Учебники / Genetics and Auditory Disorders Keats 2002

7. Mitochondrial DNA Mutations 231

that generalized neuronal dysfunction is also expressed in the auditory system.

In 1992 several families with diabetes mellitus and sensorineural hearing loss were described, and surprisingly were found to have inherited the heteroplasmic A3243G mutation in the gene for tRNAleu(UUR) the very same mutation as is associated with the systemic MELAS syndrome (Reardon et al. 1992; van den Ouweland et al. 1992). In none of these cases were other neurological symptoms present. One family had, instead of the A3243G mutation, a heteroplasmic large deletion/insertion event (Ballinger et al. 1992), and more recently the heteroplasmic point mutations T14709C in the tRNAglu gene and A8296G in the tRNAlys gene were also found to be associated with maternally inherited diabetes and deafness (Vialettes et al. 1997; Kameoka et al. 1998). This association between diabetes mellitus, hearing loss, and mtDNA mutations has been confirmed in population studies of diabetic patients (Oka et al. 1993; Alcolado et al. 1994; Kadowaki et al. 1994; Katagiri et al. 1994; Sepehrnia et al. 1995; Newkirk et al. 1997; Rigoli et al. 1997). For example, Kadowaki et al. (1994) found the A3243G mutation in 2 to 6% of diabetic patients in Japan, and in 3 out of 5 patients with diabetes and deafness. Of their 44 patients with diabetes and the A3243G mutation, 27 had hearing loss. The hearing loss is sensorineural and usually develops only after the onset of diabetes. In the non-Japanese populations that have been examined, the A3243G mutation accounts for less than 1% of the diabetic patients (Sepehrnia et al. 1995; Newkirk et al. 1997; Rigoli et al. 1997). More recently, several patients have been described with maternally inherited diabetes and deafness and also either macular dystrophy or adrenal insufficiency (Harrison et al. 1997; Nicolino et al. 1997; Souied et al. 1998). In one of these cases, the heteroplasmic A3243G mutation was found (Harrison et al. 1997), while in the other cases a heteroplasmic large deletion was identified (Nicolino et al. 1997; Souied et al.

1998).

The A7445G mutation was initially described as a nonsyndromic deafness mutation, but was subsequently found to be also associated with the skin condition palmoplantar keratoderma (PPK) in at least some of the cases (Reid et al. 1994a; Fischel-Ghodsian et al. 1995; Sevior et al. 1998). It is discussed more fully in the next section.

3.2 Mitochondrial DNA Mutations and Nonsyndromic

Hearing Loss

The first mutation associated with nonsyndromic deafness was identified in an Arab-Israeli pedigree, when the striking pattern of transmission only through mothers was noted (Jaber et al. 1992; Prezant et al. 1993). Most of the deaf family members had onset of severe to profound sensorineural hearing loss during infancy, but a minority of family members had onset during childhood, or even adulthood (Braverman et al. 1996). The homo-

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plasmic A1555G mutation in the mitochondrial 12S ribosomal RNA gene was identified as the pathogenic mutation (Prezant et al. 1993).

While subsequently many pedigrees and individual patients have been described with the same A1555G mutation, in all these cases the hearing loss occurred only after aminoglycoside exposure (Hutchin et al. 1993; Fischel-Ghodsian et al. 1993; Matthijs et al. 1996; Fischel-Ghodsian et al. 1997b; Pandya et al. 1997; Gardner et al. 1997). However, recently a significant number of pedigrees in Spain, with family members who went deaf with and without aminoglycosides, have been described (El-Schahawi et al. 1997; Estivill et al. 1998). In particular, the study by Estivill et al. (1998) is remarkable for two reasons, both of which indicate a higher than previously expected frequency of this mutation. First, 19 families were found to have the A1555G mutation out of the total of 70 families with sensorineural hearing loss that were included in the study. Even if the selection criteria led to a bias towards families with multiple affected individuals, and even when only the individuals without aminoglycoside exposure are considered, the frequency of familial sensorineural hearing loss due to the A1555G mutation is still unexpectedly high. Second, the fact that the mutation was identified on different haplotypes indicates that it is likely that this mutation exists in other populations as well, and may not be rare. It is also interesting to notice that the age of onset of hearing loss in the Spanish families was rarely congenital, which is different from the Arab-Israeli pedigree. Two similar families were identified recently in Italy, with 17 deaf family members who have the A1555G mutation and no exposure to aminoglycosides (Casano et al. 1998).

