Beta + thalassemia: aberrant splicing results from a single point mutation in an intron

We have analyzed the molecular basis of beta + thalassemia by studying the expression of a cloned beta-globin gene in HeLa cells. This beta-globin gene was isolated from a beta + thalassemic patient and differs from the normal beta-globin genes by only a single point mutation within the first intron. The beta + thalassemic and the normal beta-globin genes were cloned into an SV40-pBR328 vector and introduced into HeLa cells by calcium phosphate coprecipitation. We assayed the RNA from these transfected HeLa cells by S1 nuclease mapping and cDNA sequencing to detect the nature of the defect in beta-globin gene expression. While the transcripts of the normal beta-globin gene are processed correctly, the first intron of the beta + thalassemic beta-globin gene is incorrectly spliced in about 90% of the mRNA because of an additional 3; splice site that has been created by the point mutation. This incorrectly spliced mRNA is effectively exported to the cytoplasm, where it would conceivably be translated to give a truncated globin chain of 35 amino acids. The remaining 10% of the mRNA transcribed from the beta+ thalassemic globin gene is correctly spliced and can therefore be translated to give normal beta-globin. In addition to the incorrect splicing of the first intron, the splicing of both introns is retarded, which results in the accumulation of unspliced pre-mRNA. This suggests that removal of the first intron might facilitate splicing of the second intron.