Background: Mutations in the USH2A gene are strongly associated with hereditary sensorineural loss. However, the cellular mechanisms linking USH2A deficiency to cochlear hair cell vulnerability remain incompletely understood. Aims: This study aimed to investigate the effects of USH2A knockdown on Prestin expression and apoptosisrelated signaling in a cochlear hair cell model. Methods: Stable USH2A knockdown HEI-OC1 cells were generated using lentiviral shRNA transduction followed by puromycin selection (2 mu g/mL for 7 days). Gene and protein expression levels of Prestin, Bax, and Bcl-2 were assessed by RT-qPCR (2<^>(-Delta Delta Ct) method), Western blotting, and immunofluorescence. Apoptosis was evaluated by flow cytometry (Annexin V/PI), TUNEL staining, and cleaved caspase-3 immunofluorescence. Results: USH2A knockdown reduced Prestin mRNA and protein expression by approximately 60% compared with the sh-NC group (P < 0.01). Bax expression increased more than 2.5-fold, whereas Bcl-2 levels decreased by similar to 50%, indicating disruption of apoptotic balance. Flow cytometry revealed increased early apoptotic (2.16% -* 4.58%) and late apoptotic (16.3% -* 22.0%) populations. TUNEL-positive cells increased from 2.8 +/- 0.6% to 12.4 +/- 2.1% (P < 0.01), and cleaved caspase-3 fluorescence intensity was approximately threefold higher in the knockdown group. Conclusion: USH2A knockdown significantly reduces Prestin expression and promotes apoptosis through modulation of the Bax/Bcl-2 axis in HEI-OC1 cells. These findings provide mechanistic insights into how USH2A deficiency may increase cochlear hair cell vulnerability and potentially contribute to auditory dysfunction. Further in vivo validation is warranted.

Olfactory dysfunction is highly prevalent in the elderly population, severely affecting nutritional status, environmental safety perception, and quality of life, and is recognized as an early warning marker of neurodegenerative diseases. Compared with populations living at low altitudes, residents at high altitudes are chronically exposed to combined extreme environmental conditions including hypoxia, low temperature, and low humidity, which may lead to more complex and severe degenerative changes in the olfactory system. However, existing evidence remains fragmented, and a systematic synthesis is lacking. This narrative review aims to systematically integrate the epidemiological characteristics, pathophysiological mechanisms, diagnostic assessment tools, intervention strategies, and health risks associated with olfactory decline in elderly residents at high altitudes. Epidemiological evidence shows a significant negative correlation between altitude and olfactory function, and the effects of aging are substantially amplified under high-altitude conditions. The underlying mechanisms primarily involve chronic hypoxia interacting with aging and genetic susceptibility through damage to olfactory ensheathing cells, suppression of olfactory neurogenesis, and remodeling of synaptic plasticity in olfactory-related brain regions. Olfactory behavioral testing combined with functional magnetic resonance imaging and emerging biomarkers provides multidimensional tools for early diagnostic stratification. Although oxygen therapy, olfactory training, and environmental management have shown potential efficacy, evidence-based data specific to this population remain limited. The core value of this review lies in adopting an integrated perspective of "environment-aging" interactions to systematically elucidate the combined effects of high-altitude stress and age-related degenerative changes on the olfactory system, revealing the unique patterns and intervention targets for olfactory dysfunction in this vulnerable population. Future research urgently needs large-scale longitudinal cohort studies, establishment of altitude-specific normative olfactory assessment tools, and development of precision interventions targeting hypoxic injury to improve overall health outcomes in this population.

Background:Usher syndrome (USH) is an autosomal recessive disorder characterized by hearing loss, retinitis pigmentosa, and variable vestibular dysfunction. USH2A is one of the causative genes of USH. This study is aimed at exploring the mechanism of hearing loss induced by USH2A gene knockout. Method:USH2A knockout (Ush2a-/-) mice were used, and auditory brainstem response testing was performed on WT, Ush2a-/-, and Ush2a+/- mice. Then, the cochlea tissues were used to carry out immunofluorescence staining, hematoxylin and eosin (H&E) staining, and scanning electron microscopy (SEM). The mRNA expressions were detected by RT-qPCR. Finally, the differentially expressed genes (DEGs) in cochlear tissues of Ush2a-/- and WT mice were identified by transcriptome sequencing. Results:Compared to WT mice, Ush2a-/- and Ush2a+/- mice exhibited moderate-to-severe nonprogressive hearing loss, with more pronounced deficits at low (4 kHz) and high (32/24 kHz) frequencies. HE staining and immunofluorescence staining showed that the modiolus, stria vascularis, basilar membrane, and the number of inner hair cells and outer hair cells (OHCs) in USH2A knockout mice have not changed. However, SEM results showed that severe stereociliary collapse was evident in OHCs of the Ush2a-/- group. In addition, through transcriptomic analysis, 3632 upregulated genes and 2921 downregulated genes were obtained in the Ush2a-/- mice. Among these DEGs, the most DEGs associated with hearing loss were Scn2a, Shank2, Bsn, Fcer1g, Prkce, Tgfb1, and Irf7. Conclusion:This study demonstrates that USH2A deficiency disrupts auditory function through stereociliary instability and dysregulation of genes critical for synaptic transmission and cytoskeletal dynamics.

