Analysis of genetic determinants associated with persistent synthesis of fetal hemoglobin

One of the principal ameliorating factors in β–thalassemias is the innate ability to produce HbF. Although the majority of adults shows traces of HbF, in rare cases, named Hereditary Persistence of Fetal Hemoglobin (HPFH), its value can reach levels of 15%–30%.cis–acting variants like deletions, point mutations and SNPs within the β–cluster can explain some of the variability; however 50% of the variance is unlinked to the β–cluster.In this study, healthy HPFH individuals were analyzed: Hb profile was defined by CE–HPLC, RP–HPLC, IEF and AUT–PAGE. Structural analysis of the γ genes promoters revealed 4 ndHPFH mutations, whose strength was studied by luciferase assays.A large deletion on the β–cluster (Sicilian δβ–thalassemia) was found in one sample by MLPA and GAP–PCR.OneAγ ndHPFH sample, also showingGγ persistence, presented 2 new mutations on theGγ promoter: luciferase assays demonstrated that the persistence ofGγ is not attributable to these mutationsper se.Complete sequence of β genes showed β0(39) and β+(–87) β–thalassemia alleles; β+(–87) was found linked to acis–acting polymorphic configuration associated with high HbF.Orkin haplotype analysis showed evidence of a unique association betweenAγ –117 mutation and haplotype VII.The data presented in this thesis confirm the complexity of the globin switch and the heterogeneity of the molecular mechanisms underlying the persistence of HbF, emphasizing the importance of the molecular context of a mutation.