Suzanne de Bruijn

239 Exploring the missing heritability in subjects with hearing loss and EVA A 28 Index cases CEVA haplotype WGS Digenic inheritance M0 (n=12) M1 (n=16) M0 (n=10) M1 (n=8) M2 (n=10) M0 (n=8) Zeroallelic Monoallelic Biallelic B M1 (n=5) M0 (n=2) M2 (n=12) M1 (n=1) F F M0 (n=10) M1 (n=6) M2 (n=12) M0 M2 M1 M1/CEVA M0/FOXI1 M0/CEVA CEVA/FOXI1 Figure 1. Overview of genetic analyses performed in zeroallelic and monoallelic SLC26A4 cases. (A-B) To explain the missing heritability in zeroallelic (M0, n=12) and monoallelic (M1, n=16) SLC26A4 cases, different genetic analyses were performed. Firstly, individuals were screened for the presence of the CEVA haplotype (M0/CEVA, n=2; M1/CEVA, n=10). Secondly, whole genome sequencing (WGS) was performed in all monoallelic cases (M0/CEVA, M1) to identify potential structural, splice (M2, n=2) or regulatory variants. Lastly, sequencing data were screened for potentially pathogenic variants in the EPHA2 , FOXI1 and KCNJ10 genes. Digenic inheritance has been previously suggested for variants in these genes and the SLC26A4 gene. In three cases (M0/FOXI1 (M0 F ), n=2, CEVA/FOXI1 (M1 F ), n=1), a potentially pathogenic variant in FOXI1 (NM_012188.4, c.677C>T) was identified. The CEVA haplotype has an AF of 2.8% in the 1000G database (28 in 1006 alleles) 22,54 , and an AF of 3.3% in an in-house control cohort consisting of 322 healthy unrelated individuals (21 in 644 unphased alleles). This implies a significant enrichment of the CEVA haplotype in our M1 cohort (8 in 32 alleles) compared to the 1000G database (p-value 5.419*10 -6 ) and the control cohort (p-value 2.187*10 -5 ) as determined by a two-sided Fisher’s exact test). The two M1 cases with the V1-CEVA haplotype were not included in this statistical analysis. Also this V1-CEVA allele is significantly enriched in our M1 cohort as only a single V1-CEVA allele is reported in the 1000G database (1 in 1006 alleles) 22 (p-value 0.0027). The CEVA haplotype was not found to be significantly enriched in the M0 cohort (2 in 24 alleles). Although the pathogenicity of the CEVA haplotype is unclear, the significant enrichment of the haplotype within this M1 patient cohort and the patient cohorts (M1 and M0) previously described by Chattaraj and co-workers strongly suggests that a pathogenic defect resides within this haplotype. 22 Because of this strong association of the CEVA haplotype with HL and EVA, we considered the M1 individuals carrying the CEVA or the V1-CEVA haplotype as genetically explained (M1/CEVA), and M0 individuals with the CEVA haplotype (M0/CEVA) as monoallelic in further steps of this study. For six M1 individuals, it could not be conclusively determined whether the CEVA haplotype was present in trans with the pathogenic SLC26A4 variant, as the genetic material of family members was not available ( Table 1 ).