GENE MAPPING. Genetica per Scienze Naturali a.a prof S. Presciuttini

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1 GENE MAPPING Questo documento è pubblicato sotto licenza Creative Commons Attribuzione Non commerciale Condividi allo stesso modo

2 Genetic mapping in humans In principle, genetic mapping in humans is exactly the same as genetic mapping in any other sexually reproducing diploid organism. The aim is to discover how often two loci are separated by meiotic recombination. In this pedigree, alleles at two loci (locus A, alleles A and A ; 1 2 locus B, alleles B and B ) are 1 2 segregating in this family. Where this can be deduced, the combination of alleles a person received from his or her father is boxed. Persons in generation III who received either A 1 B 1 or A 2 B 2 from their father are the product of nonrecombinant sperm; persons who received A 1 B or A B 2 are recombinant The information shown does not enable us to classify any of the individuals in generations I and II as recombinant or nonrecombinant, nor does it identify recombinants arising from oogenesis in individual II 2.

3 The difficulty of building genetic maps in human Human genetic maps could not easily be constructed using classical genetic mapping Classical genetic maps for experimental organisms such as Drosophila and mouse are based on genes.. They have been available for decades, and have been refined continuously. They are constructed by crossing different mutants in order to determine whether the two gene loci are linked or not. For much of this period, human geneticists were envious spectators, because the idea of constructing a human genetic map was generally considered unattainable. Unlike the experimental organisms, the human genetic map was never going to be based on genes because the frequency of mating between two individuals suffering from different genetic disorders is extremely small. The first autosomal linkage in man was demonstrated in 1951, using Penrose's sib-pair method, between the Lutheran blood group and the Secretor gene (causing the AB0 antigens to be present in saliva in addition to blood)

4 The need of polymorphic markers The only way forward for a human genetic map was to base it on polymorphic markers which were not necessarily related to disease or to genes. As long as the markers showed Mendelian segregation and were polymorphic enough so that recombinants could be scored in a reasonable percentage of meioses, a human genetic map could be obtained. The problem here was that, until recently, suitably polymorphic markers were just not available. Classical human genetic markers consisted of protein polymorphisms, notably blood group and serum protein markers,, which are both rare and not very informative. By 1981, only very partial human linkage maps had been obtained, and then only in the case of a few chromosomes.

5 The development of human genetic markers Marker type When used No of available loci Blood groups and HLA ~20 Protein Electromorphs ~30 DNA RFLPs 1975 <10 5 DNA minisatellites 1985 <10 4 DNA microsatellites 1989 <10 5 DNA SNPs 1998 <10 6

6 A simple measure for marker polymorphism Observed Heterozygosity (HO). The simplest way of measuring the level of polymorphism of a locus is to calculate the proportion of individuals that are heterozygous. This is easily done for all codominant markers, and marker heterozygosities can be averaged over many loci to obtain the overall value. Expected Heterozygosity (HE). The proportion of heterozygotes of a locus in a population can also be predicted based on the known allele frequencies and assuming random mating using the equation H = 1 Σ p, E i i2 where p i is the frequency of allele i,, and corresponds to the probability that two alleles taken at random from a population are different one from the other. It is also often called Gene Diversity.

7 The microsatellites The advent of PCR finally made mapping relatively quick and easy. The microsatellites are mostly (CA) n repeats. Tri- and tetranucleotide repeats are gradually replacing dinucleotide repeats as the markers of choice because they give cleaner results - dinucleotide repeat sequences are peculiarly prone to replication slippage during PCR amplification. Much effort has been devoted to producing compatible sets of microsatellite markers that can be amplified together in a multiplex PCR reaction and give nonoverlapping allele sizes, so that they can be run in the same gel lane. With fluorescent labeling in several colors, it is possible to score perhaps ten markers on a sample in a single lane of an automated gel

8 Microsatellites may be highly polymorphic The figure below shows a number of subjects typed with a typical (CA)/(TG) marker (D17S800). Seven different alleles are recognizable. Individual alleles show a strong upper band followed by two lower 'shadow bands', one of intermediate intensity immediately underneath the strong upper band, and one that is very faint and is located immediately below the first shadow band. Genotypes are indicated below each individual. Microsatellites used in gene mapping show heterozygosities between 70% and 90%

9 Dense human genetic maps The discovery of microsatellite markers has enabled the construction of a human genetic map at an average density of one marker per cm. Although such resolution is a remarkable achievement, a cm is still a huge segment of DNA, estimated in humans to be 1 Mb. Currently, even higher resolution genetic maps are being developed on the basis of single-nucleotide polymorphisms (SNPs). A SNP is a single base-pair site within the genome at which more than one of the four possible base pairs is commonly found in natural populations. Several hundred thousand SNP sites are being identified and mapped on the sequence of the genome, providing the densest possible map of genetic differences.

