Suzanne de Bruijn

168 Chapter 4 ABSTRACT The cause of autosomal dominant Retinitis Pigmentosa (adRP), which leads to loss of vision andblindness, was investigated in families lacking amolecular diagnosis. A refined locus for adRP on Chr17q22 (RP17) was delineated through genotyping and genome sequencing, leading to the identification of structural variants (SVs) that segregate with disease. Eight different complex SVs were characterized in 22 adRP families with >300 affected individuals. All RP17-SVs had breakpoints within a genomic region spanning YPEL2 to LINC01476. To investigate the mechanism of disease, fibroblasts from affected individuals and controls were reprogrammed into induced pluripotent stem cells (iPSCs) and differentiated into photoreceptor precursor cells (PPCs) or retinal organoids (ROs). Hi-C was performed on ROs, and differential expression of regional genes and a retinal enhancer RNA at this locus was assessed by qPCR. The epigenetic landscape of the region, and Hi-C RO data, showed that YPEL2 sits within its own topologically associating domain (TAD), rich in enhancers with binding sites for retinal transcription factors. The Hi-C map of RP17 ROs revealed creation of a neo-TAD with ectopic contacts between GDPD1 and retinal enhancers, and modelling of all RP17-SVs was consistent with neo-TADs leading to ectopic retinal specific enhancer- GDPD1 accessibility. qPCR confirmed increased expression of GDPD1 , and increased expression of the retinal enhancer that enters the neo-TAD. Altered TAD structure resulting in increased retinal expression of GDPD1 is the likely convergent mechanism of disease, consistent with a dominant gain-of-function. Our study highlights the importance of SVs as a genomic mechanism in unsolved Mendelian diseases.