Suzanne de Bruijn

241 Exploring the missing heritability in subjects with hearing loss and EVA Whole genome sequencing reveals potential SLC26A4 splice and regulatory variants in M1 subjects without the CEVA haplotype To detect any potentially missed coding or unidentified intronic SLC26A4 variants or variants located in cis regulatory elements of the gene, WGS analysis was performed for all six M1 individuals who could not be genetically explained by the presence of the CEVA haplotype. Additionally, WGS analysis was performed for the two M0/CEVA individuals. In none of these eight cases, SVs overlapping with the SLC26A4 gene were identified by WGS. To identify any variants with a potential effect on splicing, the deep-learning algorithm SpliceAI was employed. 42 In two M1 individuals (SLC048 and SLC085), a rare heterozygous potentially splice altering SLC26A4 variant was identified ( Table 2 ). For both variants, the predicted splice defect was investigated using an in vitro splice assay performed in HEK293T cells. For SLC048, a canonical splice site variant (c.1342- 2A>C), that was overlooked during prescreening efforts, was predicted to remove the splice acceptor site. This variant was previously reported in a study performed by Van Beeck Calkoen and coworkers and in ClinVar. 55 Indeed, the splice assay revealed loss of the acceptor site and usage of an alternative splice acceptor site located thirteen nucleotides downstream ( Figure S1A ). This leads to the formation of an out-of-frame exon 12 and premature protein truncation (p.(Ser448Leufs*3)). Based on these results, the variant was classified as pathogenic according to the ACMG guidelines. 51 In SLC085, a synonymous variant (c.471C>T, classified as likely benign in ClinVar) was identified in exon 5. SpliceAI predicts that this variant strengthens an alternative splice acceptor site (27 nucleotides downstream of the variant). Indeed, an in vitro splice assay confirmed that the alternative splice acceptor site is used, which leads to the partial deletion of exon 5 and a truncated protein (p.(Gly139Alafs*6)) ( Figure S1B ) . Therefore, this variant is now classified as pathogenic according to the ACMG classification guidelines. 51 The observed splice defect resulting from this synonymous variant underlines the importance of evaluating potential splice effects of all rare variants in coding sequences, using in silico prediction splice tools. We considered the two identified splice variants as pathogenic and the HL of the two individuals as genetically explained, thus M2. Toexplorevariants that arepotentially locatedwithina cis regulatoryelement of SLC26A4 , we extracted all (predicted) human enhancer and promoter elements that are associated with the SLC26A4 gene fromtheGeneHancer 56 andEnhancerAtlas 57 databases ( TableS5 ). GeneHancer V5 is a collection of both predicted and experimentally validated enhancer- to-gene and promoter-to-gene interactions, based on information integrated from multiple resources: ENCODE 58 , Ensembl 59 , FANTOM5 60 , VISTA 61 , dbSuper 62 , EPDnew 63 ,