To investigate genetic etiologies of conductive/mixed hearing loss (CHL/MHL) and guide management through genotype–phenotype correlations. Retrospective study of 98 patients (29 syndromic, 69 non-syndromic) with CHL/MHL. Clinical evaluations and next-generation sequencing were performed. Surgical outcomes and familial patterns were analyzed. Syndromic cases showed high genetic confirmation (96.6

Accurate quantitative assessment of temporal bone microanatomy is essential for otologic research and surgical planning. However, existing measurement approaches, including manual tools, ex vivo microscopy, and high-resolution computed tomography, are limited by contact requirements, indirect measurement, insufficient spatial resolution, or the inability to provide efficient quantitative feedback under microscopic visualization. To address these limitations, this study developed an in situ, non-contact three-dimensional (3D) microscopic measurement system for temporal bone anatomy based on stereo imaging. The system integrates microscopic stereo calibration, deep learning-based disparity estimation, 3D reconstruction, and an interactive measurement workflow. Reconstruction accuracy was evaluated using a high-precision scanned LEGO model, while measurement accuracy was validated using a custom-designed 3D-printed module with known geometric dimensions. The feasibility of the system was further demonstrated through measurements of representative anatomical structures in four temporal bones from two fresh-frozen cadaver heads. The results showed that the root mean square error of 3D reconstruction was 0.05-0.13 mm. For geometric measurements, the average error was below 1.7%, with a maximum error below 4.0% across multiple metrics. The system enabled in situ, non-contact measurement of various anatomical parameters under microscopic conditions. These findings indicate that the proposed system provides accurate and reliable quantitative 3D measurement of temporal bone microstructures. As a technical prototype, it provides a practical tool for anatomical research and supports future development toward intraoperative, non-contact, in situ quantitative assessment in otomicrosurgery.

Background Hearing loss (HL) is genetically heterozygous, making its genetic diagnosis challenging. Identification of novel HL-associated genes and variants will enhance our understanding of the molecular mechanisms and improve genetic diagnosis. TMPRSS3, encoding a transmembrane serine protease, is implicated in autosomal recessive nonsyndromic hearing loss (ARNSHL), designated as DFNB8 (postlingual onset) or DFNB10 (prelingual onset). Although over 100 pathogenic TMPRSS3 variants have been reported, only seven splice-site variants have been documented to date. Aims/objectives This study investigates the molecular etiology of ARNSHL in three Chinese family and functionally characterizes five novel TMPRSS3 variants, including one non-canonical splice-site variant, two canonical splice-site variants, and two missense variants. Material and methods Whole-exome sequencing and gene panel sequencing identified candidate variants, followed by validation with Sanger sequencing. Functional analyses included minigene splicing assays to evaluate the variants' effect on mRNA splicing and a yeast-based functional assay to assess their impacts on protease activity. Results Three families carrying compound heterozygous TMPRSS3 variants were identified. The proband with c.205 + 5G>C/c.923T>C (p.Met308Thr) presented with progressive, postlingual, high-frequency-predominant, nonsyndromic sensorineural hearing loss, consistent with DFNB8. Probands with c.572 + 1G>A/c.967G>A (p.Val323Met) and c.1348-2A>G/c.271C>T (p.Arg91Ter) exhibited prelingual, nonsyndromic sensorineural hearing loss. Functional studies revealed that all five novel variants (c.205 + 5G>C, c.923T>C, c.572 + 1G>A, c.967G>A, and c.1348-2A>G) led to reduced protease activity. Notably, the splice-site variants caused exon skipping and resulted in a more pronounced loss of activity compared to the missense variants. Conclusion and significance This study expands the spectrum of TMPRSS3 variants and provides mechanistic insights into their pathogenicity. Intriguingly, splice-site variants led to a more severe impairment of enzymatic activity compared to missense variants, providing molecular evidence for the considerable genotype-phenotype heterogeneity observed in TMPRSS3-associated hearing loss.

