The most frequently observed congenital inner ear malformation is enlarged vestibular aqueduct (EVA). It is often accompanied with incomplete partition type 2 (IP2) of the cochlea and a dilated vestibule, which together constitute Mondini malformation. Pathogenic SLC26A4 variants are considered the major cause of inner ear malformation but the genetics still needs clarification. The aim of this study was to identify the cause of EVA in patients with hearing loss (HL). Genomic DNA was isolated from HL patients with radiologically confirmed bilateral EVA (n = 23) and analyzed by next generation sequencing using a custom HL gene panel encompassing 237 HL-related genes or a clinical exome. The presence and segregation of selected variants and the CEVA haplotype (in the 5′ region of SLC26A4) was verified by Sanger sequencing. Minigene assay was used to evaluate the impact of novel synonymous variant on splicing. Genetic testing identified the cause of EVA in 17/23 individuals (74%). Two pathogenic variants in the SLC26A4 gene were identified as the cause of EVA in 8 of them (35%), and a CEVA haplotype was regarded as the cause of EVA in 6 of 7 patients (86%) who carried only one SLC26A4 genetic variant. In two individuals with a phenotype matching branchio-oto-renal (BOR) spectrum disorder, cochlear hypoplasia resulted from EYA1 pathogenic variants. In one patient, a novel variant in CHD7 was detected. Our study shows that SLC26A4, together with the CEVA haplotype, accounts for more than half of EVA cases. Syndromic forms of HL should also be considered in patients with EVA. We conclude that to better understand inner ear development and the pathogenesis of its malformations, there is a need to look for pathogenic variants in noncoding regions of known HL genes or to link them with novel candidate HL genes.

Autosomal dominant hearing loss (ADHL) is a highly heterogeneous Mendelian disorder with numerous causative genes, yet large well-characterized European cohorts remain limited. We investigated 108 families of Polish origin with confirmed dominant inheritance using a tiered strategy integrating targeted sequencing of 237 hearing loss genes, genome-wide linkage analysis, genome sequencing, segregation studies in 437 individuals, and functional validation by minigene splicing assays. This approach established a molecular diagnosis in 52% of families, with 67% of identified pathogenic variants being previously unreported. The genetic architecture proved highly heterogeneous, with recurrent contributions from MYO6, TBC1D24, WFS1, GSDME, and POU4F3 alongside numerous single-family findings. While missense variants predominated, splice-altering and truncating changes were also frequent. Importantly, genome-based analyses uncovered pathogenic mechanisms beyond canonical coding regions, including the first deep intronic EYA4 variant associated with ADHL and a large 3'UTR deletion in ATP11A affecting alternative transcripts. Functional assays confirmed aberrant splicing induced by synonymous, non-canonical splice-site, and deep intronic variants. These findings refine the gene-specific contribution to dominant hearing loss, expand the noncoding mutational landscape, and underscore the importance of integrating genomic and functional analyses for accurate variant interpretation and improved molecular diagnostics of hereditary hearing loss in clinical practice.