Identification of Three Novel Hearing Loss Mouse Strains with Mutations in the Tmc1 Gene

  • Shehnaaz S.M. Manji
    Correspondence
    Address reprint requests to Shehnaaz S.M. Manji, Ph.D., Department of Otolaryngology, University of Melbourne, Royal Victorian Eye and Ear Hospital, Level 2, 32 Gisborne St, East Melbourne, Victoria 3002, Australia
    Affiliations
    Genetic Hearing Research, Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, Australia

    Department of Otolaryngology, Audiology, Hearing and Speech Sciences, University of Melbourne, Victoria, Australia

    HEARing Cooperative Research Centre, Audiology, Hearing and Speech Sciences, University of Melbourne, Victoria, Australia
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  • Kerry A. Miller
    Affiliations
    Genetic Hearing Research, Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, Australia
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  • Louise H. Williams
    Affiliations
    Genetic Hearing Research, Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, Australia
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  • Hans-Henrik M. Dahl
    Affiliations
    Genetic Hearing Research, Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, Australia

    Department of Pediatrics, Audiology, Hearing and Speech Sciences, University of Melbourne, Victoria, Australia
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Published:February 13, 2012DOI:https://doi.org/10.1016/j.ajpath.2011.12.034
      We report the identification of three new mouse models, baringo, nice, and stitch, with recessively inherited sensorineural deafness due to novel mutations in the transmembrane channel-like gene 1 (Tmc1). These strains were generated by N-ethyl-N-nitrosourea mutagenesis. DNA sequence analysis revealed changes in c.545A>G, c.1345T>C, and c.1661G>T, causing p.Y182C, p.Y449H, and p.W554L amino acid substitutions in baringo, nice, and stitch mutants, respectively. The mutations affect amino acid residues that are evolutionarily conserved across species. Similar to the previously reported Beethoven Tmc1 mutant, both p.Y182C and p.W554L are located outside a predicted transmembrane domain, whereas the p.Y449H mutation resides in the predicted transmembrane domain 4. Homozygous stitch-mutant mice have severe hearing loss at the age of 4 weeks and are deaf by the age of 8 weeks, whereas both baringo and nice mutants are profoundly deaf at the age of 4 weeks. None of the strains displays signs of vestibular dysfunction. Scanning electron microscopy revealed degeneration of outer hair cells in the basal region of baringo, nice, and stitch mutants. Immunolocalization studies revealed expression of TMC1 protein in the hair cells, spiral ganglion neurons, supporting cells, and stria ligament in the inner ear. Reduced levels of TMC1 protein were observed in the spiral ligament of mutants when compared with wild-type animals. These three allelic mutants provide valuable models for studying nonsyndromic recessive sensorineural hearing loss (DFNB7/11) in humans.
      Deafness is the most common sensory impairment in humans. In developed countries, approximately 1 in 800 children are born deaf and another 1 in 1000 children will develop a permanent hearing loss within the first two decades of life.
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      The need for universal neonatal hearing screening: some aspects of epidemiology and identification.
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      A genetic approach to understanding auditory function.
      Age-related or progressive hearing loss (presbycusis) affects more people; by the age of 75 years, >50% of the population will have a noticeable hearing impairment. Hearing loss significantly affects speech and language development, incurring lifelong educational, social development, and economic costs.
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      The societal costs of severe to profound hearing loss in the United States.
      Hearing loss is a multifactorial disorder, caused by environmental, genetic, or a combination of factors. Environmental factors include infections, noise exposure, premature birth, and exposure to ototoxic drugs.
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      • Nance W.E.
      Newborn hearing screening: a silent revolution.
      Genetic forms of hearing loss are classified as syndromic or nonsyndromic and account for 15% and 35% of all hearing loss in young children, respectively. There are >450 genetic syndromes that have hearing loss as a clinical feature, including Usher and Pendred syndromes.
      • Friedman T.B.
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      • Ahmed Z.M.
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      • Griffith A.J.
      Recent advances in the understanding of syndromic forms of hearing loss.
      Genetic forms of nonsyndromic hearing loss can be transmitted as autosomal recessive, autosomal dominant, maternally (mitochondrial), and X- or Y-linked inherited conditions.
      • Van Laer L.
      • Cryns K.
      • Smith R.J.
      • Van Camp G.
      Nonsyndromic hearing loss.
      Currently, 28 genes have been identified in people with recessively inherited hearing loss that accounts for most (80%) of the inherited nonsyndromic deafness. Twenty-four genes have been implicated in dominantly inherited nonsyndromic hearing loss (http://hereditaryhearingloss.org, last accessed December 11, 2011).
      The mammalian auditory and vestibular structures are highly conserved across species and, consequently, data from animal studies can be extrapolated across to the human system. Animal models, including mouse models, have proved useful in the study of auditory processes and in the identification of genes for deafness.
      • Ahituv N.
      • Avraham K.B.
      Mouse models for human deafness: current tools for new fashions.
      Having well-characterized and accurate mouse models for human genetic hearing loss is also a prerequisite for the development of novel treatments and for studying the relationship between environmental and/or genetic factors in causing deafness, including age-related hearing impairment.
      N-ethyl-N-nitrosourea (ENU) mutagenesis programs provide a powerful approach in the discovery and understanding of genes associated with several human disorders, including deafness.
      • Hoyne G.F.
      • Goodnow C.C.
      The use of genomewide ENU mutagenesis screens to unravel complex mammalian traits: identifying genes that regulate organ-specific and systemic autoimmunity.