PURPOSE:Accurate subtype classification of inner ear malformations (IEMs) on temporal bone high-resolution computed tomography (HRCT) is important for preoperative risk assessment and surgical planning, but depends heavily on subspecialty expertise. This study aimed to develop and externally validate a deep learning model for multiclass IEM diagnosis on temporal bone HRCT. METHODS:We developed and externally validated a weakly supervised deep learning framework for one-stage classification of automatically extracted temporal bone regions of interest from HRCT in a multicenter cohort of 9,182 ears (3,161 IEMs and 6,021 normal ears). The framework was based on an improved Transformer-based multiple instance learning architecture. Performance was evaluated on internal and independent external datasets. An independent reader study compared the model with five senior and five junior otolaryngologists. Explainability was assessed using attention-based slice ranking and Grad-CAM. The study was approved by an institutional review board, and informed consent was waived. RESULTS:On the independent external test set, the proposed model demonstrated strong multiclass performance, with an accuracy of 93.9 % (95 % CI, 92.4 %-95.3 %), a macro-F1 score of 88.2 % (95 % CI, 84.3 %-91.4 %), and a multiclass AUC of 0.990 (95 % CI, 0.985-0.995). Performance was consistent on the internal validation set, with an accuracy of 96.5 % (95 % CI, 95.6 %-97.4 %), a macro-F1 score of 87.5 % (95 % CI, 83.0 %-91.0 %), and a multiclass AUC of 0.994 (95 % CI, 0.989-0.998). In the reader study, the model obtained an accuracy of 94.0 % and a macro-F1 score of 89.9 %, outperforming both senior otolaryngologists (mean macro-F1, 75.7 %, Holm-adjusted p = 0.0046) and junior otolaryngologists (mean macro-F1, 62.9 %, Holm-adjusted p < 0.001). The model also provided interpretable outputs by identifying the top three highest-contributing slices and generating Grad-CAM heatmaps. CONCLUSION:This weakly supervised framework enabled accurate and generalizable subtype-level diagnosis of IEMs on temporal bone HRCT in a large multicenter cohort and may serve as a practical decision-support tool for standardized preoperative assessment, particularly in settings with limited subspecialty expertise.

Abstract Pathogenic variants in ATP6V1B2, which encodes a critical subunit of vacuolar-type H+-ATPases (V-ATPases), disrupt lysosomal acidification via haploinsufficiency and clinically manifest as intellectual disability and seizure disorders. Despite significant morbidity, mechanism-based therapies remain an unmet need. Through integrated clinical analysis of a Chinese cohort and systematic literature review, we delineated genotype-phenotype correlations in ATP6V1B2-related syndromes. Isogenic HEK293T models (ATP6V1B2 R506X/+ and ATP6V1B2 R506X/R506X) were generated using CRISPR/Cas9 for dynamic lysosomal pH monitoring via ratiometric RpH-LAMP1-3×flag imaging to evaluate pathophysiological mechanisms. Parallel investigations in Atp6v1b2 R506X/R506X mice incorporated continuous video-EEG monitoring, behavioral assessments, western blot analyses, and transmission electron microscopy to evaluate therapeutic responses. Drug concentrations in plasma and brain homogenates were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Clinical analysis revealed central nervous system manifestations (epilepsy, intellectual disability, developmental delay) as primary morbidity determinants. Cellular studies demonstrated significant increase of lysosomal pH in mutant cells compared to wild-type control. Remarkably, treatment with the cAMP analog CPT-cAMP restored lysosomal acidification in a concentration-dependent manner. In vivo studies confirmed spontaneous seizure activity in mutant mice and CPT-cAMP’s penetration of the BBB was confirmed by LC-MS/MS. Intraperitoneal CPT-cAMP administration (20 mg/kg) exerted triple therapeutic effects: (1) significant reduction in seizure frequency, (2) improved cognitive performance in behavioral paradigms, and (3) restoration of autophagic flux through resolution of autophagosome accumulation. These findings establish proof-of-concept for cAMP-mediated lysosomal pH modulation as a viable therapeutic strategy. Our results position CPT-cAMP as a promising candidate for addressing both neurological and cognitive manifestations in ATP6V1B2-related disorders.

