tim aitman t.aitman@csc.mrc.ac.uk genome resources and identification of complex trait genes...

Tim Aitmant.aitman@csc.mrc.ac.uk

Genome Resources and Identification of Complex Trait Genes

Physiological Genomics and MedicineMRC Clinical Sciences CentreFaculty of Medicine, Imperial CollegeHammersmith HospitalLondon

Identification of Genes underlying Mendelian and Complex Traits

1980-2002

Glazier, Nadeau, Aitman, 2002

No. of Mendelian traits

No. of Complex Traits

Mendelian traits

All complex traits

Human complex traits

1980 1985 1990 1995 2000

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"

InsulinResistance

Beta cell failure

Diabetes

Salt retention

Hypertension

Hepatic lipoprotein overproduction

Dyslipidaemia

The Human Insulin Resistance Syndromes(Metabolic Syndrome X)

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

Identification of Cd36 as Rat Insulin Resistance Gene

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

0

2

4

6

8Lod

0

1

2

3

4

Wox21Ae2

Arb13Il6

Wox7Mgh4

Mgh17 Mgh8

10 cM

Ae2Arb13Il6

Wox7Wox21Mgh4

Mgh17 Mgh8

10 cM

+

Microarray to Detect Differential Gene Expression between Tissues from

Affected and Control Animals

Aitman et al, Nature Genet 1997 Aitman et al, Nature Genet 1999

F2 cross Backcross

Chromosome 4 Congenic Strain Confirms Linkage and Defines Physical Limit of QTL -

to 36 cM (= 72 Mbp)__________________________________________________________________________________________

Fatty Acid Secretion

BN SHR SHR.4

Glucose Uptake

BN SHR SHR.4

P = 0.0002 P = 0.01 P = 0.003 P = 0.009

Radiation Hybrid Mapping Places Cd36 in the Centre of the Chromosome 4 QTL

Asn102SerAla145Val

Thr150Gln

Val154Phe

Glu240LysAla401Pro

Ser468Phe

Lys183Glu

Ser160IleLeu161Phe

Val190Ile

5’ 3’

G397A G601A

G791A

C871T C1339T T1450C

C619T

"Exon 6"

cDNA Sequencing Identifies Multiple Mutations in SHR Cd36 Coding Region

“Exon 6”

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

______________________________________________________________________________________________________________________________________________________

Cd36 Cd36-ps1

Chimaeric Cd36 in SHR

I II III IV V VI VII VIII IX X XI XII XIII XIV XV

I

Cd36-ps2

II III IV V VI VII VIII IX X XI XII XIII XIV XV II III IV V VI VII VIII IX X XI XII XIII XIV XVII II III IV V VI VII VIII IX X XI XII XIII XIV XV

: Promoter

Normal rat Cd36 locus

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, 1999J Biol Chem 274:19055

CLINICAL PROFILES OF HUMAN CD36 DEFICIENCY

_____________________________________________

CD36 Deficiency Control

(N=26) (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, 2001Nature 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

1% salt solutionfor drinking

1% salt solutionfor drinking

100

110

120

130

140

150

80 90 100 110 120(days of age)

mm

Hg

(days of age)

150

160

170

180

190

90 100 110 12080

mm

Hg

Night-time, diastolic160

100

110

120

130

140

150

80 90 100 110 120(days of age)

1% salt solutionfor drinking

mm

Hg

150

160

170

180

190

200

210

80

mm

Hg

90 100 110 120(days of age)

1% salt solutionfor drinking

Night-time, systolic

Day-time, systolicDay-time, diastolic

SHR

SHR.18

24 Hour Blood Pressure Measured by Telemetry in SHR and SHR.18 Congenic Strain

SHR

SHR.18

SHR

SHR.18

SHR

SHR.18

D18Rat32

Ttr

D18Rat12Adrb

10cM*

Human BP QTL (Krushkal et al, 1999)Mouse BP QTL (Wright et al, 1999)*

Ga

rre

tt e

t a

l 1

99

8,

Da

hl

S/L

ew

Ja

co

b e

t a

l 1

99

1,

SH

RS

P/W

KY

Co

wle

y e

t a

l 2

00

0,

BN

/Da

hl

S

Ko

va

cs

et

al

19

97

, S

HR

/BB

OK

Previously Mapped Rat BP QTLs (95% C.l.)

S

HR

.18

Co

ng

enic

in

terv

al

Rat Chr. 18

Differentially Expressed Genes and BP QTLs on Rat Chromsome 18

D18Rat32DE4

Ttr

D18Rat12Adrb

DE1

DE2

DE3

GeneticMarkers

Differentiallyexpressed genes

10cM*

Human BP QTL (Krushkal et al, 1999)Mouse BP QTL (Wright et al, 1999)*

Ga

rre

tt e

t a

l 1

99

8,

Da

hl

S/L

ew

Ja

co

b e

t a

l 1

99

1,

SH

RS

P/W

KY

Co

wle

y e

t a

l 2

00

0,

BN

/Da

hl

S

Ko

va

cs

et

al

19

97

, S

HR

/BB

OK

Previously Mapped Rat BP QTLs (95% C.l.)

