Учебники / Genetics and Auditory Disorders Keats 2002

By making specific assumptions about these factors, Hardy (1908) and Weinberg (1908) independently formulated what is now known as the Hardy–Weinberg law. This fundamental principle of population genetics states that, if there are two alleles at a single autosomal locus with population frequencies p and q, then, for a random mating population in equilibrium, the frequencies are p2 and q2 for the two homozygous genotypes and 2pq for the heterozygous genotype. Moreover, these frequencies remain the same from one generation to the next. Thus, if the frequency of a recessive disorder in a population is 1 in 10,000 (that is, q2 = 0.0001), then q = 0.01 (and p = 0.99 because p + q = 1), and the frequency of carriers of the deleterious gene can be calculated as 2pq, which is 1 in 50.

The Hardy–Weinberg law does not hold if mating is not random in the population. For example, if mating is consanguineous (between relatives) or assortative (between individuals with the same phenotype, such as deafness, as discussed by Nance and Pandya in Chapter 5), then the frequency of heterozygotes is less than 2pq, and the frequencies of homozygotes are increased. Also, evolutionary forces such as mutation, migration, and selection change allele frequencies. However, Hardy–Weinberg equilibrium at an autosomal locus will be restored in one generation after the force is no longer disturbing the allele frequencies.

Now consider two autosomal loci, both with two alleles (A1, A2 and B1,

B2). During meiosis (cell division that results in the haploid germ cells), crossing-over may take place between a pair of chromosomes. Suppose that one chromosome has the A1 allele at the first locus and the B1 allele at the second locus, while the other chromosome has A2 and B2, respectively. If crossing-over occurs, then the haplotype (the set of alleles on the same chromosome) in a germ cell may be A1B2, or A2B1. If crossing-over does not occur between these two loci, then all the germ cells will have the haplotypes, A1B1, or A2B2. A recombination event is a result of crossing-over that can be observed in the offspring and is explained in detail by Mueller, Van Camp, and Lench in Chapter 4.

Linkage disequilibrium is present if the product of the population frequencies of the A1B1 and A2B2 haplotypes is not equal to the product of population frequencies of the A1B2 and A2B1 haplotypes. In general, this means that not enough recombination events between the two loci have yet taken place in the population to equalize these products. For two loci that are close together, many thousands of generations may be required to reach equilibrium at the two loci considered jointly.

The estimation of the frequency of recombination events is the basis of linkage analysis, which is a method for mapping genes to chromosomes. To detect linkage, family data (not population data) must be analyzed. Two loci are said to be linked if the frequency of recombination events among offspring is less than 50%. If recombination events are very rare

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between a disease gene and a genetic marker, then it is likely that the chromosomal location of the disease gene is very close to that of the genetic marker.

3.1 Localizing and Identifying Genes

One goal of genetic research is to localize, identify, and establish the base sequence of all genes. Genetic linkage analyses of families in which some members have an inherited disorder provide the chromosomal location of the deleterious gene. A linkage study may require typing hundreds of genetic markers before the disease gene is localized, but once this step is accomplished, research to identify candidate genes in the region begins. For recessive hearing impairment, affected individuals whose parents are related are likely to have the same homozygous genotype. Thus, the search for the location of the gene may be accelerated by screening pooled DNA samples from these individuals and selecting markers for which they are all homozygous for the same allele (Sheffield et al. 1994). If pooling is feasible, the number of samples that need to be typed can be reduced dramatically. For example, Scott et al. (1995) used this approach in their study of DFNB1 Bedouin families (Mueller, Van Camp, and Lench, Chapter 4; Griffith and Friedman, Chapter 6). When the gene is identified and mutations are detected, diagnostic tests that examine the DNA within the gene itself are applicable to all individuals. Mueller, Van Camp, and Lench describe the methodology used to identify genes and detect mutations in Chapter 4.

As well as providing the approximate location of a disease gene, linkage analysis permits more precise genetic counseling (Arnos and Oerlich, Chapter 9). For example, by examining the transmission in a family of a closely linked genetic marker, relatives of individuals affected with a recessive disorder can be informed as to whether they are likely to be carriers.

The development of high-resolution genetic and physical maps, together with the construction of genomic and cDNA libraries, and the availability of sequence databases for many species, provide the tools for finding genes once they have been localized by linkage analysis. Giersch and Morton (Chapter 3) describe some of these tools. Detecting a mutation in a candidate gene in affected individuals may mean that the search for the gene responsible for the hearing impairment is over, particularly if that mutation is not found in a sample of unaffected individuals. However,

DNA sequence varies from one individual to another, so a sequence difference may not necessarily make the gene deleterious; it may simply reflect normal variation. Some mutations, though, such as a deletion or insertion of several bases, or a point mutation (single base change)

that gives rise to a codon that does not correspond to an amino acid (e.g., a stop codon), are very likely to be causal (Avraham and Hasson, Chapter 2).

