.6±13.4, p Our findings reveal an increase in the expression of markers relevant to the adhesion of erythrocytes to MCs in all MC subsets from PV patients along with more frequent erythrocyte phagocytosis in circulating cells. Further studies should yield better understanding of the role of MCs in SE and in the formation of EBIs, providing future targets for the treatment of chronic SE in PV patients. Disclosures Fertrin: Sanofi Genzyme: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Costa: Novartis: Consultancy.

Pacientes com β-talassemia homozigótica e genótipo β0β0 produzem apenas hemoglobina fetal (HbF) e uma pequena proporção de hemoglobina A2. No entanto, na medula óssea desses pacientes foram descritas duas subpopulações de eritroblastos, uma com alta produção de HbF (High HbF) e outra com baixa produção de HbF (Low HbF). As células High HbF são provavelmente aquelas que sobrevivem à eritropoiese e dão origem aos glóbulos vermelhos viáveis. Entretanto, os mecanismos moleculares envolvidos nessa alta produção de HbF ainda não foram elucidados. Portanto, o objetivo do nosso projeto foi estudar, em cultura de células CD34+ de pacientes β0β0, diferenciadas para eritrócitos in vitro, as características moleculares das células relacionadas à alta produção de HbF (High HbF), frente aquelas com baixa produção de HbF (Low HbF). Para tanto, foi feita a coleta de sangue periférico dos pacientes e foram identificadas as células CD34+, essas foram diferenciadas in vitro e isoladas por citometria de fluxo (FACS Aria) de acordo com o conteúdo intracelular de HbF. Após a separação dos grupos High HbF e Low HbF, as células foram avaliadas morfologicamente por microscopia óptica, confocal e citometria de imagem. Em seguida, o RNA total foi extraído do pool de células isoladas e realizado o ensaio de sequenciamento de RNA (RNAseq), de forma que fosse possível analisar os transcritos que estavam diferencialmente expressos entre cada um dos grupos. Por fim, validamos os dados de sequenciamento por PCR em tempo real. O isolamento foi realizado no 10°dia de cultivo celular, quando as células apresentavam duplo perfil de marcação para anticorpos anti-transferrina (CD71) e anti-glicoforina (CD235), a intensidade média de fluorescência (MIF) dos marcadores foi de 1058 e 1365 para CD71 em grupos de controle saudável e β-talassemia, respectivamente, e para CD235 foi de 430 em controles e 516 em β0β0. As células apresentavam-se em estágio intermediário de maturação. Após o isolamento das subpopulações de células de High HbF e Low HbF, a microscopia confocal mostrou uma clara diferença nos níveis de HbF intracelular das células analisadas, confirmando que o isolamento por citometria havia ocorrido conforme o esperado. Ao analisarmos, por citometria de imagem, as subpopulações das células isoladas, obtivemos um MIF de 7,8 × 105 para células High HbF e 1,7 × 105 para células Low HbF. Quando fizemos as análises para single cell, esses valores variaram de 2,2 × 105 para as células Low HbF e 1,9 × 106 de MIF para as células High HbF. Após todos os dados gerados pelo RNAseq serem filtrados, obtivemos uma lista de 16 genes enriquecidos e diferencialmente expressos entre células High HbF e Low HbF. A partir desses genes, foi possível identificar dois que aparentemente estavam associados ao aumento da produção de hemoglobina fetal, os genes SMARCA1 e LIN28B. O gene LIN28B codifica uma proteína de ligação ao RNA, que é descrita como um possível regulador dos níveis de HbF durante o desenvolvimento fetal, por aumentar a expressão de gama globina. Já o gene SMARCA1 contribui para o complexo de remodelação da cromatina durante o processo de transcrição e pode estar envolvido com os níveis de expressão de HbF. Esses resultados não apenas contribuem para um melhor entendimento dos mecanismos de regulação gênica envolvidos na produção de HbF, mas indicam uma potencial nova abordagem terapêutica para aumentar a produção de HbF em hemoglobinopatias.

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..

Key Clinical Message This manuscript describes the case of a patient with sickle cell anemia who died of fulminant hepatitis after therapy with the iron chelator Deferasirox. The patient was homozygous for the −1774delG polymorphism in the Abcc2 gene, which raises the concern about the use of hepatotoxic drugs in this specific context.

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.

