{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,13]],"date-time":"2026-10-13T14:29:40Z","timestamp":1791901780097,"version":"4.3.4"},"reference-count":23,"publisher":"MDPI AG","issue":"15","license":[{"start":{"date-parts":[[2024,7,26]],"date-time":"2024-07-26T00:00:00Z","timestamp":1721952000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000615","name":"Fight For Sight UK Early Career Investigator Award","doi-asserted-by":"publisher","award":["5045\/46"],"award-info":[{"award-number":["5045\/46"]}],"id":[{"id":"10.13039\/501100000615","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000615","name":"Fight For Sight UK Early Career Investigator Award","doi-asserted-by":"publisher","award":["206619\/Z\/17\/Z"],"award-info":[{"award-number":["206619\/Z\/17\/Z"]}],"id":[{"id":"10.13039\/501100000615","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000615","name":"Fight For Sight UK Early Career Investigator Award","doi-asserted-by":"publisher","award":["P20GM139769"],"award-info":[{"award-number":["P20GM139769"]}],"id":[{"id":"10.13039\/501100000615","id-type":"DOI","asserted-by":"publisher"}]},{"name":"National Institute of Health Research Biomedical Research Centre (NIHR-BRC) at Moorfields Eye Hospital","award":["5045\/46"],"award-info":[{"award-number":["5045\/46"]}]},{"name":"National Institute of Health Research Biomedical Research Centre (NIHR-BRC) at Moorfields Eye Hospital","award":["206619\/Z\/17\/Z"],"award-info":[{"award-number":["206619\/Z\/17\/Z"]}]},{"name":"National Institute of Health Research Biomedical Research Centre (NIHR-BRC) at Moorfields Eye Hospital","award":["P20GM139769"],"award-info":[{"award-number":["P20GM139769"]}]},{"name":"UCL Institute of Ophthalmology, NIHR-BRC at the Great Ormond Street Hospital Institute for Child Health and Moorfields Eye Charity","award":["5045\/46"],"award-info":[{"award-number":["5045\/46"]}]},{"name":"UCL Institute of Ophthalmology, NIHR-BRC at the Great Ormond Street Hospital Institute for Child Health and Moorfields Eye Charity","award":["206619\/Z\/17\/Z"],"award-info":[{"award-number":["206619\/Z\/17\/Z"]}]},{"name":"UCL Institute of Ophthalmology, NIHR-BRC at the Great Ormond Street Hospital Institute for Child Health and Moorfields Eye Charity","award":["P20GM139769"],"award-info":[{"award-number":["P20GM139769"]}]},{"DOI":"10.13039\/100004440","name":"Wellcome Trust","doi-asserted-by":"publisher","award":["5045\/46"],"award-info":[{"award-number":["5045\/46"]}],"id":[{"id":"10.13039\/100004440","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100004440","name":"Wellcome Trust","doi-asserted-by":"publisher","award":["206619\/Z\/17\/Z"],"award-info":[{"award-number":["206619\/Z\/17\/Z"]}],"id":[{"id":"10.13039\/100004440","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100004440","name":"Wellcome Trust","doi-asserted-by":"publisher","award":["P20GM139769"],"award-info":[{"award-number":["P20GM139769"]}],"id":[{"id":"10.13039\/100004440","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000057","name":"NIH","doi-asserted-by":"publisher","award":["5045\/46"],"award-info":[{"award-number":["5045\/46"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000057","name":"NIH","doi-asserted-by":"publisher","award":["206619\/Z\/17\/Z"],"award-info":[{"award-number":["206619\/Z\/17\/Z"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000057","name":"NIH","doi-asserted-by":"publisher","award":["P20GM139769"],"award-info":[{"award-number":["P20GM139769"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Biallelic variants in USH2A are associated with retinitis pigmentosa (RP) and Type 2 Usher Syndrome (USH2), leading to impaired vision and, additionally, hearing loss in the latter. Although the introduction of next-generation sequencing into clinical diagnostics has led to a significant uplift in molecular diagnostic rates, many patients remain molecularly unsolved. It is thought that non-coding variants or variants of uncertain significance contribute significantly to this diagnostic gap. This study aims to demonstrate the clinical utility of the reverse transcription\u2013polymerase chain reaction (RT-PCR)\u2013Oxford Nanopore Technology (ONT) sequencing of USH2A mRNA transcripts from nasal epithelial cells to determine the splice-altering effect of candidate variants. Five affected individuals with USH2 or non-syndromic RP who had undergone whole genome sequencing were recruited for further investigation. All individuals had uncertain genotypes in USH2A, including deep intronic rare variants, c.8682-654C&gt;G, c.9055+389G&gt;A, and c.9959-2971C&gt;T; a synonymous variant of uncertain significance, c.2139C&gt;T; p.