COCH-related autosomal dominant nonsyndromic hearing loss: a phenotype-genotype study.

This phenotype-genotype study aimed to investigate the extent of audioprofile variability related to cochlin major domains and to identify potential ethnic-specific differences associated with COCH-related hearing loss. Eight Korean families (26 cases) were diagnosed with COCH-related hearing loss by exome sequencing. Audiometric test results were combined with those from nine published East Asian families (20 cases) and compared with those from 38 European-descent families (277 cases). Audioprofiles were created by grouping audiometric test results into age ranges by age at testing and then averaging hearing loss thresholds by frequency within age ranges. The functional impact of the identified variants was assessed in vitro by examining the intracellular trafficking, secretion, and cleavage of cochlin. In both East Asian and European-descent families segregating COCH-related hearing loss, deafness-associated variants in non-LCCL domains of cochlin were associated with hearing loss that was more severe earlier in life than hearing loss caused by variants in the LCCL domain. Consistent with this phenotypic difference, functional studies demonstrated distinct pathogenic mechanisms for COCH variants in a domain-dependent manner; specifically, a cytotoxic effect was observed for the p.Phe230Leu variant, which is located in the vWFA1 domain. No ethnic-specific differences in hearing loss progression were observed, except for those attributable to an overrepresentation of presymptomatic cases in the European-descent cohort.

Differential genetic diagnoses of adult post-lingual hearing loss according to the audiogram pattern and novel candidate gene evaluation.

Ski-slope hearing loss (HL), which refers to increased auditory threshold at high frequencies, is common in adults. However, genetic contributions to this post-lingual HL remain largely unknown. Here, we prospectively investigated deafness-associated and novel candidate genes causing ski-slope HL. We analyzed 192 families with post-lingual HL via gene panel and/or exome sequencing. With an overall molecular diagnostic rate of 35.4% (68/192) in post-lingual HL, ski-slope HL showed a lower diagnostic rate (30.7%) compared with other conditions (40.7%). In patients who showed HL onset before the age of 40, genetic diagnostic probability was significantly lower for ski-slope HL than for other conditions. Further analysis of 51 genetically undiagnosed patients in the ski-slope HL group identified three variants in delta-like ligand 1 (DLL1), a Notch ligand, which presented in vitro gain-of-function effects on Notch downstream signaling. In conclusion, genetic diagnostic rates in post-lingual HL varied according to audiogram patterns with age-of-onset as a confounding factor. DLL1 was identified as a candidate gene causing ski-slope HL.

Inflammatory Monocytes Infiltrate the Spiral Ligament and Migrate to the Basilar Membrane After Noise Exposure.

OBJECTIVES: Noise exposure leads to an increase in the macrophage population. This increment is thought to be caused by the transformation of infiltrated monocytes into macrophages rather than by proliferation of the cochlear resident macrophages. However, studies on infiltrated monocytes in the cochlea are scarce. Thus, we aimed to investigate the infiltration of monocytes and their transformation into macrophages after noise exposure. METHODS: In wild-type and CX3CR1+/GFP C57/B6 mice, inflammatory monocytes were identified by immunofluorescence of mouse cochlear cells. The findings were confirmed and quantitated by flow cytometry. RESULTS: One day after noise exposure, monocytes were identified in the spiral ligament. Flow cytometric analysis confirmed that the monocyte population peaked on post-noise exposure day 1 and decreased thereafter. On day 3 after noise exposure, amoeboid-type macrophages increased in the crista basilaris, and on day 5, they spread to the basilar membrane. CONCLUSION: Infiltrated monocytes were successfully observed 1 day after noise exposure, preceding the increase in the macrophage population. This finding supports the proposal that infiltrated monocytes transform into macrophages.

Clinical Heterogeneity Associated with MYO7A Variants Relies on Affected Domains.

