**2. USH genes**

USH is genetically diverse besides its clinical heterogeneity. Currently, eleven loci have been identified (Hereditary hearing loss homepage and Hmani-Aifa et al., 2009), and nine genes on these loci are known. Among these genes, five are involved in USH1, three in USH2 and one in USH3 (Reiners et al., 2006; Williams, 2008; Millan et al., 2011). Although the functions of some USH genes are relatively clear now in the inner ear (see section 6), extensive work is still necessary to elucidate the functions of USH genes in both the inner ear and the retina.

Usher Syndrome: Genes, Proteins, Models, Molecular Mechanisms, and Therapies 295

Mutations in SANS are rare in USH1 patients. Some mutations, such as c.1373 A>T and c.163\_164 + 13del15, cause the clinical symptoms close to USH2 (Kalay et al., 2005; Bashir et al., 2011). The protein of this gene consists of several putative protein-protein interaction domains, including three ankyrin –like (ANK) repeats, a central (CEN) domain, a sterile alpha motif (SAM) and a PBM (Figure 1E). Therefore, like harmonin, SANS is believed to be

Four USH2 loci were originally defined, *USH2A*-*D*. The genes responsible for *USH2A*, *USH2C*, and *USH2D* are *USH2A* (usherin) (Eudy et al., 1998), *GPR98* (G Protein-coupled Receptor 98) (Weston et al., 2004), and *WHRN* (whirlin) (Ebermann et al., 2006), respectively. The gene for *USH2B* was once considered to be *NBC3* (sodium bicarbonate cotransporter)

Fig. 1. Domain structures of USH1 proteins

a putative scaffold protein.

**2.2 USH2 genes** 

#### **2.1 USH1 genes**

In the past 20 years, seven loci have been assigned to USH1. They are *USH1B-H*. *USH1A* was first localized on 14a32.1 from a study in nine USH1 families in the Poitou-Charentes region of France, and was recently withdrawn due to the discovery that most of these families in fact carry mutations on the *USH1B* locus (Gerber et al., 2006). The genes underlying *USH1B*, *USH1C*, *USH1D*, *USH1F*, and *USH1G* have been identified as *MYO7A* (myosin VIIa) (Weil et al., 1995), *USH1C* (harmonin) (Bitner-Glindzicz et al., 2000; Verpy et al., 2000), *CDH23* (cadherin 23) (Bolz et al., 2001; Bork et al., 2001), *PCDH15* (protocadherin 15) (Ahmed et al., 2001; Alagramam et al., 2001b), and *USH1G* (SANS) (Weil et al., 2003), respectively. Among them, *MYO7A*, *USH1C*, *CDH23* and *PCDH15* are also the causative genes for nonsyndromic deafness, *DFNB2*/*DFNA11* (Liu et al., 1997; Weil et al., 1997), *DFNB18* (Ahmed et al., 2002), *DFNB12* (Bork et al., 2001), and *DFNB23* (Ahmed et al., 2003), respectively. The *USH1E* and *USH1H* loci were mapped to chromosome 21q21 and 15q22-23 (Chaib et al., 1997; Ahmed et al., 2009 ). However, the genes at these loci have not yet been pinpointed.

*MYO7A* is the most prevalent gene causing USH1 (Astuto et al., 2000). It encodes an unconventional actin-based motor protein with the conserved motor domain and five IQ motifs (Figure 1A). These domains are responsible for binding to actin, ATP, and myosin light chain. Therefore, MYO7A may move its cargos along the actin filaments using the energy generated from the hydrolysis of ATP. However, the motor domain of MYO7A shows a strong affinity to ADP and, thus, stays bound to actin filament for a long time (Heissler and Manstein, 2011). In this case, MYO7A may be involved in generating tensions between two proteins or cellular structures. The tail of MYO7A has a series of proteinprotein interaction domains, including a single α-helix (SAH), a coiled-coil domain (CC), two myosin tail homology 4 domains (MyTH4), two band 4.1, ezrin, radixin, moesin domains (FERM), and a src homology 3 domain (SH3) (Figure 1A). These domains are thought to be engaged in binding to cargos and/or anchoring to proteins.

Harmonin (also known as AIE-75 or PDZ-73) is expressed in many different tissues (Kobayashi et al., 1999; Scanlan et al., 1999). Nine transcripts have so far been discovered (Verpy et al., 2000; Reiners et al., 2003). They are categorized into three groups, isoforms a, b and c (Figure 1B). All these isoforms have multiple PDZ (postsynaptic density 95; discs large; zonula occludens-1) domains and at least one CC domain. The CC domain is reported to participate in harmonin dimerization (Adato et al., 2005b), and the PDZ domain is well known to interact with PDZ-binding motifs (PBMs) in other proteins (Sheng and Sala, 2001). Isoform b specifically has a proline, serine and threonine-rich (PST) domain. This domain has been demonstrated to bind and bundle actin filaments (Boeda et al., 2002). In summary, harmonin may organize a multi-protein complex and attach this complex to actin filaments.

