Suzanne de Bruijn

234 Chapter 5 analyses performed in family members of M1 subjects, however, do suggest that in 98% of M1 subjects an unidentified or unrecognized variant is present on the trans SLC26A4 allele. 20,21 In line with this hypothesis, Chattaraj and coworkers reported a haplotype, referred to as the Caucasian EVA (CEVA) haplotype, that was present in 13 of 16 (81%) of the studied M1 families and that was also enriched in M0 subjects. 22 The haplotype is defined by the combination of 12 single nucleotide polymorphisms (SNPs; allele frequency (AF) 1.9-4.0%) spanning a 613 kb region. The 12 SNPs are located within a region of linkage disequilibrium that extends from upstream of PRKAR2B to intron 3 of SLC26A4 and are either intergenic or intronic of the genes SLC26A4 , BCAP29 , DUS4L, COG5 , GPR22 , HBP1 , PRKAR2B and PIK3CG . 22 The true genetic defect of the CEVA allele has not been identified yet, but it cannot be excluded that a potential defect was missed due to the technical limitations of short-read sequencing and other standard-of-care tests. The CEVA haplotype was reported to be associated with a less severe HL phenotype as compared to variants in the protein-coding or splice site regions of SLC26A4 . 23 We investigated a Dutch cohort of M1 and M0 subjects with HL and a unilateral or bilateral EVA. All subjects were tested for the presence of the CEVA haplotype, and whole genome sequencing (WGS) was performed to detect potentially missed single nucleotide variants (SNVs), structural variants (SVs), and regulatory or deep-intronic variants. Long-read sequencing and optical genome mapping were performed to reveal a potentially missed SV located on the CEVA haplotype. Variants located within the haplotype were subjected to in silico analyses to investigate potential effects on the regulation of SLC26A4 expression or on splicing. With this study, we provided further insights into SLC26A4 -associated disease. MATERIAL AND METHODS Inclusion criteria and clinical evaluation This study was approved by the medical ethics committee of the Radboud University Medical Center (registration number: NL33648.091.10) and was carried out according to the Declaration of Helsinki. Subjects diagnosed with unilateral or bilateral HL and a unilateral or bilateral EVA on CT or MRI and for whom medical genetic testing only revealed a heterozygous (M1, n=16) or no pathogenic variant (M0, n=12) in SLC26A4 were eligible to participate in this study. A retrospective cohort of nine subjects with confirmed pathogenic (biallelic) variants in SLC26A4 was added as a reference cohort ( Table S1 ).