Genome-Wide Association Study Identifies <i>Nox3</i> as a Critical Gene for Susceptibility to Noise-Induced Hearing Loss

<div><p>In the United States, roughly 10% of the population is exposed daily to hazardous levels of noise in the workplace. Twin studies estimate heritability for noise-induced hearing loss (NIHL) of approximately 36%, and strain specific variation in sensitivity has been demonstrated in mice. Based upon the difficulties inherent to the study of NIHL in humans, we have turned to the study of this complex trait in mice. We exposed 5 week-old mice from the Hybrid Mouse Diversity Panel (HMDP) to a 10 kHz octave band noise at 108 dB for 2 hours and assessed the permanent threshold shift 2 weeks post exposure using frequency specific stimuli. These data were then used in a genome-wide association study (GWAS) using the Efficient Mixed Model Analysis (EMMA) to control for population structure. In this manuscript we describe our GWAS, with an emphasis on a significant peak for susceptibility to NIHL on chromosome 17 within a haplotype block containing NADPH oxidase-3 (<i>Nox3</i>). Our peak was detected after an 8 kHz tone burst stimulus. <i>Nox3</i> mutants and heterozygotes were then tested to validate our GWAS. The mutants and heterozygotes demonstrated a greater susceptibility to NIHL specifically at 8 kHz both on measures of distortion product otoacoustic emissions (DPOAE) and on auditory brainstem response (ABR). We demonstrate that this sensitivity resides within the synaptic ribbons of the cochlea in the mutant animals specifically at 8 kHz. Our work is the first GWAS for NIHL in mice and elucidates the power of our approach to identify tonotopic genetic susceptibility to NIHL.</p></div

Genotypic effects of the peak SNP (rs33652818) at the <i>Nox3</i> locus.

<p>Comparison between alleles GG and AA across the various frequencies. There is a statistically significant difference between the alleles at 4 and 8 kHz. * = p value < 0.001. Error bars +/- 1 SE.</p

8 kHz synaptic cochleogram.

<p>Synaptic ribbon count measured at the 8 kHz tonotopic position within the cochlea. Despite the absence of a statistical difference in OHC counts, the wild-type mice demonstrated significantly greater post-noise exposure synaptic ribbon density per IHC (9A). Projections (60x, 3x zoom, oil immersed) of confocal stacks (9B) of immunostained pre and post-noise exposure mouse IHC synaptic ribbons (green, mouse anti-CtBP2).-/- = <i>Nox3</i>^<i>het</i>/<i>Nox3</i>^<i>het</i>:-/+ = <i>Nox3</i>^<i>het</i>/+: +/+ = wild-type.</p

Regional plot of the 8 kHz ABR post noise-exposure at Chr 17 association in the HMDP centered on the lead SNP at the Nox3 locus (rs33652818).

<p>The blue diamond represents the most significant SNP (p = 9.63E-06) and SNPs are colored based on their LD with the most significant SNP being: red SNPs in LD at r²>0.8, orange SNPs in LD at r²>0.6 and green SNPs in LD at r²>0.4. The positions of all RefSeq genes are plotted using genome locations (NCBI’s Build37 genome assembly).</p

Detailed analysis of the 8 kHz frequency stimulus.

<p>Dissection of the 8 kHz frequency by DPOAE I/O function (7A) and ABR wave 1 amplitudes (7B) consistently indicate more impairment in the <i>Nox3</i>^<i>het</i>/<i>Nox3</i>^<i>het</i> and <i>Nox3</i>^<i>het</i>/+ than wild-type. One-way ANOVA (Tukey multiple comparisons). * = p < 0.05. Homozygous = <i>Nox3</i>^<i>het</i>/<i>Nox3</i>^<i>het</i>: Heterozygous = <i>Nox3</i>^<i>het</i>/+: Wild-type = C57BL/6JEiJ strain.</p

Topographical analysis of the auditory pathway at different frequencies (post-noise exposure).

