Suzanne de Bruijn

55 The impact of modern technologies on molecular diagnostics of a gene, or can cause changes in gene expression levels or patterns when overlapping with regulatory elements, such as enhancers. Additionally, SVs that are limited to the non-coding regions of the DNA can interfere with the 3D genome structure and disrupt cis regulatory architecture. 125 Each chromosome is compartmentalized in regulatory units, so called topologically associating domains (TADs). Within each TAD, enhancers and gene promoters can interact. Neighboring TADs are shielded from each other by boundaries, which are typically occupied by the transcription factor CTCF. 157 Disruption ofTAD-architectureby SVs canhave severepathogenic consequences. Deletions can lead to the fusion of neighboring TADs, inversions can result in the exchange of regulatory sequences, and duplications can generate novel TAD compartments leading to ectopic enhancer-promoter contacts (neo-TADs). 147,158-160 These genomic rearrangements can result inpathogenic alterations of gene expression levels. Recently, itwas shown thatTAD rearrangements caused by SVs are an important cause of autosomal dominant retinitis pigmentosa (RP17). 155 Additional studies focused on the identification of copy number variants involved in HL or RD have also suggested a prominent role for pathogenic SVs. 161,162 To predict the potential consequences of structural rearrangements, the epigenetic landscape of the region, including the presence of CTCF-sites, -interactions and -directionality, should be evaluated. Segregation analysis Once a candidate disease-causing variant is identified, segregation analysis should be performed, if possible, to confirm that the observed inheritance of the variant matches the family history. If a variant is segregating with the phenotype within the family, this could serve as supportive evidence for linkage of the identified variant to the disorder. However, the variant might still be in linkage equilibrium with the true pathogenic variant and the genetic locus should always be carefully screened for missed variants. Additionally, a careful clinical evaluation of all family members is essential to exclude mild symptoms of reportedly unaffected individuals as well as possible phenocopies whosephenotype canbe explainedby other (non-genetic) factors. The latter is especially relevant for cases diagnosed with inherited HL, as both genetic and environmental factors are significant contributors to the development of HL. 163