SISTEMA FINANCIERO EN CUBA

The first stage to identifying a disease gene by its position is to identify the affected location within the genome and to construct a map of the area. Genome maps are linear representations of DNA that show organisation based on a particular system of landmarks. There are three types of genomic map: cytogenetic maps, physical maps and genetic maps.

Cytogenetic mapping

A cytogenetic map is a representation of an individual chromosome when examined microscopically. The earliest method used to locate a gene to a particular chromosome made use of somatic cell hybrids. When a hybrid cell is made between a human cell and a rodent cell, there is progressive loss of the human chromosomes as the cells are grown in culture. Ultimately, only one or two human chromosomes will be left. Panels of hybrid cells have been made covering the full human chromosome spectrum. Probes of interest can be mapped to a particular chromosome by hybridisation to Southern blots of DNA from these hybrid cells (Watsonera/., 1992). The HLA gene cluster was first assigned to chromosome 6 using a human-Chinese hamster hybrid cell panel (Jongsma et al., 1973).

Simpler techniques designed to examine chromosomes directly have now been developed. When a metaphase spread of chromosomes from peripheral lymphocytes is stained with the dye giemsa, each chromosome exhibits a characteristic pattern of light and dark bands that distinguish it from other chromosomes. The dark bands (G bands) are A-T rich and are late replicating (Korenberg and Rykowski, 1988). These can be seen by giemsa staining after proteolytic digestion. 0 bands are rich in LINE repeats and are relatively gene deplete. The light bands (R) are G-C rich and are early replicating. These can be visualised with giemsa staining after heat dénaturation in saline (Craig and Bickmore, 1994). In contrast to G bands, R bands are gene rich areas of the chromosome and are rich in Alu repeats (Korenberg and Rykowski, 1988). A cytogenetic map of chromosome 13 based on banding techniques can be seen in figure 1.6. The region of deletion inB cell malignancy (DBM) has been located to band 13ql4,3 (R band) as discussed later.

A karyotype is a representation of all the chromosomes for an individual. By studying chromosomal banding, this karyotype may reveal abnormalities such as translocations, duplications, deletions, inversions etc. The location of the Duchenne muscular dystrophy gene at Xp21 was determined by the discovery of such a translocation using this method (Ray et a l, 1985), as was the location of the Kallmann gene at Xp22.3 (Franco et al, 1991; Legouis et a l, 1991). To increase the sensitivity of this mapping by banding, additional fluorescent in situ

hybridisation (FISH) techniques can be used. Probes for each whole chromosome are available commercially and can each be assigned a different fluorescing colour. By 'painting' a karyotype with these chromosome paints, subtle abnormalities not

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13 q 1 4 . 3

Figure 1.6: Cytogenetic map o f Chromosome 13. The region deleted in B cell malignancy (DBM) is schematically represented at band

13ql4.3 between two microsatellite markers, D13S25 and D13S319. RB 1 - retinoblastoma gene

seen with banding, such as balanced translocations, can be identified by a change in colour of a chromosome. FISH techniques can also be used to map individual probes to this cytogenetic map (Trask, 1991). Clones containing fragments of DNA e.g. cosmids and PACs (described in more detail below), can be labelled with a fluorescent dye and hybridised to a chromosomal spread. By viewing under a fluorescent microscope the probes can be visualised and mapped to a specific chromosome (Trask, 1991). Two probes can be distinguished jfrom each other within 1Mb using metaphase FISH (Heiskanen, 1996). If interphase cells are used in addition to metaphase (the chromatin is less condensed in interphase) then probes can be distinguished from each other when they are as close as 50kb (Trask

et aL, 1989). The order of probes along a chromosome can be assessed by labelling probes in different colours and comparing their localisation to that o f a known centromeric or telomeric probe. With the advent of fibre FISH techniques, cytogenetic mapping is becoming ever more sophisticated (Heiskanen, 1996).

