Chapter Three

Recombination and linkage

3.1 INTRODUCTION

In the good old days, disease genes were identified by luck or by judgement. From the nature of the disease, the function of mutant gene was deduced. Thus, the alpha, beta, gamma and delta chains were implicated directly in the thalasemias and

hemoglobinopathies.

This type of observation, though of fundamental importance in establishing the principles of clinical genetics, nevertheless had limited potential and majority of disease genes remained unknown. Even the approximate chromosomal address of the gene in question was also not identified. Thus, there was a need to change the approach of investigation.

Therefore, a quite different strategy was introduced according to which no prior insight into the biochemical properties of the gene was required. The only requirement was that the disease was to have a genetic basis.

So, the DNA studies began full scale and a wealth of information was scooped out from the human genome. The key to this approach was complete understanding of genetic linkage phenomenon.

In genetics we study phenotypes, and in human genetics the phenotypes are often diseases. Genetic maps are used to associate genes with phenotypes, so that human maps are of great medical importance.

Map construction is now big science, run by scientist administrators and factory managers. Why are these maps cause for celebration? Because it has not previously been possible to produce complete maps of human genome.

Making a map of the genome entails estimating the distance between pairs of genes or other genomic landmarks, which (classify) must be done by inference from comparison of the

occurence of recognizable phenotypes in successive generations.

When the genes are on different chromosomes, they segregate into germ cells independently. But genes on the same chromosome do not. If they are close together, they behave as if they are linked; further apart, and the linkage may be disrupted by the process of recombination at meiosis.

So, the distance between the genes is a function of the probability that linked genes (or other genomic landmarks) will be seperated by recombination.

In the course of oogenesis or spermatogenesis, the total chromosome complement of the cell is halved. Homologous chromosomes pair and exchange the genetic material in a process termed as crossing over.

The crossed over segments may contain genes, ie. the active portion of DNA, or alternatively may lodge inactive sequences.

Genes very close to each other have a strong probability of being included in the segment that is exchanged for a corresponding one. Thus, the likelihood of any two genetically linked genes being carried together is directly determined by the distance that seperates them. More the distance less is the probability and vice versa.

Thus, if two genes are jointly transmitted through many generations, they are said to be linked. For studying the inheritance of many genes simultaneously, statistical tests are applied and probability of transmission computed.

Since recombination, cannot be observed directly under the microscope, scientist have to rely upon naturally occuring phenotypic variants in human beings, at least.

In humans on an average, 1 percent frequency of recombination equals 1 million base pairs of DNA. Recombination events can, however, be manipulated in laboratory animals which are mated with genetically defined partners.

In humans, however, pedigrees or family charts are prepared to reveal hidden details about genetic linkage. Any gene about which we have sufficient information can be used as

"marker" to explore the genome further.

Through linkage analysis, markers can be assorted to prepare genetic maps of human chromosomes. A genetic marker can be a gene (normal or mutated) or a small sequence of DNA. Linked markers have a great diagnostic potential. They may also help in the isolation of a gene.

Few characteristics of recombination are: 1. it results from an odd number of crossing over, 2. each chromosome has its independent way of undergoing recombination - bigger chromosomes have more crossovers than small ones, 3. crossing over tends to inhibit another exchange in its neighbourhood, it is called as positive interference, 4. chromatid exchange occurs mostly in germ tissue and rather infrequently in somatic cells, and 5. the unit of measuring crossing over between any two positions or loci of chromosome is "Morgan". In one Morgan segment of chromosome, an average of one crossing over per chromatid is found.

3.2 SOURCE OF LINKAGE ANALYSIS

Source of linkage analysis are: (a) genetic polymorphism, and (b) pedigrees. Existence of diverse forms of a given sequence is termed polymorphism. The simplest form of sequence variation occurs every few hundred base pairs in the DNA and can be detected by southern blotting.

Using a battery of restriction enzymes, DNA sequences are cleaved and run on electrophoretic gel.

The DNA fragments, thus, separated are blotted to a nylon or nitrocellulose membrane and permanently fixed. The immobilized DNA is hybridized to a labeled probe for detection of polymorphs.

When a given enzyme reveals difference in the length of fragments extracted from wild and mutant cells, the process is called RFLP (restriction fragment length polymorphism).

By destruction or creation of new site for the restriction enzyme, DNA fragments of varying lengths seperate out during electrophoresis.

This technique can reveal submicroscopic changes in the genome like inversions and deletions. It can be used as a genetic marker in exactly the same way as any other marker.

Instead of examining some features of the phenotype, we directly assess the genotype, as revealed by restriction map. The majority of DNA sequence polymorphisms so far identified in humans have been due to single base substitutions, although other anomalous conditions have also been encountered but rather infrequently.

Once an RFLP has been assigned to a linkage group, it can be placed in specific position of the genetic map, and "map distances" to its flanking markers can be determined. An effort to map RFLPs in humans has led to the construction of a linkage map for the entire human genome.

Most human chromosomes now can be represented in the form of a continious linkage map of RFLP's.