Background The clinical aspects of sickle cell anemia ( SCA ) are heterogeneous, and different patients may present significantly different clinical evolutions. Almost all organs can be affected, particularly the central nervous system. Transient ischemic events, infarcts, and cerebral hemorrhage can be observed and affect ≈25% of the patients with SCA . Differences in the expression of molecules produced by endothelial cells may be associated with the clinical heterogeneity of patients affected by vascular diseases. In this study, we investigated the differential expression of genes involved in endothelial cell biology in SCA patients with and without stroke. Methods and Results Endothelial progenitor cells from 4 SCA patients with stroke and 6 SCA patients without stroke were evaluated through the polymerase chain reaction array technique. The analysis of gene expression profiling identified 29 differentially expressed genes. Eleven of these genes were upregulated, and most were associated with angiogenesis (55%), inflammatory response (18%), and coagulation (18%) pathways. Downregulated expression was observed in 18 genes, with the majority associated with angiogenesis (28%), apoptosis (28%), and cell adhesion (22%) pathways. Remarkable overexpression of the MMP 1 (matrix metalloproteinase 1) gene in the endothelial progenitor cells of all SCA patients with stroke (fold change: 204.64; P =0.0004) was observed. Conclusions Our results strongly suggest that angiogenesis is an important process in sickle cell stroke, and differences in the gene expression profile of endothelial cell biology, especially MMP 1 , may be related to stroke in SCA patients.

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.

Background (TA) n repeat sequence (rs8175347) of UGT1A1 gene promoter polymorphism is associated with serum bilirubin levels and gallstones among different sickle cell anaemia (SCA) populations. There are no data on UGT1A1 polymorphisms and their impact on Nigerian SCA patients. In this study, we determined the distribution of the UGT1A1 (TA) n genotypes among a group of young Nigerian SCA patients and healthy controls. In addition, the influence of UGT1A1 (TA) n genotypes on the laboratory and clinical events among the patients was determined. Methods The distribution of the UGT1A1 (TA) n genotypes among 101 young Nigerian SCA patients and 64 normal appropriate controls were determined and studied. The UGT1A1 (TA) n genotypes were further classified into subgroups and used to differentiate the clinical events and laboratory parameters of the patients. Results Four (TA) n alleles:(TA)5, 6, 7, and 8 were found. These were associated with 10 genotypes: TA5/5, 5/6, 5/7, 5/8, 6/6, 6/7, 6/8, 7/7, 7/8, 8/8. The normal (wild-type)-(TA) 6/6), low- (TA) 7/7, 7/8, 8/8), intermediate- (TA) 5/7, 5/8, 6/7, 6/8), and high-activity (TA) 5/5, 5/6,) genotypes were found in 24.8, 24.8, 41.5, and 8.9% patients and 20.3, 15.6, 61, and 3.1% controls respectively. The general genotype distribution of the patients and control group were not significantly different. There were significant differences in serum bilirubin and lactate dehydrogenase (LDH) of the patients when differentiated by the UGT1A1 (TA) n genotypes (pp = 0.0033. Although, bilirubin and fetal hemoglobin (HbF) of patients with gallstones were significantly different from those without gallstone, only the serum bilirubin was associated with UGT1A1 (TA) n genotypes on multivariate analysis (p Conclusion This study highlights the contribution of UGT1A1 polymorphisms, a non-globin genetic factor, to the laboratory and clinical manifestations of young Nigerian SCA patients for the first time. It also shows that children with co-inheritance of low UGT1A1 (TA) n affinity genotypes may be at risk of gallstone, hence the need to follow them up.

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.

OBJECTIVE: To evaluate hematological, cytogenetic and molecular responses as well as the overall, progression-free and event-free survivals of chronic myeloid leukemia patients treated with a third tyrosine kinase inhibitor after failing to respond to imatinib and nilotinib/dasatinib. METHODS: Bone marrow karyotyping and real-time quantitative polymerase chain reaction were performed at baseline and at 3, 6, 12 and 18 months after the initiation of treatment with a third tyrosine kinase inhibitor. Hematologic, cytogenetic and molecular responses were defined according to the European LeukemiaNet recommendations. BCR-ABL1 mutations were analyzed by Sanger sequencing. RESULTS: We evaluated 25 chronic myeloid leukemia patients who had been previously treated with imatinib and a second tyrosine kinase inhibitor. Nine patients were switched to dasatinib, and 16 patients were switched to nilotinib as a third-line therapy. Of the chronic phase patients (n=18), 89% achieved a complete hematologic response, 13% achieved a complete cytogenetic response and 24% achieved a major molecular response. The following BCR-ABL1 mutations were detected in 6/14 (43%) chronic phase patients: E255V, Y253H, M244V, F317L (2) and F359V. M351T mutation was found in one patient in the accelerated phase of the disease. The five-year overall, progression-free and event-free survivals were 86, 54 and 22% (p

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.