Another close to homoplasmic, inherited mutation leading to hearing loss is the A7445G mutation. It was first described in a family from Scotland, and confirmed in two unrelated pedigrees from New Zealand and Japan (Reid et al. 1994a; Fischel-Ghodsian et al. 1995; Sevior et al. 1998). In the

New Zealand and Japanese pedigrees, the skin condition palmoplantar keratoderma also segregated in the maternal line (Sevior et al. 1998). Interestingly, the penetrance of this mutation for hearing loss in the Scottish pedigree is quite low, while in the New Zealand and Japanese pedigrees it is very high. Thus, in similarity to the Arab-Israeli pedigree, the mtDNA mutation by itself does not appear to be sufficient to cause hearing loss, but requires additional genetic or environmental factors, which seem to be rare in the Scottish pedigree and common in the New Zealand and Japanese pedigrees. The difference in penetrance in this situation appears to be due to a difference in mitochondrial haplotype. In the New Zealand pedigree, complete sequencing of the mtDNA revealed three additional sequence changes in complex I protein genes, two of which have been also labeled as secondary Leber’s hereditary optic neuroretinopathy mutations (FischelGhodsian et al. 1995). Since these or similar sequence changes are not present in the Scottish pedigree (Reid et al. 1994b), the mitochondrial haplotype appears to account for the differences in penetrance.

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A third mtDNA mutation, a cytosine insertion at position 7472 (Cins7472) in the tRNAser(UCN) gene, was identified in one large Dutch family, with 27 deaf individuals (Verhoeven et al. 1999). The same mutation had been previously described in a Sicilian family with 13 members with hearing loss, seven of whom had other neurological symptoms, such as ataxia and myoclonus (Tiranti et al. 1995). In the Dutch family, only a single individual had neurological symptoms, and the hearing loss was sensorineural-progressive with onset in early adulthood. Most of the individuals over 30 years of age were deaf, indicating that the penetrance in this family is high. The mutation is heteroplasmic, although most individuals had over 90% of abnormal mitochondrial chromosomes in the tissue examined.

Recently, a large African-American pedigree with maternal inheritance of nonsyndromic hearing loss has been identified. The A1555G, A7445G, and Cins7472 mutations, as well as large deletions/rearrangements in the mtDNA were excluded, suggesting the existence of a fourth mitochondrial mutation associated with nonsyndromic hearing impairment (Friedman et al. 1999). This was confirmed with the identification of a close-to- homoplasmic mutation, T7511C, in the tRNAser(UCN) gene (Sue et al. 1999).

3.3 Mitochondrial DNA Mutations and Ototoxic

Hearing Loss

Aminoglycoside ototoxicity is one of the most common causes of acquired deafness. Although vestibulocochlear damage is nearly universal when high drug levels are present for prolonged periods, at lower drug levels there appears to be a significant genetic component influencing susceptibility to aminoglycoside ototoxicity. The existence of families with multiple individuals with ototoxic deafness induced by aminoglycoside exposure was noticed early on. The first families with more than two members with streptomycin-induced hearing loss were described in the Japanese literature in 1957 (for review, see Higashi 1989). Prazic et al. (1964) described a family with four sisters who developed hearing loss after streptomycin injections. Tsuiki and Murai (1971) described 16 families in which two or more members had aminoglycoside ototoxicity. Konigsmark and Gorlin (1976) summarized most existing descriptions of familial aminoglycoside ototoxicity and concluded that inheritance of the predisposition is probably autosomal dominant with incomplete penetrance. However, they also noted that no male-to-male transmission has been seen, and suggested that the inheritance pattern might be multifactorial. Higashi (1989) reviewed the literature, and concluded that the most likely explanation for the maternal inheritance observed is a mitochondrial DNA defect. Hu et al. (1991) described another 36 families in China with maternally transmitted predisposition to aminoglycoside ototoxicity, and concluded also that a

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mitochondrial DNA defect may be responsible. Additional evidence for a genetic basis for aminoglycoside susceptibility comes from animal studies. Macaque monkeys are resistant to dihydrostreptomycin, while the patas monkeys are highly sensitive to that drug (Stebbins et al. 1981).