CDH23 and PCDH15 both have multiple transcripts and are grouped into isoforms a, b and c for CDH23 (Lagziel et al., 2005; Lagziel et al., 2009) and isoforms CD1, CD2, CD3 and SI for PCDH 15 (Ahmed et al., 2006) (Figures 1C and 1D). As the distant members of the classical cadherin superfamily, the proteins of these two genes have various repeats of extracellular cadherin (EC) domains in their extracellular regions. Accordingly, it has been proposed and supported by many studies in hair cells (see below) that the two proteins function in cell adhesion through their homophilic and heterophilic interactions. The two proteins probably anchor to the intracellular structures through the PBMs in their cytoplasmic regions (Figures 1C and 1D).

Fig. 1. Domain structures of USH1 proteins

Mutations in SANS are rare in USH1 patients. Some mutations, such as c.1373 A>T and c.163\_164 + 13del15, cause the clinical symptoms close to USH2 (Kalay et al., 2005; Bashir et al., 2011). The protein of this gene consists of several putative protein-protein interaction domains, including three ankyrin –like (ANK) repeats, a central (CEN) domain, a sterile alpha motif (SAM) and a PBM (Figure 1E). Therefore, like harmonin, SANS is believed to be a putative scaffold protein.

#### **2.2 USH2 genes**

294 Hearing Loss

In the past 20 years, seven loci have been assigned to USH1. They are *USH1B-H*. *USH1A* was first localized on 14a32.1 from a study in nine USH1 families in the Poitou-Charentes region of France, and was recently withdrawn due to the discovery that most of these families in fact carry mutations on the *USH1B* locus (Gerber et al., 2006). The genes underlying *USH1B*, *USH1C*, *USH1D*, *USH1F*, and *USH1G* have been identified as *MYO7A* (myosin VIIa) (Weil et al., 1995), *USH1C* (harmonin) (Bitner-Glindzicz et al., 2000; Verpy et al., 2000), *CDH23* (cadherin 23) (Bolz et al., 2001; Bork et al., 2001), *PCDH15* (protocadherin 15) (Ahmed et al., 2001; Alagramam et al., 2001b), and *USH1G* (SANS) (Weil et al., 2003), respectively. Among them, *MYO7A*, *USH1C*, *CDH23* and *PCDH15* are also the causative genes for nonsyndromic deafness, *DFNB2*/*DFNA11* (Liu et al., 1997; Weil et al., 1997), *DFNB18* (Ahmed et al., 2002), *DFNB12* (Bork et al., 2001), and *DFNB23* (Ahmed et al., 2003), respectively. The *USH1E* and *USH1H* loci were mapped to chromosome 21q21 and 15q22-23 (Chaib et al., 1997; Ahmed et

*MYO7A* is the most prevalent gene causing USH1 (Astuto et al., 2000). It encodes an unconventional actin-based motor protein with the conserved motor domain and five IQ motifs (Figure 1A). These domains are responsible for binding to actin, ATP, and myosin light chain. Therefore, MYO7A may move its cargos along the actin filaments using the energy generated from the hydrolysis of ATP. However, the motor domain of MYO7A shows a strong affinity to ADP and, thus, stays bound to actin filament for a long time (Heissler and Manstein, 2011). In this case, MYO7A may be involved in generating tensions between two proteins or cellular structures. The tail of MYO7A has a series of proteinprotein interaction domains, including a single α-helix (SAH), a coiled-coil domain (CC), two myosin tail homology 4 domains (MyTH4), two band 4.1, ezrin, radixin, moesin domains (FERM), and a src homology 3 domain (SH3) (Figure 1A). These domains are

Harmonin (also known as AIE-75 or PDZ-73) is expressed in many different tissues (Kobayashi et al., 1999; Scanlan et al., 1999). Nine transcripts have so far been discovered (Verpy et al., 2000; Reiners et al., 2003). They are categorized into three groups, isoforms a, b and c (Figure 1B). All these isoforms have multiple PDZ (postsynaptic density 95; discs large; zonula occludens-1) domains and at least one CC domain. The CC domain is reported to participate in harmonin dimerization (Adato et al., 2005b), and the PDZ domain is well known to interact with PDZ-binding motifs (PBMs) in other proteins (Sheng and Sala, 2001). Isoform b specifically has a proline, serine and threonine-rich (PST) domain. This domain has been demonstrated to bind and bundle actin filaments (Boeda et al., 2002). In summary, harmonin may organize a multi-protein complex and attach this complex to actin filaments. CDH23 and PCDH15 both have multiple transcripts and are grouped into isoforms a, b and c for CDH23 (Lagziel et al., 2005; Lagziel et al., 2009) and isoforms CD1, CD2, CD3 and SI for PCDH 15 (Ahmed et al., 2006) (Figures 1C and 1D). As the distant members of the classical cadherin superfamily, the proteins of these two genes have various repeats of extracellular cadherin (EC) domains in their extracellular regions. Accordingly, it has been proposed and supported by many studies in hair cells (see below) that the two proteins function in cell adhesion through their homophilic and heterophilic interactions. The two proteins probably anchor to the intracellular structures through the PBMs in their cytoplasmic regions (Figures

al., 2009 ). However, the genes at these loci have not yet been pinpointed.

thought to be engaged in binding to cargos and/or anchoring to proteins.