Pathogenic variants in the LIM-homeodomain transcription factor LMX1A represent a rare yet critical etiology for autosomal dominant nonsyndromic hearing loss 7 (DFNA7) and less frequently, its autosomal recessive counterpart (ARNSHL). Here, we describe a novel heterozygous frameshift variant, LMX1A c.405delT (p.Phe135LeufsTer3), identified in a three-generation Chinese family, cosegregating with progressive and asymmetric sensorineural hearing loss (ASNHL). Clinical manifestations exhibited significant intrafamilial phenotypic variability, with hearing loss (HL) severity ranging from mild to profound, and onset varying from infancy to mid-adulthood. High-resolution imaging revealed bilateral cochlear aperture stenosis (CAS) in the severely affected proband. Whole-exome sequencing (WES) and cosegregation analysis confirmed this novel variant. Structural modeling predicted the truncation of both the DNA-binding homeodomain and the C-terminus. Subsequent reporter assays demonstrated a significant loss of transcriptional activity. Furthermore, plasmid titration experiments and Actinomycin D chase assays functionally corroborated the haploinsufficiency mechanism and excluded the dominant-negative effect. Integrative multiomics profiling (RNA-seq and DIA-based proteomics) of in vitro HEI-OC1 model revealed molecular perturbations following Lmx1a deficiency, primarily involved in synaptic signaling and immune-inflammatory cascades. This study broadens the LMX1A mutational landscape, refines the clinical phenotypic spectrum of DFNA7 and establishes insufficient LMX1A dosage as the primary disease driver.

MYO15A gene, located on chromosome 17p11.2, is a major causative gene for autosomal recessive non-syndromic hearing loss (ARNSHL). It encodes myosin XVa (MYO15A), a motor protein critical for stereocilia elongation and staircase-architecture formation in cochlear and vestibular hair cells. MYO15A exhibits highly specific subcellular localization at stereociliary tips, positioning it as an ideal candidate for targeted gene therapy. Gene therapy, which involves correcting defective genes or introducing functional gene sequences, has emerged as a highly promising approach for treating hereditary hearing loss in clinical trials, particularly for specific genetic mutations. While clinical trials have demonstrated the efficacy of adeno-associated virus (AAV)-mediated gene therapy for OTOF-related HL, therapeutic strategies for MYO15A mutations remain in the ‌preclinical stage‌, primarily utilizing mouse models (e.g., MYO15A p.R819*). ‌This review elaborates current understanding of MYO15A-related pathogenesis, summarizes advances in MYO15A mutant mouse models and their therapeutic interventions, and discusses evolving ‌preclinical‌ gene therapy approaches to address this genetically heterogeneous disorder.

GJB2 gene is a common pathogenic gene for non-syndromic hearing loss, located on chromosome 13q12.11, and primarily encodes connexin 26 (Cx26). Cx26, a member of the gap-junction protein family, is mainly expressed in the supporting cells of the cochlea, where it is responsible for intercellular material transfer and signal exchange. Gene therapy, a treatment method that repairs or reconstructs genetic material, has emerged as the most effective approach for hereditary hearing loss. During the initial stages of exploration, researchers need to conduct animal experiments first. By elucidating the mechanisms of GJB2 gene-induced congenital hearing loss, we summarize the commonly used experimental animals (zebrafish, mice) for current research on the Gjb2 gene, and further promote the advancement of gene therapy strategies.

