**2. Molecular genetics**

Most individuals with inherited hearing loss suffer from profound deafness. It is hypothesized that the degree of hearing loss is profound when mutations affect genes which cause hair cell loss while it may be less severe or progressive in nature when mutations disrupt genes which affect hair cell function or that of the tectorial membrane (Grillet et al., 2009). In other instances, presence of missense or nonsense mutations in the same gene have been associated with variability in degree of hearing loss (Pennings et al., 2004). Additionally, some mutations creating new cryptic splice sites within genes have also been associated with intra-familial variability of hearing loss (Lopez-Bigas et al., 1999). However, in some instances there are no genotype-phenotype correlations. For example individuals who are homozygous for the c.35delG mutation in *GJB2* have phenotypes ranging from congenital and profound to early onset and mild hearing loss (Denoyelle et al., 1997; Snoeckx et al., 2005). There are a few other reports of identical mutations in the same gene in different subjects causing a significantly dissimilar hearing loss. This is illustrated by individuals with mutations in *CDH23*, *CLDN14* or *TRIC*. Individuals who are homozygous for identical mutations in *CDH23*, *CLDN14* or *TRIC* exhibit different degrees of hearing loss (Bashir, Fatima & Naz, 2010b; Riazuddin et al., 2006; Schultz et al., 2005).

Less severe degree of hearing loss may also result when mutations create hypomorphic alleles or affect particular domains of proteins. For example, a hypomorphic allele is hypothesized to be responsible for the moderate to profound hearing loss in affected individuals at the *DFNB73* locus (Riazuddin et al., 2009). It has also been shown that mutations that specifically disrupt the long isofrom of MYO15A cause less severe hearing loss in contrast to mutations which disrupt function of both isoforms (Bashir et al., in press, Cengiz et al., 2010; Nal et al., 2010).

Intra- or inter- familal phenotypic variability is also observed due to progression of hearing loss (*DFNB7*, *DFNB8*, *DFNB25*, *DFNB30*, *DFNB59, DFNB72/95, DFNB77, DFNB79*, *DFNB84*  and *DFNB91*) (Charizopoulou et al., 2011; de Heer et al., 2011; Ebermann et al., 2007b; Grillet et al., 2009; Li et al., 2010; Rehman et al., 2011; Schraders et al., 2010a; Schraders et al., 2010b; Sirmaci et al., 2010; Walsh et al., 2002; Weegerink et al., 2011). Younger individuals with mutations in *BSND* (*DFNB73*) also have a less severe degree of hearing loss which suggests a progressive nature of their hearing loss (Riazuddin et al., 2009). Other loci for which hearing loss is reported as less than profound are based on data from single families and the causative genes are unknown (*DFNB32*, *DFNB33*, *DFNB71, DFNB89, DFNB93*) (Basit et al., 2011; Belguith et al., 2009; Chishti et al., 2009; Masmoudi et al., 2003; Medlej-Hashim et al., 2002; Mustapha et al., 1998; Tabatabaiefar et al., 2011). For some deafness loci the degree of hearing loss was reported to be moderate to severe or severe in degree, but audiograms were not provided (*DFNB13*, *DFNB22, DFNB32*, *DFNB33* and *DFNB89*) (Basit et al., 2011; Belguith et al., 2009; Masmoudi et al., 2004; Masmoudi et al., 2003; Zwaenepoel et al., 2002). The known instances in which the degree of hearing loss is less than profound or can progress to different degrees are summarized in Table 1.

a role for specific additional epistatic interactions which can modify the hearing loss in

Most individuals with inherited hearing loss suffer from profound deafness. It is hypothesized that the degree of hearing loss is profound when mutations affect genes which cause hair cell loss while it may be less severe or progressive in nature when mutations disrupt genes which affect hair cell function or that of the tectorial membrane (Grillet et al., 2009). In other instances, presence of missense or nonsense mutations in the same gene have been associated with variability in degree of hearing loss (Pennings et al., 2004). Additionally, some mutations creating new cryptic splice sites within genes have also been associated with intra-familial variability of hearing loss (Lopez-Bigas et al., 1999). However, in some instances there are no genotype-phenotype correlations. For example individuals who are homozygous for the c.35delG mutation in *GJB2* have phenotypes ranging from congenital and profound to early onset and mild hearing loss (Denoyelle et al., 1997; Snoeckx et al., 2005). There are a few other reports of identical mutations in the same gene in different subjects causing a significantly dissimilar hearing loss. This is illustrated by individuals with mutations in *CDH23*, *CLDN14* or *TRIC*. Individuals who are homozygous for identical mutations in *CDH23*, *CLDN14* or *TRIC* exhibit different degrees of hearing loss