Physical mapping

Physical maps of DNA consist of an order of landmarks with distances between them measured in base pairs. The useful physical maps for the ’gene hunter’ are of small stretches of DNA encompassing a region known to contain a gene implicated in a particular disease. These original locations may well have been discovered from cytogenetic mapping. Several types of landmark are used in physical mapping:

Sites for restriction enzymes (recognised sequences) can be used as landmarks in physical mapping. When DNA is digested with a given restriction enzyme, a pattern of fragments of varying sizes will be produced that is dependent on the position of these sites throughout the genome. If the enzyme used cuts infrequently, e.g. BssH II, then the fragments produced can be separated by pulse field gel electrophoresis (PFGE) (Schwartz and Cantor, 1984). If the enzyme cuts more frequently, e.g. EcoR I, then the fragments can be separated on an agarose gel by standard gel electrophoresis. The DNA from these gels can be transferred to nylon membranes by Southern blotting (Southern, 1975). Probes from the area of interest can then be hybridised to these filters and the jigsaw pieces can gradually be assembled by repeating the process for a series of restriction enzymes and probes. This type of physical map is referred to as a restriction map. If the area thought to contain the gene of interest is disrupted in

affected patients e.g. by a deletion or translocation, then the pattern of fragments produced by restriction enzyme digest of their DNA will be different i.e. a fragment may be completely absent or may be rearranged and seen as a different size. This further adds to the definition of the map in relation to the location of the gene. The map of the region containing the Wilms tumour gene was initially constructed using long range restriction maps (Call et al, 1990).

Another type of landmark used for physical mapping are sequence tagged sites (STS). These are unique sequences of approximately 300 base pairs (60-1000 bp) which can by amplified with known primers using the polymerase chain reaction (PGR) (Watson et a l, 1992). STSs can be mapped relative to each other in terms of both order and spacing within a given region of the genome. These markers can either be used as hybridisation probes or in PGR amplifications. Data regarding STSs, including the primers used to amplify them and their chromosomal location can be found in various public databases e.g. Genome database (http://www.gdb.org/) and the Whitehead Institute for Biomedical Research (http://www-genome.wi.mit.edu/). Similar to the STSs are the expressed sequence tags (ESTs ). These are unique sequences isolated from cDNA libraries that can be amplified by known PGR primers, as for the STSs. These markers are of particular interest to the 'positional cloner' because they potentially represent pieces of genes. Hundreds of thousands of ESTs have been generated to date and

are available from public databases e.g. dbEST (see

http://www.ncbi.nlm.nih.gov/dbEST/index.html). Unfortunately, very few have been positioned on physical maps and so their use is limited in the construction of a localised map. This will gradually change as more of the human genome is sequenced and mapped.

The greatest advantage of these markers (STSs and ESTs) is that they can be used to assemble maps of regions of the genome represented in smaller and smaller clones (contigs). For the identification of coding sequences within a given area, not only does the region need to be well defined but the DNA is much easier to manipulate for further experiments if it is available in short pieces i.e. digested and cloned into vectors such as yeast artificial chromosomes (YAGs), PI derived artificial chromosomes (PAGs), bacteriophage derived artificial chromosomes (BAGs) and cosmids. YAGs are an excellent starting point for the construction of these contigs because they contain inserts of DNA from lOOkb - 1.5Mb and, therefore, even a relatively large region can be quickly covered in YAG clones (Schlessinger, 1990). Public human genomic YAG libraries are available (e.g.

Centre D’Etude du Polymorphisme Humaine - CBPH) for screening with STS and EST markers to identify relevant clones. If different clones contain the same markers then they can be assumed to overlap. Techniques are available to clone the end sequences of YACs (Riley et al^ 1990). This generates more specific sequence (additional STSs) from the region and further YAC clones can be identified. This method of cloning a genomic region is known as chromosome walking. The size of the YAC inserts can be assessed by PFGE and the distances within the physical map can be accurately defined. The depth of the map contig can be increased by constructing similar contigs in bacteriophage (PAC and BAC) derived clones and cosmids. PACs are derived from the bacteriophage PI cloning system (Pierce et a l, 1992) and contain inserts of DNA between 100-150kb. Methods for obtaining the sequence of the insert ends are available for these clones as well (Wang and Keating, 1994). BACs (bacterial artificial chromosomes) (Shizuya et a l, 1992) take slightly larger inserts than PACs e.g. in the region of 300kb, whilst cosmids are circular vectors able to take up to 40kb of insert. PAC libraries are publicly available as for the YACs (Human Genome Mapping Project-HGMP, Cambridge) but cosmid libraries are not widely available and need to be specifically constructed, often from corresponding YAC clones.

