Suzanne de Bruijn

254 Chapter 5 which cellular localizations assays, are warranted to evaluate the effect of the c.677C>T FOXI1 variant. We did not identify likely pathogenic variants in KCNJ10 (AF ≤5%) in our cohort. WGS did not reveal strong candidate regulatory variants based on data derived from enhancer databases and transcription factor binding site predictions. Nevertheless, interpretation of regulatory variants is still considered complex and is limited by the lack of available epigenetic datasets for the inner ear. Also, no SVs overlapping with SLC26A4 were detected using WGS, suggesting a limited contribution of SVs to the mutational landscape of SLC26A4 . This is in line with earlier observations described in literature. 67,81 For the monoallelic cases (M1, M0/CEVA), no long-read sequencing or optical genomemapping was performed. As it is generally accepted that most SVs could not be accurately detected using short-read sequencing approaches only 72 , it cannot be excluded that causative SVs are present but missed due to technical limitations. The present study did not confirm that the CEVA allele is associated with a milder HL compared to SLC26A4 variants affecting the protein-coding sequences, as indicated by Chao et al. 23 They discerned a significantly milder HL in their cohort of M1/CEVA subjects (n=20 ears, median 47.5 dB HL) than we have seen in our cohort of M1/CEVA subjects (n=16 ears, median 84 dB HL). A possible explanation for this discrepancy could be the progression of HL combined with a ~5-year difference in average age between the cohorts (7.5 and 12.8 years, respectively). Progression of HL is seen in up to 39.6% of EVA-ears 82 , with progression rates of ~3.5 – ~5.5 dB/y. 83,84 On the other hand, the older subjects in our M1/CEVA cohort show less severe HL than the younger subjects, which is questioning the relationship with age. Furthermore, there is also an average age difference of 5 years between the M2 groups in both studies (13.2 years and 18.4 years, respectively), while the severity of HL is comparable (85 and 86.3 dB HL, respectively). The reported variability of the auditory phenotype associated with EVAs 18,85,86 may be another explanation for the observed differences in severity of HL in both studies. In literature, many prognostic factors such as genotype, EVA size and morphology, age, head trauma, and gender are reported as underlying explanations for this variability, although some of these studies draw contradicting conclusions. 82,85,87-91 In the same line, Song et al. reported intrafamilial differences in the severity of hearing loss in siblings with the same biallelic variants in SLC26A4 . 92 Larger sample sizes are needed to confirm or reject the hypothesis that the CEVA haplotype is associated with a milder HL phenotype. The significant difference in HL severity between the M2 and M1/CEVA