Genetics of Cardiovascular Diseases

Jacques Genest MD

Cardiovascular Genetics Laboratory

McGill University Health Center

Genetics loads the gun, environment pulls the trigger

Elliott Joslin

The older I get, the better I was

A Cowboy

Age is a major cardiovascular risk factor

Framingham Heart Study

Human Biochemical Genetics 2009Genetics of Cardiovascular Diseases

General Principles Historical Aspects Monogenic Disorders Genome-wide Association (GWA)Studies Mendelian Randomization Epigenetics

Genetics of CAD

Monogenic Disorders

Candidate genes association studies

Genome-wide scan associations

Rare, extremes traits

Biased, often not replicated

Unbiased, weak clinical relevance

Watkins et al. Nature Reviews Genetics published online 07 February 2006

Genetics of CAD

Genetics and CAD

Genetics of CAD is complex.

Family Hx of premature CAD increases risk > 2.0 fold <55 for father; <65 for mother Corrected for other RF

Association weaker in INTERHEART (case ascertainment of familial CAD weaker than in FHS).

Lloyd-Jones D et al. Lancet 2004;291:2204

Genetics and Survival

0 50 100

Age

Genes

Environment

General Principles

Chromosomes and DNAChromosomes and DNA

Single Gene (Mendelian) Disorders- e.g. sickle cell disease, familial hypercholesterolemia, cystic fibrosis

Multifactorial or Complex Diseases- e.g. diabetes, asthma, heart disease

-Family/Twin/Adoption Studies

Chromosomal Disorders- e.g. trisomy 21 (Down syndrome), XO Turner syndrome

Types of Genetic DiseaseTypes of Genetic Disease

Copy Number of Variant Repeats

Nature 23 Nov 2006

Genetics of Complex Traits

Historical Aspects

Cholesterol Synthesis Pathway

Istvan E and Deisenhofer J. Science 2001;292:1160-1164.

Risk Factors for CAD

Cigarette Hypertension LDL-cholesterol (apo B) HDL-cholesterol Diabetes Age Atherosclerosis

Circulation 2000;101:111-116

Risk Factor OR Population Attributable Risk

Smoking 2.87 35.7%

Apo B/AI 3.25 49.2%

Hypertension 1.91 17.9%

Diabetes 2.37 9.9%

Abd. Obesity 1.62 20.1%

Fruits / Vegetables 0.70 13.7%

Alcohol 0.91 6.7%

Physical Activity 0.86 12.2%

INTERHEART: Conclusions

Monogenic Disorders

Genetics of Complex Traits

Case 1

• Familial Hypercholesterolemia

• Heterozygous

• Frequency 1:500 (up to 1:80 in Lac St-Jean)

• Tendinous xanthomas

• LDL-Receptor gene defect

• LDL-C 2x ULN

Case 2b

• Familial Hypercholesterolemia

• Homozygous

• Very rare (1:106)

• Premature CAD in childhood

• Extracorporeal LDL removal

Familial Hypercholesterolemia

• Most frequent genetic disorder associated with premature CAD (3-5%) of patients.

• LDL-receptor defects underlie the majority of cases

• CAD develops in men 35-55 years, in women 45-65 years.

• Respond to statins (+ bile acid binding resins) (+ intestinal cholesterol absorption inhibitors ezetimibe)

Within intestinal cells (and other body cells) some of the absorbed cholesterol is esterified to fatty acids, forming cholesteryl esters. (R = fatty acid chain)

The enzyme that catalyzes cholesterol esterification in plasma is LCAT (Lecithin:Cholesterol Acyl Transferase) and intra-

cellularly, ACAT (Acyl CoA: Cholesterol Acyl Transferase).