3.2 Heterogeneity

The underlying cause of most forms of hereditary hearing loss is a mutation in a single gene. Results from linkage studies of several families are often pooled to increase the probability that the correct location has been found for the gene. However, the gene in one family may be different from that in another (e.g., at least nine different genes cause Usher syndrome). This is known as locus heterogeneity. In this situation, pooling families will hinder rather than help the analysis. Studying endogamous populations or large pedigrees minimizes the chance of locus heterogeneity, but does not necessarily eliminate it.

A phenotype such as nonsyndromic hearing impairment is caused by many different genes, and the etiology may not be the same even for affected members of the same family. In some cases, auditory testing (particularly if it covers more than pure tone air conduction audiometry) may detect phenotype differences among members of the same kindred. Such findings may be critical for linkage studies. For example, in the family studied by Vahava et al. (1998) auditory testing showed that the hearing impairment in one member was different from that in the other members. Knowing that this individual was a phenocopy (same clinical phenotype, but different etiology) facilitated the linkage analysis.

Unlike locus heterogeneity, allelic heterogeneity does not confound linkage studies. In this case, different alleles (that is, different mutations in the same gene) are responsible for the phenotypes, which are generally similar. However, different alleles do not necessarily result in the same phenotype, and the mode of inheritance may even be different. For example, most mutations in the human myosin VIIa gene (MYO7A) cause Usher syndrome type IB, but a few result in recessive nonsyndromic hearing loss, and others in dominant nonsyndromic hearing loss.

4. Mouse Models

Finding a mutation that probably makes a gene deleterious is a critical step, but it does not prove that the defect actually causes the hearing impairment. However, if a transgenic or knockout mouse is hearing impaired, then the argument that the defect causes the abnormal phenotype is convincing. To obtain a transgenic mouse, copies of the gene are injected into mouse

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oocytes just after fertilization. The oocytes are then implanted into a foster mother whose uterus has been prepared for pregnancy by treatment with hormones. The transgene may incorporate anywhere in the genome, and several copies (sometimes as many as 200 copies) are usually found at a single location. In general, between 10% and 30% of the progeny are found to have the injected gene in their germline DNA, and can therefore pass it on to their offspring, thus allowing the development of a colony of transgenic mice.

The construction of a knockout mouse is a much more controlled and precise experiment than generating a transgenic mouse. The first step is to replace the normal copy of the gene with a copy containing the mutation of interest. This procedure is carried out in embryonic stem (ES) cells derived from mouse blastocysts (an early stage of embryonic development) and grown in tissue culture. The mutant gene may insert anywhere in the genome, but only the cells in which the normal copy is replaced by the mutant copy are selected for the next step. The selected ES cells are then injected into a recipient blastocyst, which is implanted into a foster mother. The resulting offspring will be chimeric, meaning that some of their cells are derived from the ES cell line, and some are from the recipient blastocyst. Mating experiments may then be set up in order to determine if the ES cells have contributed to the germline and to generate a colony of knockout mice. Knockout mice provide excellent animal models for studying the effects of gene mutations associated with human disorders.

Because of the high degree of similarity (orthology) of genes in humans and mice, studies of mouse mutants have made many valuable contributions to human disease gene identification (Meisler 1996), and hearing impairment is no exception (Brown and Steel 1994). Major advantages of using the mouse for finding disease genes are the ability to set up specific matings, and the relative ease of obtaining large numbers of informative progeny to localize the genes by linkage analysis. A relevant example is human USH1B and the mouse deafness mutant shaker-1 (sh1), which were hypothesized to be caused by mutations in orthologous genes because they had been mapped by linkage to a conserved region on human chromosome 11q13 and mouse chromosome 7. This hypothesis was proven to be correct when Gibson et al. (1995) showed that the sh1 gene encoded myosin VIIa, and Weil et al. (1995) found mutations in the human MYO7A gene in Usher type IB patients soon thereafter. Note that symbols for human genes are uppercase, while those for mouse genes are lowercase. Thus, the USH1B gene is MYO7A, and the sh1 gene is myo7a. Steel, Erven, and Kiernan

(Chapter 8) cover the application of mouse models to studies of human hearing impairment.

Mapping and sequencing of genes in non-mammalian species is also proving to be useful for studies of human diseases. In particular, the

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the patient is packaged into a vector and introduced to the cells. The gene is not incorporated into the germline cells. The purpose of gene therapy is to treat the patient, not to change the genetic material that is passed to the next generation. Many research studies are in progress to determine effective vectors and suitable approaches for delivering genetic material to specific tissues. Certain classes of viruses may be used as vectors, and ongoing investigations are exploring their potential in gene therapy. Nonviral strategies such as coating the gene in a lipid layer are also being developed.

