Role of paraoxonase 1 activity and PON1 gene polymorphisms in sickle cell disease.

Sickle cell disease (SCD) patients often exhibit a dyslipidemic sub-phenotype. Paraoxonase 1 (PON 1) is a serum glycoprotein associated with the high-density lipoproteins cholesterol (HDL-C), and variability in PON1 activity depends on the PON1 genotypes. We investigated the influence of PON1c.192Q > R and PON1c.55L > M polymorphisms on PON1 activity and laboratory parameters and the association between PON1 activity and clinical manifestations in SCD patients. We recruited 350 individuals, including 154 SCD patients and 196 healthy volunteers, which comprised the control group. Laboratory parameters and molecular analyses were investigated from the participants' blood samples. We have found increased PON1 activity in SCD individuals compared to the control group. In addition, carriers of the variant genotype of each polymorphism presented lower PON1 activity. SCD individuals carrying the variant genotype of PON1c.55L > M polymorphism had lower platelet and reticulocyte counts, C-reactive protein, and aspartate aminotransferase levels; in addition to higher creatinine levels. SCD individuals carrying the variant genotype of PON1c.192Q > R polymorphism had lower triglyceride, VLDL-c, and indirect bilirubin levels. Furthermore, we observed an association between PON1 activity history of stroke and splenectomy. The present study confirmed the association between PON1c.192Q > R and PON1c.55L > M polymorphisms and PON1 activity, in addition to demonstrate their effects on markers of dislipidemia, hemolysis and inflammation, in SCD individuals. Moreover, data suggest PON1 activity as a potential biomarker related to stroke and splenectomy.

Evaluation of 2D and 3D Erythroid Differentiation Protocols Using Sickle Cell Disease and Healthy Donor Induced Pluripotent Stem Cells.

BACKGROUND: Sickle cell disease (SCD) is a highly prevalent genetic disease caused by a point mutation in the HBB gene, which can lead to chronic hemolytic anemia and vaso-occlusive events. Patient-derived induced pluripotent stem cells (iPSCs) hold promise for the development of novel predictive methods for screening drugs with anti-sickling activity. In this study, we evaluated and compared the efficiency of 2D and 3D erythroid differentiation protocols using a healthy control and SCD-iPSCs. METHODS: iPSCs were subjected to hematopoietic progenitor cell (HSPC) induction, erythroid progenitor cell induction, and terminal erythroid maturation. Differentiation efficiency was confirmed by flow cytometry analysis, colony-forming unit (CFU) assay, morphological analyses, and qPCR-based gene expression analyses of HBB and HBG2. RESULTS: Both 2D and 3D differentiation protocols led to the induction of CD34+/CD43+ HSPCs. The 3D protocol showed good efficiency (>50%) and high productivity (45-fold) for HSPC induction and increased the frequency of BFU-E, CFU-E, CFU-GM, and CFU-GEMM colonies. We also produced CD71+/CD235a+ cells (>65%) with a 630-fold cell expansion relative to that at the beginning of the 3D protocol. After erythroid maturation, we observed 95% CD235a+/DRAQ5- enucleated cells, orthochromatic erythroblasts, and increased expression of fetal HBG2 compared to adult HBB. CONCLUSION: A robust 3D protocol for erythroid differentiation was identified using SCD-iPSCs and comparative analyses; however, the maturation step remains challenging and requires further development.

Association of laboratory markers and cerebral blood flow among sickle cell anemia children.

Background: Stroke is one of the highest complications of sickle-cell anemia (SCA). The Transcranial Doppler (TCD) has been adopted worldwide as a gold standard method for detecting alterations in the blood velocity in cerebral arteries. In this study, we investigated the association between laboratory parameters and increased cerebral blood flow velocity in Brazilian SCA pediatric patients. Methods: The study included 159 pediatric patients with SCA, submitted to TCD velocity screening, and the time-averaged maximum mean velocity (TAMMV) was determined in the middle cerebral artery (MCA), anterior cerebral artery (ACA), and distal intracranial internal carotid artery (ICA). We compared cerebral blood flow in patients stratified by the following: TCD1-defined as normal, with TAMMV inferior to 170 cm/s; TCD2-conditional, with TAMMV above 170 cm/s, but less than 199 cm/s; TCD3-altered, with TAMMV greater than or equal to 200 cm/s. Results: TAMMV was negatively correlated with age and weight (p < 0.05). Moreover, TAMMV was associated or correlated with reductions in HbF, RBC, hemoglobin, hematocrit, HDL, and haptoglobin and, increases in MCV, MCH, RDW, reticulocytes, WBC, lymphocytes, monocytes, eosinophils, total and indirect bilirubin, LDH, AST, ALT, glucose, ferritin, and AAT (p < 0.05). Conclusion: The current study highlights the importance of the investigation of hemolytic and inflammatory biomarkers for monitoring the clinical outcome of SCA pediatric patients, to avoid acute or chronic stroke. Moreover, glucose and HDL-C appear useful for predicting higher TAMMV.

