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Comprehensive screening of the PAX6 locus, including the non-coding regions, by next-generation sequencing revealed four deep-intronic variants with potential effects on pre-RNA splicing. Nevertheless, without a functional analysis, their pathogenicity could not be established. We aimed to decipher their impact on the canonical PAX6 splicing using in vitro minigene splicing assays and nanopore-based long-read sequencing. Two multi-exonic PAX6 constructs were generated, and minigene assays were carried out. An aberrant splicing pattern was observed for two variants in intron 6, c.357+136G&gt;A and c.357+334G&gt;A. In both cases, several exonization events, such as pseudoexon inclusions and partial intronic retention, were observed due to the creation or activation of new\/cryptic non-canonical splicing sites, including a shared intronic donor site. In contrast, two variants identified in intron 11, c.1032+170A&gt;T and c.1033-275A&gt;C, seemed not to affect splicing processes. We confirmed the high complexity of alternative splicing of PAX6 exon 6, which also involves unreported cryptic intronic sites. Our study highlights the importance of integrating functional studies into diagnostic algorithms to decipher the potential implication of non-coding variants, usually classified as variants of unknown significance, thus allowing variant reclassification to achieve a conclusive genetic diagnosis.<\/jats:p>","DOI":"10.3390\/ijms24021562","type":"journal-article","created":{"date-parts":[[2023,1,13]],"date-time":"2023-01-13T02:29:57Z","timestamp":1673576997000},"page":"1562","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["Minigene Splicing Assays and Long-Read Sequencing to Unravel Pathogenic Deep-Intronic Variants in PAX6 in Congenital Aniridia"],"prefix":"10.3390","volume":"24","author":[{"given":"Alejandra","family":"Tamayo","sequence":"first","affiliation":[{"name":"Department of Genetics & Genomics, Instituto de Investigaci\u00f3n Sanitaria-Fundaci\u00f3n Jim\u00e9nez D\u00edaz University Hospital, 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Unit, Instituto de Investigaci\u00f3n Sanitaria-Fundaci\u00f3n Jim\u00e9nez D\u00edaz University Hospital, Universidad Aut\u00f3noma de Madrid (IIS-FJD, UAM), 28240 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carolina","family":"Ruiz","sequence":"additional","affiliation":[{"name":"Department of Genetics & Genomics, Instituto de Investigaci\u00f3n Sanitaria-Fundaci\u00f3n Jim\u00e9nez D\u00edaz University Hospital, Universidad Aut\u00f3noma de Madrid (IIS-FJD, UAM), 28040 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julie","family":"Plaisancie","sequence":"additional","affiliation":[{"name":"Centre de R\u00e9f\u00e9rence des Affections Rares en G\u00e9n\u00e9tique Ophtalmologique (CARGO), H\u00f4pital Purpan, CHU Toulouse, 31000 Toulouse, France"},{"name":"INSERM U1214, Universit\u00e9 Toulouse III, 31000 Toulouse, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1886-8257","authenticated-orcid":false,"given":"Alejandra","family":"Damian","sequence":"additional","affiliation":[{"name":"Department of Genetics & Genomics, Instituto de Investigaci\u00f3n Sanitaria-Fundaci\u00f3n Jim\u00e9nez D\u00edaz University Hospital, Universidad Aut\u00f3noma de Madrid (IIS-FJD, UAM), 28040 Madrid, Spain"},{"name":"Center for Biomedical Network Research on Rare Diseases (CIBERER), Instituto de Salud Carlos III, 28029 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jennifer","family":"Moya","sequence":"additional","affiliation":[{"name":"Department of Genetics & Genomics, Instituto de Investigaci\u00f3n Sanitaria-Fundaci\u00f3n Jim\u00e9nez D\u00edaz University Hospital, Universidad Aut\u00f3noma de Madrid (IIS-FJD, UAM), 28040 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicolas","family":"Chassaing","sequence":"additional","affiliation":[{"name":"Centre de R\u00e9f\u00e9rence des Affections Rares en G\u00e9n\u00e9tique Ophtalmologique (CARGO), H\u00f4pital Purpan, CHU Toulouse, 31000 Toulouse, France"},{"name":"INSERM U1214, Universit\u00e9 Toulouse III, 31000 Toulouse, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Patrick","family":"Calvas","sequence":"additional","affiliation":[{"name":"Centre de R\u00e9f\u00e9rence des Affections Rares en G\u00e9n\u00e9tique Ophtalmologique (CARGO), H\u00f4pital Purpan, CHU Toulouse, 31000 Toulouse, France"},{"name":"INSERM U1214, Universit\u00e9 Toulouse III, 31000 Toulouse, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9242-7065","authenticated-orcid":false,"given":"Carmen","family":"Ayuso","sequence":"additional","affiliation":[{"name":"Department of Genetics & Genomics, Instituto de Investigaci\u00f3n Sanitaria-Fundaci\u00f3n Jim\u00e9nez D\u00edaz University Hospital, Universidad Aut\u00f3noma de Madrid (IIS-FJD, UAM), 28040 Madrid, Spain"},{"name":"Center for Biomedical Network Research on Rare Diseases (CIBERER), Instituto de Salud Carlos III, 28029 Madrid, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4099-9421","authenticated-orcid":false,"given":"Pablo","family":"Minguez","sequence":"additional","affiliation":[{"name":"Department of Genetics & Genomics, Instituto de Investigaci\u00f3n Sanitaria-Fundaci\u00f3n Jim\u00e9nez D\u00edaz University Hospital, Universidad Aut\u00f3noma de Madrid (IIS-FJD, UAM), 28040 Madrid, Spain"},{"name":"Center for Biomedical Network Research on Rare Diseases (CIBERER), Instituto de Salud Carlos III, 28029 Madrid, Spain"},{"name":"Bioinformatics Unit, Instituto de Investigaci\u00f3n Sanitaria-Fundaci\u00f3n Jim\u00e9nez D\u00edaz University Hospital, 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