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Genetic Hearing Loss
3.07 Genetic Hearing Loss B J B Keats, Louisiana State University Health Sciences Center, New Orleans, LA, USA ª 2008 Elsevier Inc. All rights reserved.
3.07.1 3.07.2 3.07.2.1 3.07.2.2 3.07.2.3 3.07.3 3.07.3.1 3.07.3.2 3.07.3.3 3.07.4 3.07.4.1 3.07.4.2 3.07.4.3 3.07.5 3.07.5.1 3.07.5.2 3.07.5.3 3.07.6 References
Introduction USH1C Structure, Expression, and Function Mutations Mouse Models GJB2 Structure, Expression, and Function Mutations Mouse Models OTOF Structure, Expression, and Function Mutations Mouse Models TECTA Structure, Expression, and Function Mutations Mouse Models Outlook for the Future
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Glossary codon A set of three bases that specifies a single amino acid or signals the termination of the amino acid sequence. compound heterozygote An individual with a recessive disorder who has a different mutation in the two copies of the gene (cf., homozygote). cryptic splice site A DNA sequence that is similar to a consensus splice site sequence but is not usually used unless there is a mutation in the normal splice site, or a mutation in the cryptic splice site results in it being preferred over the normal splice site. dominant negative The abnormal product of a mutant copy of a gene disrupts the function of the product of the normal copy of the gene. exon Segment of a gene that is transcribed and present in mature mRNA. founder effect A mutation that is present in a small ancestral population, which increases in frequency by chance due to the initial small population size. frameshift mutation An insertion or deletion of a number of base pairs that is not a multiple of three, and therefore changes the codons that follow.
genome The complete DNA sequence. For example, the nuclear genome of a human germ cell (sperm or ovum) consists of 3 million kb of DNA. genotype The two copies of the gene (one of each chromosome) that are present at a locus. haploinsufficiency The product of the normal copy of a gene is not adequate to prevent the disease when the mutant copy of the gene does not have a functional product. heterozygote An individual whose genotype consists of two different copies of the gene. homozygote An individual whose genotype consists of two identical copies of the gene. intron Segment of a gene that is not present in the mature mRNA. isoform Alternate forms of a protein. kilobase (kb) A unit of 1000 bases in a DNA sequence. linkage A statistical method of analysis of family phenotype and genotype data that provides the probability that two or more loci are not assorting independently (i.e., they are linked).
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locus The position of a gene on a chromosome. messenger RNA (mRNA) An RNA transcribed from the DNA of a gene, which directs the sequence in which amino acids are added to the product encoded by that gene. missense mutation A substitution of one base by another such that the amino acid specified by the codon is changed. modifier gene A gene that alters the phenotype associated with mutations in another gene. nonsense mutation A substitution of one base by another such that the resulting codon specifies termination of the amino acid sequence. penetrance The probability that a given genotype will result in a particular phenotype.
3.07.1 Introduction The human genome contains about 30 000 genes, and mutations in the DNA sequences of more than 100 of these genes are associated with hearing loss. The degree of genetic hearing loss may range from mild to profound and some frequencies may be affected more than others; for most individuals the loss is sensorineural, bilateral, and symmetric. Onset is often congenital (present at birth), but it can be later in childhood or in adulthood. Many different genetic forms of hearing loss may give similar pure tone air conduction audiograms. Thus, ideally, a battery of physiological measurements that include tympanometry, middle ear muscle reflexes, otoacoustic emissions (OAEs), speech audiometry, and auditory brainstem response/electrocochleography (ABR/EcochG) should be obtained for each patient in order to categorize the hearing loss accurately and determine the most effective management (Keats, B. J. B. and Berlin, C. I., 2002). About 30% of genetic hearing loss is syndromic, meaning that there are associated abnormalities in other organs such as the eye (Usher syndrome), kidney (Alport syndrome), skin (Waardenburg syndrome), heart (Jervell and Lange-Nielsen syndrome), and thyroid (Pendred syndrome). For the remaining 70%, the hearing loss is nonsyndromic. Griffith A. and Friedman T. (2002) and Toriello H. V. et al. (2004) provide comprehensive descriptions of syndromic and nonsyndromic hearing loss. The mode of inheritance of hearing loss in a family may be dominant or recessive, and the
phenocopy A phenotype that looks the same as one produced by a specific genotype, but has a different etiology. phenotype The observed characteristics of an individual. splice site The DNA sequence that specifies the boundary between an intron and an exon in a gene. The introns are removed in the generation of the mature mRNA. stop codon One of three codons specifying the termination of the product encoded by a gene. transcription The synthesis of a single-stranded mRNA molecule (transcript) from a DNA template. translation The synthesis of a sequence of amino acids from the mature mRNA template.
mutation may be in a gene on one of the autosomes (chromosomes 1–22) or on the X-chromosome; these chromosomes are in the nucleus of the cell. A pedigree showing who is affected and who is unaffected in the family helps to determine the inheritance pattern. Figure 1 shows an example of autosomal dominant inheritance in which individuals with one normal
Unaffected male Unaffected female Affected male Affected female
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Figure 1 A pedigree showing an autosomal-dominant pattern of inheritance with incomplete penetrance. N is the normal form of the gene and D is the abnormal (mutant) form of the gene. Individuals with the NN genotype are unaffected and those with the ND genotype are expected to be affected. However, the sixth individual in the middle generation is unaffected but has an affected mother and two affected children. Thus, her genotype must be ND. This is an example of incomplete penetrance meaning that for individuals with the ND genotype, the probability of being affected is less than 1.
Genetic Hearing Loss
copy (N) and one abnormal copy (D) of the gene are usually affected; their genotype is ND and each of their offspring has a 50% chance of inheriting the D. However, the phenotype may be variable, and penetrance may be incomplete, meaning that some ND individuals may show no signs of being affected. The hearing loss is autosomal recessive if only individuals with the DD genotype are affected. Those with the ND genotype are unaffected and are referred to as carriers (or heterozygotes). Each offspring of two carrier parents has a 25% chance of inheriting the DD genotype and being affected. Family history is quite likely to be negative for a child with autosomal recessive hearing loss. Conversely, both parents may have autosomal recessive hearing loss but all their children may have normal hearing because mutations in different genes are responsible for the hearing loss in the parents. If all the affected individuals in a pedigree are male, and none of the affected males have affected sons, then the inheritance pattern is likely to be X-linked recessive. In this case, each son of a carrier mother has a 50% chance of being affected and all the daughters of an affected male will be carriers. The symbols, DFNA, DFNB, and DFN, are used to designate autosomal dominant, autosomal recessive, and X-linked hearing loss, respectively, where the asterisk represents a number. The symbol is assigned when the chromosomal location of the hearing loss gene is determined, usually through genetic linkage analysis. For example, DFNB21 means the twenty-first autosomal recessive locus mapped. When the abnormal gene at the locus is identified, a gene symbol is assigned. For human genes, the symbol is written in uppercase and italics. As a generalization, recessive hearing losses tend to be prelingual while dominant forms are postlingual and progressive, but there are exceptions. In some families the phenotype may vary from one affected child to another. Such findings may be examples of variable expression, but the possibility of phenocopies must be considered. As well as the DNA in the nucleus, each of the several hundred mitochondria in a cell have multiple copies of their own DNA, and some forms of hearing loss are associated with mutations in this mitochondrial genome (mtDNA). Quite often the mutation is present in some copies of the mtDNA, but not others, a condition known as heteroplasmy. The number of base pairs of mtDNA is only about 16 000 compared with the three billion in the nuclear genome. Mitochondria are maternally inherited, so the pattern
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of inheritance is likely to be mitochondrial if all the offspring of an affected mother (but none of those of an affected father) are affected. However, because of heteroplasmy, the severity of the phenotype among the offspring may vary considerably. The majority of genetic hearing loss is autosomal recessive, but about 20% is autosomal dominant and 1–2% is X-linked or mitochondrial. Throughout the body, the set of genes that are expressed varies among cell types, and from one stage of development to another. The characteristics of genetic forms of hearing loss are likely to differ depending on the cellular and subcellular localizations, and the functions of the proteins encoded by the associated genes. These proteins must be essential for normal hearing. They include cytoskeletal proteins, extracellular matrix proteins, transcription factors, and proteins required for ion homeostasis. Because of the similarity between the mouse and human genomes, studies of mouse models are often quite informative in advancing understanding of genetic hearing loss. Many of the available mouse models are the result of spontaneous mutations, while others have been created using transgenic or gene knockout experimental approaches. The symbols for mouse genes are written in lowercase to differentiate them from human genes. This perspective will focus on four genes (USH1C, GJB2, OTOF, and TECTA) that are associated with hearing loss and typify the range of phenotypes and modes of inheritance. They encode the proteins harmonin, connexin 26, otoferlin, and -tectorin, which are expressed in four different cellular/subcellular locations in the inner ear and have diverse functions. Harmonin and otoferlin are cytoskeletal proteins, while connexin 26 is critical for maintaining ion homeostasis in the cochlear duct, and -tectorin is an extracellular matrix protein that is an essential component of the tectorial membrane. Both dominant and recessive inheritance patterns have been found in families with mutations in GJB2 and TECTA, while mutations in GJB2 and USH1C are associated with both syndromic and nonsyndromic hearing loss, and mutations in OTOF have been found in children with auditory neuropathy/auditory dyssynchrony (AN/AD).
