Adult-onset hearing loss is very common, but we know little about the underlying molecular pathogenesis impeding the development of therapies. We took a genetic approach to identify new molecules ...involved in hearing loss by screening a large cohort of newly generated mouse mutants using a sensitive electrophysiological test, the auditory brainstem response (ABR). We review here the findings from this screen. Thirty-eight unexpected genes associated with raised thresholds were detected from our unbiased sample of 1,211 genes tested, suggesting extreme genetic heterogeneity. A wide range of auditory pathophysiologies was found, and some mutant lines showed normal development followed by deterioration of responses, revealing new molecular pathways involved in progressive hearing loss. Several of the genes were associated with the range of hearing thresholds in the human population and one, SPNS2, was involved in childhood deafness. The new pathways required for maintenance of hearing discovered by this screen present new therapeutic opportunities.
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
mouse mutant known to show this defect. By activating
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
led to less effective recovery of ABR thresholds, suggesting that there is a critical period for intervention. Early activation of
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.
Diaphanous related formins are regulatory cytoskeletal protein involved in actin elongation and microtubule stabilization. In humans, defects in two of the three diaphanous genes (DIAPH1 and DIAPH3) ...have been associated with different types of hearing loss. Here, we investigate the role of the third member of the family, DIAPH2, in nonsyndromic hearing loss, prompted by the identification, by exome sequencing, of a predicted pathogenic missense variant in DIAPH2. This variant occurs at a conserved site and segregated with nonsyndromic X-linked hearing loss in an Italian family. Our immunohistochemical studies indicated that the mouse ortholog protein Diaph2 is expressed during development in the cochlea, specifically in the actin-rich stereocilia of the sensory outer hair cells. In-vitro studies showed a functional impairment of the mutant DIAPH2 protein upon RhoA-dependent activation. Finally, Diaph2 knock-out and knock-in mice were generated by CRISPR/Cas9 technology and auditory brainstem response measurements performed at 4, 8 and 14 weeks. However, no hearing impairment was detected. Our findings indicate that DIAPH2 may play a role in the inner ear; further studies are however needed to clarify the contribution of DIAPH2 to deafness.
Planar cell polarity (PCP) refers to the polarization of cells within the plane of a cell sheet. A distinctive epithelial PCP in vertebrates is the uniform orientation of stereociliary bundles of the ...sensory hair cells in the mammalian cochlea. In addition to establishing epithelial PCP, planar polarization is also required for convergent extension (CE); a polarized cellular movement that occurs during neural tube closure and cochlear extension. Studies in
Drosophila and vertebrates have revealed a conserved PCP pathway, including Frizzled (Fz) receptors. Here we use the cochlea as a model system to explore the involvement of known ligands of Fz, Wnt morphogens, in PCP regulation. We show that Wnt5a forms a reciprocal expression pattern with a Wnt antagonist, the secreted frizzled-related protein 3 (Sfrp3 or Frzb), along the axis of planar polarization in the cochlear epithelium. We further demonstrate that Wnt5a antagonizes Frzb in regulating cochlear extension and stereociliary bundle orientation in vitro, and that
Wnt5a
−/−
animals have a shortened and widened cochlea. Finally, we show that Wnt5a is required for proper subcellular distribution of a PCP protein, Ltap/Vangl2, and that Wnt5a interacts genetically with
Ltap/Vangl2 for uniform orientation of stereocilia, cochlear extension, and neural tube closure. Together, these findings demonstrate that Wnt5a functions in PCP regulation in mice.
Age-related hearing loss in humans (presbycusis) typically involves impairment of high frequency sensitivity before becoming progressively more severe at lower frequencies. Pathologies initially ...affecting lower frequency regions of hearing are less common. Here we describe a progressive, predominantly low-frequency recessive hearing impairment in two mutant mouse lines carrying different mutant alleles of the Klhl18 gene: a spontaneous missense mutation (Klhl18lowf) and a targeted mutation (Klhl18tm1a(KOMP)Wtsi). Both males and females were studied, and the two mutant lines showed similar phenotypes. Threshold for auditory brainstem responses (ABR; a measure of auditory nerve and brainstem neural activity) were normal at 3 weeks old but showed progressive increases from 4 weeks onwards. In contrast, distortion product otoacoustic emission (DPOAE) sensitivity and amplitudes (a reflection of cochlear outer hair cell function) remained normal in mutants. Electrophysiological recordings from the round window of Klhl18lowf mutants at 6 weeks old revealed 1) raised compound action potential thresholds that were similar to ABR thresholds, 2) cochlear microphonic potentials that were normal compared with wildtype and heterozygous control mice and 3) summating potentials that were reduced in amplitude compared to control mice. Scanning electron microscopy showed that Klhl18lowf mutant mice had abnormally tapering of the tips of inner hair cell stereocilia in the apical half of the cochlea while their synapses appeared normal. These results suggest that Klhl18 is necessary to maintain inner hair cell stereocilia and normal inner hair cell function at low frequencies.
Hearing impairment or vestibular dysfunction in humans often results from a permanent loss of critical cell types in the sensory regions of the inner ear, including hair cells, supporting cells, or ...cochleovestibular neurons. These important cell types arise from a common sensory or neurosensory progenitor, although little is known about how these progenitors are specified. Studies have shown that Notch signaling and the transcription factor Sox2 are required for the development of these lineages. Previously we and others demonstrated that ectopic activation of Notch can direct nonsensory cells to adopt a sensory fate, indicating a role for Notch in early specification events. Here, we explore the relationship between Notch and SOX2 by ectopically activating these factors in nonsensory regions of the mouse cochlea, and demonstrate that, similar to Notch, SOX2 can specify sensory progenitors, consistent with a role downstream of Notch signaling. However, we also show that Notch has a unique role in promoting the proliferation of the sensory progenitors. We further demonstrate that Notch can only induce ectopic sensory regions within a certain time window of development, and that the ectopic hair cells display specialized stereocilia bundles similar to endogenous hair cells. These results demonstrate that Notch and SOX2 can both drive the sensory program in nonsensory cells, indicating these factors may be useful in cell replacement strategies in the inner ear.
