Suzanne de Bruijn

170 Chapter 4 leads to ectopic retinal enhancer-gene interactions, consistent with a dominant gain- of-function. Our study highlights the pathogenicity of SVs that alter 3D chromatin organization and gene expression by rearranging TAD structures, and the need to revisit rare Mendelian diseases where genes and variants have not been substantiated in other cohorts. MATERIALS AND METHODS Study cohort The study was approved by the medical ethics committee of the ErasmusMC Rotterdam, Radboudumc Nijmegen and Moorfields Eye Hospital, and was performed in accordance with the principles of the World Medical Association Declaration of Helsinki. Informed consent was obtained from all participants or their legal representatives. Genetic analyses SNP genotyping was performed for index families NL1 and UK1 to define and refine the RP17-locus. Genomic DNA from affected individuals and their family members was analyzed by whole exome and genome sequencing. Sequence data was aligned to the Human Reference Genome build hg19. Variants were prioritized based on a minor allele frequency (MAF) ≤0.0001 in gnomAD. SVs were called using ExomeDepth, Manta Structural Variant Caller, Canvas Copy Number Variant Caller and Control-FREEC. Details of genotyping, sequencing and analysis pipelines are provided in Supplementary Materials and Methods . Characterization and validation of structural variants SV breakpoint junctions were PCR amplified and validated with Sanger sequencing. Primer sequences and coordinates are listed in Table S2 . SV breakpoint regions were assessed for the presence of microhomology and repetitive elements. To validate a triplicated region for UK-SV6, quantitative real-time PCR (qPCR) was performed on genomic DNA from affected individuals from family UK13 and unaffected controls ( Supplementary Materials and Methods ). Clinical analysis Available clinical notes of cases for the pedigrees identified at Radboudumc, Moorfields Eye Hospital, University of Cape Town and McGill University Health Centre were reviewed, as well as detailed retinal imaging, fundus autofluorescence and optical coherence tomography. Age of onset is defined as the age at which symptoms were first experienced.