*TPRN (DFNB79)*

258 Hearing Loss

*GIPC3* encodes a PDZ domain containing protein which is important in peripheral auditory signal transmission. Mutations in this gene cause both profound deafness and progressive hearing loss. In a small Dutch hearing loss followed a different course in the two affected siblings. In the oldest affected individual, the loss in hearing was 70 dB HL at 11 months which progressed to 110 dB HL by 12 years of age. The hearing loss in second individual was 80 dB HL at 3 months which seemed to be stable as it only progressed to 90 dB HL by age of 14 years. (Charizopoulou et al., 2011). The mutation in *GIPC3* identified in the Dutch family introduces a stop codon in exon 6 (p.W301X) truncating the C-terminal. It is hypothesized that since this mutation is in the last exon it will not cause nonsense mediated

*GIPC3* mutations have also been reported in seven other large families from Pakistan which include one framsehift and and six missense mutations (Rehman et al., 2011). The younger affected individuals in these families (7 and 9 years old) had better hearing as compared to the older affected individuals (20 and 25 years old). However, information is not available as

A mutation affecting PDZ domain of GIPC3 has also been reported in mice which causes progressive hearing loss, *ahl5* (Charizopoulou et al., 2011). The mice have a moderate hearing loss at one month of age which progresses to profound in degree by 1 year of age. The higher frequencies are affected first with eventual involvement of all frequencies. DPOAE and the endocochlear potential are gradually affected as well. The stereocilia are

*BSND* encodes barttin which is an essential subunit for two chloride channels. Heteromeric channels formed by barttin and the chloride channels play an important role in potassium recycling in the inner ear (Estevez et al., 2001)*.* A missense mutation, p.I12Tof BSND segregates with hearing loss in three families from Pakistan while in a fourth family it is present with a nonsense mutation in compound heterozygosity (Riazuddin et al., 2009). The younger individuals have a less severe hearing loss as compared to the older affected individuals suggesting that *BSND* may be involved in aetiology of progressive deafness. Functional analysis of the p.I12T mutation of *BSND* in HEK293T cells have demonstrated that function of the channels is unaffected. However, the number of channels on the surface membrane is reduced which results in a decreased current amplitude (Riazuddin et al., 2009).

The 15 PLAT (polycystin/lipoxygenase/a-toxin) domains encoding gene *LOXHD1* was found to be mutated in an Iranian family with postlingual onset of hearing loss at ages ranging from 7 to 8 years. Affected individuals in the family had preserved hearing at low frequencies in the beginning. The loss was mild to moderate at frequencies of 0.5 to 2 KHz. Hearing loss worsened during childhood and adolescence to moderate to severe at mid and high frequencies. All frequencies were affected eventually (Grillet et al., 2009). A nonsense mutation, p.R670X was identified which introduces a premature stop codon at the Cterminal end of f the fifth PLAT domain. This could either lead to a protein lacking 10 PLAT

decay and will allow production of a mutant protein retaining some function.

yet whether hearing loss in any of the affected individuals is stable or progressive.

defective and degenerate together with the spiral ganglion cells.

*GIPC3 (DFNB72/15/95)* 

*BSND (DFNB73)* 

*LOXHD1 (DFNB77)* 

Mutations of *TPRN* cause different degrees of hearing loss in humans. In a Dutch family, hearing loss is documented as moderate to severe in degree till 11 years of age and by 15 years it progresses to profound deafness (Li et al., 2010). In contrast, hearing loss in a Moroccan family is severe in degree even in the third decade of life (Li et al., 2010). Affected members of the Dutch family had a nonsense mutation in exon 1 of *TPRN* while the affected individuals in the Moroccan family were homozygous for a frameshift mutation in exon 1. It is interesting to note that the same frameshift mutation as observed in the Moroccan family was also identified in a Pakistani family. However, individuals in this family had profound degree of deafness (Khan et al., 2009; Rehman et al., 2010).

TPRN is present in the supporting cells as well as at the base of hair cells' stereocilia in the organ of Corti at the taper region of each stereocilium just above the cuticular plate (Rehman et al., 2010).