Tasmanian devil (tde) mice are deaf and exhibit circling behaviour. Sensory hair cells of mutants show disorganised hair bundles with abnormally thin stereocilia. The origin of this mutation is the ...insertion of a transgene which disrupts expression of the Grxcr1 (glutaredoxin cysteine rich 1) gene. We report here that Grxcr1 exons and transcript sequences are not affected by the transgene insertion in tde homozygous (tde/tde) mice. Furthermore, 5'RACE PCR experiments showed the presence of two different transcripts of the Grxcr1 gene, expressed in both tde/tde and in wild-type controls. However, quantitative analysis of Grxcr1 transcripts revealed a significantly decreased mRNA level in tde/tde mice. The key stereociliary proteins ESPN, MYO7A, EPS8 and PTPRQ were distributed in hair bundles of homozygous tde mutants in a similar pattern compared with control mice. We found that the abnormal morphology of the stereociliary bundle was associated with a reduction in the size and Ca2+-sensitivity of the mechanoelectrical transducer (MET) current. We propose that GRXCR1 is key for the normal growth of the stereociliary bundle prior to the onset of hearing, and in its absence hair cells are unable to mature into fully functional sensory receptors.
Considerable progress has been made in identifying deafness genes, but still little is known about the genetic basis of normal variation in hearing function. We recently carried out a Genome Wide ...Association Study (GWAS) of quantitative hearing traits in southern European populations and found several SNPs with suggestive but none with significant association. In the current study, we followed up these SNPs to investigate which of them might show a genuine association with auditory function using alternative approaches. Firstly, we generated a shortlist of 19 genes from the published GWAS results. Secondly, we carried out immunocytochemistry to examine expression of these 19 genes in the mouse inner ear. Twelve of them showed distinctive cochlear expression patterns. Four showed expression restricted to sensory hair cells (Csmd1, Arsg, Slc16a6 and Gabrg3), one only in marginal cells of the stria vascularis (Dclk1) while the others (Ptprd, Grm8, GlyBP, Evi5, Rimbp2, Ank2, Cdh13) in multiple cochlear cell types. In the third step, we tested these 12 genes for replication of association in an independent set of samples from the Caucasus and Central Asia. Nine out of them showed nominally significant association (p<0.05). In particular, 4 were replicated at the same SNP and with the same effect direction while the remaining 5 showed a significant association in a gene-based test. Finally, to look for genotype-phenotype relationship, the audiometric profiles of the three genotypes of the most strongly associated gene variants were analyzed. Seven out of the 9 replicated genes (CDH13, GRM8, ANK2, SLC16A6, ARSG, RIMBP2 and DCLK1) showed an audiometric pattern with differences between different genotypes further supporting their role in hearing function. These data demonstrate the usefulness of this multistep approach in providing new insights into the molecular basis of hearing and may suggest new targets for treatment and prevention of hearing impairment.
MicroRNAs (miRNAs) bind to complementary sites in their target mRNAs to mediate post-transcriptional repression, with the specificity of target recognition being crucially dependent on the miRNA seed ...region. Impaired miRNA target binding resulting from SNPs within mRNA target sites has been shown to lead to pathologies associated with dysregulated gene expression. However, no pathogenic mutations within the mature sequence of a miRNA have been reported so far. Here we show that point mutations in the seed region of miR-96, a miRNA expressed in hair cells of the inner ear, result in autosomal dominant, progressive hearing loss. This is the first study implicating a miRNA in a mendelian disorder. The identified mutations have a strong impact on miR-96 biogenesis and result in a significant reduction of mRNA targeting. We propose that these mutations alter the regulatory role of miR-96 in maintaining gene expression profiles in hair cells required for their normal function.
Age-related hearing loss is a common, heterogeneous disease with a strong genetic component. More than 100 loci have been reported to be involved in human hearing impairment to date, but most of the ...genes underlying human adult-onset hearing loss remain unknown. Most genetic studies have focussed on very rare variants (such as family studies and patient cohort screens) or very common variants (genome-wide association studies). However, the contribution of variants present in the human population at intermediate frequencies is hard to quantify using these methods, and as a result, the landscape of variation associated with adult-onset hearing loss remains largely unknown.
Here we present a study based on exome sequencing and self-reported hearing difficulty in the UK Biobank, a large-scale biomedical database. We have carried out variant load analyses using different minor allele frequency and impact filters, and compared the resulting gene lists to a manually curated list of nearly 700 genes known to be involved in hearing in humans and/or mice. An allele frequency cutoff of 0.1, combined with a high predicted variant impact, was found to be the most effective filter setting for our analysis. We also found that separating the participants by sex produced markedly different gene lists. The gene lists obtained were investigated using gene ontology annotation, functional prioritisation and expression analysis, and this identified good candidates for further study.
Our results suggest that relatively common as well as rare variants with a high predicted impact contribute to age-related hearing impairment and that the genetic contributions to adult hearing difficulty may differ between the sexes. Our manually curated list of deafness genes is a useful resource for candidate gene prioritisation in hearing loss.
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