Download Lectures 2010

GENETIC ANALYSIS
Dr Dan Bradley, Dept Genetics
LECTURE NOTES
http://www.tcd.ie/Biology_Teaching_Centre/local/senior-freshman/
1. 
2. 
3. 
4. 
Mendelian inheritance
Mapping Mendelian traits
Quantitative genetic variation
Genetics of common disease
Note in 1:
Dominance and recessivity
Ratios are consistent with 2
alleles determining a trait;
segregating independently
These are transmitted to
offspring unchanged, the
recessive appears in the F2
generation.
Each gamete receives one of
each pair of alleles
Two characters are crossed
simultaneously
Each character shows a 3:1
ratio
These assort independently –
9:3:3:1 results from two
superimposed 3:1 ratios
Bateson and Punnet
discovered departures from
9:3:3:1 in sweetpea. This was
carried further by Morgan in
Drosophilia melonogaster.
Linkage: tendency of genes
from the same chromosome to
remain together when they
enter the gamete.
Recombination The
appearance of new
combinations of alleles.
Mutation rates are higher in males
Bateson and Punnet discovered
departures from 9:3:3:1 in sweetpea.
This was carried further by Morgan in
Drosophilia melonogaster.
Linkage: tendency of genes from the
same chromosome to remain together
when they enter the gamete.
Recombination The appearance of
new combinations of alleles.
Here:
The expected ratio of 1:1:1:1
(under independent assortment in a
backcross) was not found. There is
no independence and the
characters are somehow linked.
Parental combination of alleles
appear much more frequently than
non-parentals
(1339+1195)/2839 = 89%
i.e. 89% linkage, with recombinant
(non-parental) types at 11%.
Each type of non-parental appears
at equal frequency
Sturtevants X chromosome map
1911
•  Genes assort into
linkage groups
•  These were in linear
arrays, discernible
using linkage mapping
•  Confirmed
chromosome theory of
inheritance
•  More powerful 3 point
cross. Can see double
crossovers.
•  % recombn does not equal
map units exactly because
of double cross overs. •  0.5 is maximum
recombination.
The pattern of transmission in a
pedigree indicates the mode of
inheritance:
Dominant (e.g. Huntingdon disease.
The affected individual usually
has at least one affected parent.
The disease may be transmitted
by either sex. Usually ~50% of
siblings have the disease.
Irish retinitis pigmentosa pedigree
Autosomal dominant form
Recessive (e.g. cystic fibrosis).
Affected people are usually born
to unaffected parents who are
both carriers. Children in these
matings have a 25% chance of
being affected.
X-linked (e.g. muscular dystrophy)
Usually males are affected. The
mother is an unaffected carrier.
50% of male children are affected
and there is no male-male
transmission.
An X linked recessive
Gene -Haemophilia
Retinitis pigmentosa
(recessive form)
Consanguineous pedigree
Not always the case for
recessive disorders!
2
Markers – blood is the only accessible
human organ and yields only a limited
number of proteins.
Human Gene Mapping
With experimental organisms
we can
-generate a large number of
markers by inducing
mutations
-design special crosses and
generate large numbers of
progeny
Cannot do either in humans
so there is a need for both
new types of markers and
special methods for analyzing
the sparse data from complex
human pedigrees.
..TCGA…
..AGCT..
..TCCA…
..AGGT..
A medium density map only emerged
in the 1980s when polymorphisms
started to be assayed directly in the
DNA sequence, primarily from
restriction fragment length
polymorphisms (RFLPs).
Restriction enzymes only recognize
specific DNA sequences, and will not
cut if these are mutated. There are
many, thousands of single base pair
polymorphisms in the human genome
which may be assayed by the cutting
(or not) of enzymes at particular sites.
These are visualized using a technique
called Southern blotting.