Objective:To evaluate the perioperative safety and long-term complications of simultaneous bilateral cochlear implantation（BCI） in young infants, providing reference data for clinical BCI in young children. Methods:Seventy-four infants aged 6-23 months with congenital severe to profound sensorineural hearing loss who were candidates for cochlear implantation at the Department of Otolaryngology, Chinese PLA General Hospital between August 2018 and August 2019 were consecutively enrolled. Parents made the decision to implant either unilaterally or bilaterally. Participants were divided into unilateral cochlear implantation（UCI） group（before and after 12 months of age） and simultaneous BCI group（before and after 12 months of age）. Safety indicators, including perioperative risk variables, complications, and other postoperative adverse events were monitored, with complications followed up for 5-6 years. Comparisons were made between the BCI and UCI, as well as between implantation before and after 12 months of age regarding perioperative safety and long-term complications. Results:A total of 40 BCI patients（23 before 12 months, 17 after 12 months） and 34 UCI patients（20 before 12 months, 14 after 12 months） were included in the study. Regarding perioperative risk variables, the BCI group showed significantly longer anesthesia duration, operative time, and greater blood loss compared to the UCI group, though less than twice that of the UCI group; no anesthetic complications occurred in either group; and there was no significant difference in postoperative hospital stay between the groups. Regarding surgical complications during the 5-year follow-up period, the BCI group experienced 7 complications（2 major, 5 minor）, while the UCI group had 7 complications（1 major, 6 minor）, with no statistical differences between groups. Regarding other postoperative adverse events, the BCI group demonstrated significantly higher total adverse event rates than the UCI group（80.0% vs 38.2%）, with higher rates of moderate to severe anemia（60.0% vs 20.6%） and lower mean hemoglobin levels[（92.35±12.14） g/L vs（102.39±13.09） g/L]. No significant differences were found in postoperative fever rates（50.0% vs 52.9%） or C-reactive protein levels between groups. Within the BCI group, patients implanted before 12 months indicated notably higher rates of total adverse events（91.3% vs 64.7%）, high fever（26.1% vs 0）, and moderate to severe anemia（78.3% vs 35.3%） compared to those implanted after 12 months. Conclusion:Simultaneous BCI in young children under 2 years of age demonstrates controllable overall risks. Compared to UCI, while it shows no increase in anesthetic or surgical complications, it presents higher perioperative risks and adverse event rates, especially in patients implanted before 12 months of age, warranting special attention from medical staff.

Genetic variants, many of which are in mitochondrial DNA (mtDNA), contribute to hearing loss. Screening for these variants facilitates the identification of potential carriers, as many people with these mutations do not show hearing loss at birth but show late-onset hearing loss. We conducted a population-based cohort study involving 180,458 neonates born in Beijing, China. Hearing screening was performed through detailed counseling and systematic health assessment. The patient's peripheral blood was collected, and the variants was screened by next generation sequencing. An investigation of 142 probands and their matrilineal family members indicated a homoplasmic or heteroplasmic mtDNA mutation (adenine-to-guanine mutation at position 1555 in mtDNA 12S rRNA [A1555G] or cytosine-to-thymine mutation at position 1494 in mtDNA 12S rRNA) incidence of 0.227% (409 individuals). In total, 71.8% of the probands carried the homoplasmic A1555G mutation, 21.1% had the heteroplasmic A1555G mutation, and the remaining 7.1% had the homoplasmic cytosine-to-thymine mutation at position 1494 in mtDNA 12S rRNA mutation. A mtDNA haplotype analysis showed that 50.8% of the cases belonged to haplogroup D, the predominant haplotype in this Chinese population. Individuals with haplogroups B and M, which accounted for 10.7% and 9.0% of all cases, respectively, tended to have lower hearing thresholds at higher frequencies. We found no significant difference in the rate of hearing loss between vaccinated and unvaccinated individuals with mtDNA mutations, suggesting that the amounts of aminoglycoside antibiotics contained in vaccines were insufficient to induce hearing loss. This study reveals the incidence of mtDNA variants in the largest population studied to date and establishes that carriers of mtDNA variants can safely receive vaccines with no or low aminoglycoside antibiotic levels. This study provides a paradigm for studying the impact of these mtDNA variants on disease pathogenesis and the effects of mediation strategies.

