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(Investigative Ophthalmology and Visual Science. 2001;42:770-778.)
© 2001 by The Association for Research in Vision and Ophthalmology, Inc.

Electroretinographic Anomalies in Mice with Mutations in Myo7a, the Gene Involved in Human Usher Syndrome Type 1B

Richard T. Libby and Karen P. Steel

From the Medical Research Council Institute of Hearing Research, Nottingham, United Kingdom.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. In humans, mutations in the gene encoding myosin VIIa can cause Usher syndrome type 1b (USH1B), a disease characterized by deafness and retinitis pigmentosa. Myosin VIIa is also the gene responsible for the inner ear abnormalities at the shaker1 (sh1) locus in mice. To date, none of the sh1 alleles examined have shown any signs of retinal degeneration. In the present study, electroretinograms (ERGs) were recorded from sh1 mice to determine whether they have any physiological abnormalities.

METHODS. ERGs were recorded from mice homozygous for one of nine mutant alleles of Myo7a ranging in age from postnatal day (P)20 to approximately 1 year. All mice were dark adapted for 30 minutes, and all the mutant mice were paired with an appropriately age- and strain-matched control animal. A presumptive null allele of myosin VIIa, Myo7a4626SB, was used to determine whether mice without myosin VIIa had an increased threshold, as assessed by the light level required to elicit a 15-µV b-wave.

RESULTS. At the maximum light intensity used, five of the nine alleles examined had significantly reduced a- and b-wave amplitudes. For example, Myo7a4626SB mutant mice had a 20% reduction in a-wave amplitude at the maximum light intensity, and this reduction was the same for mice ranging in age from P20 through 7 months. The b-wave thresholds of the Myo7a4626SB mutant mice were not significantly different from those of the control mice. Furthermore, whereas most of the alleles’ a-wave implicit times were the same in mutant and control mice, mutant mice with two of the alleles had significantly faster a-wave implicit times.

CONCLUSIONS. Mutations in myosin VIIa in mice can lead to decreased ERG amplitudes while threshold remains normal. This is the first report of a physiological anomaly in a mouse model with a mutation in the same gene as involved in USH1B.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Myosin VIIa is an unconventional myosin that presumably functions as an actin-based motor. In humans, mutations in the gene encoding myosin VIIa (MYO7A) cause nonsyndromic deafness1 2 3 and Usher syndrome.4 5 Usher syndrome (USH) is one of the most common forms of syndromic deafness and is characterized by hearing loss, retinitis pigmentosa, and in some types, vestibular dysfunction. Mutations in MYO7A are responsible for two forms of USH, atypical USH and USH type 1b (USH1B). USH1B is by far the most common form of USH caused by MYO7A mutations and is characterized by profound congenital deafness and the prepubertal onset of retinal degeneration.6 Presently, there are 57 mutations in MYO7A spread throughout the molecule that ultimately result in retinitis pigmentosa.4 5 7 8 9 10 11 12 13

Myosin VIIa (Myo7a) has also been identified as the gene involved in deafness at the shaker1 (sh1) locus in mice.14 To date, 10 Myo7a mutant alleles have been identified, and all of them have inner ear defects15 16 (Steel and Self, unpublished observation, 2000). In fact, none of the hair cells of Myo7a mutant mice develop normally, and all of them result eventually in profound deafness. Therefore, the sh1 mouse appears to be a good animal model for the inner ear disease in humans resulting from mutations in MYO7A. In contrast to the good correlation between the mouse and human inner ear phenotypes, the retinas of Myo7a mutant mice (including presumptive null mutations) show no signs of retinal degeneration.17 18 Therefore, the sh1 mouse does not appear to be a good model for the visual defects in USH1B.

