Howard slide

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Physiological Genomics from Rats to Human
Monika Stoll, Ph.D Director, Genetic
Epidemiology of vascular disorders Leibniz-Institu
te for Arteriosclerosis Research, Münster
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Genome-oriented Medicine
Genetic Variation influences - disease
susceptibility - disease progression -
therapeutic response - unwanted drug effects
The use of genetic variation for
diagnostic purposes and targeted treatment
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Heterogeneity of complex diseases
polygenic with genetic Heterogeneity
Gene
Gene -
Epistasis
Gene
Gene
complex phenotype
Gene -
Gene
Salt intake
others
Psychosocial Stress
Diet
Environmental factors
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Gene-environment interactions and CVD
Genetic factors
Environment
Diet, Smoking, Stress
Hypertension, Diabetes, Obesity, Age, Lipids,
Genetic Background
Risk factors
Atherosclerosis
Trait
Myocardial infarction
Stroke
Peripheral vascular disease
Phenotype
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Complex Diseases do not have a clear phenotype
but may or may not share some features
Example metabolic syndrome (syndrome X)
athero-sclerosis
hypertension
vascular disease
dislipidemia
hyperglycemia
Insulin resistance
obesity
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Genetics of Multifactorial Diseases
Difficulties
Difficulties
Disease Etiology
Human Linkage Analysis
Polygenic modest effects of single
genes Incomplete penetrance Age-of-onset Environm
ental component Genetic Heterogeneity
Family studies/ Sib-Pair Analysis large number
of patients (2,500 sibpairs) Modest
resolution Multiple Genes Interaction, Epistasis
High Complexity
Lack of Power
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Genetics of Multifactorial Diseases
Solutions
Solutions
Reduction of complexity
Association studies
Comparative Maps
Positional candidate loci for high density
genotyping
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Comparative Genomics with Biology
Human
Genes and Genetic Manipulation relevant to human
disease
Genes, Physiology and Pharmacology relevant to
human disease
Mouse Rat
Ability to avoid many biological barriers unique
to one species
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Why Comparative Genomics? Take advantage of
the wealth of genome information from the various
Genome Projects Genomic regions are
evolutionary conserved between mammalian
species (Synteny) Sequence is highly conserved
between species (Homology) The genomic sequence
of human, rat and mouse genomes are
available QTLs/Genes identified in rodent models
are predictive for human loci Rodent models can
help to elucidate the function of novel disease
genes e.g. implicated by human linkage studies or
expression profiling
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Strategies for comparative genomics

• Map novel genes identified e.g. in expression
  profiling and anchor on existing comparative maps
  (www.rgd.edu/VCMap)
• Sequence positional candidate genes in mouse, rat
  and human to identify conserved mutations and/or
  regulatory elements
• Predict potential target regions for human
  linkage studies based on model organisms
• Characterize candidate genes from human studies
  in representative experimental model (inbred
  strains, congenics, transgenics, conditional
  knock-outs)

