By 3 weeks of age, BUB/BnJ mice exhibit ABR thresholds about 40 dB higher than the normal thresholds exhibited by SWR/Bm mice tested at 15 weeks of age, and this hearing loss progresses to deafness by 20 weeks of age (). The SWR/Bm strain is genetically very similar to BUB/BnJ [13
] but does not have the Mass1frings
] and thus can serve as a control strain for the effects of this mutation. Also shown in are the combined ABR results for nine mice of the CAST/EiJ strain and nine mice of the MOLD/RkJ strain that were tested at 24–60 weeks of age. Because these wild-derived mice retain normal hearing well beyond 1 year of age [1
], they were used to make F1 hybrids with hearing-impaired BUB/BnJ mice for the linkage backcross analysis.
Fig. 1 Average ABR thresholds (dB SPL, with standard error bars) of BUB/BnJ, SWR/Bm, CAST/EiJ, and MOLD/RkJ inbred strain mice. BUB/BnJ mice were tested at ages 3 weeks (N = 6), 8 – 13 weeks (N = 17), and 20 weeks (N = 5). SWR/Bm mice (N = 6) were tested (more ...)
The (CAST/EiJ × BUB/BnJ) × BUB/BnJ backcross mice (N = 76) were tested twice for ABR thresholds, at 3 and 6 months of age. Mice of the (MOLD/RkJ × BUB/BnJ) × BUB/BnJ backcross were tested three times, at 5 (N = 44), 8 (N = 64), and 12 (N = 89) months of age. Frequency distributions of backcross mice with different ABR thresholds are shown in . The distributions are strongly bimodal at the youngest ages tested, suggesting the major influence of a single gene locus. The overall progression of hearing loss can be seen as increasing percentages of mice with higher ABR thresholds at older ages. For example, only 9% of (MOLD/RkJ × BUB/BnJ) × BUB/BnJ backcross mice have click thresholds greater than 100 dB sound pressure level (SPL) at 5 months of age compared with 23% at 12 months.
Fig. 2 Frequency distributions of backcross mice ABR thresholds. The percentages of N2 backcross mice exhibiting click thresholds within each 10-dB SPL increment are shown as histograms. The vertical dotted line demarcates the border between normal and hearing (more ...)
Quantitative trait loci (QTL) linkage analysis of ABR thresholds of backcross mice revealed highly significant associations with D13Mit9, a marker located in IVS89 within the Mass1gene (). To test formally if the maximum linkage association occurs at this locus, we typed six additional Chr 13 markers: D13Mit19, D13Mit157, D13Mit255, D13Mit159, D13Mit228,and D13Mit77. As expected, the peak association was with D13Mit9 and strongly supports the Mass1frings
mutation as the likely molecular basis for the early onset hearing loss of BUB/BnJ mice. D13Mit9
variation is most closely linked with click thresholds of backcross mice that are younger than 6 months of age (). In contrast, D10Mit138
, a marker near the Cdh23
locus, is most highly associated with high-frequency pure tones in mice older than 10 months of age [3
].At 5 months of age, the Mass1
locus (marked by D13Mit9
) can account for about 80% of the total ABR threshold variation, whereas the Cdh23
locus (marked by D10Mit138
) accounts for less than 15% (). At 12 months of age, each of the two loci contributes about 40% of the total variation. Together, the two loci can account for 75–85% of the total ABR threshold variation among the backcross mice at all ages tested, and they likely account for more than 90% of the genetic variation, considering that variation in ABR measurements and other environmental variations are also included in the total variance estimate.
Fig. 3 Linkage of the Mass1 locus with ABR thresholds. For each of the two backcrosses, average ABR thresholds in dB SPL (and their standard errors) are shown for Mass1frings/Mass1frings mice and for +/Mass1frings mice. Lod scores of linkage associations and (more ...)
Fig. 4 Percentage of total ABR threshold variation among (MOLD/RkJ ×BUB/BnJ) × BUB/BnJ backcross mice that can be explained by a QTL at the Mass1 locus (circles), a QTL at the Cdh23 locus (triangles), or both QTLs combined(squares). D13Mit9 was (more ...)
Hearing loss susceptibility alleles of both loci are recessive and originate from the BUB/BnJ strain, and resistance to hearing loss is conferred by dominant alleles (+) inherited from the wild-derived strains CAST/EiJ and MOLD/RkJ. The effects of the two loci on ABR thresholds of backcross mice are primarily additive, as shown in . No statistically significant interactive effects were detected between D13Mit9
with the Map Manager QTX computer program [14
] for any of the auditory stimuli or ages that were tested. For example, analysis of the click threshold variation for 12-month-old backcross mice gave a lod score of only 1.5 for interaction, whereas the main effect for D13Mit9
is 12.6 and the main effect for D10Mit138
is 6.9. Compound homozygotes for susceptibility alleles (Mass1frings/Mass1frings Cdh23ahl/Cdh23ahl
) exhibit the highest ABR thresholds and are already nearly deaf at the youngest ages tested (thresholds >60 dB above normal by 5 months of age). Compound heterozygotes (+/Mass1frings +/Cdh23ahl
) retain normal hearing levels at the oldest ages tested(12 months), and singly homozygous mice (Mass1frings
) have intermediate ABR thresholds. The hearing loss of +/Mass1frings Cdh23ahl/Cdh23ahl
mice is progressive and does not reach the level of Mass1frings/Mass1frings +/Cdh23ahl
mice (about 40 dB above normal)until 12 months of age.