In vertebrates, myosin VIIa is expressed in the retinal pigment epithelium (RPE) and photoreceptor cells. Both cell types contain the components of the visual cycle, and abnormal function of either can lead to retinal degeneration.19 Myosin VIIa is present at the apical surface of the RPE20 21 22 and in Myo7ash1 mutant mice, the melanosomes in the RPE do not invade the apical process.23 This abnormality is not thought to result in retinal degeneration, because other mice with melanosome abnormalities do not normally undergo retinal degeneration.23 The localization of myosin VIIa to the apical border of the microvilli and the fact that a member of the myosin VII family is involved in phagocytosis in the amoeba Dictyostelium discoideum24 suggests a possible involvement of myosin VIIa in phagocytosis of outer segments; however, no evidence of a disruption of phagocytosis has been found.23

In human and mouse photoreceptors, myosin VIIa is localized in the connecting cilium of rods and cones.22 Despite the expression of myosin VIIa in the connecting cilium of photoreceptors, no ultrastructural defects in these cilia were found in Myo7a mutant mice (Cable and Steel, manuscript in preparation). Autoradiographic studies of outer segment disc synthesis showed that the rate of new disc synthesis was significantly reduced in Myo7a mutant mice; however, it is important to note that the length of Myo7a mutant outer segments is not affected by the slow process of disc renewal.18 Also this same study showed that opsin abnormally accumulates in the connecting cilium of Myo7a mutant mice. Together these data suggest that myosin VIIa is not only present in the connecting cilium of mouse photoreceptors, but also is involved with the transport of opsin and possibly other molecules through the cilium and into the outer segment. Therefore, the sh1 mouse still appears to be a model for USH1B and also a useful model organism for determining the role of myosin VIIa in the mammalian retina.

We examined by means of electroretinography the retinas of Myo7a mutant mice. Their electroretinograms (ERGs) may elucidate a functional problem within the retinas, and if they have an abnormal ERG, the specific defect may be useable as predictor of future disease in humans. We found that five of the nine sh1 alleles examined had decreased ERG amplitudes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Animals were kept in a 14:10-hour light–dark cycle. All animal procedures were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and in accordance with United Kingdom’s Home Office regulations. Strains carrying nine different Myo7a alleles were used (Table 1) . For six of the alleles, the mutation in Myo7a has been determined.14 25 Myo7a mutant mice were distinguished from their control littermates (+/+ or +/sh1) by their head-shaking and circling behavior. A total of 122 pairs of mutant mice and control littermates were used in these experiments.


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  		Table 1. Myo7a Mutant Alleles

 
The genetic background of each stock is presented in Table 1 . The N-ethyl-N-nitrosourea (ENU)-induced mutations were derived from ENU treatment of BALBc males, and were repeatedly backcrossed to the BS inbred strain. These mice were kindly provided by Oak Ridge National Laboratories26 (Oak Ridge, TN). They have since been crossed, once to the CBA/Ca inbred strain to introduce the wild-type allele at the albino locus and intercrossed since then, with most experimental animals reported in this study being derived from the fourth or fifth generation of intercrossing. Selection during intercrossing was based only on the albino and shaker1 loci. As a result, there has been some opportunity for different alleles of other potentially modifying loci to become fixed in the different stocks.

Electroretinography
Mice were anesthetized with urethane (intraperitoneal injection of approximately 2.2 mg/g) and were prepared under room light. The animal was placed into a head holder in a light-tight Faraday cage, and the reference electrode was attached to the head holder. The eyelid was removed, and the pupil was dilated with atropine. After aligning the animal with the light source, a cotton wick electrode coupled to a silver-silver chloride half cell, was placed onto the animal’s cornea in a position that minimized any attenuation of the light flash. A test flash was presented to the animal to test the electrode placement. After a 30-minute dark adaptation, animals were presented with one of two series of flashes.

