Molecular Biotechnology: Principles and Applications - Glick, B. R., & Pasternak, J. J. 2002

Molecular Biotechnology of Microbiological Systems
Human Molecular Genetics
Construction of Multilocus Human Chromosome Maps

The Use of many thousands of polymorphic markers distributed across the entire genome has made it possible to determine both the order of loci and the distances between them on each chromosome. A linkage map of polymorphic regions is invaluable for localizing genes associated with various diseases. To identify such genes, researchers can use probes specific to the sequences flanking the Gene of interest.

Ideal families for mapping polymorphic loci are three-generation families in which both great-grandmothers and both great-grandfathers are alive, and the parents have A large number of children (>8). Based on the genotypes of the grandparents, it is possible to establish the genetic phase of the studied loci in each parent, while a large number of children increases the probability that recombination will occur. The Centre d'Etude du Polymorphisme Humain (CEPH) in Paris has collected data and DNA samples from members of 65 families, predominantly three-generation families with an average of 8.5 children each (see, for example, Fig. 20.17). This family bank (CEPH families) provides genotype information for all members to mapping laboratories worldwide. In practice, it consists of lymphoblastoid Cell line cultures from most CEPH family members, serving as a ready DNA source for mapping new polymorphic loci as they are discovered.

Constructing a multilocus genetic map (linkage map) of a human chromosome is a challenging task; it is accomplished using specialized computer software that determines the locus order best supported by recombination data. Structure/149.html">The problem of ordering loci becomes increasingly complex as the number of loci to be mapped grows. For $N$ loci, there are $N!/2$ possible arrangements. For instance, for 10 loci, this number equals 1,814,400. Although certain combinations are clearly implausible even upon visual inspection of the data, the number of potential arrangements remains very large. Typically, researchers first determine the most probable arrangement of a few linked loci, and then combine these "best" options to construct a statistically reliable linkage map for all loci. The criterion for whether one locus is located adjacent to another is the logarithm of odds (LOD) score; a score of +3.00 or higher is considered positive evidence of linkage. Generally, map construction is carried out in stages. First, several polymorphic markers located on the same chromosome are selected. Next, DNA samples obtained from multiple CEPH families are genotyped for each polymorphic marker. The structure of CEPH families is such that determining the genotypes of every single DNA sample is unnecessary. Involving additional families does not yield an improvement in map quality that would justify the extra workload. Usually, 15, and sometimes 40, families are used. Genotyping 40 CEPH families for 20 polymorphic markers requires performing approximately 10,000 assays. Each individual's genotype for every locus is entered into a database. At this stage, the database is checked for errors. A computer program scans for parent-offspring genotype inconsistencies, which typically arise during data entry or genotyping. Occasionally, re-typing is performed to clarify ambiguous results. Errors can lead to incorrect Conclusions regarding locus order and inter-locus distances; therefore, erroneous data are excluded from the analysis whenever possible. For the genotyped CEPH families, all two-locus LOD scores and recombination fractions (recombination indices, $ heta$) are determined, and based on these data, a dedicated computer program constructs the genetic (linkage) map.

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Fig. 20.17. CEPH family K1331.

Human chromosome linkage maps are constantly updated as additional polymorphic loci are identified. As the number of loci increases, map resolution improves and the distance between loci decreases. By 1994, the genotypes of CEPH family members had been determined for approximately 6,000 polymorphic markers, and multilocus mapping had established the positions of roughly 1,000 loci across the entire Human Genome, with an average inter-locus distance of about 4 cM. The objective of large-scale mapping projects is to use additional polymorphic markers to construct a map of each chromosome with an inter-locus distance of 1–2 cM.

Localizing a Disease Gene on a Linkage Map

To accomplish this task, members of families affected by a specific genetic disorder are genotyped using polymorphic markers that, according to mapping data, reside on the same chromosome arm as the disease gene. The approaches used are similar to those for calculating two-point LOD scores in linkage analysis. In this case, the disease gene locus is arbitrarily positioned among four ordered loci, and the LOD score is calculated for each position. In multilocus mapping, the LOD score is defined as the logarithm of The ratio of: 1) the probability that the disease gene occupies a specific position on the map of four ordered loci, to 2) the probability that the disease gene is unlinked to any of the considered polymorphic markers. The use of exactly four polymorphic loci is dictated by the fact that a larger number makes calculations overly complex. The disease gene may be located before the first locus, in various intervals between loci, or beyond the final locus. After calculating the LOD score for each possible position of the gene within various sets of four loci, the maximum value exceeding +3.00 is selected, yielding the most probable localization of the gene.

