A case study of Duchenne muscular dystrophy caused by Alu element insertion in DMD gene and analysis of its gray-hair symptoms.

Duchenne muscular dystrophy (DMD) is a severe X-linked recessive genetic disorder caused by mutations in the DMD gene, which leads to a deficiency of the dystrophin protein. The main mutation types of this gene include exon deletions and duplications, point mutations, and insertions. These mutations disrupt the normal expression of dystrophin, ultimately leading to the disease. In this study, we reported a case of DMD caused by an insertion mutation in exon 59 (E59) of the DMD gene. The affected child exhibited significant abnormalities in related biochemical markers, early symptoms of DMD, and multiple gray hair. His mother and sister were carriers with slightly abnormal biochemical markers. The mother had mild clinical symptoms, while the sister had no clinical symptoms. Other family members were genetically and physically normal. Sequencing and sequence alignment revealed that the inserted fragment was an Alu element from the AluYa5 subfamily. This insertion produced two stop codons and a polyadenylate (polyA) tail. To understand the impact of this insertion on the DMD gene and its association with clinical symptoms, exonic splicing enhancer (ESE) prediction indicated that the insertion did not affect the splicing of E59. Therefore, we speculated that the insertion sequence would be present in the mRNA sequence of the DMD gene. The two stop codons and polyA tail likely terminate translation, preventing the production of functional dystrophin protein, which may be the mechanism leading to DMD. In addition to typical DMD symptoms, the child also exhibited premature graying of hair. This study reports, for the first time, a case of DMD caused by the insertion of an Alu element into the coding region of the DMD gene. This finding provides clues for studying gene mutations induced by Alu sequence insertion and expands the understanding of DMD gene mutations.

Ototoxicity of polystyrene nanoplastics in mice, HEI-OC1 cells and zebrafish.

Polystyrene nanoplastics are a novel class of pollutants. They are easily absorbed by living organisms, and their potential toxicity has raised concerns. However, the impact of polystyrene nanoplastics on auditory organs remains unknown. Here, our results showed that polystyrene nanoplastics entered the cochlea of mice, HEI-OC1 cells, and lateral line hair cells of zebrafish, causing cellular injury and increasing apoptosis. Additionally, we found that exposure to polystyrene nanoplastics resulted in a significant elevation in the auditory brainstem response thresholds, a loss of auditory sensory hair cells, stereocilia degeneration and a decrease in expression of Claudin-5 and Occludin proteins at the blood-lymphatic barrier in mice. We also observed a significant decrease in the acoustic alarm response of zebrafish after exposure to polystyrene nanoplastics. Mechanistic analysis revealed that polystyrene nanoplastics induced up-regulation of the Nrf2/HO-1 pathway, increased levels of malondialdehyde, and decreased superoxide dismutase and catalase levels in cochlea and HEI-OC1 cells. Furthermore, we observed that the expression of ferroptosis-related indicators GPX4 and SLC7A11 decreased as well as increased expression of ACLS4 in cochlea and HEI-OC1 cells. This study also revealed that polystyrene nanoplastics exposure led to increased expression of the inflammatory factors TNF-α, IL-1ß and COX2 in cochlea and HEI-OC1 cells. Further research found that the cell apoptosis, ferroptosis and inflammatory reactions induced by polystyrene nanoplastics in HEI-OC1 cells was reversed through the pretreatment with N-acetylcysteine, a reactive oxygen species inhibitor. Overall, our study first discovered and systematically revealed the ototoxicity of polystyrene nanoplastics and its underlying mechanism.

Caffeine Ameliorates Age-Related Hearing Loss by Downregulating the Inflammatory Pathway in Mice.