The first ERG light stimulation protocol was designed to limit light adaptation. In this case, recordings were made over 7.8 log units at 0.6-log-unit intervals. The stimulation protocol was: four 50-msec flashes (PS22 Photopic stimulator; Grass, Quincy, MA) separated by 15 seconds for the lowest light levels (7.8–5.4 log units of attenuation); four 50-msec flashes separated by 30 seconds for the intermediate light levels (4.8–3.0 log units of attenuation); and two 50-msec flashes separated by 60 seconds for the brightest light levels (2.4–0 log units of attenuation).

The second ERG light stimulation protocol caused adaptation to occur at the higher light intensities. This protocol consisted of ten 50-msec flashes, that were separated by 3 seconds for every light intensity. A set of recordings for each animal was made over 7.8 log units at 0.3-log-unit intervals, starting with the least bright intensity, and recordings at increasing light levels were made immediately after those at the lower light intensities. In all cases the responses were amplified, recorded, and averaged by computer. The unattenuated flash was 466 candelas (cd)/m2. The a-wave amplitude was measured from the prestimulus baseline to the minimum value of the first negative deflection, and the b-wave value was measured from the trough of the a-wave (when present) to the maximum positive value. The a- and b-wave implicit times (latencies) were measured from the time of flash onset to either the minimum value of the first negative deflection (a-wave) or to the maximum positive value (b-wave). Because the maximum ERG amplitudes varied with age (see results), two-tailed paired t-tests were used to determine whether there was a significant difference of a- and b-wave amplitudes between the mutant and control animals. In all cases, ERGs were performed on a mutant and littermate control consecutively (randomly choosing whether a mutant or a control was performed first). Because there was not a significant relationship between an animal’s age and either the a- or b-wave implicit time (see the Results section), two-tailed unpaired t-tests were used in implicit time analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Variation in ERG Amplitudes with Age in Normal Mice
Because mice in a wide age range were examined in this study, a post hoc analysis of the relationship between ERG amplitude and age was performed on mice that were examined with the first (nonadapting) of the two protocols used in the study. Amplitudes increased with age up to approximately postnatal day (P)30 and thereafter declined, reaching a steady level by approximately P90 (Fig. 1) .



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  		Figure 1. The a-wave amplitudes change with age in mice. The a-wave amplitudes at maximum light intensity were plotted versus the age of the mouse for all the control mice from the first (nonadapting) exposure protocol. X, control mice for the Myo7a4626SB allele, the largest sample size. In general, the amplitude increased until it reached a peak at approximately P30. After its peak, the amplitude steadily decreased until leveling off at approximately P90. At P90 the amplitude remained steady until at least P233. The line on the graph is the best fit line between these time points for all the control mice (R2 = 0.0352, P = 0.312). A similarly shaped curve has been reported for rat.27

 
A regression analysis of a-wave amplitude obtained from all the control mice at maximum light intensity versus age (n = 76, P20–P233) showed that a-wave amplitude did not become stable until P90 (Fig. 1 ; no significant correlation of age with amplitude from P90 through P233; R2 = 0.035, P = 0.313). The b-wave amplitude reached its adult level slightly earlier at P85 (R2 = 0.021, P = 0.410). The implicit times (latencies) for both the a- and b-waves did not appear to change with age (a-wave R2 = 0.005, P = 0.528; b-wave R2 = 0.002, P = 0.720), at least over the age range examined in this study. Because several different mouse lines were included in our analysis, a separate analysis that included only mice heterozygous for Myo7a4626SB (n = 28; P20–P233) was performed. Myo7a4626SB heterozygote control animals had results similar to those of the entire control population (Fig. 1) . The a-wave and b-wave amplitudes were stable from P100 (no Myo7a4626SB control mice were examined between P80 and P99; R2 = 0.201, P = 0.124 and R2 = 0.004, P = 0.835, respectively). Similar to the total population of control animals, no differences with age were apparent in a- or b-wave implicit times (R2 = 0.049, P = 0.256 and R2 = 0.015, P = 0.501, respectively). Grossly, the relationship between age and a- and b-wave amplitudes (Fig. 1) is similar to that described by Fulton et al.27