### *PTPRQ (DFNB84)*

Congenital hearing loss which has been shown to be progressive in nature is associated with mutations in *PTPRQ*, a gene which encodes a phosphatase specific for phoshatidyl inositol, PI(4,5)P2 (Schraders et al., 2010b; Seifert et al., 2003). PI(4,5)P2 plays an important part in actin remodeling. Hearing loss due to mutations *PTPRQ* is severest in a Dutch family with a p.Y497X mutation and hearing loss was self reported to have progressed to profound by the third to fourth decade of life. However in affected individuals of a Moroccan family, a p.R457G mutation in PTPRQ caused a less severe loss in hearing. The loss in hearing was moderate which had deteriorated with age (Schraders et al., 2010b). Otoacoustic emissions were normal at the age of 13 months. The 1 to 2 KHz frequencies seemed to be more affected comparable to that observed in members of a Palestinian family with moderate to severe hearing loss also with a mutation of *PTPRQ* (Shahin et al., 2010a). In the Palestinian family, there were four affected individuals with considerable variation of hearing loss. A nonsense mutation, p.Q429X, was observed in *PTPRQ* for this family (Shahin et al., 2010a). Data is not available about progression of deafness in the Palestinian family*.* 

*Ptprq* is transiently expressed over a period of first three weeks in mouse hair cells at the basal turn of the cochlea (Goodyear et al., 2003). MYO6 plays a role in localization of PTPRQ

Genetics of Nonsyndromic Recessively Inherited

where widespread loss of outer hair cells is observed.

complex.

*USH2A (USH2A)* 

*GPR98 (USH2C)* 

(McGee et al., 2006). *WHRN (USH2D)* 

Moderate to Severe and Progressive Deafness in Humans 261

Hearing loss for USH2 can vary from mild to severe in degree and may also show interfamilial variation (Abadie et al., 2011). The four Usher genes are expressed in hair cells where except for CLRN1, all proteins interact at the ankle link in the stereocilia and form a

Usher type 2A is the most frequent cause of Usher syndrome. A large number of missense, nonsense, insertion and deletion mutations have been identified in *USH2A*. The mutations were first described in North European, Spanish and African American patients (Eudy et al., 1998). *USH2A* encodes a large transmembrane protein, usherin, with an intracellular PDZ binding domain. Usherin is present in both outer and inner hair cells stereocilia and cannot be detected by 2 months of age in mice (Liu et al., 2007). *Ush2a-/-* transgenic mice have a moderate, non-progressive hearing loss which is more noticeable at high frequencies, mimicking the phenotype observed in humans (Liu et al., 2007). In the ears of the *Ush2a-/* mice the inner hair cells and stereocilia are intact in all but at the basal turn of the cochlea

Mutations of *GPR98* or *VLGR1* were first described in USH2 patients from United States and Sweden (Weston et al., 2004). *Gpr98* is expressed transiently during the first week in mice and is no longer detectable by P11*.* GPR98 forms the ankle links that connect the stereocilia of hair cells at their base in the developing hair cell bundles*.* Transgenic *Vlgr1/del7TM* mice which lack transmembrane and cytoplasmic domains of the protein are severely deaf by third week of life and lack ankle links (McGee et al., 2006). The stereocilia are arranged in a more rounded shape as compared to the "V" like pattern present in the wild type mice. A gradual loss of both type of hair cells and pillar cells is observed at the base of the cochlea

The description of *USH2D (WHRN)* is based on only two families from Germany and Portugal. The patients in the German family had mild to moderate hearing loss (Ebermann et al., 2007a) while the Portuguese family had variable degree of hearing loss with post lingual onset which was progressive in nature (Audo et al., 2011). In the German family two mutations in *WHRN* were detected in compound heterozygosity, involving a nonsense mutation in exon 1 and a splice site mutation in intron 2. The splice site mutation causes an in-frame skipping of the second exon and is predicted to result in production of an aberrant protein (Ebermann et al., 2007a). In the Portuguese family a deletion mutation was detected in exon 2 which is predicted to truncate the protein or mark the message for nonsense mediated decay (Audo et al., 2011). These mutations disrupt only the long isoform of WHRN. Mice with a targeted deletion of the long isoform of whirlin have some partial hearing (75 dB SPL) which is non-progressive in nature (Yang et al., 2010) . The inner hair cells stereocilia are unaffected while the outer hair cells stereocilia loose their characteristic "V" shaped formation and assume a "U" shape. Some of the stereocilia are missing from the innermost row in outer hair cells. However, unlike *whirler* mutant mice who are deaf due to a deletion in *Whrn* disrupting both isoforms of the protein, mice with targeted ablation of

the long isoform of whirlin do not have abnormally short stereocilia.

in plasma membranes (Sakaguchi et al., 2008). PTPRQ has been shown to be required for formation of shaft connectors and the taper of the stereocilia (Goodyear et al., 2003; Sakaguchi et al., 2008). Two transgenic mouse mutants, *Ptprq-TM-KO* and *Ptprq-CAT-KO* were generated in which the alleles encode proteins lacking transmembrane and catalytic domains of PTPRQ respectively (Goodyear et al., 2003). Cochlear development was normal in both mice and there was progressive deterioration of the sensory epithelium. Inner hair cells abnormalities were apparent by P1. Some stereocilia were misaligned or missing in the basal turn of cochlea. Stereocilia eventually fused and ultimately the organ of Corti disappeared in some mice by the age of three months (Goodyear et al., 2003).