The extracellular matrix（ECM）is a complex network structure composed of collagen，glycoproteins，and proteoglycans.It not only provides structural support and viscoelasticity to tissues but also participates in cell signaling，responding to environmental forces and signals to mediate tissue remodeling in response to environmental cues. Due to the intricate and precise functions of the inner ear，the perception and transmission of sound rely on the complex interactions between cochlear cell structures and the ECM. In the inner ear，the ECM not only constitutes key structures such as the basilar membranes（BM）and tectorial membranes（TM），which are essential for sound perception，but also regulates cell shape，adhesion，and migration.Certain ECM components interact with cell surface receptors to activate signaling pathways that regulate gene expression.Additionally，the ECM modulates the storage and diffusion of ions and secreted factors, creating concentration gradients.These functions are critical for inner ear development，repair，and function.Thus，the ECM plays a vital role in auditory processes，and abnormalities in ECM are a cause of certain hereditary hearing loss.This review primarily summarizes the ECM genes that lead to hearing loss.

Importance:Enlarged vestibular aqueduct (EVA), the most prevalent inner ear malformation causing hearing loss (HL) in various populations, is predominantly genetically mediated. Despite advancements in genetic diagnostics, the comprehensive phenotypic and genotypic spectrum of EVA remains insufficiently characterized. Objectives:To characterize the natural history, clinical outcomes, phenotype, and genotype of EVA. Design, Setting, and Participants:This single-center, longitudinal, retrospective cohort study was conducted from March 2003 to October 2022, with follow-up until July 1, 2024. Patients with EVA who were seeking medical advice at the Chinese PLA General Hospital were included. Main Outcomes and Measures:This study presents a 21-year longitudinal analysis of Chinese patients with EVA, providing a systematic analysis of the natural history, phenotypic diversity, and molecular etiology of EVA. Results:Of 2774 patients, 1453 (52.4%) were female individuals, and the median (range) age was 8 (4 months to 45 years) years. This study identified that 124 of 341 patients (36.36%) with EVA received passing newborn hearing screening results, while 375 of 597 (62.8%) received a diagnosis through combined audiological and radiological assessments. Recurrent vertigo (256 of 597 [42.9%]) and goiter (38 of 597 [6.4%]) were common comorbidities. Genetic analysis revealed that 2661 of 2774 patients (95.9%) carried biallelic SLC26A4 variants, with 70 (2.5%) attributable to copy number variants and 13 (0.5%) to a deep-intronic variant (c.304 + 941C>T) that affected splicing. A de novo heterozygous FOXI1 variant (c.483_485delCAA) was identified in an EVA family, indicating an autosomal dominant inheritance pattern. A stepped genomic analysis strategy was associated with an improved molecular diagnosis rate of 95.9%, highlighting the necessity of comprehensive genetic testing beyond traditional coding regions. Conclusions and Relevance:The results of this cohort study underscore the importance of periodic hearing surveillance and tailored genetic counseling for patients with EVA, offering substantial implications for prevention, management, and future gene therapy approaches. This study provides an extensive phenotypic and genotypic characterization of EVA, potentially advancing an understanding of its molecular underpinnings and clinical heterogeneity.