Objective:This study aims to delineate the mechanisms through which intraperitoneal injection of gentamicin (GEN) influences the inner ear cells of mice by employing single-cell RNA sequencing (scRNA-seq) technology. Methods:Eight-week-old Kunming mice were randomly assigned to three groups: a normal control group, a GEN group, and a GEN + dexamethasone (DEX) group. The mice received continuous intraperitoneal injections of the corresponding drugs for 10 days. Auditory brainstem response (ABR) was assessed to evaluate hearing threshold shifts, and cochlear tissues were harvested for scRNA-seq. The Seurat analysis workflow was employed for data quality control, dimensionality reduction clustering, and differential gene expression analysis. Results:ABR results demonstrated a significant elevation in hearing thresholds in the GEN group, whereas thresholds in the DEX group showed improvement but remained elevated compared to the NOR group (P < 0.05). Single-cell sequencing revealed notable alterations in the populations of outer hair cells, supporting cells, and immune cells in the GEN group. Analysis of differentially expressed genes identified significant downregulation of cell-specific genes Gbp6, Ppfia4 in hair cells of the GEN group, alongside upregulation of inflammation-related genes Nnat, Gh, indicating that hair cell damage and enhanced immune responses may be pivotal mechanisms underlying GEN-induced ototoxicity. Conclusion:Utilizing scRNA-seq technology, this study uncovers substantial transcriptional changes induced by GEN in cochlear hair cells, supporting cells, and immune cells in mice, highlighting the role of inflammation and oxidative stress, TNF signaling pathways in its ototoxicity. DEX partially ameliorates hair cell damage.

The KCNQ4 gene is closely associated with autosomal dominant nonsyndromic hearing loss (DFNA2) and encodes a potassium channel crucial for potassium ion circulation in the inner ear. This study identified a novel KCNQ4 variant, c.825G>T (p.Trp275Cys), associated with progressive hereditary hearing loss in a family. In vitro experiments and structural predictions revealed that this mutation did not affect KCNQ4 channel localization, subunit assembly, or pore size. However, the mutation induced longitudinal extension of the channel, reduced protein stability, and impaired potassium ion selectivity, thereby disrupting potassium ion homeostasis in the inner ear and ultimately leading to hearing loss. Zebrafish models further validated the critical role of the kcnq4 gene in inner ear development. We used morpholino to knock down kcnq4 in zebrafish and rescued the phenotype by reintroducing wild-type kcnq4 mRNA. This approach revealed significant changes in otolith morphology, a marked reduction in hair cell numbers, and abnormal motor responses. Additionally, we cloned and completed the coding sequence (CDS) of kcnq4 mRNA from the AB strain of zebrafish, enriching the available database information. Our findings provide new insights into the molecular mechanisms underlying KCNQ4-related hereditary hearing loss and lay the groundwork for developing precision treatments and early intervention strategies.

Presbycusis, or age-related hearing loss (ARHL), is a prevalent sensory disorder in the elderly, driven by genetic factors, oxidative stress, inflammation responses, and cellular senescence. Despite its significance, the molecular mechanisms underlying ARHL remain poorly defined. In this study, we employed bioinformatic analysis of public gene expression datasets to identify differentially expressed genes in ARHL. Protein-protein interaction network analysis further nominated LCN2 as a hub gene. Experimental validation in aging C57BL/6J mice and HEI-OC1 auditory cells revealed that elevated LCN2 expression promotes cellular senescence, while its knockdown delays this phenotype. Mechanistically, LCN2 drives senescence by activating the NF-κB signaling pathway, and its inhibition alleviates senescence induced by tert-butyl hydroperoxide (TBHP). Our findings establish LCN2 as a key pro-senescence factor in ARHL and demonstrate that it regulates auditory cell senescence through the NF-κB pathway, providing new mechanistic insights and revealing potential therapeutic targets for ARHL intervention.

OBJECTIVE:This study examined the mutation spectrum and frequency of three prevalent pathogenic genes in patients with non-syndromic hearing loss (NSHL) from Gansu Province, China. METHODS:We analyzed 452 NSHL patients from five special education schools across several cities in Gansu Province using SNPscan technology to determine the mutation spectrum of mtDNA 12S rRNA, GJB2, and SLC26A4 genes. RESULTS:Among the 452 patients evaluated, mutations in the GJB2 gene were observed in 91 cases (20.13 %), mutations in the SLC26A4 gene in 81 cases (17.92 %), and homoplasmic mutations in mtDNA 12S rRNA in 26 cases (5.75 %). Significant differences in GJB2 mutations were observed between Han patients and those of Hui, Tibetan and Mongolian ethnicity (χ2 = 4.554, p = 0.033; χ2 = 3.987, p = 0.046; χ2 = 4.041, p = 0.044), as well as in SLC26A4 gene mutations between Han patients and both Hui and Tu patients (χ2 = 4.247, p = 0.039; p = 0.035, two-sided). MT-RNR1 mutations were exclusively identified in Tibetans, Han, and Hui patients. CONCLUSION:Our findings demonstrate variations in the mutation spectra of the GJB2, SLC26A4, and mtDNA 12S rRNA genes across different ethnic groups, highlighting ethnic variations in mutation prevalence. This study expands the understanding of the genetic mutation spectrum associated with deafness in Gansu and supports the enhancement of molecular diagnostic accuracy for diverse ethnic populations in the region.