<p>Although no significant difference was seen for the DPOAE thresholds (6A), the wild-type controls show a higher wave 1 amplitude (p = 0.010) only at 8 kHz compared to <i>Nox3</i>^<i>het</i>/+ and <i>Nox3</i>^<i>het</i>/<i>Nox3</i>^<i>het</i> (6B). One-way ANOVA (Tukey test for multiple comparisons). * = p < 0.05. Homozygous = <i>Nox3</i>^<i>het</i>/<i>Nox3</i>^<i>het</i>: Heterozygous = <i>Nox3</i>^<i>het</i>/+: Wild-type = C57BL/6JEiJ strain.</p

Cytocochleogram of wild-type and mutant <i>Nox3</i> mice (8A).

<p>No significant difference in OHC counts were detected among wild-type, <i>Nox3</i>^<i>het</i>/+ and <i>Nox3</i>^<i>het</i>/<i>Nox3</i>^<i>het</i>. OHC preparations of immunostained (40x) post-noise exposure cochleae (8B) at the 8 kHz tonotopic location (red, rabbit anti-myosin6) demonstrate a small impact with the loss of apical OHC (2 weeks post-noise exposure). Homozygous = <i>Nox3</i>^<i>het</i>/<i>Nox3</i>^<i>het</i>: Heterozygous = <i>Nox3</i>^<i>het</i>/+: Wild-type = C57BL/6JEiJ strain.</p

Genes within NIHL 5 association peaks regulated by local eQTL in the cochlea.

<p>^atxStart, location of transcription (NCBI Build37 genome assembly) start.</p><p>^btxEnd, location of transcription (NCBI Build37 genome assembly) end.</p><p>^cStatistically significant p value ≤ 5.1E-04 (Bonferroni corrected for the number of probes tested)</p><p>Genes within NIHL 5 association peaks regulated by local eQTL in the cochlea.</p

Characterization of post-exposure thresholds in the HMDP.

<p>Mean ± SEM for 8 kHz post-noise exposure hearing thresholds in 64 HMDP inbred mouse strains. The difference between the strains with the lowest and the highest values were 3.22-fold.</p

Role of Neuropilin-1/Semaphorin-3A signaling in the functional and morphological integrity of the cochlea

<div><p><i>Neuropilin-1 (Nrp1)</i> encodes the transmembrane cellular receptor neuropilin-1, which is associated with cardiovascular and neuronal development and was within the peak SNP interval on chromosome 8 in our prior GWAS study on age-related hearing loss (ARHL) in mice. In this study, we generated and characterized an inner ear-specific <i>Nrp1</i> conditional knockout (CKO) mouse line because <i>Nrp1</i> constitutive knockouts are embryonic lethal. <i>In situ</i> hybridization demonstrated weak <i>Nrp1</i> mRNA expression late in embryonic cochlear development, but increased expression in early postnatal stages when cochlear hair cell innervation patterns have been shown to mature. At postnatal day 5, <i>Nrp1</i> CKO mice showed disorganized outer spiral bundles and enlarged microvessels of the stria vascularis (SV) but normal spiral ganglion cell (SGN) density and presynaptic ribbon body counts; however, we observed enlarged SV microvessels, reduced SGN density, and a reduction of presynaptic ribbons in the outer hair cell region of 4-month-old <i>Nrp1</i> CKO mice. In addition, we demonstrated elevated hearing thresholds of the 2-month-old and 4-month-old <i>Nrp1</i> CKO mice at frequencies ranging from 4 to 32kHz when compared to 2-month-old mice. These data suggest that conditional loss of <i>Nrp1</i> in the inner ear leads to progressive hearing loss in mice. We also demonstrated that mice with a truncated variant of <i>Nrp1</i> show cochlear axon guidance defects and that exogenous semaphorin-3A, a known neuropilin-1 receptor agonist, repels SGN axons <i>in vitro</i>. These data suggest that Neuropilin-1/Semaphorin-3A signaling may also serve a role in neuronal pathfinding in the developing cochlea. In summary, our results here support a model whereby Neuropilin-1/Semaphorin-3A signaling is critical for the functional and morphological integrity of the cochlea and that <i>Nrp1</i> may play a role in ARHL.</p></div