Suzanne de Bruijn

252 Chapter 5 We anticipated that a pathogenic variant co-segregates with the CEVA haplotype. Therefore, we subjected the shared genomic region to extensive genomic analyses that included WES, short- and long-read WGS, and optical genome mapping, to reveal any potential variants missed or misinterpreted in earlier studies. None of the applied sequencing or imaging techniques revealed rare SVs that overlap or are present within the CEVA haplotype. In the light of the proven accuracy and efficacy of especially optical genome mapping and long-read sequencing in SV detection 72 , we deem it unlikely that any SVs within the CEVA region escaped detection. Additionally, we evaluated all SNVs with an AF ≤5% (gnomAD) present within the region for predicted regulatory or splice altering effects but for none of the 20 SNVs a potential effect was predicted by SpliceAI. Two SNVs overlap with a potential regulatory element of SLC26A4 (GeneHancer, EnhancerAtlas), and one variant is present within the intronic regions of this gene. However, all three variants are located within a highly repetitive element (LINE). Although little is known about the effects of genetic variation within LINE elements, a potential effect on the methylation landscape and consequently gene expression levels has been suggested 73 and such an effect can therefore not be excluded for the three indicated variants. For the remaining SNVs, no potential effects on transcript splicing or gene regulation were predicted. Nevertheless, we cannot rule out combinatory effects of the SNVs, since they are all located in cis . A thorough experimental (multi-omic) analysis is required to optimally assess the effects of the identified variants. RNA studies can be performed to detect quantitative or qualitative changes affecting the SLC26A4 transcripts. A defect observed on the RNA level could provide valuable insights that may point towards the true pathogenic defect, and prioritize one, or a combination, of the variants on the CEVA allele. However, SLC26A4 is not or at extremely low levels expressed in readily accessible patient cell types (e.g., fibroblasts and blood cells). The same holds true for induced pluripotent stem cells or otic progenitor cells. 74 However, Hosoya and co-workers have successfully developed a protocol that allows the differentiation of otic progenitor cells into outer sulcus-like cells that express SLC26A4 at high levels. This protocol could potentially be a powerful tool to evaluate the consequences of CEVA haplotype at the RNA level. SLC26A4 is not the only gene present within the CEVA haplotype, which also spans BCAP29 , COG5 , DUS4L , HBP1 , PIK3CG, and PRKAR2B . For none of these genes, pathogenic variants associated with (syndromic) HL have been reported, nor has a function in the inner ear been described. The majority of the CEVA-associated SNVs (16/20) are located within an intronic region of these genes, however, for none of these variants a splice altering effect is predicted by SpliceAI.