AIF translocation into nucleus caused by Aifm1 R450Q mutation: generation and characterization of a mouse model for AUNX1.

Mutations in AIFM1, encoding for apoptosis-inducing factor (AIF), cause AUNX1, an X-linked neurologic disorder with late-onset auditory neuropathy (AN) and peripheral neuropathy. Despite significant research on AIF, there are limited animal models with the disrupted AIFM1 representing the corresponding phenotype of human AUNX1, characterized by late-onset hearing loss and impaired auditory pathways. Here, we generated an Aifm1 p.R450Q knock-in mouse model (KI) based on the human AIFM1 p.R451Q mutation. Hemizygote KI male mice exhibited progressive hearing loss from P30 onward, with greater severity at P60 and stabilization until P210. Additionally, muscle atrophy was observed at P210. These phenotypic changes were accompanied by a gradual reduction in the number of spiral ganglion neuron cells (SGNs) at P30 and ribbons at P60, which coincided with the translocation of AIF into the nucleus starting from P21 and P30, respectively. The SGNs of KI mice at P210 displayed loss of cytomembrane integrity, abnormal nuclear morphology, and dendritic and axonal demyelination. Furthermore, the inner hair cells and myelin sheath displayed abnormal mitochondrial morphology, while fibroblasts from KI mice showed impaired mitochondrial function. In conclusion, we successfully generated a mouse model recapitulating AUNX1. Our findings indicate that disruption of Aifm1 induced the nuclear translocation of AIF, resulting in the impairment in the auditory pathway.

Reversal of an existing hearing loss by gene activation in Spns2 mutant mice.

Hearing loss is highly heterogeneous, but one common form involves a failure to maintain the local ionic environment of the sensory hair cells reflected in a reduced endocochlear potential. We used a genetic approach to ask whether this type of pathology can be reversed, using the Spns2tm1a mouse mutant known to show this defect. By activating Spns2 gene transcription at different ages after the onset of hearing loss, we found that an existing auditory impairment can be reversed to give close to normal thresholds for an auditory brainstem response (ABR), at least at low to mid stimulus frequencies. Delaying the activation of Spns2 led to less effective recovery of ABR thresholds, suggesting that there is a critical period for intervention. Early activation of Spns2 not only led to improvement in auditory function but also to protection of sensory hair cells from secondary degeneration. The genetic approach we have used to establish that this type of hearing loss is in principle reversible could be extended to many other diseases using available mouse resources.

The dynamics of actin protrusions can be controlled by tip-localized myosin motors.

Class III myosins localize to inner ear hair cell stereocilia and are thought to be crucial for stereocilia length regulation. Mutations within the motor domain of MYO3A that disrupt its intrinsic motor properties have been associated with non-syndromic hearing loss, suggesting that the motor properties of MYO3A are critical for its function within stereocilia. In this study, we investigated the impact of a MYO3A hearing loss mutation, H442N, using both in vitro motor assays and cell biological studies. Our results demonstrate the mutation causes a dramatic increase in intrinsic motor properties, actin-activated ATPase and in vitro actin gliding velocity, as well as an increase in actin protrusion extension velocity. We propose that both "gain of function" and "loss of function" mutations in MYO3A can impair stereocilia length regulation, which is crucial for stereocilia formation during development and normal hearing. Furthermore, we generated chimeric MYO3A constructs that replace the MYO3A motor and neck domain with the motor and neck domain of other myosins. We found that duty ratio, fraction of ATPase cycle myosin is strongly bound to actin, is a critical motor property that dictates the ability to tip localize within filopodia. In addition, in vitro actin gliding velocities correlated extremely well with filopodial extension velocities over a wide range of gliding and extension velocities. Taken together, our data suggest a model in which tip-localized myosin motors exert force that slides the membrane tip-ward, which can combat membrane tension and enhance the actin polymerization rate that ultimately drives protrusion elongation.

Biallelic variants in Plexin B2 (PLXNB2) cause amelogenesis imperfecta, hearing loss and intellectual disability.

