Effect of hydroxyurea therapy on intravascular hemolysis and endothelial dysfunction markers in sickle cell anemia patients.

Intravascular hemolysis (IH) contributes to the development of endothelial dysfunction (ED) in sickle cell anemia (SCA), and the effects of hydroxyurea (HU, the only approved drug that decreases the frequency and severity of vaso-oclussive crises) on IH and ED in SCA remain unclear. We evaluated and compared the markers of IH among steady-state adult Brazilians with SCA and HbAA individuals. Overall, this cross-sectional study enrolled 30 SCA patients not receiving HU therapy (HbSS), 25 SCA patients receiving HU therapy (HbSS_HU), and 32 HbAA volunteers (HbAA). The IH markers evaluated were serum Lactate Dehydrogenase (LDH), total heme, plasma hemoglobin (pHb), and soluble CD163 (sCD163). The ED markers analyzed were plasma von Willebrand factor (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo) levels, antigen of VWF-cleaving protease (ADAMTS13:Ag), thrombospondin-1, endothelin-1 levels, and ADAMTS13 Activity (ADAMTS13:Act). The levels of VWF:Ag, VWF:RCo, total heme, thrombospondin-1, and endothelin-1 were significantly higher in SCA patients (HbSS and HbSS_HU) compared to HbAA individuals. Also, pHb, LDH, and thrombospondin-1 levels were significantly higher in the HbSS group than in the HbSS_HU group. Contrarily, the levels of sCD163, ADAMTS13:Ag, and ADAMTS13:Act were significantly lower in both groups of SCA patients than HbAA controls, and ADAMTS13:Act levels were significantly lower in HbSS compared to HbSS_HU patients. The higher ADAMTS13 activity levels in those on HU therapy may be attributed to lower pHb and thrombospondin-1 levels as previously shown by in vitro studies that thrombospondin-1 and pHb are bound to VWF. Thus, VWF is restrained from ADAMTS13 activity and cleavage.

Alpha thalassemia, but not ßS-globin haplotypes, influence sickle cell anemia clinical outcome in a large, single-center Brazilian cohort.

Alpha thalassemia and beta-globin haplotype are considered classical genetic disease modifiers in sickle cell anemia (SCA) causing clinical heterogeneity. Nevertheless, their functional impact on SCA disease emergence and progression remains elusive. To better understand the role of alpha thalassemia and beta-globin haplotype in SCA, we performed a retrospective study evaluating the clinical manifestations of 614 patients. The univariate analysis showed that the presence of alpha-thalassemia -3.7-kb mutation (αα/-α and -α/-α) decreased the risk of stroke development (p = 0.046), priapism (p = 0.033), and cholelithiasis (p = 0.021). Furthermore, the cumulative incidence of stroke (p = 0.023) and cholelithiasis (p = 0.006) was also significantly lower for patients carrying the alpha thalassemia -3.7-kb mutation. No clinical effects were associated with the beta-globin haplotype analysis, which could be explained by the relatively homogeneous haplotype composition in our cohort. Our results reinforce that alpha thalassemia can provide protective functions against hemolysis-related symptoms in SCA. Although, several genetic modifiers can impact the inflammatory state of SCA patients, the alpha thalassemia mutation remains one of the most recurrent genetic aberration and should therefore always be considered first.

Evaluation of the mechanisms of heme-induced tissue factor activation: Contribution of innate immune pathways.

Hemolytic diseases such as Sickle Cell Disease (SCD) are characterized by a natural propensity for both arterial and venous thrombosis. The ability of heme to induce tissue factor (TF) activation has been shown both in animal models of SCD, and in human endothelial cells and monocytes. Moreover, it was recently demonstrated that heme can induce coagulation activation in the whole blood of healthy volunteers in a TF-dependent fashion. Herein, we aim to further explore the cellular mechanisms by which heme induces TF-coagulation activation, using human mononuclear cells, which have been shown to be relevant to in vivo hemostasis. TF mRNA expression was evaluated by qPCR and TF procoagulant activity was evaluated using a 2-stage assay based on the generation of activated factor X (FXa). Heme was capable of inducing both TF expression and activation in a TLR4-dependent pathway. This activity was further amplified after TNF-α-priming. Our results provide additional details on the mechanisms by which heme is involved in the pathogenesis of hypercoagulability in hemolytic diseases.

Hemograms and serial hemogram-derived ratios in survivors and non-survivors of COVID-19 in Campinas, Brazil.