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Experimentelles Modell
Monogene Erkrankung
Geschwisterpaar-Untersuchungen Bestätigung
Kandidatengen-Locus
Assoziationsstudien Identifizierung von
Kandidatengen-Polymorphismen
(polygene) komplexe Erkrankung
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Cross design
SHR-SP
SHR or WKY
Backcross
F1
F2
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SHR or WKY
SHR-SP
x
F1
F2
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Human Chromosome Regions Implicated
in Hypertension via a Cross-Species Comparison
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Blood Pressure Phenotypes
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Rat Models for Genetic Hypertension
Spontaneously Hypertensive Rat (SHR) High blood
pressure Cardiovascular disease
SHR x WKY SHR x DNY SHR x BN
Genetically Hypertensive Rat (GH) Hypertension,
cardiac hypertrophy Vascular disease, not
salt-sensitive
GH x BN
Dahl Salt-Sensitive Rat (SS) Salt-sensitive
hypertension Hyperlipidemia, insulin resistance
SS x BN
Lyon Hypertensive Rat (LH) Mild hypertension,
hyperlipidemia
LH x LN
Fawn-hooded Hypertensive Rat (FHH) Systolic
hypertension Renal failure
FHH x ACI
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Linkage Analysis for Blood Pressure QTLs
Independent total genome scans in 7 intercrosses
representing a model for genetic hypertension
200-300 SSLP markers 10-20 cM spacing 57- 390
animals
Linkage analysis using MAPMAKER/QTL computer
package
LOD score gt2.8 suggestive LOD score gt4.3
significant
Integration of QTLs on integrated map based on
genotyping information from crosses used for
linkage analysis
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Analysis of QTL Clustering
QTL 1
QTL 2
QTL 3
Drop of 1.6 LOD units 95 confidence interval
QTL cluster
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Establishment of Syntenic Regions in Human Genome
Identification of syntenic regions and
evolutionary breakpoints using comparative maps
between rat, mouse and human
Definition of positional candidate regions in
human genome based on QTLs identified in rat
models of hypertension
Designation of first priority and second
priority regions
second priority region based on QTLs from
single rat cross
first priority region based on QTLs
from multiple rat crosses
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QTLs identified in Rat
68 blood pressure QTLs total
Baseline BP 2 Max. response 7 MAP, DBP,
SBP, PP 19 Salt MAP, DBP, SBP, PP 22 Drug
challenge 7 Delta BP 11
LOD score gt 4.3 13 LOD score 2.8-4.3 44 LOD score
2.5-2.8 11
13 QTL clusters total 7 QTL clusters 2 or more
crosses 6 QTL clusters within one cross 10 single
QTLs
Coverage of rat genome in cM 500 cM (31)
First priority regions Second priority regions
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Syntenic Regions in Human
36 syntenic regions total
Confidence level
Classification
highest 7 regions (14 QTLs) high 20 regions (38
QTLs) moderate 5 regions (10 QTLs) conversion
incomplete or impossible 6 QTLs
23 first priority regions 13 second priority
regions
Coverage of human genome in cM 800 cM (24)
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Identification of Syntenic Regions and
Evolutionary Breakpoints
RATMAP server http//ratmap.gen.gu.se Oxford
Maps http//www.well.ox.ac.uk MIT
Maps http//www.genome.wi.mit.edu/rat/
RATMAP server Mouse Genome Database http//www.inf
ormatixs.jax.org UniGene http//www.ncbi.nlm.nih.
gov/ UniGene/index.html Genome Database http//gdb
www.gdb.org
Framework comparative maps
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VC-MAP Bioinformatics-Tool for comparative
maps
Stoll et al., Genome Res. 10 473 482,
2000 http//www.genome.org/cgi/content/full/10/4/4
73 Free access Kwitek et al. Genome Res. 11
1935 1943, 2001 http//www.genome.org/cgi/conten
t/full/11/11/1935 Free access
www.rgd.mcw.edu
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Comparative Mapping
Human chr. 22 and its homologies to rat chr. 11,
20, 6, 14 and 7
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Comparative mapping of BP QTLs
D18Rat85
MBP MC5R FECH
19.0 cM
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D18Mgh3
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D18Rat9
ADRB2 DRD1 PDGFRB GRL1 FGF1 EGR1
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GJA1, D18Mit16
5q
1.5
D18Mit8
D HS - LS SBP D HS - LS MAP HS basaler DBP HS
aktiver MAP Tag 2 DBP TPM Alpha2 HS Prot
Excr HDL
16.2
D18Rat57
6.7
D18Rat18
2.5
D18Mit5
6.7
D18Mgh9
2.7
D18Mgh7
2.5
D18Mit3
1.6
Humane Homologie
D18Mit14, D18Mgh8
3.4
D18Mit1
Ratte Chr. 18
7.6
D18Mit12
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Predicted susceptibility loci in the human genome
Mouse
Rat
39,40,41,42
30,31,32,33,38 34,35,36,37
45,46,47,48
20,21,22,23, 24,25,26
51,52,53,54
27,28,29
13,14,15,16 17,18,19
Krushkal et al.
20,21,22,23, 24,25,26
13,14,15,16 17,18,19
51,52,53,54
Mansfield et al.
Chr.4
Chr.3
Stoll et al. Genome Res. 10 473 482,
2000 http//www.genome.org/cgi/content/full/10/4/4
73 Free access
Chr.2
Chr.1
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Conclusion
The regions in the human genome implicated
for hypertension may be useful as primary
targets 1. Large scale testing in human
populations Association studies TDT,
Sib-TDT Linkage studies 2. High density
mapping Targeted genome scans Single
Nucleotide Polymorphisms (SNPs)
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• Genetic studies in
• human populations

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Is there a genetic component ?
Mendelian Disease Exhibits Mendelian mode of
inheritance Complex Disease Appears to
cluster in families Family, twin, adoption
studies show greater risk to relatives of
affecteds than the population incedence Segregatio
n analysis can provide estimates of genetic
and environmental contribution to disease
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Where is the gene ?
Linkage analysis Cosegregation of mapped marker
with the disease Fine mapping to narrow the
region In Complex Disease Requires a defined
genetic model Requires classifying people as
affects and unaffecteds Allele sharing methods
(sib pairs etc.) Population association studies
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Genetic Methods
Genomwide linkage (ca. 400 Mikrosatellites, 10cM)
Traditional
Fine mapping (Saturation with Mikrosatellites,
1cM)
Association and Linkage Disequilibrium (SNPs,
3-50kB, Transmission Disequilibrium, LD,
Haplotype analysis)
Association in Case/Control Design (SNPs,
Haplotype Case/Controls, ethnically divergent
populations)
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Linkage analysis Linkage Disequilibrium
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Linkage analysis
Problem late onset of CAD
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Non-Parametric Linkage Analysis
Chromosome
LOD log10 L(?)/L(1/2)
log10 Prob. Linkage/Prob. No Linkage
m1
Disease gene
m2
See Figure 1 from Broekel et al. Nature Genetics
30, 210 - 214 (2002) http//www.nature.com/ng/jou
rnal/v30/n2/full/ng827.html Free access
m3
m4
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• Several examples for hypertension linkage
  in human study populations

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• How to get from linkage to
• the causative gene variant ?