Less severe degree of hearing loss may also result when mutations create hypomorphic alleles or affect particular domains of proteins. For example, a hypomorphic allele is hypothesized to be responsible for the moderate to profound hearing loss in affected individuals at the *DFNB73* locus (Riazuddin et al., 2009). It has also been shown that mutations that specifically disrupt the long isofrom of MYO15A cause less severe hearing loss in contrast to mutations which disrupt function of both isoforms (Bashir et al., in press,

Intra- or inter- familal phenotypic variability is also observed due to progression of hearing loss (*DFNB7*, *DFNB8*, *DFNB25*, *DFNB30*, *DFNB59, DFNB72/95, DFNB77, DFNB79*, *DFNB84*  and *DFNB91*) (Charizopoulou et al., 2011; de Heer et al., 2011; Ebermann et al., 2007b; Grillet et al., 2009; Li et al., 2010; Rehman et al., 2011; Schraders et al., 2010a; Schraders et al., 2010b; Sirmaci et al., 2010; Walsh et al., 2002; Weegerink et al., 2011). Younger individuals with mutations in *BSND* (*DFNB73*) also have a less severe degree of hearing loss which suggests a progressive nature of their hearing loss (Riazuddin et al., 2009). Other loci for which hearing loss is reported as less than profound are based on data from single families and the causative genes are unknown (*DFNB32*, *DFNB33*, *DFNB71, DFNB89, DFNB93*) (Basit et al., 2011; Belguith et al., 2009; Chishti et al., 2009; Masmoudi et al., 2003; Medlej-Hashim et al., 2002; Mustapha et al., 1998; Tabatabaiefar et al., 2011). For some deafness loci the degree of hearing loss was reported to be moderate to severe or severe in degree, but audiograms were not provided (*DFNB13*, *DFNB22, DFNB32*, *DFNB33* and *DFNB89*) (Basit et al., 2011; Belguith et al., 2009; Masmoudi et al., 2004; Masmoudi et al., 2003; Zwaenepoel et al., 2002). The known instances in which the degree of hearing loss is less than profound or can

(Bashir, Fatima & Naz, 2010b; Riazuddin et al., 2006; Schultz et al., 2005).

some instances.

**2. Molecular genetics** 

Cengiz et al., 2010; Nal et al., 2010).

progress to different degrees are summarized in Table 1.


Genetics of Nonsyndromic Recessively Inherited

Δ*ENT/*Δ

inactivating *TECTA* mutations (Naz et al. 2003, unpublished data).

*TECTA (DFNB21)* 

*OTOA (DFNB22)* 

(Legan et al., 2000). *Tecta*

noncollagenous matrix is missing. *Tecta*

Moderate to Severe and Progressive Deafness in Humans 251

Inactivating mutations of *TECTA* lead to moderate to severe recessively inherited hearing loss in humans which can be more severe in the mid frequencies leading to a flat or U shaped audiogram (Meyer et al., 2007; Naz et al., 2003). *TECTA* is the major protein of the tectorial membrane which lies over the organ of Corti within the cochlea. The tectorial membrane is in contact with the tallest stereocilia of the outer hair cells and acts as a cochlear amplifier which was elegantly shown in mice with a targeted mutation in *Tecta* 

> Δ*ENT/*Δ

However DPOAE can be elicited at high threshold sounds of 65 dB SPL in *Tecta*

between sites of attachment of tectorial membrane and underlying sensory epithelia.

There are five chromosomal regions which have been implicated in genetics of moderate to severe hearing loss and the genes are currently unknown. Although *DFNB32* and *DFNB82* were mapped to overlapping regions on chromosome 1, the identification of *GPSM2*  mutations for the latter (Walsh et al., 2010) excluded this as the causative gene for the former

Five loci for less severe hearing loss have been mapped to chromosomes 1p13.3-22.1, 10p11.23-q21.1, 8p22-21.3, 16q21-q23.2 and 11q12.3-13.3 respectively (Belguith et al., 2009; Chishti et al., 2009; Masmoudi et al., 2003; Tabatabaiefar et al., 2011). All loci have been described in single families except for *DFNB89* for which two families were reported. Deafness was described to be moderate to severe in degree in all affected individuals in these two families but no audiometric data was provided. Similarly, hearing loss is described as being severe in degree for families linked to both *DFNB32* and *DFNB33* without provision of audiometric data. Patients in families described for *DFNB71* and *DFNB93* have severe and moderate to severe hearing loss respectively as documented by 1 and 4 audiograms respectively. The identification of genes involved in pathogenesis due to mutations at these loci will shed light on their essential functions in the auditory pathways.