OBJECTIVE: Age-related hearing loss (ARHL), also known as presbycusis, is a debilitating sensory impairment that affects the elderly population. There is currently no ideal treatment for ARHL. Long-term caffeine intake was reported to have anti-aging effects in many diseases. This study is to identify whether caffeine could ameliorate ARHL in mice and analyze its mechanism. METHODS: Caffeine was administered in drinking water to C57BL/6J mice from the age of 3 months to 12 months. The body weight, food intake and water intake of the mice were monitored during the experiment. The metabolic indicators of serum were detected by ELISA. The function of the hearing system was evaluated by ABR and hematoxylin and eosin staining of the cochlea. Genes' expression were detected by Q-PCR, immunofluorescencee and Western blot. RESULTS: The results showed that the ARHL mice exhibited impaired hearing and cochlear tissue compared with the young mice. However, the caffeine-treated ARHL mice showed improved hearing and cochlear tissue morphology. The expression of inflammation-related genes, such as TLR4, Myd88, NF-κB, and IL-1ß, was significantly increased in the cochleae of ARHL mice compared with young mice but was down-regulated in the caffeine-treated cochleae. CONCLUSIONS: Inflammation is involved in ARHL of mice, and long-term caffeine supplementation could ameliorate ARHL through the down-regulation of the TLR4/NF-κB inflammation pathway. Our findings provide a new idea for preventing ARHL and suggest new drug targets for ARHL treatment.

Association between CT-based adipose variables, preoperative blood biochemical indicators and pathological T stage of clear cell renal cell carcinoma.

Background: Clear cell renal cell carcinoma (ccRCC) is corelated with tumor-associated material (TAM), coagulation system and adipocyte tissue, but the relationships between them have been inconsistent. Our study aimed to explore the cut-off intervals of variables that are non-linearly related to ccRCC pathological T stage for providing clues to understand these discrepancies, and to effectively preoperative risk stratification. Methods: This retrospective analysis included 218 ccRCC patients with a clear pathological T stage between January 1st, 2014, and November 30th, 2021. The patients were categorized into two cohorts based on their pathological T stage: low T stage (T1 and T2) and high T stage (T3 and T4). Abdominal and perirenal fat variables were measured based on preoperative CT images. Blood biochemical indexes from the last time before surgery were also collected. The generalized sum model was used to identify cut-off intervals for nonlinear variables. Results: In specific intervals, fibrinogen levels (FIB) (2.63-4.06 g/L) and platelet (PLT) counts (>200.34 × 109/L) were significantly positively correlated with T stage, while PLT counts (<200.34 × 109/L) were significantly negatively correlated with T stage. Additionally, tumor-associated material exhibited varying degrees of positive correlation with T stage at different cut-off intervals (cut-off value: 90.556 U/mL). Conclusion: Preoperative PLT, FIB and TAM are nonlinearly related to pathological T stage. This study is the first to provide specific cut-off intervals for preoperative variables that are nonlinearly related to ccRCC T stage. These intervals can aid in the risk stratification of ccRCC patients before surgery, allowing for developing a more personalized treatment planning.

RNA-seq analysis highlights DNA replication and DNA repair associated with early-onset hearing loss in the cochlea of DBA/2J mice.

AIMS: Age-related hearing loss (ARHL) is a significant health concern, and DBA/2J (D2) and C57BL/6 (B6) mouse strains serve as valuable models for its study. B6 mice, harboring a homozygous ahl allele in Cdh23, manifest high-frequency hearing loss at 3 months. In contrast, D2 mice, carrying the R109H variant of the Fascin-2 gene (Fscn2), experience early-onset hearing loss by 3 weeks. Yet, the underlying molecular mechanisms driving early-onset hearing loss in D2 mice remain elusive. This study aimed to identify novel genes and regulatory pathways as therapeutic targets for early deafness. MAIN METHODS: This study employs RNA-sequencing (RNA-seq) to analyze cochlear mRNA expression at two different ages in D2 and B6 mice, respectively. The differentially expressed genes (DEGs) are uniquely associated with D2 mice by Venn diagram analysis. A protein-protein interaction (PPI) network is further constructed, followed by module analysis utilizing MCODE. Enrichment analysis of GO and KEGG pathways revealed biological functions and molecular pathways. The PPI network and VarElect analysis are conducted for genes within these pathways, facilitating the identification of pivotal genes based on scoring criteria. Subsequently, five genes are meticulously selected and validated through qRT-PCR. KEY FINDINGS: Notably, 1181 DEGs are uniquely associated with D2 mice by Venn diagram analysis. GO and KEGG pathway enrichment analyses shed light on distinctive pathways in D2 mice, encompassing DNA replication, mismatch repair, base excision repair, and nucleotide excision repair, which are associated with apoptosis. Five genes involved in these pathways were finally selected and validated by qRT-PCR. Their down-regulation with age is consistent with RNA-seq result. SIGNIFICANCE: Our study underscores the potential implication of down-regulated genes associated with DNA replication and DNA damage repair in the early-onset hearing loss observed in D2 mice.

