부산대학교 도서관

We report three novel deletions involving the Multispecies Conserved Sequences (MCS) R2, also known as the Major Regulative Element (MRE), in patients showing the α-thalassemia phenotype. The three new rearrangements showed peculiar positions of the breakpoints. 1) The (αα)ES is a telomeric 110 kb deletion ending inside the MCS-R3 element. 2) The (αα)FG, 984 bp-long, ends 51 bp upstream to MCS-R2; both are associated with a severe α-thalassemia phenotype. 3) The (αα)CT, 5058 bp-long starts at position +93 of MCS-R2 and is the only one associated to a mild α-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 (αα)ES was unable to produce α2-globin mRNA, while a high level of expression of the α2-globin genes (56%) was detected in (αα)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 (αα)CT and (αα)FG, showed comparable activity both for MCS-R2 and the boundary region (-682/-8). Considering that the (αα)CT deletion, almost entirely removing MCS-R2, has a less severe phenotype than the (αα)FG α0thalassemia 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 α-globin genes. The genotype-phenotype relationship of other previously published MCS-R2 deletions strengthened our hypothesis. Author summary: The regulation of gene expression occurs through the physical interaction between the promoters, regions preceding the genes, and the enhancer, short regions placed even at a considerable distance, with a mechanism defined as long-range regulation. Globin genes are a good model of gene regulation, due to the easy access to the target tissue (blood) and to the analyses that can be performed on it. In a study of patients with alpha-thalassemia, we identified three new deletions involving different parts of the enhancer named Major Regulative Element MCS-R2 associated with severe phenotype, and one of them, with mild. These deletions have given us the opportunity to gain insight into long-range regulation in vivo, using transcriptional analysis on reticulocytes from patients to analyze the role of individual regulatory elements. Our results highlight for the first time a functional enhancer role associated to the boundary MCS-R2 regions and shed light on aspects related to long-range regulation. The data could be useful to better understand the pathophysiology of deletions involving the enhancer MCS-R2 and also for constructing expression vectors for gene therapy and for identifying drug target sequences to modulate gene expression. [ABSTRACT FROM AUTHOR]