Sickle cell anemia is a recessive inherited disease caused by a single nucleotide polymorphism in the β-globin gene the resulting substitution of glutamic acid by valine causes red blood cell sickling when deoxygenated. Some hypomethylating agents are able to induce the expression of γ-globin by inhibiting DNA methylation at the gene promoter. However, it is not completely understood how this regulation occurs and which genes are involved in this process. To understand fetal hemoglobin (HbF) regulation, CD34+ cells were treated with 1µM of decitabine on the 9th day of culture to induce HbF production. The expression levels of transcription factors and chromatin modifiers were evaluated through the PCR Array platform (Qiagen™ Germany). The transcription factor, HNF4A (Hepatocyte Nuclear Factor 4 Alpha), was highly upregulated in cells treated with decitabine, compared to the control cells, and was chosen as a candidate for CRISPR/Cas9 knockout in HUDEP-2 cells (immortalized human erythroid progenitor cells). The HNF4A gene has been reported as a transcription factor, which regulates the expression of several hepatic genes, and is able to play a role in the development of the liver, kidney, and intestines. Moreover, HNF4A is expressed in the hematopoietic tissue. To the best of our knowledge, the association between HNF4A and gamma globin gene synthesis has not been previously described. HUDEP-2 cells were cultured and then treated with 50nM decitabine. After 72 hours, HbF levels were measured with anti-HbF antibody by flow cytometry in three biological replicates. The percentage of cells positive for HbF in decitabine-treated HUDEP-2 were 12.27 ± 0.7%, N=3, while in control cells the percentage was 1.0 ± 0.06%, N=3 (p Disclosures No relevant conflicts of interest to declare.

Dendritic cells (DCs) are the sentinels of the immune system able to recognize pathogen- and damage-associated molecular patterns (PAMPs and DAMPs), promoting a bridge between the innate and adaptive immune systems. They form a heterogeneous group of cells with different development, phenotype and functions, and they are mainly classified in conventional DCs 1 (cDC1) and 2 (cDC2), plasmacytoid DCs (pDC) and inflammatory DCs. Changes in the development of DCs, in the ratio of the subsets or in the maturation and activation can impair immunity or tolerance, inducing susceptibility to infections, as well as the development of inflammatory and autoimmune diseases. Sickle cell disease (SCD), one of the most common hemoglobinophaties throughout the world, can be considered as a chronic inflammatory disease, with systemic release of TNF-α, IL-6, IL-1β and IL-8. Despite presenting more inflammation, SCD patients have dysfunction of lymphocytes T and B responses, and are more susceptible to infections. Although DCs are the main responsible for the activation and polarization of lymphocytes function and are able to produce the pro-inflammatory cytokines present in the serum of SCD patients, very little attention has been giving to these cells so far. In the present study, we characterized the subpopulations of circulating DCs in SCD patients. Our data of flow cytometry show that SCD patients (SS) have higher percentage (AA: 0.6±0.09, n=20; SS: 1.25±0.2 n=22; p Isolated circulating monocytes from patients are able to differentiate in vitro in DCs with the expression of the characteristic markers CD1c and CD209, however, unlike healthy individuals, a great percentage of these cells also express CD14 (AA: 5.5±2.2, n=16; SS: 17.6±6.1, n=14; p=0.02), indicating the presence of inflammatory DCs. These cells are more mature and activated in steady state as shown by CD83 (p=0.007), CD86 (p=0.0001) and HLA-DR (p Monocyte-derived DCs (mo-DCs) from patients are able to induce more proliferation of CD4+ and CD8+ T lymphocytes than mo-DCs from healthy individuals (AA: n=4; SS: n=4), and CD14+ DCs induce higher proliferation of T lymphocytes than CD14- DCs from the same patient (n=2 pairs of CD14- and CD14+ DCs). We have also seen that SCD patients have reduced percentage of regulatory T cells, as shown by Foxp3+ in the gate of CD3+CD4+ cells (AA: 4.0±0.26, n=17; SS: 3.1±0.3, n=17; p=0.01), and increased IL-17-producing CD4+ (AA: 1.1±0.08, n=18; SS: 1.7±0.18, n=17; p=0.05) and CD8+ (AA: 0.24±0.02; SS: 0.68±0.1, n=17; p=0.0001) T lymphocytes in the blood. Whether DCs are responsible for this skewing of T lymphocyte phenotype in SCD is still to be determined. Our data so far show that there are differences in circulating DC populations in SCD patients and they could explain the changes observed on T cell responses and the susceptibility to infections. The present data add new knowledge about the chronic inflammation in SCD, which is one of the major events in the pathophysiology of the disease. Disclosures No relevant conflicts of interest to declare.

