The ABCA4 gene is the most frequently mutated Mendelian retinopathy-associated gene. Biallelic variants lead to a variety of phenotypes, however, for thousands of cases the underlying variants remain unknown. Here, we aim to shed further light on the missing heritability of ABCA4-associated retinopathy by analyzing a large cohort of macular dystrophy probands. A total of 858 probands were collected from 26 centers, of whom 722 carried no or one pathogenic ABCA4 variant, while 136 cases carried two ABCA4 alleles, one of which was a frequent mild variant, suggesting that deep-intronic variants (DIVs) or other cis-modifiers might have been missed. After single molecule molecular inversion probes (smMIPs)-based sequencing of the complete 128-kb ABCA4 locus, the effect of putative splice variants was assessed in vitro by midigene splice assays in HEK293T cells. The breakpoints of copy number variants (CNVs) were determined by junction PCR and Sanger sequencing. ABCA4 sequence analysis solved 207 of 520 (39.8%) naive or unsolved cases and 70 of 202 (34.7%) monoallelic cases, while additional causal variants were identified in 54 of 136 (39.7%) probands carrying two variants. Seven novel DIVs and six novel non-canonical splice site variants were detected in a total of 35 alleles and characterized, including the c.6283-321C>G variant leading to a complex splicing defect. Additionally, four novel CNVs were identified and characterized in five alleles. These results confirm that smMIPs-based sequencing of the complete ABCA4 gene provides a cost-effective method to genetically solve retinopathy cases and that several rare structural and splice altering defects remain undiscovered in Stargardt disease cases.

Blueprint Genetics Espoo Finland

Department of Human Genetics Radboud University Medical Center Nijmegen the Netherlands

Department of Human Genetics Radboud University Medical Center Nijmegen the Netherlands; Max Planck Institute for Psycholinguistics Nijmegen the Netherlands

Department of Human Genetics Radboud University Medical Center Nijmegen the Netherlands; University Lille Inserm CHU Lille U1172 LilNCog Lille Neuroscience and Cognition 59000 Lille France

Department of Ophthalmology Columbia University New York NY USA

Department of Ophthalmology Columbia University New York NY USA; Department of Pathology and Cell Biology Columbia University New York NY USA

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

Department of Ophthalmology Radboud University Medical Center Nijmegen the Netherlands

Department of Ophthalmology Radboud University Medical Center Nijmegen the Netherlands; Department of Epidemiology Erasmus Medical Center Rotterdam the Netherlands; Department of Ophthalmology Erasmus Medical Center Rotterdam the Netherlands; Institute of Molecular and Clinical Ophthalmology Basel Switzerland

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

Institute of Human Genetics University of Regensburg Regensburg Germany

Institute of Human Genetics University of Regensburg Regensburg Germany; Institute of Clinical Human Genetics University Hospital Regensburg Regensburg 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; Department of Ophthalmology 1st Faculty of Medicine Charles University and General University Hospital Prague Prague Czech Republic

The School of Genetics and Microbiology Trinity College Dublin Dublin Ireland

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Towards Uncovering the Role of Incomplete Penetrance in Maculopathies through Sequencing of 105 Disease-Associated Genes