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

Abstract

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 approximately 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.

Deconstructing Pompe Disease by Analyzing Single Muscle Fibers: “To See a World in a Grain of Sand…”

“…All were compound heterozygous and shared the most common mutation associated with adult-onset disease in Caucasian patients, c.-32-13T → G. 25,26 None had been treated with recombinant enzyme. The clinical spectrum ranged from fatigability without muscle weakness to severe hypotonia, limited mobility, and wheelchair dependence.…”

Splicing mutations in glycogen-storage disease type II: evaluation of the full spectrum of mutations and their relation to patients’ phenotypes

“…30,32,33,35 It is worth mentioning that the mutation c.-32-13T4G, the most common one among the Caucasian LO GSDII patients, 3 has been previously studied in vitro using a minigene system assay. 51 The results have shown that this mutation does not completely prevent normal splicing, as low levels of the correctly spliced mRNA were generated with the mutant construct. In fact, three splice variants (SV1, SV2 and SV3-Table2) were observed with both the wild-type and the mutant constructs, indicating that these forms represent normal alternative spliced products.…”

Papaya fruits extracts enhance the antioxidant capacity and modulate the genotoxicity and oxidative stress in the kidney of rats fed ochratoxin A-contaminated diet

“…The ethidium bromidestained gel bands were scanned and the signal intensities were quantified by the computerized Gel-Pro (version 3.1 for window 3). The ratio between the levels of the target gene amplification product and the β-actin (internal control) was calculated to normalize for initial variation in the sample concentration as a control for reaction efficiency ( Raben et al, 1996). All PCRs were independently replicated three times.…”