GENECURE GENECURE

Mike W. Zuurman, PhDMike W. Zuurman, PhD

Impact of genetic variation in the chemokine system Impact of genetic variation in the chemokine system Possibilities for GENECURE Possibilities for GENECURE

November 9 2006November 9 2006

Genetic epidemiology Genetic epidemiology -Patient cohortsPatient cohorts-General populationGeneral population-Sequence variantsSequence variants

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

Functional analysesFunctional analyses-In vitro/In vivoIn vitro/In vivo-MolecularMolecular-Fundamental Fundamental

BioinformastatisticaBioinformastatistica-Utilization of tools-Utilization of tools-Development of tools-Development of tools-Solutions-Solutions

BreedteBreedtestrategiestrategie

Gene-environment interaction of phenotypic risk factors with Gene-environment interaction of phenotypic risk factors with genetic variation in chemokine pathwaysgenetic variation in chemokine pathways

CCR2 genotype X Framingham risk scoreCCR2 genotype X Framingham risk score– Impact on CV outcome PREVEND Impact on CV outcome PREVEND

– Impact on CV benefits of antihypertensives (PREVEND)Impact on CV benefits of antihypertensives (PREVEND)

CCR5 genotype X inflammatory statusCCR5 genotype X inflammatory status– Impact on outcome in ESRD (Necosad) Impact on outcome in ESRD (Necosad)

CCR2 : G-protein coupled chemokine receptor CCR2 : G-protein coupled chemokine receptor

CCR2V64I mutationCCR2V64I mutation

Valine to isoleucineValine to isoleucine Stabilization CCR2A Stabilization CCR2A Isoform: Isoform: Impaired downregulationImpaired downregulation

Associated with Associated with CV diseaseCV disease

Conflicting resultsConflicting results

Effect of CCR2 on CV events in PREVENDEffect of CCR2 on CV events in PREVEND

• PREVEND (general population) N=8592PREVEND (general population) N=8592

•CCR2 genotype: VV/VI/II: 84/15/1 % CCR2 genotype: VV/VI/II: 84/15/1 %

•CV events : 442 (7 year follow up)CV events : 442 (7 year follow up)

•Baseline characteristics similar for genotypesBaseline characteristics similar for genotypes •Framingham risk score (FRS) predicts CV eventsFramingham risk score (FRS) predicts CV events

FRS:FRS: CV eventsCV events

0-10% 0-10% : : 2.4% 2.4% 10-20%10-20% :: 12.3% 12.3% 20-30%20-30% :: 19.1% 19.1% 30%30% :: 26.7% 26.7%

CV hazard ratio by FRS and I-alleleCV hazard ratio by FRS and I-allele

Multivariate hazard analyses:Multivariate hazard analyses:

CCR2 VI+II vs VVCCR2 VI+II vs VV:: 4.89 (4.89 (PP=0.006)=0.006)FRSFRS :: 2.20 (2.20 (PP<0.00001)<0.00001)CCR2 * FRSCCR2 * FRS :: 1.69 (1.69 (PP=0.005)=0.005)

FRS is a better predictor of CV events in I-carriersFRS is a better predictor of CV events in I-carriers

Sensitivity=specificitySensitivity=specificity AUC AUC

83% (VI+II)83% (VI+II) 0.87 (0.84-0.90) 0.87 (0.84-0.90)

73% (VV)73% (VV) 0.80 (0.78-0.82) 0.80 (0.78-0.82)

CCR2 genotype and CV benefits of antihypertensive treatmentCCR2 genotype and CV benefits of antihypertensive treatment

HRHR WaldWald PPAHTAHT 1.33 (1.05-1.70)1.33 (1.05-1.70) 5.445.44 0.0200.020CCR2 mutationCCR2 mutation 0.58 (0.39-0.86)0.58 (0.39-0.86) 7.357.35 0.0070.007AHT * CCR2AHT * CCR2 3.13 (1.92-5.39)3.13 (1.92-5.39) 16.9116.91 <0.0001<0.0001

Interference with RAAS-blockade ?Interference with RAAS-blockade ?

Antihypertensive treatment (AHT) Antihypertensive treatment (AHT)

Conclusions CCR2Conclusions CCR2

Risk of CV morbidity conferred by phenotypic risk factors usRisk of CV morbidity conferred by phenotypic risk factors usmodified by genetic variation in CCR2.modified by genetic variation in CCR2.

Possible genetic interference with therapeutic efficacy RAAS-blockadePossible genetic interference with therapeutic efficacy RAAS-blockade

Lit: Lit: AngII induces CCR expression on monocytesAngII induces CCR expression on monocytesARB reduces CCR2 expression on monocytesARB reduces CCR2 expression on monocytes

Possibilities for GENECUREPossibilities for GENECURE::

Effects of CCR2 mutation in various populations (WP4)Effects of CCR2 mutation in various populations (WP4)

Effects of CCR2 on therapeutic benefit RAAS blockade (WP6)Effects of CCR2 on therapeutic benefit RAAS blockade (WP6)

Pharmaco-economic implications?? (WP9)Pharmaco-economic implications?? (WP9)

CCR5 delta32 and inflammation-associated mortality CCR5 delta32 and inflammation-associated mortality in ESRD patients (NECOSAD)in ESRD patients (NECOSAD)

CCR5CCR5

Involved in atherogenesis and vascular inflammationInvolved in atherogenesis and vascular inflammation

CCR5 CCR5 ΔΔ32:32:

