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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 \u03b1-glucosidase (<jats:italic>GAA<\/jats:italic>). However, this assay could miss mRNA that is subjected to degradation. Here, we inhibited mRNA degradation using cycloheximide and performed\u00a0unbiased splicing analysis of all <jats:italic>GAA<\/jats:italic> 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\u00a0<jats:italic>GAA<\/jats:italic> 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&gt;T allowed the development of an antisense oligonucleotide that promoted canonical <jats:italic>GAA<\/jats:italic> pre-mRNA splicing and elevated GAA enzymatic activity. Our results indicate that this\u00a0extended 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.<\/jats:p>","DOI":"10.1038\/s41431-020-00751-3","type":"journal-article","created":{"date-parts":[[2020,11,9]],"date-time":"2020-11-09T09:09:41Z","timestamp":1604912981000},"page":"422-433","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["A generic assay for the identification of splicing variants that induce nonsense-mediated decay in Pompe disease"],"prefix":"10.1038","volume":"29","author":[{"given":"Atze J.","family":"Bergsma","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Stijn L. 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Mol Genet Metab Rep. 2018;14:3\u20139.","journal-title":"Mol Genet Metab Rep"}],"container-title":["European Journal of Human Genetics"],"original-title":[],"language":"en","link":[{"URL":"http:\/\/www.nature.com\/articles\/s41431-020-00751-3.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"http:\/\/www.nature.com\/articles\/s41431-020-00751-3","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"http:\/\/www.nature.com\/articles\/s41431-020-00751-3.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,5,20]],"date-time":"2023-05-20T21:01:02Z","timestamp":1684616462000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/s41431-020-00751-3"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,11,9]]},"references-count":36,"journal-issue":{"issue":"3","published-print":{"date-parts":[[2021,3]]}},"alternative-id":["751"],"URL":"https:\/\/doi.org\/10.1038\/s41431-020-00751-3","relation":{},"ISSN":["1018-4813","1476-5438"],"issn-type":[{"value":"1018-4813","type":"print"},{"value":"1476-5438","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,11,9]]},"assertion":[{"value":"25 February 2020","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"10 September 2020","order":2,"name":"revised","label":"Revised","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"20 October 2020","order":3,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"9 November 2020","order":4,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Compliance with ethical standards"}},{"value":"ATvdP provided consultancy services for various industries. All other authors declare that they have no conflict of interest.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Conflict of interest"}}]}}