The overall goals of our research program are to identify molecules and pathways that are important in the development and physiology of the ear. We study mouse mutations that disrupt these processes and develop these mutations as models of human deafness disorders and age-related hearing loss. We have established a screening program to identify inbred and mutant mice with hearing impairment, which we assess by auditory brainstem response (ABR) analysis. In collaboration with colleagues we identified the first known gene in the mouse to cause age-related hearing loss (AHL), the most common type of human hearing impairment. We have mapped gene loci that contribute to AHL and have identified some of the underlying genes. Our research team is also investigating new mouse mutations that cause hearing impairment. By identifying the genes underlying these mutations and studying their functions, we hope to gain a better understanding of the molecular mechanisms involved in the auditory process. The mutant mice also provide valuable models for studying human non-syndromic deafness disorders and human syndromes with associated deafness.
Genetic studies of hearing impairment in mice
We have established a screening program to identify inbred and mutant mice with hearing impairment, which we assess by auditory brainstem response (ABR) analysis. Since 1996, we have screened more than 18,000 mice from The Jackson Laboratory's large collection of inbred and mutant strains. Information from this screening project is accessible on the Hereditary Hearing Impairment in Mice website (http://hearingimpairment.jax.org/index.html). This web site also presents updated, comprehensive information on genes and mutations responsible for mouse and human hearing disorders. Once hearing impaired mice are identified, we examine their inner ears for pathological abnormalities and undertake a genetic analysis to map, identify, and characterize the responsible genes.
Age-related hearing loss in inbred strains of mice
Age-related hearing loss (AHL), or presbycusis, is the most common type of human hearing impairment, affecting about half the population by age 80. Little is known about the genetic causes of human presbycusis because of confounding factors such as noise trauma, disease, or ototoxic drugs. Our approach for investigating the genetic basis of human presbycusis is to use inbred mouse strains that exhibit progressive hearing loss as models (Noben-Trauth and Johnson, 2009). We previously showed that a locus (ahl) on Chromosome 10 is a major contributor to AHL in several inbred mouse strains and, in collaboration with Dr. Konrad Noben-Trauth (NIDCD), showed that ahl is likely a splice site variant of the cadherin 23 gene. To formally test this hypothesis, we are using a recombineering approach to produce reciprocal “knockin” mice, one in which the wildtype splice site sequence of the 129S1 strain (Cdh23753G, which is associated with increased resistance to AHL) is replaced with the splice site variant of the C57BL/6J strain (Cdh23753A, which is associated with an increased susceptibility to AHL), and the other in which the splice site variant of C57BL/6J is replaced with the wildtype splice site sequence of the 129S1 strain.
We have mapped additional AHL loci by analyses of linkage crosses, recombinant inbred strains, and chromosome substitution strains. We mapped a locus on Chromosome 5 (ahl2) that contributes to hearing loss in NOD/LtJ mice, a locus on distal Chromosome 10 (ahl4) that contributes to hearing loss in A/J mice (Zheng et al., 2009), and a locus on distal Chromosome 11 (ahl8) that contributes to hearing loss in DBA/2J mice (Johnson et al., 2008). We have refined these map positions, produced congenic strains to isolate the effects of the individual AHL loci, and tested candidate genes that may underlie these effects. We recently identified a missense mutation of the fascin 2 gene (Fscn2) as the underlying cause of the ahl8 phenotype of DBA/2J mice (Shin et al., 2010) and a missense mutation of the citrate synthase gene (Cs) as the underlying cause of the ahl4 phenotype of A/J mice (Johnson et al. 2011).