Variable number of tandem repeats (VNTRs) are a useful source of linkage analysis through RFLP technique.

VNTRs are short DNA sequences that are distributed throughout the genome in polymorphic forms. Thus, they can serve as important markers for diagnostic purposes and gene isolation.

The familial history or pedigree is the second important source for linkage analysis. Human families have to be studied, as they occur in nature rather than at the design of the investigator.

By recording phenotypic variation in members of a large family of several generations, recombinants can be identified and recombination rate estimated.

Human beings are not ideal biological materials for linkage analysis because of small family size and rarity of appropriate pedigrees. It has been calculated that there are 56 chiasmata per cell during meiosis, ie. 2.44 per chromosome (56/23).

In order to be transmitted as a single linkage unit, the genes must be within at least 30 to 50 map units.

3.3 STATISTICAL METHODS

Mathematical techniques have came very handy in solving linkage-related problems. The aim is to show that a readily identifiable genetic marker (usually a DNA polymorphism) segregates along with the disease gene during meiosis.

In practice this means demonstrating that the marker and the disease locus coincide within a family or a number of families. At meiotic division, the chance of any two genes passing together into the same daughter cell is never less than 50 percent.

If linkage is perfect (for example if the marker happens to be within gene itself), the chance of cosegregation will be 100 percent.

Any value greater than 50 percent but less than 100 percent indicates linkage with some chance of crossing over (recombination) between the marker and the gene. In analyzing the distribution of marker and disease gene in a family, we ask "What is the likelihood of observing this pattern if the two are linked giving 0, 1, and 10 percent recombination and so on.Similarly, "What is the likelihood of observing this distribution if there is no linkage between the gene and marker?" The ratio of odds calculated under the conditions (linkage and no linkage) is an estimate of the statistical probability of linkage.

Presently, standard statistical packages like LINKAGE and LIPED are used for speedy analysis of pedigree data.

3.3.1 Maximum Likelihood

Estimation of the recombination rate for two loci is usually done by the method of maximum likelihood. The maximum likelihood estimate of the recombination rate is that

value r for which the probability is maximum.

This means that the greatest chance of linkage occurs where the probability of observations is maximum. But in order to label two genes as linked, one must first adjust the effect of independent segregation.

3.3.2 LOD Score

Since 1955, Logarithm of Odds (LOD) introduced by American geneticist Newton E Morton has found wide acceptance.

It effectively differentiates linkage that is suspected to be due to physical proximity of genes in comparision to one that occurs by independent assortment.

The ratio of two possibilities obtained is normally quoted as logarithm (to the base 10) and is called as LOD score.

A LOD score (Z) of +3 or greater at a recombination fraction (0) of 0.2 or less is accepted as strong evidence of linkage, while a score of -2 or less excludes linkage.

This is very powerful test and can analyze two or more loci simultaneously. LOD scores are convinient to use because data collected from different families can simply be added to derive the cumulative LOD score(Z).

The value 0 at which Z is greatest is accepted as the best estimate of the recombination fraction and is often called the maximum likelihood estimate.

3.4 APPLICATIONS OF LINKAGE MAPPING

With the progressive accumulation of linkage data in the form of pairwise recombination estimates or LOD score tables, a number of linkage groups have been established for the human genome.

Linkage studies offer an investigation into clinical and biological conditions of doubtful etiology, where genetic or enviornmental factors may be at work.

A final product will be, of course, complete dissection of human genome into well-defined and closely-placed landmarks.

Linkage mapping is quite helpful in identifying the "disease" genes. If a locus responsible for a genetic disease is found closely linked to a previously characterized marker, the latter could greatly help in early diagnosis of the disease.

By knowing which markers are most consistently inherited along with the disease, the transmission of gene can be studied.

For prospective genetic counselling, it would be very effective tool. Not only high-risk fetus could be identified at an early stage, but also chances of same disease occuring again in the family could be predicted with a fair degree of accuracy.

Thus, linkage studies are useful in prenatal diagnosis, presymptomatic diagnosis and heterozygote detection. Genes for human disorders like cystic fibrosis, Deuchenne's muscular dystrophy (DMD), Becker's muscular dystrophy (BMD), fragile X mental retardation syndrome, and hemophilia have been marked by linkage-mapping techniques, that has led to their successful isolation.

Advances in recombinant DNA technology have removed the principal limitation of human linkage studies, permitting a large number of polymorphic genetic systems to be defined.

Homologous genetic recombination is exploited as a tool to order these markers on chromosomes and to measure their degree of physical seperation in genetic terms.

Finally, powerful computing methods are available for analyzing the joint segregation of many genes and for constructing genetic linkage maps of the human chromosomes.

As detailed maps of human chromosomes become available, a wealth of new questions may be effectively addressed. Recent discoveries in molecular genetics of cancer are dramatic outcomes of developing a gene map.

Thus, the human genetic map, primarily constructed as a tool for medical research and exploiting homologous recombination without formal knowledge of its intimate mechanisms, could ultimately lead to a deeper understanding of the behavior of chromosomes at meosis.