Col1a1 mediates the focal adhesion pathway affecting hearing in miR-29a mouse model by RNA-seq analysis.

Age-related hearing loss (ARHL) is a common neurodegenerative disease. Its molecular mechanisms have not been fully elucidated. In the present study, we obtained differential mRNA expression in the cochlea of 2-month-old miR-29a+/+ mice and miR-29a-/- mice by RNA-seq. Gene ontology (GO) analysis was used to identify molecular functions associated with hearing in miR-29a-/- mice, including being actin binding (GO: 0003779) and immune processes. We focused on the intersection of differential genes, miR-29a target genes and the sensory perception of sound (GO:0007605) genes, with six mRNA at this intersection, and we selected Col1a1 as our target gene. We validated Col1a1 as the direct target of miR-29a by molecular and cellular experiments. Total 6 pathways involved in Col1a1 were identified by through Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. We selected the focal adhesion pathway as our target pathway based. Their expression levels in miR-29a-/- mice were verified by qRT-PCR and Western blot. Compared with miR-29a+/+ mice, the expression levels of Col1a1, Itga4, Itga2, Itgb3, Itgb7, Pik3r3 and Ptk2 were different in miR-29a-/- mice. Immunofluorescence was used to locate genes in the cochlea. Col1a1, Itga4 and Itgb3 were differentially expressed in the basilar membranes and stria vascularis and spiral ganglion neurons compared to miR-29a+/+ mice. Pik3r3 and Ptk2 were differentially expressed in the basilar membranes and stria vascularis, but not at the s spiral ganglion neurons compared to miR-29a+/+ mice. Our results show that when miR-29a is knocked out, the Col1a1 mediates the focal adhesion pathway may affect the hearing of miR-29a-/- mice. These findings may provide a new direction for effective treatment of age-related hearing loss.

MiR-29a-deficiency causes thickening of the basilar membrane and age-related hearing loss by upregulating collagen IV and laminin.

Age-related hearing loss (ARHL) is the most common sensory degenerative disease and can significantly impact the quality of life in elderly people. A previous study using GeneChip miRNA microarray assays showed that the expression of miR-29a changes with age, however, its role in hearing loss is still unclear. In this study, we characterized the cochlear phenotype of miR-29a knockout (miR-29a-/-) mice and found that miR-29a-deficient mice had a rapid progressive elevation of the hearing threshold from 2 to 5 months of age compared with littermate controls as measured by the auditory brainstem response. Stereocilia degeneration, hair cell loss and abnormal stria vascularis (SV) were observed in miR-29a-/- mice at 4 months of age. Transcriptome sequencing results showed elevated extracellular matrix (ECM) gene expression in miR-29a-/- mice. Both Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that the key differences were closely related to ECM. Further examination with a transmission electron microscope showed thickening of the basilar membrane in the cochlea of miR-29a-/- mice. Five Col4a genes (Col4a1-a5) and two laminin genes (Lamb2 and Lamc1) were validated as miR-29a direct targets by dual luciferase assays and miR-29a inhibition assays with a miR-29a inhibitor. Consistent with the target gene validation results, the expression of these genes was significantly increased in the cochlea of miR-29a-/- mice, as shown by RT-PCR and Western blot. These findings suggest that miR-29a plays an important role in maintaining cochlear structure and function by regulating the expression of collagen and laminin and that the disturbance of its expression could be a cause of progressive hearing loss.

Auditory Discrimination Elicited by Nonspeech and Speech Stimuli in Children With Congenital Hearing Loss.

PURPOSE: Congenital deafness not only delays auditory development but also hampers the ability to perceive nonspeech and speech signals. This study aimed to use auditory event-related potentials to explore the mismatch negativity (MMN), P3a, negative wave (Nc), and late discriminative negativity (LDN) components in children with and without hearing loss. METHOD: Nineteen children with normal hearing (CNH) and 17 children with hearing loss (CHL) participated in this study. Two sets of pure tones (1 kHz vs. 1.1 kHz) and lexical tones (/ba2/ vs. /ba4/) were used to examine the auditory discrimination process. RESULTS: MMN could be elicited by the pure tone and the lexical tone in both groups. The MMN latency elicited by nonspeech and speech was later in CHL than in CNH. Additionally, the MMN latency induced by speech occurred later in the left than in the right hemisphere in CNH, and the MMN amplitude elicited by speech in CHL produced a discriminative deficiency compared with that in CNH. Although the P3a latency and amplitude elicited by nonspeech in CHL and CNH were not significantly different, the Nc amplitude elicited by speech performed much lower in CHL than in CNH. Furthermore, the LDN latency elicited by nonspeech was later in CHL than in CNH, and the LDN amplitude induced by speech showed higher dominance in the right hemisphere in both CNH and CHL. CONCLUSION: By incorporating nonspeech and speech auditory conditions, we propose using MMN, Nc, and LDN as potential indices to investigate auditory perception, memory, and discrimination.

