Suzanne de Bruijn

183 Structural variants cause ectopic enhancer-gene contact in retinitis pigmentosa supported by the presence of breakpoint “hotspots”, as seen in LINC01476 intron 2 and YPEL2 intron 4, with some breakpoints only differing by a small number of base pairs (e.g. for UK-SV2 and UK-SV7). None of the genes implicated in the RP17-SVs have been previously associated with retinal disease. YPEL2 is expressed in retina, and single cell RNA sequencing of human and primate retina revealed expression in photoreceptors, with highest expression in rod photoreceptors. 24,25 Although the function of YPEL2 in the retina is unknown, we show that retinal expression is controlled by a number of retinal TF binding sites, including NRL which is predominantly expressed in rod photoreceptors. Furthermore, Hi-C data show that YPEL2 and the retinal enhancer binding sites are insulated from the surrounding region in a structured YPEL2 TAD in control ROs and other tissues. Hi-C analyses of UK-SV2 ROs revealed the generation of new chromatin domains (neo-TADs), with altered structure and repositioning of the boundaries enabling GDPD1 promoter-retinal enhancer contacts and consequent GDPD1 misexpression in the retina. The molecular disease mechanism in these cases is similar to the reported duplications at the SOX9/KCNJ2 locus. 15 As described for the rearrangements reported here, the duplications at the SOX9 locus also encompass a regulatory domain (of SOX9 ), a boundary (between the SOX9 and the KCNJ2 TADs) and the neighboring gene ( KCNJ2 ). This results in the formation of a novel chromatin domain (neo-TAD) containing the SOX9 regulatory elements and the new target gene ( KCNJ2 ) that are now free to interact. In the SOX9 case, this leads to misexpression of KCNJ2 in a SOX9 pattern and consecutive limb malformation, whereas in the RP cases the interaction of GDPD1 with YPEL2 enhancers leads to misexpression in the retina. However, in some of the RP cases, such as UK-SV2, the situation is more complex because the duplications are inverted. Inversions can lead to the exchange of regulatory material fromone end of the breakpoint to the other (also calledTAD-shuffling). 14 InUK-SV2 theduplication creates twoneo-TADs but the content is reorganized by the inversion. Again, the GDPD1 gene and retinal enhancers are brought together in one new TAD. Thus, the pathogenetic principle remains the same, as all the RP17 SVs are predicted to create new TADs allowing access of the retinal enhancers to GDPD1. This suggests that increased expression of GDPD1 in photoreceptors is the convergent mechanism of disease. Consistent with this hypothesis, PPCs from NL-SV1 and ROs fromUK-SV2 showed significant increased expression of GDPD1 in RP17 families with different SVs compared to controls. In UK-SV2 ROs, an increased expression of SMG8 was observed, which is also introduced into the active neo-TAD of UK-SV2. Conversely, YPEL2 shows upregulation in NL-SV1, which is in line with the complete duplication of