R C O

O

C holesteryl E ster

HO

Cholesterol

O

Cholesteryl Ester

LCAT

H 2C

HC

H 2C

O

O

O

C R 1

O

C

C R 3

OR 2

O

O C R 3

OH 2C

HC

H 2C

O

O

OH

C R 1

O

C R 2

OH 2O

triac ylg lycero l 1 ,2 -d iac ylg lyce ro l fa tty ac id

Lipoprotein Lipase

Triglycerides

Phospholipids

CH2

O

O=P-O

O

CH2-CH-CH2

O O

O=C C=O

R2

CH3-N-CH3

CH2

CH3

R1

Choline

Phosphate

Glycerol

Acyl Chains(Fatty acids)

Phospholipid

Cholesteryl ester

Apolipoprotein

Triglyceride

Cholesterol

CHYLOMICRONRENNANTS

VLDL

IDL

LDL

HDL2

HDL3

0.95-

1.006-

1.02-

1.06-

1.10-

1.20-

Den

sity

(g

/ml)

Diameter (nm)

5 10 20 40 60 80 1000

Intestine

Liver

Lipoprotein Metabolism LDL-R

EndogenousPathway

FFA

VLDL

ApoA-I, A-IIApoC-I, C-II, C-IIIPhospholipidsFree cholesterol

ApoA-I, A-IIApoC-I, C-II, C-IIIPhospholipidsFree cholesterol

NascentHDL

PeripheralCells

FFA

FreeCholesterol

HL

Liver

LCAT

HL SteroidogenicCells

ExogenousPathwayChylomicron

ChyloRemnant

HDL2 LDL

IDL

LPL

LPL

CETPPLTP

CE

Tg

HDL3

3Liver

HL

X

X

LDL Receptor

Cells take up LDL by receptor-mediated endocytosis.

The cholesterol in LDL is then used by cells, e.g., for synthesis of cellular membranes.

The LDL receptor was identified by M. Brown & J. Goldstein, who were awarded the Nobel prize for this achievement.

Lipoprotein assembly and secretion

Cholesterol

Fatty acids Cholesteryl esters

VLDL

Bile acids

LDL-R LDL

ApoBVLDL-RLRP

Endosome

sER

HMG CoA Red ACAT

VLDL IDL

Hepatic Cell

ApoB

ApoEApoB

ApoE

LDL-R Pathway Animation

Familial Hypercholesterolemia

LDL-R gene (19p13) (Familial Hypercholesterolemia) LDL-Receptor Defects

Apo B gene (2q23) (Familial Defective apo B) Apolipoprotein B Mutations

PCSK9 (proprotein convertase subtilisin/kexin type 9) (1p32) Autosomal Dominant Hypercholesterolemia

ARH gene (1p35-36.1) (Autosomal Recessive Hypercholesterolemia) LDL-R internalization defect

LDL Overproduction Defects (1q21)(Familial Combined Hyperlipidemia)

Molecular Causes of Familial Hypercholesterolemia (FH)

LDL-R: Primary familial hypercholesterolemia

ARH:Autosomal recessive familial Hypercholesterolemia

PCSK9:Proprotein convertase subtilisin/kexin type 9

ApoB:Familial defective Apo B

Mea

n L

DL

-C (

mm

ol/

L)

Time (years)

+ Atorvastatin

Apheresis19

92

1993

1994

1995

1996

1997

1998

400

300

200

100

Mea

n L

DL

-C (

mg

/dL

)500

1999

LDL Apheresis

Genest J. NEJM 1999;341:490

PCSK9

Life-long exposure to risk factor:

Principles of Mendelian Randomization

PCSK9 Gene Mutation(African-Americans)

Cohen J. et al. NEJM 2006;354:1264

Mendelian randomization.The effect of life-long genetic variability of risk factor (exposure) on the disease process.

HDL

High-Density Lipoproteins

Case: Tangier Disease

• Tangier Disease (Familial HDL Deficiency)

• Very rare

• Orange tonsils

• Hepatosplenomegaly

• Neuropathy

• Premature CAD

• Lymphoid tissue foam cells (incl. intestinal mucosa)