Ferroptosis is involved in PGPS-induced otitis media in C57BL/6 mice.

Otitis media (OM) is a common disease that can cause hearing loss in children. Currently, the main clinical treatment for OM is antibiotics, but the overuse of antibiotics might lead to bacterial resistance, which is a worldwide public health challenge. Studying the pathogenesis of OM will help us develop new effective treatments. Ferroptosis is one type of programmed cell death characterized by the occurrence of lipid peroxidation driven by iron ions. Many studies have shown that ferroptosis is associated with infectious diseases. It is presently unclear whether ferroptosis is involved in the pathogenesis of OM. In this study, we explored the relationship between ferroptosis and OM by PGPS-induced OM in C57BL/6 mice and treating the induced OM with ferroptosis inhibitors deferoxamine (DFO), Ferrostatin-1 (Fer-1), and Liperoxstatin-1 (Lip-1). We examined the expression of ferroptosis-related proteins acyl-CoA synthetase long chain family member 4 (ACSL4) and prostaglandin-endoperoxide synthase 2 (Cox2), glutathione peroxidase 4 (GPX4) protein as well as lipid peroxidation markers 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA). The results showed that in PGPS-induced OM model mice, several ferroptosis-related proteins including ACSL4 and Cox2 were up-regulated compared to mice treated with saline. Meanwhile, a ferroptosis-related protein GPX4 was down-regulated upon PGPS treatment. The DFO treatment in PGPS-inoculated mice effectively inhibited the development of OM. The inhibitors treatment caused a significant decrease in the expression of ACSL4, Cox2, 4 HNE, MDA, reduction in free iron. Meanwhile, the ferroptosis inhibitors treatment caused increase in the expression of inflammation-related factors tumor necrosis factor-α (TNF-α) and antioxidant protein GPX4. Our results suggest that there is a crosstalk between ferroptosis signaling pathway and the pathogenesis of OM. Ferroptosis inhibition can alleviate PGPS-induced OM.

Hearing Loss in Id1-/-; Id3+/- and Id1+/-; Id3-/- Mice Is Associated With a High Incidence of Middle Ear Infection (Otitis Media).

Inhibitors of differentiation/DNA binding (Id) proteins are crucial for inner ear development, but whether Id mutations affect middle ear function remains unknown. In this study, we obtained Id1-/-; Id3+/- mice and Id1+/-; Id3-/- mice and carefully examined their middle ear morphology and auditory function. Our study revealed a high incidence (>50%) of middle ear infection in the compound mutant mice. These mutant mice demonstrated hearing impairment starting around 30 days of age, as the mutant mice presented elevated auditory brainstem response (ABR) thresholds compared to those of the littermate controls. The distortion product of otoacoustic emission (DPOAE) was also used to evaluate the conductive function of the middle ear, and we found much lower DPOAE amplitudes in the mutant mice, suggesting sound transduction in the mutant middle ear is compromised. This is the first study of the middle ears of Id compound mutant mice, and high incidence of middle ear infection determined by otoscopy and histological analysis of middle ear suggests that Id1/Id3 compound mutant mice are a novel model for human otitis media (OM).

Effectiveness of salvage intratympanic dexamethasone treatment for refractory sudden sensorineural hearing loss classified by audiogram patterns.

