**3. Phenotypic variations of hearing caused by genetic backgrounds in mouse inbred strains**

Laboratory mice are one of the best experimental models to investigate the genetic background effect for ARHL as mentioned in Section 1. Moreover, the classical inbred strains have been established in large numbers and exhibit variable hearing ability and onset time of ARHL caused by genetic background [21, 22]. **Figure 3** shows the means of ABR thresholds to tone-pip stimuli at 4, 8, 16, and 32 kHz in mice from MSM/Ms, C3H/HeN, C57BL/6J, A/J, and DBA/2J at 4 months of age, as cited in our previous studies [23–25]. The hearing phenotypes of these strains can be classified into two groups: normal hearing and early onset ARHL. The MSM/Ms and C3H/HeN comprise the normal hearing group. The ABR thresholds are stable at all frequencies. The C57BL/6J mice also show normal ABR thresholds at 4, 8, and 16 kHz. However, the ABR threshold at 32 kHz of C57BL/6J mice is significantly higher than that of MSM/Ms and C3H/HeN mice, indicating that C57BL/6J developed high-frequency-specific ARHL. The A/J mice exhibit an ARHL that is more severe for high-frequency stimuli. In addition, the ABR thresholds of the A/J mice at other frequencies are clearly increased when compared with those of C57BL/6J mice. Moreover, the DBA/2J mice developed more severe hearing loss. The ABR thresholds of the DBA/2J mice exhibited levels of severe (71–90 dB SPL) and profound (<91 dB SPL) hearing loss in sound stimuli at 4/8 and 16/32 kHz, respectively, which were significantly higher than those of A/J mice at 4, 8, and 16 kHz.

The difference between the normal hearing and early onset ARHL groups can be explained by a mutation of the Cadherin 23 gene (*Cdh23*). The responsible *Cdh23c.753G>A* mutation was identified at one base before the splice-donor site, leading to partial skipping of a single exon [21, 24, 26] and age-related stereocilia degeneration in cochlear hair cells [23, 24, 27]. The A/J mice have another strain-specific mutation (p.His55Asn) in the citrate synthase gene (*Cs*) [28]. By identifying *Csp.His55Asn* mutation, A/J mice were shown to have developed severe ARHL

additive or epistatic interaction with the *Cdh23c.753G>A* and *Fscn2p.Arg109His* mutations. Thus, the differences of hearing levels among the inbred strains are regulated by background effects

We have described in the previous section that several mouse inbred strains developed ARHL by genetic background effects. In this section, we introduce approaches to identify genetic

The forward genetics approach is phenotype-driven, with a foundation that associates the detection of chromosomal location with phenotype by linkage analysis. The start of the experiments

ceptible and resistant strains in phenotype (**Figure 4A**). The linkage analysis is based on meiotic

erozygous chromosomes derived from both the susceptible and resistant strains. Accordingly,

 mice to one parental strain, respectively, and inherited chromosomes that underwent recombination events. The recombinant region on the chromosomes was detected by using a genetic marker, such as microsatellites and SNPs, which recognized genetic polymorphisms compared

been a powerful and productive method to identify QTL-associated ARHL. Johnson et al. [37] detected the first QTL *ahl* locus for ARHL that displayed a *Cdh23c.753G>A* mutation by using N2

As mentioned earlier, there are many modifiers in the genome of inbred strains. To evaluate the effect of a single QTL identified in the mapping by avoiding the effects from other modifiers, congenic mice have become a powerful tool. Congenic mice are defined as having part of the mutation or a chromosomal segment from one inbred genetic background (donor) to another (host) [38] (**Figure 4B**). The creation of congenic mice is based on backcrossing the system for at least seven times. Although this process is long, the resolution of the phenotype

and N2

strategy is the study of *moth1* locus [39, 40]. The *tubby* mice, which are a mutant of tubby bipartite transcription factor gene (*Tub*), exhibit severe hearing loss caused by cochlear degen-