10 Detecting linkage by microsatellites A a family with six children segregating a dominant disease is typed for a microsatellite (M). This pattern is interpreted at the top of the illustration with the use of four different-sized alleles, M through M, one of which (M ) is probably linked in coupling to the disease allele P.

11 Major steps in cloning human genes

12 An example of successful gene cloning: BRCA1

13 First evidence of linkage Science :1684 Linkage of early-onset familial breast cancer to chromosome 17q21. Hall JM, Lee MK, Newman B, Morrow JE, Anderson LA, Huey B, King MC. Chromosome 17q21 appears to be the locale of a gene for inherited susceptibility to breast cancer in families with early-onset disease. Genetic analysis yields a lod score (logarithm of the likelihood ratio for linkage) of 5.98 for linkage of breast cancer susceptibility to D17S74 in early-onset families and negative lod scores in families with late-onset disease. Likelihood ratios in favor of linkage heterogeneity among families ranged between 2000:1 and greater than 10(6):1 on the basis of multipoint analysis of four loci in the region.

14 Identification of the gene Science :66 A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Miki Y, Swensen J, Shattuck-Eidens D, Futreal PA, Harshman K, Tavtigian S, Liu Q, Cochran C, Bennett LM, Ding W, et al. A strong candidate for the 17q-linked BRCA1 gene, which influences susceptibility to breast and ovarian cancer, has been identified by positional cloning methods. Probable predisposing mutations have been detected in five of eight kindreds presumed to segregate BRCA1 susceptibility alleles. The mutations include an 11- base pair deletion, a 1-base pair insertion, a stop codon, a missense substitution, and an inferred regulatory mutation. The BRCA1 gene is expressed in numerous tissues, including breast and ovary, and encodes a predicted protein of 1863 amino acids. Identification of BRCA1 should facilitate early diagnosis of breast and ovarian cancer susceptibility in some individuals as well as a better understanding of breast cancer biology.

15 Linkage map of human chromosome 1 The total length of the chromosome 1 is 356 cm,, and it spans about 246 million nucleotide base pairs (exactly 245,522,847). Chromosome 1 is the largest of the human chromosomes, containing approximately 8% of all human genetic information; ; it embraces 3,141 genes. Over 350 human diseases are associated with disruptions in the sequence of this chromosome including cancers, neurological and developmental disorders, and Mendelian conditions for which many of the corresponding genes are unknown.

16 Physical mapping of genomes A further increase in mapping resolution is accomplished by manipulating cloned DNA fragments directly. Because DNA is the physical material of the genome, the procedures are generally called physical mapping. One goal of physical mapping is to identify a set of overlapping cloned fragments that together encompass an entire chromosome or an entire genome. The resulting physical map is useful in three ways: First, the genetic markers carried on the clones can be ordered and hence contribute to the overall genome mapping process. Second, when the contiguous clones have been obtained, they represent an ordered library of DNA sequences that can be exploited for future genetic analysis (for example, to correlate mutant phenotypes with disruptions of specific molecular regions). Third, these clones form the raw material that are sequenced in large-scale genome projects.

17 The Completion of Human Chromosome 1 May of 2006 saw the publication of a paper in Nature describing the finalised, annotated sequence of human chromosome 1. With this paper, the Human Genome Project was finally completed. The reference sequence for each human chromosome provides the framework for understanding genome function, variation and evolution. Here we report the finished sequence and biological annotation of human chromosome 1. Chromosome 1 is gene-dense, with 3,141 genes and 991 pseudogenes, and many coding sequences overlap. Rearrangements and mutations of chromosome 1 are prevalent in cancer and many other diseases. Patterns of sequence variation reveal signals of recent selection in specific genes that may contribute to human fitness, and also in regions where no function is evident. Fine-scale recombination occurs in hotspots of varying intensity along the sequence, and is enriched near genes. These and other studies of human biology and disease encoded within chromosome 1 are made possible with the highly accurate annotated sequence, as part of the completed set of chromosome sequences that comprise the reference human genome.