Title: Tim Aitman t.aitman@csc.mrc.ac.uk
1Tim Aitmant.aitman_at_csc.mrc.ac.uk
- Genome Resources and Identification of Complex
Trait Genes
Physiological Genomics and Medicine MRC Clinical
Sciences Centre Faculty of Medicine, Imperial
College Hammersmith Hospital London
2Identification of Genes underlying Mendelian and
Complex Traits 1980-2002
1980
1985
1990
1995
2000
Glazier, Nadeau, Aitman, 2002
3Genome 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
4Criteria 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"
5 The Human Insulin Resistance Syndromes(Metabolic
Syndrome X)
Predominant causes of coronary heart disease
6THE SPONTANEOUSLY HYPERTENSIVE RATA 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
7Combined 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
4
F2 cross
Backcross
6
3
4
2
2
1
0
0
Il6
Il6
Ae2
Ae2
Wox7
Wox7
Arb13
Mgh4
Arb13
Mgh4
Wox21
Wox21
Aitman et al, Nature Genet 1997
Aitman et al, Nature Genet 1999
Identification of Cd36 as Rat Insulin Resistance
Gene
8Chromosome 4 Congenic Strain Confirms Linkage and
Defines Physical Limit of QTL - to 36 cM ( 72
Mbp)_____________________________________________
_____________________________________________
Fatty Acid Secretion
Glucose Uptake
BN SHR SHR.4
BN SHR SHR.4
9Radiation Hybrid Mapping Places Cd36 in the
Centre of the Chromosome 4 QTL
10cDNA Sequencing Identifies Multiple Mutations in
SHR Cd36 Coding Region
Lys183Glu
Val154Phe
Thr150Gln
Val190Ile
Ser468Phe
Glu240Lys
Ala145Val
Ala401Pro
Asn102Ser
5
3
G397A
T1450C
G601A
C871T
C1339T
G791A
C619T
Exon 6
"Exon 6"
11Western Blot Analysis Cd36 Protein is
Undetectable in Plasma Membrane/Microsomes from
SHR Adipose Tissue
12Chromosomal Deletion at Cd36 Locus Underlies Cd36
Deficiency in SHR ________________________________
__________________________________________________
__________________________________________________
__________________
Glazier et al, Mamm Genome, 2001
13How do you prove that a candidate gene, at the
peak of linkage and with strong biological
candidacy, underlies a QTL?
14Proof 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)
15Proof 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)
16PLASMA 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 __________________________________________
Plt0.005 Febbraio et al, 1999 J Biol
Chem 27419055
17CLINICAL PROFILES OF HUMAN CD36 DEFICIENCY
- _____________________________________________
- CD36 Deficiency Control
- (N26) (N96)
- 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 - __________________________________________________
_____________ - plt0.01 Miyaoka et al 2001, Lancet 357686
18CORRECTION OF METABOLIC AND CARDIOVASCULAR
PHENOTYPES IN Cd36 TRANSGENIC SHR
__________________________________________________
____________________________
- TG10 TG19 SHR
- Plasma NEFA (mM) 0.300.01 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 Plt0.05
Pravenec et al, 2001 Nature Genet 27156
19Lines 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
20How successful is the combined linkage and
microarray approach?
