Учебники / Genetics and Auditory Disorders Keats 2002

FIGURE 3.3. Diagrammatic representation of homozygosity for the disease allele

(D) and flanking markers to a disease locus in the offspring of a first cousin union in autozygosity mapping.

1995). This approach has been utilized to identify the majority of the loci for autosomal recessive nonsyndromic sensorineural hearing impairment, e.g., mapping of the first locus for autosomal recessive nonsyndromic sensorineural hearing impairment, DFNB1 (Guilford et al. 1994; Brown et al.

1996).

4. Genome Searches

In most instances, there are no clues to the chromosomal location of a gene, and genetic mapping involves the “shotgun” approach of a total genome search. This involves analyzing DNA samples from family members for a standard battery of approximately 300 polymorphic DNA markers evenly spread throughout the human genome. Until recently, this approach was fairly time-consuming and laborious. The advent of semi-automated computer-driven gel analysis of fluorescently labelled microsatellite markers means that, if the necessary number of samples from the families have been collected and the reaction conditions for the marker set are optimized, a genome search can be carried out in a matter of weeks, rather than years.

that contain polymorphic markers known to map to the interval in the region of interest. Now, because of the progress of the Human Genome Project, YAC contigs of most regions of the genome are available online. Identification of unique DNA sequences in the YAC clones allows their order to be determined leading to a physically contiguous series of overlapping YAC clones (contig).

A concern with YACs is that the DNA inserts are often chimeric, i.e., contain DNA fragments from noncontiguous regions of the genome. In addition, YAC clones are often unstable, undergoing rearrangements in propagation. For this reason, once the region containing a gene of interest has been narrowed down sufficiently, bacterial artificial chromosomes (BACs) and/or bacteriophage P1 artificial chromosomes (PACs), which replicate stably, are used to establish a contig of the interval of interest.

Identification of a gene in a contig may occur quite rapidly if the region of interest has previously been screened in a different study. For example, the DFNB3 locus mapped to chromosome 17p11.2, a region that was known to contain the gene for hereditary motor sensory neuropathy type 1a (CMT1A) (Murakami and Lupski 1996) and was also the location of the microdeletions responsible for Smith–Magenis syndrome (Juyal et al. 1996). This allowed screening of genes that had previously been found in that region (Murakami et al. 1997), one of which was the homologue of the gene responsible for the mouse deafness mutant shaker-2 (Liang et al. 1998).

6.2 Transcript Mapping

Once a physical contig is established for a particular region known to contain a gene of interest, the next step in the mapping process is the identification of expressed gene sequences or transcripts within that region

(Giersch and Morton, Chapter 3).

6.2.1 Expressed Sequence Tags (ESTs)

Partial sequences of genes identified from cDNAs are known as expressed sequence tags (ESTs) (Adams et al. 1991). More than 50,000 human ESTs have been identified, many of which have been mapped and deposited in the DNA sequence database, dbEST (Boguski 1993; Boguski and Schuler 1995; Schuler et al. 1996). Recently 30,000 of these ESTs have been assembled in an integrated map (Deloukas et al. 1998), nearly twice as many as three years earlier (Berry et al. 1995). This resource contains most of the genes that encode proteins of known function. By selecting the chromosomal region of interest in the database, ESTs mapping to that region can be identified.

ESTs, however, represent only partial sequences of genes and it is necessary to identify the full sequence to be able to reliably screen for mutations in a specific gene. This is done by screening a cDNA library with the

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ESTs to identify close to full-length cDNA clones of the transcript. These will usually be in the region of one to four kilobases in length, as opposed to most ESTs, which are generally 300 to 400 bp in length. The technique of rapid amplification of cDNA ends (RACE) may then be applied to generate the missing portions of the cDNA. This technique takes advantage of the polymerase chain reaction (PCR) to amplify the missing sequence using mRNA from a specific tissue in which the gene of interest is expressed (Frohman et al. 1988).

