A generic assay for the identification of splicing variants that induce nonsense-mediated decay in Pompe disease

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.

IGF2-tagging of GAA promotes full correction of murine Pompe disease at a clinically relevant dosage of lentiviral gene therapy

“…For analysis of secretion, HEK 293T cells were transfected as previously shown by Bergsma et al. 112 In short, GAAco or IGF2.GAAco cDNAs were cloned into pcDNA3.1 expression vector using XhoI/XbaI restriction enzymes and transfected into HEK 293T cells. Two-hundred milliliters of medium were sampled every 24 h and up until 96 h post transfection for GAA enzyme activity analysis.…”

“…Transfection efficiency was measured based on mRNA expression of the Neomycine resistance cassette present in the pcDNA3.1 backbone using RT-qPCR, as previously described. 112 …”

Tagged IDS causes efficient and engraftment-independent prevention of brain pathology during lentiviral gene therapy for Mucopolysaccharidosis type II

“…For analysis of secretion, HEK293T cells were transfected as shown previously. 25 , 77 In short, IDSco , IDS.IGF2co , IDS.ApoE2co , IDS.RAP12x2co , or GFP lentiviral transfer plasmids were transfected into HEK293T cells via calcium phosphate transfection in complete medium (1% PS, Gibco 15070) and 10% fetal bovine serum (FBS-12A) (Capricorn Scientific) in high-glucose DMEM (Gibco). Twenty-four hours after transfection, the medium was replaced.…”

Translating RNA Splicing Analysis into Diagnosis and Therapy

“…The availability of cultured cells in particular provides an opportunity to examine samples for splice isoforms subject to nonsense-mediated decay (NMD). Through the application of NMD inhibitors such as cycloheximide or anisomycin to such cultures, the otherwise degraded splicing products of pathogenic splicing mutations can subsequently be detected and quantified, as has been demonstrated in both fibroblasts and lymphocytes [24,25].…”