Analysis of three Chinese families, in which several individuals developed deafness after the use of aminoglycosides, identified the A1555G mutation in the 12S ribosomal RNA gene in all three of them, but not in hundreds of controls (Prezant et al. 1993). In addition, a small proportion of “sporadic” Chinese patients, without a positive family history for aminoglycoside ototoxicity, exhibit this particular mutation (Fischel-Ghodsian et al. 1993). These findings were confirmed in two Japanese families and additional Chinese sporadic cases (Hutchin et al. 1993). Subsequently, the same mutation was found in families and sporadic patients with aminoglycoside ototoxicity from Zaire, the United States, Mongolia, Spain, South Africa, and Israel (Matthijs et al. 1996; Fischel-Ghodsian et al. 1997; Pandya et al. 1997; El-Shahawi et al. 1997; Estivill et al. 1998; Gardner et al. 1997; Shohat et al. 1999). Interestingly, in one streptomycin-induced deaf individual with a strong familial history of aminoglycoside-induced hearing loss and the A1555G mutation, detailed vestibular examination revealed severe hearing loss, but completely normal vestibular function (Braverman et al. 1996).

Because the A1555G mutation in the mitochondrial 12S rRNA gene accounts only for a minority of patients with aminoglycoside ototoxicity, it is possible that other susceptibility mutations may be found in the same gene. Mitochondrial DNA from 35 Chinese sporadic patients with aminoglycoside ototoxicity and without the A1555G mutation was analyzed for sequence variations in the 12S rRNA gene. Three sequence changes were found, but only one of them, an absence of a thymidine at position 961 with varying numbers of cytosines inserted (DT961Cn), appeared likely to be a pathogenic mutation (Bacino et al. 1995). Analysis of 34 similar patients in the United States, of varying ethnic backgrounds, did not reveal this mutation, but recently an Italian family was reported in which five maternally related members became deaf after aminoglycoside treatment; they were all found to have the DT961Cn mutation (Casano et al.1999). This sequence change was not found in 799 control individuals (Bacino et al. 1995).

3.4 Mitochondrial DNA Mutations and Presbycusis

Another condition associated with acquired heteroplasmic mutations and hearing loss is presbycusis. Presbycusis is the hearing loss that occurs with age in a significant proportion of individuals. Because mtDNA mutations, and the resulting loss of oxidative phosphorylation activity, seem to play an important role in the aging process (reviewed by Nagley et al. 1993), it seems likely that mtDNA mutations in the auditory system might lead to presbycusis. Recently, the spiral ganglion and membranous labyrinth from archival temporal bones of five patients with presbycusis were examined for mutations within the mitochondrially encoded cytochrome oxidase II

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gene (Fischel-Ghodsian et al. 1997a). When compared with controls, the results indicated that at least a proportion of people with presbycusis have a significant load of mtDNA mutations in auditory tissue, and that there is great individual variability in both quantity and cellular location of these mutations. Similar data were obtained by Bai et al. (1997) when screening cochlear tissue from temporal bones for the presence of large deletions, although the ages of the presbycusis and control groups were not well matched. The greatest advantage of studying acquired mutations in the ear relates to the availability of temporal bone tissue banks, through which functional audiological data are available. Thus, measurable functional status, histology, immunohistochemistry of oxidative phosphorylation complexes, and mtDNA analysis can be correlated.

There are, however, two problems with the mtDNA data from patients with presbycusis. One problem relates to the fact that a sufficient number of patients has not yet been examined to draw the firm conclusion that there is indeed a clear difference between presbycusis patients and normalhearing controls of the same age. Part of the problem is that temporal bone libraries have only the very rare patient with documented normal hearing and advanced age. But even if the data will eventually unequivocally show that presbycusis patients as a group have an increased number of mtDNA mutations, the second problem is one of causality. Are these mutations the cause or the result of the cochlear process leading to presbycusis? Two models are possible. The first model assumes that some genetic and/or environmental factors combine to cause acquired mtDNA mutations, which then lead to hearing loss. This would imply a primary causative role for mtDNA mutations. Alternatively, it is possible that the genes and environment cause presbycusis directly, and that the resulting cell death and response mechanisms lead to increased oxygen radicals and an increased rate of mtDNA mutations. In this case, the mtDNA mutations would only be a sign of presbycusis, but not a cause. To differentiate between these two models, it is necessary to use animal models in which the temporal relationship between mutations and hearing loss can be studied, as well as the response to specific interventions.