3.07.2 USH1C Usher syndrome is clinically and genetically heterogeneous. Three clinical types have been described
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based on the degree of hearing loss, vestibular function, and age of onset of retinal degeneration. Usher syndrome type I is the most severe, with profound hearing loss from birth, abnormal vestibular function, and retinal degeneration beginning with night blindness by the end of the first decade. As many as 6% of children born with profound hearing loss may have Usher syndrome type I. The pattern of inheritance is autosomal recessive, and mutations in at least six different genes are associated with Usher syndrome type I (Keats, B. J. B. and Savas, S., 2004). One of these genes is USH1C. It was mapped to the short arm of chromosome 11 in families of Acadian ancestry in south Louisiana. The original Acadians were French fishermen who left France in the early 1600s and settled in the Nova Scotia region (Acadia) of Canada. Their population size expanded dramatically until 1755 when they were forced to leave Canada. Over the next 40 years about 3000 Acadians moved to south Louisiana. Today more than 600 000 Louisiana residents consider themselves to be Acadians, and the frequency of Usher syndrome type IC is significantly higher than in any other population. In 2000, the USH1C gene was identified and shown to encode a PDZ domain-containing cytoskeletal protein known as harmonin (Bitner-Glindzicz, M. et al., 2000; Verpy, E. et al., 2000). One mutation is specific to the Acadian population probably due to founder effect, but several others have been reported in different populations and some are associated with autosomal recessive nonsyndromic hearing loss (DFNB18) rather than Usher syndrome. 3.07.2.1 Structure, Expression, and Function The USH1C gene contains 28 coding exons spanning approximately 50 kb of genomic DNA. As a result of alternative splicing of eight exons, multiple isoforms that are divided into three classes (a, b, c) are found. They vary in size from approximately 400 to 900 amino acids. Both a and b isoforms contain three PDZ domains while c isoforms have only two. The b isoforms are the longest and use exons that are not translated in either of the two shorter isoforms. The human and mouse proteins are 96% identical. USH1C transcripts are present in many tissues, including colon, small intestine, kidney, brain, muscle, heart, and pancreas, in addition to the ear, eye, and vestibule. In the ear and eye, USH1C is expressed in the stereocilia of the hair cells and the
photoreceptor cell synapses (Reiners, J. et al., 2003). Isoforms a and c are widely expressed, but the longest isoforms (b) are found mostly in the stereocilia of the vestibular and cochlear hair cells. Developmental expression studies in mice detected harmonin b in the stereocilia from E15 until P30, suggesting that it is important for the development of the hair bundle (Boeda, B. et al., 2002). PDZ domain-containing proteins act as adapters for organizing protein complexes that usually involve transmembrane proteins. The protein products of other Usher type I genes (myosin VIIa, cadherin 23, protocadherin 15, and sans) cluster with harmonin via the first PDZ domain. This clustering, together with the localization of these proteins to the synaptic regions of the hair cells and photoreceptor cells, suggests this protein network may be involved in the organization or function of hair cell and photoreceptor cell synapses (Adato, A. et al., 2005). 3.07.2.2
Mutations
Mutations in USH1C that are associated with Usher syndrome type IC include two frameshift mutations (insertions), four splice site mutations, and a nonsense mutation. All of these mutations are expected to result in a severely truncated transcript and no protein product. The causal mutation in Acadian Usher IC patients is a substitution (216G!A) that introduces a cryptic splice site in exon 3 and results in the deletion of 35 bases in the mature messenger RNA (mRNA; Lentz, J. et al., 2005). The most common USH1C mutation, 238–239insC in exon 3, has been found in several different populations (Zwaenepoel, I. et al., 2001). The ability to diagnose Usher syndrome early enables appropriate management and provides time for the development of communication skills that do not rely on vision. In particular, an Acadian child with profound sensorineural hearing loss should be tested for the 216G!A mutation to determine if the correct diagnosis is actually Usher syndrome type IC. Some mutations in USH1C are associated with hearing loss but not retinal degeneration. Because the b isoforms of harmonin are expressed at high levels only in the inner ear, and use some exons that are not translated in isoforms a and c, mutations in these exons would be predicted to cause nonsyndromic hearing loss. In fact, Ouyang X. et al. (2002) found four missense mutations in exons B and D, which are used only in isoform b, in patients with nonsyndromic hearing loss (DFNB18).
Genetic Hearing Loss
In contrast, Ahmed Z. M. et al. (2002) reported members of an Indian family with nonsyndromic hearing loss who were homozygous for a splice site mutation (IVS12þ5G!C) in USH1C. In vitro studies showed that the presence of this mutation resulted in a mixture of both normal and aberrantly spliced mRNA. It is unlikely that the abnormal transcript forms a protein product; thus, the amount of harmonin that is produced must be adequate for normal retinal function, but not enough to preserve the cochlear hair cells. 3.07.2.3
Mouse Models
The deaf circler (dfcr) and deaf circler 2 Jackson (dfcr 2J) mice have spontaneous mutations in the USH1C gene and as with other mouse models for Usher syndrome type I, the stereocilia are disorganized (Johnson, K. R. et al., 2003). However, the mouse models do not develop retinitis pigmentosa. The dfcr mutation is a large deletion that includes several exons, but does not change the reading frame, while the dfcr 2J mutation is a one base deletion in exon C. Only harmonin b is affected in dfcr 2J, but all isoforms are expected to be abnormal in dfcr. ABR testing demonstrated that both dfcr and dfcr 2J mice were completely deaf (no response at 100 dB sound pressure level), and scanning electron microscopy revealed disorganized stereocilia by 3 weeks of age. A slight peripheral retinal degeneration was observed in three dfcr mice at 9 months of age, but electroretinograms (ERGs) were normal.
3.07.3 GJB2 Linkage analysis of two consanguineous families from Tunisia localized the DFNB1 locus to chromosome 13q12. The GJB2 (Gap Junction Protein Beta 2) gene had been mapped to the same location, and Kelsell D. P. et al. (1997) identified mutations in this gene in children with nonsyndromic recessive deafness. The phenotype is usually severe-to-profound congenital hearing loss, but it may be mild-to-moderate and progressive. Although not common, syndromic (usually associated with skin abnormalities) and nonsyndromic (DFNA3) autosomal dominant families have been described with mutations in GJB2. In some populations, GJB2 mutations explain as much as 50% of autosomal recessive hearing loss. Pandya A. et al. (2003) estimated that, on average, GJB2 mutations account for about 22% of
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hearing loss in children, but pointed out that the percentages differ significantly among ethnic groups. 3.07.3.1 Structure, Expression, and Function The GJB2 gene consists of two exons, with exon 2 encoding the 208 amino acid protein. Exon 1 is not translated. The promoter region of the mouse and human genes is highly conserved, and includes GC boxes, GT boxes, and a TTAAAA box (Kiang, D. T. et al., 1997). Because of the relatively short coding sequence, identification of mutations by sequencing is efficient and often provides a definitive diagnosis. GJB2 is expressed in many tissues, including skin, liver, breast, bladder, and pancreas. In the inner ear, GJB2 is expressed in both the epithelial and the connective cellular networks. These include the supporting cells of the hair cells, as well as the spiral limbus, spiral ligament, and stria vascularis. The protein encoded by GJB2 is usually called connexin 26. There are 20 different types of human connexins; two of these (connexin 26 and connexin 30) co-localize in the cochlea (Marziano, N. K. et al., 2003). Each connexin consists of four transmembrane domains connected by one intracellular loop, two extracellular loops, and cytoplasmic N and C terminals. Connexons, which are comprised of six connexins, join with those in an adjacent cell to form homotypic, heterotypic, or heteromeric channels between the cells. The connexons are formed intracellularly and are trafficked to the plasma membrane. These gap junctions allow intercellular diffusion of small molecules and metabolites with a molecular mass up to about 1 kDa. The turnover time of connexins is very rapid, indicating that gap junction channels are replaced several times every day. They are thought to be important for maintaining high concentrations of Kþ in the endolymph via recycling from the perilymph. In turn, this recycling is necessary for the maintenance of the endocochlear potential, which is essential for normal auditory function. Normal forms of both connexin 26 and connexin 30 appear to be required for formation of functional gap junction channels in the support cells of the cochlea, indicating that they do not functionally compensate for one another (Ahmad, S. et al., 2003). 3.07.3.2
Mutations
More than 90 different mutations have been reported in exon 2, the most common in the Caucasian
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population (particularly those from southern Europe) being 35delG with a carrier frequency of about 2%. This deletion of a guanine in codon 12 means that codon 13 becomes a stop codon, and translation is terminated. Other mutations have been reported with increased frequencies in particular populations, such as 167delT and 235delC in the Ashkenazi Jewish and Asian populations, respectively. Although most of the mutations in GJB2 are associated with recessive hearing loss, some have been found in families with dominant hearing loss (DFNA3). All are missense mutations and for a few, the phenotype is syndromic with skin abnormalities in addition to the hearing loss. Many variants that do not segregate with hearing loss have been reported. The M34T allele is controversial. Individuals with normal hearing based on pure-tone audiograms and 167delT/M34T and 35delG/M34T genotypes have been reported, but other studies suggest that M34T contributes to recessive mild-to-moderate- or high-frequency hearing loss. Often, interpretation of diagnostic test results is not straight forward because only one mutation is found. Several explanations are possible: the second mutation may have been missed or the individual may be a carrier (by chance), with the hearing loss having nothing to do with the mutation. In some cases, it has been shown that the second mutation is a 309 kb deletion or a 232 kb deletion that encompasses all or part of the GJB6 gene, which encodes connexin 30 (del Castillo, F. J. et al., 2005). GJB2 and GJB6 are less than 50 kb apart, and share 77% identity in amino acid sequence, with connexin 30 having 37 more amino acids at its C-terminus than connexin 26. The nonsyndromic recessive phenotype can vary from mild to profound hearing loss, even in individuals with the same GJB2 mutations. This finding suggests that modifier genes may be involved, but a systematic study to find such genes has not yet been done. 3.07.3.3
Mouse Models
The knockout mouse is an embryonic lethal, but studies of conditional knockout mice suggest that interference with potassium recycling may explain the hearing loss that results when connexin 26 is missing in the inner ear (Cohen-Salmon, M. et al., 2002). Mice do not hear until about 2 weeks after birth and the inner ears of the mutant mice were
found to develop normally until P14 when apoptotic cell death began to occur. Supporting cells of the inner hair cells were the first to die, followed by outer hair cells and their supporting cells. However, the inner hair cells were generally preserved, although ultrastructural analysis revealed anomalies. The mice showed a significant hearing loss with an average of 30 dB at 8 kHz to 36 dB at 32 kHz relative to heterozygous and control mice. The endocochlear potential in adult mutants was significantly lower than in control mice as was the Kþ concentration in the endolymph. In contrast, the mutant mice did not show any vestibular dysfunction, suggesting that connexin 26 is not required for normal function of gap junctions in the vestibular system. Additionally, connexin 30 expression was not affected by the absence of connexin 26.
