Suzanne de Bruijn

253 Exploring the missing heritability in subjects with hearing loss and EVA Since the genetic defect on the CEVA haplotype could not be pinpointed by the genetic analyses, we could not determine whether the AF of the defect is lower than that of the CEVA haplotype and more in line with the expected frequency based on the prevalence of HL (1: 1,000 newborns 75 ) and the genetic heterogeneity of the condition. Alternatively, the CEVA haplotype could be considered a hypomorphic allele, of which the penetrance depends on the contribution of other co-existing (common) variants. Not all M0 or M1 SLC26A4 cases could be genetically explained by the presence of the CEVA haplotype. Therefore, digenic inheritance with variants in EPHA2, FOXI1, and KCN10 was also explored as a potential explanation for the missing heritability. Digenic inheritance of SLC26A4 and EPHA2 has recently been reported in two Japanese Pendred syndrome cases. 70 A c.1063G>A (p.(Gly355Arg)) and a c.1532C>T (p.(Thr511Met)) variant in EPHA2 were each found in trans with a reported pathogenic variant in SLC26A4 (Deafness Variation Database 50 ). EPHA2 was identified as a binding partner of pendrin, with a crucial role in regulating pendrin localization. 70 The identified variants in EPHA2 were predicted to be pathogenic by several in silico predictions tools. However, the c.1532C>T variant has a relatively high allele frequency of 3.03% in the East Asian population, including 11 homozygotes (gnomAD). Yet, in the present study, we did not obtain indications for digenic inheritance of variants in SLC26A4 and EPHA2 in subjects with HL and EVA. Besides for EPHA2 , a digenic mechanism has also been reported and debated for variants in SLC26A4 and KCNJ10 or FOXI1 , with currently no consensus. 67-69,76,77 FOXI1 is a transcriptional regulator of SLC26A4. 69 We identified a c.677C>T (p.(Thr226Ile)) FOXI1 variant in three subjects (2 M0/ FOXI1 and 1 M0/CEVA/ FOXI1 ). This variant was previously detected in an individual diagnosed with Pendred syndrome and a monoallelic pathogenic SLC26A4 variant. 67 The variant has an allele frequency of 0.71% in non-Finnish Europeans (gnomAD) and affects an amino acid residue located outside the DNA-binding domain but close to the nuclear localization signal (NucPred 78 ). Previously reported pathogenic FOXI1 variants have been shown to affect the DNA-binding properties of the protein. 79 We speculate that a variant affecting the localization motif of the protein could potentially have a loss of function effect as well. Although the variant is classified as likely benign according to the ACMG classification guidelines, we identified the variant three times in our cohort of genetically unexplained SLC26A4 cases and combined with the fact that it has been reported in a previous study 67 , this suggests that the variant might actually contribute to the etiology of HL and EVA although not in a monogenic pattern. Interestingly, in Foxi1 -/- mice, the expansion of the endolymphatic compartment and an audio-vestibular phenotype was observed. 80 In situ hybridization of the endolymphatic duct and sac of these mice revealed complete absence of Slc26a4 mRNA expression. Functional studies, among