4. Clinical Relevance of Mitochondrial DNA Mutations Associated with Hearing Impairment

The major clinical relevance of mtDNA mutations to hearing loss remains the prevention of aminoglycoside-induced hearing loss. In countries where aminoglycosides are used commonly, aminoglycoside-induced ototoxicity is a major cause of hearing loss. For example, in a study that reviewed all deafmutes in a district of Shanghai, 21.9% had aminoglycoside-induced hearing loss, representing 167 individuals in a population of nearly half a million

(Hu et al. 1991). The A1555G mutation accounted for at least 30% of these.

In the United States, the A1555G mutation accounts for about 15% of all

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aminoglycoside-induced deafness cases (Fischel-Ghodsian et al. 1997b). The difference in frequency may reflect the fact that aminoglycosides are generally used in the United States only for severe in-hospital infection. These patients receive significantly higher levels for more prolonged periods, and are more likely to have other medical conditions that cause or exacerbate the hearing loss. The frequency of the DT961Cn predisposing mutation is unknown, but it appears to be significantly lower.

Whatever the precise frequency, prevention of a major cause of aminoglycoside-induced ototoxicity is now possible. Physicians need to inquire about a family history of aminoglycoside-induced hearing loss prior to the administration of systemic aminoglycosides, as well as prior to the local administration of aminoglycosides into the cochlea as a treatment for Meniere’s disease. In addition, every individual with aminoglycosideinduced hearing loss should probably be screened at least for the presence of the G1555A and DT961Cn mutations because presence of a mutation will allow counseling to all maternally related relatives to avoid aminoglycosides. Similarly, the study by Estivill et al. (1998) indicates that it might be reasonable to screen every individual with nonsyndromic hearing loss for the mutation, unless maternal inheritance can clearly be excluded. Because the test is easily done, and prevention of hearing loss in maternal relatives can easily be accomplished, this may be cost-effective medical practice. The tragic and avoidable hearing loss of at least forty patients in the report by Estivill et al. (1998) is a case in point.

With the exception of not administering aminoglycosides to patients with mtDNA mutations in the 12S rRNA gene, there are no proven preventive or therapeutic interventions for mitochondrially related hearing impairments. The diagnosis of such defects is, however, useful for genetic counseling (Arnos and Oelrich, Chapter 9) and is indicated in all families with an inheritance pattern of hearing loss that is consistent with maternal transmission, and possibly in all patients who have both diabetes mellitus and adult-onset hearing loss.

5. Pathophysiology of Mitochondrial DNA

Deafness Mutations

For aminoglycoside ototoxicity due to the A1555G mutation, it is interesting to notice that this mutation lies exactly in the region of the gene for which the resistance mutations in yeast and Tetrahymena have been described, and in which aminoglycoside binding has been documented in bacteria (Li et al. 1982; Spangler and Blackburn 1985; Gravel et al. 1987). In addition, the mutation makes the mitochondrial RNA gene in this region more similar to the bacterial ribosomal RNA gene (Prezant et al. 1993). Because aminoglycosides are concentrated within cochlear cells, and remain there for prolonged periods (Henley and Schacht 1988), it has been

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proposed that susceptible individuals with the A1555G mutation have increased binding to aminoglycosides, leading to altered protein synthesis in the mitochondria (Prezant et al. 1993), and that the tissue specificity is due to the concentration of the drug in those cells. Subsequent binding experiments have proved that increased binding to the mitochondrial 12S ribosomal RNA occurs (Hamasaki and Rando 1997). However, analysis of lymphoblastoid cell lines of individuals with the A1555G mutation indicated that exposure of the cell lines to high concentrations of neomycin or paromomycin led to a decreased rate of growth in glucose medium, but no mutant proteins were detected (Guan et al. 1996). Similar results of decreased protein synthesis, but no mutant proteins were obtained using mitochondrial transfer from human skin fibroblast lines with the A1555G mutation to human r0 cells (which lack mtDNA) exposed to very high levels of streptomycin (Inoue et al. 1996). This may indicate that the effect of aminoglycosides in these cell lines could be nonspecific and be different than in the cochlea, perhaps because of different transport of the antibiotic into the mitochondria.

For nonsyndromic hearing loss, at a first glance it is possible to speculate that mitochondrial mutations interfere with energy production, that the cochlea is highly dependent on sufficient energy production, and that insufficient energy production leads to degeneration of cochlear cells. However, the cochlea is not a particularly energy-dependent tissue, and in the systemic neuromuscular disorders listed in Section 3.1, the extraocular muscles appear to be the most energy-sensitive cells; hearing loss is not a prominent clinical sign in any of these mitochondrial disorders. Thus, in order to understand the pathophysiological pathways leading from the mtDNA mutations to hearing loss, two major biological questions need to be answered: Why does the same mutation cause severe hearing loss in some family members but not in others? And: Why is the ear the only organ affected?

