Suzanne de Bruijn

297 General discussion and perspectives binding (e.g. reported for Stargardt disease 89 ) or alter chromatin modifications such as DNA methylation (e.g. reported for age-related macular degeneration 90 ). Epigenomic profiling should be performed to recognize and detect the cis regulatory elements that are affected by these variants. Chromosome conformation capture techniques, such as 3C or Hi-C allow the investigation of interactions between genomic elements. With a 3C approach, putative interactions between two specific regions of interest can be investigated (one-to-one), whereas in a Hi-C approach genome-wide interactions between all elements can be mapped (all-to-all). The organization of the genome in TADs, enriched for intra-domain interactions, is crucial for maintaining promoter-enhancer interaction specificity. Using standard “C” techniques, (long-distance) interactions between two independent elements can be captured. Latest developments reporting ligation-free methods even allow the investigation of multi-region interactions (>2 elements) at an increased resolution (reviewed in (91)). An important proof-of-concept, illustrating the value of chromosome conformation caption techniques is described in chapter 4 of this thesis. Using a WGS approach, eight unique SVs affecting the RP17 locus were revealed in 22 families diagnosed with dominant RP. None of the genes implicated in the SVs were previously associated with retinal function or retinal disease, and without further investigation, the variants would be deemed variants of unknown significance. Instead, a low-C analysis on wildtype and patient-derived retinal organoids was performed to elucidate the 3D organization of the locus. Low-C, an adapted form of Hi-C, is optimized for low cell input which is necessary to allow its application in retinal organoids. Only after the incorporation of this technique, the pathogenic mechanism behind the RP17-associated SVs could be elucidated. Low-C profiles of retinal organoids carrying an RP17 SV indicated rewiring of enhancer-promoter contacts, leading to ectopic gene expression which was confirmed by RNA analyses. This describes a novel disease mechanism, that includes the ectopic and toxic expression of a gene. This mechanism of disease could also be involved in other unsolved Mendelian diseases. However, since this mechanism cannot be detected using a genomics-only approach, the implementation of multi-omics technologies is an important prerequisite.