Experiment
Noise-induced hearing loss (NIHL) is a prevalent health risk.
Inbred mouse strains 129S6/SvEvTac (129S6) and MOLF/EiJ (MOLF) show strong NIHL resistance (NR) relative to CBA/CaJ (CBACa) mice in response to noise exposure. The goal of the current study was to identify genes that contribute to NR.
Noninvasive auditory brainstem response (ABR) testing was used to provide a detailed characterization of NIHL for 129S6, MOLF, CBACa and their F1 And N2 offspring. Using QTL mapping followed by the construction of consomic and congenic strains refined the chromosomal locations of ten NR QTL and discovered epistatic interactions between the NR regions.
CBACa mice were crossed with either 129S6 or MOLF to generate (CBACa x 129S6)F1 or (CBACa x MOLF)F1 hybrids that were then backcrossed to the respective parental strain. Mice were noise exposed at 4-6 weeks and held without further treatment for an additional 2 weeks before physiological testing to quantify noise-induced permanent threshold shifts (PTS). An age matched CBACa mouse was included in each exposure session as a control. Cochlear function was tested in unexposed and exposed mice using ABR. Thresholds were determined by visual inspection of stacked waveforms, as the lowest intensity evoking a reproducible wave.
Preexposure and post-exposure ABR values were used as the phenotype for QTL analysis. To characterize and compare NR between CBACa, 129S6, MOLF and the F1 and N2 mice, the mice were separated into pre and post exposure groups and PTS values were generated by comparing the pre- and post- exposure values between the groups. Tail DNA was isolated from 232 ((129S6 x CBACa) x 129S6) (N2-129S6) mice and from 225 ((MOLF x CBACa) x MOLF) (N2-MOLF) mice using standard protocols. The respective DNAs were analyzed with 84 SSLP markers that were polymorphic between CBACa, 129S6 and MOLF, providing a full genome scan at app 20 cM resolution. Additional markers in chromosomal areas of interest were subsequently genotyped.
The QTL analysis was performed using binary scores to look for associations at each individual frequency (8, 12, 16, 24 and 32 KHz). Significance thresholds were obtained by permutation in which data from the genome scan was reanalyzed 10,000 times, randomly shuffling phenotypes while keeping genotypes fixed. Genome scans and permutations were performed using R/qtl package.
QTL for the noise-induced hearing loss resistant trait in the 12936 inbred strain were detected in interval mapping of the ((129S6 x CBACa) x 129S6) backcrossed mice. Table 1:
QTL Nrq1 (noise-induced hearing loss resistant QTL 1) mapped to Chromosome 17 peaking at marker D17Mit113 (9.62 cM, 11.98 Mb GRCm38)) with a LOD score of 3.13 and a p value of 0.0169. The 95% confidence interval spanned 5-44 cM. Nrq1 was associated with resistance at all tested frequencies, 8, 12, 16, 24 and 32 kHz.
A second, highly significant QTL Nrq1a (noise-induced hearing loss resistant QTL 1a), specific to NR at 12 kHz, mapped within the Nrq1 QTL on Chromosome 17 with a LOD score of 7.07 and a p value of <0.001 at marker D17Mit113. This locus spanned from 7 to 16 cM.
QTL Nrq2 (noise-induced hearing loss resistant QTL 2) mapped to Chromosome 18 peaking at 21.0 cM nearest marker D18Mit194, (43.66 Mb GRCm38)) with a LOD score of 3.91 and a p value of 0.0027. The 95% confidence interval spanned 6-41 cM. Nrq2 was associated with resistance at all tested frequencies, 8, 12, 16, 24 and 32 kHz.
QTL Nrq3 (noise-induced hearing loss resistant QTL 3) mapped to Chromosome 14 peaking at 35.5 cM nearest marker D14Mit39, (68.55 Mb GRCm38)) with a LOD score of 3.18 and a p value of 0.0152. The 95% confidence interval spanned 23-57 cM. Nrq3 was associated with resistance at frequencies 24 and 32 kHz.