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      ENU mutagenesis, a way forward to understand gene function.
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      A forward genetics screen in mice identifies recessive deafness traits and reveals that pejvakin is essential for outer hair cell function.
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      • Miller D.
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      • Yoo T.J.
      • Goldowitz D.
      • Zuo J.
      Identification of 17 hearing impaired mouse strains in the TMGC ENU-mutagenesis screen.
      • Manji S.S.
      • Miller K.A.
      • Williams L.H.
      • Andreasen L.
      • Siboe M.
      • Rose E.
      • Bahlo M.
      • Kuiper M.
      • Dahl H.H.
      An ENU-induced mutation of Cdh23 causes congenital hearing loss, but no vestibular dysfunction, in mice.
      • Manji S.S.
      • Williams L.H.
      • Miller K.A.
      • Ooms L.M.
      • Bahlo M.
      • Mitchell C.A.
      • Dahl H.H.
      A mutation in synaptojanin 2 causes progressive hearing loss in the ENU-mutagenised mouse strain Mozart.
      Treatment of mice with ENU mainly generates point mutations, often in gene-coding regions. Therefore, ENU mutants frequently display similar clinical features to those seen in corresponding human conditions. We screened ENU mouse libraries for mice with recessively inherited hearing loss and identified three mutants with novel mutations in the transmembrane channel-like gene 1 (Tmc1) gene. Mutations in TMC1 cause dominant progressive (DFNA36) and recessive (DFNB7/11) sensorineural hearing loss in humans and mice.
      • Kurima K.
      • Peters L.M.
      • Yang Y.D.
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      • Ahmed Z.M.
      • Naz S.
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      • Sullivan S.L.
      • Battey J.F.
      • Keats B.J.B.
      • Wilcox E.R.
      • Friedman T.B.
      • Griffith A.J.
      Dominant and recessive deafness caused by mutations of a novel gene, TMC1, required for cochlear hair-cell function.
      • Vreugde S.
      • Erven A.
      • Kros C.J.
      • Marcotti W.
      • Fuchs H.
      • Kurima K.
      • Wilcox E.R.
      • Friedman T.B.
      • Griffith A.J.
      • Balling R.
      • Hrabé De Angelis M.
      • Avraham K.B.
      • Steel K.P.
      Beethoven, a mouse model for dominant, progressive hearing loss DFNA36.
      Tmc1 is a member of the TMC gene family that is thought to act as ion channels, pumps, or transporters.
      • Keresztes G.
      • Mutai H.
      • Heller S.
      TMC and EVER genes belong to a larger novel family, the TMC gene family encoding transmembrane proteins.
      • Kurima K.
      • Yang Y.
      • Sorber K.
      • Griffith A.J.
      Characterization of the transmembrane channel-like (TMC) gene family: functional clues from hearing loss and epidermodysplasia verruciformis.
      The TMC gene family encodes transmembrane proteins that contain 6 to 10 transmembrane domains (TMDs) and a unique conserved 120–amino acid region, designated the TMC domain, with no homology to other known genes.
      • Kurima K.
      • Yang Y.
      • Sorber K.
      • Griffith A.J.
      Characterization of the transmembrane channel-like (TMC) gene family: functional clues from hearing loss and epidermodysplasia verruciformis.
      Tmc1 mRNA is expressed in mouse cochlea and vestibular hair cells, but its function remains unknown. The mutants identified in this study have sensorineural recessively inherited hearing loss, similar to that previously reported in the Tmc1 mouse mutant, deafness, and in contrast to the Tmc1 mutant Beethoven, which has a dominant progressive hearing loss (Table 1).
      • Manji S.S.
      • Miller K.A.
      • Williams L.H.
      • Andreasen L.
      • Siboe M.
      • Rose E.
      • Bahlo M.
      • Kuiper M.
      • Dahl H.H.
      An ENU-induced mutation of Cdh23 causes congenital hearing loss, but no vestibular dysfunction, in mice.
      • Manji S.S.
      • Williams L.H.
      • Miller K.A.
      • Ooms L.M.
      • Bahlo M.
      • Mitchell C.A.
      • Dahl H.H.
      A mutation in synaptojanin 2 causes progressive hearing loss in the ENU-mutagenised mouse strain Mozart.
      In this study, we present the characterization of the three novel Tmc1 mutants designated baringo, nice, and stitch and demonstrate that the TMC1 protein is expressed in the cochlea.
      Table 1Reported Allelic Variants of Tmc1 in Mice
      Mouse modelNucleotide changeProtein changeExonPhenotypePredicted effectReference
      baringo545A>GY182C8DFNB7/B11Disruption of TMD1Present study
      nice1345T>CY449H13DFNB7/B11Disruption of TMD4Present study
      stitch1661G>TW554L15DFNB7/B11Abnormal protein foldingPresent study
      Beethoven1235T>AM412K13DFNA36Abnormal protein folding
      • Manji S.S.
      • Williams L.H.
      • Miller K.A.
      • Ooms L.M.
      • Bahlo M.
      • Mitchell C.A.
      • Dahl H.H.
      A mutation in synaptojanin 2 causes progressive hearing loss in the ENU-mutagenised mouse strain Mozart.
      deafnessNAIVS13_IVS14del1.6kb14DFNB7/11In-frame deletion of exon 14
      • Manji S.S.
      • Miller K.A.
      • Williams L.H.
      • Andreasen L.
      • Siboe M.
      • Rose E.
      • Bahlo M.
      • Kuiper M.
      • Dahl H.H.
      An ENU-induced mutation of Cdh23 causes congenital hearing loss, but no vestibular dysfunction, in mice.
      NA, not applicable.

      Materials and Methods

      Identification of baringo, nice, and stitch Mutants

      This project was approved by the Royal Children's Hospital Animal Ethics Committee (Animal Ethics application numbers A488 and A585). We screened C57BL/6 ENU mutagenized mouse libraries generated at the Australian Phenomics Facility for recessively inherited hearing loss. The click-box hearing test elicits a Preyer reflex or startle response in hearing mice and provides a convenient, fast, low-cost, and, thus, suitable high-throughput phenotypic screen. The click box produces an 18.9-kHz burst of 106-dB sound pressure levels (SPLs) at a distance of 10 cm (Institute of Hearing Research, Nottingham, UK). Mice that failed the initial click-box hearing test underwent an evoked auditory brainstem response hearing test (AEP; Bio-logic Systems Corp, Chicago, IL), as previously described.
      • Manji S.S.
      • Williams L.H.
      • Miller K.A.
      • Ooms L.M.
      • Bahlo M.
      • Mitchell C.A.
      • Dahl H.H.
      A mutation in synaptojanin 2 causes progressive hearing loss in the ENU-mutagenised mouse strain Mozart.
      Mice were also screened for vestibular dysfunction, evident by the display of circling, head tossing/tilting, and star-gazing behavior.

