Suzanne de Bruijn

195 Structural variants cause ectopic enhancer-gene contact in retinitis pigmentosa associated haplotype on chromosome 17 spanning 8Mb (17q22-17q24.1) was identified ( Figure 1E ). No shared rare (MAF ≤0.0001) coding or splice-site variants were identified within the haplotype ( Table S5 ). A deep intronic shared rare (absent from gnomAD) variant (g.56293716G>A; c.262-112C>T; NM_001321269.1), in the ciliopathy gene MKS1 , was initially considered a candidate. This variant was assessed for its potential to alter splicing using lymphoblast RNA extracted from affected individuals and controls; however, no difference in pre-mRNA splicing was observed (data not shown). This rare variant was used as a flag SNV to detect this haplotype in other families. Twelve UK adRP families were found to carry the same founder haplotype ( Figure 1B and Figure 1C ). We then refined the adRP locus, by genotyping SNPs in the extended pedigrees, to a 4.4 Mb interval on Chr17q22 (chr17:55,139,138-59,536,883) ( Figure 1E ). Identification of structural variants within the RP17-locus We analyzed the genome and exome data for CNVs and SVs using Manta, Control- FREEC, Canvas and ExomeDepth. In all families, SVs within the RP17-locus were identified. Triplication was suspected from read depth of SNVs observed in IGV for UK- SV6. To validate the presence of a triplicated region, qPCR was performed on affected and control genomic DNA for genes and genomic regions implicated in this SV, and proximal and distal genomic regions (as additional controls for copy number). For all families that harbor SVs in the RP17-locus, reanalysis of sequencing data was performed to exclude other potentially pathogenic variants in IRD-associated genes. No pathogenic heterozygous coding or splice site variants were observed in genes that have been associated with IRDs (MAF ≤0.0001). NL2 consists of distantly related affected individuals, who were identified as having a common ancestor following the identification of the NL-SV5. In the middle branch of this pedigree, a plausible candidate variant in ZNF513 was described previously. 29 This variant was absent in WGS data of the other two affected individuals of this family, and therefore does not segregate with disease and is no longer a candidate variant. A combination of mutational mechanisms created the RP17- SVs Different mutational mechanisms have been described for the formation of complex SVs in the genome; including replication-based mechanisms, such as microhomology- mediated break-induced replication. 30,31 Therefore, we analyzed all breakpoint junction sequences to investigate the potential mechanism(s) that created RP17-SVs. Analysis of breakpoint sequences in the reference genome using the algorithm RepeatMasker identified an enrichment for long repetitive elements (e.g. Alu -elements) in all SVs