3.07.4 OTOF The DFNB9 locus was mapped to chromosome 2p22–p23 in a large Lebanese family in which congenital profound sensorineural hearing loss followed an autosomal recessive pattern of inheritance. Yasunaga S. et al. (1999) showed that the affected individuals in this family were homozygous for a nonsense mutation in the OTOF gene. This gene encodes a cytoskeletal protein called otoferlin that may be involved in synaptic vesicle trafficking. Expression of otoferlin in the inner ear was found to be mostly in the inner hair cells. Because of this finding, Varga R. et al. (2003) searched for otoferlin mutations in children with recessive forms of AN/ AD without peripheral neuropathy. These children had absent ABRs, but their OAEs were preserved, suggesting normal function of their outer hair cells. (For some of these children, OAEs disappeared with time.) In general, they did not benefit from hearing aids and their speech comprehension was poorer than expected based on their pure-tone audiograms, which ranged from moderate to profound. Several of these children received cochlear implants and found them helpful. 3.07.4.1 Structure, Expression, and Function OTOF consists of 48 exons spanning about 90 kb of genomic DNA. Yasunaga S. et al. (2000) detected numerous alternate splice forms involving exons 6, 31, 47, and 48, and several translation initiation sites.
Genetic Hearing Loss
There are two classes of otoferlin isoforms: long ones with approximately 2000 amino acids and six C2-like domains, and short ones (encoded by exons 20–48) with about 1200 amino acids and only the last three C2-like domains. C2 domains bind Ca2þ and are often found in proteins involved in signal transduction, vesicle trafficking, and membrane fusion. Numerous adult human and mouse tissues including the cochlea, vestibule, eye, brain, placenta, liver, heart, lungs, pancreas, skeletal muscle, and kidney, as well as total human fetus, showed OTOF expression, with the strongest expression being in the cochlea and brain. The short isoforms were not detected in mouse tissues, whereas the only human tissue in which the long isoforms were detected was the brain. Additionally, the 60 C-terminal amino acids in brain otoferlin are encoded by exon 47, whereas in the cochlea and other tissues, exon 47 is skipped and the 59 part of exon 48 is used to encode the final 60 amino acids. The strongest expression of OTOF in the inner ear of the mouse is in the cochlear inner hair cells and vestibular type I sensory hair cells; it was detected at E19.5 and persisted until at least P20. OTOF expression was also detected in the cochlear outer hair cells and spiral ganglion cells of mouse embryos, but it was found only in inner hairs cells of mature cochleas (Yasunaga, S. et al., 1999; 2000). Otoferlin is related to the C. elegans FER-1 protein, as are two other proteins with C2 domains, dysferlin and myoferlin. They are all predicted to be C-terminal membrane-anchored cytosolic proteins, which interact with phospholipids and other proteins via their C2 domains. Otoferlin function is unknown, but the C2-like domains suggest that it may be involved in synaptic vesicle–plasma membrane fusion. b0145
3.07.4.2
Mutations
In the original DFNB9 family, Yasunaga S. et al. (1999) showed that the affected individuals were homozygous for a nonsense mutation in exon 39. Of the17 frameshift, splice site, and missense mutations that have been reported so far, five are found in exons that are included only in the long isoforms, which are restricted to the brain. No mutations have been found in exon 47, but three are in exon 48, which is encoded in the cochlea but not in the brain. In the Spanish population, Migliosi V. et al. (2002) found that 4.4% of those without GJB2 mutations have OTOF mutations (in particular, Q829X in exon 22), and Rodriguez-Ballesteros M. et al. (2003) reported that
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OTOF mutations account for at least 3.5% of prelingual hearing loss. 3.07.4.3
Mouse Models
A mouse model should provide some insight into clarifying the function of otoferlin. However, no spontaneous mutations have yet been reported, and a knockout mouse has not been created.
3.07.5 TECTA Both dominant (DFNA8/12) and recessive (DFNB21) forms of hearing loss have been associated with mutations in the TECTA gene, which encodes -tectorin, a noncollagenous extracellular matrix protein. The three loci were mapped to chromosome 11q22–q24 in three different families from Austria, Belgium, and Lebanon. TECTA was considered a likely candidate gene based on its location (Verhoeven, K. et al., 1998). The dominant phenotype in the Belgian and Austrian families was nonsyndromic, prelingual, stable, moderate-to-severe hearing loss at all frequencies. This is in contrast to most dominant forms of hearing loss, which are progressive and have a postlingual onset. However, affected members of other DFNA8/12 families have the typical progressive loss starting in the high frequencies. The recessive phenotype in the family from Lebanon had a prelingual onset and was in the severe-toprofound range (Mustapha, M. et al., 1999). 3.07.5.1 Structure, Expression, and Function TECTA has 23 exons encoding a 2155 amino acid protein, -tectorin. The human DNA sequence is predicted to have 95% identity with the mouse sequence. Splice variants may exist, but have not yet been systematically investigated. The protein has hydrophobic N-terminal signal sequences that are characteristic of secreted proteins. The C-terminal is also hydrophobic and is predicted to have a glycosyl-phosphatidylinositol (GPI) anchor, which is considered to be a signal for directing the protein to the apical surface of the inner ear sensory epithelia. It also has von Willebrand factor (vWF) type D repeats, which are similar to those found in the sperm membrane protein, zonadhesin, and are rich in cysteines, and a C-terminal ZP domain, which is found in proteins in the zona pellucida, the
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extracellular matrix that surrounds the unfertilized egg. Zonadhesin is known to bind to the zona pellucida (Legan, P. K. et al., 1997). TECTA is expressed only in the inner ear, and, in particular, it is found in the tectorial membrane of the cochlea. Developmental studies in the mouse demonstrate that tecta mRNA is expressed in the basal cochlear duct by E12.5, throughout the cochlea by E14.5, and reaches its highest level at P3. After that, expression decreases with minimal detectable mRNA at P15 and none at P67 (Maeda, Y. et al., 2001). These results indicate that TECTA is expressed at high levels only briefly during the development of the cochlea and suggest that -tectorin is stable with a long half-life. Tectorins are pepsin-sensitive glycoproteins that are resistant to digestion with bacterial collagenase. The tectorial membrane is a ribbonlike sheet of extracellular matrix that spirals along the length of the cochlea, and connects to the tips of the stereocilia of the outer hair cells. It is composed of collagens (types II, V, and IX) and a noncollagenous matrix, consisting mostly of -tectorin and -tectorin. These two proteins crosslink, and they appear to be critical for attachment of the tectorial membrane to the cochlear epithelium.
3.07.5.2
Mutations
Missense mutations have been found in affected individuals in DFNA8/12 families, and a dominant negative mechanism has been proposed, although haploinsufficiency is also considered possible (Verhoeven, K. et al., 1998). The reasons for the differences in age of onset and phenotypes among affected individuals in DFNA8/12 families is not clear. However, three missense mutations that change cysteine residues at positions 1057, 1619, and 1837 are associated with progressive loss. So far, seven different missense mutations have been reported and to some extent it appears that mutations in the vWF regions cause high-frequency hearing loss, while those in the ZP domain cause mid-frequency loss (Naz, S. et al., 2003). In the Lebanese DFNB21 family, the affected individuals were homozygous for a splice site mutation. More recently, Naz S. et al. (2003) reported exon 5 and exon 20 frameshift mutations in two consanguineous DFNB21 families from Iran and Pakistan. In both families the hearing loss was prelingual and in the moderate-to-severe range.
