What’s Known

Gap junction protein β-6 (GJB6) encodes connexin 30, a gap junction protein essential for cochlear ion homeostasis and normal hearing. Large deletions in GJB6 are well-established causes of non-syndromic hearing loss, but single-nucleotide variants in this gene are rare, and their pathogenic significance remains unclear.

What’s New

A novel GJB6 gene variant (c.446C>T) was identified by whole-exome sequencing (WES) and confirmed by Sanger sequencing in the family. The integration of clinical and in silico analyses suggests a potential impact on protein structure. Literature review confirming the rarity of GJB6 single-nucleotide variants, particularly in Iranian populations.

Introduction

Hearing, which involves the conversion of physical sound waves into electrochemical signals, is a highly coordinated and dynamic event involving intricate biological mechanisms.^{1, 2} The cochlea, a fluid-filled sensory organ of the inner ear, plays a central role in auditory transduction and depends on the precise regulation of ionic gradients, particularly potassium (K⁺) homeostasis. Gap junction channels composed of connexin proteins, such as gap junction protein β-6 (GJB6), in cochlear supporting cells facilitate K⁺ recycling and enable direct intercellular communication necessary for maintaining the endocochlear potential. Moreover, GJB6 has been implicated in the repair and maintenance of cochlear epithelial integrity following sensory cell damage, highlighting its essential role in inner ear homeostasis and normal hearing.^{1, 3} While large GJB6 deletions are well characterized, single-nucleotide variants (SNVs) are rarely reported, and their pathogenic significance remains unclear.^1-3

In this study, we report a novel homozygous variant of the GJB6 gene in a woman with moderate to severe bilateral sensorineural hearing loss (SNHL) and no ectodermal dysplasia or palmoplantar keratoderma. Using in silico analyses, we evaluated the structural and functional impacts of the identified variant and compared them with those of other previously reported SNVs in GJB6.

Case Presentation

A 32-year-old woman, born to healthy consanguineous parents, was referred to the Medical Genetics Laboratory in Tehran, Iran, in March 2025. She was born at full term via an uneventful vaginal delivery with no perinatal complications, neonatal hospitalization, or significant medical issues during childhood. Developmental milestones were reportedly normal, except for an early-onset hearing impairment diagnosed before 6 months of age. Pure-tone audiometry and the Weber test revealed moderate to severe bilateral SNHL. Speech test scores indicated reduced speech clarity compared to normal, even with amplification of the sound. The patient has been using hearing amplification devices since childhood. At the most recent evaluation, no progression of hearing loss was observed. Comprehensive clinical and paraclinical evaluations ruled out external ear malformations, dermatological findings (e.g., eczema or psoriasis), visual abnormalities, and signs suggestive of metabolic or syndromic involvement. No other systemic abnormalities were observed. Based on the clinical findings and family pedigree analysis, the case was classified as non-syndromic sensorineural hearing loss (NSHL) with an autosomal recessive inheritance pattern (figure 1). Informed written consent was acquired from the proband and her parents for the release of clinical and genetic data. The study was approved by the Ethics Committee of the University of Social Welfare and Rehabilitation Sciences (IR.USWR.REC.1403.199).

Figure 1.Multiple molecular analyses that were conducted in the family to further understand the etiology of the hearing loss are shown. a) The family’s pedigree indicates autosomal recessive inheritance. b) Sanger sequencing confirmed the presence of homozygous and heterozygous variants in the proband and her parents, respectively. c) Pure-tone audiogram of the proband indicates moderate to severe bilateral sensorineural hearing loss. d) Alanine and its surrounding amino acids in gap junction protein β-6 are highly conserved in mammals, as shown by evolutionary conservation studies.

Molecular Evaluation: Whole-Exome Sequencing and Copy Number Variant Analysis

Genomic DNA was extracted from whole blood samples of the proband and her parents using the standard salting-out method.⁴ Since no pathogenic variants were identified in GJB2, whole-exome sequencing (WES) was performed to detect additional candidate genes associated with hereditary hearing loss. The patient’s DNA was fragmented and enriched using the Agilent SureSelect All Exon V7 kit (Illumina genome sequencing service in Macrogen, Seoul, South Korea). Variant calling was carried out by Genome Analysis Toolkit (GATK), and the resulting VCF file was annotated using wANNOVAR. Variant filtering was based on the assumed autosomal-recessive inheritance pattern, clinical significance databases, and population allele frequencies from the Genome Aggregation Database (gnomAD) and Iranome (minor allele frequency <0.01). All candidate variants were manually inspected in the Integrative Genomics Viewer to eliminate false positives. Copy number variations (CNVs) were analyzed using GATK (v.4.0.10.1).