Abnormal ERGs in Myo7a4626SBMutant Mice
The mutation in the Myo7a4626SB allele results in a stop codon within the head domain, and no protein has been detected in Myo7a4626SB mutant mice17 18 25 ; thus, Myo7a4626SB is thought to be a null allele. Myo7a4626SB homozygotes also have some of the most severe hair cell abnormalities among all the alleles (Steel and Self, unpublished observations, 2000). ERGs were recorded from adult (>P100) Myo7a4626SB mutant mice and control littermates (n = 13 pairs). The general shape of the ERGs in the mutant mice were the same as in the control animals (Fig. 2A ). However, the a-wave amplitude of Myo7a4626SB mutant mice was significantly reduced at the higher light intensities compared with control mice (Fig. 2B) . A reduced a-wave amplitude for the mutant mice could first be seen at 2.4 log units of attenuation (becoming statistically significant at 1.8 log units; P < 0.05) and from this point was approximately only 80% of the control values. As might be predicted, because the b-wave is driven by the a-wave, a reduced b-wave amplitude was seen in the Myo7a4626SB mutant mice, although this reduction was not significant until the highest light intensities (Fig. 2C) . There was no observable difference in implicit times of the a- or b-waves in mutant mice (Table 2) .



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  		Figure 2. Myo7a4626SB mutant mice have attenuated a- and b-waves. (A) The columns are the averaged responses from an individual mouse using the first (nonadapting) light exposure protocol. The dimmest flash was attenuated by 6.0 log units (bottom trace) and was increased at 1.2-log-unit steps until the unattenuated flash (top trace). Left: Responses from a heterozygous mouse (+/sh1) with the response closest to average of all the adult Myo7a4626SB control mice; right: responses from a Myo7a4626SB mutant mouse with the response closest to average for all the Myo7a4626SB mutant mice (sh1/sh1). Gray line: Maximum amplitude of the a-wave at the highest two light intensities in the control mouse, clearly showing that the mutant mice had attenuated a-wave responses. Light onset is at the beginning of the traces. Intensity response curves for a-waves (B) and b-waves (C) for adult Myo7a4626SB mutant and control mice. Mutant a-waves were significantly attenuated from 2.4 log units (*P < 0.05), and the b-waves were attenuated from approximately 3.6 log units, but significantly only at the brightest light intensity.

 

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  		Table 2. Analysis of ERGs of Myo7a Alleles

 
No Deterioration with Age of a-Wave Amplitudes of Myo7a4626SBMutant Mice
To determine whether the phenotype (reduced a- and b waves) of the Myo7a4626SB mutant mouse changes with age, a series of ERGs were performed on mice ranging from P20 through P229. Because ERG amplitudes varied with age in control animals (Fig. 1) , a ratio of the mutant response at the maximum light intensity to that of a control littermate was calculated (n = 28 pairs). Throughout the period examined, Myo7a4626SB mutant mice had a similar reduction in a-wave amplitude of approximately 20% (Fig. 3) . Linear regression of the ratio (mutant response over control response) with age showed no significant correlation (R2 = 0.001, P = 0.901). Thus, the ERG phenotype of the mutant mice does not worsen with age.



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  		Figure 3. The amount of a-wave attenuation of the Myo7a4626SB mutant mice did not change during development. ERGs were recorded from 28 pairs of Myo7a4626SB mutant mice (sh1/sh1) and control littermates (+/sh1) ranging in age from P20 to P233. To see whether there is a trend in the amount of attenuation of the mutant mice with age, the pairs of mutant and control mice were split into six age-defined bins. The average a-wave amplitudes at maximum light levels for each bin for the mutant and control mice were plotted along with the ratio between them for each bin. There appeared to be no difference in the ratio throughout the periods analyzed (R2 = 0.001; P = 0.901). Error bars for the ratios are ± SEM.