6.2.2 Exon Amplification/Trapping

Not all genes are represented in sequence databases. Thus, alternate approaches may be necessary to identify candidates in the region of interest. Exon amplification/trapping relies on the fact that the coding regions of most eukaryotic genes, exons, are separated by noncoding intervening sequences, introns. The production of mature mRNA involves the removal of the introns by the process of mRNA splicing to produce continuous coding sequence for protein translation. The exon/intron boundaries contain 5¢ donor and 3¢ acceptor splice site consensus sequences. Exon trapping utilizes these sequences to identify clones containing exons. Expression cloning vectors containing the DNA sequence are used to transfect a suitably modified eukaryotic cell line, resulting in transcription of the inserted target DNA into RNA, which then undergoes splicing. The production of an abnormal-sized splice product after insertion of a target DNA fragment indicates the presence of an exon in the cloned DNA fragment (Duyk et al. 1990).

A major advantage of exon trapping is that it is not dependent on the expression of the gene. Exons from genes expressed in specific tissues, or at particular stages of development, are isolated with the same efficiency as genes that are more widely expressed (Church et al. 1994). Disadvantages of exon trapping are that genes with single exons and exons in the 3¢ and 5¢ portions of the gene will be missed. Specialized vectors have been devised to help overcome this problem (Krizman et al. 1995).

6.2.3 Phenotype Rescue

More recently, “phenotype rescue” in the mouse has helped to rapidly narrow the interval within which a gene of interest is located. In this procedure, BAC clones from the contig to which the gene has been mapped are injected into fertilized mouse oocytes that are homozygous for a mutation in this gene (e.g., a gene causing hearing impairment). Identification of a transgenic mouse with normal hearing (i.e., an offspring for which the phenotype reverts to normal or is “rescued”), suggests that the injected BAC clone contains the gene. This approach was used to identify Myo15 as the defective gene in the shaker-2 mouse (Probst et al. 1998). Almost immediately, mutations in the human homologue, MYO15, were shown to be

associated with one form of nonsyndromic sensorineural hearing impairment/deafness, DFNB3 (Wang et al. 1998a).

7. Positional Candidate Gene Cloning

If a gene for a disorder is mapped to a specific chromosomal region, a known gene mapping to that interval is a positional candidate by virtue of its location. One of the first examples of the use of this approach was in Waardenburg syndrome type 1 (WS1). It was recognized that the region of human homology for the mouse mutant known as Splotch (Sp) (which is deaf and has areas of depigmentation similar to that seen in persons with Waardenburg syndrome) was 2q35–q37.3. A structural rearrangement of this region of chromosome 2 had previously been reported in an individual with WS1 and subsequent linkage analysis confirmed this location (Foy et al. 1990). The Pax3 gene in the mouse and the human homologue PAX3 mapped to the same intervals as the Sp and WS1 loci suggesting positional candidate genes for both disorders. This was confirmed by the identification of mutations in the Pax3 gene in the Sp mutant and in the PAX3 gene in WS1 patients (Tassabehji et al. 1992; Baldwin et al. 1992).

8. Mutation Detection

One of the essential prerequisites for confirmation that a gene causes an inherited disorder or disease is the identification of DNA sequence differences that are present in affected individuals, but not in unaffected individuals from the general population. In other words, differences that are normal sequence variation between individuals need to be distinguished from deleterious mutations that result in inherited disorders.

8.1 Prioritizing Candidate Genes for Mutation Detection/Screening

Determining which candidate genes to screen for mutations may not be obvious. A first step is to examine those that have been characterized. The function of the proteins encoded by these genes may suggest that some are more likely candidates than others. For example, MYO15 was considered a probable candidate gene for DFNB3 because mutations in another unconventional myosin, MYO7A had been associated with inherited hearing impairment (Wang et al. 1998a). If function is unknown, evidence of expression in the inner ear helps in prioritizing candidates for mutation screening (Giersch and Morton, Chapter 3). For example, a transcript (COCH) from a fetal cochlear cDNA library (Robertson et al. 1994) was localized to the region of the long arm of chromosome 14 to which the DFNA9 locus had

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been mapped (Robertson et al. 1997). Subsequent sequence analysis identified mutations in COCH in hearing impaired DFNA9 family members (Robertson et al. 1998).

8.2 Mutation Detection/Screening Methods

Once a potential candidate gene for a disorder has been identified, a variety of different complementary methods can be used to screen for mutations.

Each method has its own advantages and disadvantages.