Study of the mtDNA mutations leading to hearing loss has led to three possible precipitating factors modulating phenotypic expression, and it is likely that a combination of them also plays a significant role in the phenotypic expression of acquired mitochondrial disorders. The first such factor involves environmental agents, and aminoglycosides are the prime example as a triggering event in the case of the A1555G mutation. Other, as yet unrecognized, environmental factors could play similar, but perhaps less dramatic, roles. Diet and drugs affecting oxygen-radical formation and break-down come to mind. The second factor involves the mitochondrial haplotype, and, as noted above, the A7445G mutation provides a dramatic example of the effect of haplotype differences. The third factor involves nuclear genes. The Arab-Israeli pedigree, and some of the Spanish and Italian pedigrees, are good examples of the role of nuclear genes. For example, the entire Arab-Israeli family lives in a small Arab village in Israel, and all maternal relatives share the same mitochondrial haplotype. Bio-

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chemical differences between lymphoblastoid cell lines of hearing and deaf family members with the identical mitochondrial chromosomes provide direct support for the role of nuclear factors (Guan et al. 1996). An extensive genome-wide search has led to the conclusion that this nuclear effect is unlikely to be due to a single nuclear gene (Bykhovskaya et al. 1998). Thus, the model that emerges for explaining penetrance is a threshold model, where a combination of environmental, mitochondrial, and nuclear factors can push a cell over a threshold, with dramatic clinical differences on either side of this threshold.

The second major biological question relates to tissue specificity: If a homoplasmic mutation affects oxidative phosphorylation (the only known function of the human mitochondrial chromosome and an essential process in every nucleated cell of the human body), it is unclear how the clinical defect remains confined to the cochlea, rather than affecting every tissue.

6. Experimental Approaches to Elucidate the Pathophysiology of Mitochondrial DNA Deafness Mutations

The experimental approaches to elucidate the pathophysiological pathways of the mtDNA deafness mutations are limited by the absence of a spontaneous or induced animal model for any inherited mitochondrial disorder. This is mainly due to the lack of experimental ways to manipulate the mitochondrial chromosome and transfer it into the mitochondrial cytoplasm. A collaborative study involving the Jackson Laboratories has been established to screen all hearing impaired mice for differences in their mitochondrial genomes. Pending the identification of such a spontaneous mutant, and given the lack of success with the genetic approach to identify a nuclear factor influencing the phenotype of the A1555G mutation (Bykhovskaya et al. 1998), a direct approach is proposed. It is hypothesized that degree of penetrance and tissue specificity depend on components of mitochondrial RNA processing and translation, and that cochlea-specific proteins or splice variants that interact with mutated mitochondrial RNA can be identified. Four lines of evidence implicate mitochondrial RNA processing and translation in the pathophysiological pathway between mtDNA mutation and hearing loss. Thus, components of these systems may be responsible for the tissue specificity and penetrance variability observed in patients:

(1) All the mtDNA mutations associated with nonsyndromic hearing loss involve ribosomal or transfer RNA, or in the case of the A7445G mutation, noncoding DNA. That is, none of the known mtDNA mutations associated with hearing loss causes a structural change in any of the 13 proteins

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encoded by the mitochondrial genome (Table 7.1). This situation should be contrasted with other mitochondrial disorders such as Leber’s hereditary optic neuroretinopathy and Leigh’s syndrome, in which missense mutations are the rule (MITOMAP, 1995).

(2)Biochemical analysis of the effect of these mutations demonstrates a RNA processing defect or a decrease in translational efficiency. While the effect of these mutations has not been studied in the cochlea, analysis of lymphoblastoid cell lines has given support to the idea that the mutations affect RNA processing or translation. The A1555G mutation in the 12S ribosomal RNA gene, which is part of the mitochondrial ribosome, causes a generalized decrease in mitochondrial protein synthesis (Guan et al. 1996). The A7445G mutation causes a processing defect of the light strand of the polycistronic mRNA, leading to a 70% reduction in tRNASer(UCN) and ND6 mRNA, which is cotranscribed with the tRNASer(UCN). The decrease in the tRNA leads to a general decrease in protein synthesis, which is exacerbated for ND6 by the decrease of the mRNA for this protein (Guan et al. 1998). The Cins7472 mutation has not been biochemically analyzed in detail, but involves a nucleotide deletion in the TxC loop of the same tRNASer(UCN) that the A7445G mutation affects (Tiranti et al. 1995; Verhoeven et al. 1998). The main mitochondrial mutation associated with syndromic hearing loss, the A3243G mutation in the tRNALeu(UUR) gene, has been proposed to interfere with transcription termination (Hess et al. 1991), although the precise mechanism of the pathogenicity of this mutation remains unclear (Moraes et al. 1992; Flierl et al. 1997).