 
Normal Thresholds in Myo7a4626SBMutant Mice
Threshold intensities for b-waves of Myo7a4626SB were determined using a different light exposure paradigm to that used above. ERGs were recorded from adult Myo7a4626SB mutant and control mice (for these experiments control mice were either +/+ or +/sh1). In this series of ERGs, 10 flashes at 3-second intervals were averaged at each light intensity, and the light intensity was stepped at 0.3 log units instead of the 0.6 log units used for the 1st paradigm (see the Materials and Methods section for details). This protocol provides average traces with lower noise and finer incremental steps than those of the first protocol, allowing a more precise determination of b-wave threshold. When this protocol was used, adaptation occurred, but not until approximately 2.4 log units of attenuation, and from this point both the a- and b-wave amplitudes of Myo7a4626SB mutant mice were significantly less than in the control animals (Fig. 4A ). Using this protocol, at maximum responses the mutant amplitude was only 71% and 76% of the control amplitude for the a- and b-wave, respectively (n = 16 pairs; aged between P85 and P344) and both the a- and b-waves were significantly attenuated (P < 0.05) from 2.4 to 0.0 log units. The mutant and control mice had similar b-wave amplitudes in response to the dimmest light intensities (Fig. 4B) . These findings are similar to results obtained using the first ERG protocol. Furthermore, the mutant and control mice required similar levels of light to reach a 15-µV response: 5.79 ± 0.12 log units for mutant and 5.87 ± 0.08 log units for control animals (P = 0.56). Therefore, although Myo7a4626SB mutant mice had attenuated a- and b-wave responses at the higher intensity flashes, they did not appear to have significantly increased thresholds (reduced sensitivities).



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  		Figure 4. Myo7a4626SB mutant (sh1/sh1) thresholds did not appear to be different from those of the control mice (+/?). The second light exposure protocol was used to determine whether b-wave thresholds are raised for the Myo7a4626SB mutant mice. In this light exposure protocol 10 flashes with a 3-second interstimulus interval were averaged, and the light intensity was increased in 0.3-log-unit steps. The average intensity response curve for both the a- and b-waves are shown in (A). With this protocol the b-wave responses began to be attenuated because of the rapid stimulus repetition at approximately 2.4 log units. The a-wave was significantly different between mutant and control mice from 2.4 to 0 log units and the b-wave from 2.7 to 0 log units. A higher resolution plot of initial stages of mutant and control b-wave response (B) shows that in the mutant mice the b-wave mean threshold was the same as in the control animals.

 
Analysis of Eight sh1 Alleles
In addition to Myo7a4626SB, eight of the remaining nine sh1 alleles were also analyzed by electroretinography. (Myo7a3336SB appears to have a retinal degeneration in its background, unrelated to the Myo7a gene, and therefore was not analyzed.) Myo7a816SB, Myo7a7J, Myo7a8J, Myo7a9J, Myo7a4494SB, Myo7a6J, and Myo7ash1, were all analyzed using the first (nonadapting) ERG protocol. Several sh1 alleles, Myo7a4494SB, Myo7a6J, and Myo7ash1, did not show any difference in ERGs (with the exception of a significantly faster b-wave implicit time in Myo7a4494SB mutant mice; Table 2 ). However, at the brightest light intensity Myo7a816SB, Myo7a7J, Myo7a8J, and Myo7a9J mutant mice, similar to Myo7a4626SB mutant mice, had significantly reduced a-wave amplitudes (Fig. 5A ; Table 2 ). These animals also had significantly reduced b-wave wave amplitudes (with the exception of Myo7a816SB, which was reduced but not significantly), and normal implicit times (with exception of the a-wave implicit time in Myo7a816SB and the b-wave implicit time in Myo7a8J, which were both significantly faster; Table 2 ). Also, the alleles that showed a significant difference in a-wave amplitudes at maximum light intensity all showed decreased a-wave amplitudes from 2.4 log units of attenuation (Fig. 5B) ; the decreased a-wave responses in these mutant mice were significant for all these alleles from 1.8 log units of attenuation up to zero.