BACKGROUND:Hearing loss, a major public health issue, affects 1.33 per 1,000 live births worldwide. Genetic factors contribute to over half of congenital cases, with X-linked inheritance accounting for 1-5%. POU3F4 mutations are associated with approximately 50% of X-linked non-syndrome hearing loss cases. POU3F4 plays a critical role in cochlear development by regulating otic mesenchyme cell differentiation. The study investigates the impact of a novel POU3F4 p.E294G mutation on cochlear structure and function using cellular and animal model. METHODS:The study utilized immortalized lymphoblastoid cell lines, POU3F4 overexpressed HEK293 cells and generated Pou3f4 knock-in (Pou3f4KI) mice via CRISPR/Cas9 to introduce the p.E294G mutation. Alterations in expression and subcellular localization of POU3F4 were detected at the cellular level. Auditory function was assessed using auditory brainstem response testing. Cochlear structure was analyzed through histology, immunohistochemistry, scanning electron microscopy, and transmission electron microscopy. RNA sequencing, qPCR and Western blot were conducted to evaluate gene expression and mitochondrial function. RESULTS:The transcription of POU3F4 was abnormal and the expression was normal in lymphoblastoid cell lines. Abnormal nuclear localization of POU3F4 p.E294G was found in overexpressed HEK293 cells. Pou3f4KI mice exhibited cochlear malformations, including modiolus hypoplasia and reduced stria vascularis cell populations. Auditory testing revealed progressive hearing loss. Pou3f4 affect mitochondrial protein expression by affecting the expression of TFAM. Mitochondrial dysfunction was evident, with reduced oxidative phosphorylation (OXPHOS) complex assembly and activity, decreased ATP levels. The level of reactive oxygen species, mitochondrial fission and apoptosis in cochlea were elevated. CONCLUSIONS:The POU3F4 p.E294G resulted in abnormal nuclear localization. Pou3f4 mutant disrupts cochlear development and function, impairs mitochondrial integrity, induces oxidative stress, and promotes apoptosis, leading to progressive hearing loss. The findings enhance the understanding of POU3F4-related hearing loss mechanisms and highlight the importance of early genetic screening and audiological monitoring.

RATIONALE:Retropharyngeal abscesses (RPAs) following fish bone ingestion are extremely rare in infants under 2 years, with limited cases reported worldwide. Given the anatomical vulnerability and diagnostic challenges faced by infants, this gap in knowledge is critical. No previous reports have described life-threatening delayed RPA caused by endoscopic pharyngeal injury without a retained foreign body, which is a critical omission in the postoperative safety protocols. Our study details a life-threatening delayed RPA caused by posterior pharyngeal wall injury after endoscopic removal of a fish bone, highlights the critical role of postoperative imaging, which is frequently overlooked in pediatric practice, and contributes to the sparse literature on severely delayed RPAs in infants after fish bone ingestion. PATIENT CONCERNS:An 18-month-old girl presented with 6 months of recurrent hoarseness, stridor, and progressive respiratory distress. DIAGNOSES:The diagnostic evaluations included laboratory studies and imaging examinations, and imaging revealed a large RPA causing significant tracheal compression and pneumonia. INTERVENTIONS:Emergency management included intubation, mechanical ventilation, broad-spectrum antibiotics, and surgical drainage. OUTCOMES:Surgical drainage and targeted antibiotic therapy resolved inflammation. LESSONS:This novel case of delayed RPA after endoscopic foreign-body removal emphasizes the need for postoperative imaging, urgent surgical intervention, and enhanced caregiver education to prevent infant complications.