BACKGROUND: Plexins are large transmembrane receptors for the semaphorin family of signalling proteins. Semaphorin-plexin signalling controls cellular interactions that are critical during development as well as in adult life stages. Nine plexin genes have been identified in humans, but despite the apparent importance of plexins in development, only biallelic PLXND1 and PLXNA1 variants have so far been associated with Mendelian genetic disease. METHODS: Eight individuals from six families presented with a recessively inherited variable clinical condition, with core features of amelogenesis imperfecta (AI) and sensorineural hearing loss (SNHL), with variable intellectual disability. Probands were investigated by exome or genome sequencing. Common variants and those unlikely to affect function were excluded. Variants consistent with autosomal recessive inheritance were prioritised. Variant segregation analysis was performed by Sanger sequencing. RNA expression analysis was conducted in C57Bl6 mice. RESULTS: Rare biallelic pathogenic variants in plexin B2 (PLXNB2), a large transmembrane semaphorin receptor protein, were found to segregate with disease in all six families. The variants identified include missense, nonsense, splicing changes and a multiexon deletion. Plxnb2 expression was detected in differentiating ameloblasts. CONCLUSION: We identify rare biallelic pathogenic variants in PLXNB2 as a cause of a new autosomal recessive, phenotypically diverse syndrome with AI and SNHL as core features. Intellectual disability, ocular disease, ear developmental abnormalities and lymphoedema were also present in multiple cases. The variable syndromic human phenotype overlaps with that seen in Plxnb2 knockout mice, and, together with the rarity of human PLXNB2 variants, may explain why pathogenic variants in PLXNB2 have not been reported previously.

Bi-allelic variants in SPATA5L1 lead to intellectual disability, spastic-dystonic cerebral palsy, epilepsy, and hearing loss.

Spermatogenesis-associated 5 like 1 (SPATA5L1) represents an orphan gene encoding a protein of unknown function. We report 28 bi-allelic variants in SPATA5L1 associated with sensorineural hearing loss in 47 individuals from 28 (26 unrelated) families. In addition, 25/47 affected individuals (53%) presented with microcephaly, developmental delay/intellectual disability, cerebral palsy, and/or epilepsy. Modeling indicated damaging effect of variants on the protein, largely via destabilizing effects on protein domains. Brain imaging revealed diminished cerebral volume, thin corpus callosum, and periventricular leukomalacia, and quantitative volumetry demonstrated significantly diminished white matter volumes in several individuals. Immunofluorescent imaging in rat hippocampal neurons revealed localization of Spata5l1 in neuronal and glial cell nuclei and more prominent expression in neurons. In the rodent inner ear, Spata5l1 is expressed in the neurosensory hair cells and inner ear supporting cells. Transcriptomic analysis performed with fibroblasts from affected individuals was able to distinguish affected from controls by principal components. Analysis of differentially expressed genes and networks suggested a role for SPATA5L1 in cell surface adhesion receptor function, intracellular focal adhesions, and DNA replication and mitosis. Collectively, our results indicate that bi-allelic SPATA5L1 variants lead to a human disease characterized by sensorineural hearing loss (SNHL) with or without a nonprogressive mixed neurodevelopmental phenotype.

Norrie disease protein is essential for cochlear hair cell maturation.

Mutations in the gene for Norrie disease protein (Ndp) cause syndromic deafness and blindness. We show here that cochlear function in an Ndp knockout mouse deteriorated with age: At P3-P4, hair cells (HCs) showed progressive loss of Pou4f3 and Gfi1, key transcription factors for HC maturation, and Myo7a, a specialized myosin required for normal function of HC stereocilia. Loss of expression of these genes correlated to increasing HC loss and profound hearing loss by 2 mo. We show that overexpression of the Ndp gene in neonatal supporting cells or, remarkably, up-regulation of canonical Wnt signaling in HCs rescued HCs and cochlear function. We conclude that Ndp secreted from supporting cells orchestrates a transcriptional network for the maintenance and survival of HCs and that increasing the level of ß-catenin, the intracellular effector of Wnt signaling, is sufficient to replace the functional requirement for Ndp in the cochlea.

Apparent diffusion coefficient values of the white matter in magnetic resonance imaging of the neonatal brain may help predict outcome in congenital cytomegalovirus infection.

