Multi-omics profiling, in vitro and in vivo enhancer assays dissect the cis-regulatory mechanisms underlying North Carolina macular dystrophy, a retinal enhanceropathy

North Carolina macular dystrophy (NCMD) is a rare autosomal dominant disease affecting macular development. With the identification of non-coding single nucleotide variants (SNVs) near PRDM13 and duplications overlapping a DNase I hypersensitive site (DHS) near PRDM13 or IRX1 as its underlying genetic cause, we hypothesize that NCMD is a retinal enhanceropathy. Here we aim to provide insight into the cis -regulatory mechanisms of NCMD by integrating multi-omics profiling of human retina with in vitro and in vivo enhancer assays. First, we established RegRet (), a genome-wide multi-omics retinal database. Next, UMI-4C profiling was performed on adult human retina to fine-map chromatin interactions of cis -regulatory elements (CREs) with the PRDM13 and IRX1 promoters. Multi-omics analysis including the UMI-4C data revealed sixteen candidate CREs (cCREs), seven for the PRDM13 and nine for the IRX1 region. Subsequently, the activity of cCREs was investigated by in vitro luciferase assays and by in vivo enhancer assays in Xenopus laevis and tropicalis . Four cCREs showed in vivo eye- and brain-specific activity in Xenopus . Furthermore, we expanded the genetic architecture of NCMD with two novel non-coding SNVs (V15, V16) with a likely effect on PRDM13 regulation. Luciferase assays showed that the non-coding SNVs that are located in the two hotspot regions of PRDM13 have an effect on cCRE activity. Interestingly, cCRE4 in which V16 is located was shown to interact with the PRDM13 promoter and demonstrated in vivo activity in Xenopus . This cCRE is active at a specific developmental stage (d103) compatible with the timepoint when retinal progenitor cells of the central retina exit mitosis. Mining of single-cell (sc) transcriptional data of embryonic and adult retina revealed the highest expression of PRDM13 and IRX1 when amacrine cells start to emerge and begin to synapse with retinal ganglion cells. This supports the hypothesis that altered PRDM13 or IRX1 expression impairs synaptic interactions between amacrine and ganglion cells during retinogenesis. Overall, this study gained insight into the cis -regulatory mechanisms of NCMD and supports that NCMD is a retinal enhanceropathy. ### Competing Interest Statement The authors have declared no competing interest.