Учебники / Genetics and Auditory Disorders Keats 2002
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2
Genes and Mutations in Hearing Impairment
KAREN B. AVRAHAM and TAMA HASSON
1. Introduction
In the last five years, scientists have made great advances in deciphering the genetic basis of hearing impairment. A fundamental knowledge of the structure of chromosomes and genes is required to appreciate the events leading to mutations causing hearing loss. This chapter covers the organization of chromosomes and genes, discusses the flow of information from DNA to RNA to protein, and delineates how different types of mutations lead to abnormal gene expression or gene products. The mutations described are a representative sampling, not an exhaustive list, of mutations in genes that lead to hearing loss. Many more examples are described in the literature and the list is growing monthly. The emphasis will be on mutations associated with human deafness (Griffith and Friedman, Chapter 6), although examples of mouse mutations will also be mentioned, because of the relevance of mouse models to human hearing loss (Steel, Chapter 8).
For additional background information regarding the structure of chromosomes and genes, the reader is referred to Lodish et al. (1995), Klug and
Cummings (1997), and Lewis (1999).
2. Chromosome Structure
Eukaryotic cells contain large linear chromosomes, which carry the genetic material in the form of genes composed of DNA (Fig. 2.1). Most somatic cells contain two copies of each chromosome derived from the germ cells of each parent. Exceptions include: the haploid gametes, the sperm and egg cells, which contain only one set of chromosomes; the platelets and red blood cells, which lack a nucleus; and polyploid cells, such as liver regenerating cells and bone marrow megakaryocytes.
Chromosome size and number vary with species. In general, there is a correlation between the complexity of the species and the genome size. The yeast genome size is 14 megabases (Mb), whereas the genomes of the mouse
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FIGURE 2.1. Individual genes lie next to one another (and in some cases, even overlap) on chromosomes. The ability of DNA to be compacted in the nucleus of each cell is a remarkable feat. DNA molecules wrap themselves around histone proteins to form chromatin, then further condensed to form the chromosomes. A nucleosome is composed of histones and 2 1/2 turns of DNA. When DNA is transcribed to RNA, it unwinds from the histone only in the particular site where transcription is occurring. The RNA is then translated into a functional protein. The gene that encodes the gap junction protein connexin 26 lies on human chromosome 13. Thirty to fifty percent of NSHL is due to mutations in the connexin 26 (GJB2) gene.
and human are identical in size at 3,000 Mb. There are exceptions to this rule, however; the onion genome size is 15,000 Mb.
Human cells have 23 sets of chromosomes. In the haploid human cell, 22 chromosomes are autosomal, or non-sex chromosomes, while the remaining one is an X or Y sex chromosome. The fusion of two haploid cells to form a diploid cell during fertilization enables one homologue from each pair of chromosomes to join to form paired chromosomes containing nearly identical DNA. The basic material of chromosomes is termed chromatin, and it is composed of DNA and its associated histone proteins.
The histone proteins serve to compact the DNA so that it may fit into a nucleus. An assemblage of eight histone proteins is encircled by a stretch
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of 146 DNA nucleotides to form an 11 nm nucleosome (1 nm is a billionth of a meter). The nucleosomes, like beads on a string, further wind around each other to create the 30 nm chromatin fibers (Fig. 2.1). This compaction is necessary, since the human haploid genome, if stretched out, would reach the height of a person. A copy of this genome is present in each cell.
2.1 Chromosomal Abnormalities Leading to Deafness
Chromosome abnormalities can either be numerical or structural in nature, and usually lead to extreme phenotypes (Table 2.1). They often affect a large genomic DNA region, and can result in the loss or gain of partial or whole chromosomes. Large chromosome abnormalities can lead to embryonic lethality from the loss of one or more essential genes, or to a disease with several phenotypes, as is seen with syndromic hearing loss. The most common chromosome abnormalities are numerical and involve the loss or gain of whole chromosomes. Polyploidy, which describes the presence of an extra copy of the entire set of chromosomes, is not viable in humans. In contrast, some forms of aneuploidy (the loss or gain of one chromosome) are compatible with life (Giersch and Morton, Chapter 3). The most common form is the gain of an additional chromosome 21 (trisomy 21), which leads to Down syndrome. Some trisomy 21 patients exhibit conductive and/or sensorineural hearing loss, although the gene(s) contributing to the hearing loss are unknown (Gorlin et al. 1995).