BACKGROUND: White matter change is a well-known abnormality in congenital cytomegalovirus (cCMV) infection, but grading remains challenging and clinical relevance unclear. OBJECTIVE: To investigate if quantitative measurement of white matter apparent diffusion coefficient (ADC) values in magnetic resonance imaging (MRI) of the neonatal brain can predict outcome in cCMV. MATERIALS AND METHODS: A retrospective, single-center observational study, including patients with cCMV who had a neonatal brain MRI with diffusion-weighted imaging, was performed between 2007 and 2020. Regions of interest were systematically placed in the white matter on the ADC maps. Two pediatric radiologists independently scored additional brain abnormalities. Outcome measures were neonatal hearing and cognitive and motor development. Statistical analysis included simple and penalized elastic net regression. RESULTS: Neonatal brain MRI was evaluated in 255 patients (median age 21 days, 25-75 percentiles: 14-28 days, 121 male). Gyral abnormalities were noted in nine patients (3.5%), ventriculomegaly in 24 (9.4%), and subependymal cysts in 58 (22.7%). General white matter ADC was significantly higher in patients with neonatal hearing loss and cognitive and motor impairment (P< 0.05). For neonatal hearing loss, simple logistic regression using only general white matter was the best prediction model, with a receiver operating characteristic area under the curve (AUC)=0.76. For cognitive impairment, interacting elastic net regression, including other brain abnormalities and frontoparietal white matter ADC, performed best, with AUC=0.89. For motor impairment, interacting elastic net regression, including other brain abnormalities and deep anterior frontal white matter performed best, with AUC=0.73. CONCLUSION: Neonatal white matter ADC was significantly higher in patients with clinical impairments. Quantitative ADC measurement may be a useful tool for predicting clinical outcome in cCMV.

The Stria Vascularis: Renewed Attention on a Key Player in Age-Related Hearing Loss.

Age-related hearing loss (HL), or presbycusis, is a complex and heterogeneous condition, affecting a significant portion of older adults and involving various interacting mechanisms. Metabolic presbycusis, a type of age-related HL, is characterized by the dysfunction of the stria vascularis, which is crucial for maintaining the endocochlear potential necessary for hearing. Although attention on metabolic presbycusis has waned in recent years, research continues to identify strial pathology as a key factor in age-related HL. This narrative review integrates past and recent research, bridging findings from animal models and human studies, to examine the contributions of the stria vascularis to age-related HL. It provides a brief overview of the structure and function of the stria vascularis and then examines mechanisms contributing to age-related strial dysfunction, including altered ion transport, changes in pigmentation, inflammatory responses, and vascular atrophy. Importantly, this review outlines the contribution of metabolic mechanisms to age-related HL, highlighting areas for future research. It emphasizes the complex interdependence of metabolic and sensorineural mechanisms in the pathology of age-related HL and highlights the importance of animal models in understanding the underlying mechanisms. The comprehensive and mechanistic investigation of all factors contributing to age-related HL, including cochlear metabolic dysfunction, remains crucial to identifying the underlying mechanisms and developing personalized, protective, and restorative treatments.

The miR-182-5p/GPX4 Pathway Contributes to Sevoflurane-Induced Ototoxicity via Ferroptosis.

Our study aimed to investigate the role of ferroptosis in sevoflurane-induced hearing impairment and explore the mechanism of the microRNA-182-5p (miR-182-5p)/Glutathione Peroxidase 4 (GPX4) pathway in sevoflurane-induced ototoxicity. Immunofluorescence staining was performed using myosin 7a and CtBP2. Cell viability was assessed using the CCK-8 kit. Fe2+ concentration was measured using FerroOrange and Mi-to-FerroGreen fluorescent probes. The lipid peroxide level was assessed using BODIPY 581/591 C11 and MitoSOX fluorescent probes. The auditory brainstem response (ABR) test was conducted to evaluate the hearing status. Bioinformatics tools and dual luciferase gene reporter analysis were used to confirm the direct targeting of miR-182-5p on GPX4 mRNA. GPX4 and miR-182-5p expression in cells was assessed by qRT-PCR and Western blot. Ferrostatin-1 (Fer-1) pretreatment significantly improved hearing impairment and damage to ribbon synapses in mice caused by sevoflurane exposure. Immunofluorescence staining revealed that Fer-1 pretreatment reduced intracellular and mitochondrial iron overload, as well as lipid peroxide accumulation. Our findings indicated that miR-182-5p was upregulated in sevoflurane-exposed HEI-OC1 cells, and miR-182-5p regulated GPX4 expression by binding to the 3'UTR of GPX4 mRNA. The inhibition of miR-182-5p attenuated sevoflurane-induced iron overload and lipid peroxide accumulation. Our study elucidated that the miR-182-5p/GPX4 pathway was implicated in sevoflurane-induced ototoxicity by promoting ferroptosis.