Fscn2 lies within the Chr 11 candidate gene region for ahl8. Fscn2 encodes an actin bundling protein that we found by RT-PCR to be expressed in the inner ear, though others had reported it to be specific to the retina. Our collaborators Peter Gillespie and Jung Bum Shin (Oregon Health & Science University) independently confirmed the presence of FSCN2 in the hair cell bundle by proteomic analysis. Abnormal FSCN2 in hair cell bundles could potentially affect their mechanical properties and integrity. SNP analysis identified a unique nucleotide change in the protein coding sequence of Fscn2 in DBA/2J mice that would change a highly conserved arginine residue to histidine. The closely related substrains DBA/1J, DBA/2HaSmnJ, DBA/2DaJ, and DBA/2NCr do not have this Fscn2 variant and mice of these strains exhibit a later onset and slower progression of hearing loss than do DBA/2J mice. We produced a D2.B6-Fscn2 congenic strain and developed transgenic mice in which a C57BL/6J-derived BAC containing the wildtype Fscn2 gene is integrated into the genome of DBA/2J mice. We found that the C57BL/6J-derived Fscn2 congenic region and the C57BL/6J-derived Fscn2 transgene improved the hearing of mice with an otherwise DBA/2J genome. These rescue experiments confirmed the causative nature of the R109H Fscn2 variant in conferring an increased susceptibility to age-related hearing loss in DBA/2J mice (Shin et al., 2010).
We refined the map position of ahl4 to the distal-most 7 Mb of Chromosome 10 by analysis of a new linkage backcross of C57BL/6J-Chr 10A/J/NaJ and C57BL/6J strain mice and then further narrowed the interval to 5.5 Mb by analysis of eight C57BL/6J congenic lines with different A/J-derived segments of Chr 10. A nucleotide variant in exon 3 of Cs is the only known DNA difference within the 5.5 Mb ahl4 candidate gene interval that is unique to the A/J strain and that causes a nonsynonymous codon change. Multiple lines of evidence implicate this missense mutation (H55N) as the underlying cause of ahl4-related hearing loss in A/J mice, likely through its effects on mitochondrial ATP and free radical production in cochlear hair cells (Johnson et al., 2011). The A/J mouse thus provides a new model system for in vivo studies of mitochondrial dysfunction and hearing loss.
Mouse mutations causing hearing impairment
Mouse deafness mutations provide valuable models of human hereditary hearing disorders and entry points into molecular pathways important to the hearing process. We assess hearing in all abnormal mice that are chosen from the Laboratory's Deviant Search program, from mutagenesis programs or from our own research colonies. If the deviant mice have an inherited hearing impairment, genetic crosses are set up to map the mutation. We have a total of more than 50 hearing-related mutations with research still in progress. Each new mutation is characterized for inner ear pathologies and, when the underlying gene is identified, its temporal and spatial expression patterns are assessed along with those of other genes that may act in the same pathways of inner ear development or maintenance. We use the genetic mapping and candidate gene approach to identify the genes underlying new mutations that cause hearing impairment and below describe five such genes and their corresponding mutant phenotypes that we have recently characterized.
Tmhs: We discovered that a mutation in a previously uncharacterized gene was responsible for the deafness and circling phenotype of a new mouse mutation we called hurry-scurry (hscy). We named the gene “tetraspan membrane protein of hair cell stereocilia” (Tmhs) because of its predicted protein structure and inner ear localization (Longo-Guess et al., 2005). Subsequently, mutations in the human TMHS gene were shown to be responsible for DFNB67, an autosomal recessive nonsyndromic deafness locus. We then genetically engineered a mouse with a targeted null allele and lacZ reporter gene that demonstrated high expression in the cochlear and vestibular hair cells of the inner ear, from E15.5 to P15, a duration that supports the involvement of Tmhs in stereocilia development (Longo-Guess et al. 2007b).
Clic5: We showed that the spontaneous jitterbug (jbg) mutation, which causes deafness and vestibular dysfunction when homozygous, is a 97-base pair intragenic deletion of the chloride intracellular channel 5 gene, Clic5 (Gagnon et al., 2006). Using an antibody provided by our collaborator Mark Berryman, Ohio University, we detected specific CLIC5 immunofluorescence in stereocilia of both cochlear and vestibular hair cells and in microvilli on the apical surface of Kolliker's organ during inner ear development, consistent with the known association of this protein with actin-based cytoskeletal structures of other polarized epithelial cells. Further supporting these results, our collaborator Peter Gillespie, Oregon Hearing Research Center, showed by mass spectrometry that CLIC5 is expressed at high levels in hair cell bundles isolated from the chicken utricle.
