Genomevariationamongindividualsandmolecular

analysisoftraits(Chpt.11)
DNAvariationallowsdirectdetectionofgenotype
SNPs
InDels
Microsatellite
CNV
DetectionofallelicDNApolymorphisms.
Positionalcloningofsinglegenetraits
Geneticheterogeneity
GenomeWideAssociationStudies(GWAS)
283
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Pairwise comparison of three
personal genomes
Single nucleotide polymorphisms in the genomes of three individuals [(Craig
Venter, James Watson, and a Chinese man (anonymous, YH)]
2
Fig. 11.2
Differences across
the entire genome
Amino acid-changing
substitutions
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Categories of genetic variants
Five categories based on size, frequency within individual
genomes, and method used for detection
3
Table 11.1
284
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SNP:singlenucleotidepolymorphisms
SNPs in a 400 kb region of human chromosome 7 that contains the CFTR gene
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Restriction site-altering SNPs
detected by Southern blots
In this example, the SNP affects an EcoRI restriction site
Allele 1 has an EcoRI site that is not present in allele 2
After digestion with EcoRI, the two SNP alleles produce different-sized DNA
fragments
5
Fig. 11.4
289
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SNPdetectionbyPCR
Musthavesequenceoneither
sideofpolymorphism
Amplifyfragment
Exposetorestrictionenzyme
Gelelectrophoresis
e.g.,sicklecellgenotypingwitha
PCRbasedprotocol
Fig. 11.5
290
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SNPdetectionbyASO
Veryshortprobes(<21bp)specificwhichhybridizetoonealleleorother
Suchprobesareallelespecificoligonucleotides(ASOs)
Fig. 11.8
291
Fig. 11.6
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8
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SSRs are highly polymorphic because of their
potential for faulty replication
Alteration of a 15 (CA) repeat allele to a 17 (CA) repeat allele
9
Fig. 11.9
(c) continued
(d)
(e)
(a)
(b)
(c)
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Example of a population with three SSR alleles detected by PCR and
gel electrophoresis
10
Fig. 11.10d
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Mutations at the Huntington disease (HD) locus are
caused by expansion of an SSR in a coding region
Autosomal dominant disorder
Normal allele has < 34 CAG
repeats
Disease-causing alleles have 42
or more CAG repeats
11
Fig. 11.11
Triplet repeat expansions
in different genes also
cause some other
neurological disorders
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Deletions or duplications of a DNA region
Minisatellites repeat unit sizes are 500 bp to 20 kb
Repeat sequences occur at multiple genomic loci
Detected using restriction digests and hybridization of Southern blots with cross-
hybridizing minisatellite probe
Ideal for DNA fingerprinting
Copy number variants (CNVs) large blocks of duplication
or deletion with population frequency of < 1%
Copy number polymorphisms (CNPs) if frequency is > 1%
Can affect large blocks (up to 1 Mb) of DNA without having any phenotypic
consequences
Detected on arrays (increase or decrease in hybridization)
12
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Minisatellite
analysis
Fig. 11.12
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DNA fingerprint analysis confirmed
that Dolly was cloned from an adult
udder cell
Using DNA fingerprinting in
forensics:
Men accused of rape
Since 1993, > 150 men
imprisoned for rape have been
released from jail because of DNA
fingerprint analysis
Plant DNA as murder evidence
Identification of skeletal remains
14
Fig. 11.13
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Chromosomal locations of CNPs or CNVs identified in
multiple individuals
Results of DNA microarray analysis of 88 samples
Array has several hundred thousand non-polymorphic oligonucleotide probes
(NPOs) spaced evenly across entire genome
15
Fig. 11.14a
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CNVs in the olfactory receptor (OR) family
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Huntington disease (HD) was the first human disease
gene to be mapped by positional cloning
Detection of linkage between the DNA marker G8 and the
HD locus
Segregation of the G8 DNA marker (four alleles - A, B, C, and D) in a
large Venezuelan pedigree affected with HD
19
Fig. 11.18
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PositionalCloning
Findextendedfamiliesinwhichdiseaseis
segregating
Usepanelofpolymorphicmarkersspacedat10cM
intervalsacrossallchromosomes
300markerstotal
Determinegenotypeforallindividualsinfamiliesfor
eachDNAmarker
Lookforlinkagebetweenamarkeranddisease
phenotype
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Onceregionof
chromosomeis
identified,ahigh
resolutionmappingis
performedwith
additionalmarkersto
narrowdownregion
wheregenemaylie
Fig. 11.17
300
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LogofOdds(LOD)Scores
LogofRatiooftwoprobabilities
P(L)=probabilityoflinkage
P(NL)=probabilityofnolinkage
Odds=P(L)/P(NL)
ByusinglogofOddswecanaddeachfamilytoobtain
overallLODscore.
Tobeacceptedaslinked,LODScoremustbeover3(=
1000Linked/1notlinked)
301
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302
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Maximum Likelihood of Odds Ratio (pL/pNL) for u values from 0 to 0.5
u
0.0 0.1 0.2 0.3 0.4 0.5 0.6
O
d
d
s