**2.1 USH1 genes** 

1C and 1D).

Four USH2 loci were originally defined, *USH2A*-*D*. The genes responsible for *USH2A*, *USH2C*, and *USH2D* are *USH2A* (usherin) (Eudy et al., 1998), *GPR98* (G Protein-coupled Receptor 98) (Weston et al., 2004), and *WHRN* (whirlin) (Ebermann et al., 2006), respectively. The gene for *USH2B* was once considered to be *NBC3* (sodium bicarbonate cotransporter)

Usher Syndrome: Genes, Proteins, Models, Molecular Mechanisms, and Therapies 297

laminin (Lam) and fibronectin III (FN3) functional domains common in cell adhesion proteins and extracellular matrix proteins. Its cytoplasmic region has a PBM. Isoform A is an Nterminal 1546-aa fragment of isoform B. USH2A is thought to be involved in cell adhesion.

The *GPR98* gene, also known as *VLGR1* (Very Large G protein-coupled Receptor 1) and *MASS1* (Monogenic Audiogenic Seizure Susceptibility 1), exists only in the vertebrate (Gibert et al., 2005) and is one of the largest genes, with 90 exons (McMillan et al., 2002). Its mRNA is present mostly in the brain and spinal cord during development (McMillan et al., 2002; Weston et al., 2004), but it can also be found in many other tissues (Nikkila et al., 2000; Skradski et al., 2001; McMillan et al., 2002; Weston et al., 2004). *GPR98* expresses multiple mRNA transcripts, including isoforms a, b and c in humans and isoforms b, d, e and Mass1 in rodents (Figure 2B) (Nikkila et al., 2000; Skradski et al., 2001; McMillan et al., 2002; Yagi et al., 2005). Mutations in the longest isoform, isoform b, have been identified in patients with USH2C (Weston et al., 2004; Ebermann et al., 2009; Hilgert et al., 2009). Additionally, different mutations along the murine *Gpr98* gene share common phenotypes in vision and hearing (Skradski et al., 2001; McMillan and White, 2004; Johnson et al., 2005; Yagi et al., 2005; McGee et al., 2006; Michalski et al., 2007; Yagi et al., 2007). These findings suggest that isoform b is the major isoform in both the retina and the inner ear and is essential for vision and hearing. This isoform is 6306 aa long in humans. It has signature domains of family B of G protein-coupled receptors (GPCRs), i.e., a GPCR proteolytic site (GPS) and a 7 transmembrane domain (7TM). Therefore, GPR98 may function in signal transduction. GPR98 also has a PBM at its C-terminus. Along its long extracellular region, it has a laminin globular-like domain (LamG\_L), an epilepsy associated repeat (EAR)/epitempin (EPTP) domain, and multiple tandem-arranged Calxβ domains. While the function of EAR/EPTP is unknown, LamG\_L is a cell adhesion domain, and the Calxβ domain is able to bind to Ca2+

with low affinity in vitro (Nikkila et al., 2000; McMillan and White, 2011).

subcellular locations, similar to harmonin.

**2.3 USH3 and USH related genes** 

Mutations of whirlin cause either USH2D or nonsyndromic deafness, *DFNB31*. Interestingly, mutations at the N-terminal half of the gene, such as p.P246HfxX13 and compound heterozygosity of p.Q103X and c.837+1G>A, are manifested as USH2D (Ebermann et al., 2006; Audo et al., 2011), while mutations at the C-terminal half, such as p.R778X and c.2423delG, were found in patients with *DFNB31* (Mburu et al., 2003; Tlili et al., 2005). Whirlin has multiple mRNA transcripts in the inner ear and the retina (Mburu et al., 2003; Belyantseva et al., 2005; van Wijk et al., 2006; Yang et al., 2010), which can be conceptually translated into three groups of proteins, the long, N-terminal, and C-terminal isoforms (Figure 2C). The long isoform contains three PDZ domains and a proline-rich region (PR). Thus, whirlin is a homolog of harmonin. At the protein level, whirlin mainly expresses the long isoform in the retina and the long and C-terminal isoforms in the inner ear (Yang et al., 2010). Because both the PDZ domain and PR region are protein interaction modules, whirlin is believed to be implicated in the assembly of multi-protein complexes at specific

The only gene currently identified in USH3 is clarin-1 for the *USH3A* locus (Joensuu et al., 2001; Adato et al., 2002; Fields et al., 2002). Like other USH genes, clarin-1 has multiple transcript variants due to different splicings and usages of transcription start sites (Vastinsalo et al., 2010). The primary transcript encodes a protein with four predicted

