An ENU-Induced Mutation of Cdh23 Causes Congenital Hearing Loss, but No Vestibular Dysfunction, in Mice
2011; Elsevier BV; Volume: 179; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2011.04.002
ISSN1525-2191
AutoresShehnaaz S.M. Manji, Kerry A. Miller, Louise H. Williams, Lotte Andreasen, Maria Siboe, Elizabeth Rose, Melanie Bahlo, Michael J. Kuiper, Hans‐Henrik M. Dahl,
Tópico(s)Connexins and lens biology
ResumoMutations in the human cadherin 23 (CDH23) gene cause deafness, neurosensory, autosomal recessive 12 (DFNB12) nonsyndromic hearing loss or Usher syndrome, type 1D (characterized by hearing impairment, vestibular dysfunction, and visual impairment). Reported waltzer mouse strains each harbor a Cdh23-null mutation and present with hearing loss and vestibular dysfunction. Two additional Cdh23 mouse mutants, salsa and erlong, each carry a homozygous Cdh23 missense mutation and have progressive hearing loss. We report the identification of a novel mouse strain, jera, with inherited hearing loss caused by an N-ethyl-N-nitrosourea–induced c.7079T>A mutation in the Cdh23 gene. The mutation generates a missense change, p.V2360E, in Cdh23. Affected mice have profound sensorineural deafness, with no vestibular dysfunction. The p.V2360E mutation is semidominant because heterozygous mice have milder and more progressive hearing loss in advanced age. The mutation affects a highly conserved Ca2+-binding motif in extracellular domain 22, thought to be important for Cdh23 structure and dimerization. Molecular modeling suggests that the Cdh23V2360E/V2360E mutation alters the structural conformation of the protein and affects Ca2+-binding properties. Similar to salsa mice, but in contrast to waltzer mice, hair bundle development is normal in jera and hearing loss appears to be due to the loss of tip links. Thus, jera is a novel mouse model for DFNB12. Mutations in the human cadherin 23 (CDH23) gene cause deafness, neurosensory, autosomal recessive 12 (DFNB12) nonsyndromic hearing loss or Usher syndrome, type 1D (characterized by hearing impairment, vestibular dysfunction, and visual impairment). Reported waltzer mouse strains each harbor a Cdh23-null mutation and present with hearing loss and vestibular dysfunction. Two additional Cdh23 mouse mutants, salsa and erlong, each carry a homozygous Cdh23 missense mutation and have progressive hearing loss. We report the identification of a novel mouse strain, jera, with inherited hearing loss caused by an N-ethyl-N-nitrosourea–induced c.7079T>A mutation in the Cdh23 gene. The mutation generates a missense change, p.V2360E, in Cdh23. Affected mice have profound sensorineural deafness, with no vestibular dysfunction. The p.V2360E mutation is semidominant because heterozygous mice have milder and more progressive hearing loss in advanced age. The mutation affects a highly conserved Ca2+-binding motif in extracellular domain 22, thought to be important for Cdh23 structure and dimerization. Molecular modeling suggests that the Cdh23V2360E/V2360E mutation alters the structural conformation of the protein and affects Ca2+-binding properties. Similar to salsa mice, but in contrast to waltzer mice, hair bundle development is normal in jera and hearing loss appears to be due to the loss of tip links. Thus, jera is a novel mouse model for DFNB12. Deafness is the most common sensory impairment in humans, often incurring lifelong educational, social developmental, and economic costs. Hearing loss is a multifactorial disorder, caused by environmental or genetic factors or a combination of both. It is estimated that there are 100 mapped loci that may cause monogenic nonsyndromic hearing loss and >400 syndromes that include hearing as a feature.1Petit C. Genes responsible for human hereditary deafness: symphony of a thousand.Nat Genet. 