Учебники / Genetics and Auditory Disorders Keats 2002

tively, if the addition or deletion is not in multiples of three, a frameshift occurs, since the codon reading frame is now altered. Frameshifts result in the addition of incorrect amino acids to the elongating protein. Ultimately, frameshifts will terminate at a stop codon farther downstream. Because of the addition of incorrect amino acids, in some cases, the mutated protein is recognized by the cell as nonfunctional. This may be because the protein is not targeted to its correct location within the cell, or because the protein is not exhibiting the correct enzymatic activity. In these cases, the mutated protein is often destabilized and is degraded by the cell.

In addition to DNA mutations that directly alter protein sequence, point mutations, insertions and deletions can also affect sequences essential for the initiation of transcription and translation. Ultimately, these types of mutations have the same result as mutations in the coding regions, namely that the encoded protein is not correctly expressed.

Silent mutations, or variants, occur when a substitution in a nucleotide occurs without altering the amino acid. Single-nucleotide polymorphisms (SNPs), the most frequent type of variation in the human genome, may explain susceptibility to common genetic traits and diseases and are currently being identified on a large scale as part of the Human Genome Project (Wang et al. 1998a).

4.1.1 Mutations in the Coding DNA Sequence Affect the Translation of RNA into Protein and Cause Hearing Loss

There are numerous examples of missense, nonsense, deletion, insertion, and frameshift mutations in NSHL (Table 2.3), all of which affect translation of RNA into protein. One example of a locus that has multiple missense mutations that affect protein translation is the DFNA2 region. DFNA2 contains at least two deafness genes, KCNQ4, which codes for a potassium channel (Kubisch et al. 1999), and GJB3, which codes for a gap junction protein (Xia et al. 1998). One French family, exhibiting dominant profound hearing loss, was found to harbor a single copy of a missense mutation in KCNQ4. In this family, although the remainder of the protein is translated properly, a single glycine at amino acid 285 is changed to a serine. This glycine residue is normally located in the potassium channel pore, a highly conserved region of the protein. Replacement of this glycine with a serine results in a dysfunctional potassium channel. Three additional missense mutations have been found in KCNQ4 in Dutch and American DFNA2 families, and in all cases, the missense mutations alter conserved and presumably essential regions in the protein (Coucke et al. 1999).

For two dominant loci, DFNA8 and DFNA12, missense mutations in TECTA lead to dominant NSHL (Verhoeven et al. 1998). Interestingly, in one Belgian family, there are two missense mutations 12 bases apart. The mutations may interact, or only one may contribute to the disease, with the second mutation being a rare polymorphism, since it was not detected in 40

unaffected family members, or 100 non-related individuals. In all cases, the conserved zona pellucida domain of the a-tectorin protein is compromised by the replacement of alternate amino acids, suggesting this region has a crucial function for this protein (Verhoeven et al. 1998).

Connexin 26 is a transmembrane protein that forms cylindrical channels in gap junctions used to transfer small molecules between cells. This gap junction protein is highly expressed between supporting cells of the inner ear, and in the spiral lamina facing the endolymphatic duct and may play a role in K+ recycling in the inner ear (Spicer and Schulte 1996). All connexin 26 mutations characterized to date are either nucleotide substitutions (missense/nonsense/splicing mutations), or insertions and deletions ranging from small (1 bp) to large (38 bp) in the coding region (mutations updated regularly in the Connexin 26 (GJB2) Deafness Homepage (World Wide Web URL: http://www.iro.es/cx26deaf.html). The common 35delG mutation is a one-base-pair deletion in a stretch of six G nucleotides in codon 10 that results in a frameshift; a glycine is converted to a valine at codon 12 and a stop codon is formed at codon 13 (Zelante et al. 1997). In other families, an insertion has been detected at the same site, 35insG, leading to a frameshift and stop codon at codon 47 (Estivill et al. 1998). In both cases, it is highly unlikely that any protein is translated. A mutation involving both an insertion and deletion has been identified in a family with recessive NSHL (Sobe et al. 2000). A deletion of 12 bp occurred, with the insertion of one nucleotide, to form a 51del12insA mutation. This mutation occurs in the first intracellular amino terminus domain, leading to a stop codon at the beginning of the first extracellular domain. A frameshift is formed in the amino-terminal portion of the protein, resulting in 26 additional novel amino acids followed by a premature termination.

Additional examples of mutations in the DNA sequence affecting the translation of RNA into protein and causing NSHL are shown in Table 2.3.