21Successful 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)
22Future 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
gt 3
23Current Gene Identification Projects
- Salt-sensitive hypertension in SHR
- Pulmonary hypertension in F344 rats
- Nephrotoxic nephritis in WKY rat
2424 Hour Blood Pressure Measured by Telemetry in
SHR and SHR.18 Congenic Strain
Night-time, diastolic
Night-time, systolic
210
160
SHR
200
SHR
150
190
140
mm Hg
mm Hg
180
130
170
120
SHR.18
SHR.18
110
160
100
150
80
90
100
110
120
90
80
100
110
120
(days of age)
(days of age)
1 salt solution for drinking
1 salt solution for drinking
Day-time, systolic
Day-time, diastolic
SHR
150
SHR
190
140
180
130
mm Hg
mm Hg
170
120
SHR.18
110
160
SHR.18
100
150
80
90
100
110
120
90
100
110
120
80
(days of age)
(days of age)
1 salt solution for drinking
1 salt solution for drinking
25Differentially Expressed Genes and BP QTLs on Rat
Chromsome 18
Rat Chr. 18
D18Rat12
Adrb
Ttr
D18Rat32
26Differentially Expressed Genes and BP QTLs on Rat
Chromsome 18
Rat Chr. 18
Differentially expressed genes
Genetic Markers
DE1
D18Rat12
Adrb
DE2
DE3
Ttr
DE4
D18Rat32
27Differentially Expressed Genes and BP QTLs on Rat
Chromsome 18
Rat Chr. 18
Differentially expressed genes
Genetic Markers
DE1
D18Rat12
Adrb
DE2
DE3
28 bp insertion identified in 3' region of DE4
cDNA
Ttr
DE4
D18Rat32
28Hypoxia-induced Pulmonary Hypertension(Sebkhi,
Wilkins, Zhao)___________________________________
__________________________________________________
_______________________
VARWKY 736 VARF344
843 VARF1 833 VARF2
2336 VARGENETIC 1532 Heritability 65
29RV Weight QTL on Chromosome 17
Zhao et al 2000, Circulation
30RV Weight QTL on Chromosome 17
31Rat Experimental Nephrotoxic Nephritis (Cook,
Duda, Smith)
WKY NTN Day 6
WKY NTN Day 28
WKY Normal
HE
HE
HE
ED1
32Renal histology
100
75
Crescents ()
50
25
0
F2
LEW
LewxWKY
WKYxLew
WKY
F1
33Rat NTN shows linkage to chromosomes 13 and 16
Lod
Lod
6
6
4
4
2
2
Chromosome 13
Chromosome 16
34Identification of Genes underlying Mendelian and
Complex Traits 1980-2002
No. of Mendelian traits
No. of Complex Traits
1980
1985
1990
1995
2000
Glazier, Nadeau, Aitman, 2002
35Complex Trait Genes Identified During 2003
- CTLA4, autoimmune disease in mice and humans
- Ueda, Nature 423506
- Ncf1, rat pristane-induced arthritis
- Olofsson, Nature Genet 3325
- PHF11, Human asthma and IgE
- Zhang et al, Nature Genet 34181
36Identification of Genes underlying Mendelian and
Complex Traits 1980-2005
No. of Mendelian traits
No. of Complex Traits
1980
1985
1990
1995
2000
2005
Glazier, Nadeau, Aitman, 2002
37Molecular lesions in genes underlying Mendelian
and complex trait genes identified to date
Missense/ nonsense Splice Ins/del Regu-latory Others
Mendelian (n30,900) 58 10 31 1 lt1
Data from Human Gene Mutation Database, Cardiff
38Molecular lesions in genes underlying Mendelian
and complex trait genes identified to date
Missense/ nonsense Splice Ins/del Regu-latory Others
Mendelian (n30,900) 58 10 31 1 lt1
Complex (n27) 55 0 16 23 6
Data from Human Gene Mutation Database, Cardiff
39ACKNOWLEDGEMENTS
- IC/Clinical Sciences Centre
- Anne Glazier
- Caroline Wallace
- Saira Ali
- Kelly Sheehan-Rooney
- Penny Norsworthy
- James Scott
-
- Nephrology
- Terry Cook
- Mark Duda
- Jenny Smith
-
- Clinical Pharmacology
- Karim Sebkhi
- Martin Wilkins
-
- Microarray Centre
- Helen Causton
SUNY Nada Abumrad Prague Michal
Pravenec Vaclav Zidek Vladimir Kren San
Francisco Ted Kurtz Scios Larry
Stanton Funding MRC, Wellcome Trust BHF,
Affymetrix
40(No Transcript)