1996; 14: 385-391Crossref PubMed Scopus (232) Google Scholar Nevertheless, when investigating a specific family affected by inherited hearing loss, the condition is usually transmitted as a monogenic disorder. It has become clear that different mutations in a gene can cause a variety of genetic and clinical features (eg, dominantly or recessively inherited hearing loss or syndromic or nonsyndromic hearing loss). In humans, mutations in the cadherin 23 (CDH23) gene cause autosomal recessively inherited nonsyndromic congenital deafness (DFNB12; Online Mendelian Inheritance of Man no. 601386; http://www.ncbi.nlm.nih.gov/omim, last accessed March 29, 2011) or Usher syndrome, type 1D (USH1D; Online Mendelian Inheritance of Man no. 601067; http://www.ncbi.nlm.nih.gov/omim, last accessed March 29, 2011). USH1 is characterized by congenital hearing impairment, vestibular dysfunction, and visual impairment. DFNB12 deafness is usually caused by missense mutations in CDH23 alleles and USH1D by null mutations.2Bork J.M. Peters L.M. Riazuddin S. Bernstein S.L. Ahmed Z.M. Ness S.L. Polomeno R. Ramesh A. Schloss M. Srisailpathy C.R. Wayne S. Bellman S. Desmukh D. Ahmed Z. Khan S.N. Kaloustian V.M. Li X.C. 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Usher syndrome type 1 due to missense mutations on both CDH23 alleles: investigation of mRNA splicing.Hum Mutat. 2008; 29: 452Crossref PubMed Scopus (19) Google Scholar A synonymous single-nucleotide polymorphism, Cdh23753A, is linked to age-related hearing loss in mice.8Noben-Trauth K. Zheng Q.Y. Johnson K.R. Association of cadherin 23 with polygenic inheritance and genetic modification of sensorineural hearing loss.Nat Genet. 2003; 35: 21-23Crossref PubMed Scopus (347) Google Scholar Null mutations in mouse Cdh23 lead to the waltzer phenotype, characterized by hearing loss and vestibular dysfunction.9Di Palma F. Holme R.H. Bryda E.C. Belyantseva I.A. Pellegrino R. Kachar B. Steel K.P. Noben-Trauth K. Mutations in Cdh23, encoding a new type of cadherin, cause stereocilia disorganization in waltzer, the mouse model for Usher syndrome type 1D.Nat Genet. 2001; 27: 103-107Crossref PubMed Scopus (366) Google Scholar, 10Di Palma F. Pellegrino R. Noben-Trauth K. Genomic structure, alternative splice forms and normal and mutant alleles of cadherin 23 (Cdh23).Gene. 2001; 281: 31-41Crossref PubMed Scopus (73) Google Scholar, 11Yonezawa S. Yoshizaki N. Kageyama T. Takahashi T. Sano M. Tokita Y. Masaki S. Inaguma Y. Hanai A. Sakurai N. Yoshiki A. Kusakabe M. Moriyama A. Nakayama A. Fates of Cdh23/CDH23 with mutations affecting the cytoplasmic region.Hum Mutat. 2006; 27: 88-97Crossref PubMed Scopus (12) Google Scholar, 12Wada T. Wakabayashi Y. Takahashi S. Ushiki T. Kikkawa Y. Yonekawa H. Kominami R. A point mutation in a cadherin gene, Cdh23, causes deafness in a novel mutant, Waltzer mouse niigata.Biochem Biophys Res Commun. 2001; 283: 113-117Crossref PubMed Scopus (40) Google Scholar, 13Wilson S.M. Householder D.B. Coppola V. Tessarollo L. Fritzsch B. Lee E.C. Goss D. Carlson G.A. Copeland N.G. Jenkins N.A. Mutations in Cdh23 cause nonsyndromic hearing loss in waltzer mice.Genomics. 2001; 74: 228-233Crossref PubMed Scopus (89) Google Scholar Two N-ethyl-N-nitrosourea (ENU)–generated mouse strains, salsa and erlong, carry a recessive Cdh23 missense mutation.14Schwander M. Xiong W. Tokita J. Lelli A. Elledge H.M. Kazmierczak P. Sczaniecka A. Kolatkar A. Wiltshire T. Kuhn P. Holt J.R. Kachar B. Tarantino L. Muller U. A mouse model for nonsyndromic deafness (DFNB12) links hearing loss to defects in tip links of mechanosensory hair cells.Proc Natl Acad Sci U S A. 2009; 106: 5252-5257Crossref PubMed Scopus (70) Google Scholar, 15Han F. Yu H. Tian C. Chen H.E. Benedict-Alderfer C. Zheng Y. Wang Q. Han X. Zheng Q.Y. A new mouse mutant of the