The Result of Molecular and Bioinformatics Analyses in the Family

Analysis of WES data identified a novel homozygous variant, NM_001110219.3:c.446C>T (p.A149V), located in exon 5 of GJB6 (figure 1). No CNVs were detected in the proband across the known hearing loss-associated genes. According to the American College of Medical Genetics guidelines,⁵ the detected variant was classified as a variant of uncertain significance (VUS). This classification was based on PM1 (moderate), as the missense variant is found in a recognized protein functional domain, and PM2 (moderate), due to its very low allele frequency across large population databases. No additional strong or supporting criteria were fulfilled; therefore, the currently available evidence remains insufficient to establish pathogenicity (figure 1, table 1). The variant at this position had a combined annotation dependent depletion (CADD [v1.7]) score of 13.13, placing it within the top 5-10% of potentially deleterious variants but below the commonly used pathogenicity threshold (CADD≥20). Furthermore, the A149V substitution in GJB6 was modeled and compared with the wild-type structure using the SWISS-MODEL server. The resulting models were subsequently loaded and visualized in PyMOL (v2.5.5). Global backbone alignment showed no measurable deviation (Cα RMSD≈0.02 Å), indicating the preservation of the overall fold. The major effect was local and side-chain specific: the solvent-accessible surface area (SASA) at residue 149 nearly doubled (WT side chain≈36 Ų vs A149V≈70 Ų), with a moderate increase in the local segment (residues 146–152, +22 Ų). These changes reflect the bulkier and more hydrophobic side chain of valine, which protrudes further into the surrounding environment (figure 2). While these changes suggest potential local effects, their functional consequences remain speculative and require experimental validation.

Figure 2.Bioinformatics analyses by several tools were performed to further understand the function of NM_001110219.3:c.446C>T (p.Ala149Val). a) A multiple sequence alignment of GJB6 and GJB2 was conducted using CLUSTAL O (1.2.4) through the EMBL-EBI platform. The study indicated that alanine at position 149 is conserved in both proteins. b) The hydrophobicity and side-chain volume of valine are stronger than those of alanine, indicating its tendency to be buried in the protein structure.

Review of Literature and Genotype-Phenotype Correlation

A total of 47 patients with GJB6 have been reported across 14 papers, representing 20 variants (including 17 missense, 2 synonymous, and 1 insertion variant) from different ethnicities (table 2).^{1, 2, 6-17} Syndromic hearing loss was reported in 13 patients (27.6%). The remaining cases exhibited isolated hearing loss. The identified variants were distributed across multiple domains of the GJB6 protein. Most mutations were located within the extracellular loop 1 (EL1), cytoplasmic loop (CL), transmembrane domain 3 (TM3), and extracellular loop 2 (EL2) (figure 3). Variants located within the transmembrane domains included seven distinct substitutions, collectively reported in 12 individuals (25.5%).^{7, 9-11, 13, 16} Functional studies indicate that the T5M mutation exerts a dominant-negative effect, causing profound hearing loss in heterozygous humans, whereas in mice, heterozygotes remain unaffected and homozygotes exhibit only mild hearing impairment (table 2).^18-21 While reported GJB6 variants span multiple protein domains and are associated with both syndromic and non-syndromic phenotypes, clear genotype–phenotype correlations remain limited, particularly for rare missense variants.

Figure 3.According to UniProt, GJB6 protein (O95452 CXB6_HUMAN) comprises five topological domains and four transmembrane segments: TM1 (residues 23-45), TM2 (residues 76-98), TM3 (residues 132-154), and TM4 (residues 193-215). These transmembrane segments (TM) are interspersed with an N-terminal cytoplasmic domain, one cytoplasmic loop (CL), two extracellular loops (EL1 and EL2), and a C-terminal cytoplasmic domain (https://www.uniprot.org/). The asterisk marked the position of the variant (TM3) found in the proband.