**2.1b Loci involved in moderate to severe or severe hearing loss** 

*DFNB32, DFNB33, DFNB71, DFNB89, DFNB93* 

since it lies outside the linkage interval of *DFNB32* (Masmoudi et al., 2003).

mice (Lukashkin et al., 2004) though these are absent in humans with homozygous

Two Palestinian families have been reported in which the affected individuals had hearing loss due to deleterious mutations in the gene encoding otoancorin, *OTOA* (Shahin et al., 2010b; Zwaenepoel et al., 2002*)*. The hearing loss was reported to be moderate to severe in one family while it was profound in the other. One of the families had a splice site mutation in *OTOA* (Zwaenepoel et al., 2002) while the second family with members affected with profound deafness had a complete deletion of the gene (Shahin et al., 2010b; Zwaenepoel et al., 2002). *OTOA* has sequence similarity to *STRC* (Jovine, Park & Wassarman, 2002) although its expression pattern is different*.* Both OTOA and STRC are predicted to be superhelical lectins which can bind the carbohydrate moieties of extracellular glycoproteins (Sathyanarayana et al., 2009). In mice, *Otoa* expression is restricted to specific regions

*ENT* mice have detached tectorial membranes and the

*ENT* mutants are 35 dB less sensitive to sound*.*

Δ*ENT/*Δ*ENT*


Table 1. List of nonsyndromic recessive deafness loci associated with less than profound deafness

The table lists autosomal nonsyndromic recessive deafness loci for which at least two individuals are reported to have a less severe hearing loss (<80 dB). HL, Hearing loss. \* Only one patient has a dramatically less severe degree of hearing loss.

### **2.1a Genes involved in moderate to severe hearing loss**

Three genes have been identified in which mutations exclusively cause recessively inherited moderate to severe hearing loss in humans. Interestingly, they are either part of the tectorial membrane, or are in direct contact with it. The tectorial membrane acts as the cochlear amplifier and results in gain in sound intensity by 30 dB. No progression has been documented for hearing loss due to mutations in the three genes, *STRC*, *TECTA* and *OTOA*, although some variability in degree of auditory thresholds is observed in affected individuals with identical mutations in these genes.

#### *STRC (DFNB16)*

Mutations in *STRC* encoding stereocilin are reported to cause mild to severe deafness in humans with an onset in childhood at 3-5years with increased involvement of high frequencies (Verpy et al., 2001; Villamar et al., 1999). Additionally, mice with a targeted deletion of *Strc* become progressively deaf by P60 (Verpy et al., 2008). The DPOAE cannot be recorded at P14 in *Strc-/-* mice though hearing thresholds are almost normal at that age. *Strc-/-* mice lack horizontal top connectors of outer hair cells' stereocilia. It is interesting to note that stereocilin may establish contact with tectorial membrane as inferred by lack of characteristic ring like staining of STRC from tallest row of outer hair cell stereocilia in *Tecta*Δ*ENT/*Δ*ENT* mice which have disrupted tectorial membranes due to loss of TECTA (Verpy et al., 2008).

### *TECTA (DFNB21)*

250 Hearing Loss

(Charizopoulou et al., 2011; Rehman et al.,

(Riazuddin et al., 2009)

(Schraders et al., 2010a; Shahin et al., 2010a)

(Li et al., 2010)

(Basit et al., 2011)

2011)

2011)

**LOCUS GENE PHENOTYPE REFERENCE**  *DFNB71* Unknown Severe HL (One audiogram provided) (Chishti et al., 2009)

> Individuals with a C terminal mutation have progressive HL

*DFNB77 LOXHD1* Progressive HL in one family (Grillet et al., 2009)

Moderate to severe HL in another

*DFNB91 SERPINB6* Progressive HL (Sirmaci et al., 2010) *DFNB93* Unknown Moderate to Severe HL (Tabatabaiefar et al.,

Table 1. List of nonsyndromic recessive deafness loci associated with less than profound

The table lists autosomal nonsyndromic recessive deafness loci for which at least two individuals are reported to have a less severe hearing loss (<80 dB). HL, Hearing loss. \* Only