Introduction: The hemogram and hemogram-derivative ratios (HDRs) are becoming markers of the severity and mortality of COVID-19. We evaluated the hemograms and serial weekly HDRs [neutrophil-lymphocyte ratio (NLR), monocyte-lymphocyte ratio (MLR), platelet-lymphocyte ratio (PLR), neutrophil-platelet ratio (NPR) and systemic immune-inflammatory index (SII)] in the survivors and non-survivors of COVID-19. Methods: We retrospectively reviewed the medical notes and serial hemograms of real-time reverse-transcription polymerase chain reaction (RT-PCR)-confirmed COVID-19 adults hospitalized from April 2020 to March 2021 from the time of diagnosis to the 3rd week of diagnosis. Results: Of the 320 adults, 257 (80.3%) were survivors and had a lower mean age than the non-survivors (57.73 vs. 64.65 years, p < 0.001). At diagnosis, the non-survivors had lower lymphocyte (p = 0.002) and basophil (p = 0.049) counts and the hematocrit showed a p-value (Is this what you meant???) of 0.021); higher NLR (p < 0.001), PLR (p = 0.047), NPR (p = 0.022) and SII (p = 0.022). Using general linear models, the survivors and non-survivors showed significant variations with weekly lymphocyte count (p < 0.001), neutrophil count (p = 0.005), NLR (p = 0.009), MLR (p = 0.010) and PLR (p = 0.035). All HDRs remained higher in the non-survivors in the 2nd week and 3rd week of diagnosis and the HDRs were higher in the intubated patients than in the non-intubated patients. The NLR and SII were more efficient predictors of mortality in COVID-19 patients. Conclusions: This study shows that serial lymphocyte and neutrophil counts, NLR, PLR, MLR, NPR and SII could serve as good and easily accessible markers of severity and predictors of outcomes in COVID-19 patients and should be used for the monitoring of treatment response.

Polymorphisms in the heme oxygenase-1 and bone morphogenetic protein receptor type 1b genes and estimated glomerular filtration rate in Brazilian sickle cell anemia patients.

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.

Neutrophil extracellular trap regulators in sickle cell disease: Modulation of gene expression of PADI4, neutrophil elastase, and myeloperoxidase during vaso-occlusive crisis.

BACKGROUND: Recent evidence suggests that generation of neutrophil extracellular traps (NETosis), one of the components of immunothrombosis, is associated with the pathogenesis of both venous thromboembolism and sickle cell disease (SCD). NETosis is a complex process regulated by several proteins such as peptidyl arginine deaminase 4 (PADI4), neutrophil elastase (ELANE), and myeloperoxidase (MPO). Among these regulators, PADI4 is responsible of histone citrullination, an essential step for NETosis. Accordingly, its inhibition has been recently cited as a promising therapeutic strategy for diseases such as SCD. Although attractive, this strategy requires supportive evidence of its role in the pathogenesis of SCD. PATIENTS AND METHODS: Patients from two independent cohorts were enrolled in this study. Samples were obtained at steady state (53 patients) or during acute episodes of vaso-occlusive crisis (VOC; 28 patients) in patients from cohort 1. mRNA was extracted from granulocytes to analyze PADI4, ELANE, and MPO expression by qPCR. Furthermore, plasma activity of PADI4 was assessed from an independent cohort in 15 patients, within 24 hours from admission for VOC. Race-matched healthy individuals from the same geographic regions were used as controls for each cohort. RESULTS AND CONCLUSIONS: Higher levels of gene expression of PADI4 and ELANE were observed during VOC. Furthermore, plasma activity of PADI4 was higher in acute VOC when compared to healthy individuals. These results demonstrate that NETosis regulators are modulated during acute VOC, and pave the way for studies of PADI4 inhibition as a therapeutic strategy for acute VOC in SCD.

Haptoglobin Gene Polymorphism in Patients with Sickle Cell Anemia: Findings from a Nigerian Cohort Study.

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.

Serum folate and cytokines in heterozygous ß-thalassemia.