Whole exome sequencing reveals pathogenic variants in MYO3A, MYO15A and COL9A3 and differential frequencies in ancestral alleles in hearing impairment genes among individuals from Cameroon.

There is scarcity of known gene variants of hearing impairment (HI) in African populations. This knowledge deficit is ultimately affecting the development of genetic diagnoses. We used whole exome sequencing to investigate gene variants, pathways of interactive genes and the fractions of ancestral overderived alleles for 159 HI genes among 18 Cameroonian patients with non-syndromic HI (NSHI) and 129 ethnically matched controls. Pathogenic and likely pathogenic (PLP) variants were found in MYO3A, MYO15A and COL9A3, with a resolution rate of 50% (9/18 patients). The study identified significant genetic differentiation in novel population-specific gene variants at FOXD4L2, DHRS2L6, RPL3L and VTN between HI patients and controls. These gene variants are found in functional/co-expressed interactive networks with other known HI-associated genes and in the same pathways with VTN being a hub protein, that is, focal adhesion pathway and regulation of the actin cytoskeleton (P-values <0.05). The results suggest that these novel population-specific gene variants are possible modifiers of the HI phenotypes. We found a high proportion of ancestral allele versus derived at low HI patients-specific minor allele frequency in the range of 0.0-0.1. The results showed a relatively low pickup rate of PLP variants in known genes in this group of Cameroonian patients with NSHI. In addition, findings may signal an evolutionary enrichment of some variants of HI genes in patients, as the result of polygenic adaptation, and suggest the possibility of multigenic influence on the phenotype of congenital HI, which deserves further investigations.

Diabetes mellitus and hearing loss.

Diabetes mellitus (DM) is a major disease threatening human health and its incidence is increasing year on year. As a chronic complication of DM, hearing loss mostly occurs undetectably. However, the mechanism of this diabetes-related hearing loss (DRHL) remains unclear and there is no effective clinical treatment. Studies of animal or human pathology show that DM causes damage to the blood vessels, spiral ganglion neurons, afferent nerve fibers, the organ of Corti, and the stria vascularis of the inner ear. In recent years, more advances in pathological research have revealed the possible mechanism of DRHL. In addition, a large number of clinical studies suggest that the duration and severity of DM are closely related to the incidence and severity of DRHL. This review focuses on the relationship between DM and hearing loss. The clinical audiological characteristics of diabetic patients, risk factors for DRHL, typical pathology, and potential interventions of DRHL are summarized. This will help reveal the pathogenesis and intervention approaches for DRHL.

Epithelial dynamics shed light on the mechanisms underlying ear canal defects.

Defects in ear canal development can cause severe hearing loss as sound waves fail to reach the middle ear. Here, we reveal new mechanisms that control human canal development and highlight for the first time the complex system of canal closure and reopening. These processes can be perturbed in mutant mice and in explant culture, mimicking the defects associated with canal atresia. The more superficial part of the canal forms from an open primary canal that closes and then reopens. In contrast, the deeper part of the canal forms from an extending solid meatal plate that opens later. Closure and fusion of the primary canal was linked to loss of periderm, with failure in periderm formation in Grhl3 mutant mice associated with premature closure of the canal. Conversely, inhibition of cell death in the periderm resulted in an arrest of closure. Once closed, re-opening of the canal occurred in a wave, triggered by terminal differentiation of the epithelium. Understanding these complex processes involved in canal development sheds light on the underlying causes of canal atresia.