3.07.5.3
Mouse Models
Homozygous knockout mice have moderate-tosevere hearing loss and abnormal and detached tectorial membranes with no known noncollagenous components. The tectorial membrane is synthesized, but within a few weeks after birth it is no longer attached at either end and the collagen fibers are disorganized. In contrast, the structure and orientation of the stereocilia of the cochlear hair cells are unaffected, although the cochlear microphonic is smaller and differs in phase and symmetry from the wild-type mice. The heterozygous mice appear to be normal (Legan, P. K. et al., 2000). These findings demonstrate the importance of the tectorial membrane in amplifying auditory stimuli, presumably because it is essential for deflection of outer hair cell stereocilia.
3.07.6 Outlook for the Future Progress in our understanding of genetic hearing loss has been enormous over the past decade. We have made great strides in determining many of the different molecular mechanisms that contribute to the genetic heterogeneity of hearing loss. However, more genes remain to be identified, and elucidating the function of the proteins encoded by these genes is challenging. The four genes discussed in this perspective provide examples of the phenotypic and genetic heterogeneity of hearing loss and the progress that has been made in understanding their function in the inner ear. It is clear that different mutations in the same gene can be associated with both nonsyndromic and syndromic forms of hearing loss, and in some cases a mutation in one copy of the gene (dominant) is sufficient while in others mutations in both copies (recessive) are required for hearing loss. These four genes show diverse expression patterns and the proteins they encode have quite different functions. But they are all critical for normal hearing. Genetic diagnostic testing for mutations in the GJB2 gene is becoming routine and other genes are likely to be added in the near future. In particular, testing for the Q829X mutation in the OTOF gene might be considered for those who do not have GJB2 mutations. Ruling out a syndrome is important, especially for one such as Usher syndrome where the first signs of retinal degeneration may not appear for 10 or more years after the diagnosis of severe-to-profound
Genetic Hearing Loss
hearing loss. Because of the genetic heterogeneity of Usher syndrome, a genetic test for all mutations is not yet feasible, but Acadian children should be screened for the USH1C 216G!A mutation, and a mutation in the protocadherin 15 gene (R245X) should be considered in those with Ashkenazi Jewish ancestry who do not have mutations in the GJB2 gene. A genetic diagnosis is relevant not just to the affected individual but to all family members, some of whom may choose to be tested before making reproductive decisions. In many cases, knowing the genotype is not enough to give a precise phenotype and prognosis, but ongoing research is elucidating some of these relationships. The development of effective therapies based on knowledge of the underlying genetic abnormality is just beginning; however, the potential for molecular intervention strategies as a treatment for hearing loss is promising.
References Adato, A., Michel, V., Kikkawa, Y., Reiners, J., Alagramam, K., Weil, D., Yonekawa, H., Wolfrum, U., El-Amraoui, A., and Petit, C. 2005. Interactions in the network of Usher syndrome type 1 proteins. Hum. Mol. Genet. 14, 347–356. Ahmad, S., Chen, S., Sun, J., and Lin, X. 2003. Connexins 26 and 30 are co-assembled to form gap junctions in the cochlea of mice. Biochem. Biophys. Res. Comm. 307, 362–368. Ahmed, Z. M., Smith, T. N., Riazuddin, S., Makishima, T., Ghosh, M., Bokhari, S., Menon, P. S. N., Deshmukh, D., Griffith, A. J., Riazuddin, S., Friedman, T. B., and Wilcox, E. R. 2002. Nonsyndromic recessive deafness DFNB18 and Usher syndrome type IC are allelic mutations of USHIC. Hum. Genet. 110, 527–531. Bitner-Glindzicz, M., Lindley, K. J., Rutland, P., Blaydon, D., Smith, V., Milla, P., Hussain, K., Furth-Lavi, J., Cosgrove, J., Shepherd, R., Barnes, P., O’Brien, R., Farndon, P., Sowden, J., Liu, X. Z., Scanlan, M., Malcolm, S., Dunne, M., Aynsley-Green, A., and Glaser, B. 2000. A recessive contiguous gene deletion causing infantile hyperinsulinism, enteropathy and deafness identifies the Usher type 1C gene. Nat. Genet. 26, 56–60. Boeda, B., El-Amraoui, A., Bahloul, A., Goodyear, R., Daviet, L., Blanchard, S., Perfettini, I., Fath, K. R., Shorte, S., Reiners, J., Houdusse, A., Legrain, P., Wolfrum, U., Richardson, G., and Petit, C. 2002. Myosin VIIa, harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair cell bundle. EMBO J. 21, 6689–6699. Cohen-Salmon, M., Ott, T., Michel, V., Hardelin, J., Perfettini, I., Eybalin, M., Wu, T., Marcus, D., Wangemann, P., Willecke, K., and Petit, C. 2002. Targeted ablation of connexin 26 in the inner ear epithelial gap junction network causes hearing impairment and cell death. Curr. Biol. 12, 1106–1111. del Castillo, F. J., Rodriguez-Ballesteros, M., Alvarez, A., Hutchin, T., Leonardi, E., de Oliveira, C. A., Azaiez, H., Brownstein, Z., Avenarius, M. R., Marlin, S., Pandya, A., Shahin, H., Siemering, K. R., Weil, D., Wuyts, W., Aguirre, L. A., Martin, Y., Moreno-Pelayo, M. A., Villamar, M., Avraham, K. B., Dahl, H. M., Kanaan, M., Nance, W. E., Petit, C., Smith, R., Van Camp, G., Sartorato, E., Murgia, A.,
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Moreno, F., and del Castillo, I. 2005. A novel deletion involving the connexin-30 gene, del(GJB6-d13s1854), found in trans with mutations in the GJB2 gene (connexin-26) in subjects with DFNB1 non-syndromic hearing impairment. J. Med. Genet. 42, 588–594. Griffith, A. and Friedman, T. 2002. Autosomal and X-linked Auditory Disorders. In: Genetics and Auditory Disorders (eds. B. Keats, A. Popper, and R. Faye), pp. 121–227. Springer-Verlag. Johnson, K. R., Gagnon, L. H., Webb, L. S., Peters, L. L., Hawes, N. L., Chang, B., and Zheng, Q. Y. 2003. Mouse models of USH1C and DFNB18: phenotypic and molecular analyses of two new spontaneous mutations of the Ush1c gene. Hum. Mol. Genet. 12, 3075–3086. Keats, B. J. B. and Berlin, C. I. 2002. Introduction and Overview: Genetics in Auditory Science and Clinical Audiology. In: Genetics and Auditory Disorders (eds. B. Keats, A. Popper, and R. Faye), pp. 1–22. Springer. Keats, B. J. B. and Savas, S. 2004. Genetic heterogeneity in Usher syndrome. Am. J. Med. Genet. 130A, 13–16. Kelsell, D. P., Dunlop, J., Stevens, H. P., Lench, N. J., Liang, J. N., Parry, G., Mueller, R. F., and Leigh, I. M. 1997. Connexin 26 mutations in hereditary non-syndromic sensorineural deafness. Nature 387, 80–83. Kiang, D. T., Jin, N., Tu, Z. J., and Lin, H. H. 1997. Upstream genomic sequence of the human connexin 26 gene. Gene 199, 165–171. Legan, P. K., Lukashkina, V. A., Goodyear, R. J., Kossl, M., Russell, I. J., and Richardson, G. P. 2000. A targeted deletion in alpha-tectorin reveals that the tectorial membrane is required for the gain and timing of cochlear feedback. Neuron 28, 273–285. Legan, P. K., Rau, A., Keen, J. N., and Richardson, G. P. 1997. The mouse tectorins: modular matrix proteins of the inner ear homologous to components of the sperm–egg adhesion system. J. Biol. Chem. 272, 8791–8801. Lentz, J., Savas, S., Ng, S., Athas, G., Deininger, P., and Keats, B. 2005. The USH1C 216G>A splice site mutation results in a 35 base pair deletion. Hum. Genet. 116, 225–227. Maeda, Y., Fukushima, K., Kasai, N., Maeta, M., and Nishizaki, K. 2001. Quantification of TECTA and DFNA5 expression in the developing mouse cochlea. Neuroreport 12, 3223–3226. Marziano, N. K., Casalotti, S. O., Portelli, A. E., Becker, D. L., and Forge, A. 2003. Mutations in the gene for connexin 26 (GJB2) that cause hearing loss have a dominant negative effect on connexin 30. Hum. Mol. Genet. 12, 805–812. Migliosi, V., Modamio-Hoybjor, S., Moreno-Pelayo, M. A., Rodriguez-Ballesteros, M., Villamar, M., Telleria, D., Menendez, I., Moreno, F., and del Castillo, I. 2002. Q829X, a novel mutation in the gene encoding otoferlin (OTOF), is frequently found in Spanish patients with prelingual nonsyndromic hearing loss. J. Med. Genet. 39, 502–506. Mustapha, M., Weil, D., Chardenoux, S., Elias, S., El-Zir, E., Beckmann, J. S., Loiselet, J., and Petit, C. 1999. An alphatectorin gene defect causes a newly identified autosomal recessive form of sensorineural pre-lingual non-syndromic deafness, DFNB21. Hum. Mol. Genet. 8, 409–412. Naz, S., Alasti, F., Mowjoodi, A., Riazuddin, S., Sanati, M. H., Friedman, T. B., Griffith, A. J., Wilcox, E. R., and Riazuddin, S. 2003. Distinctive audiometric profile associated with DFNB21 alleles of TECTA. J. Med. Genet. 40, 360–363. Ouyang, X. M., Xia, X. J., Verpy, E., Du, L. L., Pandya, A., Petit, C., Balkany, T., Nance, W. E., and Liu, X. Z. 2002. Mutations in the alternatively spliced exons of USH1C cause non-syndromic recessive deafness. Hum. Genet. 111, 26–30. Pandya, A., Arnos, K. S., Xia, X. J., Welch, K. O., Blanton, S. H., Friedman, T. B., Sanchez, G. G., Jiu, X. Z., Morell, R., and Nance, W. E. 2003. Frequency and distribution of GJB2
