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An ENU-induced mutation of miR-96 associated with progressive hearing loss in mice

Abstract

Progressive hearing loss is common in the human population, but little is known about the molecular basis. We report a new N-ethyl-N-nitrosurea (ENU)-induced mouse mutant, diminuendo, with a single base change in the seed region of Mirn96. Heterozygotes show progressive loss of hearing and hair cell anomalies, whereas homozygotes have no cochlear responses. Most microRNAs are believed to downregulate target genes by binding to specific sites on their mRNAs, so mutation of the seed should lead to target gene upregulation. Microarray analysis revealed 96 transcripts with significantly altered expression in homozygotes; notably, Slc26a5, Ocm, Gfi1, Ptprq and Pitpnm1 were downregulated. Hypergeometric P-value analysis showed that hundreds of genes were upregulated in mutants. Different genes, with target sites complementary to the mutant seed, were downregulated. This is the first microRNA found associated with deafness, and diminuendo represents a model for understanding and potentially moderating progressive hair cell degeneration in hearing loss more generally.

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Figure 1: Scanning electron micrographs of diminuendo inner ear.
Figure 2: miR-96, miR-182 and miR-183 in littermates at P5.
Figure 3: Ocm, Slc26a5, Pitpnm1, Ptpr1 and Gfi1 expression in diminuendo.
Figure 4: Microarray analysis showing enrichment and depletion of heptamers in 3′ UTRs.

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Acknowledgements

We thank P. Ellis, K. James and R. Andrews for assistance with the microarrays, H. Williams for help with the quantitative RT-PCR, A. Rodriguez for helpful discussions on miRNAs, A. Taylor for help with bioinformatics, S. Rose, M. Drummond, K. Hawker and A. Lucas for help with mapping the mutation, F. Zhu for her work on Gfi1 and Ptprq, R. Lacey and D. Savage for animal care, A. El-Amraoui and C. Petit for the anti-MyRIP antibody, G. Richardson for the anti-Ptprq antibody, and A. Rzadzinska for high-resolution scanning electron microscopy analysis. This work was supported by Deafness Research UK, the Wellcome Trust, Medical Research Council (UK), EC (CT-97-2715, LSHG-CT-2004-512063), Spanish Ministerio de Ciencia y Tecnología (SAF2005-06355), Spanish Fondo de Investigaciones Sanitarias (FIS PI-051942; G03/203, CP03/00014, PI08/0045), NGFN (Germany), and charitable donations from individuals affected by deafness.

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Contributions

The mutagenesis programme was carried out by H.F. and M.H.D.A.; E.Q. analyzed the behavior, the middle and inner ear and the ultrastructural phenotype of the mutant and mapped the mutation. M.A.L., E.Q. and A.M.G. sequenced the region. Microarrays were run by C.L., and bioinformatic analysis was carried out by S.v.D., C.A.-G. and A.J.E. Motif analysis was done by M.P.; N.R. and T.D. carried out the luciferase assays. Literature searches, in situ hybridization, immunohistochemistry, quantitative RT-PCR and data analyses were done by M.A.L.; M.A.M.-P. shared data and ideas. K.P.S. conceived and devised the screen for new deaf mutants, obtained the funding, managed the programme, carried out the electrophysiology and interpreted the data. The paper was written by M.A.L., E.Q. and K.P.S.

Corresponding author

Correspondence to Karen P Steel.

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Supplementary Figures 1–5, Supplementary Methods and Supplementary Tables 1–5, Supplementary Note (PDF 3162 kb)

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Lewis, M., Quint, E., Glazier, A. et al. An ENU-induced mutation of miR-96 associated with progressive hearing loss in mice. Nat Genet 41, 614–618 (2009). https://doi.org/10.1038/ng.369

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