A budding yeast model for human disease mutations in the EXOSC2 cap subunit of the RNA exosome complex.

RNA exosomopathies, a growing family of diseases, are linked to missense mutations in genes encoding structural subunits of the evolutionarily conserved, 10-subunit exoribonuclease complex, the RNA exosome. This complex consists of a three-subunit cap, a six-subunit, barrel-shaped core, and a catalytic base subunit. While a number of mutations in RNA exosome genes cause pontocerebellar hypoplasia, mutations in the cap subunit gene EXOSC2 cause an apparently distinct clinical presentation that has been defined as a novel syndrome SHRF (short stature, hearing loss, retinitis pigmentosa, and distinctive facies). We generated the first in vivo model of the SHRF pathogenic amino acid substitutions using budding yeast by modeling pathogenic EXOSC2 missense mutations (p.Gly30Val and p.Gly198Asp) in the orthologous S. cerevisiae gene RRP4 The resulting rrp4 mutant cells show defects in cell growth and RNA exosome function. Consistent with altered RNA exosome function, we detect significant transcriptomic changes in both coding and noncoding RNAs in rrp4-G226D cells that model EXOSC2 p.Gly198Asp, suggesting defects in nuclear surveillance. Biochemical and genetic analyses suggest that the Rrp4 G226D variant subunit shows impaired interactions with key RNA exosome cofactors that modulate the function of the complex. These results provide the first in vivo evidence that pathogenic missense mutations present in EXOSC2 impair the function of the RNA exosome. This study also sets the stage to compare exosomopathy models to understand how defects in RNA exosome function underlie distinct pathologies.

Aldh inhibitor restores auditory function in a mouse model of human deafness.

Genetic hearing loss is a common health problem with no effective therapy currently available. DFNA15, caused by mutations of the transcription factor POU4F3, is one of the most common forms of autosomal dominant non-syndromic deafness. In this study, we established a novel mouse model of the human DFNA15 deafness, with a Pou4f3 gene mutation (Pou4f3Δ) identical to that found in a familial case of DFNA15. The Pou4f3(Δ/+) mice suffered progressive deafness in a similar manner to the DFNA15 patients. Hair cells in the Pou4f3(Δ/+) cochlea displayed significant stereociliary and mitochondrial pathologies, with apparent loss of outer hair cells. Progression of hearing and outer hair cell loss of the Pou4f3(Δ/+) mice was significantly modified by other genetic and environmental factors. Using Pou4f3(-/+) heterozygous knockout mice, we also showed that DFNA15 is likely caused by haploinsufficiency of the Pou4f3 gene. Importantly, inhibition of retinoic acid signaling by the aldehyde dehydrogenase (Aldh) and retinoic acid receptor inhibitors promoted Pou4f3 expression in the cochlear tissue and suppressed the progression of hearing loss in the mutant mice. These data demonstrate Pou4f3 haploinsufficiency as the main underlying cause of human DFNA15 deafness and highlight the therapeutic potential of Aldh inhibitors for treatment of progressive hearing loss.

Preimplantation genetic testing for hereditary hearing loss in Chinese population.

PURPOSE: To evaluate the clinical validity of preimplantation genetic testing (PGT) to prevent hereditary hearing loss (HL) in Chinese population. METHODS: A PGT procedure combining multiple annealing and looping-based amplification cycles (MALBAC) and single-nucleotide polymorphisms (SNPs) linkage analyses with a single low-depth next-generation sequencing run was implemented. Forty-three couples carried pathogenic variants in autosomal recessive non-syndromic HL genes, GJB2 and SLC26A4, and four couples carried pathogenic variants in rare HL genes: KCNQ4, PTPN11, PAX3, and USH2A were enrolled. RESULTS: Fifty-four in vitro fertilization (IVF) cycles were implemented, 340 blastocysts were cultured, and 303 (89.1%) of these received a definite diagnosis of a disease-causing variant testing, linkage analysis and chromosome screening. A clinical pregnancy of 38 implanted was achieved, and 34 babies were born with normal hearing. The live birth rate was 61.1%. CONCLUSIONS AND RELEVANCE: In both the HL population and in hearing individuals at risk of giving birth to offspring with HL in China, there is a practical need for PGT. The whole genome amplification combined with NGS can simplify the PGT process, and the efficiency of PGT process can be improved by establishing a universal SNP bank of common disease-causing gene in particular regions and nationalities. This PGT procedure was demonstrated to be effective and lead to satisfactory clinical outcomes.

