The Genetic Architecture of Hearing Impairment in Mice: Evidence for Frequency-Specific Genetic Determinants

Abstract

Genome-wide association studies (GWAS) have been successfully applied in humans for the study of many complex phenotypes. However, identification of the genetic determinants of hearing in adults has been hampered, in part, by the relative inability to control for environmental factors that might affect hearing throughout the lifetime, as well as a large degree of phenotypic heterogeneity. These and other factors have limited the number of large-scale studies performed in humans that have identified candidate genes that contribute to the etiology of this complex trait. To address these limitations, we performed a GWAS analysis using a set of inbred mouse strains from the Hybrid Mouse Diversity Panel. Among 99 strains characterized, we observed approximately two-fold to five-fold variation in hearing at six different frequencies, which are differentiated biologically from each other by the location in the cochlea where each frequency is registered. Among all frequencies tested, we identified a total of nine significant loci, several of which contained promising candidate genes for follow-up study. Taken together, our results indicate the existence of both genes that affect global cochlear function, as well as anatomical- and frequency-specific genes, and further demonstrate the complex nature of mammalian hearing variation.

Keywords: ABR; Hybrid Mouse Diversity Panel (HMDP); cochlear function; genetics; genome-wide association study (GWAS); genomics; hearing.

Figure 1

Variation in ABR in the HMDP. The auditory brainstem response (ABR) to tone burst stimuli at six different frequencies exhibits two-fold to five-fold variation among 99 HMDP strains. Each dot represents an individual mouse from the respective strain and the mean values are indicated by the horizontal black bars. Female 5-wk-old mice were exposed to auditory signals at frequencies of 4 kHz, 8 kHz, 12 kHz, 16 kHz, 24 kHz, and 32 kHz. The ability of individual mice to hear these signals was assessed using an ABR test. ABR is represented by the decibel level at which hearing threshold was reached, determined visually by an ABR waveform.

Figure 1

Variation in ABR in the HMDP. The auditory brainstem response (ABR) to tone burst stimuli at six different frequencies exhibits two-fold to five-fold variation among 99 HMDP strains. Each dot represents an individual mouse from the respective strain and the mean values are indicated by the horizontal black bars. Female 5-wk-old mice were exposed to auditory signals at frequencies of 4 kHz, 8 kHz, 12 kHz, 16 kHz, 24 kHz, and 32 kHz. The ability of individual mice to hear these signals was assessed using an ABR test. ABR is represented by the decibel level at which hearing threshold was reached, determined visually by an ABR waveform.

Figure 2

Manhattan plots of GWAS results for ABR at each tone burst frequency. (A) ABR at 4 kHz was significantly associated with a locus on chromosome 9 and a region on chromosome 19. (B) A GWAS for the 8-kHz tone burst revealed two significantly associated loci on chromosomes 10 and 19. (C) Three significantly associated loci on chromosomes 3, 13, and 19 were identified in the GWAS analysis for the 12-kHz tone burst. (D) One significant locus on chromosome 10 was associated with ABR at the 16-kHz tone burst. (E) The GWAS for ABR at 24 kHz identified two different significant loci on chromosome 13. (F) Two significant loci on chromosomes 4 and 13 were associated with ABR at 32 kHz. For each significantly associated locus, the gene(s) nearest to the peak SNP is indicated. The GWAS analyses for each of the six tone burst stimuli included 182,270 SNPs, whose genomic positions are shown along the x-axis with their corresponding -log10 P values indicated by the y-axis. The genome-wide thresholds for significant (P=4.1×10⁻⁶) and suggestive (P=4.1×10⁻⁴) evidence of association are indicated by the horizontal red and blue lines, respectively.

Figure 2

Manhattan plots of GWAS results for ABR at each tone burst frequency. (A) ABR at 4 kHz was significantly associated with a locus on chromosome 9 and a region on chromosome 19. (B) A GWAS for the 8-kHz tone burst revealed two significantly associated loci on chromosomes 10 and 19. (C) Three significantly associated loci on chromosomes 3, 13, and 19 were identified in the GWAS analysis for the 12-kHz tone burst. (D) One significant locus on chromosome 10 was associated with ABR at the 16-kHz tone burst. (E) The GWAS for ABR at 24 kHz identified two different significant loci on chromosome 13. (F) Two significant loci on chromosomes 4 and 13 were associated with ABR at 32 kHz. For each significantly associated locus, the gene(s) nearest to the peak SNP is indicated. The GWAS analyses for each of the six tone burst stimuli included 182,270 SNPs, whose genomic positions are shown along the x-axis with their corresponding -log10 P values indicated by the y-axis. The genome-wide thresholds for significant (P=4.1×10⁻⁶) and suggestive (P=4.1×10⁻⁴) evidence of association are indicated by the horizontal red and blue lines, respectively.

Figure 3

In situ images exhibiting cochlear mRNA expression of GWAS candidate genes. (A) Antisense RNA probe for Prpf19 in P1 cochlea demonstrates expression in the cochlear epithelium, represented by the black circle, and spiral ganglion, denoted by the black arrow. Stria vascularis indicated by the yellow star. (B) Antisense RNA probe for Fbp1 in P1 cochlea demonstrates expression in the basal layer of the stria vascularis, represented by the black oval.

Figure 4

ABR at 16 kHz varies by genotype at rs29362366. (A–C) The effect of Otogl peak SNP rs29362366 on 16 kHz ABR. (A) Genotype effect when all strains are included in analysis. (B) Genotype effect when DBA/2J and BXD RI strains derived from C57BL/6J and DBA/2J matings are excluded from analysis. (C) Genotype effect when only C57BL/6J, DBA/2J, and BXD RI strains are included in analysis. Haplotype containing allele C is present in C57BL/6J and haplotype containing allele T is present in DBA/2J.