Autosomal dominant hearing loss (ADHL) manifests as an adult-onset disease or a progressive disease. MYO7A variants are associated with DFNA11, a subtype of ADHL. Here, we examined the role and genotype-phenotype correlation of MYO7A in ADHL. Enrolled families suspected of having post-lingual sensorineural hearing loss were selected for exome sequencing. Mutational alleles in MYO7A were identified according to ACMG guidelines. Segregation analysis was performed to examine whether pathogenic variants segregated with affected status of families. All identified pathogenic variants were evaluated for a phenotype-genotype correlation. MYO7A variants were detected in 4.7% of post-lingual families, and 12 of 14 families were multiplex. Five potentially pathogenic missense variants were identified. Fourteen variants causing autosomal dominant deafness were clustered in motor and MyTH4 domains of MYO7A protein. Missense variants in the motor domain caused late onset of hearing loss with ascending tendency. A severe audiological phenotype was apparent in individuals carrying tail domain variants. We report two new pathogenic variants responsible for DFNA11 in the Korean ADHL population. Dominant pathogenic variants of MYO7A occur frequently in motor and MyTH4 domains. Audiological differences among individuals correspond to specific domains which contain the variants. Therefore, appropriate rehabilitation is needed, particularly for patients with late-onset familial hearing loss.

Neutrophils infiltrate into the spiral ligament but not the stria vascularis in the cochlea during lipopolysaccharide-induced inflammation.

It has been challenging to apply intravital imaging for monitoring the inner ear, as the anatomical location and intricate structure hamper the access of imaging instruments to the inner ear of live mice. By employing intravital imaging of the cochlea in live mice with two-photon microscopy, we investigated neutrophil infiltration into the cochlea tissue and its characteristics under a lipopolysaccharide (LPS)-induced inflammatory state. Methods: Cochlea inflammation was induced by LPS injection to the middle ear. Using two-photon intravital microscopy with specifically designed surgical exteriorization of the cochlea in live mice, we investigated the dynamic features of neutrophils in the lateral wall of the cochlea. The molecular expression pattern of the cochlea lateral wall was also investigated during the LPS-induce inflammation. Results: Despite the contention of whether neutrophils are recruited to the spiral ligament (SL) during inflammation, we observed that LPS-induced inflammation of the middle ear, which mimics acute otitis media, triggered neutrophil migration to the SL in the lateral wall. Notably, massive neutrophil infiltration to the SL occurred 2 days after LPS inoculation, but there was no neutrophil infiltration into the stria vascularis (SV) region. At 1 day after LPS-induced cochlear inflammation, increased mRNA expression of interleukin-1ß, interleukin-6 were identified in both the SL and SV, while the ICAM-1 mRNA expression increased only in the SL. The differential reactivity of ICAM-1 is likely responsible for the different neutrophil recruitment pattern in the cochlea. Conclusion: Intravital imaging of the cochlea revealed that neutrophil recruitment and infiltration during inflammation are spatially controlled and exclusively observed in the SL but not in the SV and organ of Corti.

Three-Dimensional Distribution of Cochlear Macrophages in the Lateral Wall of Cleared Cochlea.

OBJECTIVES: Resident macrophages are well known to be present in the cochlea, but the exact patterns thereof in spiral ligaments have not been discussed in previous studies. We sought to document the distribution of macrophages in intact cochleae using three-dimensional imaging. METHODS: Cochleae were obtained from C-X3-C motif chemokine receptor 1+/GFP mice, and organ clearing was performed. Three-dimensional images of cleared intact cochleae were reconstructed using two-photon microscopy. The locations of individual macrophages were investigated using 100-µm stacked images to reduce bias. Cochlear inflammation was then induced by lipopolysaccharide (LPS) inoculation into the middle ear through the tympanic membrane. Four days after inoculation, three-dimensional images were obtained. RESULTS: Macrophages were scarce in areas adjacent to the stria vascularis, particularly the area just beneath it even though many have suspected macrophages to be abundant in this area. This finding remained consistent upon LPS-induced cochlear inflammation, despite a significant increase in the number of macrophages, compared to non-treated cochlea. CONCLUSION: Resident macrophages in spiral ligaments are scarce in areas adjacent to the stria vascularis.

LCCL peptide cleavage after noise exposure exacerbates hearing loss and is associated with the monocyte infiltration in the cochlea.

