Suzanne de Bruijn

169 Structural variants cause ectopic enhancer-gene contact in retinitis pigmentosa INTRODUCTION Despite recent advances in next generation sequencing, approximately 30-40% individuals with inherited retinal diseases (IRDs) lack a molecular diagnosis. This is probably due to a combination of rare novel disease genes, which require large cohorts for validation, and previously intractable mutation classes such as intronic variants, structural variants (SVs), and variants in regulatory regions. 1,2 The most common form of IRD is Retinitis Pigmentosa (RP), which is genetically heterogeneous, with a prevalence of 1 in 4,000. 3 RP is defined as a retinal degeneration that primarily affects rod photoreceptors, resulting in night blindness and progressive loss of peripheral vision, often progressing into the central retina and affecting cone photoreceptors, leading to severe visual impairment or blindness. 4 Autosomal dominant RP (adRP) accounts for 25-40% of cases, depending on the population studied, and has been associated with mutations in 30 genes, including CA4 on Chr17q23.1 (RP17). 5-7 Following initial publications defining this locus 8,9 a variant in CA4 was implicated as the cause of adRP in families of South African origin, however pathogenicity of the reported variant has been questioned as it has a population frequency of 4% in healthy controls in Northern Sweden. 10-12 Subsequently reported CA4 variants in individuals with RP were identified by targeted Sanger sequencing, and do not fully exclude variants in other genes as a cause of disease ( Table S1 ). We investigated the cause of adRP in unsolved families, including the first pedigree (GC1, referred to as UK1) drawn up at Moorfields Eye Hospital over 35 years ago, and the original Dutch family (W97-079, referred to as NL1), which showed linkage to the RP17- locus, but lacked a mutation in CA4 . 9 Here, we report identification and characterizationof complex SVs onChr17q22, through whole genome sequencing (WGS), as the genomic cause of adRP at the RP17-locus in a large number of families including the families of South African origin. To explore a convergent mechanism of disease, we investigated the effect of RP17-SVs on three- dimensional (3D) chromatin organization that results in the compartmentalization of the genome into topologically associating domains (TADs), and the epigenetic landscape of the region. TADs are chromatin domains within the genome that facilitate enhancer promoter contacts within the nuclear 3D space. 13 Disruption of TAD structures can lead to loss of chromosomal contact between regulatory regions and their target genes, or the formation of novel active domains with ectopic contacts occurring between regulatory regions and a new target gene, resulting in pathogenic alterations in gene expression. 14-17 We demonstrate that altered TAD structure at the RP17-locus