      Heritability and Genetic Mapping

      Mouse mutants that failed the click-box and auditory brainstem response hearing tests were intercrossed to the congenic strains C57BL/10 or C57BL/6 for heritability testing. We expected approximately 25% of the offspring mice to be deaf if the mutation was recessively inherited and fully penetrant. When this was the case, deaf mice were outcrossed to the CBA/H or BALB/c strains and the F2 progeny were generated for homozygosity mapping and subsequent identification of candidate regions. Genomic-wide homozygosity mapping using 120 microsatellite markers (AGRF, Melbourne, Australia) was performed on genomic DNA from 10 affected mice from the baringo/CBA/H cross. Fine mapping was performed using Amplifluor-based single-nucleotide polymorphism assays (Australian Phenomics Facility, Canberra, Australia) on 45 additional affected mice. For our statistical analysis, we used the normal approximation to the binomial test for proportions of homozygous C57BL/6 genotypes (hearing loss mutants) to localize the deafness loci. Linkage intervals were examined for known or putative deafness genes using the University of California, Santa Cruz, genome browser (http://genome.ucsc.edu, last accessed July 10, 2011), and candidates were sequenced. Nice and stitch mutants were directly screened for mutations in known deafness genes, including Cdh23, Tmc1, and Myo7a, by sequencing genomic DNA (Tmc1 and Myo7a) or cDNA (Cdh23) from brain.

      Tissue Collection

      Mice (aged ≥2 weeks) were anesthetized with isoflurane and culled by cervical dislocation, whereas mice aged <2 weeks were euthanized by 2,2,2-tribromoethanol (Avertin) overdose, according to the National Health and Medical Research Council Australian code of practice for the care and use of animals for scientific purposes. Intact cochleae were surgically removed using a posterolateral approach, and the brain was dissected from mice of the same strain, age, and sex. Brains were stored in RNAlater buffer (Qiagen, GmbH, Hilden, Germany) at −70°C for total RNA isolation. Adult mouse cochleae were processed for H&E staining and immunohistochemistry, as previously described.
      • Manji S.S.
      • Sorensen B.S.
      • Klockars T.
      • Lam T.
      • Hutchison W.
      • Dahl H.H.
      Molecular characterization and expression of maternally expressed gene 3 (Meg3/Gtl2) RNA in the mouse inner ear.
      On postnatal day 5 (P5), mouse half heads were fixed in 4% paraformaldehyde at room temperature for 2 hours, followed by three 20-minute washes with PBS, before the sensory epithelium (SE) was dissected from the inner ear for whole-mount immunofluorescence analysis.

      Mutation Analysis

      The GenBank accession numbers are as follows: Tmc1 cDNA, NM 028953; and TMC1 protein, NP 083229. To scan for mutations in the Tmc1 gene, genomic DNA was isolated from tail biopsy specimens of deaf and hearing littermates. Twenty-one primer pairs were designed to amplify all 21 Tmc1 exons (including intron-exon boundaries and most 5′ and 3′ untranslated regions). Genomic DNA fragments were PCR amplified using HotStar Taq polymerase (Qiagen, Hilden) or GoTaq polymerase (Promega, Madison, WI). PCR cycling conditions were as follows: 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 45 seconds, for 35 cycles. The PCR products were sequenced in both directions using a Big Dye Sequencing kit, version 3.1 (ABI, Carlsbad, CA), and analyzed on an Applied Biosystems DNA Analyzer (model 3730xl; ABI). DNA sequences from deaf and hearing littermates were compared using Mutation Surveyor version 2.60 software (SoftGenetics, State College, PA).

      Immunohistochemical Staining for TMC1 Protein

      Processed cochleae were embedded in opitmal cutting temperature compound (Sakura, Torrance, CA), divided (10-μm–thick sections) using a cryostat (Leica Microsystems, Wetzler, Germany) onto Superfrost*Plus microscope slides (Gerhard Menzel GmbH, Braunschwig, Germany), and stored at -20°C until use. Tissue sections were processed for H&E staining or immunohistochemical studies. Cochlear SE samples, isolated from P5 mice, were processed for immunohistochemical staining with a TMC1 antibody (OST 00104W; Osenses, Adelaide, Australia). Briefly, both the SE tissue and cochlear sections were rinsed three times in PBS for 15 minutes and then permeabilized for 30 minutes with 0.3% to 1.0% Triton X-100 in PBS. Sections were washed three times in PBS for 10 minutes and blocked in 5% to 10% goat serum (with or without 2% bovine serum albumin) in PBS for 1 to 2 hours. Isolated SEs and the cochlear sections were incubated with the TMC1 antibody (1:900 and 1:750, respectively) in 1% goat serum/PBS overnight at 4°C in a humidified chamber. SE and cochlear sections were rinsed three times for 10 minutes in PBS, followed by incubation with an Alexa Fluor 594–conjugated goat anti-rabbit IgG (Invitrogen-Molecular Probes, Eugene, OR) in 1% to 10% goat serum in PBS (1:2500) at room temperature for 2 hours and washed three times with PBS for 10 minutes. Cochlear and SE tissues were counterstained with Alexa 488–phalloidin (1:250; Invitrogen-Molecular Probes) in PBS for 20 minutes and then washed three times with PBS for 10 minutes. SE and cochlear sections, incubated with rabbit serum, were used as controls. SEs (positioned on slides) and cochlear sections were mounted with Prolong Gold Antifade reagent with DAPI (Invitrogen-Molecular Probes) and viewed using a Carl Zeiss Imager.M1 (MTB2004 AXIO; Carl Zeiss MicroImaging GmbH, Gottingen, Germany) microscope or a Leica TCS SP2 laser scanning confocal microscope. Images were generated using AxioVision Rel. 4.7 (Carl Zeiss MicroImaging) or Leica Confocal Software (Leica Microsystems).

