Elsevier

Hearing Research

Volume 220, Issues 1–2, October 2006, Pages 76-86
Hearing Research

Research paper
Identification of 17 hearing impaired mouse strains in the TMGC ENU-mutagenesis screen

https://doi.org/10.1016/j.heares.2006.07.011Get rights and content

Abstract

The Tennessee Mouse Genome Consortium (TMGC) employed an N-ethyl-N-nitrosourea (ENU)-mutagenesis scheme to identify mouse recessive mutants with hearing phenotypes. We employed auditory brainstem responses (ABR) to click and 8, 16, and 32 kHz stimuli and screened 285 pedigrees (1819 mice of 8–11 weeks old in various mixed genetic backgrounds) each bred to carry a homozygous ENU-induced mutation. To define mutant pedigrees, we measured ⩾12 mice per pedigree in ⩾2 generations and used a criterion where the mean ABR threshold per pedigree was two standard deviations above the mean of all offspring from the same parental strain. We thus identified 17 mutant pedigrees (6%), all exhibiting hearing loss at high frequencies (⩾16 kHz) with an average threshold elevation of 30–35 dB SPL. Interestingly, four mutants showed sex-biased hearing loss and six mutants displayed wide range frequency hearing loss. Temporal bone histology revealed that six of the first nine mutants displayed cochlear morphological defects: degeneration of spiral ganglia, spiral ligament fibrocytes or inner hair cells (but not outer hair cells) mostly in basal turns. In contrast to other ENU-mutagenesis auditory screens, our screen identified high-frequency, mild and sex-biased hearing defects. Further characterization of these 17 mouse models will advance our understanding of presbycusis and noise-induced hearing loss in humans.

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)

  • S.P. Cordes

    N-ethyl-N-nitrosourea mutagenesis: boarding the mouse mutant express

    Microbiol. Mol. Biol. Rev.

    (2005)
  • T.B. Friedman et al.

    Human nonsyndromic sensorineural deafness

    Annu. Rev. Genomics Hum. Genet.

    (2003)
  • Gao, J., Maison, S.F., Jones, S.M., Hirose, K., Bayazitov, I., Wu, X., Tian, Y., Mittleman, G., Matthews, D.,...
  • A. Goldfarb et al.

    Genetics of deafness: recent advances and clinical implications

    J. Basic Clin. Physiol. Pharmacol.

    (2002)
  • S. Hequembourg et al.

    Spiral ligament pathology: a major aspect of age-related cochlear degeneration in C57BL/6 mice

    J. Assoc. Res. Otolaryngol.

    (2001)
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