Integrative genetic and functional analysis of autosomal dominant hearing loss in 108 multigenerational families

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.