PURPOSE: Missing heritability in human diseases represents a major challenge, and this is particularly true for ABCA4-associated Stargardt disease (STGD1). We aimed to elucidate the genomic and transcriptomic variation in 1054 unsolved STGD and STGD-like probands. METHODS: Sequencing of the complete 128-kb ABCA4 gene was performed using single-molecule molecular inversion probes (smMIPs), based on a semiautomated and cost-effective method. Structural variants (SVs) were identified using relative read coverage analyses and putative splice defects were studied using in vitro assays. RESULTS: In 448 biallelic probands 14 known and 13 novel deep-intronic variants were found, resulting in pseudoexon (PE) insertions or exon elongations in 105 alleles. Intriguingly, intron 13 variants c.1938-621G>A and c.1938-514G>A resulted in dual PE insertions consisting of the same upstream, but different downstream PEs. The intron 44 variant c.6148-84A>T resulted in two PE insertions and flanking exon deletions. Eleven distinct large deletions were found, two of which contained small inverted segments. Uniparental isodisomy of chromosome 1 was identified in one proband. CONCLUSION: Deep sequencing of ABCA4 and midigene-based splice assays allowed the identification of SVs and causal deep-intronic variants in 25% of biallelic STGD1 cases, which represents a model study that can be applied to other inherited diseases.

Augenarztpraxis Dorotheenstraße Berlin Germany

Australian Inherited Retinal Disease Registry and DNA Bank Department of Medical Technology and Physics Sir Charles Gairdner Hospital Nedlands WA Australia

Bartiméus Diagnostic Center for Complex Visual Disorders Zeist The Netherlands

Center for Medical Genetics Ghent Ghent University and Ghent University Hospital Ghent Belgium

Centre de Compétence Maladie Rare Clinique Jules Verne Nantes France

Centre for Ophthalmology and Visual Science The University of Western Australia Nedlands WA Australia

CHU Lille Institut de Génétique Médicale Lille France

Department of Epidemiology Erasmus Medical Centre Rotterdam The Netherlands

Department of Genetics IIS Fundación Jiménez Díaz CIBERER Madrid Spain

Department of Histology and Embryology Medical University of Warsaw Warsaw Poland

Department of Human Genetics David Geffen School of Medicine University of California Los Angeles Los Angeles CA USA

Department of Human Genetics Radboud University Medical Center Nijmegen The Netherlands

Department of Molecular Genetics Institute of Pathology University of Ljubljana Ljubljana Slovenia

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

Department of Ophthalmology Amsterdam University Medical Centers Amsterdam The Netherlands

Department of Ophthalmology and Visual Sciences Universidade Federal de São Paulo São Paulo SP Brazil

Department of Ophthalmology David Geffen School of Medicine Stein Eye Institute University of California Los Angeles Los Angeles CA USA

Department of Ophthalmology Erasmus Medical Centre Rotterdam The Netherlands

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

Department of Ophthalmology Leiden University Medical Center Leiden The Netherlands

Department of Ophthalmology Medical University of Warsaw SPKSO Ophthalmic University Hospital Warsaw Poland

Department of Ophthalmology New Zealand National Eye Centre Faculty of Medical and Health Sciences The University of Auckland Grafton Auckland New Zealand

Department of Ophthalmology Radboud University Medical Center Nijmegen The Netherlands

Department of Ophthalmology St Franziskus Hospital Münster Germany

Department of Ophthalmology Tel Aviv Sourasky Medical Center Tel Aviv Israel

Department of Ophthalmology The Jikei University School of Medicine Tokyo Japan

Department of Ophthalmology University Hospital University Regensburg Regensburg Germany

Department of Precision Medicine University of Campania Luigi Vanvitelli Naples and Telethon Institute of Genetics and Medicine Pozzuoli Italy

Departments of Ophthalmology and Medical Genetics University of Alberta Edmonton AB Canada

DNA Analysis Unit ŁUKASIEWICZ Research Network PORT Polish Center for Technology Development Wroclaw Poland

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

Eye Clinic Multidisciplinary Department of Medical Surgical and Dental Sciences University of Campania Luigi Vanvitelli Naples Italy

Eye Department Greenlane Clinical Centre Auckland District Health Board Auckland New Zealand

Eye Hospital University Medical Centre Ljubljana Ljubljana Slovenia

Graduate School of Health Management Keio University Tokyo Japan

Institut des Neurosciences de Montpellier INSERM Université de Montpellier Montpellier France

Institute of Human Genetics University of Regensburg Regensburg Germany

Instituto de Genética Ocular São Paulo SP Brazil

Laboratory of Molecular and Cellular Therapy Fundacion Instituto Leloir CONICET Buenos Aires Argentina

Laboratory of Visual Physiology Division of Vision Research National Institute of Sensory Organs National Hospital Organization Tokyo Medical Center Tokyo Japan

Moorfields Eye Hospital London UK

Ophthalmic Genetics Unit OMMA Ophthalmological Institute of Athens Athens Greece

Radboud Institute for Molecular Life Sciences Radboud University Medical Center Nijmegen The Netherlands

Rare Retinal Disease Center AugenZentrum Siegburg MVZ ADTC Siegburg GmbH Siegburg Germany

Research Unit for Rare Diseases Department of Paediatrics and Adolescent Medicine 1st Faculty of Medicine Charles University and General University Hospital Prague Prague Czech Republic

RetinaScience Bonn Germany

Ruth and Bruce Rappaport Faculty of Medicine Technion Israel Institute of Technology Haifa Israel

Sackler Faculty of Medicine Tel Aviv University Tel Aviv Israel

Service d'exploration de la vision et neuro ophtalmologie Centre Hospitalier Universitaire de Lille Lille France

St John of Jerusalem Eye Hospital Group East Jerusalem Palestine

The Rotterdam Eye Hospital Rotterdam The Netherlands

The Rotterdam Ophthalmic Institute Rotterdam The Netherlands

The School of Genetics and Microbiology Trinity College Dublin Dublin Ireland

UCL Institute of Ophthalmology London UK

Univ Lille Inserm CHU Lille U1172 LilNCog Lille Neuroscience and Cognition F 59000 Lille France

University Eye Hospital Hannover Medical School Hannover Germany

University of Cape Town MRC Genomic and Precision Medicine Research Unit Division of Human Genetics Department of Pathology Institute of Infectious Disease and Molecular Medicine Faculty of Health Sciences University of Cape Town Cape Town South Africa

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