Endothelial Nitric Oxide Synthase (eNOS) Gene Polymorphisms and Markers of Hemolysis, Inflammation and Endothelial Dysfunction in Brazilian Sickle Cell Anemia 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-1ß, 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-1ß (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.

Interleukin-27 and interleukin-37 are elevated in sickle cell anemia patients and inhibit in vitro secretion of interleukin-8 in neutrophils and monocytes.

BACKGROUND AND OBJECTIVE: Inflammation is implicated in the pathogenesis of most complications seen in sickle cell anemia (SCA) patients. We aimed to evaluate serum levels of two newly discovered anti-inflammatory cytokines (IL-27 and IL-37), and pro-inflammatory cytokines among Brazilian SCA patients that are not on hydroxyurea therapy (HbSS), compared with hydroxyurea-treated patients (HbSSHU) and healthy controls (HbAA). Furthermore, we demonstrated the effect of IL-27, IL-37, and heme on in vitro secretions of IL-8 in human neutrophils and monocytes. METHODS: A cross-sectional study of 82 consenting SCA (35 HbSS and 47 HbSSHU) patients in steady state and 49 HbAA consenting individuals. Clinical details were obtained from interviews and medical records. Serum levels of IL-27, IL-37, TGF-ß, TNF-α, IL-1ß, IL-6, and IL-8 were quantified by enzyme linked immunosorbent assay (ELISA). Neutrophils and monocytes were isolated from healthy controls, and cultured separately with or without cytokines (IL-27 and IL-37) and heme. Supernatant IL-8 concentration was determined by ELISA. RESULTS: Serum levels of IL-27, IL-37, IL-1ß, IL-6, and IL-8 were significantly elevated in HbSS patients compared to HbAA controls. Serum IL-8 levels were significantly higher in HbSS and HbSSHU patients than in controls. IL-27 and IL-37 were positively correlated in both HbSS and HbSSHU patients. In vitro IL-8 production by IL-27 and IL-37 pre-treated neutrophils and monocytes was significantly inhibited even after heme addition. CONCLUSIONS: Our findings show that IL-27 and IL-37, as well as the pro-inflammatory cytokines, are elevated in HbSS patients compared with controls, suggesting that the secretion of these anti-inflammatory cytokines is driven by the presence of pro-inflammatory cytokines. This role is probably sufficient in preventing further cellular or tissue damage but not potent enough to prevent inflammation. Therefore, IL-27 and IL-37 may be potential immuno-targets for ameliorating complications associated with elevated heme levels seen in SCA and other hemolytic anemias.

Expression profiles of phosphatidylinositol phosphate kinase genes during normal human in vitro erythropoiesis.

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

Association of variant alleles of MBL2 gene with vasoocclusive crisis in children with sickle cell anemia.

Vasoocclusive crisis (VOC) is the major cause of morbidity and mortality in sickle cell anemia (SCA), which is caused by the occlusion of blood vessels, followed by ischemia or infarct, resulting in progressive damage to organs. However, this clinical manifestation is variable, indicating that this process could be influenced by modifier genes. The gene MBL2 which codes for mannose-binding lectin (MBL) has been associated with modifications in the progression of infectious and inflammatory vascular diseases. The aim of this study was to determine the frequency of the polymorphisms of exon 1 (alleles A/O) and promoter region -221 (alleles Y/X) of MBL2 in children with SCA and to verify their association with VOC. The determination of the polymorphism of exon 1 and the promoter region of MBL2 was performed by SYBR GREEN((R)) and Taqman((R)) system, respectively. In the patients with SCA, the frequency of the genotype related to high production of MBL was 0.46 (YA/YA) and for intermediate/low production was 0.54 (YA/XA, XA/XA, YA/YO, XA/YO, YO/YO). The frequency of the genotypes and haplotypes of MBL2 in patients with SCA did not differ from control individuals. The populations were in Hardy-Weinberg equilibrium. The patients were divided into two groups. The groups were separated by the frequency of VOC, which was defined by the total of VOC episodes divided by the age of the children at the end of this study. Since, we choose a cut point in FVOC <1 (n=48) (which we considered of mild presentation of disease) and FVOC >or=1 (n=39) (higher severity). In children with SCA, the frequency of the genotypes of MBL2 of intermediate/low expression for MBL was associated with FVOC >or=1 (p=0.0188 OR=3.15 CI=1.19-8.50). The results suggest that MBL2 polymorphism at promoter and first exon of MBL2 associated with low serum levels and structural alterations of MBL could modify the phenotype of the child with SCA related to VOC.