148 Genetic Hearing Loss (connexin 26) and GJB6 (connexin 30) mutations in a large North American repository of deaf probands. Genet. Med. 5, 295–303. Reiners, J., Reidel, B., El-Amraoui, A., Boeda, B., Huber, I., Petit, C., and Wolfrum, U. 2003. Differential distribution of harmonin isoforms and their possible role in Usher-1 protein complexes in mammalian photoreceptor cells. Invest. Ophthalmol. Vis. Sci. 44, 5006–5015. Rodriguez-Ballesteros, M., del Castillo, F. J., Martin, Y., Moreno-Pelayo, M. A., Morera, C., Prieto, F., Marco, J., Morant, A., Gallo-Teran, J., Morales-Angulo, C., Navas, C., Trinidad, G., Tapia, M. C., Moreno, F., and del Castillo, I. 2003. Auditory neuropathy in patients carrying mutations in the otoferlin gene (OTOF). Hum. Mutat. 22, 451–456. Toriello, H. V., Reardon, W., and Gorlin, R. J. 2004. Hereditary Hearing Loss and its Syndromes, 2nd edn. Oxford University Press. Varga, R., Kelley, P. M., Keats, B. J., Starr, A., Leal, S. M., Cohn, E., and Kimberling, W. J. 2003. Non-syndromic recessive auditory neuropathy is the result of mutations in the otoferlin (OTOF) gene. J. Med. Genet. 40, 45–50. Verhoeven, K., Van Laer, L., Kirschhofer, K., Legan, P. K., Hughes, D. C., Schatteman, I., Verstreken, M., Van Hauwe, P., Coucke, P., Chen, A., Smith, R. J. H., Somers, T., Offeciers, F. E., Van de Heyning, P., Richardson, G. P.,
Wachtler, F., Kimberling, W. J., Willems, P. J., Govaerts, P. J., and Van Camp, G. 1998. Mutations in the human alpha-tectorin gene cause autosomal dominant nonsyndromic hearing impairment. Nat. Genet. 19, 60–62. Verpy, E., Leibovici, M., Zwaenepoel, I., Liu, X. Z., Gal, A., Salem, N., Mansour, A., Blanchard, S., Kobayashi, I., Keats, B., Slim, R., and Petit, C. 2000. A defect in harmonin, a PDZ domain-containing protein expressed in the inner ear sensory hair cells, underlies Usher syndrome type 1C. Nat. Genet. 26, 51–55. Yasunaga, S., Grati, M., Chardenoux, S., Smith, T. N., Friedman, T. B., Lalwani, A. K., Wilcox, E. R., and Petit, C. 2000. OTOF encodes multiple long and short isoforms: genetic evidence that the long ones underlie recessive deafness DFNB9. Am. J. Hum. Genet. 67, 591–600. Yasunaga, S., Grati, M., Cohen-Salmon, M., El-Amraoui, A., Mustapha, M., Salem, N., El-Zir, E., Loiselet, J., and Petit, C. 1999. A mutation in OTOF, encoding otoferlin, a FER-1-like protein, causes DFNB9, a nonsyndromic form of deafness. Nat. Genet. 21, 363–369. Zwaenepoel, I., Verpy, E., Blanchard, S., Meins, M., ApfelstedtSylla, E., Gal, A., and Petit, C. 2001. Identification of three novel mutations in the USH1C gene and detection of thirtyone polymorphisms used for haplotype analysis. Hum. Mutat. 17, 34–41.
3.07.1 3.07.2 3.07.2.1 3.07.2.2 3.07.2.3 3.07.3 3.07.3.1 3.07.3.2 3.07.3.3 3.07.4 3.07.4.1 3.07.4.2 3.07.4.3 3.07.5 3.07.5.1 3.07.5.2 3.07.5.3 3.07.6 References
Introduction USH1C Structure, Expression, and Function Mutations Mouse Models GJB2 Structure, Expression, and Function Mutations Mouse Models OTOF Structure, Expression, and Function Mutations Mouse Models TECTA Structure, Expression, and Function Mutations Mouse Models Outlook for the Future
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Glossary codon A set of three bases that specifies a single amino acid or signals the termination of the amino acid sequence. compound heterozygote An individual with a recessive disorder who has a different mutation in the two copies of the gene (cf., homozygote). cryptic splice site A DNA sequence that is similar to a consensus splice site sequence but is not usually used unless there is a mutation in the normal splice site, or a mutation in the cryptic splice site results in it being preferred over the normal splice site. dominant negative The abnormal product of a mutant copy of a gene disrupts the function of the product of the normal copy of the gene. exon Segment of a gene that is transcribed and present in mature mRNA. founder effect A mutation that is present in a small ancestral population, which increases in frequency by chance due to the initial small population size. frameshift mutation An insertion or deletion of a number of base pairs that is not a multiple of three, and therefore changes the codons that follow.
genome The complete DNA sequence. For example, the nuclear genome of a human germ cell (sperm or ovum) consists of 3 million kb of DNA. genotype The two copies of the gene (one of each chromosome) that are present at a locus. haploinsufficiency The product of the normal copy of a gene is not adequate to prevent the disease when the mutant copy of the gene does not have a functional product. heterozygote An individual whose genotype consists of two different copies of the gene. homozygote An individual whose genotype consists of two identical copies of the gene. intron Segment of a gene that is not present in the mature mRNA. isoform Alternate forms of a protein. kilobase (kb) A unit of 1000 bases in a DNA sequence. linkage A statistical method of analysis of family phenotype and genotype data that provides the probability that two or more loci are not assorting independently (i.e., they are linked).
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locus The position of a gene on a chromosome. messenger RNA (mRNA) An RNA transcribed from the DNA of a gene, which directs the sequence in which amino acids are added to the product encoded by that gene. missense mutation A substitution of one base by another such that the amino acid specified by the codon is changed. modifier gene A gene that alters the phenotype associated with mutations in another gene. nonsense mutation A substitution of one base by another such that the resulting codon specifies termination of the amino acid sequence. penetrance The probability that a given genotype will result in a particular phenotype.
3.07.1 Introduction The human genome contains about 30 000 genes, and mutations in the DNA sequences of more than 100 of these genes are associated with hearing loss. The degree of genetic hearing loss may range from mild to profound and some frequencies may be affected more than others; for most individuals the loss is sensorineural, bilateral, and symmetric. Onset is often congenital (present at birth), but it can be later in childhood or in adulthood. Many different genetic forms of hearing loss may give similar pure tone air conduction audiograms. Thus, ideally, a battery of physiological measurements that include tympanometry, middle ear muscle reflexes, otoacoustic emissions (OAEs), speech audiometry, and auditory brainstem response/electrocochleography (ABR/EcochG) should be obtained for each patient in order to categorize the hearing loss accurately and determine the most effective management (Keats, B. J. B. and Berlin, C. I., 2002). About 30% of genetic hearing loss is syndromic, meaning that there are associated abnormalities in other organs such as the eye (Usher syndrome), kidney (Alport syndrome), skin (Waardenburg syndrome), heart (Jervell and Lange-Nielsen syndrome), and thyroid (Pendred syndrome). For the remaining 70%, the hearing loss is nonsyndromic. Griffith A. and Friedman T. (2002) and Toriello H. V. et al. (2004) provide comprehensive descriptions of syndromic and nonsyndromic hearing loss. The mode of inheritance of hearing loss in a family may be dominant or recessive, and the
phenocopy A phenotype that looks the same as one produced by a specific genotype, but has a different etiology. phenotype The observed characteristics of an individual. splice site The DNA sequence that specifies the boundary between an intron and an exon in a gene. The introns are removed in the generation of the mature mRNA. stop codon One of three codons specifying the termination of the product encoded by a gene. transcription The synthesis of a single-stranded mRNA molecule (transcript) from a DNA template. translation The synthesis of a sequence of amino acids from the mature mRNA template.
mutation may be in a gene on one of the autosomes (chromosomes 1–22) or on the X-chromosome; these chromosomes are in the nucleus of the cell. A pedigree showing who is affected and who is unaffected in the family helps to determine the inheritance pattern. Figure 1 shows an example of autosomal dominant inheritance in which individuals with one normal
Unaffected male Unaffected female Affected male Affected female
ND
NN
NN
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NN
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ND
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Figure 1 A pedigree showing an autosomal-dominant pattern of inheritance with incomplete penetrance. N is the normal form of the gene and D is the abnormal (mutant) form of the gene. Individuals with the NN genotype are unaffected and those with the ND genotype are expected to be affected. However, the sixth individual in the middle generation is unaffected but has an affected mother and two affected children. Thus, her genotype must be ND. This is an example of incomplete penetrance meaning that for individuals with the ND genotype, the probability of being affected is less than 1.
