Transcript Document

1. Genetic mapping in humans
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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 A1 and A2; locus B, alleles B1 and B2) are
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 A1B1
or A2B2 from their father are the product of nonrecombinant sperm; persons who received
A1B2 or A2B1 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 II2.
Genetica per Scienze Naturali
a.a. 06-07 prof S. Presciuttini
2. The difficulty of building genetic maps in human
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Human genetic maps could not easily be constructed using classical
genetic mapping
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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)
Genetica per Scienze Naturali
a.a. 06-07 prof S. Presciuttini
3. The need of polymorphic markers
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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.
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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.
Genetica per Scienze Naturali
a.a. 06-07 prof S. Presciuttini
4. The development of human genetic markers
Marker type
When used
No of available loci
Blood groups and HLA
1910-1960
~20
Protein Electromorphs
1960-1975
DNA RFLPs
1975 –
<105
DNA minisatellites
1985 –
<104
DNA microsatellites
1989 –
<105
DNA SNPs
1998 –
<106
~30
Genetica per Scienze Naturali
a.a. 06-07 prof S. Presciuttini
5. A simple measure for marker polymorphism
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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
HE = 1 – Si pi2,
where pi 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.
Genetica per Scienze Naturali
a.a. 06-07 prof S. Presciuttini
6. The microsatellites
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The advent of PCR finally made mapping relatively quick and easy.
The microsatellites are mostly (CA)n repeats.
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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
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Genetica per Scienze Naturali
a.a. 06-07 prof S. Presciuttini
7. Microsatellites may be highly polymorphic
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The figure below shows a number of subjects typed with a typical
(CA)/(TG) marker (D17S800). Seven different alleles are
recognizable.
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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%
Genetica per Scienze Naturali
a.a. 06-07 prof S. Presciuttini
8. Dense human genetic maps
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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.
Genetica per Scienze Naturali
a.a. 06-07 prof S. Presciuttini
9. Detecting linkage by
microsatellites
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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.
Genetica per Scienze Naturali
a.a. 06-07 prof S. Presciuttini
10. Major steps in cloning human genes
Genetica per Scienze Naturali
a.a. 06-07 prof S. Presciuttini
11. An example of successful gene cloning: BRCA1
Genetica per Scienze Naturali
a.a. 06-07 prof S. Presciuttini
12. First evidence of linkage
Science 1990 250:1684
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.
Genetica per Scienze Naturali
a.a. 06-07 prof S. Presciuttini
13. Identification of the gene
Science 1994 266:66
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.
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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 11base 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.
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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.
Genetica per Scienze Naturali
a.a. 06-07 prof S. Presciuttini
14. Linkage map of human chromosome 1
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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.
Genetica per Scienze Naturali
a.a. 06-07 prof S. Presciuttini
15. Physical mapping of genomes
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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:
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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.
Genetica per Scienze Naturali
a.a. 06-07 prof S. Presciuttini
16. The Completion of Human Chromosome 1
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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.
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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.
Genetica per Scienze Naturali
a.a. 06-07 prof S. Presciuttini