The Mass1frings mutation underlies early onset hearing impairment in BUB/BnJ mice, a model for the auditory pathology of Usher syndrome IIC

Abstract

The human ortholog of the gene responsible for audiogenic seizure susceptibility in Frings and BUB/BnJ mice (mouse gene symbol Mass1) recently was shown to underlie Usher syndrome type IIC (USH2C). Here we report that the Mass1frings mutation is responsible for the early onset hearing impairment of BUB/BnJ mice. We found highly significant linkage of Mass1 with ABR threshold variation among mice from two backcrosses involving BUB/BnJ mice with mice of strains CAST/EiJ and MOLD/RkJ. We also show an additive effect of the Cdh23 locus in modulating the progression of hearing loss in backcross mice. Together, these two loci account for more than 70% of the total ABR threshold variation among the backcross mice at all ages. The modifying effect of the strain-specific Cdh23ahl variant may account for the hearing and audiogenic seizure differences observed between Frings and BUB/BnJ mice, which share the Mass1frings mutation. During postnatal cochlear development in BUB/BnJ mice, stereocilia bundles develop abnormally and remain immature and splayed into adulthood, corresponding with the early onset hearing impairment associated with Mass1frings. Progressive base-apex hair cell degeneration occurs at older ages, corresponding with the age-related hearing loss associated with Cdh23ahl. The molecular basis and pathophysiology of hearing loss suggest BUB/BnJ and Frings mice as models to study cellular and molecular mechanisms underlying USH2C auditory pathology.

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 at 15 weeks of age. CAST/EiJ (N =9) and MOLD/RkJ (N = 9) mice were tested at 24 – 60 weeks of age.

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 impaired values as previously defined [1]. (CAST/EiJ × BUB/BnJ) × BUB/BnJ backcross mice were tested at 3 and 6 months of age. (MOLD/RkJ × BUB/BnJ) × BUB/BnJ backcross mice were tested at 5, 8, and 12 months of age.

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 Mass1^frings/Mass1^frings mice and for +/Mass1^frings mice. Lod scores of linkage associations and percentages of total threshold variation attributable to the Mass1 locus are given for each auditory stimulus (C, click; 8, 8 kHz; 16, 16 kHz; 32, 32 kHz) and for each test age.

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 used to mark Mass1 and D10Mit138 to mark Cdh23.Mice were tested at 5, 8, and 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 Mass1^frings/Mass1^frings cdh23^ahl/cdh23^ahl mice are designated by squares, Mass1^frings/Mass1^frings +/cdh23^ahl miceby circles, +/Mass1^frings cdh23^ahl/cdh23^ahl mice by triangles, and +/Mass1^frings +/cdh23^ahl mice by horizontal lines. D13Mit9 was used to mark Mass1 and D10Mit138 to mark Cdh23. Mice were tested at 5, 8, and 12 months of age.

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 – 14 kHz [17]. (A) Representative surface view of organ of Corti showing three rows of outer hair cells; magnification bar corresponds to 4 μm. (B, C) High magnification (original magnification 20,000 ; 3 keV) of individual stereocilia bundles; magnification bar, 1 Am. (D) Stereocilia bundles from two inner hair cells are shown; magnification bar, 1 μm. (E) Shown is an organ of Corti surface view characteristic of a 41-day-old BUB/BnJ mouse showing three rows of outer hair cells; arrow points to a missing hair cell; magnification bar, 4 μm. (F) A surface view of the organ of Corti of a normal hearing C3HeBe/FeJ mouse at P10; rows of outer (OH) and inner (IH) hair cells are indicated; magnification bar, 5 μm. (G) The structure of a normal stereocilia hair bundle from a C3HeB/FeJ mouse.

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. Arrows indicate the organ of Corti (OC) and spiral ganglion cells (SGC).