Macrostructural Changes of the Acoustic Radiation in Humans with Hearing Loss and Tinnitus Revealed with Fixel-Based Analysis.

Age-related hearing loss is the most prevalent sensory impairment in the older adult population and is related to noise-induced damage or age-related deterioration of the peripheral auditory system. Hearing loss may affect the central auditory pathway in the brain, which is a continuation of the peripheral auditory system located in the ear. A debilitating symptom that frequently co-occurs with hearing loss is tinnitus. Strikingly, investigations into the impact of acquired hearing loss, with and without tinnitus, on the human central auditory pathway are sparse. This study used diffusion-weighted imaging (DWI) to investigate changes in the largest central auditory tract, the acoustic radiation, related to hearing loss and tinnitus. Participants with hearing loss, with and without tinnitus, and a control group were included. Both conventional diffusion tensor analysis and higher-order fixel-based analysis were applied. The fixel-based analysis was used as a novel framework providing insight into the axonal density and macrostructural morphologic changes of the acoustic radiation in hearing loss and tinnitus. The results show tinnitus-related atrophy of the left acoustic radiation near the medial geniculate body. This finding may reflect a decrease in myelination of the auditory pathway, instigated by more profound peripheral deafferentation or reflecting a preexisting marker of tinnitus vulnerability. Furthermore, age was negatively correlated with the axonal density in the bilateral acoustic radiation. This loss of fiber density with age may contribute to poorer speech understanding observed in older adults.SIGNIFICANCE STATEMENT Age-related hearing loss is the most prevalent sensory impairment in the older adult population. Older individuals are subject to the cumulative effects of aging and noise exposure on the auditory system. A debilitating symptom that frequently co-occurs with hearing loss is tinnitus: the perception of a phantom sound. In this large DWI-study, we provide evidence that in hearing loss, the additional presence of tinnitus is related to degradation of the acoustic radiation. Additionally, older age was related to axonal loss in the acoustic radiation. It appears that older adults have the aggravating circumstances of age, hearing loss, and tinnitus on central auditory processing, which may partly be because of the observed deterioration of the acoustic radiation with age.

Macrophages in the cochlea; an immunological link between risk factors and progressive hearing loss.

Macrophages are abundant in the cochlea; however, their role in hearing loss is not well understood. Insults to the cochlea, such as noise or insertion of a cochlear implant, cause an inflammatory response, which includes activation of tissue-resident macrophages. Activation is characterized by changes in macrophage morphology, mediator expression, and distribution. Evidence from other organs shows activated macrophages can become primed, whereby subsequent insults cause an elevated inflammatory response. Primed macrophages in brain pathologies respond to circulating inflammatory mediators by disproportionate synthesis of inflammatory mediators. This signaling occurs behind an intact blood-brain barrier, similar to the blood-labyrinth barrier in the cochlea. Local tissue damage can occur as the result of mediator release by activated macrophages. Damage is typically localized; however, if it is to structures with limited ability to repair, such as neurons or hair cells within the cochlea, it is feasible that this contributes to the progressive loss of function seen in hearing loss. We propose that macrophages in the cochlea link risk factors and hearing loss. Injury to the cochlea causes local macrophage activation that typically resolves. However, in susceptible individuals, some macrophages enter a primed state. Once primed, these macrophages can be further activated, as a consequence of circulating inflammatory molecules associated with common co-morbidities. Hypothetically, this would lead to further cochlear damage and loss of hearing. We review the evidence for the role of tissue-resident macrophages in the cochlea and propose that cochlear macrophages contribute to the trajectory of hearing loss and warrant further study.