INTRODUCTION: Folate deficiency is commonly reported in ß-thalassemia. Individuals heterozygous for ß-thalassemia may have higher folate requirements than normal individuals. OBJECTIVES: To document the concentration of serum total folate and its forms in ß-thalassemia heterozygote users (ß-TmU) and nonusers (ß-TmN) of 5 mg folic acid/d; to determine whether folic acid (FA) consumption from fortified foods allows beta-Tm patients, who do not take FA supplements, to meet their dietary folate requirements; and to investigate the association between higher serum unmetabolized folic acid (UMFA) and inflammatory cytokine concentrations. METHODS: Serum total folate and forms were measured in 42 ß-Tm (13 ß-TmU and 29 ß-TmN) and 84 healthy controls. The mononuclear leucocyte mRNA expression of relevant genes and their products and hematological profiles were determined. RESULTS: ß-TmU had higher serum total folate, 5-methyltetrahydrofolate, UMFA, and tetrahydrofolate (THF) compared with ß-TmN. The ß-TmN had lower serum total folate and THF than controls. Plasma total homocysteine (tHcy) was lower in ß-TmU compared with both ß-TmN and controls, while ß-TmN had higher tHcy than controls. ß-TmU had higher IL-8 than their controls while ß-TmN had higher IL-6 and IL-8 than their controls. ß-TmU have higher levels of serum total folate, 5- methyltetrahydrofolate, UMFA, and THF than controls. There was no association between UMFA concentrations and cytokine levels. CONCLUSIONS: Mandatory flour fortification with FA in Brazil may be insufficient for ß-TmN, since they have higher tHcy and lower serum total folate than controls. Furthermore, ß-TmN have higher IL-6 levels than ß-TmU. UMFA was not associated with inflammatory cytokine levels.

Endothelial Barrier Integrity Is Disrupted In Vitro by Heme and by Serum From Sickle Cell Disease Patients.

Free extracellular heme has been shown to activate several compartments of innate immunity, acting as a danger-associated molecular pattern (DAMP) in hemolytic diseases. Although localized endothelial barrier (EB) disruption is an important part of inflammation that allows circulating leukocytes to reach inflamed tissues, non-localized/deregulated disruption of the EB can lead to widespread microvascular hyperpermeability and secondary tissue damage. In mouse models of sickle cell disease (SCD), EB disruption has been associated with the development of a form of acute lung injury that closely resembles acute chest syndrome (ACS), and that can be elicited by acute heme infusion. Here we explored the effect of heme on EB integrity using human endothelial cell monolayers, in experimental conditions that include elements that more closely resemble in vivo conditions. EB integrity was assessed by electric cell-substrate impedance sensing in the presence of varying concentrations of heme and sera from SCD patients or healthy volunteers. Heme caused a dose-dependent decrease of the electrical resistance of cell monolayers, consistent with EB disruption, which was confirmed by staining of junction protein VE-cadherin. In addition, sera from SCD patients, but not from healthy volunteers, were also capable to induce EB disruption. Interestingly, these effects were not associated with total heme levels in serum. However, when heme was added to sera from SCD patients, but not from healthy volunteers, EB disruption could be elicited, and this effect was associated with hemopexin serum levels. Together our in vitro studies provide additional support to the concept of heme as a DAMP in hemolytic conditions.

Hemograms and serial hemogram-derived ratios in survivors and non-survivors of COVID-19 in Campinas, Brazil

Introduction The hemogram and hemogram-derivative ratios (HDRs) are becoming markers of the severity and mortality of COVID-19. We evaluated the hemograms and serial weekly HDRs [neutrophil-lymphocyte ratio (NLR), monocyte-lymphocyte ratio (MLR), platelet-lymphocyte ratio (PLR), neutrophil-platelet ratio (NPR) and systemic immune-inflammatory index (SII)] in the survivors and non-survivors of COVID-19. Methods We retrospectively reviewed the medical notes and serial hemograms of real-time reverse-transcription polymerase chain reaction (RT-PCR)-confirmed COVID-19 adults hospitalized from April 2020 to March 2021 from the time of diagnosis to the 3rd week of diagnosis. Results Of the 320 adults, 257 (80.3%) were survivors and had a lower mean age than the non-survivors (57.73 vs. 64.65 years, p < 0.001). At diagnosis, the non-survivors had lower hematocrit (p = 0.021), and lymphocyte (p = 0.002) and basophil (p = 0.049) counts and the hematocrit showed a p-value (Is this what you meant???) of 0.021); higher NLR (p < 0.001), PLR (p = 0.047), NPR (p = 0.022) and SII (p = 0.022). Using general linear models, the survivors and non-survivors showed significant variations with weekly lymphocyte count (p < 0.001), neutrophil count (p = 0.005), NLR (p = 0.009), MLR (p = 0.010) and PLR (p = 0.035). All HDRs remained higher in the non-survivors in the 2nd week and 3rd week of diagnosis and the HDRs were higher in the intubated patients than in the non-intubated patients. The NLR and SII were more efficient predictors of mortality in COVID-19 patients. Conclusions This study shows that serial lymphocyte and neutrophil counts, NLR, PLR, MLR, NPR and SII could serve as good and easily accessible markers of severity and predictors of outcomes in COVID-19 patients and should be used for the monitoring of treatment response.