#### *SERPINB6 (DFNB91)*

A single Turkish family has been described in which affected individuals have a mutation in *SERPINB6* segregating with hearing loss. The nonsense mutation, p.E245X was shown to substantially decrease the amount of the mutant transcript in blood of affected individuals (Sirmaci et al., 2010). The degree of hearing loss is moderate to severe with some residual hearing in all affected individuals. The 54 year old individual was the oldest and had the severest degree of hearing loss with greater degree of loss at high frequencies. The two younger individuals 24 and 23 years respectively, presented with moderate to severe hearing loss which was more severe in the 23 year old patient. Progressive nature of hearing loss was self reported by affected individuals but had not been documented by audiometry.

SERPINB6 is present in the hair cells in the organ of Corti as well as the greater epithelial ridge and may function as an inhibitor of proteases. It is hypothesized that SERPINB6 prevents non-specific tissue damage to inner ear tissue by inhibiting proteases. A transgenic mouse line lacking *Serpinb6* was created (Scarff et al., 2004) but the hearing status of the mice and inner ear structure was not evaluated. It will be interesting to identify if *Serpinb6-/-* mice have deafness at onset of hearing or whether they exhibit progressive deafness.

So far the results of genetic studies have revealed that although moderate to severe hearing loss is the usual finding for individuals with mutations in *STRC*, *TECTA* and *OTO* and in some individuals with mutations which cause progressive deafness, some individuals with mutations in other genes, for example *GJB2*, *CLDN14* or *TRIC* also have similarly milder phenotypes. Additionally, identical mutations in a gene may cause either progressive or stable hearing loss (Bashir et al. manuscript in preparation). Research has also shown that genes involved in progressive hearing loss may also have implications for age related hearing loss. For example, a polymorphism in *Cdh23* has been associated with age related hearing loss in different mice strains (Noben-Trauth, Zheng & Johnson, 2003).

#### **2.4a Moderate to severe or progressive deafness in Usher Syndrome**

Usher syndrome (USH) is a common syndrome of deafness-blindness and can be easily misdiagnosed as nonsyndromic hearing loss since onset of retinitis pigmentosa is gradual and the early manifestation can be very mild in some cases. So far mutations in 4 genes have been identified which cause the less severe phenotypes of hearing loss in Usher syndrome (*USH2A*, *GPR98*, *WHRN* and *CLRN1*). Additionally, some mutations of genes which cause Usher type 1 can also cause phenotypes termed as atypical usher syndromes.

Hearing loss for USH2 can vary from mild to severe in degree and may also show interfamilial variation (Abadie et al., 2011). The four Usher genes are expressed in hair cells where except for CLRN1, all proteins interact at the ankle link in the stereocilia and form a complex.

#### *USH2A (USH2A)*

260 Hearing Loss

in plasma membranes (Sakaguchi et al., 2008). PTPRQ has been shown to be required for formation of shaft connectors and the taper of the stereocilia (Goodyear et al., 2003; Sakaguchi et al., 2008). Two transgenic mouse mutants, *Ptprq-TM-KO* and *Ptprq-CAT-KO* were generated in which the alleles encode proteins lacking transmembrane and catalytic domains of PTPRQ respectively (Goodyear et al., 2003). Cochlear development was normal in both mice and there was progressive deterioration of the sensory epithelium. Inner hair cells abnormalities were apparent by P1. Some stereocilia were misaligned or missing in the basal turn of cochlea. Stereocilia eventually fused and ultimately the organ of Corti

A single Turkish family has been described in which affected individuals have a mutation in *SERPINB6* segregating with hearing loss. The nonsense mutation, p.E245X was shown to substantially decrease the amount of the mutant transcript in blood of affected individuals (Sirmaci et al., 2010). The degree of hearing loss is moderate to severe with some residual hearing in all affected individuals. The 54 year old individual was the oldest and had the severest degree of hearing loss with greater degree of loss at high frequencies. The two younger individuals 24 and 23 years respectively, presented with moderate to severe hearing loss which was more severe in the 23 year old patient. Progressive nature of hearing loss was self reported by affected individuals but had not been documented by