Cdh23 gene with early-onset hearing loss facilitates evaluation of otoprotection drugs.Pharmacogenomics J. 2010; ([Epub ahead of press])https://doi.org/10.1038/tpj.2010.60Crossref Scopus (30) Google Scholar Both mutants have progressive hearing loss, with no vestibular abnormalities, and are, therefore, models for DFNB12 deafness. Retinal degeneration is not a clinical feature in any of the mice with Cdh23 mutations. Previously reported allelic variants of Cdh23 in mice are detailed in Table 1.Table 1Allelic Variants of Cdh23 in MiceMouse modelNucleotide changeAmino acid changeExonEC domainPhenotypePredicted effectReferencejera7486T>AV2360E52EC22DFNB12Disruption of the Ca2+-binding motifPresent studyerlong208T>CS70P3EC1DFNB12Loss of protein function15Han F. Yu H. Tian C. Chen H.E. Benedict-Alderfer C. Zheng Y. Wang Q. Han X. Zheng Q.Y. A new mouse mutant of the Cdh23 gene with early-onset hearing loss facilitates evaluation of otoprotection drugs.Pharmacogenomics J. 2010; ([Epub ahead of press])https://doi.org/10.1038/tpj.2010.60Crossref Scopus (30) Google Scholarsalsa2210A>TE737V22EC7DFNB12Disruption of the Ca2+-binding motif14Schwander M. Xiong W. Tokita J. Lelli A. Elledge H.M. Kazmierczak P. Sczaniecka A. Kolatkar A. Wiltshire T. Kuhn P. Holt J.R. Kachar B. Tarantino L. Muller U. A mouse model for nonsyndromic deafness (DFNB12) links hearing loss to defects in tip links of mechanosensory hair cells.Proc Natl Acad Sci U S A. 2009; 106: 5252-5257Crossref PubMed Scopus (70) Google ScholarCdh23V834insGN279EfsX399EC3USH1DPremature stop codon13Wilson S.M. Householder D.B. Coppola V. Tessarollo L. Fritzsch B. Lee E.C. Goss D. Carlson G.A. Copeland N.G. Jenkins N.A. Mutations in Cdh23 cause nonsyndromic hearing loss in waltzer mice.Genomics. 2001; 74: 228-233Crossref PubMed Scopus (89) Google ScholarCdh23VJ3505delGE1169NfsX729EC11USH1DPremature stop codon13Wilson S.M. Householder D.B. Coppola V. Tessarollo L. Fritzsch B. Lee E.C. Goss D. Carlson G.A. Copeland N.G. Jenkins N.A. Mutations in Cdh23 cause nonsyndromic hearing loss in waltzer mice.Genomics. 2001; 74: 228-233Crossref PubMed Scopus (89) Google ScholarCdh23V-2J4104+1G>A31EC13USH1DSplice-site mutation9Di Palma F. Holme R.H. Bryda E.C. Belyantseva I.A. Pellegrino R. Kachar B. Steel K.P. Noben-Trauth K. Mutations in Cdh23, encoding a new type of cadherin, cause stereocilia disorganization in waltzer, the mouse model for Usher syndrome type 1D.Nat Genet. 2001; 27: 103-107Crossref PubMed Scopus (366) Google ScholarCdh23V-3J5291G>AW1764X40EC17USH1DLoss of functional domains10Di Palma F. Pellegrino R. Noben-Trauth K. Genomic structure, alternative splice forms and normal and mutant alleles of cadherin 23 (Cdh23).Gene. 2001; 281: 31-41Crossref PubMed Scopus (73) Google ScholarCdh23V-4J8152del9N2718del356EC25USH1DDisruption of the Ca2+-binding motif10Di Palma F. Pellegrino R. Noben-Trauth K. Genomic structure, alternative splice forms and normal and mutant alleles of cadherin 23 (Cdh23).Gene. 2001; 281: 31-41Crossref PubMed Scopus (73) Google ScholarCdh23V-5J8803C>TR2935X60EC27USH1DLoss of functional domains10Di Palma F. Pellegrino R. Noben-Trauth K. Genomic structure, alternative splice forms and normal and mutant alleles of cadherin 23 (Cdh23).Gene. 2001; 281: 31-41Crossref PubMed Scopus (73) Google ScholarCdh23V-6J904G>TE302X9EC3USH1DLoss of functional domains9Di Palma F. Holme R.H. Bryda E.C. Belyantseva I.A. Pellegrino R. Kachar B. Steel K.P. Noben-Trauth K. Mutations in Cdh23, encoding a new type of cadherin, cause stereocilia disorganization in waltzer, the mouse model for Usher syndrome type 1D.Nat Genet. 2001; 27: 103-107Crossref PubMed Scopus (366) Google ScholarCdh23V-7J3589delTY1197MfsX4730EC11USH1DPremature stop codon9Di Palma F. Holme R.H. Bryda E.C. Belyantseva I.A. Pellegrino R. Kachar B. Steel K.P. Noben-Trauth K. Mutations in Cdh23, encoding a new type of cadherin, cause stereocilia disorganization in waltzer, the mouse model for Usher syndrome type 1D.Nat Genet. 