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  		Figure 5. Five of the nine Myo7a alleles had attenuated a-wave amplitudes. (A) Normalized a-wave amplitudes for all the alleles examined are shown. The a-wave amplitudes were obtained from either the first (nonadapting, 1st) or second (adapting, 2nd) protocols, and all amplitudes were the averages of the maximum responses obtained. The a-wave amplitudes of the Myo7a4626SB, Myo7a816SB, Myo7a7J, Myo7a8J, and Myo7a9J mutant mice were all significantly less (*P < 0.05) than those of their control mates, whereas those of the Myo7a4494SB, Myo7a6J, Myo7a7sh1, and Myo7a26SB mutant mice were not significantly different from their control littermates. (B) The a-wave intensity response curves for all the Myo7a alleles that showed a significant difference at the maximum light intensity normalized to the maximum response of the control mice for each allele. The control curve is the averaged normalized response of all the control mice. The mutant mice of these alleles have a similar shape throughout their response; the mutant mice for each allele began to be attenuated in amplitude at approximately 2.4 log units.

 
The second ERG protocol (the protocol that causes light adaptation) was used on three alleles, Myo7a4626SB (discussed earlier), Myo7a4494SB, and Myo7a26SB. The second light exposure protocol produced similar results for both Myo7a4626SB and Myo7a4494SB as the first: Myo7a4626SB mutant mice had significantly decreased a- and b-wave amplitudes at maximum response (Fig. 4A ; Table 2 ) and Myo7a4494SB mutant mice had no difference in a- and b-wave maximum amplitudes compared with control animals (note that their maximum responses occurred by 1.2 log units of attenuation, not at the brightest light intensity; see Fig. 4A for the typical intensity response curve for this light-exposure paradigm). Myo7a26SB mutant mice had slightly decreased maximum a- and b-wave amplitudes when the second protocol was used for recording, but these decreases were not significant (P > 0.05; Table 2 ). However, the a- and b-wave implicit times of Myo7a26SB mutant mice were significantly faster throughout much of the intensity response curve with the mutant a-waves being significantly faster (P < 0.05), from 3.6 to 0.0 log units of attenuation, and the b-wave, from 6.0 to 0.0 log units of attenuation (Table 2 and data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This is the first report of any electrophysiological abnormalities in the retinas of sh1 mutant mice. The a-wave amplitudes of mice homozygous for five of the nine mutant Myo7a alleles examined in this study (Myo7a4626SB, Myo7a816SB, Myo7a7J, Myo7a8J, and Myo7a9J) were significantly attenuated compared with those of their control littermates. In general, the attenuated a-waves were all reduced by approximately 20% over most of stimulus intensities examined. Because the a-wave of the ERG is the result of photoreceptors responding to light,28 29 the reduction in a-wave amplitudes suggests that some Myo7a mutant mice have abnormal or subnormal photoreceptor function. All the alleles that had attenuated a-waves also had reduced b-waves, which is not surprising. The b-wave of the ERG originates from retinal interneurons30 31 32 as a result of photoreceptor activity. For at least one allele that had decreased amplitude, Myo7a4626SB, there was no change in the level of attenuation of the response between P20 and P233, suggesting that this phenotype does not worsen with age. Furthermore, Myo7a4626SB mutant mice did not appear to have raised thresholds. It is interesting that the a-wave implicit times for all the mutant alleles examined were normal, with the exception of Myo7a816SB, which was significantly faster, implying that the process of phototransduction occurs at a normal rate in most of the mutant mice.33 Therefore, the primary finding of this study is that some Myo7a mutant retinas have a smaller response to light than do normal mice.