随着现代科学技术的飞速发展,临床诊疗技术和方法不断发展和完善。本专栏的开辟旨在创建一个学术交流平台,针对本学科临床工作中的热点和难点,邀请在相关领域做出大量工作并颇有建树的专家和教授,介绍他们的见解和经验,以飨读者。圆桌论坛为个人意见不具共识性。

The solute carriers (SLCs) are important membrane-bound transporters that regulate cellular nutrition, metabolism, homeostasis and survival. Emerging evidence highlights the critical involvement of SLCs in auditory physiology. To date, over ten SLC family members have been linked to hearing function. MFSD3 (also known as SLC33A2), is a putative plasma membrane-localized acetyl-CoA transporter regulating lipid metabolism and energy homeostasis. It has been found to be associated with the pathogenesis of neurodegenerative dementia and tumor progression. Nevertheless, its potential role in hearing remains unexplored. In this study, through qRT-PCR, we demonstrated that mfsd3 was predominantly expressed during early embryonic development in zebrafish. Morpholino-mediated mfsd3 knockdown in zebrafish induced inner ear malformations (hypoplastic otic vesicles, reduced otolith size) and hair cells loss in lateral line neuromasts. Additionally, Mfsd3 deficiency led to developmental defects (pericardial edema, body axis curvature) and impaired locomotor activity in zebrafish. The qRT-PCR analysis further revealed significant upregulation of key Wnt/β-catenin pathway components (dkk1b, wnt8a, lrp6, frzb and COX2) in mfsd3 knockdown zebrafish. Our findings suggest MFSD3 as a potential participant in auditory function and embryogenesis, with implications for understanding hearing loss pathogenesis.

BACKGROUND:Hearing loss (HL) is genetically and phenotypically heterogeneous. Variants in GRHL2, which encodes the Grainyhead-like 2 transcription factor, cause autosomal dominant nonsyndromic HL (DFNA28). Only six pathogenic GRHL2 variants have been reported, predominantly associated with high-frequency HL. OBJECTIVES:To elucidate the molecular etiology of ADNSHL presenting with low-to-mid frequency HL in a four-generation Chinese Han family. MATERIAL AND METHODS:Whole exome sequencing was performed to identify the candidate variant, followed by Sanger sequencing for co-segregation analysis. Functional assessments included protein subcellular localization, luciferase assays and co-immunoprecipitation. RESULTS:A novel nonsense variant, c.648C>G (p.Tyr216Ter), in GRHL2 was identified and co-segregated with the HL phenotype. Contrary to the characteristic high-frequency HL in DFNA28, the 11-year-old proband (IV:4) displayed low-to-mid frequency HL. The variant introduces a premature termination codon, producing a truncated protein missing 409 C-terminal amino acids. Initially classified as likely pathogenic according to the ACMG/AMP guidelines, functional analysis demonstrated cytoplasmic mislocalization and abnormal transcriptional upregulation, potentially via interaction with wild-type GRHL2, prompting reclassification to pathogenic. CONCLUSION AND SIGNIFICANCE:Our findings broaden the GRHL2 mutational spectrum, support a gain-of-function mechanism in DFNA28, and establish GRHL2 as a gene associated with low-to-mid frequency HL, expanding known genotype-phenotype correlations.