Intestine

VLDL

Liver

ApoA-I, A-IIApoC-I, C-II, C-IIIPhospholipidsFree cholesterol

ApoA-I, A-IIApoC-I, C-II, C-IIIPhospholipidsFree cholesterol

NascentHDL

PeripheralCells

FFA

FFA

FreeCholesterol

HL

Liver

LCAT

HL SteroidogenicCells

ExogenousPathway

EndogenousPathway

ChylomicronChylo

Remnant

HDL2 LDL

IDL

LPL

LPL

CETPPLTP

CE

Tg

HDL3

34

1

Lipoprotein Metabolism: HDL

Liver

HL

X

Cholesterol

Cholesteryl ester Stores

LDL-R

LDL

ApoBEndosome

sER

HMG CoA Red ACAT

ABCA1

Nascent HDL

HDL3

Lipid-free apo AI

LCAT

- COOH

NH

2-

cholesterol&

phospholipid

ATP ADP+Pi

apoA-I

ABCA1

Full-transporter Phospholipid and

(cholesterol) efflux to apoA-I

Early HDL maturation LxR/RxR

ATP ADP+Pi

HDL Biogenesis

Macrophage <5%

Intestine 20%

Liver 80%

HDL-C Mass

ABCA1

ApoAI

ABCA1

ApoAI

ABCA1

ABCG1

ApoE

ABCA1 is Essential for HDL Biogenesis

LxR/RxR

ABC Transporters and Human Disease

HUGO Gene Disease Function

ABCA1 ABC1 Tangier / FHD PL, Chol transport

ABCA3 Sufractant Deficiency PL transport

ABCA4 ABCR Stargardt Disease Rod receptors

ABCB4 MDR3 Familial Intrahepatic Cholestasis 3 Biliary PL secretion

ABCB7 ABC7 X-linked sideroblastic anemia Iron transport

ABC11 BSEP Familial intrahepatic cholestasis 2 Bile acid transport

ABCC2 MRP2 Dubin-Johnson Syndrome Biliary secretion

ABCC6 Pseudoxanthoma elasticum

ABCC7 CFTR Cystic Fibrosis Electrolyte transport

ABCC8 SUR1 Persistent hyperinsulinemic hypoglycemia Insulin secretion

ABCD1 ALD X-linked adrenoleukodystrophy Fatty acid

ABCD3 PMP70 Zellweger syndrome Peroxysome formation

ABCG5,8 Sitosterolemia Sitosterol transport

ABCB3 TAP2 HSV infection

ABCA1 Mutations

HDL-C is Highly Heritable

Studies in twins (n=9) Family studies (n=14) Heritability of HDL-C 0.24 – 0.83 Canadian data: 0.58

Peackock JM ATVB 2001;21:1823

Candidate Gene Sequence Variants in Low HDL subjects

Cohen JC et al. Science 2004;305:869• ABCA1 variants seen in ~10% of low HDL-C (p<0.001)

Frikke-Schmidt R et al. J Clin Invest. 2004;114:1343 • Genetic variation in ABCA1 contributes to HDL-C (~10%)

Alrasadi M et al. Atherosclerosis 2006 • ABCA1 mutations found in 20% of French Canadians with

HDL deficiency.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 2725 26 28 3029 31 3332 34 35 36 37 38 39 40 41 42 43 44 45 46 4847 49 503’5’ UTR UTR

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 2725 26 28 3029 31 3332 34 35 36 37 38 39 40 41 42 43 44 45 46 4847 49 503’5’ UTR UTR

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 2725 26 28 3029 31 3332 34 35 36 37 38 39 40 41 42 43 44 45 46 4847 49 503’5’ UTR UTR

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 2725 26 28 3029 31 3332 34 35 36 37 38 39 40 41 42 43 44 45 46 4847 49 503’5’ UTR UTR

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 2725 26 28 3029 31 3332 34 35 36 37 38 39 40 41 42 43 44 45 46 4847 49 503’5’ UTR UTR

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 2725 26 28 3029 31 3332 34 35 36 37 38 39 40 41 42 43 44 45 46 4847 49 503’5’ UTR UTR

