A translational pipeline for classification of variants of uncertain significance to support gene therapy clinical trial in OTOF-related deafness.

OTOF gene dysfunction causes congenital deafness. OTOF gene therapy achieves promising clinical efficacy. However, OTOF variants of uncertain significance (VUS) hinder diagnosis and restrict eligibility for gene therapy in half of 135 hearing loss patients with biallelic OTOF variants in our cohort. Here, we establish a translational pipeline to reclassify VUSs and guide clinical decisions via functional analysis. We evaluate coding VUSs using an adeno-associated virus (AAV)-mediated variant delivery system into Otof-/- mice and evaluate non-coding VUSs via splicing assays. Isolated homozygous E1700Q confers normal hearing, while E1700Q consistently occurs in cis with K1992R in patients, and K1992R correlates with abnormal otoferlin subcellular localization. This pipeline reclassifies eight representative VUSs, enabling definitive molecular diagnoses for 38 patients. Seven eligible patients receive gene therapy and show auditory improvement, including one achieving near-normal hearing that was sustained for 1.5 years. This pipeline effectively expands the gene therapy eligibility for OTOF-related deafness. This study is registered with the Chinese Clinical Trial Registry: ChiCTR2200063181.