(Bok et al., 2003). However, further study of the consanguineous Tunisian family carrying the *USH2B* locus demonstrates that mixed mutations in the *GPR98* and *PDE6B* genes are responsible for the disease manifestation in the family and, thus, the *USH2B* locus was withdrawn (Hmani-Aifa et al., 2009). Moreover, a novel USH2 locus has recently been localized on the chromosome 15q, though the underlying gene has not been identified so far (Ben Rebeh et al., 2008). '

Fig. 2. Domain structures of USH2 proteins

*USH2A* is the most predominant causative gene in all USHs among different human ethnic populations (Eudy et al., 1998; Dreyer et al., 2000; Weston et al., 2000; Aller et al., 2004; van Wijk et al., 2004; Adato et al., 2005a; Hartong et al., 2006; Baux et al., 2007; Kaiserman et al., 2007; Dreyer et al., 2008; Nakanishi et al., 2009; Yan et al., 2009; McGee et al., 2010). Its mutations lead to a wide spectrum of vision and hearing defects in patients. Some *USH2A* mutations, such as p.C759F and p.G4674R, are known to cause only nonsyndromic retinitis pigmentosa (Rivolta et al., 2002; Seyedahmadi et al., 2004; Kaiserman et al., 2007). *USH2A* has 72 exons and is expressed as isoforms A and B (Figure 2A). Isoform B, the major isoform in the retina (Liu et al., 2007), is an extremely large transmembrane protein with 5202 amino acids (aa) in humans (van Wijk et al., 2004). Its long extracellular region has repeated various

(Bok et al., 2003). However, further study of the consanguineous Tunisian family carrying the *USH2B* locus demonstrates that mixed mutations in the *GPR98* and *PDE6B* genes are responsible for the disease manifestation in the family and, thus, the *USH2B* locus was withdrawn (Hmani-Aifa et al., 2009). Moreover, a novel USH2 locus has recently been localized on the chromosome 15q, though the underlying gene has not been identified so far

*USH2A* is the most predominant causative gene in all USHs among different human ethnic populations (Eudy et al., 1998; Dreyer et al., 2000; Weston et al., 2000; Aller et al., 2004; van Wijk et al., 2004; Adato et al., 2005a; Hartong et al., 2006; Baux et al., 2007; Kaiserman et al., 2007; Dreyer et al., 2008; Nakanishi et al., 2009; Yan et al., 2009; McGee et al., 2010). Its mutations lead to a wide spectrum of vision and hearing defects in patients. Some *USH2A* mutations, such as p.C759F and p.G4674R, are known to cause only nonsyndromic retinitis pigmentosa (Rivolta et al., 2002; Seyedahmadi et al., 2004; Kaiserman et al., 2007). *USH2A* has 72 exons and is expressed as isoforms A and B (Figure 2A). Isoform B, the major isoform in the retina (Liu et al., 2007), is an extremely large transmembrane protein with 5202 amino acids (aa) in humans (van Wijk et al., 2004). Its long extracellular region has repeated various

(Ben Rebeh et al., 2008). '

Fig. 2. Domain structures of USH2 proteins

laminin (Lam) and fibronectin III (FN3) functional domains common in cell adhesion proteins and extracellular matrix proteins. Its cytoplasmic region has a PBM. Isoform A is an Nterminal 1546-aa fragment of isoform B. USH2A is thought to be involved in cell adhesion.

The *GPR98* gene, also known as *VLGR1* (Very Large G protein-coupled Receptor 1) and *MASS1* (Monogenic Audiogenic Seizure Susceptibility 1), exists only in the vertebrate (Gibert et al., 2005) and is one of the largest genes, with 90 exons (McMillan et al., 2002). Its mRNA is present mostly in the brain and spinal cord during development (McMillan et al., 2002; Weston et al., 2004), but it can also be found in many other tissues (Nikkila et al., 2000; Skradski et al., 2001; McMillan et al., 2002; Weston et al., 2004). *GPR98* expresses multiple mRNA transcripts, including isoforms a, b and c in humans and isoforms b, d, e and Mass1 in rodents (Figure 2B) (Nikkila et al., 2000; Skradski et al., 2001; McMillan et al., 2002; Yagi et al., 2005). Mutations in the longest isoform, isoform b, have been identified in patients with USH2C (Weston et al., 2004; Ebermann et al., 2009; Hilgert et al., 2009). Additionally, different mutations along the murine *Gpr98* gene share common phenotypes in vision and hearing (Skradski et al., 2001; McMillan and White, 2004; Johnson et al., 2005; Yagi et al., 2005; McGee et al., 2006; Michalski et al., 2007; Yagi et al., 2007). These findings suggest that isoform b is the major isoform in both the retina and the inner ear and is essential for vision and hearing. This isoform is 6306 aa long in humans. It has signature domains of family B of G protein-coupled receptors (GPCRs), i.e., a GPCR proteolytic site (GPS) and a 7 transmembrane domain (7TM). Therefore, GPR98 may function in signal transduction. GPR98 also has a PBM at its C-terminus. Along its long extracellular region, it has a laminin globular-like domain (LamG\_L), an epilepsy associated repeat (EAR)/epitempin (EPTP) domain, and multiple tandem-arranged Calxβ domains. While the function of EAR/EPTP is unknown, LamG\_L is a cell adhesion domain, and the Calxβ domain is able to bind to Ca2+ with low affinity in vitro (Nikkila et al., 2000; McMillan and White, 2011).