ABCA1 gene variations in subjects with defective cellular lipid efflux

RDU43/45%

SBO44/62%

RPH68/71%

GOB39/58%

LBO54/66%

NL-1289/59%

R1851X42G>T R219K P312P 30G>T 17C>T 794A

14369T>C289-291CTC2522C>A

26G>A2520C>A

R219K G316G 24T>A V771M 30G>T R1587K N1800H 18T>C 794A Q2210H

8539C>T9620G>A

9996-87792C>T

9087T>A 7420T8995G>A

42G>T

238insG 14369T>CR219K P312P

270insGG616V

4152A

24T>A 30G>T R1587K 18T>C 889G>C

8995G>A

270insG 452T I883M 23G>A 30G>T

14369T>C 30G>T

1258G>Csplice site 30G>T R1587K

GTT238insG296G>C

L158L

L158L

24T>A

fs F1840L, L1869X

8995G>A9620G>A

7420T8549T8850T>G

7420T8241T>A8995G>A

9936A

8539C>T9996-8GTT

7792C>T

8995G>A9936A

9402G>A9543G>A9620G>A

coding variant Heterozygous variant

non coding variant Homozygous variant

gene defect translation affected

exon

intron

not sequenced

ABCA1 mutations found in 20% of French Canadians with HDL <5%

Intestine

Liver

EndogenousPathway

FFA

VLDL

ApoA-I, A-IIApoC-I, C-II, C-IIIPhospholipidsFree cholesterol

ApoA-I, A-IIApoC-I, C-II, C-IIIPhospholipidsFree cholesterol

NascentHDL

PeripheralCells

FFA

FreeCholesterol

HL

Liver

LCAT

HL SteroidogenicCells

ExogenousPathwayChylomicron

ChyloRemnant

HDL2 LDL

IDL

LPL

LPL

CETPPLTP

CE

Tg

HDL3

3Liver

HL

Lipoprotein Metabolism

Feces

Modulators of HDL in Humans

SR-BICellularfactors { ABCA1

ELHL

CETPLPL

PLTPs-PLA2

Extracellularfactors {

LCAT

-17.0

-12.2

- 7.1

[nm]

-9.5

ApoA-I-containinglipoproteins

-LpA-Ipre-1-LpA-I

O pre-

-17.0

-12.2

- 7.1

[nm]

-9.5

-17.0

-12.2

- 7.1

[nm]

-9.5

ApoA-I-containinglipoproteins

-LpA-Ipre-1-LpA-I

O pre-

ApoAI

SMase

Dastani Z et al. Figure 1Gene defects identified in man

Lipases

Candidate genes and HDL-C

Structural Receptor/Transport Lipases Exchange

Apo AI ABCA1 Hep Lipase (LIPC) PLTP

Apo AII NPC1 LPL CETP

Endo Lipase LCAT

S-PLA2

SMAse

The Proprotein convertase kexin/subtilisin type 5 gene (PSCK5)

affects HDL-C

PCSK5 inactivates Endothelial Lipase (EL) directly and via ANGPTL3 expression

Iatan I. Circ Cardiovasc Genet 2009 Oct

The PCSK5 gene (302 714 bp)) contains 21 exons and is located on chr 9q21.13

PCSK5 gene SNPs

SNP locations in the PCSK5 gene. Schematic representation of the human PCSK5 gene locus showing the exon structure and the location of the 19 variants (bottom panel) identified through sequencing and the 9 genetic variants associated with HDL-C (upper panel) identified by genotyping. SNPs in bold are associated with HDL-C with P<0.01. Locations are based on RefSeq NM_006200.3.

Quantitative Trait Analysis PCSK5 and HDL-C

Chromosome SNP Trait Beta P-Value  

9 rs11144782 HDL -0.07623 0.002021

9 rs11144766 HDL -0.06287 0.005061

9 rs1339246 HDL 0.05575 0.01767

9 rs1331384 HDL 0.03717 0.03825

9 rs11144688 HDL -0.05339 0.03921

9 rs11144690 HDL -0.09334 0.04026

9 rs1338746 HDL -0.03619 0.04393

9 rs4745522 HDL 0.05101 0.04487

9 rs2050833 HDL 0.04527 0.04515

9 rs11144782 TRIG 0.5019 0.04878

9 rs11144782 VLDL 0.1686 0.03858

9 rs11144782 APOB 10.72 0.02234

Iatan I et al. Figure 2

HDL2

Endothelial cells

EL Phospholipase activity

HSPG

EL PCSK5Loss of function

ANGPLT3+

_

_

HDL3

The Genetics of HDL

Genome-Wide Scans of FamiliesGenome-Wide Association Studies

Genome-Wide Scans and HDL-C

The two-point linkage results of the genome wide scan of the Canadian families with low HDL-C when allowing for heterogeneity. The alpha value for the peak marker of each chromosome is indicated for lod scores > 1.0.