S

HR

.18

Co

ng

enic

in

terv

al

Rat Chr. 18

Differentially Expressed Genes and BP QTLs on Rat Chromsome 18

D18Rat32DE4

Ttr

D18Rat12Adrb

DE1

DE2

DE3

GeneticMarkers

Differentiallyexpressed genes

10cM*

Human BP QTL (Krushkal et al, 1999)Mouse BP QTL (Wright et al, 1999)*

Ga

rre

tt e

t a

l 1

99

8,

Da

hl

S/L

ew

Ja

co

b e

t a

l 1

99

1,

SH

RS

P/W

KY

Co

wle

y e

t a

l 2

00

0,

BN

/Da

hl

S

Ko

va

cs

et

al

19

97

, S

HR

/BB

OK

Previously Mapped Rat BP QTLs (95% C.l.)

S

HR

.18

Co

ng

enic

in

terv

al

Rat Chr. 18

Differentially Expressed Genes and BP QTLs on Rat Chromsome 18

28 bp insertion identified in 3' region of DE4

cDNA

Hypoxia-induced Pulmonary HypertensionHypoxia-induced Pulmonary Hypertension(Sebkhi, Wilkins, Zhao)(Sebkhi, Wilkins, Zhao)

________________________________________________________________________________________________________________________________________________________________________________________________________________________

VARWKY = 736 VARF344 = 843VARF1 = 833VARF2 = 2336

VARGENETIC = 1532Heritability = 65%

0

50

100

150

200

250

300

350

400

450

500

RV

wei

gh

t (m

g)

WKY F344 F1 F2

RV Weight QTL on Chromosome 17RV Weight QTL on Chromosome 17

1

2

3

4

5

6

7

LO

D

D1

7R

at1

3

D1

7R

at4

7

D1

7R

at4

6

D1

7R

at3

2

D1

7R

at4

3

D1

7R

at4

1

D1

7R

at1

5

D1

7R

at1

2

D1

7M

IT7

D1

7R

at6

Zhao et al 2000, Circulation

RV Weight QTL on Chromosome 17RV Weight QTL on Chromosome 17

1

2

3

4

5

6

7

LO

D

D1

7R

at1

3

D1

7R

at4

7

D1

7R

at4

6

D1

7R

at3

2

D1

7R

at4

3

D1

7R

at4

1

D1

7R

at1

5

D1

7R

at1

2

D1

7M

IT7

D1

7R

at6

Minimal interval ~ 5cMMinimal interval ~ 5cM

Congenic 1Congenic 2

WKY NTN Day 6H&E

ED1

WKY NTN Day 28H&E

WKY NormalH&E

Rat Experimental Nephrotoxic Nephritis(Cook, Duda, Smith)

LEW LewxWKYWKYxLew WKY0

25

50

75

100

Cre

sc

ents

(%

)

F2

F1

Renal histology

Chromosome 13

Lod

2

4

6

Chromosome 16

Rat NTN shows linkage to chromosomes 13 and 16

Lod

2

4

6

No. of Mendelian traits

No. of Complex Traits

Glazier, Nadeau, Aitman, 2002

Mendelian traits

All complex traits

Human complex traits

1980 1985 1990 1995 2000

Identification of Genes underlying Mendelian and Complex Traits

1980-2002

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

No. of Mendelian traits

No. of Complex Traits

Glazier, Nadeau, Aitman, 2002

Mendelian traits

All complex traits

Human complex traits

1980 1985 1990 1995 2000 2005

Identification of Genes underlying Mendelian and Complex Traits

1980-2005

Molecular lesions in genes underlying Mendelian and complex trait genes

identified to date

Missense/

nonsense

Splice Ins/del Regu-latory

Others

Mendelian *

(n=30,900)

58% 10% 31% 1% <1%

* Data from Human Gene Mutation Database, Cardiff

Missense/

nonsense

Splice Ins/del Regu-latory

Others

Mendelian *

(n=30,900)

58% 10% 31% 1% <1%

Complex

(n=27)

55% 0% 16% 23% 6%

Molecular lesions in genes underlying Mendelian and complex trait genes

identified to date

* Data from Human Gene Mutation Database, Cardiff

ACKNOWLEDGEMENTSIC/Clinical Sciences CentreAnne GlazierCaroline WallaceSaira AliKelly Sheehan-RooneyPenny NorsworthyJames Scott

NephrologyTerry CookMark DudaJenny Smith

Clinical PharmacologyKarim SebkhiMartin Wilkins

Microarray CentreHelen Causton

SUNYNada Abumrad

PragueMichal Pravenec

Vaclav Zidek

Vladimir Kren

San FranciscoTed Kurtz

SciosLarry Stanton

FundingMRC, Wellcome Trust

BHF, Affymetrix