Ush1c: We mapped two new recessive mutations causing circling behavior and deafness to the same region on Chromosome 7 and showed they are allelic by complementation analysis. One was named “deaf circler” (allele symbol dfcr) and the other “deaf circler 2 Jackson” (allele symbol dfcr‑2J). Both were shown to be mutations of the Ush1c gene (Johnson et al. 2003), the mouse ortholog of the gene responsible for human Usher syndrome type IC and for the nonsyndromic deafness disorder DFNB18. The qualitatively different mutations provided a means to evaluate the functional domains of the USH1C protein.
Otof: We demonstrated that the ENU-induced deaf5jcs mutation, imported from John Schimenti (Cornell U., NY), is a missense mutation of the otoferlin (Otof) gene and assessed the auditory and vestibular function of OtofDeaf5jcs mutant mice by ABR and VsEP electrophysiological analyses (Longo-Guess et al., 2007a). Otoferlin is essential for vesicle exocytosis at the inner hair cell synapse. OtofDeaf5jcs mutant mice are deaf but exhibit normal outer hair cell function and normal vestibular behavior. The mice provide a new model for the human nonsyndromic deafness disorder DFNB9, which is caused by mutations of the orthologous human OTOF gene.
Duox2: We showed that a spontaneous mouse mutation causing congenital hypothyroidism with associated dwarfism and profound hearing impairment is a missense mutation (named thyroid dyshormonogenesis, thyd) in the dual oxidase 2 (Duox2) gene (Johnson et al., 2007). DUOX2 is an NADPH oxidase that generates hydrogen peroxide in thyroid follicular cells, which is used by thyroid peroxidase for the oxidation of iodide and the production of thyroid hormone. This mutant provides the first mouse model for DUOX2-related hypothyroidism.
In addition to peer-reviewed journal publications, we also post short articles on our public website (http://hearingimpairment.jax.org/screening.html). These short articles give detailed descriptions of new mouse mutations we have discovered that have associated hearing impairment, including ABR results, inner ear pathologies, genetic mapping and allelism test results, and, in some cases, molecular characterizations of the mutations. The following newly discovered deafness-causing mutant alleles have been posted on this website: Otogtwt-2J, -3J, -4J, -5J, Tmiesr-2J, Ush1cdfcr-3J, Cdh23v-7J, -11J, Slc26a4pdsm, Chd7Whll, Dfb. Ush1gjs2-J, Kcnq1vtg-2J, -4J, Myo6sv-2J, -4J, Myo15sh2-3J, Spnb4qv-10J, -11J, Pou3f4del-J, Lmx1adr-10J, -11J, and Otx1jv.
Hearing modifiers and gene interactions
Mouse mutations often show variable phenotypes on different strain backgrounds. In collaborations with other investigators, we mapped genetic modifiers of hearing in mice with mutations in several different deafness-related genes, including Atp2b2dfw, Tubtub, and Eya1bor. More recently we published a paper on Cdh23ahl modification of the Gpr98frings mutation (Johnson et al., 2005) and another paper on strain background effects on hearing in Jackson circler (jc) mutant mice (Calderon et al., 2006). In collaboration with QY Zheng (Case Western University) and XZ Liu (University of Miami), we published a paper describing a genetic interaction between mutations of the cadherin genes Cdh23 and Pcdh15 that causes a progressive hearing loss in digenic heterozygous mice and in human patients with compound mutations (Zheng et al., 2005). We wrote a collaborative review paper describing these and other hearing modifiers (Johnson et al., 2006) and are working on several additional projects to identify and evaluate new genetic modifiers and gene interactions involved in hearing. In collaboration with Sally Camper (University of Michigan), we mapped a hearing modifier of the dwarf (dw) mutation of the Pou1f1 gene that is associated with thyroid hormone deficiency (Karolyi et al., 2007; Qing et al. 2011). Antioxidant enzymes such as Cu/Zn superoxide dismutase (SOD1) protect cells from toxic, reactive oxygen species and may be involved in age-related degeneration. We previously reported that the absence of SOD1 in Sod1 knockout mice results in hearing loss at an earlier age than in wildtype mice (Keithley et al., 2005) and more recently reported on a genetic interaction between Sod1 deficiency and the ahl variant of Cdh23 (Johnson et al. 2010).
