Inner Ear Morphology Is Perturbed in Two Novel Mouse Models of Recessive Deafness

<div><p>Human <em>MYO7A</em> mutations can cause a variety of conditions involving the inner ear. These include dominant and recessive non-syndromic hearing loss and syndromic conditions such as Usher syndrome. Mouse models of deafness allow us to investigate functional pathways involved in normal and abnormal hearing processes. We present two novel mouse models with mutations in the <em>Myo7a</em> gene with distinct phenotypes. The mutation in <em>Myo7a^I487N/I487N ewaso</em> is located within the head motor domain of Myo7a. Mice exhibit a profound hearing loss and manifest behaviour associated with a vestibular defect. A mutation located in the linker region between the coiled-coil and the first MyTH4 domains of the protein is responsible in <em>Myo7a^F947I/F947I dumbo</em>. These mice show a less severe hearing loss than in <em>Myo7a^I487N/I487N ewaso</em>; their hearing loss threshold is elevated at 4 weeks old, and progressively worsens with age. These mice show no obvious signs of vestibular dysfunction, although scanning electron microscopy reveals a mild phenotype in vestibular stereocilia bundles. The <em>Myo7a^F947I/F947I dumbo</em> strain is therefore the first reported <em>Myo7a</em> mouse model without an overt vestibular phenotype; a possible model for human DFNB2 deafness. Understanding the molecular basis of these newly identified mutations will provide knowledge into the complex genetic pathways involved in the maintenance of hearing, and will provide insight into recessively inherited sensorineural hearing loss in humans.</p> </div

Immunohistochemistry of P5 sensory epithelia.

<p>Images of phalloidin (green) and Myo7a (red) stained cochlear sensory epithelium from P5 <i>Myo7a^+/+</i> (<b>A and B</b>), <i>Myo7a^I487N/I487N ewaso</i> (<b>C and D</b>) and <i>Myo7a^F947I/F947I dumbo</i> (<b>E and F</b>) mice at the basal cochlear level. No Myo7a expression is evident in <i>Myo7a^I487N/I487N ewaso</i> mutant tissue (<b>D</b>), and may be slightly reduced in <i>Myo7a^F947I/F947I dumbo</i> mutants (<b>F</b>). No difference in protein localisation was observed between wilt-type and <i>Myo7a^F947I/F947I dumbo</i> tissue. Phalloidin staining highlights abnormal IHC structure in <i>Myo7a^I487N/I487N ewaso</i> mutants (<b>C</b>) and OHC hair bundles appear misorientated and/or fragmented in <i>Myo7a^F947I/F947I dumbo</i> (arrowheads in E). Scale bar; 8 µM (A–H).</p

<i>Myo7a</i> mutations identified in <i>Myo7a^I487N/I487N ewaso</i> and <i>Myo7a^F947I/F947I dumbo</i> strains.

<p>(<b>A</b>) Direct sequencing identified a 1460T>A (I487N) missense mutation in <i>Myo7a^I487N/I487N ewaso</i> and a missense mutation 2839T>A (F947I) in the <i>Myo7a^F947I/F947I dumbo</i> mouse strain (<b>B</b>). Wildtype and heterozygote sequences are shown for comparison. (<b>C</b>) Sequence conservations of <i>Myo7a^I487N/I487N ewaso</i> (red) and <i>Myo7a^F947I/F947I dumbo</i> (blue) mutations, and the closely positioned <i>Myo7a^sh-1 shaker</i> mutation (green) showing high evolutionary conservation of our two mutant strains.</p

Reported <i>Myo7a</i> mouse models.

<p>Reported <i>Myo7a</i> mouse models.</p

Schematic diagram of Myo7a protein structure showing the location of <i>Myo7a^I487N/I487N ewaso</i> and <i>Myo7a^F947I/F947I dumbo</i> mutations (in red) in relation to reported <i>shaker</i> mutations.