BACKGROUND: Intratympanic dexamethasone is commonly conducted to treat refractory sudden sensorineural hearing loss (RSSNHL). However, no consensus has been reached on its effectiveness. OBJECTIVES: The study aimed to evaluate the effectiveness of otoendoscope-assisted salvage intratympanic dexamethasone treatment (IDT) on RSSNHL with different audiogram patterns after failure of initial therapy. MATERIAL AND METHODS: A total of 108 patients with unilateral RSSNHL were classified into 4 groups according to audiogram patterns. Hearing results were evaluated by pure-tone audiometry (PTA), which was performed at baseline and one month after otoendoscope-assisted salvage IDT. The effectiveness of otoendoscope-assisted salvage IDT was assessed in each group. RESULTS: The efficiency in low-frequency, high-frequency, flat, and deaf group was 48%, 24.1%, 46.2%, 17.9%, respectively. The efficacy did not differ between the high-frequency and deaf group. Notably, the efficacy in the low-frequency and flat group was significantly higher than that in the deaf group. CONCLUSIONS: Otoendoscope-assisted salvage IDT is a safe and effective treatment for RSSNHL. This treatment provided better results for patients with low-frequency damaged and flat curve audiogram than patients with other audiogram patterns. SIGNIFICANCE: Audiogram patterns should be considered in the clinical management of patients with RSSHNL prior to salvage IDT.

Naringin attenuates cisplatin- and aminoglycoside-induced hair cell injury in the zebrafish lateral line via multiple pathways.

Exposure to ototoxic drugs is a significant cause of hearing loss that affects about 30 thousand children with potentially serious physical, social and psychological dysfunctions every year. Cisplatin (CP) and aminoglycosides are effective antineoplastic or bactericidal drugs, and their application has a high probability of ototoxicity which results from the death of hair cells (HCs). Here, we describe the therapeutic effect of the flavonoid compound naringin (Nar) against ototoxic effects of cisplatin and aminoglycosides include gentamicin (GM) and neomycin (Neo) in zebrafish HCs. Animals incubated with Nar (100-400 µmol/L) were protected against the pernicious effects of CP (150-250 µmol/L), GM (50-150 µmol/L) and Neo (50-150 µmol/L). We also provide evidence for the potential mechanism of Nar against ototoxicity, including antioxidation, anti-apoptosis, promoting proliferation and hair cell regeneration. We found that mRNA levels of the apoptotic- and pyroptosis-related genes are regulated by Nar both in vivo and in vitro. Finally, by proving that Nar does not affect the anti-tumour efficacy of CP and antibacterial activity of aminoglycosides in vitro, we highlight its value in clinical application. In conclusion, these results unravel a novel therapeutic role for Nar as an otoprotective drug against the adverse effects of CP and aminoglycosides.

Ferrostatin-1 protects auditory hair cells from cisplatin-induced ototoxicity in vitro and in vivo.

Cisplatin is used in a wide variety of malignancies, but cisplatin-induced ototoxicity remains a major issue in clinical practice. Experimental evidence indicates that ferroptosis plays a key role in mediating the unwanted cytotoxicity effect caused by cisplatin. However, the role of ferroptosis in cisplatin-induced ototoxicity requires elucidation. Ferrostatin-1 (Fer-1) was identified as a potent inhibitor of ferroptosis and radical-trapping antioxidant with its ability to reduce the accumulation of lipid peroxides and chain-carrying peroxyl radicals. In the current study, we investigated the effects of Fer-1 in cisplatin-induced ototoxicity in in vitro, ex vivo, and in vivo models. We found, for the first time that Fer-1 efficiently alleviated cisplatin-induced cytotoxicity in HEI-OC1 cells via a concentration-dependent manner. Furthermore, Fer-1 mitigated cisplatin cytotoxicity in transgenic zebrafish sensory hair cells. In HEI-OC1 cells, Fer-1 pretreatment not only drastically reduced the generation of intracellular reactive oxygen species but also remarkably decreased lipid peroxidation levels induced by cisplatin. This was not only ascribed to the inhibition of 4-hydroxynonenal, the final product of lipid peroxides, but also to the promotion of glutathione peroxidase 4, the protein marker of ferroptosis. MitoTracker staining and transmission electron microscopy of mitochondrial morphology suggested that in HEI-OC1 cells, Fer-1 can effectively abrogate mitochondrial damage resulting from the interaction with cisplatin. In addition, Fer-1 pretreatment of cochlear explants substantially protected hair cells from cisplatin-induced damage. Therefore, our results demonstrated that ferroptosis might be involved in cisplatin ototoxicity. Fer-1 administration mitigated cisplatin-induced hair cell damage, further investigations are required to elucidate the molecular mechanisms of its otoprotective effect.