[Family screening for HBB*S gene and detection of new cases of sickle cell trait in Northeastern Brazil]. / Triagem familiar para o gene HBB*S e detecção de novos casos de traço falciforme em Pernambuco.

OBJECTIVE: To estimate the additional number of affected individuals based on the prevalence of sickle-cell syndromes among relatives of index cases. METHODS: Cross-sectional study of relatives of a random sample of index cases identified through a neonatal screening program in Northeastern Brazil, between 2001 and 2005. The extended family trial model included 463 relatives of 21 index cases. Relatives were classified as nuclear family (NF: father, mother, and siblings); first degree extended family (N1: grandparents, uncles and aunts, and first cousins); second degree extended family (N2: children of first cousins); extended family (NA: NF+N1+N2); and extended nuclear family (NA1: NF+N1). The presence of HBB*S and other abnormal hemoglobins was confirmed by high-performance liquid chromatography. The association between the presence of HBB*S and other variables was calculated using prevalence ratios and their respective 95% confidence intervals, and differences between means were calculated using Student's t test with a 5% significance level. RESULTS: Of relatives, 81% had no knowledge of sickle-cell anemia and HBB*S was present in 114 family members. A total of 53.3% of the studied population was considered as of reproductive age, and 80% of HBB*S carriers had already had children. Frequency was higher among NF (69%), but was also high in N1 (22.8%). NA1 screening resulted in the detection of 69 carriers additional (a 172% increase). CONCLUSIONS: These results indicate that family screening for the identification of sickle-cell carriers should be extended to first degree relatives.

Polymorphisms in the heme oxygenase-1 and bone morphogenetic protein receptor type 1b genes and estimated glomerular filtration rate in Brazilian sickle cell anemia patients

ABSTRACT 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.

Investigating alpha-globin structural variants: a retrospective review of 135,000 Brazilian individuals.

BACKGROUND: Brazil has a multiethnic population with a high diversity of hemoglobinopathies. While screenings for beta-globin mutations are far more common, alterations affecting alpha-globin genes are usually more silent and less well known. The aim of this study was to describe the results of a screening program for alpha-globin gene mutations in a representative sample of the Southeastern Brazilian population. METHODS: A total of 135,000 individuals, including patients with clinical suspicion of hemoglobinopathies and their family members, randomly chosen individuals submitted to blood tests and blood donors who were abnormal hemoglobin carriers were analyzed. The variants were screened by alkaline and acid electrophoreses, isoelectric focusing and cation-exchange high performance liquid chromatography (HPLC) and the abnormal chains were investigated by reverse-phase high performance liquid chromatography (RP-HPLC). Mutations were identified by molecular analyses, and the oxygen affinity, heme-heme cooperativity and Bohr effect of the variants were evaluated by functional tests. RESULTS: Four new and 22 rare variants were detected in 98 families. Some of these variants were found in co-inheritance with other hemoglobinopathies. Of the rare hemoglobins, Hasharon, Stanleyville II and J-Rovigo were the most common, the first two being S-like and associated with alpha-thalassemia. CONCLUSION: The variability of alpha-globin alterations reflects the high degree of racial miscegenation and an intense internal migratory flow between different Brazilian regions. This diversity highlights the importance of programs for diagnosing hemoglobinopathies and preventing combinations that may lead to important clinical manifestations in multiethnic populations.

Role of innate immunity-triggered pathways in the pathogenesis of Sickle Cell Disease: a meta-analysis of gene expression studies.

Despite the detailed characterization of the inflammatory and endothelial changes observed in Sickle Cell Disease (SCD), the hierarchical relationship between elements involved in the pathogenesis of this complex disease is yet to be described. Meta-analyses of gene expression studies from public repositories represent a novel strategy, capable to identify key mediators in complex diseases. We performed several meta-analyses of gene expression studies involving SCD, including studies with patient samples, as well as in-vitro models of the disease. Meta-analyses were performed with the Inmex bioinformatics tool, based on the RankProd package, using raw gene expression data. Functional gene set analysis was performed using more than 60 gene-set libraries. Our results demonstrate that the well-characterized association between innate immunity, hemostasis, angiogenesis and heme metabolism with SCD is also consistently observed at the transcriptomic level, across independent studies. The enrichment of genes and pathways associated with innate immunity and damage repair-associated pathways supports the model of erythroid danger-associated molecular patterns (DAMPs) as key mediators of the pathogenesis of SCD. Our study also generated a novel database of candidate genes, pathways and transcription factors not previously associated with the pathogenesis of SCD that warrant further investigation in models and patients of SCD.