Chromosome 1

0.00.51.01.52.02.53.0

263 cM

LO

D

Chromosome 2,0.59

0.00.51.01.52.02.53.0

265 cML

OD

Chromosome 3,0.30

0.00.51.01.52.02.53.0

225 cM

LO

D

Chromosome 6

0.00.51.01.52.02.53.0

194 cM

LO

D

Chromosome 7

0.00.51.01.52.02.53.0

192 cM

LO

D

Chromosome 8

0.00.51.01.52.02.53.0

160 cM

LO

D

Chromosome 9

0.00.51.01.52.02.53.0

150 cM

LO

D

Chromosome 10

0.00.51.01.52.02.53.0

182 cM

LO

D

Chromosome 11

0.00.51.01.52.02.53.0

154 cM

LO

D

Chromosome 12,0.31

0.00.51.01.52.02.53.0

173 cM

LO

D

Chromosome 13

0.00.51.01.52.02.53.0

132 cM

LO

D

Chromosome 14,0.23

0.00.51.01.52.02.53.0

126 cM

LO

D

Chromosome 15,0.62

0.0

0.51.0

1.5

2.02.5

3.0

130 cM

LO

D

Chromosome 16,0.51

0.00.51.01.52.02.53.0

131 cM

LO

D

Chromosome 17

0.0

0.5

1.0

1.5

2.0

2.5

3.0

133 cM

LO

D

Chromosome 18

0.0

0.5

1.0

1.5

2.0

2.5

3.0

118 cM

LO

D

Chromosome 19

0.0

0.5

1.0

1.5

2.0

2.5

3.0

113 cM

LO

D

Chromosome 21

0.00.51.01.52.02.53.0

57 cM

LO

D

Chromosome 5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

211 cM

LO

D

Chromosome 22

0.0

0.5

1.0

1.5

2.0

2.5

3.0

68 cM

LO

D

Chromosome 20

0.0

0.5

1.0

1.5

2.0

2.5

3.0

101 cM

LO

D

Chromosome 4,0.49

0.00.51.01.52.02.53.03.54.04.55.0

209 cM

LO

D

Figure 3

The two-point linkage results of the genome wide scan of the Canadian families with low HDL-C when allowing for heterogeneity. The alpha value for the peak marker of each chromosome is indicated for lod scores > 1.0.

Chromosome 1

0.00.51.01.52.02.53.0

263 cM

LO

D

Chromosome 2,0.59

0.00.51.01.52.02.53.0

265 cML

OD

Chromosome 3,0.30

0.00.51.01.52.02.53.0

225 cM

LO

D

Chromosome 6

0.00.51.01.52.02.53.0

194 cM

LO

D

Chromosome 7

0.00.51.01.52.02.53.0

192 cM

LO

D

Chromosome 8

0.00.51.01.52.02.53.0

160 cM

LO

D

Chromosome 9

0.00.51.01.52.02.53.0

150 cM

LO

D

Chromosome 10

0.00.51.01.52.02.53.0

182 cM

LO

D

Chromosome 11

0.00.51.01.52.02.53.0

154 cM

LO

D

Chromosome 12,0.31

0.00.51.01.52.02.53.0

173 cM

LO

D

Chromosome 13

0.00.51.01.52.02.53.0

132 cM

LO

D

Chromosome 14,0.23

0.00.51.01.52.02.53.0

126 cM

LO

D

Chromosome 15,0.62

0.0

0.51.0

1.5

2.02.5

3.0

130 cM

LO

D

Chromosome 16,0.51

0.00.51.01.52.02.53.0

131 cM

LO

D

Chromosome 17

0.0

0.5

1.0

1.5

2.0

2.5

3.0

133 cM

LO

D

Chromosome 18

0.0

0.5

1.0

1.5

2.0

2.5

3.0

118 cM

LO

D

Chromosome 19

0.0

0.5

1.0

1.5

2.0

2.5

3.0

113 cM

LO

D

Chromosome 21

0.00.51.01.52.02.53.0

57 cM

LO

D

Chromosome 5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

211 cM

LO

D

Chromosome 22

0.0

0.5

1.0

1.5

2.0

2.5

3.0

68 cM

LO

D

Chromosome 20

0.0

0.5

1.0

1.5

2.0

2.5

3.0

101 cM

LO

D

Chromosome 4,0.49

0.00.51.01.52.02.53.03.54.04.55.0

209 cM

LO

D

Figure 3

A Gene on Chromosome 16 affects HDL-C levels

WWOX: tumor suppressor gene contains 2 WW domains and a short-chain dehydrogenase domain (SRD). Expressed in Sterol producing tissues and in the liver

