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. Large multigenerational cohort provides new insight into autosomal dominant hearing loss (ADHL). This study analyzes 108 multigenerational families with confirmed dominant inheritance, representing one of the largest single-center European cohorts specifically focused on ADHL. Integrated genomic strategy improves molecular diagnosis. A tiered approach combining targeted sequencing of 237 hearing loss genes, genome-wide linkage analysis, genome sequencing, and segregation studies established a molecular diagnosis in 52% of families. High proportion of previously unreported pathogenic variants. Sixty seven percent of identified pathogenic variants were novel, substantially expanding the mutational spectrum of ADHL. Genetic architecture of ADHL is highly heterogeneous. Recurrent contributions from MYO6, TBC1D24WFS1, GSDME, and POU4F3 were observed alongside numerous single-family findings. Noncanonical variants contribute significantly to disease. Pathogenic mechanisms extended beyond canonical coding regions, including deep intronic and regulatory variants. First deep intronic EYA4 variant associated with ADHL. Genome-based analyses identified a novel deep intronic EYA4 variant that was functionally confirmed to cause aberrant splicing. Structural variant affecting ATP11A transcripts. A large 3’UTR deletion in ATP11A affecting alternative transcripts was identified and segregated with hearing loss in the studied family. Functional assays confirm splicing disruption. Minigene experiments demonstrated that synonymous, non-canonical splice-site, and deep intronic variants can lead to aberrant splicing. Functional validation improves variant interpretation. These findings highlight the importance of integrating genomic data with functional assays for accurate classification of rare variants. Implications for molecular diagnostics of hereditary hearing loss. The results refine variant interpretation and support improved genetic diagnostics of ADHL in clinical practice.