Possible Functions of Myosin VIIa in the Retina
No sign of retinal degeneration has ever been observed in sh1 mutant mice, even up to 744 days of age.17 18 23 34 Thus, mutations in the mouse myosin VIIa gene do not appear to cause retinal degeneration, as they do in humans. In both human and mouse retinas, myosin VIIa is expressed in both the cell types that participate in the visual cycle: RPE and photoreceptor cells. In the RPE, myosin VIIa is in the apical processes of RPE cells.20 21 22 In Myo7ash1 mutant mice the melanosomes in the RPE did not invade the apical process, suggesting that myosin VIIa functions in melanosome transport in the RPE. However, Liu et al.23 argue that this abnormality is not likely to result in retinal degeneration, because other mice with melanosome abnormalities do not normally undergo retinal degeneration. In photoreceptors, myosin VIIa is concentrated in the connecting cilium of photoreceptors21 22 and, at least in humans, in the photoreceptor synapse.21 Myosin VIIa mutant mice have abnormal opsin transport through the connecting cilium revealed by an accumulation of opsin in the cilium and have a rate of new photoreceptor disc synthesis that is decreased by approximately 13%. However, these abnormalities do not affect the total amount of opsin expressed in the sh1 retina or the length of the outer segments.18 These previous studies show that myosin VIIa functions in both the cell types that are involved in the visual cycle and also in both the cell types in which dysfunction can lead to retinitis pigmentosa.19

It is unclear whether either of the previously observed abnormalities could explain the present ERG finding. If the decreased rate of photoreceptor disc synthesis affected the amount of rhodopsin available for phototransduction, we would expect to see an increase in threshold of the ERG,35 36 but we do not. As for the abnormal accumulation of opsin in the connecting cilium, we can see no mechanism to explain how this could result in a decreased ERG amplitude. Of note, the mouse mutant pearl, similar to some of the sh1 alleles, has a lack of melanosomes in the apical microvilli of the RPE37 and has reduced a- and b-wave amplitudes.38 Pearl mice have abnormalities in the retina that are not seen in sh1 mutant mice, most notably a reduced total number of melanosomes in the RPE. The gene disrupted in the pearl mouse is the ß3A subunit of the AP-3 adapter complex, which functions in cargo-selected transport.39 Because unconventional myosins are know to be involved in intracellular transport40 and, in both pearl and sh1 mutant mice, there are clearly abnormalities in melanosome transport in the RPE, it is tempting to speculate that both myosin VIIa and the AP-3 complex have similar functions in the RPE and that disruptions of these functions lead to reduced ERG amplitudes.

Differences between Human and Mouse
There are several possible reasons that mutations in MYO7A in humans can lead to retinal degeneration, whereas in sh1 mutant mice they do not. (1) The mutations found in human MYO7A resulting in USH may be different from those found in mouse Myo7a. (2) The genetic background of the particular organism may play a significant role in determining the severity of disease that results from a given mutation in MYO7A. (3) Inherent differences between the retinas of the two species could be the reason for different levels of disease. We examine each of these possibilities in turn.

To date, 10 mutant alleles of Myo7a have been found (the mutations in 7 of them have been described14 25 ; see Table 1 ), and more than 57 mutations have been found in human MYO7A.4 5 7 8 9 10 11 12 13 Unfortunately, even with this vast array of mutations in both mice and humans, there is no case in which a patient with USH1B has had two copies of a mutation identical with that of one of the sh1 mouse lines. Until we show identical mutations in the human population and in a mouse line, we cannot be certain that the absence of retinal degeneration in the shaker1 mouse lines is not simply because we do not have the appropriate mutations in Myo7a.

