**2.4b Atypical manifestations of hearing loss in Usher syndrome**

Atypical USH is genetically heterogeneous*.* Two different mutations of *SANS* (*USH1G*) have been reported to cause a USH2 like phenotype in two consanguineous families from Turkey and Pakistan (Bashir, Fatima & Naz, 2010a; Kalay et al., 2005). Similarly, some mutations in *MYO7A* (*USH1B*) cause an USH3 phenotype (Liu et al., 1998). Additionally, two mutations in *CDH23* (*USH1D*), result in an atypical phenotype of milder USH3. These involve a missense and an intronic mutation which are compound heterozygous in a German family with two affected siblings (Astuto et al., 2002). Some USH2A mutations also cause atypical manifestations and the phenotype resembles that of USH3 (Liu et al., 1999). On the converse, it is interesting to note that though majority of the mutations in CLRN1 cause a USH3 phenotype, some mutations in the same gene cause a more severe USH1 phenotype (Aller et al., 2004; Pennings et al., 2003) or in some cases an USH2 phenotype (Sadeghi et al., 2005). The finding of identical mutations in some cases which cause both the less severe or the more profound USH phenotype suggest the importance of genetic background in manifestation of the disorder and a role of modifiers in its aetiology.

#### **2.5 Modifiers in less severe hearing loss**

To date a single modifier, *DFNM1*, has been mapped to chromosome 1 which completely ameliorates hearing loss (Riazuddin et al., 2000) but the gene is currently unknown. Targeted sequencing of the linked region from DNA of both affected and unaffected individuals using massively parallel sequencing technology may identify the causative change in future.

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

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

Atypical USH is genetically heterogeneous*.* Two different mutations of *SANS* (*USH1G*) have been reported to cause a USH2 like phenotype in two consanguineous families from Turkey and Pakistan (Bashir, Fatima & Naz, 2010a; Kalay et al., 2005). Similarly, some mutations in *MYO7A* (*USH1B*) cause an USH3 phenotype (Liu et al., 1998). Additionally, two mutations in *CDH23* (*USH1D*), result in an atypical phenotype of milder USH3. These involve a missense and an intronic mutation which are compound heterozygous in a German family with two affected siblings (Astuto et al., 2002). Some USH2A mutations also cause atypical manifestations and the phenotype resembles that of USH3 (Liu et al., 1999). On the converse, it is interesting to note that though majority of the mutations in CLRN1 cause a USH3 phenotype, some mutations in the same gene cause a more severe USH1 phenotype (Aller et al., 2004; Pennings et al., 2003) or in some cases an USH2 phenotype (Sadeghi et al., 2005). The finding of identical mutations in some cases which cause both the less severe or the more profound USH phenotype suggest the importance of genetic background in manifestation of the disorder and a role of modifiers in its

To date a single modifier, *DFNM1*, has been mapped to chromosome 1 which completely ameliorates hearing loss (Riazuddin et al., 2000) but the gene is currently unknown. Targeted sequencing of the linked region from DNA of both affected and unaffected individuals using massively parallel sequencing technology may identify the causative

intracellular vesicle transport (Zallocchi et al., 2009).

**2.5 Modifiers in less severe hearing loss** 

**2.4b Atypical manifestations of hearing loss in Usher syndrome** 

*CLRN1 (USH3A)* 

al., 2009).

aetiology.

change in future.

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 identify genetic modifiers in future.

*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 locus for at least a subset of the participants.

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 help in identification of genetic modifiers in future.

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 important for modification of hearing loss.
