Beta-Globin Cluster Cis-ACTING Sequence and BCL11A Variants in the Control of Hemoglobin F Expression

Abstract 1091 Fetal hemoglobin (Hb F: α2γ2.) is the most important modulating factor of sickle cell disease(SCD) and beta-major thalassemias (β°thal). Due to the expected benefit from an active control of gamma gene expression, studies of the regulation of Hb F expression are very intense with the recent focus on BCl11 A, a transacting factors strongly involved in the control of its expression. Studies have subsequently established that Bcl 11A is a repressor of HbF expression through different mechanisms including a direct action “in trans” on the β-globin locus. Our work aims to look for Bcl 11A coding sequence's variations in a cohort of homozygous β°thal patients whose clinical status is less severe than that expected from their globin genotype because of high Hb F level (12 patients). Interest has also been given to a cohort of patients (12 patients) displaying a small deletions of the Ψβ-δ intergenic region for which it has been noted significant Hb F rate differences in possible correlation with a Bcl 11A binding site located1.5 Kb 5 'to the δ gene previously described (Sankaran, 2008). Methods: Sequencing of Bcl 11A (promoter, XL and L isoforms exons and flanking regions) was performed and the determination of the four main SNPs cited in the literature as predictors of the rate of HbF: rs4671393 and rs11886868 (Bcl 11A), rs9399137 (HSB1L-MYB), and rs7482144-XmnI (β-globin cluster), was carried out. The precise study of deletions was performed using a custom CGH array chip and the subsequent determination of breakpoints. In both patients' cohorts, the absence of the deletion of one BCL11A allele was checked by a tailored semi-quantitative Multiplex PCR, and the hypothesis of a sequence variation inside the “Sankaran region” was screened using a High Resolution Melting procedure (HRM). Results and discussion: Analysis of BCL11A: We have not found mutation inside the coding sequence. However, A “CCG” triplet insertion within a CCG-rich region located in the 5'UTR, immediately 5' to the ATG codon was observed in three patients. That triplet insertion was not found in the other patients of our cohort of 12 patients with high expression of Hb F, and neither in another cohort of high F patients (23 patients). It was also absent in low Hb F patient ( 15) or in controls (50 patients not known to have a red cell disease), highlighting the rarity of this insertion. The effects on transcription or RNA translation could not be predicted by “in silico” analysis. The deletion analysis: We found in this cohort patients two kind of deletions: a first group where the 5' breakpoint of the deletion is located close to the δ-globin gene and for which the presumed BCL11A binding domain was not removed and a second group for which the 5' deletion breakpoint extends 5' and removes that region. Interestingly the second group has a higher Hb F expression calculated as the absolute level of Hb f (Hb value * Hb F %) than the first group with a ratio of 2.54. That result highlight the role of this domain for Hb F control. We, also, cannot found sequence variation in that domain using HRM analysis. In conclusion: The presence of BCL 11A binding sites immediately 5' of d gene appears to be necessary for Hb F control. However, investigating the role of Bcl 11A in Hb F dysregulation (ie: the absence of down regulation) either by looking at protein variants or at variations in the supposed target of Bcl11A in β globin cluster, we found that this lack of regulation is not linked to mutated Bcl 11a and nor to SNPs into its binding region. These results support the hypothesis that the control of Hb F is due to variation of the level of Bcl 11 A expression and the finding of a variation in the 5'UTR region which may interfere with the Bcl 11A RNA translation is another supporting evidence. Further studies of the consequence of this triplet insertion are ongoing to estimate its possible involvement in BCL11A transcription and translation and thus Bcl11A level. We confirmed that the “Sankaran region” is an important element of Bcl11A control of Hb F expression. A study of chromatin conformation (3C-qPCR) using patient bearing these small deletions are needed to test the hypothesis of a non-dissociation of the beta-globin LCR (locus control region) from its attachment point in the genes Gγ Aγ region confirming the major role of this site in the HbF downregulation. Disclosures: No relevant conflicts of interest to declare.