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Many non-canonical splice site variants have been reported in public databases, but an effect on pre-mRNA splicing has only been functionally verified for a subset of these variants. In this study, we aimed to extend the knowledge regarding splicing events by assessing a selected set of USH2A non-canonical splice site variants and to study their potential pathogenicity. Eleven non-canonical splice site variants were selected based on four splice prediction tools. Ten different USH2A constructs were generated and minigene splice assays were performed in HEK293T cells. An effect on pre-mRNA splicing was observed for all 11 variants. Various events, such as exon skipping, dual exon skipping and partial exon skipping were observed and eight of the tested variants had a full effect on splicing as no conventionally spliced mRNA was detected. We demonstrated that non-canonical splice site variants in USH2A are an important contributor to the genetic etiology of the associated disorders. This type of variant generally should not be neglected in genetic screening, both in USH2A-associated disease as well as other hereditary disorders. In addition, cases with these specific variants may now receive a conclusive genetic diagnosis.<\/jats:p>","DOI":"10.3390\/ijms232113343","type":"journal-article","created":{"date-parts":[[2022,11,2]],"date-time":"2022-11-02T08:15:12Z","timestamp":1667376912000},"page":"13343","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Minigene-Based Splice Assays Reveal the Effect of Non-Canonical Splice Site Variants in USH2A"],"prefix":"10.3390","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5677-9258","authenticated-orcid":false,"given":"Janine","family":"Reurink","sequence":"first","affiliation":[{"name":"Department of Human Genetics, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands"},{"name":"Donders Institute for Brain Cognition and Behaviour, Radboud University Medical Center, 6525 AJ Nijmegen, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3980-4220","authenticated-orcid":false,"given":"Jaap","family":"Oostrik","sequence":"additional","affiliation":[{"name":"Department of Otorhinolaryngology, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6069-3847","authenticated-orcid":false,"given":"Marco","family":"Aben","sequence":"additional","affiliation":[{"name":"Department of Human Genetics, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8246-1817","authenticated-orcid":false,"given":"Mariana Guimar\u00e3es","family":"Ramos","sequence":"additional","affiliation":[{"name":"Department of Human Genetics, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emma","family":"van Berkel","sequence":"additional","affiliation":[{"name":"Department of Human Genetics, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4216-9141","authenticated-orcid":false,"given":"Monika","family":"O\u0142dak","sequence":"additional","affiliation":[{"name":"Department of Genetics, Institute of Physiology and Pathology of Hearing, 02-042 Warsaw, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5702-3460","authenticated-orcid":false,"given":"Erwin","family":"van Wijk","sequence":"additional","affiliation":[{"name":"Donders Institute for Brain Cognition and Behaviour, Radboud University Medical Center, 6525 AJ Nijmegen, The Netherlands"},{"name":"Department of Otorhinolaryngology, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0841-8693","authenticated-orcid":false,"given":"Hannie","family":"Kremer","sequence":"additional","affiliation":[{"name":"Department of Human Genetics, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands"},{"name":"Donders Institute for Brain Cognition and Behaviour, Radboud University Medical Center, 6525 AJ Nijmegen, The Netherlands"},{"name":"Department of Otorhinolaryngology, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9038-0067","authenticated-orcid":false,"given":"Susanne","family":"Roosing","sequence":"additional","affiliation":[{"name":"Department of Human Genetics, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands"},{"name":"Donders Institute for Brain Cognition and Behaviour, Radboud University Medical Center, 6525 AJ Nijmegen, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4954-5592","authenticated-orcid":false,"given":"Frans P. M.","family":"Cremers","sequence":"additional","affiliation":[{"name":"Department of Human Genetics, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands"},{"name":"Donders Institute for Brain Cognition and Behaviour, Radboud University Medical Center, 6525 AJ Nijmegen, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,11,1]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"407","DOI":"10.1038\/nrg.2016.46","article-title":"Lessons from non-canonical splicing","volume":"17","author":"Sibley","year":"2016","journal-title":"Nat. Rev. Genet."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"16783","DOI":"10.1038\/s41598-017-16846-9","article-title":"Combined genetic approaches yield a 48% diagnostic rate in a large cohort of French hearing-impaired patients","volume":"7","author":"Baux","year":"2017","journal-title":"Sci. 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