(Gly713=); and a predicted loss of function duplication spanning an intron\/exon boundary, c.3812-3_3837dup p.(Met1280Ter). In silico assessment using SpliceAI provided splice-altering predictions for all candidate variants which were investigated using ONT sequencing. All predictions were found to be accurate; however, in the case of c.3812-3_3837dup, the outcome was a complex cryptic splicing pattern with predominant in-frame exon 18 skipping and a low level of exon 18 inclusion leading to the predicted stop gain. This study detected and functionally characterised simple and complex mis-splicing patterns in USH2A arising from previously unknown deep intronic variants and previously reported variants of uncertain significance, confirming the pathogenicity of the variants.<\/jats:p>","DOI":"10.3390\/cells13151261","type":"journal-article","created":{"date-parts":[[2024,7,26]],"date-time":"2024-07-26T09:22:42Z","timestamp":1721985762000},"page":"1261","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Investigating Splice Defects in USH2A Using Targeted Long-Read Sequencing"],"prefix":"10.3390","volume":"13","author":[{"given":"Shwetha","family":"Chandrasekhar","sequence":"first","affiliation":[{"name":"UCL Institute of Ophthalmology, University College London, London EC1V 9EL, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1122-8396","authenticated-orcid":false,"given":"Siying","family":"Lin","sequence":"additional","affiliation":[{"name":"UCL Institute of Ophthalmology, University College London, London EC1V 9EL, UK"},{"name":"National Institute of Health Research Biomedical Research Centre at Moorfields Eye Hospital and the UCL Institute of Ophthalmology, London EC1V 9EL, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Neringa","family":"Jurkute","sequence":"additional","affiliation":[{"name":"UCL Institute of Ophthalmology, University College London, London EC1V 9EL, UK"},{"name":"National Institute of Health Research Biomedical Research Centre at Moorfields Eye Hospital and the UCL Institute of Ophthalmology, London EC1V 9EL, UK"},{"name":"Department of Neuro-Ophthalmology, The National Hospital for Neurology and Neurosurgery, London WC1N 3BG, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kathryn","family":"Oprych","sequence":"additional","affiliation":[{"name":"UCL Institute of Ophthalmology, University College London, London EC1V 9EL, UK"},{"name":"Clinical Genetics, St George\u2019s University Hospitals NHS Foundation Trust, London SW17 0QT, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Leire","family":"Estramiana Elorrieta","sequence":"additional","affiliation":[{"name":"UCL Institute of Ophthalmology, University College London, London EC1V 9EL, UK"},{"name":"Section for Paediatrics, Department of Infectious Diseases, Faculty of Medicine, Imperial College London, London W2 1NY, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9848-1302","authenticated-orcid":false,"given":"Elena","family":"Schiff","sequence":"additional","affiliation":[{"name":"UCL Institute of Ophthalmology, University College London, London EC1V 9EL, UK"},{"name":"National Institute of Health Research Biomedical Research Centre at Moorfields Eye Hospital and the UCL Institute of Ophthalmology, London EC1V 9EL, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0020-4049","authenticated-orcid":false,"given":"Samantha","family":"Malka","sequence":"additional","affiliation":[{"name":"UCL Institute of Ophthalmology, University College London, London EC1V 9EL, UK"},{"name":"National Institute of Health Research Biomedical Research Centre at Moorfields Eye Hospital and the UCL Institute of Ophthalmology, London EC1V 9EL, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Genevieve","family":"Wright","sequence":"additional","affiliation":[{"name":"UCL Institute of Ophthalmology, University College London, London EC1V 9EL, UK"},{"name":"National Institute of Health Research Biomedical Research Centre at Moorfields Eye Hospital and the UCL Institute of Ophthalmology, London EC1V 9EL, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michel","family":"Michaelides","sequence":"additional","affiliation":[{"name":"UCL