Mutations of whirlin cause either USH2D or nonsyndromic deafness, *DFNB31*. Interestingly, mutations at the N-terminal half of the gene, such as p.P246HfxX13 and compound heterozygosity of p.Q103X and c.837+1G>A, are manifested as USH2D (Ebermann et al., 2006; Audo et al., 2011), while mutations at the C-terminal half, such as p.R778X and c.2423delG, were found in patients with *DFNB31* (Mburu et al., 2003; Tlili et al., 2005). Whirlin has multiple mRNA transcripts in the inner ear and the retina (Mburu et al., 2003; Belyantseva et al., 2005; van Wijk et al., 2006; Yang et al., 2010), which can be conceptually translated into three groups of proteins, the long, N-terminal, and C-terminal isoforms (Figure 2C). The long isoform contains three PDZ domains and a proline-rich region (PR). Thus, whirlin is a homolog of harmonin. At the protein level, whirlin mainly expresses the long isoform in the retina and the long and C-terminal isoforms in the inner ear (Yang et al., 2010). Because both the PDZ domain and PR region are protein interaction modules, whirlin is believed to be implicated in the assembly of multi-protein complexes at specific subcellular locations, similar to harmonin.

#### **2.3 USH3 and USH related genes**

The only gene currently identified in USH3 is clarin-1 for the *USH3A* locus (Joensuu et al., 2001; Adato et al., 2002; Fields et al., 2002). Like other USH genes, clarin-1 has multiple transcript variants due to different splicings and usages of transcription start sites (Vastinsalo et al., 2010). The primary transcript encodes a protein with four predicted

Usher Syndrome: Genes, Proteins, Models, Molecular Mechanisms, and Therapies 299

or during puberty (Smith et al., 1994; Petit, 2001), more and more atypical USH patients have been found (Edwards et al., 1998; Sadeghi et al., 2006; Cohen et al., 2007; Fishman et al., 2007; Sandberg et al., 2008; Malm et al, 2010.; Bashir et al., 2011). These patients have relatively late onset vision loss, which may explain the lack of retinal phenotype in most

Zebrafish models for several USH genes have also been reported, including mariner (*myo7a*), *ush1c*, sputnik (*cdh23*), and orbiter (*pcdh15*) (Phillips et al., 2011; Nicolson et al., 1998; Ernest et al., 2000; Sollner et al., 2004; Seiler et al., 2005). Defects in hearing, balance, and vision are manifested during the early life in two *ush1c* mutants. Interestingly, zebrafish has two orthologs of *PCDH15*. Disruption of one leads to the auditory and vestibular dysfunction, while disturbance of the other results in defects in the photoreceptor structure and retinal function. Mariner exhibits similar phenotypes to *Myo7a* mice in hearing, balance and vision. Sputnik has problems with the auditory and vestibular system, but its vision phenotype has not been reported. Currently, studies on other USH genes in zebrafish using the morpholino knockdown technique are being actively pursued (Ebermann et al., 2010). Moreover, a rat model with a point mutation leading to premature truncation of *Myo7a* was generated by Nethyl-N-nitrosourea mutagenesis and named Tornado (Smits et al., 2005). In this model, hearing but not vision defects have been characterized. Therefore, exploration of USH genes in more vertebrate organisms will provide additional ways to understand the biological

**Model name Mutations Phenotypes References** 

impairment

deafness

deafness

deafness

deafness

deafness, reduced ERG

deafness, reduced ERG

(Mburu et al., 1997; Libby and Steel,

(Mburu et al., 1997; Libby and Steel,

(Mburu et al., 1997; Libby and Steel,

(Mburu et al., 1997; Libby and Steel,

(Mburu et al., 1997; Libby and Steel,

(Mburu et al., 1997; Liu et al., 1999; Libby and Steel, 2001)

(Mburu et al., 1997; Libby and Steel,

2001)

2001)

2001)

2001)

2001)

2001)

*Myo7ash1* p.R502P Circling, head tossing, hearing

*Myo7a6J* p.R241P Circling, head tossing,

*Myo7a26SB* p.F1762I Circling, head tossing,

*Myo7a816SB* p.L646\_Q655del Circling, head tossing,

*Myo7a3336SB* p.C2144X Circling, head tossing,

*Myo7a4494SB* p.A246fs?X5 Circling, head tossing,

*Myo7a4626SB* p.Q720X Circling, head tossing,

USH mutant mice, whose lifespan is only about two years.

functions of these genes, in particular, in the retina.

