Research paperIdentification of 17 hearing impaired mouse strains in the TMGC ENU-mutagenesis screen
Introduction
An increasing number of naturally occurring mouse mutations and genetically engineered or chemically induced mouse mutants with hearing impairment have served as models for human deafness (Friedman and Griffith, 2003, Goldfarb and Avraham, 2002, Steel and Kros, 2001). However, only a limited number of mouse models for age-related hearing loss (AHL or presbycusis) and noise-induced hearing loss (NIHL), prevalent disorders in humans that are characteristic of high-frequency hearing loss (HFHL), have been identified (Kujawa and Liberman, 2006, Noben-Trauth et al., 2003). In addition, a significant lack of mouse models for mild hearing loss in non-syndromic genetic deafness exists. Identification of these types of mutants would require careful, maybe labor-intensive, mutagenesis and screening strategies.
N-ethyl-N-nitrosourea (ENU) is a strong chemical mutagen that introduces random single base-pair changes in the genome. ENU mutagenesis is complementary to other types of mutagenesis such as gene-trap insertional mutations or specific-gene knockouts achieved by homologous recombination in ES cells. Because approximately 70% of the 38,000 human mutations in over 1500 genes that have been identified are of the single-base pair variety (http://www.hgmd.cf.ac.uk/ac/hahaha.php), mutants identified through ENU-based screens more closely model these naturally occurring human mutations. The NIH funded neuromutagenesis program in the Tennessee Mouse Genome Consortium (TMGC) employed an ENU mutagenesis scheme in which visible or molecular markers and specific mouse strains with inverted chromosomal regions were used to easily identify mice carrying the recessive mutations (Goldowitz et al., 2004, Jablonski et al., 2005) (http://www.tnmouse.org/neuromutagenesis/).
One of the commonly used hearing tests in human and mice is far field auditory brainstem evoked responses (ABR). However, nearly all other auditory screens in various mouse ENU-mutagenesis programs employ assays other than ABR; instead the screens use acoustic startle response (ASR), pre-pulse inhibition (PPI) or click-box (Hrabe de Angelis et al., 2000, Munroe et al., 2000, Nolan et al., 2000). The stimuli used in these screens are usually at high levels (90–110 dB SPL) and at frequencies (18–20 kHz); thus most of the mutants identified are “stone” deaf (>60 dB SPL ABR threshold elevation) with severe inner ear defects.
In our auditory primary screen in the TMGC neuromutagenesis program, we employed ABR to click and 8, 16, 32 kHz pure tone stimuli. In the past 3 years, we have screened a total of 285 pedigrees (1819 mice) at the age of 8–11 weeks in mixed strain backgrounds. A total of 17 pedigrees (6%) were confirmed to display hearing loss at frequencies ⩾16 kHz; 6 were exclusively at 32 kHz; 6 were at wide-range frequencies; 4 were sex-biased. The average ABR threshold elevation at each frequency is 30–35 dB SPL. The histology of 6 of the 9 mutant pedigrees we first identified and analyzed showed degeneration of spiral ganglia (SG), spiral ligament (SL) fibrocytes or inner hair cells, but not outer hair cells, in the basal cochlea. These results in combination verified the effectiveness and feasibility of our strategy to screen for frequency-specific and mild hearing mutants using ABR and provided potential novel mouse models for the prevalent age-related and noise-induced hearing loss in humans. These mutants are available for detailed characterization to academic researchers.
Section snippets
TMGC mutagenesis and strain backgrounds
TMGC utilized three different mutagenesis schemes as outlined below:
- (1)
Visible markers-using mice with visually apparent chromosomal alterations to identify potentially mutant (test class) animals. ENU mutagenesis was conducted at the Oak Ridge National Laboratory for this group of mice and portions of chromosomes (Chr) X, 15, 10, and 7 are targets of this screen (Table 1). Mice identified by visible markers as carriers of recessive alleles are mated in the 2nd (G2) or 3rd (G3) generations to
ABR thresholds in different projects
In the auditory primary screen of the TMGC neuromutagenesis program, we recorded ABR to click (a mixed range of lower frequencies, mostly between 2 and 10 kHz) and 8, 16, and 32 kHz pure-tone stimuli. A total of 285 pedigrees (1819 mice) have been screened thus far: the first ∼300 mice were screened only with click stimuli and subsequent ∼1500 mice were screened with click and three pure tone stimuli. A small percentage (<5%) of mice were screened only with click and 32 kHz pure-tone stimuli. All
Discussion
Our 17 mutants are of interest for studying AHL and NIHL, the two common hearing disorders of the aging population. Cochlear histological analysis of the first 9 mutants provided a foundation for pathology in these mutants. Further characterization and identification of the underlying mutant genes will advance our understanding of these important pathological conditions. Hearing researchers are encouraged to further characterize these mutants in detail (see www.neuromice.org for ordering
Acknowledgments
We thank M.C. Liberman as an external advisor to the auditory screen in TMGC and other members of TMGC for their assistance. This work was supported, in part, by NIH Grants (MH61971, DC06471, DC050101, CA21765), 2005 UNCF/MERCK Postdoctoral Science Research Fellowship and American Lebanese Syrian Associated Charities (ALSAC).
References (30)
- et al.
Presbycusis
Lancet
(2005) - et al.
Large-scale mutagenesis of the mouse to understand the genetic bases of nervous system structure and function
Brain Res. Mol. Brain Res.
(2004) Males lose hearing earlier in mouse models of late-onset age-related hearing loss; females lose hearing earlier in mouse models of early-onset hearing loss
Hear. Res.
