Download Dr Tim Aitman - Complex Trait

Tim Aitman
[email protected]
Genome Resources and Identification
of Complex Trait Genes
Physiological Genomics and Medicine
MRC Clinical Sciences Centre
Faculty of Medicine, Imperial College
Hammersmith Hospital
London
Identification of Genes underlying
Mendelian and Complex Traits
1980-2002
No. of Mendelian
traits
No. of Complex Traits
Mendelian traits
All complex traits
Human complex traits
1980
1985
1990
1995
2000
Glazier, Nadeau, Aitman, 2002
Genome Resources for Identifying
Complex Trait Genes
• Genome sequences
- Annotation, homology searching, comparative
mapping, SNP resources, text mining
• Omics technologies
- Transcriptional arraying, proteomics
• Congenic, isogenic, near isogenic strains
-Rodents, plants, flies
• LD and haplotype maps
- Humans, rodents
Criteria for Establishing Proof of QTL Gene
Identity in Whole-Genome Linkage Studies
• Coarse mapping with genome-wide significance
• Fine mapping
– Congenic strains, advanced intercrosses
– LD mapping
• Sequence analysis
• Functional testing of proposed sequence
nucleotide variant(s)
– In vivo or in vitro complementation
• Circumstantial evidence
– Formal proof vs. "wealth of evidence"
The Human Insulin Resistance Syndromes
(Metabolic Syndrome X)
Hypertension
Salt
retention
Diabetes
Beta cell
failure
Insulin
Resistance
Hepatic lipoprotein
overproduction
Dyslipidaemia
Predominant causes of coronary heart disease
THE SPONTANEOUSLY HYPERTENSIVE RAT:
A model of the common insulin resistance syndromes
•
•
•
•
•
•
Spontaneous hypertension
Decreased insulin action
Hyperinsulinaemia
Central obesity
Defective fatty acid metabolism
Hypertriglyceridaemia
 Intermediate phenotype: In vitro insulin and
catecholamine resistance in SHR fat cells
Combined DNA Microarrays and Linkage Analysis to
Investigate Insulin Resistance in Hypertensive Rats
QTL Plots of Chromosome 4 for
Defects in Insulin Action and Fatty
Acid Metabolism
Microarray to Detect Differential Gene
Expression between Tissues from
Affected and Control Animals
Lod
8
F2 cross
4
6
3
4
2
Backcross
+
10 cM
2
10 cM
1
0
Il6
Ae2Arb13
Mgh17 Mgh8
Wox7
Wox21 Mgh4
0
Ae2 Il6
Arb13
Mgh17 Mgh8
Wox7
Wox21 Mgh4
Aitman et al, Nature Genet 1997
Aitman et al, Nature Genet 1999
Identification of Cd36 as Rat Insulin Resistance Gene
Chromosome 4 Congenic Strain Confirms
Linkage and Defines Physical Limit of QTL to 36 cM (= 72 Mbp)
__________________________________________________________________________________________
Glucose Uptake
P = 0.0002 P = 0.01
BN
SHR SHR.4
Fatty Acid Secretion
P = 0.003 P = 0.009
BN
SHR SHR.4
Radiation Hybrid Mapping Places Cd36 in the
RADI ATI ON HYBRI D M AP OF PROXIM AL
Centre
of the
Chromosome 4 QTL
RAT CHROM OSOM E 4
~ 8cM
~8cM
Cd36
ILG6
D4Rat133
D4Rat4
D4Arb13
D4Rat139
D4Rat2
D4Rat3
Ce10
Slc4a2
D4Bro1
Nos3
D4Rat1
Ce3
D4Rat136 Psmc2
Fgl2
D4Rat5
D4Rat6
D4Rat7
D4Rat8
D4Mgh1
Cacna2
D4Rat149
D4Rat150
50cR3000
Al-Majali et al, 1999
Mamm Genome 10:471-6
cDNA Sequencing Identifies Multiple
Mutations in SHR Cd36 Coding Region