Acoustic trauma induces an inflammatory response in the cochlea, resulting in debilitating hearing function. Clinically, amelioration of inflammation substantially prevents noise-induced hearing loss. The Limulus factor C, Cochlin, and Lgl1 (LCCL) peptide plays an important role in innate immunity during bacteria-induced inflammation in the cochlea. We aimed to investigate the LCCL-induced innate immune response to noise exposure and its impact on hearing function. METHODS: We used Coch (encodes cochlin harboring LCCL peptide) knock-out and p.G88E knock-in mice to compare the immune responses before and after noise exposure. We explored their hearing function and hair cell degeneration. Moreover, we investigated distinct characteristics of immune responses upon noise exposure using flow cytometry and RNA sequencing. RESULTS: One day after noise exposure, the LCCL peptide cleaved from cochlin increased over time in the perilymph space. Both Coch-/- and CochG88E/G88E mutant mice revealed more preserved hearing following acoustic trauma compared to wild-type mice. The outer hair cells were more preserved in Coch-/- than in wild-type mice upon noise exposure. The RNA sequencing data demonstrated significantly upregulated cell migration gene ontology in wild-type mice than in Coch-/- mice following noise exposure, indicating that the infiltration of immune cells was dependent on cochlin. Notably, infiltrated monocytes from blood (C11b+/Ly6G-/Ly6C+) were remarkably higher in wild-type mice than in Coch-/- mice at 1 day after noise exposure. CONCLUSIONS: Noise-induced hearing loss was attributed to over-stimulated cochlin, and led to the cleavage and secretion of LCCL peptide in the cochlea. The LCCL peptide recruited more monocytes from the blood vessels upon noise stimulation, thus highlighting a novel therapeutic target for noise-induced hearing loss.

Cleaved Cochlin Sequesters Pseudomonas aeruginosa and Activates Innate Immunity in the Inner Ear.

In the inner ear, endolymph fluid surrounds the organ of Corti, which is important for auditory function; notably, even slight environmental changes mediated by trauma or infection can have significant consequences. However, it is unclear how the immune response is modulated in these tissues. Here, we report the local immune surveillance role of cleaved cochlin LCCL (Limulus factor C, Cochlin, and Lgl1) during Pseudomonas aeruginosa infection in the cochlea. Upon infection, the LCCL domain is cleaved from cochlin and secreted into the perilymph. This cleaved fragment sequesters infiltrating bacteria in the scala tympani and subsequently recruits resident immune cells to eliminate the bacteria. Importantly, hearing loss in a cochlin knockout mouse model is remedied by treatment with a cochlin LCCL peptide. These findings suggest cleaved cochlin LCCL constitutes a critical factor in innate immunity and auditory function and may be a potential therapeutic target to treat chronic otitis media-induced hearing loss.

Thick airway surface liquid volume and weak mucin expression in pendrin-deficient human airway epithelia.

Pendrin is an anion exchanger whose mutations are known to cause hearing loss. However, recent data support the linkage between pendrin expression and airway diseases, such as asthma. To evaluate the role of pendrin in the regulation of the airway surface liquid (ASL) volume and mucin expression, we investigated the function and expression of pendrin and ion channels and anion exchangers. Human nasal epithelial cells were cultured from 16 deaf patients carrying pendrin mutations (DFNB4) and 17 controls. The cells were treated with IL-13 to induce mucus hypersecretion. Airway surface liquid thickness was measured and real-time polymerase chain reaction was performed targeting various transporters and MUC5AC. Anion exchanger activity was measured using a pH-sensitive fluorescent probe. Periodic acid-Schiff staining was performed on the cultured cells and inferior turbinate tissues. The ASL layer of the nasal epithelia from DFNB4 subjects was thicker than the controls, and the difference became more prominent following IL-13 stimulation. There was no difference in anion exchange activity after IL-13 treatment in the cells from DFNB4 patients, while it increased in the controls. Goblet cell metaplasia induced by IL-13 treatment seen in the controls was not observed in the DFNB4 cells. Furthermore, the periodic acid-Schiff staining-positive area was lesser in the inferior turbinate tissues from DFNB4 patients that those from controls. Pendrin plays a critical role in ASL volume regulation and mucin expression as pendrin-deficient airway epithelial cells are refractory to stimulation with IL-13. Specific blockers targeting pendrin in the airways may therefore have therapeutic potential in the treatment of allergic airway diseases.