(2004)- et al.
Ahl2, a second locus affecting age-related hearing loss in mice
Genomics
(2002) - et al.
Cu/Zn superoxide dismutase and age-related hearing loss
Hear. Res.
(2005) - et al.
Ahl3, a third locus on mouse chromosome 17 affecting age-related hearing loss
Biochem. Biophys. Res. Commun.
(2004) - et al.
Cochlear cultures as a model system for studying aminoglycoside induced ototoxicity
Hear. Res.
(1991) - et al.
Female MRL.MpJ-Fas(lpr) autoimmune mice have greater hearing loss than males
Hear. Res.
(2002) - et al.
Aminoglycoside ototoxicity in adult CBA, C57BL and BALB mice and the Sprague–Dawley rat
Hear. Res.
(2001) - et al.
Assessment of hearing in 80 inbred strains of mice by ABR threshold analyses
Hear. Res.
(1999)
N-ethyl-N-nitrosourea mutagenesis: boarding the mouse mutant express
Microbiol. Mol. Biol. Rev.
Human nonsyndromic sensorineural deafness
Annu. Rev. Genomics Hum. Genet.
Genetics of deafness: recent advances and clinical implications
J. Basic Clin. Physiol. Pharmacol.
Spiral ligament pathology: a major aspect of age-related cochlear degeneration in C57BL/6 mice
J. Assoc. Res. Otolaryngol.
Cited by (17)
Characterization of a novel enu-generated myosin vi mutant mouse strain with congenital deafness and vestibular dysfunction
2013, Hearing ResearchCitation Excerpt :ENU-mutagenesis programs have proven to be a highly valuable approach in identifying novel mutations and genes involved in the auditory pathway, as well as revealing novel functions of genes already known to be involved in hearing (Curtin et al., 2003; Hardisty et al., 2003; Hrabe de Angelis et al., 2000; Kiernan et al., 2001; Marcotti et al., 2006; Nolan et al., 2000; Rhodes et al., 2004, 2003; Tsai et al., 2001; Vreugde et al., 2002). Initially, ENU-mutagenesis programs focused on dominant disease-causing mutations, however, as the majority of human genetic deafness is recessively inherited, characterisation of recessive ENU-mutants, such as charlie, will be essential for gaining further insight into the development and functioning of the mammalian auditory system (Kermany et al., 2006; Manji et al., 2012, 2011a; 2011b; Parker et al., 2010). Given the high degree of similarity between the mouse and human inner ear, and the conserved role of Myo6 in the mammalian inner ear, charlie will be a valuable resource for gaining a more thorough understanding of the role Myo6 plays in hearing and balance, and may ultimately help to guide therapeutic approaches for patients with deafness attributable to Myo6 mutations.
Identification of three novel hearing loss mouse strains with mutations in the Tmc1 gene
2012, American Journal of PathologyEthanol-induced hyperactivity is associated with hypodopaminergia in the 22-TNJ ENU-mutated mouse
2009, AlcoholCitation Excerpt :In the field of alcohol research, it is apparent that complex genetic and environmental factors contribute of the effects of ethanol, and a variety of animal models have been created, using targeted and random genetic alterations and selective breeding, to examine and understand these effects (e.g., Bell et al., 2008; Bergstrom et al., 2003; Crabbe and Belknap, 1993; Gorwood et al., 2002; Hoplight et al., 2007; Phillips, 1993). Recently, the Integrative Neuroscience Initiative on Alcoholism and Tennesse Mouse Genome Consortia used N-ethyl-N-nitrosourea (ENU)-mutagenesis to induce random single base-pair mutations in mice and then characterized ethanol-related phenotypes in mutated mouse lines using high-throughput behavioral screens (Hamre et al., 2007; Kermany et al., 2006). In behavioral assays described by Hamre et al. (2007), the 22-TNJ mutant mouse strain was noted because it exhibited a hyperactive locomotor response to ethanol compared with 1-TNH controls.
Inheritance patterns of progressive hearing loss in laboratory strains of mice
2009, Brain ResearchCitation Excerpt :Response to a loud sound can be detected in a mouse by the Preyer reflex (ear flick), but this simple behavioral method detects only supra-threshold responses. More informative, quantitative assessments of hearing function in mice on a large scale are currently performed using electrophysiological methods: auditory-brain stem response (ABR) measurements and distortion-product-otoacoustic-emission (DPOAE) testing (Kermany et al., 2006; Martin et al., 2007; Schwander et al., 2007). ABR waveforms are recorded from the activity of the cochlear nerve and brainstem nuclei in response to an acoustic stimulus of defined frequency and amplitude and represent a measurement of the activity of all cellular structures involved in acoustic signal receiving, processing, amplification, and transmission.
A new mouse mutant for the LDL receptor identified using ENU mutagenesis
2008, Journal of Lipid ResearchCitation Excerpt :It is notable when comparing the mouse with human FH phenotypes that, in general, patients heterozygous for Ldlr mutations present severe phenotypes as a result of hyperlipidemia, whereas WHC and Ldlr KO mice require homozygosity to more closely mimic the human disease. High-throughput phenotyping of mouse ENU mutants has revealed novel, single-base mutations in disease-related genes and produced numerous robust models of human disorders (25–32). These ENU mutants are otherwise genetically identical to their nonmutagenized inbred strain counterparts, allowing highly specific analyses of the functional consequence of a single base change, and often produce distinctly different phenotypes from those observed in analogous KO models.
Auditory brainstem responses are impaired in EphA4 and ephrin-B2 deficient mice
2008, Hearing Research