2001; 27: 103-107Crossref PubMed Scopus (366) Google ScholarCdh23V-ngt145delGG49VfsX32EC1USH1DPremature stop codon12Wada T. Wakabayashi Y. Takahashi S. Ushiki T. Kikkawa Y. Yonekawa H. Kominami R. A point mutation in a cadherin gene, Cdh23, causes deafness in a novel mutant, Waltzer mouse niigata.Biochem Biophys Res Commun. 2001; 283: 113-117Crossref PubMed Scopus (40) Google ScholarCdh23V-Alb1635C>Tdel119G545G15EC5USH1DGenerates ectopic splice site9Di Palma F. Holme R.H. Bryda E.C. Belyantseva I.A. Pellegrino R. Kachar B. Steel K.P. Noben-Trauth K. Mutations in Cdh23, encoding a new type of cadherin, cause stereocilia disorganization in waltzer, the mouse model for Usher syndrome type 1D.Nat Genet. 2001; 27: 103-107Crossref PubMed Scopus (366) Google ScholarCdh23Vbus9633+1G>A67CYTOUSH1DSplice-site mutation11Yonezawa S. Yoshizaki N. Kageyama T. Takahashi T. Sano M. Tokita Y. Masaki S. Inaguma Y. Hanai A. Sakurai N. Yoshiki A. Kusakabe M. Moriyama A. Nakayama A. Fates of Cdh23/CDH23 with mutations affecting the cytoplasmic region.Hum Mutat. 2006; 27: 88-97Crossref PubMed Scopus (12) Google ScholarCdh23Ahl753G>AP251P7EC3AHLIn-frame skipping of exon 78Noben-Trauth K. Zheng Q.Y. Johnson K.R. Association of cadherin 23 with polygenic inheritance and genetic modification of sensorineural hearing loss.Nat Genet. 2003; 35: 21-23Crossref PubMed Scopus (347) Google Scholar Open table in a new tab Auditory hair cells, located within the organ of Corti, have a bundle of stereocilia with a central kinocilium, located at their apical surface. These structures act as mechanoelectrical transduction sensors and are, therefore, essential for normal hearing. Stereocilia and kinocilium are connected via transient lateral links, kinociliary links, and tip links in immature cochlear hair cells (Figure 1). Cdh23, together with protocadherin 15 (Pcdh15), is a major component of these links.16Siemens J. Lillo C. Dumont R.A. Reynolds A. Williams D.S. Gillespie P.G. Muller U. Cadherin 23 is a component of the tip link in hair-cell stereocilia.Nature. 2004; 428: 950-955Crossref PubMed Scopus (345) Google Scholar, 17Sollner C. Rauch G.J. Siemens J. Geisler R. Schuster S.C. Muller U. Nicolson T. Mutations in cadherin 23 affect tip links in zebrafish sensory hair cells.Nature. 2004; 428: 955-959Crossref PubMed Scopus (264) Google Scholar, 18Kazmierczak P. Sakaguchi H. Tokita J. Wilson-Kubalek E.M. Milligan R.A. Muller U. Kachar B. Cadherin 23 and protocadherin 15 interact to form tip-link filaments in sensory hair cells.Nature. 2007; 449: 87-91Crossref PubMed Scopus (503) Google Scholar The tip links are connected to transduction channels on the stereocilia, in which the movement of stereocilia causes the rigid tip links to pull open the transduction channels and thereby initiate mechanotransduction.19Beurg M. Fettiplace R. Nam J.H. Ricci A.J. Localization of inner hair cell mechanotransducer channels using high-speed calcium imaging.Nat Neurosci. 2009; 12: 553-558Crossref PubMed Scopus (323) Google Scholar, 20Sotomayor M. Weihofen W.A. Gaudet R. Corey D.P. Structural determinants of cadherin-23 function in hearing and deafness.Neuron. 2010; 66: 85-100Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar Therefore, Cdh23 is thought to be directly involved in this process. Also, Cdh23 may be involved in hair bundle development, as this process is defective in waltzer mice with Cdh23-null mutations.9Di Palma F. Holme R.H. Bryda E.C. Belyantseva I.A. Pellegrino R. Kachar B. Steel K.P. Noben-Trauth K. Mutations in Cdh23, encoding a new type of cadherin, cause stereocilia disorganization in waltzer, the mouse model for Usher syndrome type 1D.Nat Genet. 