CCR2 variant and cardiovascular outcome in a general population-based cohort

Mike Zuurman, PhDInternal Medicine/Nephrology UMCG, GroningenThe NetherlandsBreedtestrategie

Breedtestrategie

Complex Trait analysis in man:

Genetic basis of atherosclerotic end-organ damage

Breedtestrategie

Interdisciplinary strategy

Clinical cardiology

Clinical nephrology

Functional nephrology

Endocrinology Epidemiology

GeneticsMolecular biology

Genomics

Breedtestrategie

Three post-doc positions:

1.Genetic Epidemiology2.Genetic molecular biology3.Bioinformatics

Project duration: 4 years

QTL analysis / Mutagenesis

candidate genes

Humanpopulations

functionalstudies

Genetic basis of atherosclerotic organ damage:

From animal genetics to human cohorts

functionalstudies

atherosclerosisrenal damage

HDL cholesterolPLTP activity

Principle of QTL analysis (2)

Principle of QTL analysis (3)

Advantages of QTL analysis

QTL analysis allows identification of novel genes involved in the phenotype.

QTL mapping is more likely to find mutations in rate limiting or regulatory genes, which will be very important therapeutic targets.

P

PG

G G

EF

Environment

Individual

EF

Complex Traits Diagram

FP

EF

EF

1

1. env. response to phenotype (treatment), 2. env. influence on gene expression, 3. env. inducing internal phenotype, 4. env. influencing expression of unknown gene, 5. phenotypes combine to one detectable phenotype, 6. phenotype is only partly surfacing, 7. internal feedback mechanisms 8. unidentified gene influencing detectable phenotype, 9. un- visible/detectable/known phenotype 10. gene-phenotype-phenotype-gene interaction

2

3

4

P FP P+ + = trait

5

6

7

P9

= intermediate phenotype

10

Research Flow

Genetic epidemiology -Patient cohorts-General population-Sequence variants

Genetic determinants of Atherosclerotic (End-Stage) disease in man

Functional analyses-In vitro/In vivo-Molecular-Fundamental

Bioinformastatistica-Utilization of tools-Development of tools-Solutions

Genetical epidemiology

•Cohorts

- PREVEND (8592/40000) - NECOSAD (1000)- Zwolle Diabetes (1200)-a.o.

•Sequence variants

-Single nucleotide polymorphisms-Haplotypes-Tandem repeats-Insertion/deletion

•Cross sectional/prospective

-Major adverse cardiovascular events-Microalbuminuria -Renal dysfunction

Genetical epidemiology

•Cohorts

- PREVEND (8592/40000)

•Sequence variants

-Single nucleotide polymorphisms

•Cross sectional/prospective

-Major adverse cardiovascular events

Confounders

-associated with exposure-related to the outcome-not part of the causal pathway

Mother highly educated Child Down syndrome

Alcohol intake Lung cancer

Blood pressure UAE

Age

Smoking

Gender

PREVEND design

n=85.421 invited for pre-screening

n=40.856 responded (47.8%)

UAC < 10 mg/L

n= 30.890UAC >= 10 mg/L

n= 9.966

Exclusion -pregnancy -insuline use -No informed consent

n= 3.395 invited for screening 1n= 2.592 responded (76.3%)

n= 7.768 invited for screening 1n= 6.000 responded (77.2%)

n= 8.592screening 1 1997-98

Chemokines in pathology: atherosclerosis

CCR2

•Ligand: CCL2 (MCP-1)•Gi-protein coupled

- Inhibits cAMP•Seven-transmembrane spanning•Hetero/Homo di- and multimers•Two splice variants (A and B)

AboutBasic

•Intracellular calcium•Cellular migration (chemotaxis)•Pro-inflammatory•Atherogenesis•Angiogenesis

Functional

CCR2 p. V64I: a single nucleotide polymorphism (SNP)

Gives rise to a valine to isoleucine substitution

Nakayama et al.,AIDS 2004 Mar 26;18(5):729-38.

CCR2 p. V64I and cardiovascular outcome

• 64I is associated with reduced coronary artery calcification

• 64I is associated with higher prevalence of myocardial infarction

CCR2 p. V64I and receptor function

Nakayama et al.(2004): CCR2A not CCR2B is altered

• Increased surface expression of CCR2A

• More stable expression of CCR2A

• Increased down-regulation of CCR5

Research question

Is risk of cardiovascular events modified by CCR2 V64I genotype?

