Role of nonsense-mediated decay and nonsense-associated altered splicing in the mRNA pattern of two new alpha-thalassemia mutants

alpha-thalassemia is a common disease characterized mainly by deletion mutants. We identified two new ?-thalassemia pointform mutants: alpha1cod22 GGC > GGT Gly > Gly creating a 5' splicing sequence and alpha1cod23 GAG > TAG Glu > stop. We performed qualitative and semi-quantitative analysis of the mRNA molecules, from carriers' blood, to define the molecular mechanisms giving rise to the thalassemia phenotype. In vitro analysis using alpha-globin constructs and cycloheximide was performed to evaluate if the mutants are substrates of nonsense-mediated mRNA decay (NMD). In the alpha1cod22 GGC > GGT the new 5' splicing site in exon 1 completely substitutes the normal one. We demonstrated the presence of mRNA decay as the abnormally spliced mRNA was consistent in the nucleus, partially degraded in the cytoplasm of cultured cells, but only 2.8% in the reticulocytes. The analysis of the alphacod23 transcript showed an escape from the NMD as for the human alpha-globin transcript with nonsense mutations in the first exon: the anomalous mRNA was reduced in the nucleus, followed by only a slight lowering from 32% to 27% of the normal alpha1 mRNA in the reticulocytes. In both the mutants we showed a moderate sensitivity to the NMD assay and we speculate the activation of other RNA surveillance mechanisms for the alphacod22 mutant. No activation of cryptic splice sites was detected and no role could be assigned to the nonsense-associated altered splicing. Studies on transcripts from patient cells represent a very useful approach providing considerable information about the processes occuring in vivo.