Three genes have been identified in which mutations exclusively cause recessively inherited moderate to severe hearing loss in humans. Interestingly, they are either part of the tectorial membrane, or are in direct contact with it. The tectorial membrane acts as the cochlear amplifier and results in gain in sound intensity by 30 dB. No progression has been documented for hearing loss due to mutations in the three genes, *STRC*, *TECTA* and *OTOA*, although some variability in degree of auditory thresholds is observed in affected

Mutations in *STRC* encoding stereocilin are reported to cause mild to severe deafness in humans with an onset in childhood at 3-5years with increased involvement of high frequencies (Verpy et al., 2001; Villamar et al., 1999). Additionally, mice with a targeted deletion of *Strc* become progressively deaf by P60 (Verpy et al., 2008). The DPOAE cannot be recorded at P14 in *Strc-/-* mice though hearing thresholds are almost normal at that age. *Strc-/-* mice lack horizontal top connectors of outer hair cells' stereocilia. It is interesting to note that stereocilin may establish contact with tectorial membrane as inferred by lack of characteristic ring like staining of STRC from tallest row of outer hair cell stereocilia in

*ENT* mice which have disrupted tectorial membranes due to loss of TECTA (Verpy

*DFNB72/95 GIPC3* 2 families with mild to severe HL,

*DFNB73 BSND* Younger individuals have less severe degree of HL

*DFNB84 PTPRQ* Progressive HL or Moderate to severe HL

one patient has a dramatically less severe degree of hearing loss.

**2.1a Genes involved in moderate to severe hearing loss** 

individuals with identical mutations in these genes.

audiograms provided

*DFNB79 TPRN* Progressive HL in one family,

*DFNB89* Unknown Moderate to severe HL, no

deafness

*STRC (DFNB16)* 

*Tecta*Δ*ENT/*Δ

et al., 2008).

Inactivating mutations of *TECTA* lead to moderate to severe recessively inherited hearing loss in humans which can be more severe in the mid frequencies leading to a flat or U shaped audiogram (Meyer et al., 2007; Naz et al., 2003). *TECTA* is the major protein of the tectorial membrane which lies over the organ of Corti within the cochlea. The tectorial membrane is in contact with the tallest stereocilia of the outer hair cells and acts as a cochlear amplifier which was elegantly shown in mice with a targeted mutation in *Tecta*  (Legan et al., 2000). *Tecta*Δ*ENT/*Δ*ENT* mice have detached tectorial membranes and the noncollagenous matrix is missing. *Tecta*Δ*ENT/*Δ*ENT* mutants are 35 dB less sensitive to sound*.* However DPOAE can be elicited at high threshold sounds of 65 dB SPL in *Tecta*Δ*ENT/*Δ*ENT* mice (Lukashkin et al., 2004) though these are absent in humans with homozygous inactivating *TECTA* mutations (Naz et al. 2003, unpublished data).

#### *OTOA (DFNB22)*

Two Palestinian families have been reported in which the affected individuals had hearing loss due to deleterious mutations in the gene encoding otoancorin, *OTOA* (Shahin et al., 2010b; Zwaenepoel et al., 2002*)*. The hearing loss was reported to be moderate to severe in one family while it was profound in the other. One of the families had a splice site mutation in *OTOA* (Zwaenepoel et al., 2002) while the second family with members affected with profound deafness had a complete deletion of the gene (Shahin et al., 2010b; Zwaenepoel et al., 2002). *OTOA* has sequence similarity to *STRC* (Jovine, Park & Wassarman, 2002) although its expression pattern is different*.* Both OTOA and STRC are predicted to be superhelical lectins which can bind the carbohydrate moieties of extracellular glycoproteins (Sathyanarayana et al., 2009). In mice, *Otoa* expression is restricted to specific regions between sites of attachment of tectorial membrane and underlying sensory epithelia.

### **2.1b Loci involved in moderate to severe or severe hearing loss**

There are five chromosomal regions which have been implicated in genetics of moderate to severe hearing loss and the genes are currently unknown. Although *DFNB32* and *DFNB82* were mapped to overlapping regions on chromosome 1, the identification of *GPSM2*  mutations for the latter (Walsh et al., 2010) excluded this as the causative gene for the former since it lies outside the linkage interval of *DFNB32* (Masmoudi et al., 2003).