When a region is mapped and cloned in this way, candidate gene sequences can be identified. For example, deletion of the long arm of chromosome 5 (5q-) is a recurring abnormality seen in myeloid malignancies and is thought to contain at least one tumour suppressor gene. A physical map of the region was generated in 108 PAC, YAC and BAC clones with 11 ESTs and 97 STSs Using this highly detailed map the defined minimal region of deletion was greatly reduced and candidate tumour suppressor gene cDNAs were identified (Zhao et a l, 1997).

A further technique used in physical mapping is radiation hybrid mapping. This technique is a sophisticated version of somatic cell hybrids. Rearranged and ’broken’ chromosomes can now be artificially produced by irradiation of DNA. An individual chromosome from a somatic cell hybrid can be selected and irradiated to artificially produce fragmented DNA with translocations and deletions within the chromosomal structure (Benham et a l, 1989). The irradiated cells are then fused again with rodent cells as before so that only a fragment of abnormal human chromosome exists within a cell. A panel of cells representing one chromosome can be produced. Cells containing specific regions of DNA can be identified using STS and EST markers and the DNA can be isolated. The region of DNA containing the Wilms tumour gene was isolated with this technique (Call et al.

1990). Using further mapping and cDNA library screening, the gene was subsequently identified.

Radiation hybrid panels also allow for a powerful form of mapping. By assessing the frequencies with which various STS and EST markers are found together in radiation hybrid cells, statistical packages can be used to assess the distance between them and so assemble a map of a given region. This technique has been extensively used for mapping of many disease regions including that spanning the affected area of chromosome 15q26.1 in Blooms syndrome (Straughen et a l, 1996), an autosomal recessive disease characterised by growth retardation, male infertility and immunodeficiency. A similar technique has been used for mapping of chromosome 13 (Hawthorn and Cowell, 1995). The Human Genome Mapping Project are making extensive use of radiation hybrid mapping across the entire genome. This information can be found at web sites such as the CEPH-Genethon integrated map (http://www.cephb.fr: 80/)

Genetic mapping

A genetic map reflects the possibility that two markers that are closely spaced on a chromosome will remain together at mitosis i.e. they will not be separated in the process of homologous recombination. The greater the frequency of two markers being separated, the greater the distance between them and vice versa. The unit of measure in a genetic map is the centimorgan (cM). There is some correlation between physical and genetic distances and a cM is taken to be approximately 1Mb. To be informative, a genetic marker must display polymorphic properties i.e. its sequence must be different in the same individual on opposing alleles. If these differences affect a site for a restriction enzyme then digestion of DNA with that enzyme will provide a different length of DNA fragment for each chromosome-restriction fragment length polymorphism (RFLP). These can be detected by Southern blot analysis. An individual is said to be informative for a RFLP if their chromosomes can be differentiated on the bases of different size fragments. RFLPs are inherited in a Mendelian manner and can be traced through families. Linkage maps of the genome have been produced using large sets of families. Much of this work has been done with families from the Mormon Church and the pedigree is very extensive (Watson et a l, 1992). DNA from these families makes up the core of the collection of linkage data provided by CEPH.

Mapping of regions of the genome by RFLPs led to isolation of candidate sequences for many genes including the cystic fibrosis gene and the Duchenne muscular dystrophy gene. RFLPs were particularly important in the hunt for the cystic fibrosis gene as no chromosomal abnormality could be detected by other methods (see below).

Microsatellite markers are now superseding RFLPs for genetic mapping. These are short repeat sequences (repeated 5 to 50 times) that are polymorphic and, therefore, often informative for individual chromosomes. They are very numerous (every 30-60kb) and can be amplified by PCR. They can be followed through family pedigrees in the same way as RFLPs.

Ultimately, correlation of information from all three types of map is necessary to build a picture of the human genome and, more importantly in the context of this project, to clearly define the location of a particular disease gene. In all cases of positional cloning, it is essential to keep returning to the patient data to define and redefine the boundaries of the genome that indicate where the relevant gene may lie. As maps become more sophisticated and more markers are generated, these markers can be used to identify more genomic clones and consequently more unique markers. In this way a gradual 'walk' across the genome is performed. Many cDNAs of genes have been identified by chromosome walking e.g. retinoblastoma gene at 13ql4.3 (Friend et al, 1986).

When the area that contains a disease gene has been identified and mapped, candidate sequences for the gene need to be identified. There are several different techniques available to do this that will be discussed here.

1.4.5. Identification of candidate gene coding sequences from a mapped