A model of mRNA splicing in adult lysosomal storage disease (glycogenosis type II)

Glycogenosis type II is a recessively inherited disorder caused by mutations in the acid maltase (GAA) gene. Clinically, three different phenotypes are recognized: infantile, juvenile and adult forms, A majority 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 with wild-type and mutant minigenes expressed in a GAA deficient cell line, We have demonstrated that the mutation does not prevent normal splicing; low levels of correctly spliced mRNA are generated with the mutant construct, The data explain why the mutation is restricted to a milder, adult-onset phenotype, We also demonstrate that splicing out of exon 2 occurs with the wild-type construct, and thus represents alternative splicing which takes place in normal cells. Three splice variants (SV1, SV2 and SV3) are made with both the mutant and the wild-type constructs, Furthermore, as shown by RNAse protection assay, these mRNA 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 construct is reduced compared with the wild type. The removal of similar to 90% of the intron 1 (2.6 kb) sequence resulted in a dramatic increase in the levels of correctly spliced mRNA, indicating that the intron may contain a powerful transcriptional repressor.