4.1.2 Mutations in Components of the Translation Machinery Cause Hearing Loss

The RNA components of the ribosome are essential for translation of mRNA into protein. Hearing loss has been correlated with mutations in both tRNA genes and the 12S rRNA gene, genes found on the mitochondrial genome (Fischel-Ghodsian, Chapter 7). Unlike the deafness genes described elsewhere in this chapter, this mitochondrial-dependent hearing loss is transmitted directly from mother to child. Presumably, these mutations alter the translational machinery in the inner ear, and compromise the production of essential protein components of the mitochondria.

promoter of these genes, a region of DNA found upstream of the coding sequences. These transcription factors, in some cases, are required for proper bone development. In other cases, the transcription factors are required for sensory-cell differentiation at later stages of development. Surprisingly, a wide variety of different transcription factor types have been found to encode deafness genes. Thus far, the downstream genes activated by these transcription factors have not been identified, but clearly these genes are critical for ear development. In this section, a review of some of the transcription factors known to be required for hearing, and the phenotypes associated with these transcription mutations, is presented.

In humans, a mutation in the POU4F3 transcription factor gene (also called Brn3c and Brn-3.1) is the basis for progressive, nonsyndromic dominant hearing loss (DFNA15) (Vahava et al. 1998). This transcription factor, which is specifically expressed in the cochlea, has a conserved DNA binding domain, termed a homeodomain, found in a wide variety of transcription factors that have been shown to be important for embryonic development in all species (Semenza 1998). The mutation in POU4F3 is an 8-bp deletion within the homeodomain. This deletion results in a frameshift that produces a protein that is truncated 40 amino acids prematurely. This truncated POU4F3 protein acts in a dominant fashion to disrupt gene transcription in the cochlea.

Targeted deletion of the mouse Pou4f3 gene also leads to profound deafness, confirming the general importance of this transcription factor in mammalian inner ear development (Erkman et al. 1996). In the Pou4f3 mutant mice, the sensory cells of the inner ear, as well as their associated nerves, are not present, although the shape and development of the inner ear is normal. It is the loss of these cell types that results in deafness, and Pou4f3 protein is clearly required for terminal differentiation of these cell types.

Mutations in a related transcription factor, POU3F4, are responsible for X-linked mixed hearing loss, characterized by stapes fixation and progressive sensorineural deafness (DFN3) (de Kok et al. 1995). This homeodomain transcription factor has been implicated in bone development, since patients with POU3F4 mutations have a fixed footplate of the stapes and deficient or absent bone between the lateral end of the meatus and the basal turn of the cochlea. Patients also exhibit cochlear defects, suggesting that POU3F4 is involved in multiple stages of ear development.

POU-type transcription factors are not the only inner ear specific transcriptional regulators. In the mouse, a transcriptional regulator of the forkhead family, Foxi1 has been found to encode a nonsyndromic deafness gene

(Hulander et al. 1998). Foxi1 mutant mice have profound defects in the structure of the bones of the inner ear, and, as a result, the sensory regions of the cochlea and vestibular apparatus do not develop. The result is a “common cavity,” a malformation seen in a portion of human congenital

36 K.B. Avraham and T. Hasson

inner ear defects. Thus far, no human patients have been identified with alterations in Foxi1, but it is considered a good candidate for a human deafness gene.

Gene-targeted mutagenesis of Math1, the mouse homologue of the Drosophila transcription factor atonal, has been found to cause deafness in mice (Bermingham et al. 1999). Most significantly, the hair cells in these mice never form, indicating that this transcription factor regulates the genesis of hair cells.

Mutations in transcription factors can also result in syndromic deafness. A summary of these transcription factors and their associated syndrome genes is shown in Table 2.4. Of these diseases, the best characterized is Waardenburg syndrome (WS). Waardenburg patients have an auditorypigmentary syndrome characteristic of a defect in melanocyte development (reviewed in Read and Newton 1997). Most patients have mutations in the transcription factor PAX3 and, depending on the penetrance of the mutation, patients can exhibit dystopia canthorum (lateral displacement of eyes; WS type 1) or musculoskeletal abnormalities (WS type 3), in addition to sensorineural deafness and depigmentation. Patients with deletions of the PAX3 gene have phenotypes indistinguishable from patients that have small substitutions in the DNA-binding domains of PAX3, and it has been observed that two very similar mutations can result in different phenotypes in different families. To account for these data, it has been proposed that gene dosage may play an important role in the cause of WS, with patients having a lower effective dose of PAX3 exhibiting more severe phenotypes.

Waardenburg patients with a milder series of melanocyte defects have mutations in the transcription factor MITF (Tassabehji et al. 1994). MITF has been shown to transcriptionally transactivate the gene for tyrosinase, a key enzyme in melanogenesis and overexpression of MITF can convert fibroblasts into cells with melanocyte characteristics (Tachibana et al. 1996).

Defects in MITF likely lead to anomalies during melanocyte differentiation which cause the hearing defects and hypopigmentation seen in WS type 2 patients.

Only recently has the role of MITF in melanocyte differentiation been integrated into the phenotypes seen in patients with PAX3 mutations. PAX3 can transcriptionally transactivate the MITF promoter (Watanabe et al. 1998). These results suggest that PAX3 directly regulates MITF, and that MITF, as a gene that requires functional PAX3 for expression, is responsible for the defects seen in melanocyte differentiation in WS types 1 and 3. These studies provide the first example of the cascade of transcription factors required for inner ear development.