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.

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.

Hemoglobin (Hb) Zurich-Albisrieden (ZA) [α2 59(E8) Gly > Arg; HBA2:c.178G > C] is a rare and highly unstable α-chain variant. A few simple and compound heterozygotes (αZA α/αα and -/αZA α, respectively) have been described so far in Switzerland and China. We describe here a case of homozygosity for the Hb ZA mutation (αZA α/αZA α) in a Brazilian child with severe congenital hemolytic anemia and ineffective erythropoiesis.

Background There is paucity of data on the influence of alpha thalassemia on the clinical and laboratory parameters among Nigerian sickle cell anemia (SCA) patients. This study aimed to determine the prevalence of alpha thalassemia and the influence of alpha thalassemia on laboratory parameters and clinical manifestations in a group of young Nigerian SCA patients. Methods This was a cross-sectional retrospective study conducted on 100 patients with SCA and 63 controls. The diagnosis of SCA was confirmed by DNA studies. Alpha thalassemia genotyping was performed by multiplex gap-PCR method. Laboratory parameters including complete blood count, hemoglobin quantitation, serum lactate dehydrogenase (LDH), and bilirubin were determined with standard techniques. Results Alpha thalassemia was found in 41 (41.0%) patients compared to 24 (38.1%) controls (P = 0.744), and all were due to the 3.7 κb α-globin gene deletions. Alpha thalassemia was associated with more frequent bone pain crisis, higher hemoglobin concentration, red blood cell count, and HbA2 level among the patients. On the contrary, patients with alpha thalassemia had lower mean corpuscular volume, mean corpuscular hemoglobin, and white blood cell count (WBC) (P ˂ 0.05). There were 6 (6.0%) patients with leg ulcers, and none of them had alpha thalassemia, P = 0.04. Conclusion This study confirms that coexistence of alpha thalassemia with SCA significantly influences both the clinical and laboratory manifestations of young Nigerian SCA patients. The coexistence of this genetic modifier is associated with increased bone pain crisis and protects against sickle leg ulcers among the patients.

To identify clinical and genetic risk factors for moderate hyperbilirubinemia during the first week of life.Using univariate and multivariate multiple regression analyses, the RR for clinical factors, the African variant of glucose-6-phosphate dehydrogenase (G6PD) deficiency (G202A/A376G), and (TA)(n) UGT1A1 polymorphisms were established in a cohort of 608 Brazilian newborn infants. Hyperbilirubinemia was monitored until 134.5 ± 49.8 h of life (IQR, 111.0 to 156.7). The dependent variable was total bilirubinemia (TB) ≥12.9 mg per 100 ml estimated by transcutaneous or plasma bilirubin measurements.The African variant of G6PD deficiency and (TA)(7)/(TA)(7) and (TA)(7)/(TA)(8) polymorphisms present in 6.1 and 12.0% of newborns, respectively, were not risk factors for moderate hyperbilirubinemia. Coexpression of G6DP deficiency and UGT1A1 polymorphisms occurred in 0.49% of the subjects. Independent clinical predictors for TB≥ 12.9 mg per 100 ml were gestational age38 weeks and reference curve percentilesP40th.In this study, G6PD deficiency and UGT1A1 gene promoter polymorphisms were not risk factors for moderate hyperbilirubinemia. Genetic factors may vary considerably in importance among different populations.

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.

Hemoglobin (Hb) Indianapolis [β112 (G14) CysArg] is a rare and slightly unstable β-globin variant. All carriers described to date were clinically normal with only mild reticulocytosis. We report here a case of a Brazilian patient in whom hemolytic anemia and acute renal failure were probably caused by the presence of this variant. Am. J. Hematol., 2007. © 2007 Wiley-Liss, Inc.

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.

The presence of high levels of fetal haemoglobin (HbF) provides well-validated clinical benefits to patients with sickle cell anaemia (SCA). Nevertheless it has been difficult to show clear direct effects of the known genetic HbF modifiers, such as the enhancer polymorphisms for haematopoietic transcription factors BCL11A and MYB, on SCA severity. Investigating SCA patients from Brazil, with a high degree of European genetic admixture, we have detected strong effects of these variants on HbF levels. Critically, we have shown, for the first time, that the presence of such HbF-promoting variants leads to a reduced rate of SCA complications, especially stroke.

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.