Discussion

Gap junctions in humans and other mammals are composed of two hemichannels (connexons), each formed by six connexin subunits. Among the connexin family, GJB2 and GJB6 are the most abundant in the cochlea and work together to maintain inner ear homeostasis. These two genes share high sequence homology (approximately 76%) and contain several conserved structural domains essential for normal auditory function.¹⁹ Studies suggest that, due to the established functional interplay between GJB6 and GJB2, digenic or modifier effects should be considered even when no pathogenic GJB2 variants are detected.^{3, 9, 10} Reports of incomplete penetrance have been observed, and while GJB6 deletions in combination with GJB2 variants can exacerbate disease severity, the resulting phenotype largely depends on the deletion location and its effect on GJB2 expression.^{3, 9, 10}

In contrast to GJB2, point mutations and deletions in GJB6 are uncommon.²² Variants such as G21R, T5M, and A40V are associated with non-syndromic hearing loss,^{6, 19, 23} whereas N54K, G11R, and N14S are linked to specific forms of ectodermal dysplasia, with or without deafness.^{14, 24, 25} In GJB6, skin-related pathogenic variants are proposed to act by increasing hemichannel open probability, causing excessive efflux of vital metabolites; for example, A88V in TM2 leads to hidrotic ectodermal dysplasia without affecting hearing.²¹ However, altered permeability to signaling molecules, such as inositol 1,4,5-trisphosphate (IP3), together with impaired Ca²⁺ signaling propagation, represents a potential pathogenic mechanism by which GJB6 mutations contribute to the development of NSHL.²²

Some variants impair auditory function by reducing the number of functional gap junction channels; notably, N54K is retained in the cytoplasm, thereby blocking neurobiotin transfer, resulting in aberrant intracellular localization with subsequent loss of channel activity.¹⁴ The N54K mutation in either GJB6 or GJB2 results in a similar phenotype comprising hearing impairment and cutaneous abnormalities, highlighting the essential role of connexin genes in both epidermal and auditory systems.^{14, 22} The GJB6:p.T5M, located within the N-terminal domain,⁶ disrupts both Ca²⁺ signaling and IP₃ permeability, whereas GJB6:p.A40V,¹⁹ Alanine in this region, together with other amino acids, contributes to the formation of a hydrophobic core, which is essential for establishing a functional and stable intramolecular structure of GJB6.^{19, 22}

In another study, GJB6: p.P70L was detected in the homozygous state in a patient from a consanguineous family, in which relatives were either unaffected wild-type carriers or heterozygous for this variant. The patient presented with bilateral prelingual deafness and no family history of hearing loss.¹⁵

In our study, WES identified a homozygous NM_001110219.3:c.446C>T (p.A149V) variant in the proband, which was confirmed by Sanger sequencing in her family. Residue 149 is located within TM3 (figure 3). Previous studies have indicated that TM3 is located at the periphery of the hemichannel and is in contact with the lipid environment.^{26, 27} In connexins, TM3 primarily contributes to helix packing, inter-subunit interactions, and structural stability rather than lining the channel pore.^{26, 27} In the p.A149V variant, valine exhibits stronger hydrophobicity and side-chain volume than alanine, which is associated with its heightened propensity to be buried in the protein structure.^{26, 27} Thus, the observed increase in side-chain exposure is more consistent with a potential disturbance in helix packing, membrane embedding, or connexon assembly/trafficking than with a direct alteration in pore diameter.²⁶ Overall, the modeling suggests that A149V does not destabilize the backbone structure but introduces a local sidechain perturbation in TM3 that may affect protein folding, subunit packing, or oligomerization. These findings alone are insufficient to establish pathogenicity; functional studies (trafficking assays, gap-junction coupling, etc.) are required to clarify the biological impact.

The variant’s low frequency in Iranome, gnomAD, and other databases, including GenomeAsia (with no reported homozygotes) supports its rarity. However, its relatively higher frequency in a population-matched database (Turkish variome) may reflect reduced penetrance or population-specific variation, potentially requiring additional genetic or environmental factors for NSHL to manifest. Moreover, this study has inherent limitations. Segregation analysis was restricted by the proband being an only child, and longitudinal follow-up data were limited, although no progression was reported. The rarity of GJB6 point mutations and poorly defined genotype-phenotype correlations for missense variants limit broader generalization. Furthermore, while extensive in silico analyses were performed, these computational predictions cannot replace functional validation. Nevertheless, the findings provide valuable insight into this rare variant and contribute to expanding the current understanding of GJB6-associated hearing loss.

Conclusion

Next-generation sequencing plays a crucial role in NSHL diagnosis. In this study, we report a novel GJB6 missense variant (NM_001110219.3:c.446C>T [p.Ala149Val]) associated with NSHL, thereby expanding the known genetic spectrum of this gene.