Objectives:To explore molecular diagnoses in cochlear implantation (CI) recipients and evaluate CI outcomes in patients with PCDH15 mutations. Methods:Whole-exome sequencing and biomedical informatics were used to identify potential genetic causes in 467 individuals with congenital sensorineural hearing loss. We reviewed six CI recipients with PCDH15 mutations, assessing their CI outcomes and clinical features. Results:Nine PCDH15 variants and a heterozygous variant in CDH23 were identified in members of five families who underwent CI. Six of these were novel variants: exon 14-21 del, exon two del, exon 19 del, two splicing variants (c.2869-2A>C, c.1918-1G>A) in PCDH15, and c.209C>T in CDH23. All but one of the individuals with PCDH15 mutations exhibited autosomal recessive inheritance; one showed both digenic and autosomal recessive inheritance. Variants in PCDH15 contributed to Usher syndrome type 1F in patients 1 and 5, whereas the remaining four had isolated deafness (DFNB23). All six patients expressed satisfaction with their CI outcomes. Conclusion:CI significantly improved auditory and communication abilities in individuals with PCDH15 mutations. Early intervention is critical for achieving favorable outcomes. Preoperative genetic testing in individuals with hearing loss provides valuable insights for predicting CI success, offering potential treatments for retinal degeneration in Usher syndrome and facilitating personalized genetic counseling.

Hearing loss is the most prevalent disabling disease. Cochlear implantation（CI） serves as the primary intervention for severe to profound hearing loss. This consensus systematically explores the value of genetic diagnosis in the pre-operative assessment and efficacy prognosis for CI. Drawing upon domestic and international research and clinical experience, it proposes an evidence-based medicine three-tiered prognostic classification system（Favorable, Marginal, Poor）. The consensus focuses on common hereditary non-syndromic hearing loss（such as that caused by mutations in genes like GJB2, SLC26A4, OTOF, LOXHD1） and syndromic hereditary hearing loss（such as Jervell & Lange-Nielsen syndrome and Waardenburg syndrome）, which are closely associated with congenital hearing loss, analyzing the impact of their pathological mechanisms on CI outcomes. The consensus provides recommendations based on multiple round of expert discussion and voting. It emphasizes that genetic diagnosis can optimize patient selection, predict prognosis, guide post-operative rehabilitation, offer stratified management strategies for patients with different genotypes, and advance the application of precision medicine in the field of CI.

Haploinsufficiency of the ATP6V1B2, a subunit of V-ATPases, underlies genetic disorders including Dominant deafness-onychodystrophy (DDOD), deafness, onychodystrophy, osteodystrophy, mental retardation and seizures (DOORS), and Zimmermann-Laband syndromes, all characterized by congenital hearing loss and onychodystrophy. Effective therapies for ATP6V1B2-associated hearing loss remain elusive. The study generates a hair cell-specific knockout mouse (Atp6v1b2fl/fl;Atoh1Cre/+) recapitulating the human phenotypes, with pathological features including hair cell loss and abnormal lysosomal morphology and function. To enhance therapeutic precision and minimize toxicity, an optimized adeno-associated virus-inner ear vector incorporating promoter enhancer 3 (AAV-ie-Eh3) is engineered. A single administration of AAV-ie-Eh3-mAtp6v1b2 into the scala media at postnatal days 0-2, prevented hair cell degeneration, restored lysosome morphology, and robustly rescued auditory and vestibular function for at least 24 weeks. The findings highlight the critical role of Atp6v1b2 in lysosomal function and demonstrate AAV-ie-Eh3 as a potent gene delivery tool for inner ear therapy. This study establishes a novel therapeutic paradigm for ATP6V1B2-associated hearing loss and vestibular dysfunction, with significant clinical potential.

