Whole genome sequencing for USH2A-associated disease reveals several pathogenic deep-intronic variants that are amenable to splice correction

Janine Reurink, Nicole Weisschuh, Alejandro Garanto, Adrian Dockery, L Ingeborgh van den Born, Isabelle Fajardy, Lonneke Haer-Wigman, Susanne Kohl, Bernd Wissinger, G Jane Farrar, Tamar Ben-Yosef, Fatma Kivrak Pfiffner, Wolfgang Berger, Marianna E Weener, Lubica Dudakova, Petra Liskova, Dror Sharon, Manar Salameh, Ashley Offenheim, Elise HeonGiorgia Girotto, Paolo Gasparini, Anna Morgan, Arthur A Bergen, Jacoline B Ten Brink, Caroline C W Klaver, Lisbeth Tranebjærg, Nanna D Rendtorff, Sascha Vermeer, Jeroen J Smits, Ronald J E Pennings, Marco Aben, Jaap Oostrik, Galuh D N Astuti, Jordi Corominas Galbany, Hester Y Kroes, Milan Phan, Wendy A G van Zelst-Stams, Alberta A H J Thiadens, Joke B G M Verheij, Mary J van Schooneveld, Suzanne E de Bruijn, Catherina H Z Li, Carel B Hoyng, Christian Gilissen, Lisenka E L M Vissers, Frans P M Cremers, Hannie Kremer, Erwin van Wijk, Susanne Roosing

Research output: Contribution to journalArticleResearchpeer-review


A significant number of individuals with a rare disorder such as Usher syndrome (USH) and (non-)syndromic autosomal recessive retinitis pigmentosa (arRP) remain genetically unexplained. Therefore, we assessed subjects suspected of USH2A-associated disease and no or mono-allelic USH2A variants using whole genome sequencing (WGS) followed by an improved pipeline for variant interpretation to provide a conclusive diagnosis. One hundred subjects were screened using WGS to identify causative variants in USH2A or other USH/arRP-associated genes. In addition to the existing variant interpretation pipeline, a particular focus was put on assessing splice-affecting properties of variants, both in silico and in vitro. Also structural variants were extensively addressed. For variants resulting in pseudoexon inclusion, we designed and evaluated antisense oligonucleotides (AONs) using minigene splice assays and patient-derived photoreceptor precursor cells. Biallelic variants were identified in 49 of 100 subjects, including novel splice-affecting variants and structural variants, in USH2A or arRP/USH-associated genes. Thirteen variants were shown to affect USH2A pre-mRNA splicing, including four deep-intronic USH2A variants resulting in pseudoexon inclusion, which could be corrected upon AON treatment. We have shown that WGS, combined with a thorough variant interpretation pipeline focused on assessing pre-mRNA splicing defects and structural variants, is a powerful method to provide subjects with a rare genetic condition, a (likely) conclusive genetic diagnosis. This is essential for the development of future personalized treatments and for patients to be eligible for such treatments.

Original languageEnglish
Pages (from-to)100181
JournalHGG advances
Issue number2
Publication statusPublished - 13 Apr 2023


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