Institute of Ophthalmology, University College London, London EC1V 9EL, UK"},{"name":"National Institute of Health Research Biomedical Research Centre at Moorfields Eye Hospital and the UCL Institute of Ophthalmology, London EC1V 9EL, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1254-0832","authenticated-orcid":false,"given":"Omar A.","family":"Mahroo","sequence":"additional","affiliation":[{"name":"UCL Institute of Ophthalmology, University College London, London EC1V 9EL, UK"},{"name":"National Institute of Health Research Biomedical Research Centre at Moorfields Eye Hospital and the UCL Institute of Ophthalmology, London EC1V 9EL, UK"},{"name":"Department of Ophthalmology, St Thomas\u2019 Hospital, London SE1 7EH, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrew R.","family":"Webster","sequence":"additional","affiliation":[{"name":"UCL Institute of Ophthalmology, University College London, London EC1V 9EL, UK"},{"name":"National Institute of Health Research Biomedical Research Centre at Moorfields Eye Hospital and the UCL Institute of Ophthalmology, London EC1V 9EL, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gavin","family":"Arno","sequence":"additional","affiliation":[{"name":"UCL Institute of Ophthalmology, University College London, London EC1V 9EL, UK"},{"name":"National Institute of Health Research Biomedical Research Centre at Moorfields Eye Hospital and the UCL Institute of Ophthalmology, London EC1V 9EL, UK"},{"name":"Greenwood Genetic Center, Greenwood, SC 29646, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,7,26]]},"reference":[{"key":"ref_1","first-page":"250","article-title":"Vereizelte Beobachtungen und Bemerkungen. Exceptionnelles verhalter des Gesichts feldes bei Pigmentenarter der Netzhalt","volume":"4","year":"1858","journal-title":"Arch. Klin. Ophtalmol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1975","DOI":"10.1086\/302926","article-title":"Missense mutation in the USH2A gene: Association with recessive retinitis pigmentosa without hearing loss","volume":"66","author":"Rivolta","year":"2000","journal-title":"Am. J. Hum. Genet."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"613","DOI":"10.1002\/humu.24365","article-title":"Tissue-specific genotype-phenotype correlations among USH2A-related disorders in the RUSH2A study","volume":"43","author":"Hufnagel","year":"2022","journal-title":"Hum. Mutat."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1318","DOI":"10.1038\/ejhg.2014.283","article-title":"A detailed clinical and molecular survey of subjects with nonsyndromic USH2A retinopathy reveals an allelic hierarchy of disease-causing variants","volume":"23","author":"Lenassi","year":"2015","journal-title":"Eur. J. Hum. Genet."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"699","DOI":"10.1016\/j.oret.2024.01.012","article-title":"Spectrum of Genetic Variants in the Most Common Genes Causing Inherited Retinal Disease in a Large Molecularly Characterized United Kingdom Cohort","volume":"12","author":"Lin","year":"2024","journal-title":"Ophthalmol. Retina"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1384","DOI":"10.1016\/j.ophtha.2020.04.008","article-title":"Genetic Basis of Inherited Retinal Disease in a Molecularly Characterized Cohort of More Than 3000 Families from the United Kingdom","volume":"127","author":"Pontikos","year":"2020","journal-title":"Ophthalmology"},{"key":"ref_7","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_8","unstructured":"Ellard, S., Baple, E.L., Callaway, A., Berry, I., Forrester, N., Turnbull, C., Owens, M., Eccles, D.M., Abbs, S., and Scott, R. (2020). ACGS Best Practice Guidelines for Variant Classification in Rare Disease 2020, ACGS."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"595","DOI":"10.1093\/hmg\/ddac227","article-title":"Multidisciplinary team directed analysis of whole genome sequencing reveals pathogenic non-coding variants in molecularly undiagnosed inherited retinal dystrophies","volume":"32","author":"Varela","year":"2022","journal-title":"Hum. Mol. Genet."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"100000 Genomes Project Pilot Investigators, Smedley, D., Smith, K.R., Martin, A., Thomas, E.A., McDonagh, E.M., Cipriani, V., Ellingford, J.M., Arno, G., and Tucci, A. (2021). 