In humans, mutations in MYO7A are associated with a wide phenotypic spectrum of diseases.5 These mutations can lead to both dominant1 and recessive2 3 nonsyndromic deafness and to two clinically distinct forms of USH, one mild and one severe in effect. Based on there being such a wide range of mutations in MYO7A that can result in a fairly broad phenotypic spectrum of diseases, the fact that an identical mutation has been found in both atypical USH and USH1B5 and that the mutations causing both USH and nonsyndromic deafness are spread throughout the molecule, it has been proposed that genetic background may play an important role in determining the nature of the disease caused by MYO7A mutations.5 Because genetic background may influence the phenotype caused by mutations in MYO7A in humans and it is known to affect the inner ear phenotype in sh1 mutant mice41 as well as the rate of retinal degeneration in at least one mouse model of a human disease,42 it is possible that the genetic background is the sole reason for the difference between humans and mice. In this study and in others17 18 many different alleles of Myo7a mutant mice have been examined, but the backgrounds of the alleles are fairly similar (see Table 1 ).

It is notable that the data presented suggest the possibility that genetic background may affect the manifestation of the ERG phenotype. Both Myo7a4626SB and Myo7a4494SB are thought to be null mutant mice,17 18 whereas only Myo7a4626SB has attenuated ERG amplitudes. Both presumptive null alleles are on a largely 50% CBA/Ca, 50% BS genetic background. However, the stocks have been maintained by intercrossing within each colony for several generations since the original BS-based stocks were outcrossed to CBA/Ca, and different modifiers from the two backgrounds have therefore most likely become fixed in the stocks. If these two alleles are actually null, then the difference in the ERG amplitudes would have to be the result of the lines’ having different modifiers for myosin VIIa. Recently, the first modifiers that effect retinal degeneration have been mapped.43 44 It will interesting to place the different mutant myosin VIIa alleles onto a background containing these modifiers and determine whether they exacerbate the phenotype observed in Myo7a mutant mice.

The difference between the human and mouse phenotypes may simply be that there are intrinsic differences between the two species. One of the more obvious differences between humans and mice is that the first signs of retinal degeneration in patients with USH1B generally occur after they are several years of age, well beyond the life span of a mouse. Kedzierski et al.45 noted that at least in some retinal degenerations, the rate of degeneration appears to be determined, not by the absolute age of the photoreceptor, but by its relative age compared with the life span of the host species. This observation suggests that the relatively short life span of the mouse may not be the reason there is no degeneration. However, there are several mutant mouse strains in which retinal degeneration is dependent on exposure to light or in which the rate of degeneration can be increased by exposure to light.46 47 48 49 50 51 It is possible that the retinas of Myo7a mutant mice do not degenerate because they have not been exposed to the same total light levels as the typical human with USH1B, and/or their relatively short life span is not long enough for the necessary accumulation of insult to result in degeneration.

These data are the first evidence of a physiological abnormality in the retinas of Myo7a mutant mice. Mice homozygous for several different mutant Myo7a alleles have decreased a- and b-wave amplitudes, suggesting that their photoreceptors are not responding properly to light. The implicit time of the mutant mouse’s a- and b-wave were generally the same as in the control mouse. In humans with USH1 (there is no molecular characterization of these persons, and the underlying causes therefore may not be mutations in MYO7A), multifocal ERGs have shown that they have amplitude losses with normal implicit times.52 Patients with USH2 and other forms of retinitis pigmentosa have decreased amplitudes and increased implicit times.52 The electroretinographic findings reported in this study appear to correlate with physiological findings in humans, at least during the early stages of disease. Thus, the sh1 mouse may provide a useful model for studying at least the early stages of the retinitis pigmentosa associated with MYO7A mutations in humans.


   Acknowledgements
 
The authors thank Bill Brunken, David Williams, Grant Balkema, and Amy Kiernan for helpful comments and advice and Eugene Rinchik, Wayne Frankel, and Ken Johnson for passing on the sh1 mutants to us.


   Footnotes
 
Supported by the Medical Research Council, Defeating Deafness and the European Commission (BMH4-CT96-1324).

Submitted for publication August 4, 2000; revised November 20, 2000; accepted November 30, 2000.

Commercial relationships policy: N.

Corresponding author: Karen P. Steel, MRC Institute of Hearing Research, University Park, Nottingham NG7 2RD, UK. karen{at}ihr.mrc.ac.uk


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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