Although still in its infancy, gene therapy holds great promise for future treatment of genetic disorders, including hearing impairment. Cook et al. (2000) provide a summary of ongoing gene therapy research that targets the inner ear.

7. Summary

Genetic studies are resulting in the identification of genes for many disorders. In theory, once a disease gene is identified, diagnostic tests can be developed to detect mutations in individuals who may have the disease. Offering such diagnostic tests can be beneficial if one or two mutations are responsible for most cases. For many genes, however, unrelated affected individuals are likely to have different mutations, making detection a timeconsuming and expensive task, especially if the gene is large. Moreover, finding a sequence difference does not necessarily mean that the responsible mutation has been found; sequence variation among individuals is common and very few of the variants are associated with a disorder. Thus, setting up diagnostic tests that are commercially available may not be economically feasible. On the other hand, once a mutation is identified in one family member, related individuals can be tested for this mutation. This can be beneficial for: (1) presymptomatic diagnosis in later age at onset disorders; (2) detecting the presence of the deleterious gene when penetrance is not complete, meaning that unaffected individuals may have the gene; and (3) identifying carriers of the gene when the inheritance pattern is recessive.

Many ethical issues must also be considered. Should molecular diagnostic testing be offered routinely if the result will not provide an alternative approach to treatment and management of the disorder? What are the psychological effects of presymptomatic genetic testing? Should parents be able to have their children tested for the presence of a mutation for which the age at onset of symptoms is after they reach adulthood? What is the obligation to other family members if a deleterious gene is detected? Remember, genes are transmitted from one generation to the next, and the results for one individual are pertinent to many relatives.

7.1 A Look to the Future

Routine molecular diagnostic testing for many of the genes causing hearing impairment is likely to be available within the next few years, and genetic testing may become the method of choice for newborn hearing screening. Although the development of effective therapies based on gene identification is still in its infancy, promising new advances made possible by research are arising at a rapid rate, and the potential for developing molecular intervention strategies as a treatment, and perhaps cure, is encouraging. A prerequisite, though, is determining the normal function of proteins encoded by hearing impairment genes.

Knowing the gene mutation that is responsible for hearing impairment in an individual provides information that is relevant to all family members. Based on this information, they may choose to be tested in order to help in making reproductive decisions. In addition to genetic tests, a desirable goal is to be able to predict the phenotype based on the genotype, and vice versa. However, defining the precise phenotype and providing an accurate prognosis based on the mutation is not yet straightforward. Physiologic indices that correlate with the presence of a mutation are valuable phenotypic measures that will enhance our understanding of the function of the normal gene product (Hood 1998; Huang et al. 1996, 1998; Liu and Newton 1997; Morell et al. 1998). The development of such measures that accurately define the phenotype, together with the application of microarray technology to diagnostic testing and functional studies of hearing impairment genes and the proteins they encode, will lead to improved classification and prediction of outcomes, as well as to rational and effective therapies for all forms of hearing impairment.

Glossary

ABR/EcochG: Auditory brainstem response and electrocochleography are companion evoked-potential studies that reveal the cochlear microphonic, summating potential, action potential and synchronous discharge of neural elements in the auditory pathway from cochlea to lateral lemniscus. The tests are used as tools to evaluate both auditory and neural integrity of the auditory system. While neither is a direct test of hearing, the results are powerful predictors of hearing status, when they are normal. In contrast, the absence of a synchronous ABR or EcochG does not always mean deafness is present.

Allele: One of several alternative forms of a gene or DNA sequence at a specific chromosomal location.

ATP (adenosine triphosphate): The principal immediate-energy source in all eukaryotic cells.

Autozygosity: Homozygosity for an allele that is identical by descent.

BAC (bacterial artificial chromosome): A vector designed for cloning relatively large DNA fragments between 50 and 200 kb. BACs are propagated in a host bacterial cell.

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Centromere: Primary constriction of a chromosome, separating the short arm from the long arm. Its major function is to ensure correct segregation of homologous chromosomes during meiosis and mitosis.

Codon: Nucleotide triplet that specifies an amino acid, or a signal for terminating the synthesis of a polypeptide.

Consensus sequence: In genes or proteins, an idealized sequence in which each base or amino acid residue represents the one most frequently found at that position when many actual sequences are compared.

Contig: Continuous region of genomic DNA that has been cloned in a series of identifiable overlapping DNA clones.

Degenerate code: The genetic code is described as degenerate because more than one codon can encode the same amino acid.

Dimerization: The formation of a compound composed of two molecules. Dominant negative mutation: The abnormal product of one allele disrupts the

function of the product of the normal allele.

Endonuclease: An enzyme that can cut phosphodiester bonds that occur internally in a DNA chain.

