Digenic inheritance of deafness caused by 8J allele of myosin-VIIA and mutations in other Usher I genes

Abstract

Inherited hearing loss in mice has contributed substantially to our understanding of inner-ear function. We identified a new allele at the Myo7a locus, Myo7a(sh1-8J); genomic characterization indicated that Myo7a(sh1-8J) arose from complex deletion encompassing exons 38-40 and 42-46. Homozygous mutant mice had no detectable auditory brainstem response, displayed highly disorganized hair-cell stereocilia and had no detectable MYO7A protein. We generated mice that were digenic heterozygotes for Myo7a(sh1-8J) and one of each Cdh23(v-2J), Ush1g(js) or Pcdh15(av-3J) alleles, or an Ush1c null allele. Significant levels of age-related hearing loss were detected in +/Myo7a(sh1-8J) +/Ush1g(js), +/Myo7a(sh1-8J) +/Cdh23(v-2J) and +/Myo7a(sh1-8J) +/Pcdh15(av-3J) double heterozygous mice compared with age-matched single heterozygous animals, suggesting epistasis between Myo7a and each of the three loci. +/Pcdh15(av-3J) +/Ush1g(js) double heterozygous mice also showed elevated hearing loss, suggesting Pcdh15-Ush1g epistasis. While we readily detected MYO7A, USH1C, CDH23 and PCDH15 using mass spectrometry of purified chick utricle hair bundles, we did not detect USH1G. Consistent with that observation, Ush1g microarray signals were much lower in chick cochlea than those of Myo7a, Ush1c, Cdh23 and Pcdh15 and were not detected in the chick utricle. These experiments confirm the importance of MYO7A for the development and maintenance of bundle function and support the suggestion that MYO7A, USH1G (Sans) and CDH23 form the upper tip-link complex in adult mice, likely in combination with USH1C (harmonin). MYO7A, USH1G and PCDH15 may form another complex in stereocilia. USH1G may be a limiting factor in both complexes.

Figure 1.

Genomic structure of the 8J mutation. (A) MYO7A includes a motor head domain, five IQ domains (IQ region indicated) and a putative coiled-coil domain (CC); these are followed by two repeats of MyTH4 and FERM domains. The epitope recognized by the antibody used here (25–6790) is indicated in gray. (B) Structure of 3′ end of the WT Myo7a allele. Dashed lines between (A) and (B) indicate the relationship between Myo7a exons and protein structure. DNA missing in the 8J allele is highlighted with thick black bars. H and E indicate HindIII and EcoRI restriction sites; expected sizes resulting from HindIII or EcoRI restriction digests are indicated. (C) 8J allele. Dashed lines between (B) and (C) indicate deleted genomic DNA. (D) Genotyping for the 8J allele. DNA from five mice with indicated allele composition was separately subjected to PCR with 8J-specific and WT-specific primers. (E) Southern analysis; predicted band sizes (in kb) are indicated on right. The 3′ probe indicated in (A) and (B) was used.

Figure 2.

ABR for Usher I heterozygotes. ABR thresholds are plotted for 1–2, 3–4, 5–7 and 8–10 months for WT (n = 4–15) and the following heterozygous mice: (A) +/Myo7a^sh1–8J (n = 9–34), (B) +/Ush1c^KO (n = 2–17), (C) +/Cdh23^v-2J (n = 3–8), (D) +/Pcdh15^av-3J (n = 5–20) and (E) +/Ush1g^js (n = 3–22). Significance for pairwise comparisons using two-tailed t-tests between WT and heterozygotes at individual ages and stimuli are indicated (*P < 0.05; **P < 0.01; ***P < 0.001). Mean ± SEM are plotted.

Figure 3.

Disrupted hair bundles in Myo7a^sh1–8J homozygotes and adult MYO7A localization. (A–D) Actin detected with phalloidin. (A and B) Cochlear hair bundles from P2 and P6 +/8J heterozygote mice have normal morphology. (C and D) Cochlear hair bundles from P2 and P6 8J/8J mice are profoundly disrupted. (E) Isolated adult bullfrog hair cell stained for MYO7A. Note labeling at ankle region (asterisk) and at tips (arrow), site of mechanotransduction.

Figure 4.

No MYO7A protein in Myo7a^sh1–8J homozygotes. Equal protein concentrations of extracts from Myo7a^sh1–8J heterozygotes (±) or homozygotes (−/−) were separated by SDS–PAGE; MYO7A was detected by immunoblotting with the 25-6790 antibody. No MYO7A protein was detected in 8J/8J homozygous mutants.

Figure 5.

Digenic inheritance of age-related deafness from Myo7a and other Usher genes. ABR for Usher I heterozygotes. ABR thresholds are plotted for 1–2, 3–4, 5–7 and 8–10 months for WT and the following heterozygous mice: (A) +/Ush1c^KO +/Myo7a^sh1–8J (n = 3–7), (B) +/Cdh23^v-2J +/Myo7a^sh1–8J (n = 4), (C) +/Pcdh15^av-3J +/Myo7a^sh1–8J (n = 6–18), (D) +/Ush1g^js +/Myo7a^sh1–8J (n = 7–22) and (E) +/Ush1g^js +/Pcdh15^av-3J (n = 3–7). Significance was assessed for four genotypes at each time point and stimulus using ANOVA (*P < 0.05; **P < 0.01; ***P < 0.001). Significance is only indicated for double heterozygous mice; values for significance for other mice are listed in Supplementary Material Tables S4–S8. WT and heterozygous data from Figure 1 are replotted here, as the model tested incorporates all four genotypes at each stimulus and age. Mean ± SEM are plotted.

Figure 6.

Expression of Usher I genes in the chick utricle and cochlea. (A) Mass spectrometry sequence coverage of Usher I proteins MYO7A, USH1C, CDH23 and PCDH15 in purified hair bundles from the chick utricle. Each mark corresponds to a detected peptide, with its width equal to the corresponding sequence length and height indicating the number of different times that peptide was counted. The shade of the mark indicates the average log(e) score of the detected peptides, indicating confidence in the identification. Data are summed from 13 independent experiments. (B) Quantitation of Usher proteins. Data from 13 experiments were plotted with box plots; outliers are indicated by open circles. The median is indicated as a horizontal line within the box, while the box bottom and top correspond to the 25th and 75th percentiles; whiskers correspond to the data range exclusive of outliers. (C) Affymetrix microarray analysis of Usher I transcripts. Results from four utricle and four cochlea hybridizations are presented. Two probesets for Myo7a were present on the chicken genome chip; all other genes were represented by single probesets. Ush1g was not detected in the chick utricle, consistent with the inability to detect it by protein mass spectrometry.