Sickle Cell Anemia: Variants in the CYP2D6, CAT, and SLC14A1 Genes Are Associated With Improved Hydroxyurea Response.

Differences in hydroxyurea response in sickle cell anemia may arise due to a series of factors with genetic factors appearing to be predominant. This study aims to investigate the effects of single nucleotide polymorphisms in genes encoding drug-metabolizing enzymes and solute carriers on hydroxyurea response, in patients with sickle cell anemia. For that purpose, a total number of 90 patients with sickle cell anemia were recruited, 45 were undergoing hydroxyurea treatment, while 45 were not under the treatment. Association analyses were performed between CYP3A4 (rs2740574), CYP2D6 (rs3892097), CAT (rs7943316 and rs1001179), and SLC14A1 (rs2298720) variants and laboratory parameters. According to our findings, patients with hydroxyurea treatment demonstrated higher HbF levels and a significant improvement in hemolytic, hepatic, inflammatory, and lipid parameters in comparison to those without the treatment. We also found significant associations between the CYP2D6 (rs3892097), CAT (rs7943316 and rs1001179), and SLC14A1 (rs2298720) variants and an improvement of the therapeutic effects, specifically the hemolytic, hepatic, inflammatory, lipid, and renal parameters. In conclusion, our results highlight the importance of the investigated variants, and their strong association with hydroxyurea efficacy in patients with sickle cell anemia, which may be considered in the future as genetic markers.

Hydroxyurea Scavenges Free Radicals and Induces the Expression of Antioxidant Genes in Human Cell Cultures Treated With Hemin.

The excessive release of heme during hemolysis contributes to the severity of sickle cell anemia (SCA) by exacerbating hemoglobin S (HbS) autoxidation, inflammation and systemic tissue damage. The present study investigated the effect of hydroxyurea (HU) on free radical neutralization and its stimulation of antioxidant genes in human peripheral blood mononuclear cells (PBMC) and human umbilical vein endothelial cells (HUVEC) in the presence or absence of hemin. HU (100 and 200 µM) significantly reduced the production of intracellular reactive oxygen species (ROS) induced by hemin at 70 µM in HUVEC. HUVECs treated with HU+hemin presented significant increases in nitric oxide (NO) production in culture supernatants. HU alone or in combination with hemin promoted the induction of superoxide dismutase-1 (SOD1) and glutathione disulfide-reductase (GSR) in HUVECs and PBMCs, and glutathione peroxidase (GPX1) in PBMCs. Microarray analysis performed in HUVECs indicated that HU induces increased expression of genes involved in the antioxidant response system: SOD2, GSR, microsomal glutathione S-transferase (MGST1), glutathione S-transferase mu 2 (GSTM2), carbonyl reductase 1 (CBR1) and klotho B (KLB). Significant increases in expression were observed in genes with kinase activity: protein kinase C beta (PRKCB), zeta (PRKCZ) and phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2 beta (PIK3C2B). HU also induced a significant increase in expression of the gene p62/sequestosome (p62/SQSTM1) and a significant decrease in the expression of the transcriptional factor BACH1 in HUVECs. Upstream analysis predicted the activation of Jun, miR-155-5p and mir-141-3p. These results suggest that HU directly scavenges free radicals and induces the expression of antioxidant genes via induction of the Nrf2 signaling pathway.

Effect of N(Epsilon)-(carboxymethyl)lysine on Laboratory Parameters and Its Association with ß S Haplotype in Children with Sickle Cell Anemia.

The present study aimed to investigate the association of N ε -carboxymethyllysine (CML) with laboratory parameters and ß S haplotypes in pediatric sickle cell anemia (SCA) patients with or without hydroxyurea (HU) therapy. We included 55 children with SCA (SCAtotal), where 27 were on HU treatment (SCA-HU+) and 28 without HU treatment (SCA-HU-). Laboratory characteristics were determined using electronic methods while CML was measured using competitive ELISA. ß S haplotypes were determined by RFLP-PCR. Significant increases in MCV and MCH and significant decreases in leukocytes, eosinophils, basophils, atypical lymphocytes, lymphocytes, and monocytes were found in SCA-HU+ compared to SCA-HU-. SCA-HU+ presented significant reduction in aspartate transaminase and lactate dehydrogenase and increase in creatinine levels compared to SCA-HU-. CML levels were significantly higher in both SCA-HU+ and SCA-HU- compared to the healthy control. In addition, a negative correlation was found between CML and alanine transaminase in SCA-HU+ and SCAtotal (p < 0.01). A significant association was found between CML levels and ß S haplotypes. The results suggest that CML has a role to play in SCA complications, independent of HU therapy.