Epistasis: Phenotypic expression is the result of interaction between alleles at different loci.

EST (expressed sequence tag): A sequence of part of the coding region of a gene. Eukaryote: Each cell has a nucleus that contains the genetic material, surrounded by cytoplasm, which in turn is bounded by the plasma membrane that marks the

periphery of the cell.

Exon: Segment of a gene that is decoded to give an mRNA product.

Frameshift Mutation: A mutation in which there is a deletion or insertion of a number of nucleotides that is not a multiple of three. This causes the codon reading frame to shift.

Glutamic acid: An amino acid that is part of the cochlear afferent cycle. Haploid gametes: Cells having only a single copy of each chromosome. Haploinsufficiency: A locus shows haploinsufficiency if producing a normal

phenotype requires more gene product than the amount produced by a single copy.

Haplotype:

chromosome.

Hardy–Weinberg (equilibrium) law: The relationship between gene frequencies and genotype frequencies that is found in a population under certain conditions.

Heteromultimeric proteins: Proteins that consist of nonidentical subunits (coded by different genes).

Heteroplasmy: Two or more genetically distinct populations of mitochondria in a somatic cell tissue.

Heterozygous: The individual’s genotype at the locus consists of two different alleles.

Histones: Proteins associated with DNA in the chromosomes, rich in basic amino acids (lysine or arginine) and virtually invariant throughout eukaryote evolution.

Homeodomain: Conserved DNA binding domain consisting of the 60 amino acids encoded by a homeobox gene.

Homozygous: The individual’s genotype at the locus consists of two identical alleles.

Intron: Noncoding DNA that separates neighboring exons in a gene. Linkage disequilibrium: Nonrandom association of alleles at linked loci. Locus: The position of a gene or genetic marker on a chromosome.

Lod score: A measure of the likelihood of genetic linkage between loci. A score greater than +3 is often taken as evidence of linkage; one that is less than -2 is often taken as evidence against linkage.

Megakaryocytes: White blood cells that produce platelets by cytoplasmic budding. Missense mutation: A nucleotide substitution that results in an amino acid

change.

Multimeric complex: Structure composed of several identical or different subunits held together by weak bonds.

Nullisomy: A diploid cell missing both copies of the same chromosome. Oligonucleotide: A short DNA molecule synthesized for use as a probe.

Open Reading Frame (ORF): A significantly long sequence of DNA in which there are no termination codons.

Penetrance: The probability that a given genotype will result in a particular phenotype.

Phenocopy: A phenotype that looks the same as one produced by a specific genotype, but has a different etiology.

Pleiotropy: Multiple phenotypic effects of a single gene.

Polycistronic mRNA: Includes coding regions representing more than one gene.

Polymorphism: The occurrence together in a population of two or more alleles at a locus, none of which are at a frequency that could be maintained by recurrent mutation alone.

Polyploid: Having multiple chromosome sets.

Positional cloning: Cloning of a gene which is dependent only on knowledge of its subchromosomal location.

Purine: A nitrogen-containing compound with a double-ring structure (e.g., adenine and guanine).

Pyrimidine: A nitrogen-containing compound with a single-ring structure (e.g., cytosine, thymine, and uracil).

Radiation hybrid: A type of somatic cell hybrid in which fragments of chromosomes of one cell type are generated by exposure to X-rays, and are subsequently allowed to integrate into the chromosomes of a second cell type.

Reproductive fitness: Relative reproductive success of a genotype as measured by survival, fecundity or other life history parameters.

Splice site: The boundary between an intron and exon. The introns are removed in the generation of mature mRNA.

Stop codon: One of the three codons (UAG, UAA and UGA) that terminate synthesis of a polypeptide.

Synteny: The property of occurring on the same chromosome.

Transcription: The assembly of a complementary single-stranded molecule of RNA on a DNA template.

Transfection: Transfer of a gene, or cDNA (next to a promoter), into a cell, enabling the transfected cell to form a new gene product.

Transition: A nucleotide substitution in which one purine is replaced by another, or one pyrimidine is replaced by another.

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Translation: The assembly of a polypeptide chain from the coded information in the mRNA that directs the amino acid sequence of the chain.

Transversion: A nucleotide substitution of purine for pyrimidine, or vice versa.

VNTR (variable number of tandem repeats): A type of DNA polymorphism created by a tandem arrangement of multiple copies of short DNA sequences.

Wild type: Term used to indicate the normal allele or the normal phenotype. YAC (yeast artificial chromosome): A linear cloning vector into which a large frag-

ment of DNA can be inserted.

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Berlin CI, Hood LJ, Hurley A, Wen H (1996) Hearing aids: Only for hearingimpaired patients with abnormal otoacoustic emissions. In: Berlin CI (ed) Hair Cells and Hearing Aids. San Diego: Singular Publishing Group.

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