      SEM Findings

      Inner ears from 2-, 4-, and 8-week-old mice were isolated; the round and oval windows were cleared; and the cochlear apex was pierced to allow thorough perfusion of the fixative. Inner ears were fixed in 2.5% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer (pH 7.4) with 3 mmol/L CaCl2 for 3 hours at room temperature. Tissue was then washed in PBS and further dissected to reveal the cochlear SE by removing the bony shell of the cochlea, the stria vascularis, and the Reissner's and tectorial membranes. Cochlear specimens were incubated in osmium tetroxide for 2.5 hours, washed thoroughly, dehydrated through an ethanol gradient, and then critical point dried (CPD030; Bal-Tec GmbH, Schalksmuhle, Germany). Tissues were mounted on scanning electron microscopy (SEM) stubs using conductive silver paint, then gold coated approximately 10 nm using an Edwards S150B sputter coater (Edwards High Vacuum Inc., Crawley, West Sussex, UK), and viewed using a Philips XL30 FE Scanning Electron Microscope (Philips Electron Optics, Eindhoven, the Netherlands).

      Results

      Baringo, nice, and stitch Mutants Harbor a Point Mutation in the Tmc1 Gene

      Genome-wide homozygosity mapping, followed by fine mapping, in the baringo mutant located the deafness locus to mouse chromosome 19, between 18.37 and 22 Mb. Sequence analysis of Tmc1 revealed a novel mutation, an A-to-G transition at nucleotide position 545 in exon 8, that results in the amino acid substitution of Tyr182 to a Cys (Tmc1p.Y182C) (Figure 1B). Sequencing of nice-mutant mice uncovered a novel mutation, a T-to-C transition at nucleotide position 1345 in exon 13, that changes Tyr449 to a His (Tmc1p.Y449H) (Figure 1C). Tmc1 was also mutated in the mouse strain stitch. The mutation, a G-to-T transition at nucleotide position 1661 in exon 15, results in the amino acid substitution of Trp554 to a Leu (Tmc1p.W554L) (Figure 1A).
      Figure thumbnail gr1
      Figure 1Tmc1 mutations identified in the baringo, nice, and stitch ENU mouse strains. Wild-type and heterozygote nucleotide sequences for unaffected mice are shown for comparison. A: The stitch mouse has a c.1661G>T (p.W554L) missense mutation. B: The baringo mouse carries a c.545A>G (p.Y182C) missense mutation in Tmc1. C: A missense mutation, c.1345T>C (p.Y449H), was identified in nice. D: Alignment of TMC1 sequences from various species showing the mutated amino acids in the Tmc1 mutant strains are highly conserved. E: A schematic diagram of TMC1 protein predicted by TMHMM2.0
      • Moller S.
      • Croning M.D.
      • Apweiler R.
      Evaluation of methods for the prediction of membrane spanning regions.
      to contain six TMDs. The locations of TMC1 p.Y182C, p.Y449H, and p.W554L mutations are shown, as is the Beethoven Tmc1 p.M412K mutation residing in the linker between TMDs 3 and 4. The deafness mutant deletion del463-519 omits most of the loop between TMDs 4 and 5. Details of these mouse mutations are included in . DFNB7/B11 human mutations within close proximity to reported Tmc1 mutations are shown in parentheses.
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      A novel mutation adjacent to the Bth mouse mutation in the TMC1 gene makes this mouse an excellent model of human deafness at the DFNA36 locus.
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      Mutations in TMC1 are a common cause of DFNB7/11 hearing loss in the Iranian population.

      Loss of Auditory Function in baringo, nice, and stitch Mutants

      The stitch-homozygous Tmc1W554L/W554L mutant mice displayed a hearing loss ranging from 60 to 120 dB SPL at the age of 4 weeks (Figure 2). The homozygous Tmc1Y182C/Y182C mutant baringo mice showed severe to profound hearing loss at the age of 4 weeks, ranging from 85 to 120 dB SPL (Figure 2). The homozygous Tmc1Y449H/Y449H mutant nice mice displayed profound hearing loss at the age of 4 weeks, ranging from 100 to >120 dB SPL (Figure 2). The hearing losses are recessive because there was no difference in hearing thresholds between heterozygous Tmc1W554L/+, Tmc1Y182C/+, and Tmc1Y449H/+ mice and their respective normal wild-type littermates at the age of 24 weeks. No circling behavior or head tilting/tossing was observed in baringo Tmc1Y182C/Y182C, nice Tmc1Y449H/Y449H, and stitch Tmc1W554L/W554L mutant mice, indicating normal vestibular function.
      Figure thumbnail gr2
      Figure 2Hearing profile of stitch, baringo, and nice mutants. The homozygous mutant mice (MM) displayed severe to profound hearing loss at the age of 4 weeks in all strains. No differences in hearing thresholds were observed in heterozygotes (NM) at the age of 4 or 24 weeks compared with wild-type (NN) littermates.