Radiation Hybrid Mapping

Radiation hybrid (RH) mapping does not require collecting pedigrees or genotyping members of the CEPH family bank. The method is based on somatic cell work and the screening (using PCR probes) of cell lines containing fragments of Human Chromosomes. RH mapping of an entire chromosome or a specific region begins with the creation of a human-rodent hybrid cell line containing a single human chromosome. Cells of such a monochromosomal hybrid cell line are exposed to lethal Doses of ionizing radiation (X-rays or gamma rays), which disrupts cell membranes, inactivates Enzymes, and causes chromosome fragmentation. The unit of measurement for ionizing radiation absorbed by biological tissue is the rad (radiation absorbed dose). One rad is equal to 0.01 J per 1 kg of tissue or 100 ergs per 1 g of tissue. Typically, cultured cells perish at 3,000 rad. The higher the dose, the more severe the damage and the smaller the resulting DNA fragments. At a dose of 10,000 rad, the fragments become too small for RH mapping.

A crucial step in RH mapping is the release and preservation of human DNA fragments generated by irradiation. To achieve this, irradiated (donor) cells are fused with non-irradiated (recipient) rodent cells. Irradiated cells that have fused with one another or remained isolated are unable to grow in culture due to radiation damage. Conversely, recipient cells—whether self-fused or unfused—lack the selective marker present in donor cells that ensures their growth in the Selection medium used for fusion. Consequently, only viable donor-recipient fusion cells carrying the selective marker will proliferate in this medium, with most DNA fragments from the irradiated cells becoming integrated or translocated onto the functional chromosomes of the recipient cells. The surviving hybrid cells are co-cultivated until distinct cell lines—known as radiation hybrids (RHs)—are established. A group of radiation hybrids obtained from a single experiment is called a radiation hybrid panel (RH panel). It stores the majority of the human chromosomal DNA, derived from the monochromosomal hybrid cell line, in the form of fragments.

The DNA of each member of the RH panel is analyzed using several chromosome-specific PCR probes, many of which recognize polymorphic regions. However, polymorphism as such is not required for RH mapping. The goal of this mapping is simply to determine whether a given chromosomal segment is present in The Cell lines of the RH panel. Therefore, PCR primers specific to unique (single-copy) DNA sequences can also be used for screening. Monomorphic, PCR-identifiable chromosome-specific segments are called sequence tagged sites (STSs). All cell lines of the RH panel are tested for the presence (+) or absence (-) of such a site (Table 20.5) using the full set of probes, and hybrids whose DNA fails to amplify are discarded. Effective RH mapping requires a panel of approximately 100 RHs derived from a single monochromosomal hybrid cell line.

Table 20.5. Marker Retention Data in RH Mapping¹⁾

¹⁾ Numbers and letters represent radiation hybrids and PCR markers, respectively. Plus and minus signs indicate the presence or absence of marker sites in a given radiation hybrid.

The THEORETICAL FOUNDATIONS OF RH mapping and meiotic mapping are quite similar. The closer two loci are on a chromosome, the higher the probability that both will be retained within the same DNA fragment following irradiation. Similarly, the closer the sites are to each other, the less likely they are to be separated by recombination during meiotic mapping. The core principles underlying RH mapping are as follows: 1) radiation-induced breakage between two sites is independent of marker retention; 2) the retention of a fragment containing one marker is independent of the retention of any other fragment in the same cell.

The retention patterns (retention signatures) of presence (+) or absence (-) for each marker, obtained across all cell lines of the RH panel, are used to construct the RH map. The LOD score is calculated as the logarithm of the ratio of the probability of obtaining a specific retention pattern for two sites to the probability that these sites are always separated by a break upon irradiation. Unlike meiotic recombination, $ heta$ for radiation breakage frequency ranges from 0 to 1; $ heta = 0$ means that two marker sites are never separated at a given radiation dose (i.e., they are tightly linked). When $ heta = 1$, the markers are always separated at that radiation dose (i.e., they are completely unlinked). If the LOD score is equal to or greater than +3.00, one can confidently conclude that two markers are linked. Computer programs have been developed to order sites and determine the distances between them on the RH map.

The distance between sites on an RH map is measured in centirays (cR). Because fragment size is inversely proportional to the radiation dose, it is necessary to specify the dose at which a given RH panel was produced and the RH map constructed. For example, a distance of 1 $ ext{cR}_{8000}$ means that a breakage occurs between two markers in 1% of cases at a dose of 8,000 rad.

There is no direct relationship between centirays and the number of Base Pairs. One can only state that the higher the radiation dose in rads, the smaller the physical distance corresponding to a given centiray value. For instance, distances of 1 $ ext{cR}_{9000}$, $ ext{cR}_{8000}$, 1 $ ext{cR}_{6000}$, 1 $ ext{cR}_{5000}$, and $ ext{cR}_{3000}$ are roughly equivalent to 50, 53, 62, 90, and 100 kb, respectively. In contrast, meiotic (genetic) mapping can at best resolve sites located 1 cM apart, which corresponds to roughly 1,000 kb. RH maps not only offer higher resolution but are also more complete than genetic maps. Furthermore, RH mapping is technically simpler than meiotic mapping, and new sites can be rapidly incorporated into an existing RH map. Unfortunately, RH mapping does not allow for the localization of disease genes to specific chromosomal regions. Despite this limitation, RH mapping is likely to supersede linkage mapping based on CEPH family genotyping in the construction of multilocus maps of human chromosomes.