Structural chromosomal abnormalities involve chromosome breaks, with rejoining of breakpoints in several possible configurations. The breaks may affect one or more genes, and lead to chromosomal translocations, inversions and deletions (Fig. 2.2A). When a translocation occurs, genetic material is transferred from one chromosome to another. An inversion is a reversal in the order of a chromosomal segment; genetic information is usually not lost, but the linear sequence of the genes is altered. A pericentric inversion involves the centromere, whereas a paracentric inversion does not include the centromere. Although chromosome breaks are uncommon in nonsyndromic sensorineural hearing loss (NSHL), a paracentric inversion is known to cause NSHL in the mutant deaf mouse, Snell’s waltzer (Avraham et al. 1995). The chromosomal breakpoints of this inversion are near, but not within, the coding regions of the short ear (Bmp5) and Snell’s
TABLE 2.1. Types of chromosomal mutations
Chromosomal Abnormalities |
Consequence |
Numerical |
Aneuploidy |
|
Polyploidy |
Structural |
Translocations |
|
Deletions |
|
Inversions |
|
|
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FIGURE 2.2. (A) Structural chromosomal abnormalities may lead to translocations, inversions, or deletions. (B) A paracentric inversion on mouse chromosome 9 leads to the sesv combined phenotype of short ear (se) and Snell’s waltzer (sv, deafness and circling) (Avraham et al. 1995). Breaks occur at the dotted lines, causing the myosin VI (Myo6) gene, normally transcribed in the direction shown by the arrow, to be inverted. Upstream regulatory regions, shown in grey, are lost, leading to down-regulation of myosin VI expression.
waltzer (Myo6) genes (Fig. 2.2B). No other genes appear to be affected, since the inverted DNA remains intact (except for small deletions at each breakpoint) and the mice only harbor phenotypes representative of the two genes. The breakpoints near the Bmp5 and Myo6 genes affect the downstream and upstream regions of these genes, respectively, leading to skeletal (for Bmp5; DiLeone 1998) and hearing (for Myo6) abnormalities in these mice. The consequence of this inversion is a position effect, where the expression of a gene is altered due to the relocation of chromosomal regions.
Most chromosomal deletions causing NSHL are intragenic (within the gene) and comprise no more than a few base pairs. One exception is the X- linked DFN3 locus, with mutations in the POU3F4 gene. Several chromo-
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somal deletions encompassing this gene have been reported, ranging from 250 kb to several megabases (Huber et al. 1994). This region of the X chromosome appears to be gene-poor because hearing loss (resulting from deletion of the POU3F4 gene) is most often the only phenotype.
3. From DNA to Protein: Transcription and Translation
The flow of information begins with the genetic material, the DNA. All cells contain the same genetic material in the form of DNA, but the levels at which this material is expressed, in the form of protein, varies between each tissue. The process of “expression” is a complex one, involving many steps, all of which may be affected by genetic mutations (Fig. 2.3). The first step, DNA transcription, changes the DNA information into RNA. There are three major types of RNA molecules produced by transcription: messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA). mRNAs are utilized in the process of translation and are the template for protein synthesis. rRNAs serve as structural components of the translational machinery, whereas tRNAs serve as the carriers that bring in new amino acids for the growing peptide chain.
DNA is transcribed into mRNA in the nucleus. Sequences in the DNA, termed promoters, are recognized by transcription factors that recruit the RNA synthesis machinery to the gene and initiate RNA synthesis at that site. After RNA synthesis is initiated, the growing RNA is elongated until a termination signal is reached and the RNA synthesis machinery is
FIGURE 2.3. A schematic view of the flow of information within a cell, beginning with DNA as the genetic material and ending with protein as the “expression” of the genetic material. Mutations can occur at each of these steps. Modified with permission from Pearson Education Limited (Carter and Murphey 1999).
28 K.B. Avraham and T. Hasson
released from the DNA. The initial mRNA product, termed the pre-mRNA, contains both exons, regions that encode for proteins, and introns, intervening sequences that do not encode protein. In an essential process called mRNA splicing, the noncoding introns are removed out of the pre-mRNA to form the mature and functional mRNA (Fig. 2.4).