The CCR5Δ32 polymorphism in Brazilian patients with sickle cell disease.

BACKGROUND: Previous studies on the role of inflammation in the pathophysiology of sickle cell disease (SCD) suggested that the CCR5Δ32 allele, 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 the CCR5Δ32 polymorphism 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: The CCR5/CCR5Δ32 polymorphism was determined by allele-specific PCR. RESULTS: No homozygous patient for the CCR5Δ32 allele 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 the CCR5Δ32 allele in the population sample studied here.

JAK2 V617F mutation prevalence in myeloproliferative neoplasms in Pernambuco, Brazil.

BACKGROUND: The JAK2 V617F mutation is associated with three myeloproliferative neoplasms (MPNs): polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). It generates an unregulated clonal hematopoietic progenitor and leads to abnormal increased proliferation of one or more myeloid lineages. Subjects bearing this mutation may present more frequently with complications such as thrombosis and bleeding, and no specific treatment has yet been developed for BCR-ABL-negative JAK2 V617F-negative MPNs. AIMS: To determine the prevalence of JAK2 V617F in MPNs in Pernambuco, Brazil, and to compare it with previous studies. MATERIAL AND METHODS: 144 blood samples were collected at the Hospital of Hematology of the HEMOPE Foundation and were genotyped by polymerase chain reaction-restriction fragment length polymorphism with BsaXI enzymatic digestion. RESULTS AND DISCUSSION: 88% (46/52) of the patients with PV, 47% (39/81) with ET, and 77% (8/11) with PMF were positive for JAK2 V617F, while more than 35% of the individuals were JAK2 V617F-negative, confirming a high prevalence of this abnormality in MPNs, more frequently with a low mutated allele burden, similar to what has been reported in other Western countries, despite differences among methods used to detect this mutation. Screening for JAK2 V617F may allow specific management of these diseases with JAK2 inhibitors in the future and highlights the need for further studies on the pathogenesis of BCR-ABL-negative JAK2 V617F-negative MPNs.

Haptoglobin genotypes in sickle-cell disease.

We compared the frequencies of the haptoglobin (Hp) genotypes of 775 Brazilian patients with sickle-cell disease divided into the following age groups: 3 months-5 years, 6-10 years, 11-15 years, 16-20 years, and over 20 years. The last group (>20 years) was also compared with a healthy control group and was further divided into subgroups including only subjects aged 21-30 years (V.a and Control.a) and over 30 years (V.b and Control.b). There was no significant difference in the frequencies of the Hp genotypes between the different patient groups or between the patients and controls. However, the Hp2-2 genotype was always less frequent than the Hp1-1 genotype in the patient groups, whereas the opposite was observed in healthy controls. The frequency of Hp2-2 was 25.0% in patients in the 21-30 years age group and fell to 19.5% in those over 30 years. In the controls, the corresponding frequency was around 28%. Although our results do not allow us to conclude that Hp genotypes on their own confer greater or lesser selective advantage on sickle-cell disease patients in the population studied, this polymorphism may, when combined with other genetic and environmental factors, contribute to the clinical diversity observed in this disease.

Investigating alpha-globin structural variants: a retrospective review of 135000 Brazilian individuals

Background: Brazil has a multiethnic population with a high diversity of hemoglobinopathies. While screenings for beta-globin mutations are far more common, alterations affecting alpha-globin genes are usually more silent and less well known. The aim of this study was to describe the results of a screening program for alpha-globin gene mutations in a representative sample of the Southeastern Brazilian population. Methods: A total of 135,000 individuals, including patients with clinical suspicion of hemoglobinopathies and their family members, randomly chosen individuals submitted to blood tests and blood donors who were abnormal hemoglobin carriers were analyzed. The variants were screened by alkaline and acid electrophoreses, isoelectric focusing and cation-exchange high performance liquid chromatography (HPLC) and the abnormal chains were investigated by reverse-phase high performance liquid chromatography (RP-HPLC). Mutations were identified by molecular analyses, and the oxygen affinity, heme-heme cooperativity and Bohr effect of the variants were evaluated by functional tests. Results: Four new and 22 rare variants were detected in 98 families. Some of these variants were found in co-inheritance with other hemoglobinopathies. Of the rare hemoglobins, Hasharon, Stanleyville II and J-Rovigo were the most common, the first two being S-like and associated with alpha-thalassemia. Conclusion: The variability of alpha-globin alterations reflects the high degree of racial miscegenation and an intense internal migratory flow between different Brazilian regions. This diversity highlights the importance of programs for diagnosing hemoglobinopathies and preventing combinations that may lead to important clinical manifestations in multiethnic populations.