(3)In humans and other organisms, tissue-specific isoenzymes and splice variants of genes involved in general cellular processes, also called housekeeping genes, have been described. For example, tissue-specific isoenzymes have been identified for some of the nuclear-encoded subunits of the oxidative phosphorylation complexes (Arnaudo et al. 1992), and splice variants of a mitochondrial inner-membrane phosphate carrier have been described in bovine tissues (Dolce et al. 1996).

(4)In humans and other organisms, different processing of mitochondrial RNA and protein, leading to tissue specific defects or functions, have been described. For example, in Drosophila melanogaster, the mitochondrial large ribosomal RNA can, in germ cell precursor cells, also be processed for export into the cytoplasm, where it induces pole cell formation in embryos, a key event in the determination of the germ line (Kobayashi et al. 1993). A human example is a case of a 22-year-old patient who died from respiratory failure due to a mitochondrial myopathy. Analysis of tissues from the patient showed that the causative mutation in the mitochondrial tRNALeu(UUR) gene resulted in a RNA processing defect in skeletal muscle, but not in fibroblasts (Bindoff et al. 1993). Also, in humans it has been shown that a normally occurring RNA processing intermediate of the heavy-strand polycistronic RNA is more prevalent in lymphocytes and liver than in other tissues (Nardelli et al. 1994).

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Based on these four lines of evidence, it is proposed that cochlea-specific subunits of mitochondrial ribosomes or RNA processing proteins interact abnormally with the mitochondrial defect, leading to insufficient oxidative phosphorylation, or loss of a secondary function in the cochlea. Also, variation in the coding sequence for some of these proteins may explain the penetrance differences observed among patients. Mitochondrial RNA processing and translation is a complex process, which occurs exclusively in the matrix of the mitochondria. It requires nuclear-encoded proteins for every step, and has characteristics reminiscent of the bacterial origin of the mitochondrial genome. Estimates of the number of proteins involved range from 200 to 300, and only a small number of these proteins have been identified. Additional proteins can be identified by a variety of methods. For example, the mitochondrial ribosomal S12 gene was identified by cybercloning (Johnson et al. 1998), and this protein has been used as a bait in the yeast dihybrid system to identify a second protein that is an excellent candidate for being another mitochondrial ribosomal protein. All the new proteins identified through such methods are tested for isoforms and cochlea-specific splice variants, using cochlea-specific cDNA libraries (Giersch and Morton, Chapter 3). A more direct approach to the identification of some of these proteins is through isolation of mitochondrial ribosomes from cochlea and another tissue, resolution by 2D gel electrophoresis, and microsequencing of proteins with different electrophoretic characteristics from individual spots. The bovine model system has been established for this purpose (Matthews et al. 1982; O’Brien and Denslow, 1996), and identity correspondence to the human mitoribosomal system has been established by 2D PAGE co-electrophoretic separations of human and bovine mitoribosomal proteins (Matthews et al. 1982).

7. Summary

In conclusion, mitochondria-related hearing loss can be caused by a variety of mutations, and can present in a variety of clinical forms with different degrees of severity. These mutations are not uncommon and, owing to the susceptibility of individuals with the A1555G and DT961Cn mutations and their maternal relatives to aminoglycosides, are important to diagnose. Despite the fact that these mostly homoplasmic mitochondrial mutations represent the simplest model of a mitochondrial DNA disease, it remains unclear how mtDNA mutations lead to the clinically crucial features of penetrance and tissue specificity. Within the same family, some individuals with the mutation can have profound hearing loss, while others have completely normal hearing, and only the hearing is affected although all tissues have the mutation and are dependent on mitochondrial ATP production. Experimental approaches using spontaneous mouse models of mitochondrial hearing impairment, or direct investigation of the most likely biochemical