INTRODUCTION:Although the relationship between psychiatric disorders and common auditory diseases has been discovered in observational studies, the causal linkage between them remains inconclusive. METHODS:The bidirectional two-sample Mendelian randomization (MR) analysis was performed, drawing on the most recent and expansive genome-wide association studies (GWAS) data for seven psychiatric disorders and five common auditory diseases. Additionally, a mediation analysis was conducted using data on 731 immune cell phenotypes and 1400 metabolite levels to explore potential mediating factors influencing the causal pathways. RESULTS:Autism Spectrum Disorder (ASD) could increase the risk of presbycusis (odds ratio [OR] = 1.161 [95 % confidence interval (CI), 1.042-1.295], P-value = 0.007) through CD25 on CD45RA- CD4 not regulatory T cell (Mediation effect β = 0.017), and Major Depressive Disorder (MDD) could increase the risk of vertigo (OR = 1.215 [95 % CI, 1.043-1.415], P-value = 0.012) through CD33+ HLA DR+ CD14dim Absolute Count (Mediation effect β = 0.007). Alzheimer's Disease (AD) could reduce the risk of presbycusis through four metabolite levels related to lipid metabolism. And Bipolar Disorder I (BD I) could reduce the risk of vertigo, as well as sudden sensorineural hearing loss (SSNHL) could reduce the risk of Post Traumatic Stress Disorder (PTSD). CONCLUSION:This study underscores the intricate causal links between psychiatric disorders and auditory diseases. Mediation analyses indicate that immune cells are facilitators of positive effects, while metabolite levels play a protective role. These insights offer potential pathways for more effective clinical diagnosis and treatment.

BACKGROUND:Heterozygous variant in HOMER2 is associated with autosomal dominant nonsyndromic hearing loss (ADNSHL), designated as the locus of DFNA68. Only five pathogenic variants in HOMER2 have been identified to date. AIMS/OBJECTIVES:The aim of this study is to investigate the molecular etiology of ADNSHL in a four-generation Chinese family. MATERIAL AND METHODS:Whole exome sequencing analysis was conducted to detect the disease-causing variant. Co-segregation analysis was performed using Sanger sequencing. RESULTS:The family exhibited autosomal dominant, progressive, post-lingual, nonsyndromic sensorineural hearing loss, similar to that observed in previously reported DFNA68 families. Unlike the initially high-frequency hearing loss observed in the previously reported families, the young proband in our study (IV:4, 18 years old) exhibited typical low-frequency hearing loss. A novel frameshift variant, c.1023_1029del (p.Asp342ArgfsTer54), in HOMER2 was identified, which co-segregated with the hearing loss phenotype in the family. The variant deletes 7 nucleotides, leading to an extended incorrect protein C terminus. Based on the American College of Medical Genetics and Genomics guidelines, the variant c.1023_1029del (p.Asp342ArgfsTer54) is classified as likely pathogenic. CONCLUSIONS AND SIGNIFICANCE:These findings may help expand the spectrum of pathogenic variants of the HOMER2 gene and provide a molecular interpretation for these patients with ADNSHL.

Recessive variants in TWNK cause syndromes arising from mitochondrial DNA (mtDNA) depletion. Hearing loss is the most prevalent manifestation in individuals with these disorders. However, the clinical and pathophysiological features have not been fully elucidated. In this study, we collected five cases of hearing loss carrying bi-allelic TWNK variants from three unrelated Chinese families and identified two cases with isolated auditory neuropathy (AN) and three cases segregating with Perrault syndrome, characterized by AN, global developmental delay, and ovarian dysgenesis in females. All patients with cochlear implantation (CI) show poor speech discrimination outcomes, suggesting that the defect involves post-synaptic sites. In the mouse inner ear, Twinkle was immunolocalized to inner phalangeal cells and spiral ganglion neurons. Additionally, the broad expression pattern of Twinkle was observed in the auditory cortex, which to some extent explains the poor rehabilitation outcomes following CI. At the cellular level, Twinkle is localized at the mtDNA membrane, and the p.(Arg609AlaTer6) variant prevents the protein from reaching the mtDNA while the p.(Arg65Trp) variant exhibits a similar localization to the wild type, indicating a second mechanism of action. RT-PCR results indicated that the canonical transcript was abundant in the inner ear, while the shorter transcript was more abundant in the brain. Our findings revealed that bi-allelic TWNK variants lead to AN, which can be either syndromic or non-syndromic, with the molecular pathogenesis involving defects in mtDNA replication at post-synaptic sites. Patients with TWNK-associated conditions are not ideal candidates for CI and gene therapy may offer a solution for hearingrehabilitation.

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