Functional pathogenicity of ESRRB variant of uncertain significance contributes to hearing loss (DFNB35).

Advances in next-generation sequencing technologies have led to elucidation of sensorineural hearing loss genetics and associated clinical impacts. However, studies on the functional pathogenicity of variants of uncertain significance (VUS), despite their close association with clinical phenotypes, are lacking. Here we identified compound heterozygous variants in ESRRB transcription factor gene linked to DFNB35, specifically a novel splicing variant (NM_004452.4(ESRRB): c.397 + 2T>G) in trans with a missense variant (NM_004452.4(ESRRB): c.1144C>T p.(Arg382Cys)) whose pathogenicity remains unclear. The splicing variant (c.397 + 2T>G) caused exon 4 skipping, leading to premature stop codon formation and nonsense-mediated decay. The p.(Arg382Cys) variant was classified as a VUS due to its particularly higher allele frequency among East Asian population despite disease-causing in-silico predictions. However, functional assays showed that p.(Arg382Cys) variant disrupted key intramolecular interactions, leading to protein instability. This variant also reduced transcriptional activity and altered expression of downstream target genes essential for inner ear function, suggesting genetic contribution to disease phenotype. This study expanded the phenotypic and genotypic spectrum of ESRRB in DFNB35 and revealed molecular mechanisms underlying ESRRB-associated DFNB35. These findings suggest that variants with high allele frequencies can also possess functional pathogenicity, providing a breakthrough for cases where VUS, previously unexplored, could be reinterpreted by elucidating their functional roles and disease-causing characteristics.