#### *DFNB32, DFNB33, DFNB71, DFNB89, DFNB93*

Five loci for less severe hearing loss have been mapped to chromosomes 1p13.3-22.1, 10p11.23-q21.1, 8p22-21.3, 16q21-q23.2 and 11q12.3-13.3 respectively (Belguith et al., 2009; Chishti et al., 2009; Masmoudi et al., 2003; Tabatabaiefar et al., 2011). All loci have been described in single families except for *DFNB89* for which two families were reported. Deafness was described to be moderate to severe in degree in all affected individuals in these two families but no audiometric data was provided. Similarly, hearing loss is described as being severe in degree for families linked to both *DFNB32* and *DFNB33* without provision of audiometric data. Patients in families described for *DFNB71* and *DFNB93* have severe and moderate to severe hearing loss respectively as documented by 1 and 4 audiograms respectively. The identification of genes involved in pathogenesis due to mutations at these loci will shed light on their essential functions in the auditory pathways.

Genetics of Nonsyndromic Recessively Inherited

progressive hearing loss (Luxon et al., 2003).

hearing in Tg[E];Tg[R];*Slc26a4*<sup>Δ</sup>/<sup>Δ</sup>

*CLDN14 (DFNB29)* 

(Ben-Yosef et al., 2003).

*ILDR1 (DFNB42)* 

MYO15A isoform 1 and its function remains to be elucidated.

isoform (Mburu et al., 2003)*.*

*SLC26A4 (DFNB4/PDS)* 

Moderate to Severe and Progressive Deafness in Humans 253

interesting to note that mutations affecting the long isoform of WHRN (Ebermann et al., 2007a) also cause a less severe hearing loss in contrast to mutations which disrupt the short

Currently, no corresponding mouse carrying a mutation affecting the long N terminal extension domain has been reported. However, in *shaker 2* mice, a missense mutation affects the motor domain of MYO15A and the mice are profoundly deaf (Probst et al., 1998) . The stereocilia are extremely short and this defect can be fully rescued by transfecting *shaker 2* hair cells with MYO15A isoform 2 (Belyantseva et al., 2005). No specific role is known for

*SLC26A4* mutations may cause Pendred syndrome or non-syndromic deafness with enlarged vestibular aqueducts. Considerable residual hearing is present in some affected individuals who are homozygous for mutations creating new splice sites in *SLC26A4*  (Lopez-Bigas et al., 1999; Naz, 2001). These two described mutations create splice sites a few nucleotides away from the canonical donor sites, and support the hypothesis that variable degree of hearing loss in *SLC26A4* linked families may indicate splice site mutations. Additionally, a few missense mutations of *SLC26A4* also cause a significantly less severe hearing loss (Kitamura et al., 2000). Some mutations of *SLC26A4* result in development of a

SlC26A4 or pendrin is expressed in endolymphatic duct and sac as well as in the external sulcus in the cochlea (Everett et al., 1999)*.* SLC26A4 plays a significant role in the maintenance of the ionic balance within the inner ear and is involved in bicarbonate secretion (Wangemann et al., 2007)*.* There are three different mouse mutants of *Slc26a4* which lack pendrin but none of them model the less severe hearing loss observed in humans (Dror et al., 2010; Everett et al., 2001; Lu et al., 2011). However, recently, transgenic mice with *Slc26a4* expression inducible by doxycycline on a background of mice lacking endogenous pendrin expression were generated. It was demonstrated that expression of pendrin at early embryonic stages of E0-E17.5 was necessary and sufficient to restore normal

this critical time results in complete or partial hearing loss in these mice, recapitatulating the phenotypes documented for many patients with mutations in *SLC26A4* (Choi et al., 2011).

CLDN14 is an integral part of the tight junctions in the sensory epithelium within the inner ear. Only two mutations have been reported in this gene in four Pakistani families which cause hearing loss. The usual phenotype associated with the two mutations is severe to profound deafness (Wilcox et al., 2001). However members of one family with p.V85D mutation in *CLDN14* have hearing loss which varies from moderately severe to severe in degree (Bashir et al., 2010b). It is interesting to note that mice with a targeted deletion of *Cldn14* are profoundly deaf and no variability of hearing loss was observed in these mice

ILDR1 is a membrane protein with an Immunoglobulin-like extracellular domain, and has different motifs for interaction with other proteins. Additionally, soluble isoforms of ILDR1

mice (Choi et al., 2011). Ablating expression of *Slc26A4* at

#### **2.2 Genes involved in Intra- or inter- familial variability of hearing loss**

#### *GJB2 (DFNB1)*

In the cochlea, gap junctions are proposed to maintain K+ homeostasis by transporting K+ away from the hair cells during auditory transduction (Kikuchi et al., 1995)*. GJB2* encodes connexin 26 which oligomerizes to form a connexon (a hemichannel) which binds to a connexon from adjacent cell to form a functional gap junction in many tissues including the inner ear. *GJB2* is expressed in the supporting cells and the stria vascularis in the cochlea. The important function of *GJB2* in normal hearing is shown by the large number of mutations which have been reported in this gene from most diverse human populations which cause deafness. Although most individuals who are homozygous for c.35delG mutation in *GJB2* have severe to profound deafness, many individuals with the identical mutation have a mild or a less severe hearing loss (Murgia et al., 1999). Additionally, patients who are compound heterozygous for one truncating mutation together with a missense mutation in *GJB2* usually have a less severe hearing impairment (Liu et al., 2005; Snoeckx et al., 2005).