Characterization of alpha thalassemic genotypes by multiplex ligation-dependent probe amplification in the Brazilian population.

Alpha-thalassemia is the most common inherited disorder of hemoglobin synthesis. Genomic deletions involving the alpha-globin gene cluster on chromosome 16p13.3 are the most frequent molecular causes of the disease. Although common deletions can be detected by a single multiplex gap-PCR, the rare and novel deletions depend on more laborious techniques for their identification. The multiplex ligation-dependent probe amplification (MLPA) technique has recently been used for this purpose and was successfully used in the present study to detect the molecular alterations responsible for the alpha-thalassemic phenotypes in 8 unrelated individuals (3 males and 5 females; age, 4 months to 30 years) in whom the molecular basis of the disease could not be determined by conventional methods. A total of 44 probe pairs were used for MLPA, covering approximately 800 kb from the telomere to the MSLN gene in the 16p13.3 region. Eight deletions were detected. Four of these varied in size from 240 to 720 kb and affected a large region including the entire alpha-globin gene cluster and its upstream regulatory element (alpha-MRE), while the other four varied in size from 0.4 to 100 kb and were limited to a region containing this element. This study is the first in Brazil to use the MLPA method to determine the molecular basis of alpha-thalassemia. The variety of rearrangements identified highlights the need to investigate all cases presenting microcytosis and hypochromia, but without iron deficiency or elevated hemoglobin A2 levels and suggests that these rearrangements may be more frequent in our population than previously estimated.

Clinical and genetic risk factors for moderate hyperbilirubinemia in Brazilian newborn infants.

OBJECTIVE: To identify clinical and genetic risk factors for moderate hyperbilirubinemia during the first week of life. STUDY DESIGN: 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. RESULT: 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 age <38 weeks and reference curve percentiles >P40th. CONCLUSION: 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.

Characterization of alpha thalassemic genotypes by multiplex ligation-dependent probe amplification in the Brazilian population

Alpha-thalassemia is the most common inherited disorder of hemoglobin synthesis. Genomic deletions involving the alpha-globin gene cluster on chromosome 16p13.3 are the most frequent molecular causes of the disease. Although common deletions can be detected by a single multiplex gap-PCR, the rare and novel deletions depend on more laborious techniques for their identification. The multiplex ligation-dependent probe amplification (MLPA) technique has recently been used for this purpose and was successfully used in the present study to detect the molecular alterations responsible for the alpha-thalassemic phenotypes in 8 unrelated individuals (3 males and 5 females; age, 4 months to 30 years) in whom the molecular basis of the disease could not be determined by conventional methods. A total of 44 probe pairs were used for MLPA, covering approximately 800 kb from the telomere to the MSLN gene in the 16p13.3 region. Eight deletions were detected. Four of these varied in size from 240 to 720 kb and affected a large region including the entire alpha-globin gene cluster and its upstream regulatory element (alpha-MRE), while the other four varied in size from 0.4 to 100 kb and were limited to a region containing this element. This study is the first in Brazil to use the MLPA method to determine the molecular basis of alpha-thalassemia. The variety of rearrangements identified highlights the need to investigate all cases presenting microcytosis and hypochromia, but without iron deficiency or elevated hemoglobin A2 levels and suggests that these rearrangements may be more frequent in our population than previously estimated.