Ser160Ile
Leu161Phe
Val154Phe
Thr150Gln
Ala145Val
Asn102Ser
Lys183Glu
Val190Ile
Glu240Lys
Ser468Phe
Ala401Pro
5’
3’
G397A
G601A
C619T
“Exon
6” 6"
"Exon
C871T
G791A
C1339T
T1450C
Western Blot Analysis: Cd36 Protein is
Undetectable in Plasma Membrane/Microsomes
from SHR Adipose Tissue
Chromosomal Deletion at Cd36 Locus
Underlies Cd36 Deficiency in SHR
______________________________________________________________________________________________________________________________________________________
Normal rat Cd36 locus
I II
III
IV
V
VI VII VIII
IX X
XI XII XIII
XIV
XV
I II
Cd36
III
IV
V
VI VII VIII
IX X
XI XII XIII
XIV
XV
Cd36-ps1
I II
III
IV
V
VI VII VIII
IX X
XI XII XIII
XIV
XV
Cd36-ps2
: Promoter
I
II
III
IV
V
VI VII VIII
IX X
XI XII XIII
XIV
XV
Chimaeric Cd36 in SHR
Glazier et al, Mamm Genome, 2001
How do you prove that a candidate
gene, at the peak of linkage and with
strong biological candidacy, underlies a
QTL?
“Proof” that Cd36 underlies
SHR QTLs
•
•
•
•
Cd36 transgenic mouse (Aitman 1999)
Cd36 knockout mouse (Febbraio 1999)
Human Cd36 deficiency (Miyaoka 2001)
Cd36 transgenic SHR (Pravenec 2001)
“Proof” that Cd36 underlies
SHR QTLs
•
•
•
•
Cd36 transgenic mouse (Aitman 1999)
Cd36 knockout mouse (Febbraio 1999)
Human Cd36 deficiency (Miyaoka 2001)
Cd36 transgenic SHR (Pravenec 2001)
PLASMA LIPIDS IN Cd36 KNOCKOUT MICE
Triglycerides
NEFA
(mg/dl)
(mEq/l)
_______________________
Control
97.3 ± 5.9*
1.73 ± 0.09*
Knockout
138 ± 10.0
3.28 ± 0.12
__________________________________________
* P<0.005
Febbraio et al, 1999
J Biol Chem 274:19055
CLINICAL PROFILES OF HUMAN
CD36 DEFICIENCY
_____________________________________________
CD36 Deficiency
(N=26)
Control
(N=96)
Age (years)
64 ± 10
60 ± 7
Sex (Male, Female)
(15,11)
(68,28)
BMI (kg/m²)
23.6 ± 3.8
23.6 ± 2.7
TC (mmol/l)
5.29 ± 1.01
5.3 ± 0.83
TG (mmol/l)
2.19 ± 1.15 *
1.42 ± 0.7
HDL-C (mmol/l)
1.24 ± 0.42 *
1.61 ± 0.42
FPG (mmol/1)
6.18 ± 1.24 *
5.44 ± 1.11
Systolic BP (mmHg)
135 ± 19 *
118 ± 15
Whole Body Glucose Uptake 5.08 ± 1.4 *
8.6 ± 0.5
_______________________________________________________________
* p<0.01
Miyaoka et al 2001, Lancet 357:686
CORRECTION OF METABOLIC AND
CARDIOVASCULAR PHENOTYPES IN Cd36
TRANSGENIC SHR
______________________________________________________________________________
TG10
TG19
SHR
Plasma NEFA (mM)
0.30±0.01*
0.28 ±0.03*
0.44 ±0.04
Glucose AUC (mM.hr)
12.4 ±0.8*
11.6 ±0.4*
15.5 ±1.10
Glucose uptake (mM/g)
226 ± 16*
341 ±95*
143 ± 15
_____________________________________________________________
Values are means ± se;
*P<0.05
Pravenec et al, 2001
Nature Genet 27:156
Lines of evidence supporting
Cd36 as SHR QTL gene
•
•
•
•
Cd36 lies at peak of linkage
Linkage confirmed in congenic strains
Strong biological candidacy
Altered genomic locus with multiple coding
sequence variants
• Undetectable protein on Western blot
• Comparable phenotypes in Cd36-deficient mice
and humans
• Transgenic complementation
How successful is the combined
linkage and microarray approach?
