Suzanne de Bruijn

244 Chapter 5 The variant was not identified in any of the M1/CEVA or the two M2 cases. FOXI1 encodes the Forkhead transcription factor FOXI1, a key transcriptional regulator of SLC26A4 . 69 Segregation analysis has confirmed that the FOXI1 variant is not co-inherited with the CEVA allele in individual SLC039, which is in line with digenic inheritance . The Thr226 residue is located outside of the conserved forkhead DNA-binding domain of FOXI1 (amino acids 94-211) 69 and none of the in silico tools used for analysis predicted a deleterious effect of the c.677C>T variant. Nevertheless, the variant is enriched in individuals diagnosed with HL and EVA (3 in 56 alleles in the study cohort versus 165 in 26.590 alleles of the in-house WES cohort, p-value 0.0004), and we consider the c.677C>T FOXI1 variant as an interesting candidate for functional validation. In case SLC017, a heterozygous missense variant in EPHA2 was detected (c.2627G>A (p.(Arg876His)). Although the variant is predicted to be pathogenic by in silico prediction tools, it has a relatively high AF of 1.70% (gnomAD) and 2.36% (in-house database) and is classified as likely benign according to the ACMG classification guidelines. Because the variant was only found in an M0 SLC26A4 case, a potential digenic inheritance of pathogenic SLC26A4 variants and the newly identified EPHA2 variant could not be addressed. To summarize, the CEVA haplotype or a short CEVA haplotype (V1-CEVA) was detected in 12 of the 28 index cases (16 M1, 12 M0) that were included in our study ( Figure 1 ). In two individuals (M1), an SLC26A4 splice variant was identified using WGS. After performing these genetic analyses by which the enrichment of the (V1-)CEVA haplotype in M1 cases was demonstrated, we consider the HL in 12 individuals to be associated with SLC26A4 defects and these subjects to be genetically explained (2 M2, 10 M1/CEVA), six individuals are consideredM1 (4 M1, 2 M0/CEVA), and ten individuals are still considered M0. Additionally, in three individuals (1 M0/CEVA, 2 M0) a potentially pathogenic variant in FOXI1 was found. Determination of boundaries of CEVA haplotype To identify the true pathogenic defect located on the CEVA haplotype, an in-depth analysis of this genomic region was performed. Firstly, the exact boundaries of the genomic region shared by CEVA haplotype carriers were determined using VNTR marker analysis. For two individuals with the complete CEVA haplotype and the two subjects with the V1-CEVA haplotype, DNA samples of family members were available, allowing reliable determination of the marker alleles located within the haplotype. A shared haplotype of 0.89 Mb delimited by markers D7S501 and D7S2459 was identified ( Figure 2 and Figure S2 ). Although the V1-CEVA haplotype shares the marker alleles with the complete CEVA haplotype, the absence of SNPs 1-3 potentially delimits the