Now there are 9 million Single
nucleotide polymorphisms (SNPs)
described from the human genome
project
Analysis – this concentrates
on two major types of
analysis –
(i) 
(ii) 
Pedigree with segregation of a dominant disease
analysis of the
segregations of markers
and traits in pedigrees.
i.e. does a marker allele
show a pattern of
transmission which is
similar to that of a trait?
Analysis of coincidence
of marker alleles and
traits within populations association
11
22
Marker with alleles 1 and 2
12
22
*
12
22
12
22
22
If linked the disease should co-segregate with allele 1 and the
healthy allele with allele 2. Of 5 offspring, four show linkage and
one is recombinant (*).
0.5
0.25
0.25
0.25
0.25
0.4
0.30
0.30
0.20
0.20
0.3
0.35
0.35
0.15
0.15
0.2
0.40
0.40
0.10
0.10
This approach is good for
whole genome scans (using
~400 markers); it requires a
defined mode of
inheritance: resolution is
limited to the extent that
recombinations occur within
the life of a pedigree, and is
typically to a substantial
fraction of a chromosome.
The test statistic for linkage
analysis is the LOD score.
Probability of each chromosome type
Theta
1 AFFECTED
2 UNAFFECTED
2 AFFECTED
1 UNAFFECTED
(i) Linkage mapping in
pedigrees
Affected individuals:
Filled symbols
0.1
0.45
0.45
0.05
0.05
This is based on the log of
the ratio of the probability of a
segregation occurring under
the assumption of linkage
and the probability of it
occurring under the
assumption of independent
segregation.
P
R
Probability of observed segregation at theta=
0.5 = 0.25 X 0.25 X 0.25 X 0.25 X 0.25 X B (0.001)
More simply, an indication of
the distance between a trait
gene and a marker is given
by the fraction of recombinant
chromosomes (here 1/5 or
20%). The certainty about
the result depends on how
extensive the pedigree is and
how tight the linkage is.
0.2 = 0.40 X 0.40 X 0.40 X 0.10 X 0.40 X B (0.0026)
B=5 (combinations with 1 rec, 4 parental)
Probability of Probability
parental type Segregation odds ratio
0.450
0.002
2.10
0.400
0.003
2.62
0.350
0.002
2.30
0.300
0.002
1.66
0.250
0.001
1.00
Association mapping:
the coincidence of trait and marker allele in populations
Many diseases (eg heart
disease, stroke, several
cancers, some psychiatric
disorders) have some genetic
component but as this is an
influence, not a determinant,
their segregation in pedigrees
is complex.
These diseases arise from
the interaction of multiple
factors, and multiple genes.
Linkage analysis is not very
useful for their mapping.
11
12
12
22
12
12
22
11
12
11
12
12
12
12
Test: when a population is divided by a trait (eg disease status), are the
frequencies of marker alleles different on either side of the divide?
Association mapping.
Resolution is high, about 1
cM but coverage is low.
Therefore it is a good
technique for candidate gene
testing, i.e testing genes
which are already suspected
of involvement in a trait.
Only recently becoming
useful for whole genome
searches. An advantage is
that it does not presume a
particular pattern of
transmission; the type of
inheritance can be complex,
eg with complex traits such
as heart disease.
Disease
status
HLA-Bw53
For example, in a survey of
children in the Gambia, West
Africa, who were infected
with malaria (Adrian Hill,
Oxford, 1991), the following
genotypes were observed at
the human leucocyte antigen
class I locus.
3
Why do alleles at neighbouring loci associate?
(ie why is there “linkage disequilibrium”)
A +
A +
a +
a +
a +
16.9%
Severe
25.4%
a
A +
a
A +
a +
a
a +
A +
Descendent mutant chromosomes in same
Background, including marker allele “a”
a
A +
a +
a +
Many generations
A +
A +
a
A +
a
A +
a
A +
a
A +
a
a +
a
a +
Can test with chi-squared test
of association (p value gives
strength of evidence;
p=0.008)
a +
Few generations
A +
Mild
Chromosomes in an ancestral population
Mutation at disease locus.
a +
A +
A +
A +
There was a significant
association between allele
HLA-Bw53 and susceptibility
to the disease. Note that this
suggests a role for this gene
in the disease progression,
not that it determines the
trait.
