Учебники / Genetics and Auditory Disorders Keats 2002

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in a number of individuals with Alport syndrome (Tryggvason et al. 1993). Perhaps not surprisingly, mutations have also been detected in the COL4A3 and COL4A4 genes on chromosome 2 in persons with the autosomalrecessive forms of Alport syndrome (Boye et al. 1998).

Because of the limited knowledge of the detailed structure and function of the inner ear, the candidate gene approach has found limited use in the identification of genes responsible for inherited hearing impairment, especially in the case of genes for nonsyndromic inherited hearing impairment.

As in most other areas of inherited human diseases, the positional candidate approach has found widespread use in the identification of genes for inherited hearing impairment.

2. Positional Cloning

Positional cloning involves identifying transcripts in the interval of the region of the chromosome to which a gene has been mapped and screening those transcripts for mutations. Therefore, the first step in positional cloning is the mapping of the gene responsible through linkage analysis in family studies.

2.1 Linkage and Linkage Analysis

Mendel’s third law, the principle of independent assortment, states that members of different gene pairs assort to gametes independently of one another. While this is true of genes on different chromosomes, it will not always be true for genes that are on the same chromosome. An exchange of genetic material, or what is known as crossing-over or recombination, occurs on average two to three times in each meiosis between homologous chromosomes. However, if two loci are positioned sufficiently close together on the same chromosome, recombination between them will be a rare event (Fig. 3.1). If the alleles at two loci are inherited together more often than would occur by chance, then they are said to be linked. Linkage analysis involves studying the pattern of segregation of polymorphic DNA markers located throughout the chromosomes in families in which a disorder is segregating.

2.2 Polymorphic DNA Markers

Variation in the nucleotide sequence, or what are called DNA sequence variants, of the human genome is common. This variation is inherited in a Mendelian codominant manner and usually occurs in intergenic noncoding DNA, and therefore has no phenotypic consequences. There are different

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types of DNA sequence variants that can be used in linkage analysis. The oldest, restriction fragment length polymorphisms (RFLPs), owing to their limited variation, have almost exclusively been replaced by a subset of variable number tandem repeats (VNTRs) known as microsatellites. The latter are likely to be succeeded in the near future by single nucleotide polymorphisms (SNPs).

VNTR polymorphisms are due to the presence of a different number of tandem repeats of short DNA sequences including either di-, trior tetranucleotide repeats known as short tandem repeats (STRs). The most commonly used VNTRs are dinucleotide repeats which occur some 50,000 to 100,000 times in the genome and consist of blocks of variable numbers of tandem repeats of the dinucleotide CA : GT constituting so-called CA repeats or microsatellites (Weber and May 1989). Microsatellites are highly polymorphic and some 8,000 or more have been identified (Weissenbach et al. 1992; Gyapay et al. 1994; Dib et al. 1996) and mapped to the human genome providing a skeleton framework of the human genome for linkage analysis.

More recently, a third generation of polymorphic DNA variants based on single nucleotide polymorphisms (SNPs) have been identified (Wang et al. 1998b). Some 700 to 900 SNPs at appropriate intervals throughout the genome will allow the possibility of mapping a disorder in a single analysis by DNA chip technology (Kruglyak 1997).

2.3 Recombination Fraction

The recombination fraction is the probability that a crossing-over event will occur between two loci in meiosis. It is designated by the symbol q. If two loci are not linked, i.e. are on different chromosomes or are very far apart on the same chromosome, then the chance that they segregate independently is 50% and therefore q = 0.5. If, however, in 19 out of 20 meioses the alleles at two loci segregate together, then they are on the same chromosome and they are said to be linked with q = 0.05.

The unit of measurement of linkage map distance between two loci is the morgan. One Morgan, or 100 centiMorgans (cM), is defined as the map distance in which an average of one crossover per chromosome strand occurs. Over very small distances, the probability of more than one crossover is negligible. Thus, for example, if two loci are one cM apart, then a crossover would be expected to occur between them once in every 100 meioses, and q = 0.01. The human genome is estimated by recombination studies to be

3,000 cM in length. The physical length of the human genome is approximately 3 ¥ 109 base pairs (bp), so on average, one cM corresponds to 106 bp. The relationship between genetic distance and physical length is not, however, linear because crossing-over is a nonrandom process with some regions being recombination “hotspots” and vice versa; also, recombination occurs more frequently in female than in male meioses.

