Suzanne de Bruijn

238 Chapter 5 RESULTS Patient inclusion and genetic prescreening In this study, we included 28 Dutch index cases diagnosed with a unilateral or bilateral EVA and unilateral or bilateral HL. All individuals were prescreened for pathogenic variants in SLC26A4 (NM_000441.1) in a diagnostic setting and complete coverage of the coding and splice site (+/- 14 nucleotides) regions of SLC26A4 was confirmed. In 16 individuals, a heterozygous (likely) pathogenic SLC26A4 variant was reported and these cases were deemed M1. In the remaining 12 individuals, no potentially pathogenic variants were found in the coding or splice site regions of this gene, and these subjects were therefore considered M0. Causative variants in other genes associated with arHL 48 were addressed and excluded by analyzing available sequencing data (WES or MIPs- based) or in WGS data obtained in this study ( Table S2 ). This revealed no homozygous or compound heterozygous variants that were known or predicted to be pathogenic, except two compound heterozygous variants in OTOGL (NM_173591.3) in individual SLC012 ( Table S4 ). The c.890C>T (p.(Pro297Leu)) variant in OTOGL has, however, been reported as (likely) benign in ClinVar 49 and the Deafness Variation Database 50 and is classified as likely benign according to the ACMG guidelines. 51 The c.1369G>T (p.(Val457Leu)) is considered as a variant of unknown significance (ACMG classification). Furthermore, subject SLC012 has progressive high-frequency HL, which differs from the symmetric, moderate, and stable HL associated with OTOGL (DFNB84B). 52,53 Therefore, we considered the identified OTOGL variants as non-causative. For none of the cases, (likely) pathogenic variants (UV4/UV5, ClinVar) were identified in genes associated with autosomal dominant HL or syndromic HL. 48 The CEVA haplotype is enriched in Dutch monoallelic SLC26A4 cases In 2017, Chattaraj et al. described the ≥613-kb CEVA haplotype located centromeric of the SLC26A4 gene to be enriched in M1 SLC26A4 cases and M0 cases with HL and EVA. 22 To investigate whether this haplotype is also enriched in the selected Dutch cohort of M0 and M1 SLC26A4 cases, we screened for the presence of this haplotype using VNTR marker analysis followed by Sanger sequencing of the 12 CEVA-associated SNPs. The CEVA haplotype was detected in 8 out of 16 (50%) M1 individuals and 2 out of 12 (16.7%) M0 subjects ( Figure 1 , Table 1 ). In two additional M1 individuals (SLC040 and SLC071), only a partial CEVA haplotype was found, harboring 9/12 SNPs. We will refer to this smaller haplotype as the variant 1-CEVA (V1-CEVA) haplotype.