Fig. 5 Average ABR thresholds (dB SPL, with standard error bars) of (MOLD/RkJ × BUB/BnJ) × BUB/BnJ backcross mice sorted by their two-locus Mass1 and Cdh23 genotypes. ABR thresholds of Mass1frings/Mass1frings cdh23ahl/cdh23ahl mice are designated (more ...)
Retinitis pigmentosa, as well as deafness or hearing impairment, is a characteristic of the Usher syndromes. Mouse tissue sources for ESTs indicate that the Mass1 gene is expressed in the retina and expression has been detected in eyes of mouse embryos [9
]. However, the effect of the Mass1frings
mutation on retinal function could not be evaluated with certainty because BUB/BnJ is homozygous for the retinal degeneration mutation on Chr 5 (Pde6brd1
).Because all of the Usher syndrome type IIc patients with MASS1
mutations that have been identified so far are female [12
], we also examined the effect of sex on hearing loss in the BUB/BnJ backcross mice. We sorted backcross mice by sex and Mass1 (D13Mit9
) genotypes but found no statistically significant ABR threshold differences between male and female mice at any age in either backcross.
BUB/BnJ mice show audiogenic seizures only when tested at young ages (<25 days of age), whereas Frings mice retain seizure sensitivity well into adulthood [6
]. Because both BUB/BnJ and Frings mice share the Mass1frings
], we reasoned that a modifier locus, affecting progressive hearing loss, might explain this difference. To test this hypothesis, Frings mice were crossed with BUB/BnJ mice to generate F1 hybrids, and these were then intercrossed to generate F2 animals. All (50/50) F1 hybrids retained audiogenic seizure susceptibility well after 25 days of age. All F2 intercross mice seized at 19 days of age (n = 161); however, 43/161 (26.7%) lost seizure susceptibility after 25 days of age. When tested for ABR thresholds at 42 days of age, seizing (n = 118) and nonseizing (n = 43) F2 mice had mean 16-kHz thresholds of 50.0 and 66.0 dB SPL, respectively ( p < 0.0001). The 3:1 segregation ratio of seizing to nonseizing F2 mice and the elevated ABR thresholds of nonseizing mice are consistent with a single recessive gene that contributes to progressive hearing loss in homozygous mice and that consequently eliminates the audiogenic seizure susceptibility of these mice at older ages.
The Chr 10 Ahl gene (Cdh23ahl
) previously was shown to influence hearing loss progression in BUB/BnJ backcross mice [3
]. To see if Cdh23ahl
could be responsible for the differences in audiogenic seizure susceptibility and hearing thresholds between BUB/BnJ and Frings mice, we sequenced the region of Cdh23
containing the exon 7 splicing variant thought to underlie the Ahl phenotype.Sequence analysis showed that Frings and the closely related SWR/BmJ strains have the 753G variant of Cdh23
, which confers resistance to hearing loss, whereas the BUB/BnJ strain has the 753A variant, which increases hearing loss susceptibility. Taken together, seizure segregation and sequence data are consistent with a causative role for Cdh23
in the audiogenic seizure differences observed between Frings and BUB/BnJ mice.
To ascertain the cellular pathology associated with the hearing loss of BUB/BnJ mice we examined surface preparations of organs of Corti by scanning electron microscopy. At postnatal day 14 (P14) the stereocilia bundle of outer hair cells along the length of the neuroepithelium is largely immature and underdeveloped (). Lateral and apical aspects of the bundle appear rounded and deformed, and the bundle fails to form its characteristic V-shaped structure. In addition, stereocilia are disconnected and detached, are sometimes found outside their unit, and in some instances are bent (). The most severely affected bundles have entirely lost their polarity and graded height (), although the pointing direction of the majority of bundles is toward the nonneural side of the organ of Corti. Hair bundles of inner hair cells show a similar phenotype: stereocilia are splayed, disorganized, and of irregular height (). Interestingly, with increasing age the morphology of the bundle changes little: at 7 weeks of age (P41) stereocilia maintain their overall height, location, and immature appearance although sporadically there are gaps in the cellular array (). At 15 weeks of age, degeneration of hair cells and spiral ganglion cells is pronounced in the basal turn, as seen in cochlear cross sections of BUB/BnJ mice but not SWR/Bm mice ().
Fig. 6 Stereocilia pathology associated with early onset hearing loss of BUB/BnJ mice. (A – C) Taken from P14 BUB/BnJ mice from an area located 1 – 3 mm distal to the apex, which corresponds to a frequency spectrum of approximately 2 – (more ...)
Fig. 7 Cochlear pathology associated with age-related hearing loss of BUB/BnJ mice. Both cross sections are from the basal turn of cochleae from 15-week-old mice. The genetically related SWR/Bm strain is shown for comparison to illustrate normal cochlear structure. (more ...)