Principal Investigator: Kenneth R. Johnson, Ph.D.
Research Assistant III: Leona H. Gagnon, B.A., Chantal Longo-Guess, B.S.
Research Assistant I: Sandra Gray, Kelly L. Kane, B.S.
Research Administrative Assistant: Maxine Friend
Johnson KR, Longo-Guess CM, Gagnon LH. 2012. Mutations of the mouse ELMO domain containing 1 gene (Elmod1) link small GTPase signaling to actin cytoskeleton dynamics in hair cell stereocilia. PLoS One. 7:e36074.
Kane KL, Longo-Guess CM, Gagnon LH, Ding D, Salvi RJ, Johnson KR. 2012. Genetic background effects on age-related hearing loss associated with Cdh23 variants in mice. Hear Res. 283:80-88.
Fang Q, Longo-Guess C, Gagnon LH, Mortensen AH, Dolan DF, Camper SA, Johnnson KR. 2011. A modifier gene alleviates hypothyroidism-induced hearing impairment in Pou1f1dw dwarf mice. Genetics. 189:665-673.
Qing F, Longo-Guess C, Gagnon LH, Mortensen AH, Dolan DF, Camper SA, Johnson KR. 2011. A locus on Chromosome 2 modifies the severity of hearing impairment in hypothyroid Pou1f1dw dwarf mice. Genetics (In press).
Johnson KR, Gagnon LH, Longo-Guess C, Kane KL. 2011. Association of a citrate synthase missense mutation with age-related hearing loss in A/J mice. Neurobiol Aging (In Press).
Shin B-B, Longo-Guess CM, Gagnon LH, Saylor KW, Dumont RA, Spinelli KJ, Pagana JM, Wilmarth PA, David LL, Gillespie PG, Johnson KR. 2010. The R109H variant of fascin-2, a developmentally regulated actin crosslinker in hair-cell stereocilia, underlies early-onset hearing loss of DBA/2J mice. J Neurosci 30:9683-9694.
Johnson KR, Yu H, Ding D, Jiang H, Gagnon LH, Salvi RJ . 2010. Separate and combined effects of Sod1 and Cdh23 mutations on age-related hearing loss and cochlear pathology in C57BL/6J mice. Hear Res 268:85-92.
Noben-Trauth K, Johnson KR. 2009. Inheritance patterns of progressive hearing loss in laboratory strains of mice. Brain Res 1277:42-51.
Zheng QY, Ding DL, Yu H, Salvi RJ, Johnson KR. 2009. A locus on distal Chromosome 10 (ahl4) affecting age-related hearing loss in A/J mice. Neurobiol Aging 30:1693-1705.
Johnson KR, Longo-Guess C, Gagnon LH, Yu H, Zheng QY. 2008. A locus on distal Chromosome 11 (ahl8) and its interaction with Cdh23ahl underlie the early onset, age-related hearing loss of DBA/2J mice. Genomics 92:219-225.
Longo-Guess CM, Gagnon LH, Bergstrom DE, Johnson KR. 2007a. A missense mutation in the conserved C2B domain of otoferlin causes deafness in a new mouse model of DFNB9. Hear Res 234: 21-28.
Longo-Guess CM, Gagnon LH, Fritzsch B, Johnson KR. 2007b. Targeted knockout and lacZ reporter expression of the mouse Tmhs deafness gene and characterization of the hscy-2J mutation. Mamm Genome 18: 646-656.