After the mRNAs are fully processed in the nucleus, they are transported through the nuclear pores into the cytoplasm. Here the mRNAs are assembled onto ribosomes, the translation machinery that converts the information encoded in each mRNA into protein. Each ribosome consists of two major subunits, one large and one small, and these subunits are made up of both protein and ribosomal RNA (rRNA) components. The process of translation, like that of transcription, can be divided into three steps: initiation, elongation and termination. All three of these processes are catalyzed by the ribosomal proteins, but are dependent on proper rRNA function.
Translation initiation begins with the binding of the small ribosomal subunit to a recognition sequence on the mRNA (Fig. 2.5). All 20 amino acids are encoded by three base sequences of mRNA termed codons. The translational recognition sequence includes the sequences surrounding the “start codon,” usually an AUG codon encoding the amino acid methionine. After binding to the recognition sequence, a methionine-charged tRNA is brought into place, the large subunit of the ribosome joins the complex, and translation is initiated. Elongation of the peptide occurs catalytically with the ribosome moving along the mRNA in the 5¢ to 3¢ direction. The open reading frame refers to the subset of nucleotides of the mRNA that are used as the template to create protein. The ribosome shifts to the next three bases in the sequence (the next codon) and adds the appropriate amino acid to the growing peptide. The process of translation is halted when a stop codon—UAA, UAG, or UGA—is encountered.
FIGURE 2.4. The coding sequence of genes are composed of exons that are separated by intervening sequences, introns. The full length of the gene is transcribed into a primary RNA transcript that undergoes RNA splicing. Introns are removed, allowing the RNA segments to be joined at the splice junctions to form mRNA.
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FIGURE 2.5. Translation of the mRNA begins with the formation of an initiation complex. The first step in the formation of the initiation complex is the binding of the small ribosomal subunit to a recognition sequence on the mRNA. The translational recognition sequence includes the sequences surrounding the “start codon,” usually an AUG codon encoding the amino acid methionine. After binding to the recognition sequence, a methionine-charged tRNA is brought into place, the large subunit of the ribosome joins the complex, and translation is initiated. Elongation of the peptide occurs catalytically with the ribosome moving along the mRNA in the 5¢ to 3¢ direction.
4. Mutations
Mutations are defined as changes in the chemical composition of DNA and can manifest themselves either during transcription or translation. The end result, in either case, is altered expression of the protein. Mutations can occur in somatic cells, in which case they only affect the individual in whose cells the mutation is present. Mutations in the germ line (sperm or ovum), however, can be passed on to subsequent generations.
4.1 Mutations at the Level of the Gene
Mutations at the level of DNA can ultimately result in damage at the level of the protein. There are three major types of DNA mutations: point mutations, insertions and deletions (Fig. 2.6; Table 2.2). A point mutation, or substitution, is a change in a single nucleotide. Point mutations may be due to transitions or transversions of the nucleotide. A transition occurs when a purine replaces a purine (A to G or G to A), or a pyrimidine replaces a pyrimidine (C to T or T to C). In a transversion, a purine replaces a pyrimidine and vice versa (A or G to T or C).
As each amino acid is encoded by a three-nucleotide codon, single nucleotide changes in the DNA often lead to an amino acid change. This change from one amino acid to another is termed a missense mutation. When a missense mutation occurs, the structure and function of the protein can be affected. In some cases, the change in the DNA sequence does not alter the encoded amino acid. This change is termed a polymorphism or a
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FIGURE 2.6. Mutations may be in the form of deletions, insertions or substitutions. In (A) and (B), a deletion or insertion of one nucleotide forms a frameshift, changing the subsequent amino acids. In (C), a transversion leads to a missense mutation, affecting only one amino acid. In (D), a transition leads to a nonsense mutation, forming a stop codon.
TABLE 2.2. Types of DNA mutations
DNA mutation |
Examples of resulting mutation |
Point (substitution, transversion or transition) |
Missense—altered amino acid |
|
Nonsense—stop codon |
Deletion/ insertion |
|
Multiple of 3 |
Deletion/insertion of amino acid(s) |
Not a multiple of 3 |
Frameshift, truncation |
|
|
variant and may occur naturally in the population, rather than be the cause of a disease. Finally, when a DNA mutation changes the amino-acid codon to a stop codon (UAA, UAG, or UGA in RNA), a nonsense mutation occurs. This mutation can lead to protein truncation and can clearly affect protein function.
Insertions and deletions of DNA nucleotides also change the encoded protein. The addition or deletion of three nucleotides will add or delete an amino acid, which may have dire consequences for the protein. Alterna-