Acknowledgment

The authors thank the Medical Genetics Laboratory of the Hope Generation Institute for their support in patient and family coordination.

Authors’ Contribution

F.ZA: Conceptualization, study design, experimental procedures, genetic testing, and drafting; M.M: Data interpretation and processing, and reviewing the manuscript; E.E: Data gathering and reviewing the manuscript; H.Gh: Data gathering and reviewing the manuscript, HR.KhKh: Data interpretation and reviewing the manuscript; R.N: Conceptualization, Study design, experimental procedures, genetic testing, and drafting; All authors have read and approved the final manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Declaration of AI

No AI tools were used for data analysis or manuscript preparation.

Conflict of Interest

None declared.

References

1. Alkhidir S, El-Akouri K, Al-Dewik N, Khodjet-El-Khil H, Okashah S, Islam N. The genetic basis and the diagnostic yield of genetic testing related to nonsyndromic hearing loss in Qatar. Sci Rep. 2024; 14:4202. Publisher Full Text | DOI | PubMed [ PMC Free Article ]
2. Morgan A, Lenarduzzi S, Cappellani S, Pecile V, Morgutti M, Orzan E. Genomic Studies in a Large Cohort of Hearing Impaired Italian Patients Revealed Several New Alleles, a Rare Case of Uniparental Disomy (UPD) and the Importance to Search for Copy Number Variations. Front Genet. 2018; 9:681. Publisher Full Text | DOI | PubMed [ PMC Free Article ]
3. Limmon R, Punagi AQ, Gaffar M, Idris I, Halik H. The variants and prevalence of the GJB2 and GJB6 in patients with non-syndromic congenital sensorineural hearing loss in Maluku, Indonesia. The Egyptian Journal of Otolaryngology. 2025; 41:167. DOI
4. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988; 16:1215. Publisher Full Text | DOI | PubMed [ PMC Free Article ]
5. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015; 17:405-24. Publisher Full Text | DOI | PubMed [ PMC Free Article ]
6. Grifa A, Wagner CA, D’Ambrosio L, Melchionda S, Bernardi F, Lopez-Bigas N. Mutations in GJB6 cause nonsyndromic autosomal dominant deafness at DFNA3 locus. Nat Genet. 1999; 23:16-8. DOI | PubMed
7. Yang JJ, Huang SH, Chou KH, Liao PJ, Su CC, Li SY. Identification of mutations in members of the connexin gene family as a cause of nonsyndromic deafness in Taiwan. Audiol Neurootol. 2007; 12:198-208. DOI | PubMed
8. Nemoto-Hasebe I, Akiyama M, Kudo S, Ishiko A, Tanaka A, Arita K. Novel mutation p.Gly59Arg in GJB6 encoding connexin 30 underlies palmoplantar keratoderma with pseudoainhum, knuckle pads and hearing loss. Br J Dermatol. 2009; 161:452-5. DOI | PubMed
9. Asma A, Ashwaq A, Norzana AG, Atmadini AM, Ruszymah BH, Saim L. The association between GJB2 mutation and GJB6 gene in non syndromic hearing loss school children. Med J Malaysia. 2011; 66:124-8. PubMed
10. Battelino S, Repic Lampret B, Zargi M, Podkrajsek KT. Novel connexin 30 and connexin 26 mutational spectrum in patients with progressive sensorineural hearing loss. J Laryngol Otol. 2012; 126:763-9. DOI | PubMed
11. Oh SK, Choi SY, Yu SH, Lee KY, Hong JH, Hur SW. Evaluation of the pathogenicity of GJB3 and GJB6 variants associated with nonsyndromic hearing loss. Biochim Biophys Acta. 2013; 1832:285-91. DOI | PubMed
12. Miyagawa M, Naito T, Nishio SY, Kamatani N, Usami S. Targeted exon sequencing successfully discovers rare causative genes and clarifies the molecular epidemiology of Japanese deafness patients. PLoS One. 2013; 8:e71381. Publisher Full Text | DOI | PubMed [ PMC Free Article ]