8.2.1 Single-Stranded Conformational Polymorphism (SSCP)

Following denaturation, PCR products fold into a three-dimensional structure in non-denaturing conditions through the influence of sequencespecific intramolecular bonds. If there is a sequence difference between DNA strands, the resulting three-dimensional structure (conformation) may have a different mobility through a nondenaturing polyacrylamide electrophoretic gel than if the DNA strands are identical (Orita et al. 1989). Sequence variation detected in this way is known as single-stranded conformational polymorphism (SSCP) (Fig. 3.4). The optimal DNA fragment size for mutation detection by this technique is 200 to 300 bp in length. The sensitivity of SSCP for detecting mutations varies with factors such as temperature, the strength of cross-linking, and ionic strength of the gel. In addition, SSCP is not efficient at detecting mutations in DNA fragments that are larger than 350 bp.

8.2.2 Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing gradient gel electrophoresis (DGGE) relies upon differences in mobility between normal and mutant DNA on an electrophoretic gel with a gradient of increasing denaturant concentration. Migration of double-stranded DNA fragments through the gel will continue until the strands of the DNA duplexes begin to separate because of the presence of the denaturant (Fischer and Lerman 1979). Addition of a 30 to 40 nucleotide GC-rich sequence (a GC clamp) to the 5¢ end of the primers used to generate the DNA fragments for analysis makes DGGE a highly sensitive mutation-detection method (Myers et al. 1985; Sheffield et al. 1989), but also results in its becoming relatively expensive because of the need to synthezise longer PCR primers.

8.2.3 Heteroduplex Analysis

In heteroduplex analysis, PCR products generated from both mutant and normal DNA templates are mixed, heat denatured, and allowed to cool to room temperature. This results in the formation of heteroduplexes of the wild type and mutant DNA sequences. These heteroduplexes often differ

FIGURE 3.4. SSCP analysis of PCR products of exon 2 of the coding region of the Cx26 gene of DNA samples from persons with nonsyndromic hearing impairment showing a mobility shift/doublet in an individual with the 310de114 mutation in the Cx26 gene in the sample in Lane 2.

in mobility from homoduplexes on polyacrylamide gel analysis (Nagamine et al. 1989). This is thought to be due to sequence-dependent conformational changes in the double-stranded DNA duplex. Heteroduplex analysis has a similar sensitivity to SSCP for DNA fragments less than 300 bp in size (White et al. 1992).

8.2.4 Cleavage Mismatch

Cleavage mismatch involves adding the chemicals hydroxylamine and osmium tetroxide, which react with free cytosine and thymine nucleotides, respectively. The DNA template is denatured, then hybridized with a singlestranded radiolabeled DNA probe. Any mismatched cytosine or thymine nucleotides will be exposed and susceptible of reaction with the hydroxy-

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lamine and osmium tetroxide. Such sites are detected by the addition of piperidine, which results in cleavage of the DNA at these sites. The technique identifies mismatches on either strand, and thereby indirectly detects adenine and guanine mismatches through their complementary nucleotides on the other strand (Cotton et al. 1988). Chemical cleavage works for longer DNA fragments than SSCP, DGGE, and heteroduplex analysis and it also has the advantage that it provides information about the location of the mutation. Adaptation to fluorescent-based detection systems (Ellis et al.

1998), as well as use of less toxic chemicals (Roberts et al. 1997) and enzymatic methods of chemical cleavage (Rzhetsky et al. 1996; Smith and Modrich 1996; Kortenkamp et al. 1997), may increase the use of chemical cleavage as a mutation-detection method.

8.2.5 Sequence Analysis

DNA sequencing is the gold standard of mutation detection, but it is expensive and laborious, especially if the gene being sequenced is large. However, advances in automated DNA sequencing technology are making sequence analysis the method of choice for mutation detection. In addition, ongoing developments in DNA chip technology will permit rapid, automated, largescale mutation detection once a gene has been identified.

9. Approaches to Confirming that a Detected Mutation Causes Hearing Impairment

While identification of mutations in affected individuals in a candidate gene provides strong evidence that it is responsible for inherited hearing impairment, further evidence can be provided in a number of different ways.