32bp deletion leads to CCR5 deficiency/dysfunctionality32bp deletion leads to CCR5 deficiency/dysfunctionalityAssociated with improved renal survival in IgA nephropathy Associated with improved renal survival in IgA nephropathy and delayed onset of coronary heart disease in womenand delayed onset of coronary heart disease in women

CRPCRP(Micro)inflammation(Micro)inflammation

C-reactive protein in NECOSAD and CCR5C-reactive protein in NECOSAD and CCR5

Single or repeated measures of serum CRP are associated with all-cause Single or repeated measures of serum CRP are associated with all-cause and cardiovascular mortalityand cardiovascular mortality

Hypothesis: Hypothesis:

MortalityMortality

Pro-inflammationPro-inflammation

CCR5CCR5

CCR5 and inflammation-associated mortalityCCR5 and inflammation-associated mortality

•NECOSAD populationNECOSAD population

•CCR5 CCR5 wt/wtwt/wt wt/wt/ΔΔ32 32 ΔΔ32/32/ΔΔ32 32 383 (79.5%) 383 (79.5%) 98 (18.2%)98 (18.2%) 11 (2.3%)11 (2.3%)

HW: slight overrepresentation of homozygote HW: slight overrepresentation of homozygote ΔΔ32 32

•Dominant model (wt/Dominant model (wt/ΔΔ32 + 32 + ΔΔ32/32/ΔΔ32 are carriers)32 are carriers)

•Age differenceAge difference

wt/wtwt/wt carriers carriers 61.40 (48.30-70.80)61.40 (48.30-70.80) 65.15 (50.50-72.30) 65.15 (50.50-72.30)

•High CRP defined as CRP>= 10 mg/LHigh CRP defined as CRP>= 10 mg/L

CCR5+CRPCCR5+CRP Crude overall mortalityCrude overall mortality Adjusted overall mortalityAdjusted overall mortality

wt/wt & CRP<10wt/wt & CRP<10 11 11wt/wt & CRP>10wt/wt & CRP>10 2.25 (1.65-3.05)2.25 (1.65-3.05) 1.85 (1.35-2.54)1.85 (1.35-2.54)carriers & CRP<10carriers & CRP<10 1.29 (0.84-1.99)1.29 (0.84-1.99) 1.17 (0.75-1.81)1.17 (0.75-1.81)carriers & CRP>10carriers & CRP>10 1.14 (0.67-1.94)1.14 (0.67-1.94) 0.89 (0.52-1.53)0.89 (0.52-1.53)

CCR5CCR5 Crude CV mortalityCrude CV mortality Adjusted CV mortalityAdjusted CV mortality

wt/wt & CRP<10wt/wt & CRP<10 11 11wt/wt & CRP>10wt/wt & CRP>10 2.65 (1.64-4.30)2.65 (1.64-4.30) 2.29 (1.38-3.78)2.29 (1.38-3.78)carriers & CRP<10carriers & CRP<10 1.42 (0.72-2.81)1.42 (0.72-2.81) 1.31 (0.66-2.61)1.31 (0.66-2.61)carriers & CRP>10carriers & CRP>10 0.99 (0.39-2.54)0.99 (0.39-2.54) 0.82 (0.32-2.12)0.82 (0.32-2.12)

Hazard ratios for mortality by CRP and CCR5 genotype Hazard ratios for mortality by CRP and CCR5 genotype

All-cause mortality

0 1 2 3 4 5

0

20

40

60

80

100CRP<10, INSCRP<10, DELCRP>10, INSCRP>10, DEL

Follow-up (years)

Cu

mu

lativ

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urv

iva

l(%

)

Survival for ACM by CRP and CCR5 genotype Survival for ACM by CRP and CCR5 genotype

YearsYears 00 11 22 33 44 55wt/wt+<=10wt/wt+<=10 260260 248248 209209 157157 107107 5656carriers+<=10carriers+<=10 5959 5858 4747 3838 2525 1313wt/wt+>10wt/wt+>10 123123 108108 8585 5555 3232 1919carriers+>10carriers+>10 3939 3636 2727 1919 1717 1111

ConclusionConclusion

Association between CRP (Association between CRP (markermarker of inflammation) and CV/overall mortality of inflammation) and CV/overall mortality depends on CCR5 delta32 genotype. depends on CCR5 delta32 genotype.

CRPCRP(Micro)inflammation(Micro)inflammation

MortalityMortality

Pro-inflammationPro-inflammation

CCR5CCR5

CCR5 delta32 as a protective genetic variant supported by CCR5 delta32 as a protective genetic variant supported by

> Elder age of onset dialysis of carriers > Elder age of onset dialysis of carriers > Slight overrepresentation of homozygote carriers (selection?)> Slight overrepresentation of homozygote carriers (selection?)> Better survival despite current inflammation> Better survival despite current inflammation

Treatment with CCR5 antagonists attenuates atherogenesis in Treatment with CCR5 antagonists attenuates atherogenesis in APOE -/- mice ! APOE -/- mice !

Conclusion/implicationsConclusion/implications

Possibilities for GENECURE:Possibilities for GENECURE:

Test CCR5 in other populations (WP4)Test CCR5 in other populations (WP4)Test modulation by CCR5 for risk associated with other markers (WP4)Test modulation by CCR5 for risk associated with other markers (WP4)

Study (altered) expression of CCR5 in blood vessels (WP2)Study (altered) expression of CCR5 in blood vessels (WP2)