SERPINB6 is present in the hair cells in the organ of Corti as well as the greater epithelial ridge and may function as an inhibitor of proteases. It is hypothesized that SERPINB6 prevents non-specific tissue damage to inner ear tissue by inhibiting proteases. A transgenic mouse line lacking *Serpinb6* was created (Scarff et al., 2004) but the hearing status of the mice and inner ear structure was not evaluated. It will be interesting to identify if *Serpinb6-/-* mice

So far the results of genetic studies have revealed that although moderate to severe hearing loss is the usual finding for individuals with mutations in *STRC*, *TECTA* and *OTO* and in some individuals with mutations which cause progressive deafness, some individuals with mutations in other genes, for example *GJB2*, *CLDN14* or *TRIC* also have similarly milder phenotypes. Additionally, identical mutations in a gene may cause either progressive or stable hearing loss (Bashir et al. manuscript in preparation). Research has also shown that genes involved in progressive hearing loss may also have implications for age related hearing loss. For example, a polymorphism in *Cdh23* has been associated with age related

Usher syndrome (USH) is a common syndrome of deafness-blindness and can be easily misdiagnosed as nonsyndromic hearing loss since onset of retinitis pigmentosa is gradual and the early manifestation can be very mild in some cases. So far mutations in 4 genes have been identified which cause the less severe phenotypes of hearing loss in Usher syndrome (*USH2A*, *GPR98*, *WHRN* and *CLRN1*). Additionally, some mutations of genes which cause

have deafness at onset of hearing or whether they exhibit progressive deafness.

hearing loss in different mice strains (Noben-Trauth, Zheng & Johnson, 2003).

**2.4a Moderate to severe or progressive deafness in Usher Syndrome** 

Usher type 1 can also cause phenotypes termed as atypical usher syndromes.

disappeared in some mice by the age of three months (Goodyear et al., 2003).

*SERPINB6 (DFNB91)* 

audiometry.

Usher type 2A is the most frequent cause of Usher syndrome. A large number of missense, nonsense, insertion and deletion mutations have been identified in *USH2A*. The mutations were first described in North European, Spanish and African American patients (Eudy et al., 1998). *USH2A* encodes a large transmembrane protein, usherin, with an intracellular PDZ binding domain. Usherin is present in both outer and inner hair cells stereocilia and cannot be detected by 2 months of age in mice (Liu et al., 2007). *Ush2a-/-* transgenic mice have a moderate, non-progressive hearing loss which is more noticeable at high frequencies, mimicking the phenotype observed in humans (Liu et al., 2007). In the ears of the *Ush2a-/* mice the inner hair cells and stereocilia are intact in all but at the basal turn of the cochlea where widespread loss of outer hair cells is observed.

#### *GPR98 (USH2C)*

Mutations of *GPR98* or *VLGR1* were first described in USH2 patients from United States and Sweden (Weston et al., 2004). *Gpr98* is expressed transiently during the first week in mice and is no longer detectable by P11*.* GPR98 forms the ankle links that connect the stereocilia of hair cells at their base in the developing hair cell bundles*.* Transgenic *Vlgr1/del7TM* mice which lack transmembrane and cytoplasmic domains of the protein are severely deaf by third week of life and lack ankle links (McGee et al., 2006). The stereocilia are arranged in a more rounded shape as compared to the "V" like pattern present in the wild type mice. A gradual loss of both type of hair cells and pillar cells is observed at the base of the cochlea (McGee et al., 2006).

#### *WHRN (USH2D)*

The description of *USH2D (WHRN)* is based on only two families from Germany and Portugal. The patients in the German family had mild to moderate hearing loss (Ebermann et al., 2007a) while the Portuguese family had variable degree of hearing loss with post lingual onset which was progressive in nature (Audo et al., 2011). In the German family two mutations in *WHRN* were detected in compound heterozygosity, involving a nonsense mutation in exon 1 and a splice site mutation in intron 2. The splice site mutation causes an in-frame skipping of the second exon and is predicted to result in production of an aberrant protein (Ebermann et al., 2007a). In the Portuguese family a deletion mutation was detected in exon 2 which is predicted to truncate the protein or mark the message for nonsense mediated decay (Audo et al., 2011). These mutations disrupt only the long isoform of WHRN.

Mice with a targeted deletion of the long isoform of whirlin have some partial hearing (75 dB SPL) which is non-progressive in nature (Yang et al., 2010) . The inner hair cells stereocilia are unaffected while the outer hair cells stereocilia loose their characteristic "V" shaped formation and assume a "U" shape. Some of the stereocilia are missing from the innermost row in outer hair cells. However, unlike *whirler* mutant mice who are deaf due to a deletion in *Whrn* disrupting both isoforms of the protein, mice with targeted ablation of the long isoform of whirlin do not have abnormally short stereocilia.