**USH1**  *Myo7a* 

transmembrane domains and a C-terminal potential PBM (Figure 3). Clarin-1 shows a sequence homologous to stargazin, an auxiliary subunit of ion channels in the synapse (Osten and Stern-Bach, 2006; Tomita et al., 2007). Presently, several research groups are intensively focusing on understanding this gene (Aarnisalo et al., 2007; Geller et al., 2009; Geng et al., 2009; Tian et al., 2009; Zallocchi et al., 2009). However, the biological function and cellular expression of clarin-1 still remain elusive.

#### Fig. 3. Domain structure of USH3A

Recently, *PDZD7* was shown to be a modifier gene for the retinal symptom in USH2A patients and, together with *USH2A* or *GPR98*, to contribute to a digenic USH form (Ebermann et al., 2010). Interestingly, this newly identified USH modifier and contributor gene is also a homolog of harmonin. It has several isoforms (Schneider et al., 2009; Ebermann et al., 2010). The long isoform has three PDZ domains and one PR region. The two short isoforms are the N-terminal fragments of the long isoform with only two PDZ domains. However, the short isoforms have not been confirmed at the protein level. Similar to both harmonin and whirlin, different mutations in *PDZD7* are involved in either USH or nonsyndromic deafness. A homozygous reciprocal translocation, 46,XY,t(10;11)(q24;q23), was found to disrupt the *PDZD7* gene at intron 10, which causes nonsyndromic congenital hearing impairment (Schneider et al., 2009). A heterozygous p.R56PfsX mutation of *PDZD7* was found to exacerbate retinal degeneration in an USH2A patient, compared to her sibling carrying the same *USH2A* mutation. Additionally, the heterozygous mutations of *PDZD7*, c.1750-2A>G and p.C732LfsX, are present in USH patients with a heterozygous *USH2A* mutation, p.R1505SfsX, and with a heterozygous *GPR98* mutation, p.C732LfsX, respectively (Ebermann et al., 2010).

### **3. Animal models**

Numerous spontaneous and transgenic USH animal models are now available. Table 1 lists the detailed information about the mouse models. The majority of these models show congenital hearing loss as expected. However, only a few of them, *Ush1c* knockin*, Ush2a* knockout, and whirlin knockout mice, manifest obvious widespread retinal degeneration. *Ush1cdfcr* mice on some specific genomic background and *Myo7a4626SB* and *Cdh23V* double mutant mice show only slight retinal degeneration (Johnson et al., 2003; Lillo et al., 2003; Williams et al., 2009). Among the rest of the USH mouse models, some *Myo7a, Cdh23, Pcdh15, and Grp98* mutant strains show abnormal electroretinogram (ERG) responses but no retinal degeneration (Libby and Steel, 2001; Libby et al., 2003; Haywood-Watson et al., 2006; McGee et al., 2006), indicating that the function of photoreceptors is compromised. The reasons for the discrepancy between USH patient symptoms and USH mutant mouse phenotypes are largely unclear. Many factors could contribute to this, such as the gene isoform composition, mutation type and position in the genes, genomic background, redundant protein compensation, photoreceptor structure and physiology, influence of nongenetic factors, sensitivity of diagnostic measures, etc. (El-Amraoui and Petit, 2005). Additionally, although retinitis pigmentosa in USH is characterized to have an onset before

transmembrane domains and a C-terminal potential PBM (Figure 3). Clarin-1 shows a sequence homologous to stargazin, an auxiliary subunit of ion channels in the synapse (Osten and Stern-Bach, 2006; Tomita et al., 2007). Presently, several research groups are intensively focusing on understanding this gene (Aarnisalo et al., 2007; Geller et al., 2009; Geng et al., 2009; Tian et al., 2009; Zallocchi et al., 2009). However, the biological function

Recently, *PDZD7* was shown to be a modifier gene for the retinal symptom in USH2A patients and, together with *USH2A* or *GPR98*, to contribute to a digenic USH form (Ebermann et al., 2010). Interestingly, this newly identified USH modifier and contributor gene is also a homolog of harmonin. It has several isoforms (Schneider et al., 2009; Ebermann et al., 2010). The long isoform has three PDZ domains and one PR region. The two short isoforms are the N-terminal fragments of the long isoform with only two PDZ domains. However, the short isoforms have not been confirmed at the protein level. Similar to both harmonin and whirlin, different mutations in *PDZD7* are involved in either USH or nonsyndromic deafness. A homozygous reciprocal translocation, 46,XY,t(10;11)(q24;q23), was found to disrupt the *PDZD7* gene at intron 10, which causes nonsyndromic congenital hearing impairment (Schneider et al., 2009). A heterozygous p.R56PfsX mutation of *PDZD7* was found to exacerbate retinal degeneration in an USH2A patient, compared to her sibling carrying the same *USH2A* mutation. Additionally, the heterozygous mutations of *PDZD7*, c.1750-2A>G and p.C732LfsX, are present in USH patients with a heterozygous *USH2A* mutation, p.R1505SfsX, and with a heterozygous *GPR98* mutation, p.C732LfsX, respectively

