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.

Center for Human Genetics University Hospitals Leuven Leuven Belgium

Center for Integrative Human Physiology University of Zurich Zurich Switzerland

Centre de Biologie Pathologie Génétique CHU de Lille Lille France

CRO Oftalmic Moscow Russia

Department of Clinical Genetics Amsterdam UMC University of Amsterdam 1105 Amsterdam the Netherlands

Department of Clinical Genetics The Kennedy Center Copenhagen University Hospital 2600 Glostrup Denmark

Department of Epidemiology Erasmus Medical Center Rotterdam the Netherlands

Department of Human Genetics Radboud University Medical Center Nijmegen the Netherlands

Department of Medical Genetics University Medical Center Groningen University of Groningen Groningen the Netherlands

Department of Medicine Surgery and Health Sciences University of Trieste 34149 Trieste Italy

Department of Ophthalmology 1st Faculty of Medicine Charles University and General University Hospital Prague Prague Czech Republic

Department of Ophthalmology Amsterdam UMC University of Amsterdam 1105 Amsterdam the Netherlands

Department of Ophthalmology Erasmus Medical Center Rotterdam the Netherlands

Department of Ophthalmology Radboud University Medical Center Nijmegen the Netherlands

Department of Ophthalmology Rijnstate Hospital Arnhem the Netherlands

Department of Paediatrics and Inherited Metabolic Disorders 1st Faculty of Medicine Charles University and General University Hospital Prague Prague Czech Republic

Department of Pediatrics Amalia's Children Hospital Radboud University Medical Center Nijmegen The Netherlands

Departments of Ophthalmology and Vision Sciences The Hospital for Sick Children The University of Toronto Toronto ON Canada

Division Laboratories Pharmacy and Biomedical Genetics Department of Genetics University Medical Center of Utrecht Utrecht the Netherlands

Division of Human Genetics Center for Biomedical Research Faculty of Medicine Diponegoro University Semarang Indonesia

Division of Ophthalmology Hadassah University Medical Center Faculty of Medicine The Hebrew University of Jerusalem Jerusalem Israel

Donders Institute for Brain Cognition and Behaviour Radboud University Medical Center Nijmegen the Netherlands

Hearing and Genes Department of Otorhinolaryngology Radboud University Medical Center Nijmegen the Netherlands

Institute for Maternal and Child Health 1 R C C S Burlo Garofolo 34137 Trieste Italy

Institute of Clinical Medicine University of Copenhagen 2200 Copenhagen Denmark

Institute of Medical Molecular Genetics University of Zurich Schlieren Switzerland

Molecular Genetics Laboratory Institute for Ophthalmic Research Centre for Ophthalmology University of Tübingen Tübingen Germany

Neuroscience Center Zurich University and ETH Zurich Zurich Switzerland

Radboud Institute of Molecular Life Sciences Radboud University Medical Center Nijmegen the Netherlands

The Rappaport Faculty of Medicine Technion Israel Institute of Technology Haifa Israel

The Rotterdam Eye Hospital Rotterdam the Netherlands

The School of Genetics and Microbiology Smurfit Institute of Genetics Trinity College Dublin Dublin 2 Ireland

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