1. A non-invasive diagnostic assay for rapid detection and characterization of aberrant mRNA-splicing by nonsense mediated decay inhibition.
- Author
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Häuser F, Gökce S, Werner G, Danckwardt S, Sollfrank S, Neukirch C, Beyer V, Hennermann JB, Lackner KJ, Mengel E, and Rossmann H
- Subjects
- Alleles, Alternative Splicing drug effects, Anisomycin pharmacology, Cells, Cultured, Child, Preschool, Chromatography, Liquid, Codon, Nonsense, Exons, Female, Glycogen Storage Disease Type II blood, Glycogen Storage Disease Type II physiopathology, Heterozygote, Humans, Infant, Lymphocytes drug effects, Male, Mutation, Nonsense Mediated mRNA Decay genetics, Protein Synthesis Inhibitors pharmacology, RNA, Messenger drug effects, Tandem Mass Spectrometry, alpha-Glucosidases blood, alpha-Glucosidases genetics, Alternative Splicing genetics, Glycogen Storage Disease Type II diagnosis, Glycogen Storage Disease Type II genetics, Lymphocytes metabolism, Nonsense Mediated mRNA Decay drug effects, RNA, Messenger genetics
- Abstract
Background: Interpretation of genetic variants detected by sequencing of genomic DNA, which may cause splicing defects, regularly requires mRNA analysis. Usually, only bioinformatic testing is provided, because simple and non-invasive assay protocols are lacking. Furthermore, the detection of mis-splicing is often hampered by nonsense mediated mRNA decay (NMD)., Methods: Starting from a case of Pompe disease with two potential splicing variants an assay for the analysis of splice defects in general was developed. We analyzed the transcripts from the gene of interest by standard methods after short-term culture of the patient's lymphocytes in the presence and absence of a NMD inhibitor. Variant and wild type transcript expression were quantified by allele specific PCR in the patient and both parents and the expression ratio with/without NMD inhibition was calculated for each transcript., Results: NMD detection in lymphocytes was optimized and evaluated by analyzing a naturally occurring NMD transcript. Several compounds inhibited NMD successfully, including potential therapeutic agents. Sample storage for up to 4 days at room temperature prior to lymphocyte isolation did not affect results. In a proof of concept we identified two candidate variants as severe splicing variants in a patient with Pompe disease, but the strategy can also be used to screen for any mis-spliced transcripts prone to NMD., Conclusions: We developed a simple, non-invasive assay for the detection and characterization of potential splicing variants. This is essential, because early and near-term diagnosis and disease classification is required to facilitate therapy in many genetic diseases., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2020
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