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What is Linkage Disequilibrium ?
Linkage - property of the relative position of
loci, not their alleles. Linkage is the
cosegregation of a disease or trait with a
specific genomic region in multiple families (it
can involve any allele at the marker locus in
a given family) Association - property of
alleles a specific allele of a gene or marker
is found with a disease or trait in a
population Linkage Disequilibrium the presence
of linkage AND association Cosegregation of a
specific allele with the disease in a
significant number of families
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Why do we care about Linkage Disequilibrium ?
It is a tool for fine mapping Affected sib pair
analysis may not be sensitive enough to detect
minor genes Association test may be sensitive
but the association detected may not be due to
linkage disequilibrium. It could be caused by
population stratification (confounding due to
race, admixture, heterogeneity in the
population for some other reason)
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How do you analyze for Linkage Disequilibrium ?
Transmission Disequilibrium Test (TDT) TDT
tests for equal numbers of transmissions of
specific alleles and all others from
heterozygous parents to an affected offspring
GENEHUNTER Transmitted vs. Untransmitted
alleles TRANSMIT Expected vs. Observed
alleles TDT test is McNemars Chi-square test
(b-c)2/(bc) Trans Untrans Allele
1 211 138 Chi-square 15.27 Allele
2 138 211 p0.000093 Limitations locus
heterogeneity, allelic heterogeneity, need for
specific polymorphisms, can only detect linkage
in the presence of association, need to be
very close to disease gene
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Whats all that Fuzz about Haplotypes ?
Linkage Disequilibrium decays with time (No. of
recombinations)
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Size of Haplotype blocks depends on population
history

• L. Kruglyak (1998) need 1 SNP/3kb for genomewide
  association
• D. Reich (2001) haplotype block size in
  Caucasians 60-120kb due to
• bottle neck in population history 50,000
  years ago
• haplotype block size in Africans 10-30 kb
• M. Daly (2001) haplotype block structure in
  human genome
• 2003 haplotype structure varies. Blocks of
  long range LD
• interspersed with recombination hot spots
• Human Haplotype Map will be finished in 2005

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Hierachical Linkage Disequilibrium Mapping
See figures from Stoll et al. Nature Genetics 36
(5) 476-480, 2004 http//www.nature.com/ng/journa
l/v36/n5/index.html Subscription access only
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ALOX5AP is a susceptibility gene for MI and stroke
296 multiplex icelandic families (713
individuals) Linkage on 13q12-13 LOD score
2.86 14 additional microsatellites LOD score
2.48 (p0.0036) at D13S289 Haplotype based
case-control association using 150
microsatellites Haplotype with association to MI
(p0.00004) Gene within haplotype ALOX5AP 144
SNPs identified by resequencing 97 individuals 2
haplotype blocks in strong LD Association
testing in case/control study design
See figure from Helgadottir A. et al. Nature
Genetics 36 (3) 233-239 (2004) http//www.nature.
com/ng/journal/v36/n3/index.html Subscription
required
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ALOX5AP is a susceptibility gene for MI and stroke
See Table 1 from Helgadottir A. et al. Nature
Genetics 36 (3) 233-239 (2004) http//www.nature.
com/ng/journal/v36/n3/index.html Subscription
required
See Table 2 from Helgadottir A. et al. Nature
Genetics 36 (3) 233-239 (2004) http//www.nature.
com/ng/journal/v36/n3/index.html Subscription
required
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Conclusion
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Success stories for Comparative Genomics
Obesity Discovery of Leptin as the human
homologue of the mouse (ob) mutant Leptin
receptor and db/db mice (diabetes and obesity
phenotype) Melanocortin-4 receptor and severe
obesity in mice and man Diabetes Cd36 as a
susceptibility factor for insuline resistance in
the SHR rat Cblb (ubiquitin-protein ligase) as
susceptibility factor for Type I
Diabetes Atherosclerosis APOAI/CIII/AIV gene
cluster and lipid metabolism in mice and
man Hypertension Predictive power of QTLs from
rodents for human hypertension
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Total Genome Scan
Case-control Studies
Phenotype
Candidate Gene Approach
Positional Cloning
Transgenics Knock-outs Knock-ins
Congenics Consomics ENU-Mutagenesis
Gene