Nonsense mutations in the human beta-globin gene lead to unexpected levelsof cytoplasmic mRNA accumulation

Generally, nonsense codons 50 bp or more upstream of the 3'-most intron of the human beta-globin gene reduce mRNA abundance. In contrast, dominantly i nherited beta-thalassemia is frequently associated with nonsense mutations in the last axon. In this work, murine erythroleukemia (MEL) cells were sta bly transfected with human beta-globin genes mutated within each of the 3 e xons, namely at codons 15(TGG-->TGA), 39 (C-->T), or 127 (C-->T), Primer ex tension analysis after erythroid differentiation induction showed codon 127 (C-->T) mRNA accumulated in the cytoplasm at approximately 20% of the norm al mRNA level. Codon 39 (C-->T) mutation did not result in significant mRNA accumulation. Unexpectedly, codon 15 (TGG-->TGA) mRNA accumulated at appro ximately 90%. Concordant results were obtained when reticulocyte mRNA from 2 carriers for this mutation was studied. High mRNA accumulation of codon 1 5 nonsense-mutated gene was revealed to be independent of the type of nonse nse mutation and the genomic background in which this mutation occurs. To i nvestigate the effects of other nonsense mutations located in the first exo n on the mRNA level, nonsense mutations at codons 5, 17, and 26 were also c loned and stably transfected into MEL cells, After erythroid differentiatio n induction, mRNAs with a mutation at codon 5 or 17 were detected at high l evels, whereas the mutation at codon 26 led to low mRNA levels. These findi ngs suggest that nonsense-mediated mRNA decay is not exclusively dependent on the localization of mutations relative to the 3'-most intron, Other fact ors may also contribute to determine the cytoplasmic nonsense-mutated mRNA level in erythroid cells. (Blood, 2000;96:2895-2901) (C) 2000 by The Americ an Society of Hematology.