QTL Nrq4 (noise-induced hearing loss resistant QTL 4) mapped to Chromosome 11 peaking at marker D11Mit126 (92.9 cM) with a LOD score of 3.15 and a p value of 0.0193. The 95% confidence interval spanned 39-105 cM. Nrq4 was associated with resistance at frequencies 16, 24 and 32 kHz.
QTL Nrq5 (noise-induced hearing loss resistant QTL 5) mapped to Chromosome 4 peaking at 76.2 cM nearest marker D4Mit74, (114.35 Mb GRCm38)) with a LOD score of 2.71 and a p value of 0.0471. The 95% confidence interval spanned 67-97 cM. Nrq5 was associated with resistance at frequencies 16, 24 and 32 kHz.
QTL Nrq1 through Nrq5 represented positive associations between noise-induced hearing loss resistance and the 129S6/129S6 homozygous genotype.
QTL for the noise-induced hearing loss resistant trait in the MOLF inbred strain were detected in interval mapping of the ((MOLF x CBACa) x MOLF) backcrossed mice. Table 2:
Curator Note: The text states that for all 4 MOLF chromosomes, the same QTL were observed when using 8 kHz or 8, 12, 16, 24, or 32 kHz. We have created unique QTL for each of the loci detected at 8 kHz since differing LOD scores and p values were reported.
QTL Nrq7 (noise-induced hearing loss resistant QTL 7) mapped to Chromosome 4 peaking at 49.2 cM nearest marker D4Mit9 (95.07 Mb GRCm38) with a LOD score of 4.34 and a p value of 0.0011. The 95% confidence interval spanned 30-62 cM. Nrq7 was associated with resistance at all tested frequencies, 8, 12, 16, 24 and 32 kHz.
A second QTL Nrq7a (noise-induced hearing loss resistant QTL 7a), specific to NR at 8 kHz, overlapped the Nrq7 QTL on Chromosome 4 with a LOD score of 5.12 and a p value of 0.0003 at 51.2 cM nearest marker D4Mit9 (95.07 Mb GRCm38). The Nrq7a locus spanned from 35-65 cM.
QTL Nrq8 (noise-induced hearing loss resistant QTL 8) mapped to Chromosome 17 peaking at 44.5 cM at marker D17Mit93 (73.80 Mb GRCm38) with a LOD score of 4.36 and a p value of 0.0011. The 95% confidence interval spanned 38-51 cM. Nrq8 was associated with resistance at all tested frequencies, 8, 12, 16, 24 and 32 kHz.
A second QTL Nrq8a (noise-induced hearing loss resistant QTL 8a), specific to NR at 8 kHz, overlapped the Nrq8 QTL on Chromosome 17 with a LOD score of 4.57 and a p value of 0.0009 at 44.5 cM at marker D17Mit93 (73.80 Mb GRCm38). The Nrq8a locus spanned from 38-52 cM.
QTL Nrq9 (noise-induced hearing loss resistant QTL 9) mapped to Chromosome 6 peaking at 67.0 cM at marker D6Mit198 with a LOD score of 3.41 and a p value of 0.0092. The 95% confidence interval spanned 56-67 cM. Nrq9 was associated with resistance at all tested frequencies, 8, 12, 16, 24 and 32 kHz.
A second QTL Nrq9a (noise-induced hearing loss resistant QTL 9a), specific to NR at 8 kHz, mapped within the Nrq9 QTL on Chromosome 6 with a LOD score of 3.99 and a p value of 0.0028 at 67.00 cM at marker D6Mit198. The Nrq9a locus spanned from 56-67 cM.
QTL Nrq10 (noise-induced hearing loss resistant QTL 10) mapped to Chromosome 12 peaking at 53.0 cM at marker D12Mit16 (109.78 Mb GRCm38)) with a LOD score of 2.31 and a p value of 0.0968. The 95% confidence interval spanned 2-53 cM. Nrq10 was associated with resistance at all tested frequencies, 8, 12, 16, 24 and 32 kHz.
A second QTL Nrq10a (noise-induced hearing loss resistant QTL 10a), specific to NR at 8 kHz, mapped within the Nrq10 QTL on Chromosome 12 with a LOD score of 2.68 and a p value of 0.0412 at 53.0 cM at marker D12Mit16 (109.78 Mb GRCm38). The Nrq10a locus spanned from 13-53 cM.