Genetic Hearing Loss
copy (N) and one abnormal copy (D) of the gene are usually affected; their genotype is ND and each of their offspring has a 50% chance of inheriting the D. However, the phenotype may be variable, and penetrance may be incomplete, meaning that some ND individuals may show no signs of being affected. The hearing loss is autosomal recessive if only individuals with the DD genotype are affected. Those with the ND genotype are unaffected and are referred to as carriers (or heterozygotes). Each offspring of two carrier parents has a 25% chance of inheriting the DD genotype and being affected. Family history is quite likely to be negative for a child with autosomal recessive hearing loss. Conversely, both parents may have autosomal recessive hearing loss but all their children may have normal hearing because mutations in different genes are responsible for the hearing loss in the parents. If all the affected individuals in a pedigree are male, and none of the affected males have affected sons, then the inheritance pattern is likely to be X-linked recessive. In this case, each son of a carrier mother has a 50% chance of being affected and all the daughters of an affected male will be carriers. The symbols, DFNA, DFNB, and DFN, are used to designate autosomal dominant, autosomal recessive, and X-linked hearing loss, respectively, where the asterisk represents a number. The symbol is assigned when the chromosomal location of the hearing loss gene is determined, usually through genetic linkage analysis. For example, DFNB21 means the twenty-first autosomal recessive locus mapped. When the abnormal gene at the locus is identified, a gene symbol is assigned. For human genes, the symbol is written in uppercase and italics. As a generalization, recessive hearing losses tend to be prelingual while dominant forms are postlingual and progressive, but there are exceptions. In some families the phenotype may vary from one affected child to another. Such findings may be examples of variable expression, but the possibility of phenocopies must be considered. As well as the DNA in the nucleus, each of the several hundred mitochondria in a cell have multiple copies of their own DNA, and some forms of hearing loss are associated with mutations in this mitochondrial genome (mtDNA). Quite often the mutation is present in some copies of the mtDNA, but not others, a condition known as heteroplasmy. The number of base pairs of mtDNA is only about 16 000 compared with the three billion in the nuclear genome. Mitochondria are maternally inherited, so the pattern
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of inheritance is likely to be mitochondrial if all the offspring of an affected mother (but none of those of an affected father) are affected. However, because of heteroplasmy, the severity of the phenotype among the offspring may vary considerably. The majority of genetic hearing loss is autosomal recessive, but about 20% is autosomal dominant and 1–2% is X-linked or mitochondrial. Throughout the body, the set of genes that are expressed varies among cell types, and from one stage of development to another. The characteristics of genetic forms of hearing loss are likely to differ depending on the cellular and subcellular localizations, and the functions of the proteins encoded by the associated genes. These proteins must be essential for normal hearing. They include cytoskeletal proteins, extracellular matrix proteins, transcription factors, and proteins required for ion homeostasis. Because of the similarity between the mouse and human genomes, studies of mouse models are often quite informative in advancing understanding of genetic hearing loss. Many of the available mouse models are the result of spontaneous mutations, while others have been created using transgenic or gene knockout experimental approaches. The symbols for mouse genes are written in lowercase to differentiate them from human genes. This perspective will focus on four genes (USH1C, GJB2, OTOF, and TECTA) that are associated with hearing loss and typify the range of phenotypes and modes of inheritance. They encode the proteins harmonin, connexin 26, otoferlin, and -tectorin, which are expressed in four different cellular/subcellular locations in the inner ear and have diverse functions. Harmonin and otoferlin are cytoskeletal proteins, while connexin 26 is critical for maintaining ion homeostasis in the cochlear duct, and -tectorin is an extracellular matrix protein that is an essential component of the tectorial membrane. Both dominant and recessive inheritance patterns have been found in families with mutations in GJB2 and TECTA, while mutations in GJB2 and USH1C are associated with both syndromic and nonsyndromic hearing loss, and mutations in OTOF have been found in children with auditory neuropathy/auditory dyssynchrony (AN/AD).
3.07.2 USH1C Usher syndrome is clinically and genetically heterogeneous. Three clinical types have been described
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based on the degree of hearing loss, vestibular function, and age of onset of retinal degeneration. Usher syndrome type I is the most severe, with profound hearing loss from birth, abnormal vestibular function, and retinal degeneration beginning with night blindness by the end of the first decade. As many as 6% of children born with profound hearing loss may have Usher syndrome type I. The pattern of inheritance is autosomal recessive, and mutations in at least six different genes are associated with Usher syndrome type I (Keats, B. J. B. and Savas, S., 2004). One of these genes is USH1C. It was mapped to the short arm of chromosome 11 in families of Acadian ancestry in south Louisiana. The original Acadians were French fishermen who left France in the early 1600s and settled in the Nova Scotia region (Acadia) of Canada. Their population size expanded dramatically until 1755 when they were forced to leave Canada. Over the next 40 years about 3000 Acadians moved to south Louisiana. Today more than 600 000 Louisiana residents consider themselves to be Acadians, and the frequency of Usher syndrome type IC is significantly higher than in any other population. In 2000, the USH1C gene was identified and shown to encode a PDZ domain-containing cytoskeletal protein known as harmonin (Bitner-Glindzicz, M. et al., 2000; Verpy, E. et al., 2000). One mutation is specific to the Acadian population probably due to founder effect, but several others have been reported in different populations and some are associated with autosomal recessive nonsyndromic hearing loss (DFNB18) rather than Usher syndrome. 3.07.2.1 Structure, Expression, and Function The USH1C gene contains 28 coding exons spanning approximately 50 kb of genomic DNA. As a result of alternative splicing of eight exons, multiple isoforms that are divided into three classes (a, b, c) are found. They vary in size from approximately 400 to 900 amino acids. Both a and b isoforms contain three PDZ domains while c isoforms have only two. The b isoforms are the longest and use exons that are not translated in either of the two shorter isoforms. The human and mouse proteins are 96% identical. USH1C transcripts are present in many tissues, including colon, small intestine, kidney, brain, muscle, heart, and pancreas, in addition to the ear, eye, and vestibule. In the ear and eye, USH1C is expressed in the stereocilia of the hair cells and the
photoreceptor cell synapses (Reiners, J. et al., 2003). Isoforms a and c are widely expressed, but the longest isoforms (b) are found mostly in the stereocilia of the vestibular and cochlear hair cells. Developmental expression studies in mice detected harmonin b in the stereocilia from E15 until P30, suggesting that it is important for the development of the hair bundle (Boeda, B. et al., 2002). PDZ domain-containing proteins act as adapters for organizing protein complexes that usually involve transmembrane proteins. The protein products of other Usher type I genes (myosin VIIa, cadherin 23, protocadherin 15, and sans) cluster with harmonin via the first PDZ domain. This clustering, together with the localization of these proteins to the synaptic regions of the hair cells and photoreceptor cells, suggests this protein network may be involved in the organization or function of hair cell and photoreceptor cell synapses (Adato, A. et al., 2005). 3.07.2.2
Mutations
Mutations in USH1C that are associated with Usher syndrome type IC include two frameshift mutations (insertions), four splice site mutations, and a nonsense mutation. All of these mutations are expected to result in a severely truncated transcript and no protein product. The causal mutation in Acadian Usher IC patients is a substitution (216G!A) that introduces a cryptic splice site in exon 3 and results in the deletion of 35 bases in the mature messenger RNA (mRNA; Lentz, J. et al., 2005). The most common USH1C mutation, 238–239insC in exon 3, has been found in several different populations (Zwaenepoel, I. et al., 2001). The ability to diagnose Usher syndrome early enables appropriate management and provides time for the development of communication skills that do not rely on vision. In particular, an Acadian child with profound sensorineural hearing loss should be tested for the 216G!A mutation to determine if the correct diagnosis is actually Usher syndrome type IC. Some mutations in USH1C are associated with hearing loss but not retinal degeneration. Because the b isoforms of harmonin are expressed at high levels only in the inner ear, and use some exons that are not translated in isoforms a and c, mutations in these exons would be predicted to cause nonsyndromic hearing loss. In fact, Ouyang X. et al. (2002) found four missense mutations in exons B and D, which are used only in isoform b, in patients with nonsyndromic hearing loss (DFNB18).