D8M

it12

LO

D=

3.5

MouseQTL

PSM

D7

GL

G1

FA

2HR

FW

D3

ML

KL

WD

R59

ZN

RF

1L

DH

DZ

FP

1C

TR

B1

AK

1270

04B

CA

R1

CF

DP

1B

X64

8484

CH

ST

6C

HS

T5

GA

BA

RA

PL

2A

DA

T1

KA

RS

TE

RF

2IP

CA

SP

R4(

CN

TP

)H

SR

G1(

MO

N1B

AD

AM

TS1

8N

UD

T7

KIA

A15

76C

LE

CS

F1

WW

OX

AF

4477

09M

AF

DN

CL

2BC

DY

L2

DC

13K

IAA

0431

BC

0027

01G

CS

HP

KD

1L2

BC

MO

1G

AN

CIM

P

tel

D1 6

S5 1

4

8.1

D1 6

S5 1

6

D1 6

S5 0

5

D1 6

S7 6

3

1.1 .6 10

D1 6

S3 1

07

D1 6

S3 0

95

D1 6

S3 1

06

D1 6

S3 0

66D

1 6S

3 018

D1 6

S5 0

4

D1 6

S3 0

40

D1 6

S5 0

7

D1 6

S3 0

98

2.6 1.1 1.4 0.8 1.4 0.8 3.2 0.8 3.2

18.1cM~7.8Mb

CH

ST6

LC

AT

CE

TP

D1 6

S5 0

3

D1 6

S5 1

5

DiscreteLOD=1.7QUE(D16S505)

LOD(QTL)=2.3QUE(95cM)

RegionforSNPsassociationinSLSJ(23Mb)

LOD(QTL)=2.55SLSJ(85cM)

D16

S31

40

1.87.4

25.5cM~18Mb

C

ABD

cen

1-1430581

7-27413

2-27587.2

8-1440369

5-27587.1

6-1435859

3-1200242

4-1434384

10-24292

9-1384255

54.9

75.1

77.7

80.262

.2

Figure 2

WWOX Protein

WW domains indicates a role in protein-protein interactions. WWOX binds the proline-rich domain PPxY. Highest expression detected in hormonally regulated tissues such as testis, ovary, prostate. Expression pattern and presence of SRD domain suggests a role in steroid metabolism. Wwox-/- mice testis and ovaries display impaired gene expression of key steroidogenesis enzymes.

Interrogation of several online databases show that WWOX is strongly associated with HDL-C (P = 0.0000225). [Willer 08]

HDL Biological Networks

SMPD1

HDL-C

ABCA1

Apo AI

EL

PCSK5

LCAT

LPL

sPLA2

CETPHL

PLTP

SR-B1 ABCG1

?

??

AGPL4

Apo E

WWOX

Genetics of HDL:Family Studies and Candidate Genes

Several genes account for ~25% of severe HDL deficiency (HDL-C <5th percentile) i.e.:

A genetic basis for HDL is identified in 1-2% of subjects

New genes (SMAse, PCSK5, WWOX) provide novel pathways in a complex network

J Clin Invest 2007:117:748

HDL are More Complex than Imagined

Copyright ©2007 American Society for Clinical Investigation

Vaisar, T. et al. J. Clin. Invest. 2007;117:746-756

Questions?

Second session 21 Jan 2010

Genome-Wide Association Studies(GWAS)

Katherisan S, Willer C, Nat Genet 2009

Genetics of Complex Traits

The yet identified genes together explain only a small amount of less than 10% of the HDLC variance, which leaves an enormous room for further yet to be identified genetic variants.

This might be accomplished by large population-based genome-wide meta-analyses and by deep-sequencing approaches on the identified genes.

The resulting findings will probably result in a re-drawing and extension of the involved metabolic pathways of HDLC metabolism.