Framingham risk score

Calculated using:

•Sex•Age categories •Total cholesterol levels•HDL levels•Smoking status•(Treated) Systolic blood pressure

Indicates:

•10-year risk assessment for CHD/CVD •Atherosclerotic risk

PREVEND CCR2-V64I encoding allele frequencies

CCR2 p. V64I Frequency ∑ χ2 p

V 0.91 0.13 0.72

I 0.09

∑ χ2: cumulative chi-square value for all genotypes (VV, VI and II).

CCR2 p. V64I N FrequencyVV 6343 0,84

VI 1177 0,16

II 57 0,01

7577

Population :

-Caucasian-No missing genotype

Baseline characteristics VI + II VV Age (years) 49,39 ± 12,73 49,56 ± 12,69 Sex (Male %) 50,57 49,14 BMI 26,14 ± 4,06 26,09 ± 4,24 Obesity (BMI > 30) 15,78 15,53 Blood Pressure (%) <120/<80 36,01 37,09 120-139/80-89 35,93 35,99 140-159/90-99 19,55 18,97 >=160/>=100 8,52 7,95 HDL (mmol/L) 1,3 ± 0,39 1,33 ± 0,4 Triglicerides (mmol/L) 1,49 ± 1,24 1,41 ± 0,95 Cholesterol (mmol/L) 5,62 ± 1,14 5,67 ± 1,12 ACD (%) 5,71 6,34 CVA history (%) 0,74 0,67 AHD (%) 13,25 11,75 MI history (%) 3,4 3,24 CRP (mmol/L) 1,38 (0,59-2,98) 1,27 (0,56-3,01) CrCl (ml/min/1,73m**2) 93,19 ± 21,14 92,17 ± 20,68 UAE (mg/24h) 9,7 (6,58-17,44) 9,4 (6,31-18,15) Smoking (%) 39,14 37,4 Diabetes Mellitus Type II (%) 3,55 3,65 Framingham score (per 10% risk) 0-10 67,32 68,31 10-20 18,8 18,9 20-30 9,91 9,59 30+ 3,98 3,2

**

VI + II VV

Major Adverse Cardiovascular Event N % N %

No Event 1167 94,57 6026 95,0

Major Adverse Cardiovascular Event 67 5,42 317 4,99

PREVEND MACE by CCR2 p. V64I

Detailed N % N %

Mycocardinal Infarction 20 30,76923 80 26,75585

Ischemic Heart Disease 15 23,07692 68 22,74247

CABG 2 3,076923 18 6,020067

PTCA 4 6,153846 35 11,70569

Subarachn Hemorrhage 0 0 5 1,672241

Intracerebrale Hemorrhage 3 4,615385 7 2,341137

Other and unspecified Intracranial Hemorrhage 1 1,538462 2 0,668896

Occlusion and Stenosis of Precerebral Arteries 2 3,076923 11 3,67893

Occlusion and Cerebral Arteries 6 9,230769 27 9,0301

Carotis Desobstructie 2 3,076923 2 0,668896

Aorta Peripheral Bypass Surgery 2 3,076923 17 5,685619

PTFA 3 4,615385 14 4,682274

MACE: Framingham risk range by CCR2 SNP

0

1

2

3

4

5

6

7

0-10 10-20 20-30 30+Framingham Risk range

O/E

MA

CE

II + IVVV

Multivariate model : P = 0.008 for interaction genotype and FRR

B Wald Sig. RR

Framingham (>30%) *CCR2 p. VV -1,00 8,35 <0.01 0,37

CCR2 p. V64I and Framingham high risk (>30%)

0

10

20

30

40

No YesFramingham high risk

% M

AC

E

VVVI + II

0

5

10

15

20

25

30

0 1 2 3 4 5 6Framingham SBP score

% M

AC

E

VI+II (Men)VV (Men)VI+II (Women)VV (Women)

MACE: Framingham SBP score by CCR2 SNP

Prevalence of cardiovascular events during follow-up. CCR264I I-carriers show morecardiovascular events (P=0.004) than VV homozygotes in subjects with a history of antihypertensiveTreatment. No difference was observed in non-hypertensives