Genetic Hearing Loss
In contrast, Ahmed Z. M. et al. (2002) reported members of an Indian family with nonsyndromic hearing loss who were homozygous for a splice site mutation (IVS12þ5G!C) in USH1C. In vitro studies showed that the presence of this mutation resulted in a mixture of both normal and aberrantly spliced mRNA. It is unlikely that the abnormal transcript forms a protein product; thus, the amount of harmonin that is produced must be adequate for normal retinal function, but not enough to preserve the cochlear hair cells. 3.07.2.3
Mouse Models
The deaf circler (dfcr) and deaf circler 2 Jackson (dfcr 2J) mice have spontaneous mutations in the USH1C gene and as with other mouse models for Usher syndrome type I, the stereocilia are disorganized (Johnson, K. R. et al., 2003). However, the mouse models do not develop retinitis pigmentosa. The dfcr mutation is a large deletion that includes several exons, but does not change the reading frame, while the dfcr 2J mutation is a one base deletion in exon C. Only harmonin b is affected in dfcr 2J, but all isoforms are expected to be abnormal in dfcr. ABR testing demonstrated that both dfcr and dfcr 2J mice were completely deaf (no response at 100 dB sound pressure level), and scanning electron microscopy revealed disorganized stereocilia by 3 weeks of age. A slight peripheral retinal degeneration was observed in three dfcr mice at 9 months of age, but electroretinograms (ERGs) were normal.
3.07.3 GJB2 Linkage analysis of two consanguineous families from Tunisia localized the DFNB1 locus to chromosome 13q12. The GJB2 (Gap Junction Protein Beta 2) gene had been mapped to the same location, and Kelsell D. P. et al. (1997) identified mutations in this gene in children with nonsyndromic recessive deafness. The phenotype is usually severe-to-profound congenital hearing loss, but it may be mild-to-moderate and progressive. Although not common, syndromic (usually associated with skin abnormalities) and nonsyndromic (DFNA3) autosomal dominant families have been described with mutations in GJB2. In some populations, GJB2 mutations explain as much as 50% of autosomal recessive hearing loss. Pandya A. et al. (2003) estimated that, on average, GJB2 mutations account for about 22% of
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hearing loss in children, but pointed out that the percentages differ significantly among ethnic groups. 3.07.3.1 Structure, Expression, and Function The GJB2 gene consists of two exons, with exon 2 encoding the 208 amino acid protein. Exon 1 is not translated. The promoter region of the mouse and human genes is highly conserved, and includes GC boxes, GT boxes, and a TTAAAA box (Kiang, D. T. et al., 1997). Because of the relatively short coding sequence, identification of mutations by sequencing is efficient and often provides a definitive diagnosis. GJB2 is expressed in many tissues, including skin, liver, breast, bladder, and pancreas. In the inner ear, GJB2 is expressed in both the epithelial and the connective cellular networks. These include the supporting cells of the hair cells, as well as the spiral limbus, spiral ligament, and stria vascularis. The protein encoded by GJB2 is usually called connexin 26. There are 20 different types of human connexins; two of these (connexin 26 and connexin 30) co-localize in the cochlea (Marziano, N. K. et al., 2003). Each connexin consists of four transmembrane domains connected by one intracellular loop, two extracellular loops, and cytoplasmic N and C terminals. Connexons, which are comprised of six connexins, join with those in an adjacent cell to form homotypic, heterotypic, or heteromeric channels between the cells. The connexons are formed intracellularly and are trafficked to the plasma membrane. These gap junctions allow intercellular diffusion of small molecules and metabolites with a molecular mass up to about 1 kDa. The turnover time of connexins is very rapid, indicating that gap junction channels are replaced several times every day. They are thought to be important for maintaining high concentrations of Kþ in the endolymph via recycling from the perilymph. In turn, this recycling is necessary for the maintenance of the endocochlear potential, which is essential for normal auditory function. Normal forms of both connexin 26 and connexin 30 appear to be required for formation of functional gap junction channels in the support cells of the cochlea, indicating that they do not functionally compensate for one another (Ahmad, S. et al., 2003). 3.07.3.2
Mutations
More than 90 different mutations have been reported in exon 2, the most common in the Caucasian
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population (particularly those from southern Europe) being 35delG with a carrier frequency of about 2%. This deletion of a guanine in codon 12 means that codon 13 becomes a stop codon, and translation is terminated. Other mutations have been reported with increased frequencies in particular populations, such as 167delT and 235delC in the Ashkenazi Jewish and Asian populations, respectively. Although most of the mutations in GJB2 are associated with recessive hearing loss, some have been found in families with dominant hearing loss (DFNA3). All are missense mutations and for a few, the phenotype is syndromic with skin abnormalities in addition to the hearing loss. Many variants that do not segregate with hearing loss have been reported. The M34T allele is controversial. Individuals with normal hearing based on pure-tone audiograms and 167delT/M34T and 35delG/M34T genotypes have been reported, but other studies suggest that M34T contributes to recessive mild-to-moderate- or high-frequency hearing loss. Often, interpretation of diagnostic test results is not straight forward because only one mutation is found. Several explanations are possible: the second mutation may have been missed or the individual may be a carrier (by chance), with the hearing loss having nothing to do with the mutation. In some cases, it has been shown that the second mutation is a 309 kb deletion or a 232 kb deletion that encompasses all or part of the GJB6 gene, which encodes connexin 30 (del Castillo, F. J. et al., 2005). GJB2 and GJB6 are less than 50 kb apart, and share 77% identity in amino acid sequence, with connexin 30 having 37 more amino acids at its C-terminus than connexin 26. The nonsyndromic recessive phenotype can vary from mild to profound hearing loss, even in individuals with the same GJB2 mutations. This finding suggests that modifier genes may be involved, but a systematic study to find such genes has not yet been done. 3.07.3.3
Mouse Models
The knockout mouse is an embryonic lethal, but studies of conditional knockout mice suggest that interference with potassium recycling may explain the hearing loss that results when connexin 26 is missing in the inner ear (Cohen-Salmon, M. et al., 2002). Mice do not hear until about 2 weeks after birth and the inner ears of the mutant mice were
found to develop normally until P14 when apoptotic cell death began to occur. Supporting cells of the inner hair cells were the first to die, followed by outer hair cells and their supporting cells. However, the inner hair cells were generally preserved, although ultrastructural analysis revealed anomalies. The mice showed a significant hearing loss with an average of 30 dB at 8 kHz to 36 dB at 32 kHz relative to heterozygous and control mice. The endocochlear potential in adult mutants was significantly lower than in control mice as was the Kþ concentration in the endolymph. In contrast, the mutant mice did not show any vestibular dysfunction, suggesting that connexin 26 is not required for normal function of gap junctions in the vestibular system. Additionally, connexin 30 expression was not affected by the absence of connexin 26.
3.07.4 OTOF The DFNB9 locus was mapped to chromosome 2p22–p23 in a large Lebanese family in which congenital profound sensorineural hearing loss followed an autosomal recessive pattern of inheritance. Yasunaga S. et al. (1999) showed that the affected individuals in this family were homozygous for a nonsense mutation in the OTOF gene. This gene encodes a cytoskeletal protein called otoferlin that may be involved in synaptic vesicle trafficking. Expression of otoferlin in the inner ear was found to be mostly in the inner hair cells. Because of this finding, Varga R. et al. (2003) searched for otoferlin mutations in children with recessive forms of AN/ AD without peripheral neuropathy. These children had absent ABRs, but their OAEs were preserved, suggesting normal function of their outer hair cells. (For some of these children, OAEs disappeared with time.) In general, they did not benefit from hearing aids and their speech comprehension was poorer than expected based on their pure-tone audiograms, which ranged from moderate to profound. Several of these children received cochlear implants and found them helpful. 3.07.4.1 Structure, Expression, and Function OTOF consists of 48 exons spanning about 90 kb of genomic DNA. Yasunaga S. et al. (2000) detected numerous alternate splice forms involving exons 6, 31, 47, and 48, and several translation initiation sites.
Genetic Hearing Loss
There are two classes of otoferlin isoforms: long ones with approximately 2000 amino acids and six C2-like domains, and short ones (encoded by exons 20–48) with about 1200 amino acids and only the last three C2-like domains. C2 domains bind Ca2þ and are often found in proteins involved in signal transduction, vesicle trafficking, and membrane fusion. Numerous adult human and mouse tissues including the cochlea, vestibule, eye, brain, placenta, liver, heart, lungs, pancreas, skeletal muscle, and kidney, as well as total human fetus, showed OTOF expression, with the strongest expression being in the cochlea and brain. The short isoforms were not detected in mouse tissues, whereas the only human tissue in which the long isoforms were detected was the brain. Additionally, the 60 C-terminal amino acids in brain otoferlin are encoded by exon 47, whereas in the cochlea and other tissues, exon 47 is skipped and the 59 part of exon 48 is used to encode the final 60 amino acids. The strongest expression of OTOF in the inner ear of the mouse is in the cochlear inner hair cells and vestibular type I sensory hair cells; it was detected at E19.5 and persisted until at least P20. OTOF expression was also detected in the cochlear outer hair cells and spiral ganglion cells of mouse embryos, but it was found only in inner hairs cells of mature cochleas (Yasunaga, S. et al., 1999; 2000). Otoferlin is related to the C. elegans FER-1 protein, as are two other proteins with C2 domains, dysferlin and myoferlin. They are all predicted to be C-terminal membrane-anchored cytosolic proteins, which interact with phospholipids and other proteins via their C2 domains. Otoferlin function is unknown, but the C2-like domains suggest that it may be involved in synaptic vesicle–plasma membrane fusion. b0145
3.07.4.2
Mutations
In the original DFNB9 family, Yasunaga S. et al. (1999) showed that the affected individuals were homozygous for a nonsense mutation in exon 39. Of the17 frameshift, splice site, and missense mutations that have been reported so far, five are found in exons that are included only in the long isoforms, which are restricted to the brain. No mutations have been found in exon 47, but three are in exon 48, which is encoded in the cochlea but not in the brain. In the Spanish population, Migliosi V. et al. (2002) found that 4.4% of those without GJB2 mutations have OTOF mutations (in particular, Q829X in exon 22), and Rodriguez-Ballesteros M. et al. (2003) reported that
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OTOF mutations account for at least 3.5% of prelingual hearing loss. 3.07.4.3
Mouse Models
A mouse model should provide some insight into clarifying the function of otoferlin. However, no spontaneous mutations have yet been reported, and a knockout mouse has not been created.