#### *MYO7A (DFNB2)*

*MYO7A* encodes a protein classified as an unconventional myosin which plays a role in intracellular trafficking. Unconventional myosins are actin-activated motor proteins with structurally conserved heads important for movement along actin filaments. The tails are highly divergent and are presumed to interact with different macromolecular components in the cell. All mutations in *MYO7A* except one cause severe to profound deafness. However, an individual with a missense mutation affecting the motor domain of MYO7A had a dramatically reduced hearing loss as compared to all other cases with *MYO7A* mutations, including those in his own family (Hildebrand et al., 2010). The onset of deafness was delayed to seven years of age and the degree of hearing loss was moderate to severe at the age of 31.

*MYO7A* is present in cytoplasm of hair cells and in the stereocilia including the upper tiplink density (Grati & Kachar, 2011). Different mutations of *Myo7a* result in profound deafness in mice (Gibson et al., 1995). However, one missense mutation affecting the kinesin and MyTH4 domains of myosin 7a leads to a severe deafness phenotype in contrast to other mice with mutations in *Myo7a* (Mburu et al., 1997).

#### *MYO15A (DFNB3)*

Mutations in *MYO15A* are a significant cause of deafness in many world populations. All pathogenic mutations in *MYO15A* except three which are located in exon 2 cause profound deafness. The four mutations were identified in three Pakistani and one Turkish families and are associated with hearing loss which can range from moderate to severe or moderate to profound in degree (Bashir et al., in press, Cengiz et al., 2010; Nal et al., 2010). The mutations in alternatively spliced exon 2 affect the class 1 isoform of MYO15A which has a long N-terminal extension. The presence of residual hearing in affected individuals who have mutations in exon 2 of *MYO15A* is probably due to the availability of normally functioning short isoform of MYO15A which remains unaffected by the mutations in exon 2 (Nal et al., 2007).

MYO15A is a motor protein present in hair cells in a cap like structure at top of the stereocilia where it is known to interact with WHRN (Belyantseva et al., 2005)*.* It is interesting to note that mutations affecting the long isoform of WHRN (Ebermann et al., 2007a) also cause a less severe hearing loss in contrast to mutations which disrupt the short isoform (Mburu et al., 2003)*.*

Currently, no corresponding mouse carrying a mutation affecting the long N terminal extension domain has been reported. However, in *shaker 2* mice, a missense mutation affects the motor domain of MYO15A and the mice are profoundly deaf (Probst et al., 1998) . The stereocilia are extremely short and this defect can be fully rescued by transfecting *shaker 2* hair cells with MYO15A isoform 2 (Belyantseva et al., 2005). No specific role is known for MYO15A isoform 1 and its function remains to be elucidated.

### *SLC26A4 (DFNB4/PDS)*

252 Hearing Loss

In the cochlea, gap junctions are proposed to maintain K+ homeostasis by transporting K+ away from the hair cells during auditory transduction (Kikuchi et al., 1995)*. GJB2* encodes connexin 26 which oligomerizes to form a connexon (a hemichannel) which binds to a connexon from adjacent cell to form a functional gap junction in many tissues including the inner ear. *GJB2* is expressed in the supporting cells and the stria vascularis in the cochlea. The important function of *GJB2* in normal hearing is shown by the large number of mutations which have been reported in this gene from most diverse human populations which cause deafness. Although most individuals who are homozygous for c.35delG mutation in *GJB2* have severe to profound deafness, many individuals with the identical mutation have a mild or a less severe hearing loss (Murgia et al., 1999). Additionally, patients who are compound heterozygous for one truncating mutation together with a missense mutation in *GJB2* usually have a less severe hearing impairment (Liu et al., 2005;