ABSTRACTBackgroundMYO15A is one of the common genes of severe‐to‐profound sensorineural deafness. Mutations in this gene can cause both pre‐ and post‐lingual hearing losses. In this study, a novel MYO15A variant (c.2482C>T) was identified to be associated with autosomal recessive non‐syndromic hearing loss (ARNSHL) in a Chinese Uighur family.MethodsTo examine the effects of the MYO15A mutation on the morphology and function of the derived hair cell‐like cells, two iPSCs were generated separately from the proband and a mutation‐negative family member and those were then induced to hair cell‐like cells.ResultsResults showed that this homozygous MYO15A mutation (PVS1 + PM2 + PP1 + PP3), which is located in the N‐terminal domain, displayed significant differences in the morphology and function of hair cell‐like cells between the proband and the normal control, although it had no effect on the totipotency of iPSCs.ConclusionOur study demonstrates that the novel variant c.2482C>T in the MYO15A gene may cause inner ear hair cell dysfunction and audiological disorders in this family.

Large vestibular aqueduct syndrome（LVAS） is a common recessive hereditary hearing loss disease, and some patients may also experience vestibular dysfunction. With the wide application of cochlear implant（CI） and the development of vestibular medicine, the pathophysiological mechanism of LVAS and the influence mechanism of CI on vestibular function are gradually elucidated. Consequently, the evaluation and rehabilitation of vestibular dysfunction function have also become research hotspots. This article reviews studies on vestibular function and related rehabilitation in patients with large vestibular aqueduct syndrome.

Defects in mitochondrial RNA metabolism have been linked to sensorineural deafness that often occurs as a consequence of damaged or deficient inner ear hair cells. In this report, we investigated the molecular mechanism underlying a deafness- associated tRNA(Phe) 593T > C mutation that changed a highly conserved uracil to cytosine at position 17 of the DHU-loop. The m.593T > C mutation altered tRNA(Phe) structure and function, including increased melting temperature, resistance to S1 nuclease-mediated digestion, and conformational changes. The aberrant tRNA metabolism impaired mitochondrial translation, which was especially pronounced by decreases in levels of ND1, ND5, CYTB, CO1, and CO3 harboring higher numbers of phenylalanine. These alterations resulted in aberrant assembly, instability, and reduced activities of respiratory chain enzyme complexes I, III, IV, and intact supercomplexes overall. Furthermore, we found that the m.593T > C mutation caused markedly diminished membrane potential, and increased the production of reactive oxygen species in the mutant cell lines carrying the m.593T > C mutation. These mitochondrial dysfunctions led to the mitochondrial dynamic imbalance via increasing fi ssion with abnormal mitochondrial morphology. Excessive fi ssion impaired the process of autophagy including the initiation phase, formation, and maturation of the autophagosome. In particular, the m.593T > C mutation upregulated the PARKIN-dependent mitophagy pathway. These alterations promoted an intrinsic apoptotic process for the removal of damaged cells. Our fi ndings provide critical insights into the pathophysiology of maternally inherited deafness arising from tRNA mutation-induced defects in mitochondrial and cellular integrity.

Background:Aminoacyl-tRNA synthetases are highly conserved proteins that catalyze the tRNA aminoacylation reaction to produce aminoacyl-tRNAs involved in protein synthesis, which are required to translate cytoplasmic and mitochondrial proteins. The mt-ARS genes encode the mitochondrial aminoacyl-tRNA synthetase (mt-ARSs), and variants in mt-ARS genes affect mitochondrial protein synthesis. This can impair the translation of mitochondrial proteins, adversely affecting oxidative phosphorylation and leading to related diseases. To date, 19 mt-ARS genes have been identified and found to be strongly associated with the development of mitochondrial disorders. Hearing loss (HL) is one of the most common chronic conditions in children and a leading cause of communication disorders. Genetic studies of sensorineural HL are critical to diagnosing and treating sensorineural HL. The relationship between mt-ARS genes and sensorineural HL is gradually surfacing as cases of HL phenotypes caused by variants in the mammalian mt-ARS genes continue to be reported. Seven mt-ARS genes have been reported to contribute to various hereditary sensorineural HL. Summary:This article reviews studies on mitochondrial aminoacyl-tRNA synthetase, mt-ARS genes, and variants associated with HL phenotypes. Investigating their genetic characteristics provides deeper insights into the pathophysiology and molecular mechanisms of sensorineural hearing loss. Key Messages:Disease phenotypes resulting from variants in mt-ARS genes exhibit significant clinical heterogeneity. The varying degrees of sensorineural HL phenotypes caused by mt-ARS gene variants warrant the attention of otologists and researchers. At least seven of the currently reported mt-ARS genes are associated with sensorineural HL. However, the molecular mechanisms by which these genes contribute to HL remain incompletely understood. Further studies of the mt-ARS genes still await additional case reports, as well as related model animal studies and combined functional studies.

