Suzanne de Bruijn

320 Chapter 7 In chapter 3 , the genetic defect underlying HL type DFNA21 was investigated. DFNA21 is a dominantly inherited type of adult-onset HL. Genetic diagnostics of adult-onset HL is severely complicated by the large involvement of environmental factors affecting hearing. These factors mask inheritance patterns in families with inherited adult-onset HL, thereby impeding the identification of potential monogenic defects. DFNA21 clearly illustrates this complexity. The implicated genetic defect of DFNA21 was previously localized to the so-called DFNA21 locus on chromosome 6. In the past, no putatively pathogenic variants could be identified within this locus. In chapter 3.1 , WES was performed to screen all coding regions for putatively pathogenic variants and an in- frame deletion in the RIPOR2 gene, located 0.9 Mb centromeric of the DFNA21 locus, was identified. Analyses revealed that the locus was wrongly delimited in the past, due to the presence of phenocopies and genocopies within the DFNA21-affected family. The newly identified RIPOR2 variant was detected in 12 Dutch families, all diagnosed with dominantly-inherited and predominantly adult-onset HL. Ex vivo protein expression studies in murine cochlear hair cells confirmed a functional effect of the RIPOR2 variant. An aberrant localization of mutant RIPOR2 could be observed in the stereocilia of these hair cells, and unlike wildtype RIPOR2, the mutant protein was unable to rescue the morphological defects in RIPOR2-deficient hair cells. Based on allele frequency datasets, the RIPOR2 in-frame deletion is presumably the most frequent cause of adult-onset HL in Northwest Europe. More than 30,000 individuals worldwide are estimated to be at risk to develop HL due to this variant. For this reason, the opportunity to design a potential genetic therapy was explored in chapter 3.2 . Based on the expected non- haploinsufficiency pathogenic mechanism of the variant, allele-specific RNaseH1- dependent antisense oligonucleotides (AONs) were designed and tested for their potency to suppress mutant RIPOR2 expression. A lead AON molecule was identified that is able to efficiently and specifically reduce mutant transcript levels as observed in patient-derived fibroblasts and HEK293T cells. Based on these results, future steps including the validation of the lead AON in animal model studies can be initiated. Despite the success stories of WES as exemplified by the results described in chapters 2 and chapter 3, an important limitation of WES remains the inability to detect structural variants (SVs). In chapter 4 , whole genome sequencing (WGS) was performed afterWES failed to identify the genetic defect associated with RP type 17 (RP17). In 22 unrelated families with autosomal dominant RP, eight distinct SVs (i.e. tandem duplications, duplication-inversion and triplication events) were identified. All SVs overlap with the RP17 locus on chromosome 17. None of the genes implicated in the SVs were previously associated with RP and therefore the genetic mechanism involved was not readily understood. To unravel this genetic disease mechanism, 3D chromosome mapping was performed in retinal organoids. By comparing the 3D maps of wildtype and RP17-