AKR/J and CAST/Ei showed normal hearing. Ikeda et al. [39] mapped the modifier, *moth1*, via

study led to the successful identification of the association of the modification of hearing loss with a strain-specific mutation in microtubule-associated protein 1 gene (*Map1a*), which was

the first elucidated causative gene caused by the background effect in hearing [40].

and N2

and N2

Effects of Genetic Background on Susceptibility and the Acceleration of Hearing Loss in Mice

mice, by crossing between the sus-

http://dx.doi.org/10.5772/intechopen.72469

9

mice were investigated to determine

mice. The most successful example of this

mice produced by intercrossing with

and N2

mice and confirmed the locus by creating congenic mice. This

hybrid, which have het-

mice and backcrossing of

mice. This approach has

modifiers and susceptibility loci of hearing loss underlying the genetic backgrounds.

through the epistatic and additive effects.

**4.1. Classical forward genetics approach**

the F<sup>2</sup>

F1

and N2

included production of chromosomal recombinants, F<sup>2</sup>

to parental strains. Finally, the phenotypes of F<sup>2</sup>

greatly improves when compared with F<sup>2</sup>

linkage analysis using both F2

eration in C57BL/6J background [39]. However, some F<sup>2</sup>

whether there is linkage with the recombinant regions of F2

recombination events that occur in sperm and egg precursor cells of F<sup>1</sup>

progenies were produced by intercrossing between F<sup>1</sup>

backcross mice between ARHL-susceptible C57BL/6J and -resistant CAST/Ei.

**4. Identification of the genetic modifiers in mice**

**Figure 3.** Comparison of hearing levels among the mouse inbred strains. The means (circles, squares, diamonds, and upper and lower triangles) and standard deviations (error bars) of ABR thresholds for 4, 8, 16, and 32 kHz sound stimuli are shown for MSM/Ms, C3H/HeN, DBA/2J, C57BL/6J, and A/J mice at 4 months of age. The graph was created by using data from our previous studies [23–25].

by interaction between *Cdh23c.753G>A* and *Csp.His55Asn* mutations. The CDH23 is a member of the calcium-dependent cell-cell adhesion and tip link component [21, 29, 30]. In contrast, CS is the first enzyme of the tricarboxylic acid cycle, generating citrate and free coenzyme A [31]. Therefore, both proteins seem to contribute different functions in the inner ear for hearing, suggesting that A/J mice develop early onset hearing loss from additive effects of different functional mutations, as described in the previous section (**Figure 1B**). In addition, A/J mice carried a single adenine insertion in the mitochondrial tRNA-Arg gene (*mt-TrArg*) [32]. CS is transported into the mitochondrial matrix and plays an important role in condensing mitochondrial acetyl-coenzyme A and oxaloacetate for transporting of acetyl-coenzyme A from the mitochondrial matrix to the cytosol [31], suggesting that mitochondrial dysfunction by epistasis (**Figure 1B**) between *Csp.His55Asn* and *mt-TrArg* mutations is accelerated in ARHL of A/J mice. The DBA/2J mice also have a strain-specific mutation (p.Arg109His) in the fascin 2 gene (*Fscn2*) [33]. DBA/2J mice exhibit progressive shortening of the stereocilia, and this phenotype only develops with homozygosity of both the *Cdh23c.753G>A* and *Fscn2p.Arg109His* mutations [34]. FSCN2 is an actin crosslinking protein and localizes along the length of stereocilia at especially high concentration around the stereocilia tips [33, 34]. Although the pathological mechanisms in the genetic interaction between the *Cdh23c.753G>A* and *Fscn2p.Arg109His* mutations are widely unknown, the degeneration of stereocilia in DBA/2J mice may be explained by epistasis. Moreover, the other QTLs related to ARHL were detected in DBA/2J mice. The QTLs, *Ahl9* [35] and *Chr5* QTL [25, 36], are likely to contribute to frequency-specific ARHL. Although the causative genes and mutations are still unknown, these QTLs lead to severe hearing loss by additive or epistatic interaction with the *Cdh23c.753G>A* and *Fscn2p.Arg109His* mutations. Thus, the differences of hearing levels among the inbred strains are regulated by background effects through the epistatic and additive effects.
