Abstract
DNA variants affecting mRNA expression and processing in genetic diseases are often missed or poorly characterized. We previously reported a generic assay to identify variants that affect mRNA expression and splicing in Pompe disease, a monogenic disorder caused by deficiency of acid α-glucosidase (GAA). However, this assay could miss mRNA that is subjected to degradation. Here, we inhibited mRNA degradation using cycloheximide and performed unbiased splicing analysis of all GAA exons using exon flanking RT-PCR and exon internal RT-qPCR. In four patients that were suspected of harboring splicing variants but for which aberrant splicing could not be detected in normally growing cells, we detected a total of 10 novel splicing events in cells treated with cycloheximide. In addition, we found that sequences of GAA introns 6 and 12 were naturally included in a subset of transcripts from patients and healthy controls, indicating inefficient canonical splicing. Identification of aberrant splicing caused by the common Asian variant c.546G>T allowed the development of an antisense oligonucleotide that promoted canonical GAA pre-mRNA splicing and elevated GAA enzymatic activity. Our results indicate that this extended generic splicing assay allows the detection of aberrant splicing in cases of mRNA degradation to enable functional analysis of unknown splicing variants and the development of targeted treatment options.
Highlights
Supplementary information The online version of this article contains supplementary material, which is available to authorized users.Department of Clinical Genetics, Erasmus MC Medical Center, Rotterdam, NetherlandsDepartment of Pediatrics, Erasmus MC Medical Center, Rotterdam, NetherlandsCenter for Lysosomal and Metabolic Diseases, Erasmus MC Medical Center, Rotterdam, NetherlandsDepartment of Laboratory Medicine, Shinshu University Hospital, Nagano, JapanDepartment of Pediatrics, Asahikawa Medical University, Hokkaido, JapanDepartment of Biomedical Laboratory Sciences, Faculty of Life Sciences, Kumamoto University, Kumamoto, JapanPompe disease (Glycogen storage disease type II, OMIM 232300) is an autosomal inherited recessive disorder caused by the partial or complete deficiency of the lysosomal enzyme acid α-glucosidase (GAA) [1]
The functional consequences of variants on pre-mRNA splicing often remain undetermined in current standard diagnostic analysis of monogenic disorders
Our study shows that inhibition of mRNA degradation and subsequent splicing analysis resulted in the identification of aberrant mRNA transcripts in four patients for which the conventional splicing assay failed to detect aberrant splicing
Summary
Clinical signs of Pompe disease are confirmed by determination of residual GAA enzymatic activity in primary fibroblasts, muscle biopsies and/or leukocytes. We reported a generic method that analyzes the functional effect of GAA variants on pre-mRNA splicing and mRNA expression in primary fibroblasts [4]. This assay has a fundamental restriction: analysis of variants that alter canonical splicing can be complicated due to possible effect of nonsense-mediated decay (NMD). Patients were diagnosed with Pompe disease based on phenotype, GAA enzymatic activity in leukocytes and fibroblasts, and genomic DNA sequence analysis. The diagnostically determined healthy control range is 45–180 nmol 4-MU/hr/mg protein and the patient range is from 0 to 20 nmol 4-MU/hr/mg protein
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