Karolyi IJ, Dootz GA, Halsey K, Probst FJ, Johnson KR, Parlow AF, Dolan DF, Camper SA. 2007. Dietary thyroid hormone replacement ameliorates hearing deficits in hypothyroid mice. Mamm Genome 18: 596-608
Johnson KR, Marden CC, Ward-Bailey P, Gagnon LH, Bronson RT, Donahue LR. 2007. Congenital hypothyroidism, dwarfism and hearing impairment caused by a missense mutation in the mouse dual oxidase 2 gene, Duox2. Mol Endocrinol 21: 1593-1602.
Gagnon LH, Longo-Guess CM, Berryman M, Shin JB, Saylor KW, Yu H, Gillespie PG, Johnson KR. 2006. The chloride intracellular channel protein CLIC5 is expressed at high levels in hair cell stereocilia and is essential for normal inner ear function. J Neurosci 26:10188-10198.
Calderon A, Derr A, Stagner BB, Johnson KR, Martin G, Noben-Trauth K. 2006. Cochlear developmental defect and background-dependent hearing thresholds in the Jackson circler (jc) mutant mouse. Hear Res 221: 44-58.
Johnson KR, Zheng QY, Noben-Trauth K. 2006. Strain background effects and genetic modifiers of hearing in mice. Brain Res 1091: 79-88.
Jones SM, Jones TA, Johnson KR, Yu H, Erway LC, Zheng QY. 2006. A comparison of vestibular and auditory phenotypes in inbred mouse strains. Brain Res 1091: 40-46.
Jones SM, Johnson KR, Yu H, Erway LC, Alagramam KN, Pollak N, Jones TA. 2005. A quantitative survey of gravity receptor function in mutant mouse strains. J Assoc Res Otolaryngol 6: 297-310.
Keithley EM, Canto C, Zheng QY, Wang X, Fischel-Ghodsian N, Johnson KR. 2005. Cu/Zn superoxide dismutase and age-related hearing loss. Hear Res 209: 76-85.
Longo-Guess CM, Gagnon LH, Cook SA, Wu J, Zheng QY, Johnson KR. 2005. A missense mutation in the previously undescribed gene Tmhs underlies deafness in hurry-scurry (hscy) mice. Proc Natl Acad Sci USA. 102: 7894-7899.
Johnson KR, Zheng QY, Weston MD, Ptacek LJ, Noben-Trauth K. 2005. The Mass1frings mutation underlies early-onset hearing impairment in BUB/BnJ mice, a model for the auditory pathology of Usher Syndrome IIC. Genomics 85: 582-590.
Zheng QY, Yan D, Ouyang XM, Li F, Du LL, Yu H, Johnson KR, Liu XZ. 2005. Digenic inheritance of deafness caused by mutations in the genes encoding cadherin 23 and protocadherin 15 in mice and humans. Hum Mol Genet 14: 103-111.
Jones SM, Erway LC, Johnson KR, Yu H, Jones TA. 2004. Gravity receptor function in mice with graded otoconial deficiencies. Hear Res 191: 34-40.
Keithley EM, Canto C, Zheng QY, Fischel-Ghodsian N, Johnson KR. 2004. Age-related hearing loss and the ahl locus in mice. Hear Res 188: 21-28.
Johnson KR, Gagnon LH, Webb LS, Peters LL, Hawes NL, Chang B, Zheng QY. 2003. Mouse models of USH1C and DFNB18: phenotypic and molecular analyses of two new spontaneous mutations of the Ush1c gene. Hum Mol Genet 12: 3075-3086.
Noben-Trauth K, Zheng QY, Johnson KR. 2003. Association of cadherin-23 with polygenic inheritance and genetic modification of sensorineural hearing loss. Nat Genet 35: 21-23.
Willott JF, Tanner L, O’Steen J, Johnson KR, Bogue MA, and Gagnon L. 2003. Acoustic startle and prepulse inhibition in forty inbred strains of mice. Behav Neurosci 117: 728-737.