Genetic architecture and phenotypic landscape of SLC26A4-related hearing loss.

Mutations of coding regions and splice sites of SLC26A4 cause Pendred syndrome and nonsyndromic recessive hearing loss DFNB4. SLC26A4 encodes pendrin, a transmembrane exchanger of anions and bases. The mutant SLC26A4 phenotype is characterized by inner ear malformations, including an enlarged vestibular aqueduct (EVA), incomplete cochlear partition type II and modiolar hypoplasia, progressive and fluctuating hearing loss, and vestibular dysfunction. A thyroid iodine organification defect can lead to multinodular goiter and distinguishes Pendred syndrome from DFNB4. Pendred syndrome and DFNB4 are each inherited as an autosomal recessive trait caused by biallelic mutations of SLC26A4 (M2). However, there are some EVA patients with only one detectable mutant allele (M1) of SLC26A4. In most European-Caucasian M1 patients, there is a haplotype that consists of 12 variants upstream of SLC26A4, called CEVA (Caucasian EVA), which acts as a pathogenic recessive allele in trans to mutations affecting the coding regions or splice sites of SLC26A4. This combination of an M1 genotype with the CEVA haplotype is associated with a less severe phenotype than the M2 genotype. The phenotype in EVA patients with no mutant alleles of SLC26A4 (M0) has a very low recurrence probability and is likely to be caused by other factors.

Directed differentiation and direct reprogramming: Applying stem cell technologies to hearing research.

Hearing loss is the most widely spread sensory disorder in our society. In the majority of cases, it is caused by the loss or malfunctioning of cells in the cochlea: the mechanosensory hair cells, which act as primary sound receptors, and the connecting auditory neurons of the spiral ganglion, which relay the signal to upper brain centers. In contrast to other vertebrates, where damage to the hearing organ can be repaired through the activity of resident cells, acting as tissue progenitors, in mammals, sensory cell damage or loss is irreversible. The understanding of gene and cellular functions, through analysis of different animal models, has helped to identify causes of disease and possible targets for hearing restoration. Translation of these findings to novel therapeutics is, however, hindered by the lack of cellular assays, based on human sensory cells, to evaluate the conservation of molecular pathways across species and the efficacy of novel therapeutic strategies. In the last decade, stem cell technologies enabled to generate human sensory cell types in vitro, providing novel tools to study human inner ear biology, model disease, and validate therapeutics. This review focuses specifically on two technologies: directed differentiation of pluripotent stem cells and direct reprogramming of somatic cell types to sensory hair cells and neurons. Recent development in the field are discussed as well as how these tools could be implemented to become routinely adopted experimental models for hearing research.

Single and Dual Vector Gene Therapy with AAV9-PHP.B Rescues Hearing in Tmc1 Mutant Mice.

AAV-mediated gene therapy is a promising approach for treating genetic hearing loss. Replacement or editing of the Tmc1 gene, encoding hair cell mechanosensory ion channels, is effective for hearing restoration in mice with some limitations. Efficient rescue of outer hair cell function and lack of hearing recovery with later-stage treatment remain issues to be solved. Exogenous genes delivered with the adeno-associated virus (AAV)9-PHP.B capsid via the utricle transduce both inner and outer hair cells of the mouse cochlea with high efficacy. Here, we demonstrate that AAV9-PHP.B gene therapy can promote hair cell survival and successfully rescues hearing in three distinct mouse models of hearing loss. Tmc1 replacement with AAV9-PHP.B in a Tmc1 knockout mouse rescues hearing and promotes hair cell survival with equal efficacy in inner and outer hair cells. The same treatment in a recessive Tmc1 hearing-loss model, Baringo, partially recovers hearing even with later-stage treatment. Finally, dual delivery of Streptococcus pyogenes Cas9 (SpCas9) and guide RNA (gRNA) in separate AAV9-PHP.B vectors selectively disrupts a dominant Tmc1 allele and preserves hearing in Beethoven mice, a model of dominant, progressive hearing loss. Tmc1-targeted gene therapies using single or dual AAV9-PHP.B vectors offer potent and versatile approaches for treating dominant and recessive deafness.