A +
a +
A +
a
a +
A
*
Descendent mutant chromosomes in same
background, now reduced by recombinations
between ancestral chromosomes. Marker allele
“a” remains associated with the mutation,
perhaps with some disruption by
recombination (*) depending on time since
mutation and distance between marker and
locus.
Notes about association between traits and genetic markers
• 
Association can occur between
neighbouring makers and the trait, not
just the causative mutation itself
Association declines with:
-  time since the mutation occurred on the
specific genetic background
-  map distance between markers and
causative variant
-  If influence of variant on trait is weak
or swamped by other factors
-  If the mutation in the gene which causes
the trait is one of a number of mutations
of similar effect
An example of gene
identification by gene
mapping.
..TCGA…
..AGCT..
..TCCA…
..AGGT..
Cystic fibrosis. This is a
genetic disease which has its
world maximum prevalence
in Ireland with 1/1800 births.
It is recessive and involves
chronic bacterial infection,
inflammation of the lungs and
high electrolyte level in
sweat.
The gene, which was
unknown was known to
influence chloride ion
transport and mucus
consistency on epithelial
surfaces. This was the first
gene which was identified
through linkage analysis.
• 
Populations which are recent
mixtures of different ancestry
require care; you could get a
significant association between
blood group O and several different
typical Irish traits if you sampled
randomly in New York!
a) Cut DNA with
restriction
enzyme, eg TaqI
b) Fragment size
separation by gel
electrophoresis.
Fragment size is
determined by
cut/not cut of
restriction
enzyme.
Individuals:
1
2
3
First, linkage analysis using
markers spread around the
genome in many nuclear
family pedigrees gave a
region on chromosome 7
which was linked to the gene.
This was bounded by
recombinations with two
markers: MET and D7S8.
c) Visualisation of
polymoprhism at
particular gene by
radioactive probing
with specific marker
DNA. Technique
used is Southern
blotting.
Allelic
association
Then, researchers generated
new markers within the this
500 Kb segment and tested
these for association with the
disease.
0.6
0.3
0.0
3 RFLPs which
showed strongest
association, all within
new CFTR gene, close
to a 3bp deletion.
Kerem et al Science 1989 245:1073-80
Sequencing of this gene
revealed that it had deletions
of 3 bp in some patients and
analysis showed that it was
involved in transmembrane
ion transport.
The association was
strongest around a specific
new gene.
(this methodology is more
successful with recessive
diseases, dominant diseases
are more likely to be
heterogenous in causal
mutations)
It was called the cystic
fibrosis transmembrane
conductance regulator
(CFTR).
Detection of the delta F 308 mutation by
oligo hybridisation
It is only present in two copies in
affected offspring, never in carrier
parents. It is common, but not the only
CF mutation.
Its distribution is primarily western
European.
There is some evidence from the
gene product’s involvement in ion
transport and water retention that it
may have been subject to natural
selection in the past, with
heterozygote advantage causing the
high incidence of disease alleles in
the present. Resistance to typhoid is
one possibility (cells expressing wt
CFTR internalise more S. typhi than
those with two copies of the common
mutant).
There is some hope for genetic
therapy, but development of this is a
slow process (especially for
sufferers!). In principle an insertion of
a wild type gene could correct the
chloride conductance defect.
Mendelian mapping
•  Over a decade, 1200 genes causing human disease
or traits identified
•  Mendelian traits are primarily associated with
alterations to coding sequence of proteins.
•  Relationship between severity of amino acid
replacement and clinical severity eg Duchenne’s
(frame-shift deletions) and Becker’s (in-frame
changes) muscular dystrophy
•  ‘simple’ Mendelian traits are not always so simple 68% of all chromosomes with
a copy of the gene causing
the disease show this same
3bp deletion.