ccr2 variant and cardiovascular outcome in a general population-based cohort mike zuurman, phd...
TRANSCRIPT
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