Exp Gerontol 2008

Genome-Wide Scans and Quantitative traits

Broad and Lund databaseWellcome-Trust Case Control

Consortium (WTCCC)Framingham Study Database Fusion/Sardinia/FHS

Common variants at 30 loci contribute to polygenic dyslipidemiaSekar Kathiresan, Cristen J Willer et al. Nat Genet 2009;41;36

Common variants at 30 loci contribute to polygenic dyslipidemiaSekar Kathiresan, Cristen J Willer et al. Nat Genet 2009;41;36

Genes you have seen before in candidate gene approach

Common variants contribute to Lipoprotein Lipid LevelsCristen J Willer et al. Available on line (www.sph.umich.edu/csg/abecasis/public/lipids/)

Trait Chrom SNP P val Gene MAF Effect (SD)

HDL 11q12 rs174547 2x10-12 FADS1-2-3 T, C (0.33) –0.09(0.02)

HDL 16q22 rs2271293 9x10-13 LCAT G, A (0.11) +0.07 (0.03

HDL 9p22 rs471364 3x10-10 TTC39B T, C (0.12) –0.08 (0.03)

HDL 20q13 rs1800961 8x10-10 HNF4A C, T (0.03) –0.19 (0.05)

HDL 20q13 rs7679 4x10-9 PLTP T, C (0.19) –0.07 (0.02)

HDL 19p13 rs2967605 1x10-8 ANGPTL4 C, T (0.16) –0.12 (0.04)

HDL 16q13 rs173539 4x10-75 CETP C, T (0.32)c +0.25 (0.02)

HDL 8p21 rs12678919 2x10-34 LPL A, G (0.10)c +0.23 (0.03)

HDL 15q22 rs10468017 8x10-23 LIPC (HL) C, T (0.30)c +0.10 (0.02)

HDL 18q21 rs4939883 7x10-15 LIPG C, T (0.17) –0.14 (0.02)

HDL 1q23 rs964184 1x10-12 A1-C3-A4-A5 C, G (0.14)c –0.17 (0.03)

HDL 12q24 rs2338104 1x10-10 MMAB, MVK G, C (0.45) –0.07 (0.02)

HDL 9q31 rs1883025 1x10-9 ABCA1 C, T (0.26)c –0.08 (0.02)

HDL 1q42 rs4846914 4x10-8 GALNT2 A, G (0.40) –0.05 (0.02)

GWAS HDL-C TraitSekar Kathiresan, Cristen J Willer et al. Nat Genet 2009;41;36

Common variants at 14 loci contribute to HDL-CSekar Kathiresan, Cristen J Willer et al. Nat Genet 2009;41;36

Based on combined GWAS analysis of >40,000 subjects, 11% of variance of HDL-C levels can be explained.

The Genetics of HDL:Impact on HDL-C or CAD?

Monogenic Disorders Genome-wide Associations Association with CAD

Mendelian randomization is a method of using non-experimental studies to examine the causal effect of a modifiable exposure on disease by making use of measured variation in genes of known function.

Mendelian Randomization

Implies causality of a gene product (intermediary phenotype) in a disease process

Gene IP

Often used, perhaps wrongfully, to dismiss a gene or its product as a causal factor

Copenhagen Heart Study: HDL-C and CVD Risk

Frikke-Schmidt R JAMA. 2008;299(21):2524-2532.

Copenhagen Heart Study: ABCA1 Mutations and CVD Risk

Frikke-Schmidt R JAMA. 2008;299(21):2524-2532.

HDL-C Epidemiology

4.0

3.0

2.0

1.0

0.65 1.17 1.68

HDL-C (mmol/L)

CHD risk ratio 2.0

1.0

0

4.0Observations:

• Risk of CHD varies continuously and inversely with HDL-C levels.

• CHD risk decreases by 50% for each 0.52 mmol/L increase in HDL-C (unproven).

Kannel WB. AJC 1983;52,9B-12B

CHD risk ratio

4.0

3.0

2.0

1.0

0.65 1.17 1.68

HDL-C (mmol/L)

2.0

1.0

0

4.0

HDL Epidemiology: Yin and YangC

V R

isk

Apo AI Milano (▼ CV Risk)

CETP Deficiency(▲ CV Risk)

3.16

ABCA1*

*Copenhagen Heart Study** Wellington S, Genest J

ABCA1**

LCAT HL (LIPC)

Genome-Wide Associations(GWA) Studies

The Genetics of Complex traits

Genome-Wide Scans of FamiliesGenome-Wide Association Studies

Genetics of Complex Traits

Genome-Wide Scans for CAD and MI

(Lack of )Validation of Genetic Markers for CAD

Morgan T et al. JAMA 2007;297:1551

The Genetics of Continuous traits

Heritability Segregation Studies Genome-Wide Scans of Families Quantitative Trait Loci (QTL) Genome-Wide Association Studies Mouse Synthenic Regions