Differential gene expression analysis of sickle cell anemia in steady and crisis state.

Sickle cell anemia is one of the most prevalent genetic diseases worldwide, showing great clinical heterogeneity. This study compared the gene expression patterns between sickle cell anemia pediatric patients in steady state and in crisis state, as compared to age-paired, healthy individuals. RNA sequencing was performed from these groups of patients/controls using Illumina HiSeq 2500 equipment. The resulting differentially expressed genes were loaded into QIAGEN's ingenuity pathway analysis. The results showed that EIF2 pathway and NRF2-mediated oxidative stress-response pathways were more highly activated both in steady state and in crisis patients, as compared to healthy individuals. In addition, we found increased activation of eIF4 and p70S6K signaling pathways in crisis state compared to healthy individuals. The transcription factor GATA-1 was found exclusively in steady state while SPI was found exclusively in crisis state. IL6 and VEGFA were found only in crisis state, while IL-1B was found exclusively in steady state. The regulator effects analysis revealed IgG1 as an upstream regulator in steady state compared to healthy individuals, resulting in invasion of prostate cancer cell lines as the disease/function outcome. For crisis-state patients versus healthy individuals, two networks of regulator effects revealed STAT1, CD40LG, TGM2, IRF7, IRF4, and IRF1 acting as upstream regulators, resulting in disease/function outcomes, including engulfment of cells and aggregation of blood cells and inflammation of joints. Our results indicated genes and pathways that can provide clues on the molecular events involved in the severity of sickle cell disease.

Laboratory and Genetic Biomarkers Associated with Cerebral Blood Flow Velocity in Hemoglobin SC Disease.

Reference values for cerebral blood flow velocity (CBFV) in hemoglobin SC disease (HbSC) have not been established. We aimed to investigate associations between laboratory and genetic biomarkers associated with CBFV in HbSC children. Sixty-eight HbSC children were included; CBFV was analyzed by transcranial Doppler, and the time-averaged maximum mean velocity (TAMMV) was estimated. Hematological, biochemical, immunological, and genetic analyses were performed. TAMMV was negatively correlated with red blood cell count (RBC) count, hemoglobin, hematocrit, and direct bilirubin (DB), yet positively correlated with monocytes and ferritin. We found that children with TAMMV ≥ 128 cm/s had decreased red blood cell distribution width (RDW) and nitric oxide metabolite (NOx) concentration. Children with TAMMV ≥ 143.50 cm/s had decreased hemoglobin and hematocrit, as well as increased ferritin levels. Decreased hemoglobin, hematocrit, RDW, and NOx and increased ferritin were detected in children with TAMMV ≥ 125.75 cm/s. The CAR haplotype was associated with higher TAMMV. In association analyses, RBC, hemoglobin, hematocrit, RDW, monocyte, DB, NOx, and ferritin, as well as the CAR haplotype, were found to be associated with higher TAMMV in HbSC children. Multivariate analysis suggested that high TAMMV was independently associated with hematocrit, RDW, and NOx. Additional studies are warranted to validate the establishment of a cutoff value of 125.75 cm/s associated with elevated TAMMV in HbSC children.

Transcranial Doppler in hemoglobin SC disease.

BACKGROUND: Stroke is a severe clinical disorder in sickle cell disease (SCD), and few studies have evaluated transcranial Doppler (TCD) flow velocities in hemoglobin SC disease (HbSC). The guidelines for stroke risk are based on evaluations in sickle cell anemia (SCA) or HbS/ß thalassemia. PROCEDURE: In this study, we compare cerebral blood flow in patients with SCD stratified by genotypes. A total of 1,664 pediatric patients with SCD underwent TCD velocity screening, and the time-averaged maximum mean velocity (TAMM) was determined in the middle cerebral artery (MCA), anterior cerebral artery (ACA), and distal intracranial internal carotid artery (ICA). RESULTS: Abnormal velocities were not identified in the ACA; therefore, we only use ICA and MCA velocities. TAMM from the left and right in the ICA and MCA was 134.3 ± 32.0 and 134.4 ± 32.6 cm/s in patients with SCA, and 105.2 ± 20.6 and 104.7 ± 20.0 cm/s in the patients with HbSC, respectively. Mean TAMM between right and left ICA/MCA was 134.5 ± 30.5 cm/s in the SCA group, and 104.9 ± 19.3 cm/s in the HbSC group. Notably, our data show that TCD velocities were significantly lower among the patients with HbSC compared to SCA. TAMM was negatively correlated with hemoglobin and hematocrit in both genotypes. CONCLUSION: These results suggest that a different cut-off value for abnormal TCD velocities could be considered for patients with HbSC. Additional studies are warranted to determine the actual risk of stroke in HbSC genotype associated with this possible TCD risk value.