      All Tmc1 Mutants Show Abnormal Cochlear Morphological Characteristics

      The structural integrity of the inner ear was investigated by H&E staining of cochlear sections from 4- and 8-week-old wild-type and mutant mice (Figure 3). Cochlear sections of Tmc1W554L/W554L stitch mice showed normal morphological characteristics at 4 weeks. However, by 8 weeks, the basal region lacked sensory or supporting cells and the organ/tunnel of Corti had collapsed (Figure 3). Extensive degeneration of hair and supporting cells, and the collapse of the tunnel of Corti, was evident in the basal to mid region in Tmc1Y182C/Y182C baringo mice at the age of 4 weeks. A more severe pathological condition to that seen in baringo mice was observed in the cochlear sections of Tmc1Y449H/Y449H nice mice, with complete degeneration of the SE in the basal and mid region at the age of 4 weeks (Figure 3).
      Figure thumbnail gr3
      Figure 3H&E staining of cochlear sections from 4- and 8-week-old stitch, baringo, and nice mice. Cochlear degeneration is evident in the basal region of the stitch mutant at 8 weeks, with lack of cellular architecture along the basilar membrane. Loss of sensory and supporting cells was observed as early as 4 weeks in baringo mice, with complete degeneration of the SE in the basal to mid region by 8 weeks. Degeneration of the SE is evident in the nice mutant at 4 weeks. Scale bar = 200 μm (10-μm–thick sections). BM, basilar membrane; OS, organ of Corti; RM, Reissner's membrane; SGN, spiral ganglion neuron.

      Differential Temporal Degeneration of Hair Cells in the Tmc1 Mutants

      SEM analysis of the Tmc1W554L/W554L stitch mutant revealed no signs of hair cell degeneration at 2 weeks and only slight indications at 4 weeks (occasional fusion of stereocilia at the ends of the hair bundles). At 8 weeks (Figure 4), only the basal turn of the cochlea showed severe degeneration with the loss of both inner hair cells (IHCs) and outer hair cells (OHCs) (Figure 4F). Close examination of IHC and OHC bundles at 4 weeks revealed that, similar to baringo and nice strains, the inner row of stereocilia of the OHC hair bundle was often irregular in length or missing (Figure 4J).
      Figure thumbnail gr4
      Figure 4SEM analysis of cochlear SE of Tmc1 mutant strains at 8 weeks. Apical, mid, and basal cochlear turns were examined in Tmc1+/+ (AC), stitch (FH), baringo (KM), and nice (PR) mice. Signs of hair cell degeneration are seen in OHCs of the basal cochlear turn (F) in stitch mutant mice. In the baringo (K and L) and nice (P and Q) mutant mice, the OHCs are completely missing from the basal to the mid cochlear region. The apical cochlear region shows a normal structure in all three mutants (H, M, and R). The IHCs and OHCs of Tmc1+/+ (D and E), stitch (I and J), baringo (N and O), and nice (S and T) mutant mice at 4 weeks were also analyzed (mid cochlear region). The innermost (smallest) row of stereocilia was often missing or of irregular height in OHC hair bundles, whereas the IHC hair bundles appear normal in stitch (I and J), baringo (N and O), and nice (S and T) mutant mice. Scale bars: 10 μm (SE); 1 μm (IHCs); 2 μm (OHCs). WT, wild-type.
      Degeneration of the SE in baringo Tmc1Y182C/Y182C mutants was observed as early as the age of 4 weeks (data not shown). By 8 weeks (Figure 4, K–M), the OHCs were completely missing from the basal through the mid cochlear region, and the lateral supporting cells show irregular morphological characteristics. Some IHCs remaining in the basal and mid region showed severe degeneration and fusion of stereocilia. At 8 weeks, the apical region of the organ of Corti still appeared largely intact. Examination of the hair cell stereocilia in baringo Tmc1Y182C/Y182C mutants at the age of 4 weeks (Figure 4, N and O) indicated that, although the IHCs appear normal, the hair bundles of OHCs were abnormal in that the inner (smallest) row of stereocilia was often missing or, if present, was of irregular height.
      In nice Tmc1Y449H/Y449H mutants, SEM analysis revealed degeneration of the organ of Corti as early as the age of 2 weeks (data not shown). By 8 weeks (Figure 4, P–R), the basal cochlear region was entirely missing IHCs and OHCs. Throughout the mid cochlear turn, the IHCs and OHCs were largely degenerated, hair bundles were absent, and cell morphological characteristics (including those of the supporting Deiters' cells) were irregular. Signs of degeneration were also apparent in the apical region, with many IHCs and OHCs lacking hair bundles or having highly fused or irregular stereocilia. An examination of hair cells in regions of the cochlea not yet showing signs of degeneration revealed that the OHCs had an irregular hair bundle, with the innermost row of stereocilia being nonuniform in height and missing many stereocilia (Figure 4T).