100,000 Genomes Pilot on Rare-Disease Diagnosis in Health Care\u2014Preliminary Report. N. Engl. J. Med., 385, 1868\u20131880.","DOI":"10.1056\/NEJMoa2035790"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"108330","DOI":"10.1016\/j.exer.2020.108330","article-title":"USH2A-retinopathy: From genetics to therapeutics","volume":"201","author":"Toualbi","year":"2020","journal-title":"Exp. Eye Res."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"2441","DOI":"10.1016\/j.ymthe.2021.04.024","article-title":"Antisense oligonucleotide-based treatment of retinitis pigmentosa caused by USH2A exon 13 mutations","volume":"29","author":"Dulla","year":"2021","journal-title":"Mol. Ther."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"114","DOI":"10.1111\/cge.12520","article-title":"Patient reported outcomes and patient empowerment in clinical genetics services","volume":"88","author":"McAllister","year":"2015","journal-title":"Clin. Genet."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1016\/j.ajhg.2016.12.003","article-title":"Comprehensive Rare Variant Analysis via Whole-Genome Sequencing to Determine the Molecular Pathology of Inherited Retinal Disease","volume":"100","author":"Carss","year":"2017","journal-title":"Am. J. Human. Genet."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1560","DOI":"10.1038\/s41588-019-0528-2","article-title":"PanelApp crowdsources expert knowledge to establish consensus diagnostic gene panels","volume":"51","author":"Martin","year":"2019","journal-title":"Nat. Genet."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"535","DOI":"10.1016\/j.cell.2018.12.015","article-title":"Predicting Splicing from Primary Sequence with Deep Learning","volume":"176","author":"Jaganathan","year":"2019","journal-title":"Cell"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1016\/0022-2836(91)90380-O","article-title":"Prediction of human mRNA donor and acceptor sites from the DNA sequence","volume":"220","author":"Brunak","year":"1991","journal-title":"J. Mol. Biol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1016\/j.exer.2014.02.018","article-title":"The effect of the Common c.2299delG mutation in USH2A on RNA splicing","volume":"122","author":"Lenassi","year":"2014","journal-title":"Exp. Eye Res."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"576","DOI":"10.1038\/s41431-019-0548-5","article-title":"Diagnostic yield of panel-based genetic testing in syndromic inherited retinal disease","volume":"28","author":"Jiman","year":"2020","journal-title":"Eur. J. Hum. Genet."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Downes, S.M., Nguyen, T., Tai, V., Broadgate, S., Shah, M., Al-Khuzaei, S., MacLaren, R.E., Shanks, M., Clouston, P., and Halford, S. (2020). Genetic and Clinical Findings in an Ethnically Diverse Cohort with Retinitis Pigmentosa Associated with Pathogenic Variants in CERKL. Genes, 11.","DOI":"10.3390\/genes11121497"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Qian, X., Wang, J., Wang, M., Igelman, A.D., Jones, K.D., Li, Y., Wang, K., Goetz, K.E., Birch, D.G., and Yang, P. (2021). Identification of Deep-Intronic Splice Mutations in a Large Cohort of Patients With Inherited Retinal Diseases. Front. Genet., 12.","DOI":"10.3389\/fgene.2021.647400"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"e381","DOI":"10.1038\/mtna.2016.89","article-title":"Antisense Oligonucleotide-based Splice Correction for USH2A-associated Retinal Degeneration Caused by a Frequent Deep-intronic Mutation","volume":"5","author":"Slijkerman","year":"2016","journal-title":"Mol. Ther. Nucleic Acids"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Reurink, J., Oostrik, J., Aben, M., Ramos, M.G., van Berkel, E., O\u0142dak, M., van Wijk, E., Kremer, H., Roosing, S., and Cremers, F.P.M. (2022). Minigene-Based Splice Assays Reveal the Effect of Non-Canonical Splice Site Variants in USH2A. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms232113343"}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/13\/15\/1261\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T15:24:07Z","timestamp":1760109847000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/13\/15\/1261"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,7,26]]},"references-count":23,"journal-issue":{"issue":"15","published-online":{"date-parts":[[2024,8]]}},"alternative-id":["cells13151261"],"URL":"https:\/\/doi.org\/10.3390\/cells13151261","relation":{},"ISSN":["2073-4409"],"issn-type":[{"value":"2073-4409","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,7,26]]}}}