*MYO7A* encodes a protein classified as an unconventional myosin which plays a role in intracellular trafficking. Unconventional myosins are actin-activated motor proteins with structurally conserved heads important for movement along actin filaments. The tails are highly divergent and are presumed to interact with different macromolecular components in the cell. All mutations in *MYO7A* except one cause severe to profound deafness. However, an individual with a missense mutation affecting the motor domain of MYO7A had a dramatically reduced hearing loss as compared to all other cases with *MYO7A* mutations, including those in his own family (Hildebrand et al., 2010). The onset of deafness was delayed to seven years of age and the degree of hearing loss was moderate to severe at the age of 31. *MYO7A* is present in cytoplasm of hair cells and in the stereocilia including the upper tiplink density (Grati & Kachar, 2011). Different mutations of *Myo7a* result in profound deafness in mice (Gibson et al., 1995). However, one missense mutation affecting the kinesin and MyTH4 domains of myosin 7a leads to a severe deafness phenotype in contrast to other

Mutations in *MYO15A* are a significant cause of deafness in many world populations. All pathogenic mutations in *MYO15A* except three which are located in exon 2 cause profound deafness. The four mutations were identified in three Pakistani and one Turkish families and are associated with hearing loss which can range from moderate to severe or moderate to profound in degree (Bashir et al., in press, Cengiz et al., 2010; Nal et al., 2010). The mutations in alternatively spliced exon 2 affect the class 1 isoform of MYO15A which has a long N-terminal extension. The presence of residual hearing in affected individuals who have mutations in exon 2 of *MYO15A* is probably due to the availability of normally functioning short isoform of MYO15A which remains unaffected by the mutations in exon 2

MYO15A is a motor protein present in hair cells in a cap like structure at top of the stereocilia where it is known to interact with WHRN (Belyantseva et al., 2005)*.* It is

**2.2 Genes involved in Intra- or inter- familial variability of hearing loss** 

*GJB2 (DFNB1)* 

Snoeckx et al., 2005). *MYO7A (DFNB2)* 

*MYO15A (DFNB3)* 

(Nal et al., 2007).

mice with mutations in *Myo7a* (Mburu et al., 1997).

*SLC26A4* mutations may cause Pendred syndrome or non-syndromic deafness with enlarged vestibular aqueducts. Considerable residual hearing is present in some affected individuals who are homozygous for mutations creating new splice sites in *SLC26A4*  (Lopez-Bigas et al., 1999; Naz, 2001). These two described mutations create splice sites a few nucleotides away from the canonical donor sites, and support the hypothesis that variable degree of hearing loss in *SLC26A4* linked families may indicate splice site mutations. Additionally, a few missense mutations of *SLC26A4* also cause a significantly less severe hearing loss (Kitamura et al., 2000). Some mutations of *SLC26A4* result in development of a progressive hearing loss (Luxon et al., 2003).

SlC26A4 or pendrin is expressed in endolymphatic duct and sac as well as in the external sulcus in the cochlea (Everett et al., 1999)*.* SLC26A4 plays a significant role in the maintenance of the ionic balance within the inner ear and is involved in bicarbonate secretion (Wangemann et al., 2007)*.* There are three different mouse mutants of *Slc26a4* which lack pendrin but none of them model the less severe hearing loss observed in humans (Dror et al., 2010; Everett et al., 2001; Lu et al., 2011). However, recently, transgenic mice with *Slc26a4* expression inducible by doxycycline on a background of mice lacking endogenous pendrin expression were generated. It was demonstrated that expression of pendrin at early embryonic stages of E0-E17.5 was necessary and sufficient to restore normal hearing in Tg[E];Tg[R];*Slc26a4*<sup>Δ</sup>/<sup>Δ</sup> mice (Choi et al., 2011). Ablating expression of *Slc26A4* at this critical time results in complete or partial hearing loss in these mice, recapitatulating the phenotypes documented for many patients with mutations in *SLC26A4* (Choi et al., 2011).

#### *CLDN14 (DFNB29)*

CLDN14 is an integral part of the tight junctions in the sensory epithelium within the inner ear. Only two mutations have been reported in this gene in four Pakistani families which cause hearing loss. The usual phenotype associated with the two mutations is severe to profound deafness (Wilcox et al., 2001). However members of one family with p.V85D mutation in *CLDN14* have hearing loss which varies from moderately severe to severe in degree (Bashir et al., 2010b). It is interesting to note that mice with a targeted deletion of *Cldn14* are profoundly deaf and no variability of hearing loss was observed in these mice (Ben-Yosef et al., 2003).

### *ILDR1 (DFNB42)*

ILDR1 is a membrane protein with an Immunoglobulin-like extracellular domain, and has different motifs for interaction with other proteins. Additionally, soluble isoforms of ILDR1

Genetics of Nonsyndromic Recessively Inherited

*TMPRSS3 (DFNB8)* 

observed in the affected individuals.