      TMC1 Is Expressed in the Developing Mouse Cochlea and Adult Inner Ear

      Immunofluorescent analysis, performed in SE of the developing cochlea at P5, showed similar levels of TMC1 expression in both IHCs and OHCs of wild-type mice in our three mutant mouse strains (Figure 5).
      Figure thumbnail gr5
      Figure 5TMC1 protein expression in the developing cochlea. A: Phalloidin staining (green) localizes to stereocilia on IHCs and OHCs of P5 SE. TMC1 protein expression (red) was detected as a punctate pattern in both IHCs and OHCs at the mid cochlear region in wild-type (WT; B), stitch (C), baringo (D), and nice (E). Similar levels of TMC1 protein were detected in WT and Tmc1 mutant IHCs and OHCs. F: Nonimmune control SE showing background staining. Scale bar = 8 μm.
      An examination of TMC1 expression in inner ear sections of 4-week-old wild-type Tmc1+/+ mice showed expression of TMC1 in hair cells, supporting cells, and the spiral ganglion neurons (Figure 6, A, C, and F). A similar cellular expression pattern was observed in stitch Tmc1W554L/W554L and baringo Tmc1Y182C/Y182C mutant cochleae in the apical region when compared with the wild-type (Figure 6, G and H). However, the level of TMC1 appeared to be reduced in the basal cochlear region in the stitch Tmc1W554L/W554L mutant (Figure 6D), whereas the collapsed tunnel of Corti in the baringo Tmc1Y182C/Y182C mutant (Figure 6E) made it difficult to determine the cellular localization of TMC1 in this region. An examination of TMC1 protein expression of nice mutant at 4 weeks (data not shown) was similar to that observed in the 4-week-old baringo mutant. We detected TMC1 expression in the spiral ligament, more specifically in type II and III fibrocytes in the Tmc1+/+ cochlear sections (Figure 7B). A gradient of TMC1 expression was observed in the type III fibrocytes of wild-type cochlea, high in the basal region and lower toward the apical region (Figure 7, D–F). Reduced levels of TMC1 protein were detected in the type III fibrocytes of all of the Tmc1 mutants in the basal to mid region (Figure 7, C, G, and H). However, the TMC1 level was significantly elevated in type III fibrocytes in the apical region of the Tmc1W554L/W554L stitch mutant only (Figure 7I).
      Figure thumbnail gr6
      Figure 6TMC1 protein expression in adult cochlear sections. A: Expression of TMC1 protein (red) was observed in the SGN and OC, including OHCs and supporting cells in cochlear sections from wild-type mice. Original magnification: ×20 (A); ×40 (C and F). B: Nonimmune control staining of a cochlear section showing background staining. Expression of TMC1 protein in stitch (D and G) and baringo mutant cochlear sections (E and H). Original magnification, ×40 (10-μm–thick cochlear sections). BM, basilar membrane; IHC, inner hair cells; OC, organ of Corti; OHC, outer hair cells; SC, supporting cell; SGN, spiral ganglion neuron; TM, tectorial membrane; ToC, tunnel of Corti. Nuclei stained blue with DAPI, and stereocilia stained green with phalloidin.
      Figure thumbnail gr7
      Figure 7Immunofluorescence staining showing TMC1 expression in the type II and III fibrocytes in the spiral ligament of 4-week-old wild-type and baringo mutant mice. A: Nonimmune serum control showing background staining. B: Wild-type cochlea shows strong TMC1 expression in the type III fibrocytes and a lower expression level in the type II fibrocytes. C: The baringo mutant cochlea shows lower levels of TMC1 expression in the spiral ligament type III fibrocytes compared with wild-type. Original magnification, ×20. Expression of TMC1 protein in the spiral ligament of basal, mid, and apical regions of wild-type (DF) and stitch-mutant mouse (G–I) cochleae. Arrows indicate the localization of Tmc1 expression in the Type II and III fibrocytes. Original magnification, ×40 (10-μm–thick cochlear sections). SL, spiral ligament; SV, stria vascularis.

      Protein Predictions

      We used several proteomic algorithms to investigate the possible consequences of the Tmc1 mutations p.Y182C, p.Y449H, and p.W554L. Both Y182 and Y449 residues are highly evolutionarily conserved. DIPro (http://scratch.proteomics.ics.uci.edu, last accessed November 26, 2011) and DiANNA (http://bioinformatics.bc.edu/clotelab/DiANNA, last accessed November 26, 2011) disulfide bond prediction programs suggested that both p.Y182C and p.Y449H mutations alter disulfide bond pairing. Polymorphism phenotyping (Polyphen) analysis (http://genetics.bwh.harvard.edu/pph, last accessed November 26, 2011) predicted that there was a high probability that all three mutations have an impact on structure and, consequently, function of the TMC1 protein by causing a conformational change, with PSCI scores of 2.884 (p.Y182C), 1.984 (p.Y449H), and 3.624 (p.W554L).

