Influência da hidroxiureia e polimorfismos na expressão de citocinas inflamatórias em pacientes portadores de Anemia Falciforme

Abstract

Introduction: Sickle cell anemia (SCA) is the most prevalent hereditary hemoglobinopathy worldwide. It results from a point mutation on chromosome 11 at position 6 of the β-globin gene, leading to the replacement of glutamic acid by valine. This alteration generates variant hemoglobin “S”. Objective: To molecularly characterize the promoter regions and quantify the cytokines IL-1β, IL-6, IL-8, IL-10, IL-18, and TNF-α in individuals with SCA with and without treatment with Hydroxyurea (HU) and healthy donors. Methods: The BDTM Cytometric Bead Array (CBA) System was used to analyze the cytokines, with the KITs: CBA Human Th1/Th2/Th17 Cytokines and CBA Human Inflammatory Cytokines. Molecular characterization was performed by conventional PCR of the promoter regions of cytokine genes, purified by the ExoSAP-IT™ methodology and subsequent sequencing by the Sanger method. Statistical analyses were performed using the SPSS vs21 and Prism vs5 programs, with p-values <0.05 considered significant. Results: A total of 214 patients (HbSS) and 240 controls (HbAA) participated in the study. Females were predominant in patients (62.15%), while males were predominant in controls (74.17%). The most frequent clinical events in patients were VOCs (85.2%), followed by hospitalizations (64.2%). All patients, regardless of clinical status, showed significant elevation of cytokines (P<0.001). Significantly higher frequencies of the wild-type c.-31CC (IL-1B), c.-251AA (IL-8), and c.-607CC (IL-18) genotypes were found in blood donors when compared to patients. The wild-type c.-251AA (IL-8) genotype was significantly more frequent in those without HU use (p=0.004 - OR: 0.22 - CI: 0.07-0.66), presenting itself as a protective genotype for comorbidities in patients. Significant associations for elevation of inflammatory cytokines in the study were found in the c-31TT (IL-1B) genotype in both groups, while the c.-607AA (IL-18) genotype only in patients. The mutant c.-251TT (IL-8) genotype showed significantly decreased concentrations in both study groups. Discussion: The heterogeneous clinical presentation of these patients is in agreement with other studies in the national literature. Occlusive crises and intense hemolysis predominated. Although the polymorphisms found in our study have been previously described, the correlations of genotypes in patients with and without HU use are described for the first time. The frequency of the wild genotypes c.-31CC, c.-251AA and c.-607CC have different expressions, increasing mainly in those inflammatory diseases. The wild genotype c.-251AA (IL-8) was shown to be a protective biomarker in individuals, since studies demonstrate high concentrations of IL-8 in patients with mild crises, corroborating our results. The finding in our study of prevalence and elevated levels of IL-18 in the c-607AA mutant genotype in patients corroborates several studies that favor the severity of the disease, which enables this SNP as an important inflammatory biomarker of risk, involving comorbidities and a high mortality rate. Conclusion: The most prevalent events in patients were VOCs (85.2%), followed by hospitalizations (64.2%). All cytokines were elevated in the study patients, with the highest prevalence of mutant genotypes. The wild-type genotype c.-251AA (IL-8) was significantly more frequent in sickle cell patients not using HU, with its concentration significantly higher in patients, while the mutant genotypes c-31TT (IL-1B) and c.-607AA (IL-18) had significantly elevated plasma concentrations in both groups.