A Mutation in Myo15 Leads to Usher-Like Symptoms in LEW/Ztm-ci2 Rats

The LEW/Ztm-ci2 rat is an animal model for syndromal deafness that arose from a spontaneous mutation. Homozygous animals show locomotor abnormalities like lateralized circling behavior. Additionally, an impaired vision can be observed in some animals through behavioral studies. Syndromal deafness as well as retinal degeneration are features of the Usher syndrome in humans. In the present study, the mutation was identified as a base substitution (T->C) in exon 56 of Myo15, leading to an amino acid exchange from leucine (Leu) to proline (Pro) within the carboxy-terminal MyTH4 domain in the proteins' tail region. Myo15 mRNA was expressed in the retina as demonstrated for the first time with the help of in-situ hybridization and PCR. To characterize the visual phenotype, rats were examined by scotopic and photopic electroretinography and, additionally, histological analyses of the retinas were conducted. The complete loss of sight was detected along with a severe degeneration of photoreceptor cells. Interestingly, the manifestation of the disease does not solely depend on the mutation, but also on environmental factors. Since the LEW/Ztm-ci2 rat features the entire range of symptoms of the human Usher syndrome we think that this strain is an appropriate model for this disease. Our findings display that mutations in binding domains of myosin XV do not only cause non-syndromic hearing loss but can also lead to syndromic disorders including retinal dysfunction

Retinal phenotypes detected by electroretinography.

<p>0 = no response, animal blind; A = significantly reduced response, animal affected; UA = normal response, animal unaffected.</p><p>*extincted ERG amplitudes recorded in just one eye of that animal, amplitudes of the second eye significantly decreased.</p

In situ hybridization in a paraffin section of a LEW/Ztm-<i>ci2</i> rat indicates the presence of <i>Myo15</i> mRNA in the retina.

<p>The insert demonstrates the same retina with the sense-control. (×80) IPL = Inner Plexiform Layer, INL = Inner Nuclear Layer, OPL = Outer Plexiform Layer, ONL = Outer Nuclear Layer, PRC = Inner and Outer Segments of Photoreceptor Cells.</p

Illustration of the LEW/Ztm-<i>ci2</i> mutation in the MyTH4 domain of the <i>Myo15</i> gene.

<p>(<b>A</b>) Polyphred view of the single point mutation found in exon 56 in the C-terminal MyTH4-domain. (<b>B</b>) The base substitution (T→C) does not cause a stop codon but leads to an exchange of the amino acid Leucin for Proline at position 3157 in the Myosin XV protein.</p

Evidence of <i>Myo15</i> expression in the rat retina.

<p>Agarose gel results of PCR products obtained from retinas of LEW/Ztm rats. <b>Lane M</b> contains the DNA ladder; <b>lane 1</b> contains the negative control reaction (no cDNA template, <i>Myo15</i> primers); <b>lane 2</b> contains cDNA (10 ng) obtained from the retina (<i>Myo15</i> primers, expected length of the fragment 125 bp); <b>lane 3</b> contains positive control, cDNA (10 ng) obtained from the pituitary gland (<i>Myo15</i> primers, expected length of the fragment 125 bp); <b>lane 4</b> contains negative control reaction (no cDNA template, <i>ß-Actin</i> primers); <b>lane 5</b> contains cDNA obtained from the retina (<i>ß-Actin</i> primers, expected length of fragment 101 bp).</p

Light intensities inside cages measured at 3 levels of the rack.

<p>Light intensities inside cages measured at 3 levels of the rack.</p

Histological examinations of retinae from unaffected and affected LEW/Ztm-<i>ci2</i> rats.

<p>(<b>A</b>.) Unaffected LEW/Ztm-<i>ci2</i> rat (MedSchool group) showing slight reduction of photoreceptor cells due to age (<b>B</b>.) Affected LEW/Ztm-<i>ci2</i> rat (VetSchool group) with distinct reduction of number and density of rod and cone somata within the ONL (<b>C</b>.) Severely affected LEW/Ztm-<i>ci2</i> rat (VetSchool group) cell bodies, inner and outer segments of photoreceptor cells are nearly absent while the remaining retinal layers are arranged in a normal pattern (×400) NF = Nerve fibres, IPL = Inner Plexiform Layer, INL = Inner Nuclear Layer, OPL = Outer Plexiform Layer, ONL = Outer Nuclear Layer, PRC = Inner and Outer Segments of Photoreceptor Cells, RPE = Retinal Pigment Epithelium.</p

Mean values of rod responses recorded by electroretinography.

<p>Average skotopic a- and b-wave maximum amplitudes elicited by a 3 cds/m² light stimulus. (<b>A</b>) a-wave amplitudes of the left eyes of LEW/Ztm-<i>ci2</i> (ci2) rats from two different hygienic units, MedSchool (MHH) and VetSchool (TiHo) and corresponding controls (K) (<b>B</b>) a-wave amplitudes of the right eyes (<b>C</b>) b-wave amplitudes of the left eyes (<b>D</b>) b-wave amplitudes of the right eyes; responses of <i>ci2</i> mutants of the VetSchool group are strongly increased compared to controls and mutant rats of the MedSchool group *significant differences compared to controls p<0.05 are indicated with an asterix.</p