9.1 Prediction of the Likely Functional Consequences

of Mutations

Analysis of the functional consequences of specific mutations can provide compelling evidence that a particular gene is responsible for inherited hearing impairment. For example, deletion of the whole gene, as has been reported in some patients with the branchio-oto-renal syndrome (Abdelhak et al. 1997; Kumar et al. 1998), would be expected to lead to loss of function. Alternatively, a nonsense mutation, or a frameshift mutation that generates a stop codon, especially if it occurs in the 5¢ portion of a gene, leads to a severely truncated protein, which is likely to have severe functional consequences. A number of mutations in the connexin 26 (Cx26) gene are of this type (Avraham and Hasson, Chapter 2).

The likely functional consequence of missense mutations cannot be predicted reliably in some instances, and may require knowledge of the

structure and function of the protein. Mutations which result in nonconservative amino acid substitutions in a part of the protein that is critical to its function, or in regions known to be conserved between species, are likely to have significant effects. Examples include a T to G missense mutation in a family with Pendred’s syndrome, which leads to substitution of Cys for Phe at a highly conserved position in the protein pendrin (PDS ) (Everett et al. 1997), and the single nucleotide missense mutations reported in the POU homeodomain of the POU3F4 gene in individuals with the X-linked form of mixed deafness DFN3 (Kok et al. 1995).

9.2 Analysis of Control Samples

Supportive evidence that a DNA sequence variant is likely to be a mutation, rather than a normal polymorphism, is provided by the absence of the sequence variant in an appropriate number of normal control individuals. The M34T sequence variant in the Cx26 gene reported in association with autosomal dominant sensorineural hearing impairment (Kelsell et al. 1997) occurs in a small but significant proportion of the normal population; this suggests that it might not be of functional significance, but instead a normal polymorphic variant (Scott et al. 1998; Kelley et al. 1998). However, in vitro functional studies (Section 9.3) provide support for the association of this mutation (101T to C) with hearing impairment.

9.3 In Vitro Functional Studies

In vitro functional assays of a protein can reveal the effect of known or introduced mutations in a gene. For example, mRNA generated from wild type and variant Cx26 gene sequences was expressed in Xenopus oocytes to measure gap junction channel activity. Analysis of the 101T to C sequence variant in the Cx26 gene (Kelsell et al. 1997) indicated that it was likely to be a mutation, rather than a polymorphic variant (White et al. 1998). However, care needs to be taken in the interpretation of the results of these types of studies, since it is possible that they might not reflect function in vivo.

9.4 Immunohistochemical/In Situ Hybridization Studies

Further evidence that a mutation in a gene may be responsible for inherited hearing impairment can be obtained by examining the expression of the gene at the mRNA or protein level in appropriate tissues using immunohistochemical or in situ hybridization studies. For example, Cx26 expression is found in the stria vascularis, basement membrane, limbus and spiral prominence of the cochlea using murine monoclonal antibodies to

Cx26 (Fig. 3.5) (Kelsell et al. 1997).

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FIGURE 3.5. Cx26 immunofluoresence staining of the cochlear duct. Rabbit polyclonal anti-Cx26 staining showing expression in the stria vascularis, basement membrane, limbus and spiral prominence (magnification, 16¥). (Reprinted with permission from Nature (387:80–83). Copyright 1997 Macmillan Magazines Limited.)

9.5 Transgenic/Knockout Models

The ability to introduce targeted mutations in a gene by homologous recombination in the mouse allows the possibility of confirming the phenotypic consequences of mutations in a gene for inherited hearing impairment or deafness (Friedman and Ryan 1992; Friedman 1996; Faerman and

Shani 1997). Examples are the analysis of the role of the POU-domain factor Brn-3c gene in auditory and vestibular hair cell development (Xiang et al. 1997) and the knockout mouse model for Alport syndrome (Cosgrove et al. 1988).

9.6 New Approaches Resulting from the Human Genome Project

The availability of human DNA sequence information has allowed the development of a new cloning strategy for identifying expressed sequences. If a gene is mapped to a relatively small interval, genomic clones from the contig covering that region can be subcloned and sequenced. The resulting sequence information can be compared with that in a number of sequence databases, such as the GenBank BLAST Search database (http://www.ncbi.nlm.nih.gov/BLAST/) to identify sequences with homol-