NM_194248.3(OTOF):c.4960G>A (p.Gly1654Ser)

OTOF c.4960G>A (p.Gly1654Ser) is a pathogenic missense mutation located in the C2F calcium-binding domain of otoferlin. The glycine-to-serine substitution introduces a hydroxyl-bearing side chain, which may disrupt the folding stability of the β-sandwich hydrophobic core of the C2F domain, leading to reduced calcium sensitivity at inner hair cell synapses and/or decreased overall otoferlin protein levels. For this G>A point mutation, an adenine base editor (ABE8e) could theoretically achieve precise A>G reverse correction, but feasibility must be confirmed by analyzing PAM accessibility at the target site and validating the editing window. Before clinical translation of base editing matures, dual-AAV OTOF gene replacement therapy (e.g., DB-OTO), as a mutation-agnostic strategy, remains the most clinically accessible therapeutic pathway at present.

The C2F domain of otoferlin is the C2 domain closest to the C-terminus and contains a calcium-dependent phospholipid-binding interface required for inner hair cell exocytosis. Gly1654 is located in the β3–β4 loop region of the C2F domain. Structural modeling (based on PDB homologous C2-domain templates) indicates that this glycine residue provides a critical conformational constraint for the β-turn; after substitution to Ser, steric hindrance from the side chain may cause local secondary-structure perturbation. ClinVar classifies this variant as pathogenic (single submitter, with sufficient criteria). Precedents for base-editing strategies come from extra-cochlear diseases such as DMD and β-thalassemia: ABE7.10 established by Gaudelli et al. (Nature 2017, PMID:29160308) and its improved version ABE8e can efficiently edit A:T→G:C (efficiency >60%). Together with relaxed-PAM variants such as SpRY and SaCas9-KKH, PAM feasibility for antisense-strand targeting at the c.4960G>A site (where the A is on the antisense strand) should be systematically evaluated using genome-wide PAM scanning tools (e.g., CRISPRscan). Delivery of base editors to inner ear hair cells can leverage existing AAV cochlear injection platforms: AAVAnc80L65 achieves >90% transduction efficiency in mouse cochlear inner hair cells (Landegger et al., Nat Biotechnol 2017, PMID:28252004), providing a mature delivery framework for cochlear delivery of base-editing vectors.

The feasibility of base editing at the c.4960G>A site has not yet been experimentally validated: (1) whether the ABE editing window (typically positions 4–8 of the protospacer) covers the target adenine requires precise modeling; (2) nearby A bases may cause bystander editing, necessitating high-fidelity ABE variants (e.g., ABE8e-V106W) to reduce off-target risk; (3) cochlear inner hair cells are terminally differentiated and non-dividing, and long-term (>2 years) intra-cochlear data supporting the efficiency and durability of AAV-mediated base editing are still lacking. The exact pathogenic mechanism of p.Gly1654Ser (protein folding instability vs. loss of calcium-binding function) has not been validated in patient-derived iPSC-differentiated cochlear inner hair cell–like cells; if it represents a pure loss of function rather than reduced protein abundance, pharmacological chaperone strategies (small molecules that stabilize misfolded proteins) are worth exploring as an adjunct, and AutoDock Vina pocket analysis around the C2F-domain variant site can serve as an initial screening tool. At present, for DFNB9 patients, dual-AAV full-length OTOF replacement therapy (DB-OTO) has entered the clinical stage; if gene-editing approaches are not yet mature, patients should be prioritized for evaluation of eligibility for enrollment in existing gene-therapy clinical trials.