(Weegerink et al., 2011).

*CDH23 (DFNB12)* 

progressive hearing loss as observed in humans.

mice have a missense mutation in *Tmc1* (Vreugde et al., 2002).

Moderate to Severe and Progressive Deafness in Humans 255

TMC1 is a transmembrane protein present in hair cells and may be involved in intracellular protein trafficking. Additionally it is also proposed to play a role in differentiation of hair cells into functional auditory receptors. The *deafness* mice mutants carry a homozygous genomic deletion in *Tmc1* and are profoundly deaf (Kurima et al., 2002). Currently there is no mouse model which mimics the recessively inherited progressive hearing loss due to mutations in *Tmc1* though a model, *Beethoven* exists for dominant deafness *DFNA36* and the

The gene encoding serine protease, TMRSS3 is expressed in supporting cells, stria vasularis as well as in the spiral ganglion in the inner ear. Mutations in *TMPRSS3* are responsible for deafness at the *DFNB8* locus. Most mutations in *TMPRSS3* result in severe to profound deafness in many world populations. However there are many mutations in *TMPRSS3*  causing less severe hearing loss which is progressive in nature. The first family with progression in hearing loss due to a *TMPRSS3* mutation was reported from Pakistan. Affected members in this family suffered from progressive deafness with onset of hearing loss in childhood and had a mutation at a splice acceptor site in intron 4 (Scott et al., 2001; Veske et al., 1996). This is predicted to create an alternative splice acceptor site which on use introduces a frameshift in the open reading frame of *TMPRSS3*. It is hypothesized that this mutation may allow limited normal splicing since the actual splice site remains unchanged. Thus some normal TMPRSS3 could be produced accounting for progressive hearing loss

There are other reports of mutations in *TMPRSS3* causing post-lingual progressive deafness in British, Turkish, German and Dutch families (Elbracht et al., 2007; Hutchin et al., 2005; Wattenhofer et al., 2005; Weegerink et al., 2011). One of the first studies reported a British family with two affected individuals who were homozygous for a missense mutation in *TMPRSS3* and suffered from moderate to severe hearing loss (Hutchin et al., 2005). A recent study involved 8 small nuclear families from Holland and affected individuals were compound heterozygous for different mutations of *TMPRSS3* including missense and frameshift mutations (Weegerink et al., 2011). The higher frequencies were affected first resulting in a distinctive "ski-slope" audiometric configuration followed by low frequency hearing loss resulting in a flat audiogram (Weegerink et al., 2011). It was hypothesized that some mutations in *TMPRSS3* result in creation of hypomorphic alleles accounting for the less severe loss in hearing and progression of deafness observed in affected individuals

*Tmprss3* was shown to be important for hearing in mice as well since mutants homozygous for a nonsense mutation in the gene suffer from deafness. The hair cells start to degenerate at P12 from basal to apical turn of the cochlea and the degeneration is complete by P14 (Fasquelle et al., 2011). However, no mouse model has been reported which mimics the

Most mutations of *CDH23* cause severe phenotypes of deafness or *USH1D*. However, a few missense mutations in *CDH23* when present together in compound heterozygosity are reported to cause moderate to severe hearing loss or severe to profound deafness which is

also exist which may be involved in lipoprotein transport (Borck et al., 2011b). Hearing loss due to mutations of this gene varies from moderate to profound in different individuals while it is severe in degree for one family with a mutation affecting the start codon of *ILDR1* (Borck et al., 2011b)*. Ildr1* is expressed in the developing mouse cochlea but the expression is low at birth. It increases gradually by P4 and P10. The pillar and Hensen cells have the highest expression of *Ildr1* while it can also be detected in other cells in organ of Corti including the hair cells (Borck et al., 2011b).

#### *TRIC (DFNB49)*

*TRIC* or *MARVELD2* encodes a tight junction protein with a ubiquitous expression in the epithelial junctions throughout the body tissues. In the inner ear, TRIC is specifically expressed in the tricellular junctions of sensory epithelia as well as those between supporting cells and the hair cells (Riazuddin et al., 2006). Affected individuals with identical mutations in *TRIC* show a wide range of variability in severity of deafness ranging from moderate to severe hearing loss to profound deafness (Chishti et al., 2008; Riazuddin et al., 2006). All mutations described in *TRIC* are predicted to produce truncated proteins and consequent inability to bind several scaffolding proteins.