So far, over eight transcription factors, each a member of a distinct transcription-factor family, have been identified as key players in the regulation of genes required for inner ear development. In most cases, the genes regulated by the transcription factors have not been precisely identified.

Ultimately, these downstream genes are required for both the development

38 K.B. Avraham and T. Hasson

of the bones in the outer, middle and inner ear, as well as the differentiation of the nerves and sensory regions found in the inner ear.

4.2.2 Mutations that Alter Splicing Patterns Lead to Inner Ear Defects

The processing of primary RNA transcripts to remove intronic sequences is essential. For proper splicing to occur, small nuclear ribonucleoproteins recognize short specific sequences at the junctions between introns and exons (usually GT at the 5¢ end of the intron and AG at the 3¢ end of the intron) (Fig. 2.4). It has been found that mutations that alter these consensus sequences alter splicing patterns. Splice-site mutations can result in exon skipping, whereby the resulting mRNA is missing a coding exon. Splice-site mutations can also lead to the inclusion of intronic sequences within the mRNA. In both cases, the resulting mRNA is often nonfunctional. The incorrectly spliced mRNA may encode a protein with an internal deletion as a result of exon skipping, in frame. Alternatively, and more commonly, the incorrectly spliced mRNA can result in the addition of improper amino acid sequences to the protein, as well as termination of translation resulting in a truncated protein product.

Mutations that affect both the 5¢ donor and 3¢ acceptor splice sites, as well as mutations that affect intermediates in the splicing process, have been observed in both syndromic and nonsyndromic deafness genes. An example of an array of splice site mutations found in one gene, myosin VIIa (MYO7A), is shown in Table 2.5. Interestingly, in this case, splice-site mutations can yield different phenotypes: in some cases the mutations result in Usher syndrome type 1B, a syndromic form of deafness with retinitis pigmentosa, whereas in other cases the mutations lead to nonsyndromic deafness (DFNB2). Clearly, different tissues have different sensitivities to alterations at splice acceptor and donor sites.

Splicing defects can also be responsible for dominant deafness. The dominantly inherited progressive hearing loss (DFNA1) seen in a large kindred from Costa Rica is due to a transversion in the splice donor of an exon of the diaphanous gene (Lynch et al. 1997). The observed G to T transversion disrupts the consensus AG sequence at the 5¢ splice donor of the next to last exon. A shift in the open reading frame results and 21 novel amino acids are added to the protein before the peptide is truncated prematurely. This truncated protein acts in a dominant fashion to disrupt hearing. Diaphanous is a member of the formin family of proteins and may have a function in the cell cytoskeleton.

4.2.3 Deafness Mutations May Result in mRNA Destabilization

One last way that gene transcription can be altered by mutation is by the destabilization of the mRNA itself. Even small mutations can produce mRNAs that are degraded in the cell, resulting in a phenotype that is equivalent to a total gene deletion. One such example is seen for the mouse

40 K.B. Avraham and T. Hasson

mutant deafwaddler (dfw). In the dfw2J allele, a two-base-pair deletion is present (Street et al. 1998). This mutation would be predicted to result in a frame shift and a truncated protein. Northern analysis, however, confirmed that this small deletion resulted in the destabilization of the mRNA. The mechanism whereby mRNAs that encode nonfunctional or truncated proteins are recognized and degraded by the cell has not been elucidated.

5. Summary

The programmed expression of proteins is required for hearing. This chapter describes the structure of chromosomes and genes and delineates how the processes of DNA transcription and RNA translation are required to form functional proteins. In cases of genetic hearing loss, mutations have occurred in either transcription or translation of the genes that encode for proteins crucial for the proper development, structure and function of the inner ear. By identifying these genes, and defining the mutation in them that cause deafness, a better understanding of the biology of hearing will be attained. These studies may one day in the future enable the “correction” of genetic mutations through gene therapy, or regeneration of hair cells. New sensory hair cells are not formed postnatally, and during aging these important cells degenerate. Studies into why and how the inner ear cells degenerate during “accelerated” hearing loss will likely provide clues to what happens during the normal aging process.

Acknowledgments. The authors are supported by grants from the NIH/ Fogarty International Center Grant 1 R03 TW01108-01 and the European Commission (QLG2-CT-1999-00988) (K.B.A.), and the Deafness Research

Foundation (T.H.).

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Adato A, Weil D, Kalinski H, Pel-Or Y, Hammadi H, Petit C, Korostishevsky M, Bonne-Tamir B (1997) Mutation profile of all 49 exons of the human myosin VIIA gene and haplotype analysis of Usher 1B families from diverse origins. Am J Hum Genet 61:813–821.

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