Successful combined use of linkage
analysis and microarray-based expression
profiling
• Complex traits
– Insulin resistance (Aitman et al 1999)
– Experimental asthma (Karp 2000)
• Mendelian traits
– Sitosterolemia (Berge 2000)
– Tangier disease (Lawn 1999)
Future combined use of microarrays and
genetic linkage analysis to define
genetics of gene expression
• Genetical genomics
– Jansen & Nap 2001
• Budding yeast
– Brem et al 2002
• Maize, mouse and man
– Schadt et al 2003
– 11,021 eQTLs (of 23,574 genes on array)
with Lod > 3
Current Gene Identification Projects
• Salt-sensitive hypertension in SHR
• Pulmonary hypertension in F344
rats
• Nephrotoxic nephritis in WKY rat
24 Hour Blood Pressure Measured by Telemetry in
SHR and SHR.18 Congenic Strain
Night-time, diastolic
Night-time, systolic
210
160
SHR
140
130
SHR
200
mm Hg
mm Hg
150
190
180
170
120
SHR.18
110
160
100
SHR.18
150
80
90
100
110
120
(days of age)
80
90
1% salt solution
for drinking
SHR
SHR
190
mm Hg
mm Hg
120
(days of age)
Day-time, systolic
140
130
120
110
110
1% salt solution
for drinking
Day-time, diastolic
150
100
SHR.18
100
180
170
160
SHR.18
150
80
90
100
110
1% salt solution
for drinking
120
(days of age)
80
90
100
110
1% salt solution
for drinking
120
(days of age)
Differentially Expressed Genes and BP QTLs on Rat Chromsome 18
Rat
Chr. 18
SHR.18 Congenic interval
Jacob et al 1991, SHRSP/WKY
Garrett et al 1998, Dahl S/Lew
Cowley et al 2000, BN/Dahl S
Kovacs et al 1997, SHR/BBOK
Previously Mapped Rat
BP QTLs (95% C.l.)

D18Rat12
Adrb
*
Ttr
D18Rat32
*
Human BP QTL (Krushkal et al, 1999)
Mouse BP QTL (Wright et al, 1999)
10cM
Differentially Expressed Genes and BP QTLs on Rat Chromsome 18
Rat
Chr. 18
Genetic
Markers
DE1
SHR.18 Congenic interval
Jacob et al 1991, SHRSP/WKY
Garrett et al 1998, Dahl S/Lew
Cowley et al 2000, BN/Dahl S
Kovacs et al 1997, SHR/BBOK
Previously Mapped Rat
BP QTLs (95% C.l.)
Differentially
expressed genes

D18Rat12
Adrb
DE2
DE3
*
D18Rat32
*
10cM
Ttr
DE4
Human BP QTL (Krushkal et al, 1999)
Mouse BP QTL (Wright et al, 1999)
Differentially Expressed Genes and BP QTLs on Rat Chromsome 18
Rat
Chr. 18
Genetic
Markers
DE1
SHR.18 Congenic interval
Jacob et al 1991, SHRSP/WKY
Garrett et al 1998, Dahl S/Lew
Cowley et al 2000, BN/Dahl S
Kovacs et al 1997, SHR/BBOK
Previously Mapped Rat
BP QTLs (95% C.l.)