Numerous spontaneous and transgenic USH animal models are now available. Table 1 lists the detailed information about the mouse models. The majority of these models show congenital hearing loss as expected. However, only a few of them, *Ush1c* knockin*, Ush2a* knockout, and whirlin knockout mice, manifest obvious widespread retinal degeneration. *Ush1cdfcr* mice on some specific genomic background and *Myo7a4626SB* and *Cdh23V* double mutant mice show only slight retinal degeneration (Johnson et al., 2003; Lillo et al., 2003; Williams et al., 2009). Among the rest of the USH mouse models, some *Myo7a, Cdh23, Pcdh15, and Grp98* mutant strains show abnormal electroretinogram (ERG) responses but no retinal degeneration (Libby and Steel, 2001; Libby et al., 2003; Haywood-Watson et al., 2006; McGee et al., 2006), indicating that the function of photoreceptors is compromised. The reasons for the discrepancy between USH patient symptoms and USH mutant mouse phenotypes are largely unclear. Many factors could contribute to this, such as the gene isoform composition, mutation type and position in the genes, genomic background, redundant protein compensation, photoreceptor structure and physiology, influence of nongenetic factors, sensitivity of diagnostic measures, etc. (El-Amraoui and Petit, 2005). Additionally, although retinitis pigmentosa in USH is characterized to have an onset before

and cellular expression of clarin-1 still remain elusive.

Fig. 3. Domain structure of USH3A

(Ebermann et al., 2010).

**3. Animal models** 

or during puberty (Smith et al., 1994; Petit, 2001), more and more atypical USH patients have been found (Edwards et al., 1998; Sadeghi et al., 2006; Cohen et al., 2007; Fishman et al., 2007; Sandberg et al., 2008; Malm et al, 2010.; Bashir et al., 2011). These patients have relatively late onset vision loss, which may explain the lack of retinal phenotype in most USH mutant mice, whose lifespan is only about two years.

Zebrafish models for several USH genes have also been reported, including mariner (*myo7a*), *ush1c*, sputnik (*cdh23*), and orbiter (*pcdh15*) (Phillips et al., 2011; Nicolson et al., 1998; Ernest et al., 2000; Sollner et al., 2004; Seiler et al., 2005). Defects in hearing, balance, and vision are manifested during the early life in two *ush1c* mutants. Interestingly, zebrafish has two orthologs of *PCDH15*. Disruption of one leads to the auditory and vestibular dysfunction, while disturbance of the other results in defects in the photoreceptor structure and retinal function. Mariner exhibits similar phenotypes to *Myo7a* mice in hearing, balance and vision. Sputnik has problems with the auditory and vestibular system, but its vision phenotype has not been reported. Currently, studies on other USH genes in zebrafish using the morpholino knockdown technique are being actively pursued (Ebermann et al., 2010). Moreover, a rat model with a point mutation leading to premature truncation of *Myo7a* was generated by Nethyl-N-nitrosourea mutagenesis and named Tornado (Smits et al., 2005). In this model, hearing but not vision defects have been characterized. Therefore, exploration of USH genes in more vertebrate organisms will provide additional ways to understand the biological functions of these genes, in particular, in the retina.


Usher Syndrome: Genes, Proteins, Models, Molecular Mechanisms, and Therapies 301

(Di Palma et al.,

(Di Palma et al.,

(Di Palma et al.,

(Di Palma et al.,

(Di Palma et al.,

(Di Palma et al., 2001b; Libby et al.,

(Yonezawa et al.,

(Noben-Trauth et al.,

(Alagramam et al., 2001a; Ball et al.,

(Alagramam et al., 2001a; Ball et al.,

(Alagramam et al., 2001a; Ball et al.,

(Washington et al., 2005; Haywood-Watson et al., 2006)

(Alagramam et al.,

(Hampton et al., 2003; Haywood-Watson et al., 2006)

(Alagramam et al., 2001a; Ball et al.,

(Kikkawa et al., 2003)

(Wada et al., 2001)

2001a)

2001a)

2001a)

2001b)

2001a)

2003)

2006)

2003)

2003)

2003)

2003)

2011)

2003)

