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

• Published in: PLoS genetics 2023-05, Vol.19 (5), p.e1010727-e1010727
• Main Authors: Capasso, Serena, Cardiero, Giovanna, Musollino, Gennaro, Prezioso, Romeo, Testa, Rosario, Dembech, Sabrina, Piluso, Giulio, Nigro, Vincenzo, Digilio, F Anna, Lacerra, Giuseppina
• Format: Article
• Language: English
• Subjects: alpha-Thalassemia - genetics; Analysis; Biology and Life Sciences; Blood diseases; Breakpoints; Causes of; Chromosomes; Conserved Sequence; Enhancer Elements, Genetic - genetics; Gene expression; Gene mutations; Genes; Genetic aspects; Genetic research; Genotype; Genotype & phenotype; Genotypes; Globins - genetics; Hematology; Humans; Medicine and Health Sciences; Mutation; Phenotype; Phenotypes; Reticulocytes; Thalassemia
• ISSN: 1553-7404; 1553-7390; 1553-7404
• DOI: 10.1371/journal.pgen.1010727

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.