2001; 27: 103-107Crossref PubMed Scopus (366) Google Scholar, 12Wada T. Wakabayashi Y. Takahashi S. Ushiki T. Kikkawa Y. Yonekawa H. Kominami R. A point mutation in a cadherin gene, Cdh23, causes deafness in a novel mutant, Waltzer mouse niigata.Biochem Biophys Res Commun. 2001; 283: 113-117Crossref PubMed Scopus (40) Google Scholar, 13Wilson S.M. Householder D.B. Coppola V. Tessarollo L. Fritzsch B. Lee E.C. Goss D. Carlson G.A. Copeland N.G. Jenkins N.A. Mutations in Cdh23 cause nonsyndromic hearing loss in waltzer mice.Genomics. 2001; 74: 228-233Crossref PubMed Scopus (89) Google Scholar, 21Alagramam K.N. Murcia C.L. Kwon H.Y. Pawlowski K.S. Wright C.G. Woychik R.P. The mouse Ames waltzer hearing-loss mutant is caused by mutation of Pcdh15, a novel protocadherin gene.Nat Genet. 2001; 27: 99-102PubMed Google Scholar, 22Gibson F. Walsh J. Mburu P. Varela A. Brown K.A. Antonio M. Beisel K.W. Steel K.P. Brown S.D. A type VII myosin encoded by the mouse deafness gene shaker-1.Nature. 1995; 374: 62-64Crossref PubMed Scopus (545) Google Scholar, 23Johnson K.R. Gagnon L.H. Webb L.S. Peters L.L. Hawes N.L. Chang B. Zheng Q.Y. Mouse models of USH1C and DFNB18: phenotypic and molecular analyses of two new spontaneous mutations of the Ush1c gene.Hum Mol Genet. 2003; 12: 3075-3086Crossref PubMed Scopus (130) Google Scholar, 24Kikkawa Y. Shitara H. Wakana S. 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Physical and functional interaction between protocadherin 15 and myosin VIIa in mechanosensory hair cells.J Neurosci. 2006; 26: 2060-2071Crossref PubMed Scopus (132) Google Scholar salsa mice confirmed the role of Cdh23 in mechanotransduction, as hair cells and hair bundles develop normally, but the function of these cells was affected.14Schwander M. Xiong W. Tokita J. Lelli A. Elledge H.M. Kazmierczak P. Sczaniecka A. Kolatkar A. Wiltshire T. Kuhn P. Holt J.R. Kachar B. Tarantino L. Muller U. A mouse model for nonsyndromic deafness (DFNB12) links hearing loss to defects in tip links of mechanosensory hair cells.Proc Natl Acad Sci U S A. 2009; 106: 5252-5257Crossref PubMed Scopus (70) Google Scholar A recent report by Lelli et al28Lelli A. Kazmierczak P. Kawashima Y. Muller U. Holt J.R. Development and regeneration of sensory transduction in auditory hair cells requires functional interaction between cadherin-23 and protocadherin-15.J Neurosci. 2010; 30: 11259-11269Crossref PubMed Scopus (45) Google Scholar provided physiological evidence to support the involvement of CDH23 and PCDH15 N-terminal regions in tip link formation. The Cdh23 gene codes for several different splice isoforms.10Di Palma F. Pellegrino R. Noben-Trauth K. Genomic structure, alternative splice forms and normal and mutant alleles of cadherin 23 (Cdh23).Gene. 2001; 281: 31-41Crossref PubMed Scopus (73) Google Scholar The longest isoform of Cdh23 in mouse comprises 71 exons and codes for a 3354–amino acid protein. It is expressed in the mouse inner ear but not in the mouse retina.29Lagziel A. Overlack N. Bernstein S.L. Morell R.J. Wolfrum U. Friedman T.B. Expression of cadherin 23 isoforms is not conserved: implications for a mouse model of Usher syndrome type 1D.Mol Vis. 2009; 15: 1843-1857PubMed Google Scholar Cadherins are adhesion molecules that mediate Ca2+-dependent cell-cell adhesion via extracellular (EC) domains. Cdh23 contains 27 EC domains; and each EC domain consists of three Ca2+-binding motifs (consensus sequences DXD, LDRE, and DXNDN) that are essential for dimerization, linearization, and rigidification.10Di Palma F. Pellegrino R. Noben-Trauth K. Genomic structure, alternative splice forms and normal and mutant alleles of cadherin 23 (Cdh23).Gene. 2001; 281: 31-41Crossref PubMed Scopus (73) Google Scholar Different EC domains are affected in waltzer, salsa, and