Differentially
expressed genes

D18Rat12
Adrb
DE2
DE3
28 bp insertion
identified in 3'
region of DE4
cDNA
*
D18Rat32
*
10cM
Ttr
DE4
Human BP QTL (Krushkal et al, 1999)
Mouse BP QTL (Wright et al, 1999)
Hypoxia-induced Pulmonary Hypertension
(Sebkhi, Wilkins, Zhao)
____________________________________________________________________________________________________________
RV weight (mg)
WKY
500
450
400
350
300
250
200
150
100
50
0
F344
F1
F2
VARWKY
VARF344
VARF1
VARF2
= 736
= 843
= 833
= 2336
VARGENETIC = 1532
Heritability = 65%
D17Rat13
D17Rat47
D17Rat46
D17Rat43
D17Rat41
D17Rat32
D17Rat15
D17Rat12
D17Rat6
D17MIT7
LOD
RV Weight QTL on Chromosome 17
7
6
5
4
3
2
1
Zhao et al 2000, Circulation
Congenic 1
Congenic 2
Minimal interval ~ 5cM
D17Rat13
D17Rat47
D17Rat46
D17Rat43
D17Rat41
D17Rat32
D17Rat15
D17Rat12
D17Rat6
D17MIT7
LOD
RV Weight QTL on Chromosome 17
7
6
5
4
3
2
1
Rat Experimental Nephrotoxic Nephritis
(Cook, Duda, Smith)
WKY Normal
H&E
WKY NTN Day 6
WKY NTN Day 28
H&E
H&E
ED1
Renal histology
Crescents (%)
100
75
50
25
0
LEW
LewxWKYWKYxLew
F1
WKY
F2
Rat NTN shows linkage to
chromosomes 13 and 16
Lod
Lod
6
6
4
4
2
2
Chromosome 13
Chromosome 16
Identification of Genes underlying
Mendelian and Complex Traits
1980-2002
No. of Mendelian
traits
No. of Complex Traits
Mendelian traits
All complex traits
Human complex traits
1980
1985
1990
1995
2000
Glazier, Nadeau, Aitman, 2002
Complex Trait Genes Identified
During 2003
• CTLA4, autoimmune disease in mice
and humans
– Ueda, Nature 423:506
• Ncf1, rat pristane-induced arthritis
– Olofsson, Nature Genet 33:25
• PHF11, Human asthma and IgE
– Zhang et al, Nature Genet 34:181
Identification of Genes underlying
Mendelian and Complex Traits
1980-2005
No. of Mendelian
traits
No. of Complex Traits
Mendelian traits
All complex traits
Human complex traits
1980
1985
1990
1995
2000
2005
Glazier, Nadeau, Aitman, 2002
Molecular lesions in genes underlying
Mendelian and complex trait genes
identified to date
Mendelian *
(n=30,900)
Missense/
nonsense
Splice
Ins/del
Regulatory
Others
58%
10%
31%
1%
<1%
* Data from Human Gene Mutation Database, Cardiff
Molecular lesions in genes underlying
Mendelian and complex trait genes
identified to date
Missense/
nonsense
Splice
Ins/del
Regulatory
Others
Mendelian *
(n=30,900)
58%
10%
31%
1%
<1%
Complex
(n=27)
55%
0%
16%
23%
6%
* Data from Human Gene Mutation Database, Cardiff
ACKNOWLEDGEMENTS
IC/Clinical Sciences Centre
SUNY
Anne Glazier
Caroline Wallace
Saira Ali
Kelly Sheehan-Rooney
Penny Norsworthy
James Scott
Nada Abumrad
Prague
Michal Pravenec
Vaclav Zidek
Vladimir Kren
Nephrology
Terry Cook
Mark Duda
Jenny Smith
San Francisco
Clinical Pharmacology
Larry Stanton
Karim Sebkhi
Martin Wilkins
Microarray Centre
Helen Causton
Ted Kurtz
Scios
Funding
MRC, Wellcome Trust
BHF, Affymetrix