      Discussion

      In this study, we identified and characterized three new ENU-induced mouse models, designated baringo, nice, and stitch, with recessively inherited hearing loss caused by mutations in the Tmc1 gene. The three Tmc1 missense variants in this study (p.Y182C, p.Y449H, and p.W554L) are highly conserved (Figure 1D) and are predicted to affect TMC1 structure and function, possibly by preventing normal folding, trafficking, or assembly of the mutated protein into multimers. Tmc1 is a member of a family of eight genes encoding transmembrane proteins. The Tmc genes are classified into three subfamilies based on their sequence and genomic structures, with Tmc1, Tmc2, and Tmc3 belonging to subfamily A.
      • Kurima K.
      • Peters L.M.
      • Yang Y.D.
      • Riazuddin S.
      • Ahmed Z.M.
      • Naz S.
      • Arnaud D.
      • Drury S.
      • Mo J.H.
      • Makishima T.
      • Ghosh M.
      • Menon P.S.N.
      • Deshmukh D.
      • Oddoux C.
      • Ostrer H.
      • Khan S.
      • Deininger P.L.
      • Hampton L.L.
      • Sullivan S.L.
      • Battey J.F.
      • Keats B.J.B.
      • Wilcox E.R.
      • Friedman T.B.
      • Griffith A.J.
      Dominant and recessive deafness caused by mutations of a novel gene, TMC1, required for cochlear hair-cell function.
      • Keresztes G.
      • Mutai H.
      • Heller S.
      TMC and EVER genes belong to a larger novel family, the TMC gene family encoding transmembrane proteins.
      Several protein prediction programs, including MemBrain, TMpred TopPred, HMMTOP, TMHMM, and SOSUI, predict between 6 and 11 putative TMDs in the TMC1 protein. By using a heterologous system, Labay et al
      • Labay V.
      • Weichert R.M.
      • Makishima T.
      • Griffith A.J.
      Topology of transmembrane channel-like gene 1 protein.
      demonstrated that mouse TMC1 is a transmembrane protein containing six TMDs with cytosolic N and C termini. This topology bears similarity to the α-subunit of the voltage-dependent K+ channels,
      • Hanlon M.R.
      • Wallace B.A.
      Structure and function of voltage-dependent ion channel regulatory beta subunits.
      with the first four TMDs of the K+ channel α-subunit acting as voltage sensors for activation gating, whereas the intervening region between TMD5 and TMD6 appears to confer channel selectivity.
      • Hanlon M.R.
      • Wallace B.A.
      Structure and function of voltage-dependent ion channel regulatory beta subunits.
      • Li-Smerin Y.
      • Hackos D.H.
      • Swartz K.J.
      Alpha-helical structural elements within the voltage-sensing domains of a K(+) channel.
      Our three mouse models support the six-TMD hypothesis if the severity of hearing loss of each of our mouse mutants is correlated to how close the mutation is to a predicted TMD (ie, nice is the most severe, with a mutation predicted within TMD4; and baringo, with a mutation predicted two amino acids intracellularly from the beginning of TMD1, is less severe than nice but more severe than stitch, which has a mutation in a predicted linker region between TMD4 and TMD5) (Figure 1E).
      Mutations in the TMC1 gene cause postlingual dominant progressive DFNA36 (Online Mendelian Inheritance in Man 606705) and prelingual recessive profound DFNB7/11 (Online Mendelian Inheritance in Man 600974) deafness.
      • Kurima K.
      • Peters L.M.
      • Yang Y.D.
      • Riazuddin S.
      • Ahmed Z.M.
      • Naz S.
      • Arnaud D.
      • Drury S.
      • Mo J.H.
      • Makishima T.
      • Ghosh M.
      • Menon P.S.N.
      • Deshmukh D.
      • Oddoux C.
      • Ostrer H.
      • Khan S.
      • Deininger P.L.
      • Hampton L.L.
      • Sullivan S.L.
      • Battey J.F.
      • Keats B.J.B.
      • Wilcox E.R.
      • Friedman T.B.
      • Griffith A.J.
      Dominant and recessive deafness caused by mutations of a novel gene, TMC1, required for cochlear hair-cell function.
      There is a similar genotype-phenotype correlation in mice carrying mutant alleles of Tmc1; the reported deafness (dn) and Beethoven (Bth) represent models for DFNB7/B11 and DFNA36, respectively. The homozygous deafness mouse model is a spontaneous mutant with a 1.6-kb genomic deletion encompassing Tmc1 exon 14. The Tmc1dn/dn mouse is profoundly deaf and displays hair cell degeneration in the postnatal period.
      • Kurima K.
      • Peters L.M.
      • Yang Y.D.
      • Riazuddin S.
      • Ahmed Z.M.
      • Naz S.
      • Arnaud D.
      • Drury S.
      • Mo J.H.
      • Makishima T.
      • Ghosh M.
      • Menon P.S.N.
      • Deshmukh D.
      • Oddoux C.
      • Ostrer H.
      • Khan S.
      • Deininger P.L.
      • Hampton L.L.
      • Sullivan S.L.
      • Battey J.F.
      • Keats B.J.B.
      • Wilcox E.R.
      • Friedman T.B.
      • Griffith A.J.
      Dominant and recessive deafness caused by mutations of a novel gene, TMC1, required for cochlear hair-cell function.
      Beethoven mice carry a semidominant Tmc1 missense mutation, p.M412K. Tmc1Bth/+ heterozygous mice have a progressive hearing loss associated with hair cell degeneration, beginning in the postnatal period and predominantly affecting IHCs. Homozygous Tmc1Bth/Bth mutants are profoundly deaf from birth.
      • Vreugde S.
      • Erven A.
      • Kros C.J.
      • Marcotti W.
      • Fuchs H.
      • Kurima K.
      • Wilcox E.R.
      • Friedman T.B.
      • Griffith A.J.
      • Balling R.
      • Hrabé De Angelis M.
      • Avraham K.B.
      • Steel K.P.
      Beethoven, a mouse model for dominant, progressive hearing loss DFNA36.
      The novel strains reported herein, baringo, nice, and stitch, when homozygous for the mutations, show a fully penetrant, severe to profound hearing loss at the age of 4 weeks, the earliest age the hearing tests could be performed, with no vestibular dysfunction.
      Histological analysis demonstrates extensive degeneration of both IHCs and OHCs as early as 4 weeks in the nice Tmc1Y449H/Y449H mutant, a phenotype similar to that observed in other mouse strains with Tmc1 mutations.
      • Kurima K.
      • Peters L.M.
      • Yang Y.D.
      • Riazuddin S.
      • Ahmed Z.M.
      • Naz S.
      • Arnaud D.
      • Drury S.
      • Mo J.H.
      • Makishima T.
      • Ghosh M.
      • Menon P.S.N.
      • Deshmukh D.
      • Oddoux C.
      • Ostrer H.
      • Khan S.
      • Deininger P.L.
      • Hampton L.L.
      • Sullivan S.L.
      • Battey J.F.
      • Keats B.J.B.
      • Wilcox E.R.
      • Friedman T.B.
      • Griffith A.J.
      Dominant and recessive deafness caused by mutations of a novel gene, TMC1, required for cochlear hair-cell function.
      • Vreugde S.
      • Erven A.
      • Kros C.J.
      • Marcotti W.
      • Fuchs H.
      • Kurima K.
      • Wilcox E.R.
      • Friedman T.B.
      • Griffith A.J.
      • Balling R.
      • Hrabé De Angelis M.