Objective: To investigate the consistency between the hearing handicap inventory (HHI) and pure-tone audiometry (PTA) scores in assessing hearing status to provide valuable insights for clinical application. Methods: Retrospective analysis of clinical data and the HHI reporting status of 6540 patients admitted between April 2020 and July 2022 for self-reported unilateral hearing loss who met the study inclusion and exclusion criteria. The kappa coefficient was used to evaluate the consistency of HHI and PTA in assessing the hearing status of the participants. Results: The PTA results showed that among the 6540 participants, 3895 exhibited normal hearing, 1434 showed mild hearing loss, 809 presented with moderate hearing loss, and 402 showed severe hearing loss. The mean hearing thresholds from 0.5 to 4 kHz in healthy ears ranged from 3.65 to 18.45 dB HL, with a mean of 10.83 +/- 5.29 dB HL; in ears affected by hearing loss, this ranged from 35 to 125 dB HL, with a mean of 69.63 +/- 28.45 dB HL. The HHI scores showed that 4820 people had normal hearing, 1245 had mild-to-moderate hearing loss, and 475 had severe hearing loss. The kappa coefficients of normal, mild-to-moderate, and severe hearing loss were 0.312, 0.223, and 0.716, respectively (P = .001). The consistency between the 2 groups was particularly significant in the assessment of severe hearing loss. Using the PTA results as a benchmark, the sensitivity, specificity, positive predictive value, and negative predictive value of the HHI were found to be 73.08%, 87.83%, 95.60%, and 70.98%, respectively. Conclusion: The HHI and PTA results were consistent in the assessment of hearing status, particularly in the assessment of severe hearing loss, and the level of consistency between the 2 methods was high. The combined use of these tools can facilitate a comprehensive assessment of the auditory status of patients with hearing loss.

Abstract Background Usher syndrome type 3 (USH3) is an autosomal recessive inherited disorder caused by pathogenic variants in the CLRN1 gene. Object To evaluate the genotype-phenotype correlation of Usher syndrome type 3 (USH3) in a deaf-blind Chinese family of 3 generations with 2 patients. Methods We collected blood samples and clinical data from all of the pedigree family members. Genomic DNA was isolated from peripheral leukocytes using standard method. Targeted next generation sequencing and Sanger sequencing were performed to find the pathogenic variants in this family. Digital PCR and plasmid overexpression assay were used to verify the pathogenicity of variant sites in different transcripts. Results All patients developed bilateral sensorineural hearing loss (SHL), progressive vision loss and nyctalopia. NGS of genes for Usher syndrome, deafness and retinal dystrophy identified a locus mutation in CLRN1 that caused completely different amino acid changes in different transcripts[CLRN1:c.474T > A(P.Cys158Ter) at NM_001256819.2 or c.302T > A(p.Val101Asp) at NM_174878.3], and plasmid overexpression experiments confirmed that the c.474T > A(P.Cys158Ter, NM_001256819.2) was a pathogenic variant which has never been associated with Usher syndrome in China, and the transcript of this mutation was not the version commonly found worldwide. Conclusions The CLRN1c.474T > A(NM_001256819.2) mutation is the causative variant in the Chinese family with USH3. The pathogenicity of different transcripts should be particularly considered in pathogenicity analysis.

Abstract Objective We aimed to evaluate the genotype–phenotype relationship in two Chinese family members with enlarged vestibular aqueduct (EVA). Methods We collected blood samples and clinical data from each pedigree family member. Genomic DNA was isolated from peripheral leukocytes using standard methods. Targeted next‐generation sequencing and Sanger sequencing were performed to find the pathogenic mutation in this family. Minigene assays were used to verify whether the novel intronic mutation SLC26A4c.765+4A>G influenced mRNA splicing. Results Hearing loss in the patients with EVA was diagnosed using auditory tests and imaging examinations. Two pathogenic mutations, c.765+4A>G and c.919‐2A>G were detected in SLC26A4. In vitro minigene analysis confirmed that c.765+4A>G variant could cause aberrant splicing, resulting in skipping over exon 6. Conclusions The SLC26A4c.765+4A>G mutation is the causative variant in the Chinese family with EVA. Particular attention should be paid to intronic variants.