The natural history and genotype-phenotype correlations of TMPRSS3 hearing loss: an international, multi-center, cohort analysis.

TMPRSS3-related hearing loss presents challenges in correlating genotypic variants with clinical phenotypes due to the small sample sizes of previous studies. We conducted a cross-sectional genomics study coupled with retrospective clinical phenotype analysis on 127 individuals. These individuals were from 16 academic medical centers across 6 countries. Key findings revealed 47 unique TMPRSS3 variants with significant differences in hearing thresholds between those with missense variants versus those with loss-of-function genotypes. The hearing loss progression rate for the DFNB8 subtype was 0.3 dB/year. Post-cochlear implantation, an average word recognition score of 76% was observed. Of the 51 individuals with two missense variants, 10 had DFNB10 with profound hearing loss. These 10 all had at least one of 4 TMPRSS3 variants predicted by computational modeling to be damaging to TMPRSS3 structure and function. To our knowledge, this is the largest study of TMPRSS3 genotype-phenotype correlations. We find significant differences in hearing thresholds, hearing loss progression, and age of presentation, by TMPRSS3 genotype and protein domain affected. Most individuals with TMPRSS3 variants perform well on speech recognition tests after cochlear implant, however increased age at implant is associated with worse outcomes. These findings provide insight for genetic counseling and the on-going design of novel therapeutic approaches.

Progress in Modeling and Targeting Inner Ear Disorders with Pluripotent Stem Cells.

Sensorineural hearing loss and vestibular dysfunction are caused by damage to neurons and mechanosensitive hair cells, which do not regenerate to any clinically relevant extent in humans. Several protocols have been devised to direct pluripotent stem cells (PSCs) into inner ear hair cells and neurons, which display many properties of their native counterparts. The efficiency, reproducibility, and scalability of these protocols are enhanced by incorporating knowledge of inner ear development. Modeling human diseases in vitro through genetic manipulation of PSCs is already feasible, thereby permitting the elucidation of mechanistic understandings of a wide array of disease etiologies. Early studies on transplantation of PSC-derived otic progenitors have been successful in certain animal models, yet restoration of function and long-term cell survival remain unrealized. Through further research, PSC-based approaches will continue to revolutionize our understanding of inner ear biology and contribute to the development of therapeutic treatments for inner ear disorders.

Hair Follicle Development in Mouse Pluripotent Stem Cell-Derived Skin Organoids.

The mammalian hair follicle arises during embryonic development from coordinated interactions between the epidermis and dermis. It is currently unclear how to recapitulate hair follicle induction in pluripotent stem cell cultures for use in basic research studies or in vitro drug testing. To date, generation of hair follicles in vitro has only been possible using primary cells isolated from embryonic skin, cultured alone or in a co-culture with stem cell-derived cells, combined with in vivo transplantation. Here, we describe the derivation of skin organoids, constituting epidermal and dermal layers, from a homogeneous population of mouse pluripotent stem cells in a 3D culture. We show that skin organoids spontaneously produce de novo hair follicles in a process that mimics normal embryonic hair folliculogenesis. This in vitro model of skin development will be useful for studying mechanisms of hair follicle induction, evaluating hair growth or inhibitory drugs, and modeling skin diseases.

Improved autologous cortical bone harvest and viability with 2Flute otologic burs.

OBJECTIVES: To determine if 2Flute (Stryker Corporation, Kalamazoo, MI) otologic burs improve the size, cellular content, and bone healing of autologous cortical bone grafts harvested during canal wall reconstruction (CWR) tympanomastoidectomy with mastoid obliteration. STUDY DESIGN: Institutional review board-approved prospective cohort study. METHODS: Human autologous cortical bone chips were harvested using various burs (4 and 6 mm diameter; multiflute, and 2Flute [Stryker Corporation]) from patients undergoing CWR tympanomastoidectomy for the treatment of chronic otitis media with cholesteatoma. Bone chip size, cell counts, cellular gene expression, and new bone formation were quantified. RESULTS: Bone chips were significantly larger when harvested with 2Flute (Stryker Corporation) bur compared to multiflute burs at both 6 mm diameter (113 ± 14 µm2 vs. 66 ± 8 µm2 ; P < 0.05) and 4 mm diameter (70 ± 8 µm2 vs. 50 ± 3 µm2 ; P < 0.05). After 2 weeks in culture, cell numbers were significantly higher when harvested with 2Flute (Stryker Corporation) bur compared to multiflute burs at both 6 mm diameter (48.7 ± 3 vs. 31.8 ± 3 cells/µg bone; P < 0.05) and 4 mm diameter (27.6 ± 1.2 vs. 8.8 ± 1.2 cells/µg bone; P < 0.05). Bone-derived cells express osteoblast markers (alkaline phosphatase, osteocalcin). Cultured cells are able to form new bone in culture, and bone formation is facilitated by the presence of bone chips. CONCLUSION: Use of 2Flute (Stryker Corporation) otologic burs for human autologous cortical bone harvest results in more viable bone fragments, with larger bone chips and more osteoblasts. Future studies are needed to determine if this leads to improved bone healing. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:E41-E46, 2018.

Proteomic identification of hair cell repair proteins in the model sea anemone Nematostella vectensis.

Sea anemones have an extraordinary capability to repair damaged hair bundles, even after severe trauma. A group of secreted proteins, named repair proteins (RPs), found in mucus covering sea anemones significantly assists the repair of damaged hair bundle mechanoreceptors both in the sea anemone Haliplanella luciae and the blind cavefish Astyanax hubbsi. The polypeptide constituents of RPs must be identified in order to gain insight into the molecular mechanisms by which repair of hair bundles is accomplished. In this study, several polypeptides of RPs were isolated from mucus using blue native PAGE and then sequenced using LC-MS/MS. Thirty-seven known polypeptides were identified, including Hsp70s, as well as many polypeptide subunits of the 20S proteasome. Other identified polypeptides included those involved in cellular stress responses, protein folding, and protein degradation. Specific inhibitors of Hsp70s and the 20S proteasome were employed in experiments to test their involvement in hair bundle repair. The results of those experiments suggested that repair requires biologically active Hsp70s and 20S proteasomes. A model is proposed that considers the function of extracellular Hsp70s and 20S proteasomes in the repair of damaged hair cells.

Cadherin-23 may be dynamic in hair bundles of the model sea anemone Nematostella vectensis.

Cadherin 23 (CDH23), a component of tip links in hair cells of vertebrate animals, is essential to mechanotransduction by hair cells in the inner ear. A homolog of CDH23 occurs in hair bundles of sea anemones. Anemone hair bundles are located on the tentacles where they detect the swimming movements of nearby prey. The anemone CDH23 is predicted to be a large polypeptide featuring a short exoplasmic C-terminal domain that is unique to sea anemones. Experimentally masking this domain with antibodies or mimicking this domain with free peptide rapidly disrupts mechanotransduction and morphology of anemone hair bundles. The loss of normal morphology is accompanied, or followed by a decrease in F-actin in stereocilia of the hair bundles. These effects were observed at very low concentrations of the reagents, 0.1-10 nM, and within minutes of exposure. The results presented herein suggest that: (1) the interaction between CDH23 and molecular partners on stereocilia of hair bundles is dynamic and; (2) the interaction is crucial for normal mechanotransduction and morphology of hair bundles.