R
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t
i
o

p
L
/
p
N
L
0
2
4
6
8
10
12
14
( )
8
7
25 . 0
2 2
) 1 ( u u

|
.
|

\
|

= z
303
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HowtoidentifyDNAsequencethatcausesphenotype?
Candidategenesfrommapposition
Correlateexpressionwithtargettissueororgan
Correlatemutationswithphenotype
Transgenicexperimentstoshowgainoffunctionor
complementation.
Knockoutgenetoshowsimilarphenotypeinmodel
organism
304
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Positionalcloning Step2
identifyingcandidategenes
Onceregionofchromosomehasbeennarroweddownbylinkageanalysis
to1000kborless,allgeneswithinthisregionareidentified.
Candidategenes
Usuallyabout17genesper1000kbfragment
Identifycodingregions
Computationalanalysistoidentifyconservedsequencesbetween
species
Computationalanalysistoidentifyexonlikesequencesbylooking
forcodonusage,ORFs,andsplicesites
AppearanceononeormoreESTclonesderivedfromcDNA
305
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NorthernblotexampleshowingSRY candidatefortestesdeterminingfactoris
expressedintestes,butnotlung,ovary,orkidney
Fig. 11.20
308
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Transgenicanalysiscanprovecandidategeneis
diseaselocus
310
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Example:PositionalCloningofCysticFibrosisGene
LinkageanalysisplacesCFonchromosome7
311
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EveryCFpatienthasamutatedalleleoftheCFTRgeneonboth
chromosome7homologs.Locationandnumberofmutations
indicatedunderdiagramofchromosome
313
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ProvingCFTRistherightgene
Diseasephenotypeeliminatesgenefunction
Cannotusetransgenictechnology
InsteadperformCFTRgeneknockoutinmousetoexamine
phenotypewithoutCRFTgene
Targetedmutagenesis
IntroducemutantCFTRintomouseembryoniccellsin
culture
Raredoublerecombinanteventswithhomologouswildtype
CFTRgeneareselectedfor
Mutantcellisintroducedintonormalmouseembryoswhere
theyincorporateintogermline
Knockoutmousecreated
315
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Fig.E.14acMouseModelforCysticFibrosisbyKnockingOutCFTRGene
316
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Fig.E.14de
317
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Fig.E.14fGeneratingChimericMousewhich
containscellswithCFTRknockoutgene
318
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Fig.E.14ghBreedingHeterozygousCFTRKnockoutmicetogeneratinghomozygoteslackingwildtype
CFTRgene
319
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Incomplete penetrance and genetic heterogeneity in the
inheritance of breast cancer
37
Fig. 11.19
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Genome-wide association studies (GWAS)
A GWAS doesn't depend on traditional pedigree analysis
Thousands of individuals make up a study group
Each individual is observed or tested for expression of one or more traits
of interest
DNA microarrays are used to obtain whole-genome profiles for each
member of the study population
Genotypes at each tag SNP are tested for association with
each trait
Only a small number of tag SNPs will show a significant association with a
trait
Reveals genomic regions that harbor alleles associated with the trait
38
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GWAS of body mass index (BMI)
P values for all SNPs tested for association with BMI
across all chromosomes
Each dot represents a single SNP test
Lowest P values are shown as the highest dots
39
Fig. 11.20a
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GWAS of BMI (cont)
Fine-scale mapping of two BMI-associated regions
40
Fig. 11.20b
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Published GWA Reports, 2005 6/2012
T
o
t
a
l

N
u
m
b
e
r
o
f

P
u
b
l
i
c
a
t
i
o
n
s
Calendar Quarter
Through 6/30/12 postings
0
200
400
600
800
1000
1200
1400
2005 2006 2007 2008 2009 2010 2011 2012
1350
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NHGRI GWA Catalog
www.genome.gov/GWAStudies
www.ebi.ac.uk/fgpt/gwas/
Published Genome-Wide Associations through 12/2012
Published GWA at p5X10
-8
for 17 trait categories
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320
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