North Carolina macular dystrophy (NCMD) is a rare autosomal-dominant disease affecting macular development. The disease is caused by non-coding single-nucleotide variants (SNVs) in two hotspot regions near PRDM13 and by duplications in two distinct chromosomal loci, overlapping DNase I hypersensitive sites near either PRDM13 or IRX1. To unravel the mechanisms by which these variants cause disease, we first established a genome-wide multi-omics retinal database, RegRet. Integration of UMI-4C profiles we generated on adult human retina then allowed fine-mapping of the interactions of the PRDM13 and IRX1 promoters and the identification of eighteen candidate cis-regulatory elements (cCREs), the activity of which was investigated by luciferase and Xenopus enhancer assays. Next, luciferase assays showed that the non-coding SNVs located in the two hotspot regions of PRDM13 affect cCRE activity, including two NCMD-associated non-coding SNVs that we identified herein. Interestingly, the cCRE containing one of these SNVs was shown to interact with the PRDM13 promoter, demonstrated in vivo activity in Xenopus, and is active at the developmental stage when progenitor cells of the central retina exit mitosis, suggesting that this region is a PRDM13 enhancer. Finally, mining of single-cell transcriptional data of embryonic and adult retina revealed the highest expression of PRDM13 and IRX1 when amacrine cells start to synapse with retinal ganglion cells, supporting the hypothesis that altered PRDM13 or IRX1 expression impairs interactions between these cells during retinogenesis. Overall, this study provides insight into the cis-regulatory mechanisms of NCMD and supports that this condition is a retinal enhanceropathy.

Center for Medical Genetics Antwerp University Hospital Antwerp Belgium

Center for Medical Genetics Ghent University Hospital Ghent Belgium; Department of Biomedical Molecular Biology Ghent University Ghent Belgium

Center for Medical Genetics Ghent University Hospital Ghent Belgium; Department of Ophthalmology Ghent University Hospital Ghent Belgium; Department of Head and Skin Ghent University Ghent Belgium; Division of Ophthalmology and Center for Cellular and Molecular Therapeutics Children's Hospital of Philadelphia Philadelphia PA USA

Centro Andaluz de Biología del Desarrollo Consejo Superior de Investigaciones Científicas and Universidad Pablo de Olavide Sevilla Spain

Department of Biomolecular Medicine Ghent University Ghent Belgium; Center for Medical Genetics Ghent University Hospital Ghent Belgium

Department of Biomolecular Medicine Ghent University Ghent Belgium; Center for Medical Genetics Ghent University Hospital Ghent Belgium; Department of Biomedical Molecular Biology Ghent University Ghent Belgium

Department of Human Genetics Amsterdam UMC Academic Medical Center 1105 AZ Amsterdam The Netherlands; Queen Emma Centre of Precision Medicine Amsterdam University Medical Centre University of Amsterdam Amsterdam The Netherlands

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

Department of Ophthalmology Amsterdam University Medical Centers University of Amsterdam Amsterdam The Netherlands; Bartiméus Diagnostic Center for Complex Visual Disorders Zeist The Netherlands

Department of Ophthalmology Amsterdam University Medical Centers University of Amsterdam Amsterdam The Netherlands; Department of Ophthalmology Leiden University Medical Center Leiden The Netherlands

Department of Ophthalmology Ghent University Hospital Ghent Belgium

Department of Ophthalmology SUNY Downstate Medical Center University Brooklyn New York USA

Department of Ophthalmology University Hospitals Leuven Leuven Belgium

Division of Molecular Medicine Leeds Institute of Medical Research University of Leeds Leeds UK

Institute of Molecular and Clinical Ophthalmology Basel Basel Switzerland; Department of Ophthalmology University of Basel Basel Switzerland; Department of Genetics and Genome Biology University of Leicester Leicester UK

Macula and Retina Institute Los Angeles and Glendale California USA

University of Lausanne Jules Gonin Eye Hospital Lausanne Switzerland

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De novo and inherited dominant variants in U4 and U6 snRNAs cause retinitis pigmentosa

Disruption of OVOL2 Distal Regulatory Elements as a Possible Mechanism Implicated in Corneal Endothelial Dystrophy