Genetics of Nonsyndromic Recessively Inherited

identify genetic modifiers in future.

locus for at least a subset of the participants.

help in identification of genetic modifiers in future.

important for modification of hearing loss.

to treat and cure deafness in future.

**3. Conclusions** 

Moderate to Severe and Progressive Deafness in Humans 263

A role of modifier genes in reducing severity of hearing loss is suspected for many other deafness loci as well, especially those showing a wide phenotypic variation in presence of identical genetic mutations. However, currently no locus has been mapped or any gene identified as a potential modifier. Usually each family has few individuals with sufficiently different phenotypes to make gene mapping studies feasible. Using whole genome sequencing approaches in either a small subset of such individuals or in larger families may

*GJB2* is the most widely sequenced deafness genes and mutations in this gene are associated with deafness which is mild in degree to more profound losses. However, it has not been possible to map a genetic modifier which reduces severity of hearing loss using either traditional linkage analysis or association studies. A whole genome association study on DNA of more than a thousand *GJB2* c.35delG homozygous individuals living across Europe and North America regions failed to identify a single locus as a modifier in individuals with mild phenotypes of hearing loss. However, some SNPs with smaller modifying effects on the phenotype were identified (Hilgert et al., 2009). It is possible that a more stringent ethnic definition and data re-analysis in a smaller group may succeed in mapping a single

The unavailability of different cell types derived from inner ear tissues in humans have inhibited direct evaluation of modifier genes in hearing loss by transcriptional analysis as for other disorders such as spinal muscular atrophy (Oprea et al., 2008). However, induced pluripotent stem cell lines can be derived from more and less severely affected deaf individuals respectively. These could be then differentiated *in vitro* into the relevant cochlear cell types followed by comparison of their mRNA expression profiles. This may

Modifiers of hearing loss identified in mice may also be eventually found to be implicated in humans. Currently, only two genes have been identified which result in complete rescue of hearing defects in mice. For example alleles of *Cdh23* and *Mtap1a* can rescue hearing loss in mice with mutations in *Atp2b2* and *Tub* respectively (reviewed by Yan & Liu, 2010). Targeted sequencing of these genes in humans may also identify comparable variants

Genes and loci continue to be identified in aetiology of moderate to severe and progressive deafness. Current research has revealed that different alleles of a deafness gene can cause less severe hearing loss or more profound deafness. Further work needs to be carried out to identify additional loci and genes for progressive deafness and those for less severe hearing loss phenotypes as well as modifiers in the genetic background that suppress or enhance hearing loss. The contribution of different genes to moderate to severe and progressive hearing loss also needs to be studied in different world populations. Additionally, it remains important to document hearing loss in families which have already been described to suffer from moderate to severe hearing loss in order to check for progression of hearing loss in future. Strategies need to be evolved for identification of modifiers which will elucidate molecular pathways involved in normal hearing. This may be of help in designing strategies

#### *CLRN1 (USH3A)*

The deafness phenotype associated with USH3 involves postlingual, progressive hearing loss. The time of onset and severity of hearing loss can be highly variable (Ness et al., 2003). So far only one gene, *CLRN1* has been identified which causes an USH3 phenotype (Joensuu et al., 2001). In contrast to other world populations, mutations in *CLRN1* are a frequent cause of Usher syndrome in the Finnish population. *Clrn1-/-* mice have early onset hearing loss and are profoundly deaf by P30 (Geng et al., 2009). *Clrn1-/-* mice have demonstrated that CLRN1 is important for normal maturation of hair cells as well as the afferent nerve synapses. Absence of CLRN1 in mice leads to gradual loss of outer hair cells and supporting cells which is most severe at the basal turn of the cochlea (Geng et al., 2009).

CLRN1 is a small 232 amino acid transmembrane protein which is present at both the basal and apical poles of the hair cells with expression being higher in outer hair cells as compared to the inner hair cells (Geng et al., 2009; Zallocchi et al., 2009). *Clrn1* is also expressed in the spiral ganglion (Geng et al., 2009; Zallocchi et al., 2009). In mice, CLRN1 cannot be detected at the apex of hair cells at P1 and continues to be expressed in both the pre- and post-synaptic regions of outer hair cell type I afferent ribbon synapses (Zallocchi et al., 2009). Additionally, CLRN1 may also have a role in actin assembly (Tian et al., 2009) and intracellular vesicle transport (Zallocchi et al., 2009).