<p>DFNA11/DFNB2/USH1B human mutations within close proximity to a reported <i>shaker</i> mutation are shown in parentheses <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051284#pone.0051284-Weston1" target="_blank">[14]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051284#pone.0051284-Bharadwaj1" target="_blank">[56]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051284#pone.0051284-Jaijo1" target="_blank">[57]</a>. Details of these mouse mutations are included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051284#pone-0051284-t001" target="_blank">Table 1</a>. IQ, isoleucine-glutamine motif; CC1, Coiled Coil domain; MyTH4, Myosin Tail Homology 4; SH3, SRC Homology 3 domain.</p

Scanning electron micrographs of P2–5 vestibular epithelia.

<p>(<b>A–C and J</b>) <i>Myo7a^+/+</i>, (<b>D–F and K</b>) <i>Myo7a^I487N/I487N ewaso</i> and (<b>G–I and L</b>) <i>Myo7a^F947I/F947I dumbo</i>. A clear line of polarity reversal can be identified in <i>Myo7a^+/+</i> saccular maculae (<b>A; dashed line</b>), and normal hair bundle morphology is apparent, with a staircase arrangement of stereocilia and kinocilium located with the tallest stereocilia (<b>B and C</b>). Hair cell bundles are affected in <i>Myo7a^I487N/I487N ewaso</i> saccules, and no clear zone of polarity is evident (<b>D</b>). These bundles do contain stereocilia of various lengths, however are generally disorientated and unstructured (<b>E and F</b>). A zone of polarity can be identified in <i>Myo7a^F947I/F947I dumbo</i> saccules (<b>G</b>), however hair bundles do appear to have a mild phenotype, with bundles missing some of the tallest (<b>H</b>) and/or shortest stereocilia (<b>I</b>). Scale bar; 10 µM (A, D and G), 2 µM (B, C, E, F, H and I).</p

Phenotypic observations of <i>Myo7a</i> mutant strains.

<p>(<b>A</b>) Hearing profile of <i>Myo7a^+/+</i>, <i>Myo7a^I487N/I487N ewaso</i> and <i>Myo7a^F947I/F947I dumbo</i> strains at 4 weeks (*p = 2.2×10⁻²⁵, **p = 4.5×10⁻¹⁰) and 24 weeks (*p = 3.7×10⁻²⁹, **p = 7.2×10⁻²⁰). (<b>B–G</b>) Video surveillance and middle ear morphology in <i>Myo7a</i> strains. Observations highlighted an increased number of turns in <i>Myo7a^I487N/I487N ewaso</i> mice (<b>C</b>), when compared to wild-type (<b>B</b>). No such behaviour was seen in <i>Myo7a^F947I/F947I dumbo</i> mutants (<b>D</b>). Middle ear bones appear largely normal in <i>Myo7a^I487N/I487N ewaso</i> (<b>F</b>) and <i>Myo7a^F947I/F947I dumbo</i> (<b>G</b>) mutants, comparable to normal morphology of the malleus, incus and stapes (<b>E</b>). M; manubrium of malleus, A; articulation surfaces of malleus and incus joint, T; tubercle, G; gonial angle, LI; attachment points of suspensory ligaments of incus, LP; lenticular process, C; capitulum of stapes, V; arched ventral crus, F; footplate. Scale bar; 1 mm (E–G).</p

Molecular modelling of the <i>Myo7a^I487N/I487N ewaso</i> mutation.

<p>(<b>A</b>) Partial ribbon representation of a 3D model of myosin V 2DFS highlighting the Ile⁴⁸⁷ amino acid residue, which is localised to the hinge region between the head and tail domains of the protein. (<b>B</b>) Schematic diagram showing superimposed images of the hinge region (<i>Myo7a^+/+</i> in red and the <i>Myo7a^I487N/I487N ewaso</i> mutation in blue) after 4 ns of molecular dynamics simulation showing a conformational change in the hinge region close to the Ile⁴⁸⁷ mutation site, showing the involvement of residues 670–673 around the hinge region causing the distortion.</p

Predictive values of neuron-specific enolase (NSE) for poor outcome after out-of-hospital cardiac arrest.

<p>Predictive values of neuron-specific enolase (NSE) for poor outcome after out-of-hospital cardiac arrest.</p

Patient inclusion.

<p>Displaying the total Targeted Temperature Management (TTM) population, Intention-To-Treat (ITT) population and outcomes by the Cerebral Performance Category (CPC) scale.</p