erlong mice. We have screened ENU mouse mutant libraries for recessively inherited hearing loss; in this study, we describe a novel mouse strain, jera, with a missense mutation in Cdh23. The hearing loss in jera is more severe than that in salsa and erlong. Similar to salsa and erlong, the development of hair bundles appears normal in jera; therefore, it is a new model for DFNB12. This project was approved by the Royal Children's Hospital Animal Ethics Committee (application numbers A488 and 585). The jera mouse was generated by ENU mutagenesis at the Australian Phenomics Facility, Canberra, in a program aimed at identifying recessive conditions. Male C57BL/6 mice were treated weekly for 3 weeks with 100 mg/kg ENU. To identify recessive phenotypes, G2 siblings were mated to generate G3 offspring homozygous for ENU mutations. We screened 5750 ENU C57BL/6 mutant G3 mice for hearing loss. The initial click box hearing test elicits a Preyer reflex or a startle response in hearing mice by producing an 18.9-kHz burst of 106-dB sound pressure level (SPL) at a distance of 10 cm (Institute of Hearing Research, Nottingham, UK). Mice that failed the initial click box hearing test were subsequently tested by an evoked auditory brainstem response (ABR) (Bio-logic Systems Corp, Chicago, IL). Subdermal active, reference, and ground electrodes were placed at the vertex, ventrolateral to the left ear, and the abdomen, respectively, of the anesthetized mouse. A specific auditory stimulus in the form of broadband clicks was delivered in a range of decibel SPLs and the ABR was recorded. Mice were also screened for the presence of vestibular dysfunction that was identified by the display of hyperactivity that manifests as circling, head tossing/tilting, and/or star-gazing behavior. Mice that failed both the click box and ABR hearing test were intercrossed to the congenic strain C57BL/10 for heritability testing. We expected 25% of the G2 offspring to be deaf if the mutation was recessively inherited and fully penetrant. Deaf mice were then outcrossed to the CBA/H strain, and the F2 progeny were generated for homozygosity mapping and subsequent identification of candidate regions. Genomic DNA samples from 10 affected mice were analyzed by genomewide scans using 120 microsatellite markers (AGRF, Melbourne, Australia). Fine mapping was performed using Amplifluor-based single-nucleotide polymorphism assays (Australian Phenomics Facility) on 53 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 map the deafness loci. In chromosome regions of linkage, the proportion of homozygous C57BL/6 genotypes would approach 1.0; at unlinked loci, this proportion would be 0.25. Linkage intervals were examined for candidate genes using the UCSC genome bioinformatics (http://genome.ucsc.edu). To scan for mutations in the Cdh23 gene (transcript reference, ENSMUST00000105464; protein reference, ENSMUSP00000101104), 1 μg of total RNA from mouse brain was reverse transcribed using random hexamers (Roche, Penzberg, Germany). cDNA products were PCR amplified using HotStar Taq polymerase (Qiagen, Hilden, Germany) with 16 primer sets, generating overlapping amplicons that covered the 10,572-bp Cdh23 RNA coding region transcript and most of the 5′ and 3′ untranslated regions. 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 (SoftGenetics, State College, PA). Mice [postnatal day (P) 14 and older] were anesthetized with isoflurane and euthanized by cervical dislocation, whereas mice younger than P14 were euthanized by 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 (Royal Children's Hospital Animal Ethics Committee approval A488 and 585). 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 prepared as described by Whitlon et al30Whitlon D.S. Szakaly R. Greiner M.A. Cryoembedding and sectioning of cochleas for immunocytochemistry and in situ hybridization.Brain Res Brain Res Protoc. 