0

10

20

30

Non-hypertensives Hypertensives

%

VV

II+IV

*

History of antihypertensive treatment by CCR2 p. V64I

P=0.004

VV VI + II

N 730 160

Age (yrs) 60.28 ± 9.74 60.57 ± 10.17

Male (%) 52.33 57.50

HDL (mmol/L) 1.22 ± .35 1.17 ± .34

(V)LDL (mmol/L) 4.71 ± 1.04 4.69 ± 1.11

TGL (mmol/L) 1.74 ± 1.01 1.90 ± 1.18

BMI (kg/m2) 28.59 ± 4.32 28.20 ± 4.08

MAP (mmHg) 102.45 ± 11.46 100.84 ± 11.45

LLD (%) 17.76 14.00

UAE (mg/24h) 15.33 (8.02 - 40.87) 14.22 (8.01 - 33.55)

Subjects on AHT drugs by CCR2 p. V64I

No differences

Conclusions

•CCR2 p. V64I modifies risk associated prevalence of MACE in the general population, independantly of other confounders

•The association is only apparent in high risk subjects- Framingham Risk Score >10 % - Hypertensive patients (treated)

General idea

The (biological) effects of SNPs or other gene variants on disease are likely to surface to detectable range only under conditions of advanced progressive pathology (i.e. when compensation no longer suffices).

Baseline characteristics and risk factors for mortality according to CCR2-V64I genotype

CCR2-V64I genotype

VV VI II

N 6343 1177 57

Age (yrs) 49.7 ± 12.7 49.3 ± 12.7 51.8 ± 13.5

Male N (%) 3117 (49.1) 594 (50.1) 30 (52.6)

Obesity N (%) 975(15.5) 180 (15.4) 13 (22.8)

Hypertension N (%) 730 (11.8) 147 (12.8) 13 (22.8)a

MI N (%) 202 (3.2) 37 (3.2) 4 (7)

MA N (%) 862 (13.6) 154 (13.1) 8 (14)

CVA N (%) 49 (0.7) 9 (0.8) 0 (0)

CCR (ml/min/1.73 m2) 92.2 ± 20.7 93.2 ± 21.2 92.9 ± 20.8

CRP (mg/L) 1.3 (0.6-3.0) 1.4 (0.6-2.9) 2.0 (0.9-3.8)

Smoking N (%) 2372 (37.4) 455 (38.7) 28 (49.1)

All-cause mortality hazard ratios of CCR2 VI subjects using Cox regression models and CCR2V64I VV-individuals as reference.

Model HR 95%CI P Wald

Crude 1.31 (1.00 - 1.72 ) 0.048 3.9

Age + Sex 1.32 (1.00 - 1.73) 0.045 4.0

Full 1.42 (1.07 - 1.89) 0.015 5.90 500 1000 1500 2000 2500 3000

Follow-up time (days)

0,975

0,980

0,985

0,990

0,995

1,000

Surv

ival

rat

e

VV

IV

Conclusions: 1. CCR2 p. V64I associates with CV-events when subjects already suffer from hypertension

2. CCR2 p. V64I associates with all-cause mortality

Chemokines in pathology: renal disease

J Am Soc Nephrol 11:152-176, 2000

CCR2A / CCR2B in human renal tissue

CCR2A CCR2B

Acute rejection

CCR2A / CCR2B in human renal tissue

CCR2A

Atherosclerotic vessel

CCR2B

Future research

-Investigation of aortic/vascular sectionsDifferential expression of CCR2A/2BLocalisation by double stainingQPCR of fresh tissue

-Possible association of CCR2 p. V64I with restenosis in CV patient cohort

-In vitro study of effect of CCR2 p. V64I polymorphism on vascular smooth muscle cells

Department of Internal Medicine Subsection Nephrology

Mike ZuurmanGerjan Navis Paul de Jong

Trial Coordination Centre

Hans Hillege

Cardiology / Clinical Pharmacology

Wiek van Gilst

Pathologie

Harry van Goor

Department of Medical BiologySubsection Intergrative Genomics

Elvira Oosterom Jelle Conradie Ron KorstanjeGerrit van der Steege