**Model name Mutations Phenotypes References** 

deafness

deafness

deafness

deafness

deafness

deafness

responses

deafness

function

function

function

responses

deafness

responses

function

deafness

A large insertion Circling, head tossing,

hearing loss

deafness, normal ERG

deafness, normal retinal

deafness, normal retinal

deafness, normal retinal

deafness, reduced ERG

deafness, reduced ERG

deafness, normal retinal

*Cdh23V-3J* p.W1764X Circling, head tossing,

*Cdh23V4J* p.N2718del3 Circling, head tossing,

*Cdh23V5J* p.R2935X Circling, head tossing,

*Cdh23V-6J* p.E302X Circling, head tossing,

*Cdh23V-7J* p.Y1197MfsX47 Circling, head tossing,

*Cdh23V-ngt* p.G49VfsX3 Circling, head tossing,

*Cdh23V-Alb* c.1635C>Tdel119 Circling, head tossing,

*Cdh23Vbus* c.9633 + 1G>A Circling, head tossing,

*Pcdh15av-J* p.A645\_K922del Circling, head tossing,

*Pcdh15av-2J* p.D31\_N57del Circling, head tossing,

*Pcdh15av-3J* p.E1373RfsX36 Circling, head tossing,

*Pcdh15av-5J* IVS14-2A>G Circling, head tossing,

*Pcdh15av-6J* p.G962\_K1008del Circling, head tossing,

*Pcdh15av-Jfb* p.D701GfsX17 Circling, head tossing,

*Ush1gjs* p.E228RfsX8 Circling, head tossing,

*Pcdh15* 

*Pcdh15av-TgN2742Rpw* 

*Sans* 

*Cdh23Ahl* c.753G>A Susceptibility to age-related


deafness, reduced ERG

deafness, reduced ERG

deafness?, reduced ERG

Circling, head tossing,

Circling, head tossing, deafness, slight retinal degeneration at 9 months of

Circling, head tossing,

deafness, retinal degeneration

Hair bundle defect (Grillet et al., 2009)

deafness

age

deafness

deafness

*jera* p.V2360E deafness (Manji et al., 2011)

deafness

deafness

responses

deafness

deafness, reduced ERG

deafness, faster ERG responses

frequency hearing impairment

(Mburu et al., 1997; Libby and Steel, 2001; Yang et al.,

(Rhodes et al., 2004)

(Mburu et al., 1997; Libby and Steel,

(Mburu et al., 1997; Libby and Steel,

(Tian et al., 2010)

(Johnson et al., 2003)

(Johnson et al., 2003)

(Lefevre et al., 2008)

(Lentz et al., 2007; Lentz et al., 2010)

(Han et al., 2010)

(Schwander et al.,

(Wilson et al., 2001; Libby et al., 2003)

(Wilson et al., 2001)

(Di Palma et al., 2001b; Libby et al.,

2009)

2003)

2011)

2001)

2001)

**Model name Mutations Phenotypes References** 

*Myo7a7J* p.A1363AfsX27 Circling, head tossing,

*Myo7aHdb* p.I178F Circling, head tossing, low-

*Myo7a8J* Not known Circling, head tossing,

*Myo7a9J* Not known Circling?, head tossing?,

exons 1-4 with βgal/neo cassette

involving exons 12-

*Ush1ctm1.1Ugds* Exon 1 deletion Circling, head tossing,

*Ush1c* knockin c.216G>A Circling, head tossing,

*erlong* p.S70P Circling, head tossing,

*salsa* p.E737V Circling, head tossing,

*Cdh23V* p.N279EfsX39 Circling, head tossing,

*Cdh23V-J* p.E1169NfsX7 Circling, head tossing,

*Cdh23V-2J* c.4104 + 1G>A Circling, head tossing,

GLG (221-223aa) in PDZ2 with AAA

15, A-D

*Ush1cdfcr-2J* One bp deletion in exon C

*Ush1c*-PDZ2AAA Replacement of

*Harmonin* 

*Cdh23* 

*Ush1c* knockout Replacement of

*Ush1cdfcr* A deletion


Usher Syndrome: Genes, Proteins, Models, Molecular Mechanisms, and Therapies 303

Defects in USH proteins result in Usher syndrome, nonsyndromic deafness, or retinitis pigmentosa, indicating that these proteins are essential in the inner ear and the retina. Therefore, extensive efforts have been put to investigate the cellular location of these proteins in these two tissues. The cellular localization of USH proteins in other tissues is relatively unclear, although some USH proteins are known to be present in the kidney, colon, brain, lung, olfactory neuron, ovary, oviduct, testes and intestine (el-Amraoui et al., 1996; Hasson et al., 1997; Wolfrum et al., 1998; Kobayashi et al., 1999; Scanlan et al., 1999;

**4. Cellular localization of USH proteins** 

Bhattacharya et al., 2002; Pearsall et al., 2002).

Fig. 4. Schematic diagrams of a rod photoreceptor and a hair cell

The inner ear is composed of the cochlea and vestibular system for hearing and balance, respectively. In the vestibular system, hair cells exist in the maculae of the saccule and utricle and the cristae ampullares of the semicircular canals. In the cochlea, one row of inner hair cells and three rows of outer hair cells exist in the organ of Corti. The inner hair cells are responsible for mechanoelectric transduction, whereas the electromotile outer hair cells also perform an electromechanical transduction, thereby amplifying the sound-evoked vibrations of the entire sensory epithelium (Leibovici et al., 2008). All types of hair cells have stereocilia on their apical surfaces, which are modified microvilli filled with bundles of actin filaments. The stereocilia are well-organized into rows of different lengths and form a staircase-like hair bundle (Figure 4). Along with the hair bundle, there exists a real cilium, called kinocilium, which is filled with microtubules. Various links have been discovered along the entire length of the stereocilia and the kinocilium during development and in

**4.1 USH proteins in the inner ear** 


MYO7A: NP\_032689, CDH23: NP\_075859, PCDH15: NP\_075604, SANS: NP\_789817 \*: our unpublished data.

Table 1. USH mutant mouse models
