A MODEL OF MESSENGER-RNA SPLICING IN ADULT LYSOSOMAL STORAGE DISEASE (GLYCOGENOSIS TYPE-II)

Glycogenosis type II is a recessively inherited disorder caused by mut ations in the acid maltase (GAA) gene. Clinically, three different phe notypes are recognized: infantile, juvenile and adult forms, A majorit y of compound heterozygous adult-onset patients carry a t-13g mutation in intron 1 associated with splicing out the first coding exon (exon 2), We have studied the mechanism of this mutation in a model system w ith wild-type and mutant minigenes expressed in a GAA deficient cell l ine, We have demonstrated that the mutation does not prevent normal sp licing; low levels of correctly spliced mRNA are generated with the mu tant construct, The data explain why the mutation is restricted to a m ilder, adult-onset phenotype, We also demonstrate that splicing out of exon 2 occurs with the wild-type construct, and thus represents alter native splicing which takes place in normal cells. Three splice varian ts (SV1, SV2 and SV3) are made with both the mutant and the wild-type constructs, Furthermore, as shown by RNAse protection assay, these mRN A variants are less abundant with the mutant construct. Thus, a major effect of the mutation appears to be a low splicing efficiency, since the total amount of all the transcripts generated from the mutant cons truct is reduced compared with the wild type. The removal of similar t o 90% of the intron 1 (2.6 kb) sequence resulted in a dramatic increas e in the levels of correctly spliced mRNA, indicating that the intron may contain a powerful transcriptional repressor.