Pathogenic variants in SLC26A4 have been associated with autosomal recessive hearing loss (arHL) and a unilateral or bilateral enlarged vestibular aqueduct (EVA). SLC26A4 is the second most frequently mutated gene in arHL. Despite the strong genotype–phenotype correlation, a significant part of cases remains genetically unresolved. In this study, we investigated a cohort of 28 Dutch index cases diagnosed with HL in combination with an EVA but without (M0) or with a single (M1) pathogenic variant in SLC26A4. To explore the missing heritability, we first determined the presence of the previously described EVA-associated haplotype (Caucasian EVA (CEVA)), characterized by 12 single nucleotide variants located upstream of SLC26A4. We found this haplotype and a delimited V1-CEVA haplotype to be significantly enriched in our M1 patient cohort (10/16 cases). The CEVA haplotype was also present in two M0 cases (2/12). Short- and long-read whole genome sequencing and optical genome mapping could not prioritize any of the variants present within the CEVA haplotype as the likely pathogenic defect. Short-read whole-genome sequencing of the six M1 cases without this haplotype and the two M0/CEVA cases only revealed previously overlooked or misinterpreted splice-altering SLC26A4 variants in two cases, who are now genetically explained. No deep-intronic or structural variants were identified in any of the M1 subjects. With this study, we have provided important insights that will pave the way for elucidating the missing heritability in M0 and M1 SLC26A4 cases. For pinpointing the pathogenic effect of the CEVA haplotype, additional analyses are required addressing defect(s) at the RNA, protein, or epigenetic level.

SLC26A4的致病性变异与常染色体隐性遗传性听力损失 (arHL) 和单侧或双侧前庭导水管 (EVA) 扩大有关。SLC26A4是 arHL 中第二常见的突变基因。尽管基因型 - 表型相关性很强，但很大一部分病例在遗传上仍未解决。在这项研究中，我们调查了一组 28 名荷兰指数病例，这些病例被诊断为 HL 并结合 EVA 但没有 (M0) 或具有SLC26A4中的单一 (M1) 致病性变异。为了探索缺失的遗传力，我们首先确定了先前描述的 EVA 相关单倍型（高加索 EVA (CEVA)）的存在，其特征在于位于SLC26A4上游的 12 个单核苷酸变体. 我们发现这种单倍型和分隔的 V1-CEVA 单倍型在我们的 M1 患者队列（10/16 例）中显着富集。CEVA 单倍型也存在于两个 M0 病例 (2/12) 中。短读长和长读长全基因组测序和光学基因组作图不能将 CEVA 单倍型中存在的任何变体列为可能的致病缺陷。没有这种单倍型的六个 M1 病例和两个 M0/CEVA 病例的短读全基因组测序仅揭示了以前被忽视或误解的剪接改变SLC26A4两种情况下的变异，现在可以从基因上解释。在任何 M1 受试者中均未发现深内含子或结构变异。通过这项研究，我们提供了重要的见解，将为阐明 M0 和 M1 SLC26A4病例中缺失的遗传力铺平道路。为了确定 CEVA 单倍型的致病作用，需要额外的分析来解决 RNA、蛋白质或表观遗传水平的缺陷。