Sickle cell anemia (SCA) occurs due to molecular changes in the beta globin gene. A point mutation results in the substitution of one base, where an adenine is substituted by a thymine. This leads to the production of an abnormal hemoglobin - HbS. The pathophysiology of this disease causes, primarily, the polymerization of HbS and subsequent sickling of red blood cells, causing shortening of the average life of the red blood cells, vaso-occlusive phenomena, episodes of pain and organ damage. SCA is a disease of great clinical heterogeneity that undergoes the genetic effects of a variety of modulators. The production of fetal hemoglobin (HbF) by cells of the erythroid lineage is a significant factor that modulates the clinical presentation of patients with SCA and sickle cell disease (SCD), since the presence of HbF in the red cells can inhibit polymerization of HbS. Three genetic loci contribute to nearly 50% of the variability in the production of HbF; the site on chromosome 11p15 XmnIG ³ contributes to 10% of the variation, the locus on chromosome 2 BCL11A contributes 15% of the variation, and the locus on chromosome 6q HMIP contributes 19%. In Brazil, few studies have been developed in order to correlate the genetic patterns of patients with sickle cell and fetal hemoglobin levels. A study investigating the frequency of a greater variety of polymorphisms in the HMIP region and the BCL11A gene in Brazilian SCD patients and healthy Brazilian individuals of African descent, and their association with the production of HbF, will provide more information about genetic variants that may contribute to changes in the production of HbF in the Brazilian population. DNA from subjects with SCA, HbSC disease and healthy controls will be extracted to perform Taqman Real Time PCR to identify 5 polymorphisms, two SNPs will be genotyped by microsatellite analysis and XmnIG³ (rs7482144) genotyping of all samples will be conducted by PCR and restriction enzyme analysis. Functional studies will also be developed in order to elucidate some of the mechanisms by which polymorphisms in the HBS1L-MYB intergenic region could be acting to modulate the levels of HbF. Culture of CD34+ cells from patients heterozygous for the selected SNPs will be harvested to identify allele-specific gene markers in MYB. All SCD patients with elevated levels of HbF will undergo KLF1 gene sequencing to identify potential new SNPs. This project will be conducted in collaboration with researchers from Hemope-Pernambuco and King's College London as part of KCL-called FAPESP (10/51459-5).
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