Genetic and physical mapping

Molecular characterization of genetic disease is based on the supposition that it is possible to identify an alteration in gene expression that causes the disease. In single-gene disorders the task is conceptually straightforward (although often technically demanding); the aim is to establish that all those with the disorder have DNA sequence changes (mutations) affecting the same gene and that such changes are not found in unaffected individuals. Enough is known about sequence codes to recognize a mutation that will disrupt gene expression, and this can be experimentally verified by looking at the production and structure of mRNA. The technical challenge is to find the one mutation in 3000 million bases which is responsible for the change in gene expression.

Genetic mapping is still the basic method for locating a disease gene to a small region of a chromosome. Before explaining what gene mapping entails, some terminology needs to be explained. A position on a chromosome is termed a locus, a general term which can refer to a gene or a segment of DNA with no known function. DNA sequences that differ at the same locus are termed allelic variants. Since we have two copies of each chromosome, by definition we have two alleles at each locus. If these alleles are identical the individual is said to be a homozygote at that locus; if they are different, the individual is a heterozygote. The number of alleles at any locus varies remarkably; at the most polymorphic loci, hundreds of alleles may be found. Polymorphism in the human genome is important in a practical sense because it permits gene mapping, and hence disease gene identification.

The basic idea of gene mapping is to track genetic recombinants in pedigrees. Recombination can occur almost anywhere on a chromosome and will break up the association of alleles between different loci along that chromosome (such an association is called a haplotype). Alleles, like most single-gene disorders, follow Mendelian laws of segregation so that it is possible to ask whether one is segregating with a disease and test the result statistically using methods described in Chapter..2.4.1. The expected result is an estimate of the probability that an allele and a disease locus will recombine; the lower the probability, the closer together the two loci are on the chromosome.

The statistical test tells us the likelihood that the estimate of recombination distance is correct. If the likelihood is acceptably high, then the next task is further genetic mapping to reduce the genetic interval as much as possible, at which point physical mapping and characterization of the region takes over as the principal tool of disease gene identification. The latter process is currently the most time consuming and expensive, but will become easier with the completion of the Human Genome Project.

Genetic mapping of disease genes is a powerful method because it does not require any knowledge of a gene's function to find the chromosomal location and then the identity of the disease gene. It is a successful method because the genome is replete with DNA sequence polymorphisms whose only known use is to enable geneticists to map disease genes. On average, every 1000 bp will contain 1.4 bases that differ between two randomly chosen individuals (although the full extent of variation is not known), almost all of which have no phenotypic consequence. In addition, there are small runs of repeated sequence (most commonly CA) which differ in length between individuals. At least one of these short tandem repeats (STRs), or microsatellites, is found every 50 kilobases (kb) and they also have no known phenotypic consequences. There are other more complex sequence polymorphisms, but single-nucleotide polymorphisms ( SNPs) and microsatellites are the most useful for identifying disease genes. The process of defining which alleles a person has at a polymorphic locus is termed genotyping.

Microsatellites are currently preferred as genetic markers in disease mapping because they can be detected using the polymerase chain reaction ( PCR) (see Fig 4).

Many thousands have already been placed on a genetic map which provides the basic cartographic resource for molecular cloning, and eventually sequencing, of the human genome. However, recent technical innovations promise to make genotyping SNPs the favoured way to map genetic disease.

Fig. 4 The polymerase chain reaction. The essential ingredients for in vitro DNA amplification are as follows: a DNA polymerase (shaded shape); a pair of oligonucleotides (referred to as primers in the figure and shown as short grey wavy lines), which are synthetic single-stranded DNA, usually between 15 and 25 bases long, complementary to two sequences on opposite strands of the target DNA; the target DNA itself; all four nucleotides, usually present in excess; appropriate buffer and cofactors for the reaction. The reaction proceeds in a cycle of three steps: (1) the mixture is heated to over 90°C for 1 min to separate the complementary strands of target DNA; (2) the mixture is cooled, to about 50°C, so that the oligonucleotides anneal to their complementary sequence in the target DNA and allow the DNA polymerase to bind (oligonucleotides are required to prime the polymerase); (3) the temperature is adjusted to allow the polymerase to function in the extension component of the reaction. Typically the polymerase is from a thermophilic bacterium with a permissive temperature of 72°C. Products from the first cycle can serve as targets for a second round of amplification and the cycle can be repeated, usually up to about 40 times. One way of visualizing the products of a pCr reaction is shown on the right of the figure. The DNA has been electrophoresed through an agarose gel, which resolves the PCR products by size so that smaller fragments are lower down the gel. The PCR products in the gel are stained with a dye that fluoresces under ultraviolet light and appear as white bands in the photograph. The gel shows samples from four individuals at a polymorphic locus. Lanes 2 and 4 are heterozygotes and lanes 1 and 3 are homozygotes.

A number of approaches are now available for genotyping many thousands of SNPs in a few hours. One method uses mass spectrometry to measure size differences between DNA fragments; another uses hybridization to a minute glass slide (smaller than a postage stamp and called a DNA chip) containing tens of thousands of small stretches of DNA (oligonucleotides). Hybridization is the process whereby two strands of genomic DNA, separated chemically or by heat (the DNA is said to be denatured), will pair to their complementary sequences, which can be either their original partner or any other DNA with the same complementary sequence. The chip technology involves hybridizing DNA labelled with a fluorescent nucleotide to oligonucleotides that have been synthesized directly onto glass.

The advantage of the new techniques is the high throughput they permit, so that it is possible to genotype very large numbers of individuals with very large numbers of markers.(9) For statistical reasons, successful genetic analysis of common psychiatric disorders may only be possible with the massive genotyping provided by SNP analysis.

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