Phenotype Distance

Genetics of complex traits: longer phenotype distance decreases specificity

Risk Factor BiomarkersSurrogate End-Points

Mortality/Morbidity

Phenotype Distance

IMT CHD death, MIBiol. PathwaysLipid Levels

Genome-Wide Scans and CAD:Chromosome 9q21 locus

• McPherson R. Science 2007;316:1488• Helgdottir A. Science 2007;316:1491• WTCCC Nature 2007;447:661• Samani NJ. NEJM 2007;357:443

• Drinking from the fire hose –Statistical issues in genome-wide association studies: Hunter DJ. NEJM 2007;357:436

Genome-Wide Scans for CAD and MI

Samani NJ. NEJM 2007;357:443

Chromosome 9q21: which gene?

Schunkert et al. Circulation 2008

Meta-analysis of 9p21 and heart disease

Mendelian Randomization

Women’s Genome Health Study GWAS for Plasma C-Reactive Protein Level

Am J Hum Genet 2008;82:1185-1192

CRP Genetics and Outcome

Zacko et al NEJM 2008;359:1897.

Mendelian Randomization

Epigenetics

• Transcriptional regulation

• Genome stabilization

• Genomic imprinting

• X-inactivation

DNMTs

DNA MethylationDNA Methylation

• Cancer

• ICF, RETT syndrome

• Prader-Willi, Angelman, Beckwith-Wiedemann syndromes

Imprinted GenesImprinted Genes

Igf2r DMR2

7 CpGs

Snrpn DMR1

16 CpGs

Peg1/Mest Promoter & exon 1

23 CpGs

Peg3 Promoter & exon 1

18 CpGs

DNA from 400 oocytes Bisulfite genomic sequencing

The Epigenome

Nature 429:457, 2004

Imprinting DiseasesImprinting Diseases

- Angelman Syndrome

- Prader-Willi Syndrome

- Beckwith-Wiedemann Syndrome

CancerCancerInactivation of tumor

suppressor genesInactivation of

DNA repair genes

CpG island hypermethylation

Normal DNA methylationNormal DNA methylation

Global hypomethylation

Chromosome instability Retrotransposon

activation

Oncogene activation

?? ?

Adapted from Strathdee et al., Expert Reviews in Molecular Medicine (2002).

ICF SyndromeICF Syndrome

Mutation in DNMT3B

hypomethylation of centromeric

chromatin

IImmunodeficiency, CCentromeric region instability, FFacial

anomalies

Human Diseases Associated with Altered Methylation

Profiles

Human Diseases Associated with Altered Methylation

Profiles

Imprinted Genes

PAR5SNURF-SNRPN

PAR-SN

ZNF217 NDN MAGEL2

IPW

PAR1UBE3A

GABRB3

GABRA5

GABRG3

UBE3A-AS

ICZNF217-AS

Angelman and Prader-Willi syndromes locus 15q11-q13

H19INS IGF2ASCL2

KCNQ1OT1

KCNQ1CDKN1C CD81 TH

IGF2-AS

TSSC5TSSC3NAP1L4 WT1

Beckwith-Wiedemann syndrome locus 11p15.5

Loss of Maternal Methylation at SNRPN and KCNQ1OT1

Female Predominance and Transmission Distortion in the Long-QT Syndrome

Imboden M et al, NEJM 2007;355:2744-2751

Long QT syndrome caused by KCNQ1 and

KCNH2 mutationsAutosomal DominantTransmission Ratio 55% Female; 45% Male

(p=0.005 from normal) Possible genomic imprinting

EPIGENETICS and CVD

Female Predominance and Transmission Distortion in the Long-QT SyndromeImbodem M. NEJM 2006; 355:2744-2751

Long-QT syndrome, types 1 and 2, are caused by mutations in the potassium-channel genes KCNQ1 and KCNH2

http://www.mgu.har.mrc.ac.uk/research/imprinting/imprin-viewmaps.html

Human Biochemical Genetics 2009Genetics of Cardiovascular Diseases

General Principles Historical Aspects Monogenic Disorders Genome-wide Association Studies (GWA) Mendelian Randomization Epigenetics