      • Avraham K.B.
      • Steel K.P.
      Beethoven, a mouse model for dominant, progressive hearing loss DFNA36.
      All three mutants display a similar sequence of degeneration, basal to apical, but differ slightly in the age of onset and severity of the degeneration (between 2 and 4 weeks in nice Tmc1Y449H/Y449H and baringo Tmc1Y182C/Y182C and approximately 4 weeks in stitch Tmc1W554L/W554L). All three Tmc1 mutant strains have abnormal OHC hair bundles, evident at the age of 2 weeks. The innermost row of stereocilia is consistently irregular, with many stereocilia missing or of uneven height. This phenotype was present before the onset of any signs of degeneration of the hair cells, suggesting that this abnormality may be directly related to TMC1 function (or lack thereof), whereas the subsequent hair cell degeneration is likely to be a general consequence of nonfunctioning hair cells.
      Tmc1 mRNA is expressed in mouse cochlear hair cells from early stages of development. TMC1 is thought necessary for maturation and survival of hair cells in the mouse cochlea.
      • Kurima K.
      • Peters L.M.
      • Yang Y.D.
      • Riazuddin S.
      • Ahmed Z.M.
      • Naz S.
      • Arnaud D.
      • Drury S.
      • Mo J.H.
      • Makishima T.
      • Ghosh M.
      • Menon P.S.N.
      • Deshmukh D.
      • Oddoux C.
      • Ostrer H.
      • Khan S.
      • Deininger P.L.
      • Hampton L.L.
      • Sullivan S.L.
      • Battey J.F.
      • Keats B.J.B.
      • Wilcox E.R.
      • Friedman T.B.
      • Griffith A.J.
      Dominant and recessive deafness caused by mutations of a novel gene, TMC1, required for cochlear hair-cell function.
      • Vreugde S.
      • Erven A.
      • Kros C.J.
      • Marcotti W.
      • Fuchs H.
      • Kurima K.
      • Wilcox E.R.
      • Friedman T.B.
      • Griffith A.J.
      • Balling R.
      • Hrabé De Angelis M.
      • Avraham K.B.
      • Steel K.P.
      Beethoven, a mouse model for dominant, progressive hearing loss DFNA36.
      • Marcotti W.
      • Erven A.
      • Johnson S.L.
      • Steel K.P.
      • Kros C.J.
      Tmc1 is necessary for normal functional maturation and survival of inner and outer hair cells in the mouse cochlea.
      In this study, we demonstrate that low levels of TMC1 protein are endogenously expressed in hair cells of developing mouse cochlea. In the adult cochlea, TMC1 expression was detected in hair cells, supporting cells, spiral ganglion neurons, and the spiral ligament, exhibiting a vesicular and filamentous distribution. The spatial localization is consistent with a potential function as an ion transporter, or modulator, of channels or transporters.
      • Kurima K.
      • Peters L.M.
      • Yang Y.D.
      • Riazuddin S.
      • Ahmed Z.M.
      • Naz S.
      • Arnaud D.
      • Drury S.
      • Mo J.H.
      • Makishima T.
      • Ghosh M.
      • Menon P.S.N.
      • Deshmukh D.
      • Oddoux C.
      • Ostrer H.
      • Khan S.
      • Deininger P.L.
      • Hampton L.L.
      • Sullivan S.L.
      • Battey J.F.
      • Keats B.J.B.
      • Wilcox E.R.
      • Friedman T.B.
      • Griffith A.J.
      Dominant and recessive deafness caused by mutations of a novel gene, TMC1, required for cochlear hair-cell function.
      • Keresztes G.
      • Mutai H.
      • Heller S.
      TMC and EVER genes belong to a larger novel family, the TMC gene family encoding transmembrane proteins.
      Furthermore, defective Slo-type, Ik,f K+ currents in IHCs of Tmc1Bth/Bth mutants suggest that TMC1 modulates ion channel activity.
      • Marcotti W.
      • Erven A.
      • Johnson S.L.
      • Steel K.P.
      • Kros C.J.
      Tmc1 is necessary for normal functional maturation and survival of inner and outer hair cells in the mouse cochlea.
      By using mice with targeted deletion of Tmc1 and Tmc2, Kawashima et al
      • Kawashima Y.
      • Géléoc G.S.
      • Kurima K.
      • Labay V.
      • Lelli A.
      • Asai Y.
      • Makishima T.
      • Wu D.K.
      • Della Santina C.C.
      • Holt J.R.
      • Griffith A.J.
      Mechanotransduction in mouse inner ear hair cells requires transmembrane channel-like genes.
      demonstrated that they are necessary for hair cell mechanotransduction and may be integral components of the mechanotransduction complex, after the observation that exogenously expressed TMC1 and TMC2 proteins localized at or near the tips of stereocilia.
      Multiple quantitative traits loci have been identified as a result of crossing Beethoven (Tmc1Bth/Bth) mice on a C3HeB/FeJ background with C57BL/6J or DBA/2J mice that are associated with cochlear hair cell degeneration and altered hearing thresholds.
      • Noguchi Y.
      • Kurima K.
      • Makishima T.
      • de Angelis M.H.
      • Fuchs H.
      • Frolenkov G.
      • Kitamura K.
      • Griffith A.J.
      Multiple quantitative trait loci modify cochlear hair cell degeneration in the Beethoven (Tmc1Bth) mouse model of progressive hearing loss DFNA36.
      We did not detect significant changes in hearing profiles and thresholds of F2 mutant progeny when baringo and nice mice on the C57BL/6J background were outcrossed to CBA/H mice. However, when stitch mice were outcrossed to the BALB/c strain, 50% of the Tmc1W554L/W554L mutant F2 progeny had a hearing threshold comparable to wild-type up to the age of 16 weeks, suggesting a modifier delaying the onset of hearing loss in these mice. This observation highlights the importance of characterizing each mutation on various genetic backgrounds, where the identification of a modifier gene may highlight a desirable genotype that reduces the severity of the hearing loss.
      Baringo, nice, and stitch are novel mutants that provide fundamental tools in elucidating the basis of TMC1-associated human hearing loss. Detailed comparative molecular and electrophysiological investigations of the allelic variants will provide valuable insights into the molecular basis underlying the pathogenic mutations and enable us to define the mechanistic role that TMC1 protein mediates in the auditory process. Future studies involving subcellular localization, identification of interacting protein partners, and mechanotransduction function will enable us to define the molecular pathway(s) that lead to DFNB7/11 and DFNA36 forms of hearing loss.

      Acknowledgments

      We thank the Australian Phenomics Facility for its contribution and Belinda Whittle, Jessica Cardwell, Melissa Arnold, Wendy Hutchison, Kim Boddum, Helene Myrtue Nielsen, Cynthia Luk, and Melanie Bahlo for their technical help and advice. The authors acknowledge the financial support from the HEARing Cooperative Research Centre, established and supported under the Australian Government’s Cooperative Research Centers Program.

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