3.07.5 TECTA Both dominant (DFNA8/12) and recessive (DFNB21) forms of hearing loss have been associated with mutations in the TECTA gene, which encodes -tectorin, a noncollagenous extracellular matrix protein. The three loci were mapped to chromosome 11q22–q24 in three different families from Austria, Belgium, and Lebanon. TECTA was considered a likely candidate gene based on its location (Verhoeven, K. et al., 1998). The dominant phenotype in the Belgian and Austrian families was nonsyndromic, prelingual, stable, moderate-to-severe hearing loss at all frequencies. This is in contrast to most dominant forms of hearing loss, which are progressive and have a postlingual onset. However, affected members of other DFNA8/12 families have the typical progressive loss starting in the high frequencies. The recessive phenotype in the family from Lebanon had a prelingual onset and was in the severe-toprofound range (Mustapha, M. et al., 1999). 3.07.5.1 Structure, Expression, and Function TECTA has 23 exons encoding a 2155 amino acid protein, -tectorin. The human DNA sequence is predicted to have 95% identity with the mouse sequence. Splice variants may exist, but have not yet been systematically investigated. The protein has hydrophobic N-terminal signal sequences that are characteristic of secreted proteins. The C-terminal is also hydrophobic and is predicted to have a glycosyl-phosphatidylinositol (GPI) anchor, which is considered to be a signal for directing the protein to the apical surface of the inner ear sensory epithelia. It also has von Willebrand factor (vWF) type D repeats, which are similar to those found in the sperm membrane protein, zonadhesin, and are rich in cysteines, and a C-terminal ZP domain, which is found in proteins in the zona pellucida, the
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extracellular matrix that surrounds the unfertilized egg. Zonadhesin is known to bind to the zona pellucida (Legan, P. K. et al., 1997). TECTA is expressed only in the inner ear, and, in particular, it is found in the tectorial membrane of the cochlea. Developmental studies in the mouse demonstrate that tecta mRNA is expressed in the basal cochlear duct by E12.5, throughout the cochlea by E14.5, and reaches its highest level at P3. After that, expression decreases with minimal detectable mRNA at P15 and none at P67 (Maeda, Y. et al., 2001). These results indicate that TECTA is expressed at high levels only briefly during the development of the cochlea and suggest that -tectorin is stable with a long half-life. Tectorins are pepsin-sensitive glycoproteins that are resistant to digestion with bacterial collagenase. The tectorial membrane is a ribbonlike sheet of extracellular matrix that spirals along the length of the cochlea, and connects to the tips of the stereocilia of the outer hair cells. It is composed of collagens (types II, V, and IX) and a noncollagenous matrix, consisting mostly of -tectorin and -tectorin. These two proteins crosslink, and they appear to be critical for attachment of the tectorial membrane to the cochlear epithelium.
3.07.5.2
Mutations
Missense mutations have been found in affected individuals in DFNA8/12 families, and a dominant negative mechanism has been proposed, although haploinsufficiency is also considered possible (Verhoeven, K. et al., 1998). The reasons for the differences in age of onset and phenotypes among affected individuals in DFNA8/12 families is not clear. However, three missense mutations that change cysteine residues at positions 1057, 1619, and 1837 are associated with progressive loss. So far, seven different missense mutations have been reported and to some extent it appears that mutations in the vWF regions cause high-frequency hearing loss, while those in the ZP domain cause mid-frequency loss (Naz, S. et al., 2003). In the Lebanese DFNB21 family, the affected individuals were homozygous for a splice site mutation. More recently, Naz S. et al. (2003) reported exon 5 and exon 20 frameshift mutations in two consanguineous DFNB21 families from Iran and Pakistan. In both families the hearing loss was prelingual and in the moderate-to-severe range.
3.07.5.3
Mouse Models
Homozygous knockout mice have moderate-tosevere hearing loss and abnormal and detached tectorial membranes with no known noncollagenous components. The tectorial membrane is synthesized, but within a few weeks after birth it is no longer attached at either end and the collagen fibers are disorganized. In contrast, the structure and orientation of the stereocilia of the cochlear hair cells are unaffected, although the cochlear microphonic is smaller and differs in phase and symmetry from the wild-type mice. The heterozygous mice appear to be normal (Legan, P. K. et al., 2000). These findings demonstrate the importance of the tectorial membrane in amplifying auditory stimuli, presumably because it is essential for deflection of outer hair cell stereocilia.
3.07.6 Outlook for the Future Progress in our understanding of genetic hearing loss has been enormous over the past decade. We have made great strides in determining many of the different molecular mechanisms that contribute to the genetic heterogeneity of hearing loss. However, more genes remain to be identified, and elucidating the function of the proteins encoded by these genes is challenging. The four genes discussed in this perspective provide examples of the phenotypic and genetic heterogeneity of hearing loss and the progress that has been made in understanding their function in the inner ear. It is clear that different mutations in the same gene can be associated with both nonsyndromic and syndromic forms of hearing loss, and in some cases a mutation in one copy of the gene (dominant) is sufficient while in others mutations in both copies (recessive) are required for hearing loss. These four genes show diverse expression patterns and the proteins they encode have quite different functions. But they are all critical for normal hearing. Genetic diagnostic testing for mutations in the GJB2 gene is becoming routine and other genes are likely to be added in the near future. In particular, testing for the Q829X mutation in the OTOF gene might be considered for those who do not have GJB2 mutations. Ruling out a syndrome is important, especially for one such as Usher syndrome where the first signs of retinal degeneration may not appear for 10 or more years after the diagnosis of severe-to-profound
Genetic Hearing Loss
hearing loss. Because of the genetic heterogeneity of Usher syndrome, a genetic test for all mutations is not yet feasible, but Acadian children should be screened for the USH1C 216G!A mutation, and a mutation in the protocadherin 15 gene (R245X) should be considered in those with Ashkenazi Jewish ancestry who do not have mutations in the GJB2 gene. A genetic diagnosis is relevant not just to the affected individual but to all family members, some of whom may choose to be tested before making reproductive decisions. In many cases, knowing the genotype is not enough to give a precise phenotype and prognosis, but ongoing research is elucidating some of these relationships. The development of effective therapies based on knowledge of the underlying genetic abnormality is just beginning; however, the potential for molecular intervention strategies as a treatment for hearing loss is promising.
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Wachtler, F., Kimberling, W. J., Willems, P. J., Govaerts, P. J., and Van Camp, G. 1998. Mutations in the human alpha-tectorin gene cause autosomal dominant nonsyndromic hearing impairment. Nat. Genet. 19, 60–62. Verpy, E., Leibovici, M., Zwaenepoel, I., Liu, X. Z., Gal, A., Salem, N., Mansour, A., Blanchard, S., Kobayashi, I., Keats, B., Slim, R., and Petit, C. 2000. A defect in harmonin, a PDZ domain-containing protein expressed in the inner ear sensory hair cells, underlies Usher syndrome type 1C. Nat. Genet. 26, 51–55. Yasunaga, S., Grati, M., Chardenoux, S., Smith, T. N., Friedman, T. B., Lalwani, A. K., Wilcox, E. R., and Petit, C. 2000. OTOF encodes multiple long and short isoforms: genetic evidence that the long ones underlie recessive deafness DFNB9. Am. J. Hum. Genet. 67, 591–600. Yasunaga, S., Grati, M., Cohen-Salmon, M., El-Amraoui, A., Mustapha, M., Salem, N., El-Zir, E., Loiselet, J., and Petit, C. 1999. A mutation in OTOF, encoding otoferlin, a FER-1-like protein, causes DFNB9, a nonsyndromic form of deafness. Nat. Genet. 21, 363–369. Zwaenepoel, I., Verpy, E., Blanchard, S., Meins, M., ApfelstedtSylla, E., Gal, A., and Petit, C. 2001. Identification of three novel mutations in the USH1C gene and detection of thirtyone polymorphisms used for haplotype analysis. Hum. Mutat. 17, 34–41.