2001; 6: 159-166Crossref PubMed Scopus (54) Google Scholar and processed for H&E staining. P5 mouse half heads were fixed in 4% paraformaldehyde at 4°C for 2 hours, followed by PBS washes three times for 20 minutes, before isolation of the cochlear sensory epithelium (SE) for whole-mount immunofluorescence analysis. The P14 to P30 half heads were fixed in 4% paraformaldehyde at room temperature for 2 hours before PBS washes and fine dissection. Processed cochleae were embedded in OCT (Sakura Finetek USA, Inc., Torrance, CA) and sectioned (10 μm) using a cryostat (Leica Microsystems, Wetzler, Germany) onto SuperfrostPlus microscope slides (Gerhard Menzel GmbH, Braunschweig, Germany) and stored at −20°C until use. For H&E staining, cochlear sections were processed through H&E staining, coverslipped with Pertex mounting media, and visualized using a Leica DMI3000 microscope (Leica Microsystems). Cochlear SE isolated from P5 to P30 mice were processed for immunohistochemical staining using either a cadherin 23 antibody (provided by Prof. Thomas Friedman and Dr. Ayala Lagziel, National Institute on Deafness and Other Communication Disorders, Bethesda, MD)29Lagziel A. Overlack N. Bernstein S.L. Morell R.J. Wolfrum U. Friedman T.B. Expression of cadherin 23 isoforms is not conserved: implications for a mouse model of Usher syndrome type 1D.Mol Vis. 2009; 15: 1843-1857PubMed Google Scholar or a polyclonal rabbit anti-human Cdh23 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Briefly, SEs were rinsed three times in PBS and then permeabilized for 30 minutes with 0.3% to 1% Triton X-100 (Sigma-Aldrich, St. Louis, MO) in PBS. SEs were then blocked in 5% to 10% goat serum (with or without 2% bovine serum albumin) in PBS for 1 to 2 hours. Isolated SEs were incubated with an anti-Cdh23 antibody [1:250 (antibody from National Institute on Deafness and Other Communication Disorders) or 1:500 (antibody from Santa Cruz Biotechnology, Inc.), respectively] overnight at 4°C. SEs were rinsed three times in PBS with 10- to 20-minute intervals. This was followed by incubation with an Alexa Fluor 488– or an Alexa Fluor 594–conjugated goat anti-rabbit IgG antibody (Invitrogen-Molecular Probes, Eugene, OR) in 1% to 10% goat serum in PBS (1:2500) at room temperature for 2 hours and washes three times with PBS. SEs were counterstained with Alexa Fluor 594– or Alexa Fluor 488–phalloidin (1:250; Molecular Probes) in PBS for 20 minutes, then washed a further three times with PBS for 10 minutes. SEs were incubated with a rabbit IgG antibody (Invitrogen Corp., Carlsbad, CA) as a control. SEs were positioned on slides, mounted with Prolong Gold Antifade reagent with DAPI (Molecular Probes), and viewed using a Carl Zeiss Imager.M1 (MTB2004 AXIO; Carl Zeiss MicroImaging GmbH, Gottingen, Germany) or a Leica TCS SP2 laser scanning confocal microscope. Images were generated using AxioVision Rel. 4.7 or Leica Confocal Software (Leica Microsystems). Inner ears from P14, P30, and 13-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. Samples were then washed in PBS and further dissected by removing the bony shell, stria vascularis, and Reissner's and tectorial membranes to reveal the cochlear SE. Cochlear specimens were incubated in osmium tetroxide for 2.5 hours, washed, dehydrated through an ethanol gradient, and then critical point dried (CPD030; Bal-Tec GmbH, Schalksmuhle, Germany). Specimens were mounted on stubs using a conductive silver paint, gold sputter coated (Edwards S150B sputter coater, approximately 10 nm; Edwards High Vacuum Inc., Crawley, West Sussex, UK), and viewed using a Philips XL30 FE scanning electron microscope (Philips Electron Op
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