13. Beck C, Perez-Alvarez JC, Sigruener A, Haubner F, Seidler T, Aslanidis C. Identification and genotype/phenotype correlation of mutations in a large German cohort with hearing loss. Eur Arch Otorhinolaryngol. 2015; 272:2765-76. DOI | PubMed
14. Pandey N, Xavier DF, Chatterjee A, Mani RS, Hiremagalore R, Tharakan A. Functional Analysis of a Novel Connexin30 Mutation in a Large Family with Hearing Loss, Pesplanus, Ichthyosis, Cutaneous Nodules, and Keratoderma. Ann Hum Genet. 2016; 80:11-9. DOI | PubMed
15. Alkowari MK, Vozzi D, Bhagat S, Krishnamoorthy N, Morgan A, Hayder Y. Targeted sequencing identifies novel variants involved in autosomal recessive hereditary hearing loss in Qatari families. Mutat Res. 2017; 800-802:29-36. DOI | PubMed
16. Nonose RW, Lezirovitz K, de Mello Auricchio MTB, Batissoco AC, Yamamoto GL, Mingroni-Netto RC. Mutation analysis of SLC26A4 (Pendrin) gene in a Brazilian sample of hearing-impaired subjects. BMC Med Genet. 2018; 19:73. Publisher Full Text | DOI | PubMed [ PMC Free Article ]
17. Amritkumar P, Jeffrey JM, Chandru J, Vanniya SP, Kalaimathi M, Ramakrishnan R. Role of DFNB1 mutations in hereditary hearing loss among assortative mating hearing impaired families from South India. BMC Med Genet. 2018; 19:105. Publisher Full Text | DOI | PubMed [ PMC Free Article ]
18. Schutz M, Scimemi P, Majumder P, De Siati RD, Crispino G, Rodriguez L. The human deafness-associated connexin 30 T5M mutation causes mild hearing loss and reduces biochemical coupling among cochlear non-sensory cells in knock-in mice. Hum Mol Genet. 2010; 19:4759-73. Publisher Full Text | DOI | PubMed [ PMC Free Article ]
19. Wang WH, Liu YF, Su CC, Su MC, Li SY, Yang JJ. A novel missense mutation in the connexin30 causes nonsyndromic hearing loss. PLoS One. 2011; 6:e21473. Publisher Full Text | DOI | PubMed [ PMC Free Article ]
20. Zhang Y, Hao H. Conserved glycine at position 45 of major cochlear connexins constitutes a vital component of the Ca(2)(+) sensor for gating of gap junction hemichannels. Biochem Biophys Res Commun. 2013; 436:424-9. DOI | PubMed
21. Berger AC, Kelly JJ, Lajoie P, Shao Q, Laird DW. Mutations in Cx30 that are linked to skin disease and non-syndromic hearing loss exhibit several distinct cellular pathologies. J Cell Sci. 2014; 127:1751-64. DOI | PubMed
22. Mammano F. Inner Ear Connexin Channels: Roles in Development and Maintenance of Cochlear Function. Cold Spring Harb Perspect Med. 2019; 9Publisher Full Text | DOI | PubMed [ PMC Free Article ]
23. Sommen M, Schrauwen I, Vandeweyer G, Boeckx N, Corneveaux JJ, van den Ende J. DNA Diagnostics of Hereditary Hearing Loss: A Targeted Resequencing Approach Combined with a Mutation Classification System. Hum Mutat. 2016; 37:812-9. DOI | PubMed
24. Liu YT, Guo K, Li J, Liu Y, Zeng WH, Geng SM. Novel mutations in GJB6 and GJB2 in Clouston syndrome. Clin Exp Dermatol. 2015; 40:770-3. DOI | PubMed
25. Fujimoto A, Kurban M, Nakamura M, Farooq M, Fujikawa H, Kibbi AG. GJB6, of which mutations underlie Clouston syndrome, is a potential direct target gene of p63. J Dermatol Sci. 2013; 69:159-66. DOI | PubMed
26. Villanelo F, Escalona Y, Pareja-Barrueto C, Garate JA, Skerrett IM, Perez-Acle T. Accessing gap-junction channel structure-function relationships through molecular modeling and simulations. BMC Cell Biol. 2017; 18:5. Publisher Full Text | DOI | PubMed [ PMC Free Article ]
27. Al Mughram MH, Catalano C, Herrington NB, Safo MK, Kellogg GE. 3D interaction homology: The hydrophobic residues alanine, isoleucine, leucine, proline and valine play different structural roles in soluble and membrane proteins. Front Mol Biosci. 2023; 10:1116868. Publisher Full Text | DOI | PubMed [ PMC Free Article ]