SERS-active Au@Ag core-shell nanorod (Au@AgNR) tags for ultrasensitive bacteria detection and antibiotic-susceptibility testing.

There is a challenge to obtain an ultrasensitive and rapid approach to detect bacteria and identify resistance. As a powerful bioanalytical tool, surface-enhanced Raman scattering (SERS) in bacterial detection have attracted increasing attentions. Herein, we developed a SERS-active Au@Ag core-shell nanorod (Au@AgNR) tag platform for ultrasensitive bacteria detection and antibiotic-susceptibility testing (AST). The platform established that surface enhanced Raman scattered Rhodamine 6G (R6G) absorption at 1517 cm-1 had a good linearity (RI = 3865 + 193logC; R2 = 0.97) with logarithm of E. coli concentration over a range of 107-102 CFU (colony forming unit)/mL with limit of detection as low 102 CFU/mL. When E. coli was exposed to ampicillin at minimum inhibitory concentration (MIC, 4 µg/mL), Raman spectroscopy showed the obvious variation between ampicillin-susceptible E. coli (Amp--E. coli) and the ampicillin-resistant E. coli (Amp+-E. coli). Combined with principal component analysis (PCA) statistical analysis, the Raman intensity variation mentioned above allows to obtain rapid antibiotic resistance testing (<3.5 h). In addition, E.coli spiked into blood from C57BL/6 mice can be identified clearly, indicating the potential for point-of-care diagnostics.

Role of Endoplasmic Reticulum Stress in Otitis Media.

Endoplasmic reticulum (ER) stress occurs in many inflammatory responses. Here, we investigated the role of ER stress and its associated apoptosis in otitis media (OM) to elucidate the mechanisms of OM and the signaling crosstalk between ER stress and other cell damage pathways, including inflammatory cytokines and apoptosis. We examined the expression of inflammatory cytokine- and ER stress-related genes by qRT-PCR, Western blotting, and immunohistochemistry (IHC) in the middle ear of C57BL/6J mice after challenge with peptidoglycan polysaccharide (PGPS), an agent inducing OM. We also evaluated the effect of the suppression of ER stress with tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor. The study revealed the upregulation of ER stress- and apoptosis-related gene expression after the PGPS treatment, specifically ATF6, CHOP, BIP, caspase-12, and caspase-3. TUDCA treatment of PGPS-treated mice decreased OM; reduced the expression of CHOP, BIP, and caspase 3; and significantly decreased the proinflammatory gene expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). These results suggest that PGPS triggers ER stress and downstream proinflammatory gene expression in OM and that inhibition of ER stress alleviates OM. We propose that ER stress plays a critical role in inflammation and cell death, leading to the development of OM and points to ER stress inhibition as a potential therapeutic approach for the prevention of OM.

Identification of two novel mutations in POU4F3 gene associated with autosomal dominant hearing loss in Chinese families.

Autosomal dominant non-syndromic hearing loss is genetically heterogeneous with 47 genes identified to date, including POU4F3. In this study, by using a next-generation sequencing panel targeting 127 deafness genes, we identified a pathogenic frameshift mutation c.704_705del and a missense mutation c.593G>A in two three-generation Chinese families with late-onset progressive ADNSHL, respectively. The novel mutations of POU4F3 co-segregated with the deafness phenotype in these two families. c.704_705del caused a frameshift p.T235fs and c.593G>A caused an amino acid substitution of p.R198H. Both mutations led to an abnormal and incomplete protein structure. POU4F3 with either of the two mutations was transiently transfected into HEI-OC1 and HEK 293 cell lines and immunofluorescence assay was performed to investigate the subcellular localization of mutated protein. The results indicated that both c.704_705del (p.T235fs) and c.593G>A (p.R198H) could impair the nuclear localization function of POU4F3. The p.R198H POU4F3 protein was detected as a weak band of the correct molecular weight, indicating that the stability of p.R198H POU4F3 differed from that of the wild-type protein. While, the p.T235fs POU4F3 protein was expressed with a smaller molecular weight, implying this mutation result in a frameshift and premature termination of the POU4F3 protein. In summary, we report two novel mutations of POU4F3 associated with progressive ADNSHL and explored their effects on POU4F3 nuclear localization. These findings expanded the mutation spectrum of POU4F3 and provided new knowledge for the pathogenesis of POU4F3 in hearing loss.