QTL mapping to Chrs 6, 12 and 17 represent a positive association between resistance and the MOLF/MOLF homozygous genotype. Surprisingly the Chr 4 QTL represented a positive association between resistance and the CBACa/MOLF heterozygous genotype.
Based on the QTL findings on Chr 4 and Chr 17 in both backcrosses three consomic 129S6 strains containing either Chr 4, Chr17 or Chr4 and Chr17 in a CBACa background were created: CBA/CaJ-Chr4129S6/SvEvTac/Tem, CBA/CaJ-Chr17129S6/SvEvTac/Tem and CBA/CaJ-Chr4129S6/SvEvTac/Tem Chr17129S6/SvEvTac/Tem.
Analysis of the Chr 4 consomic showed PTS values nearly identical to CBACa at 5.6, 8, 24 and 32 kHz. At 16 and 24 kHz PTS values were significantly different from CBACa but similar to 129S6, indicating that 129S6 Chr 4 contributes strongly to resistance at 12 and 16 kHz.
Analysis of the Chr 17 consomic revealed that 129S6 Chr 17 contributes strongly to resistance in the high frequency range (24 and 32 kHz) and significantly at the low frequencies (5.6 and 8 kHz).
Analysis of the double consomic strain indicated that 129S6 Chr 4 and Chr 17 interact. The CBA/CaJ-Chr4129S6/SvEvTac/Tem Chr17129S6/SvEvTac/Tem consomic stain displayed resistance only at 24 and 32 kHz. The presence of the whole 129S6 Chr 17 in the double consomic appeared to mask the mid-frequency resistance contribution of 129S6 Chr 4.
To narrow the NR regions of 129S6 Chr 4 and Chr 17 the CBA/CaJ-Chr4129S6/SvEvTac/Tem and
CBA/CaJ-Chr17129S6/SvEvTac/Tem consomic strains were used in marker-assisted mating strategies to generate two Chr 4 congenic strains [CBACa.129S6-(D4Mit227-D4Mit74)/Tem and CBACa.129S6-(D4Mit74-D4Mit42)/Tem] and three Chr 17 congenic strains [ CBACa.129S6-(D17Mit143-D17Mit51)/Tem, CBACa.129S6-(D17Mit143-D17Mit100)/Tem and CBACa.129S6-(D17Mit119-D17Mit1)/Tem].
The CBACa.129S6-(D4Mit227-D4Mit74)/Tem strain demonstrated PTS values that were not significantly different from those of CBACa. Similarly the CBACa.129S6-(D4Mit74-D4Mit42)/Tem strain did not show PTS values significantly different from CBACa.
Strain CBACa.129S6-(D17Mit119-D17Mit1)/Tem revealed the presence of a QTL in a homozygous 129S6 region, Nrq6 (noise-induced hearing loss resistant QTL 6), that demonstrated marked NR resistance at 12 kHz on Chr 17 that was not detected in the CBA/CaJ-Chr17129S6SvEvTac/Tem strain. Nrq6 mapped to 32.9 cM on Chr 17 between flanking markers D17Mit119 and D17Mit1.
Table 4: Candidate genes for:
proximal Chr 17 QTL Nrq1 and Nrq1a are Nox3, Synj2, Cldn9, Noxo1, Axin1 and Aif1;
for Nrq2 on Chr 18 are Aqp4, Diap1, Pou4f3, Slc12a2, Tcof1 and Atp8b1;
for Nrq3 on Chr 14 are Gjb2, Gjb6, and Diap3;
for Nrq4 on Chr 11 are Col1a1, Ush1g, Actg1, Cd68 and Ccl2;
for Nrq5, Nrq7, Nrq7a on Chr 4 are Bsnd, Lepre1, Kcnq4 and Gjb3;
for Nr6, Nrq8 and Nrq8a on distal Chr 17 are Slc5a7, Slc25a41 and Slc25a23;
for Nrq9 and Nrq9a on Chr6 is Atp2b2;
and for Nrq10 and Nrq10a is Coch.
Analysis Tools