2.4 Lod Score

In order to test the hypothesis that two loci are linked, a series of likelihood ratios are calculated for different values of the recombination fraction q, ranging from q = 0 (i.e., tightly linked) to q = 0.5 (i.e., unlinked). The likelihood ratio at a given value of q equals the chance of the observed data occurring if the loci are linked at the recombination value of q divided by the chance of the observed data occurring if they are unlinked (q = 0.5). The logarithm to the base 10 of this ratio is known as the Lod or Z score, i.e., Lod (q) = log10 [Lq/L(0.5)]. Logarithms are used because they allow the results of linkage studies from different families to be added together. A Lod score of 3, which means that there is a greater than 103 to 1 chance that the observed data are due to the loci being linked rather than unlinked, is taken as the level of significance confirming linkage (Terwilliger and Ott 1994).

2.5 Multipoint Linkage Analysis

Two-point linkage analysis is used to map a disease locus to a specific chromosome region. More precise mapping can be carried out by multipoint linkage analysis of a series of ordered polymorphic markers known to map to a particular region. This allows the most likely position of the disease locus relative to known markers to be estimated based on the location score, which is a multipoint Lod score (Fig. 3.2) (Terwilliger and Ott 1994). The next step is physical mapping to isolate/identify the gene responsible (Liang et al. 1998). The limit of resolution for defining a candidate region by linkage analysis is usually about 1 cM. Analysis of recombination events in individual families may allow further refinement of the interval containing the gene.

3. Difficulties in Locating Hearing Impairment Genes

The process of locating the position of a gene by linkage analysis has been, until recently, a daunting prospect. The first requirement is to collect families in which the disorder is segregating. While this is easy with some forms of syndromic inherited hearing impairment with distinctive physical features, it is not so simple with nonsyndromic sensorineural inherited hearing impairment. The well recognized genetic heterogeneity of inherited nonsyndromic sensorineural hearing impairment, with multiple genes being responsible for both autosomal dominant and autosomal recessive forms, means that special care is needed in collecting families for linkage studies. If linkage data from unrelated small nuclear families are used, efforts to localize a gene are likely to be unsuccessful because a different gene may be responsible for the hearing impairment in each family.

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FIGURE 3.2. Multipoint linkage analysis. A, B and C represent the known linkage relationships of three polymorphic marker loci. X, Y and Z represent, in descending order of likelihood, the probable position of the disease locus. (Reprinted from Emery’s Elements of Medical Genetics, 10th ed, Meuller, RF, Fig. 3.2, Copyright 1998, by permission of Churchill Livingston.)

In the case of autosomal dominant nonsyndromic sensorineural hearing impairment, single large families have been collected in which there are affected individuals in multiple generations in order to overcome this problem. An example of this approach was the analysis of the large Puerto Rican family with autosomal dominant low-frequency hearing impairment

(Leon et al. 1981), which led to the identification of the first locus for autosomal dominant nonsyndromic sensorineural hearing impairment, DFNA1, on the long arm of chromosome 5 (Leon et al. 1992).

In the case of autosomal recessive nonsyndromic sensorineural hearing impairment, the problem is potentially even more difficult. In the majority of families with autosomal recessive nonsyndromic sensorineural hearing impairment, there are usually at most two or three affected siblings. Pooling the Lod scores from these families is very unlikely to demonstrate linkage. This problem has been overcome by use of the method of autozygosity mapping, which involves using samples from families in which the parents of affected offspring are consanguineous (Lander and Botstein 1987).

Affected individuals in such pedigrees will have regions of their genome that are homozygous for polymorphic DNA markers because they were inherited from a common ancestor (Fig. 3.3). Identification of large consanguineous families from ethnic isolates means a single family can be sufficient to establish linkage (Mueller and Bishop 1993; Kruglyak et al.