Functional analysis of three new alpha-thalassemia deletions involving MCS-R2 reveals the presence of an additional enhancer element in the 5' boundary region

We report three novel deletions involving the Multispecies Conserved Sequences (MCS) R2, also known as the Major Regulative Element (MRE), in patients showing the ?-thalassemia phenotype. The three new rearrangements showed peculiar positions of the breakpoints. 1) The (??)ES is a telomeric 110 kb deletion ending inside the MCS-R3 element. 2) The (??)FG, 984 bp-long, ends 51 bp upstream to MCS-R2; both are associated with a severe ?-thalassemia phenotype. 3) The (??)CT, 5058 bp-long starts at position +93 of MCS-R2 and is the only one associated to a mild ?-thalassemia phenotype. To understand the specific role of different segments of the MCS-R2 element and of its boundary regions we carried out transcriptional and expression analysis. Transcriptional analysis of patients' reticulocytes showed that (??)ES was unable to produce ?2-globin mRNA, while a high level of expression of the ?2-globin genes (56%) was detected in (??)CT deletion, characterized by the presence of the first 93 bp of MCS-R2. Expression analysis of constructs containing breakpoints and boundary regions of the deletions (??)CT and (??)FG, showed comparable activity both for MCS-R2 and the boundary region (-682/-8). Considering that the (??)CT deletion, almost entirely removing MCS-R2, has a less severe phenotype than the (??)FG ?0thalassemia deletion, removing both MCS-R2 almost entirely and an upstream 679 bp, we infer for the first time that an enhancer element must exist in this region that helps to increase the expression of the ?-globin genes. The genotype-phenotype relationship of other previously published MCS-R2 deletions strengthened our hypothesis.