Connexin 26 in Hearing Health and Disease: StructuralFoundations, Mutation Mechanisms, and Therapeutic Perspectives

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript reviews mutations in GJB2, the most common genetic cause of hereditary hearing loss, with a focus on connexin 26 protein structure, disease-associated mutation mechanisms, and current therapeutic strategies. The review is well written, clearly organized, and appropriately referenced. Overall, it provides a comprehensive and accurate overview, and only minor revisions are recommended.

Minor changes:

1. References 6 & 7 cite GeneReviews chapters, which are a continuously updated resource and does not use edition numbers. Chapter 6 has undergone substantive updates (notably in 1998 and 2023). While I am not certain of IJMS’s preferred approach for referencing resources like this, I think it would be appropriate to indicate the most recent update date and include the internet access date in the reference. https://www.ncbi.nlm.nih.gov/books/NBK1272/
2. Reference 17 should be 2019 instead of 2018. https://pmc.ncbi.nlm.nih.gov/articles/PMC6601451/
3. Reference 18 has an extra period after the question mark in the title.
4. Reference 23 and 34 are the same reference.
5. In section 3, the authors should consider including a figure of the GJB-2 gene indicating where deafness-associated mutations occur along the gene.

Author Response

Please see the attachment below.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The logical flow from Cx26 architecture to gating mechanisms, mutation classification, and therapeutic perspectives is clear, and Table 1 together with Figures 1 and 2 effectively support the narrative. The discussion of population-specific founder mutations (c.35delG, c.235delC, V37I, c.167delT, IVS1+1G>A) is particularly well balanced, and the inclusion of AAV-SCpro delivery, miniaturized SaCas9-NNG-ABE8e base editing, and the GJB2 knockout pig model gives the review strong contemporary relevance.To strengthen the manuscript further, I respectfully suggest the following revisions.Major points. Please re-verify all reference dates, as several entries (e.g., Refs 11, 145, 150) and the Figure 2 credit are listed as 2026; mark any in-press papers explicitly. All metadata placeholders in the front matter should be finalized. In Sections 2.2 and 3.2, please propose an explicit integrated model that reconciles K⁺ recycling failure with developmental ATP–Ca²⁺ signaling deficits, since K⁺ recycling alone does not account for profound congenital phenotypes. In Section 4.2.2, please expand the discussion of CRISPR safety, including off-target analysis (Cas-OFFinder, GUIDE-seq, CIRCLE-seq), bystander editing, and long-term in vivo follow-up. In Section 2, the persistent disorder of NT, CT, and CL domains would benefit from a forward-looking paragraph on AlphaFold3, RoseTTAFold-AA, and integrative modeling approaches. Given the authors' expertise, a deeper comparison of native-lipid strategies (SMA copolymers, nanodiscs, native membrane extraction) would also distinguish this review. Finally, a new comparative table summarizing mouse, rat, non-human primate, and porcine models for Cx26 gene therapy would be highly informative.Minor points. Please define the "DFN" prefix (DFNB1, DFNA3) at first use; clarify atomic-resolution limits for Ca²⁺ site assignment in Section 2.1.2.1; add a brief clinical-management note for heterozygous Cx26⁺/⁻ carriers in Section 3.1.4; include at least one paragraph on antisense oligonucleotide (ASO) strategies, currently absent from Section 4; balance the cochlear implant discussion in Section 4.1 with known limitations (music perception, bilateral synchronization); ensure Figure 1 panel color coding is fully explained in the legend; consider adding the KCNQ1/KCNE1 K⁺ secretion pathway to Figure 2; and update genetic interpretation guidelines to include the ClinGen Hearing Loss VCEP updates (2022–2024). Section 5 would benefit from explicitly noting the need for calmodulin–Cx26 complex structures. Please also complete the Conflicts of Interest, Author Contributions, Funding, and IRB Statement sections.

Author Response

Please see the attachment below.

Author Response File: Author Response.pdf