CCR2 V64I polymorphism: morbidity and mortality in PREVEND

18-04-2005 Mike Zuurman, Department of Internal Medicine, Section NephrologyUniversity Medical Centre Groningen, The Netherlands

Breedtestrategie

Chemokines and their receptors: families

Classified based on position of conserved cystein residuesin the protein structure

International union of pharmacology. XXII. Nomenclature for chemokine receptors.Murphy et al. , Pharmacol Rev. 2000 Mar;52(1):145-76.

CCR1 Ccr1 Ccr1 CMKBR1, CC-CKR1, HM145, MIP-1a-R, RANTES-R, LD78-R

- Ccr1l1 - CMKBR1L1, CC-CKR1-like 1, MIP-1a-R-like 1

CCR2 Ccr2 Ccr2 CMKBR2, CC-CKR2, CCR2A, CCR2B, MCP-1-R, mJE-R

CCR3 Ccr3 Cmkbr3 CMKBR3, CC-CKR3, CC-CKR1-like 2, MIP-1a-R-like 2, eotaxin receptor

CCR4 Ccr4 Ccr4 CMKBR4, CC-CKR-4, K5-5

CCR5 Ccr5 Ccr5 CMKBR5, CC-CKR5, ChemR13, CD195, HIV-1 fusion coreceptor

CCR6 Ccr6 Ccr6 CMKBR6, STRL22, GPR29, GPR-CY4, CKR-L3, DRY6, LARC-R

CCR7 Ccr7 Ccr7 CMKBR7, BLR2, EBI1, CDw197, AMG1

CCR8 Ccr8 - CMKBR8, CMKBRL2, CKR-L1, TER1, GPR-CY6, ChemR1, CC CKR-type 2

CCR9 Ccr9 Ccr9 CMKBR9, GPR-9-6, CC-CKR-9, CCR9A, CCR9B<

GPR2 Gpr2 - CCR10, CCR9, CCR10A, CCR10B

CCRL1 Ccrl1 - CCR11, VSHK1, CCBP2, CCX-CKR, GPCR14

CCRL1P - - -

CCRL2 Ccrl2 - CC CKR-like 2, HCR, CRAM-A, CRAM-B, CKRX, E01, L-CCR

HRH4 Hrh4 Hrh4 histamine receptor H4, HH4R, GPRv53, GPCR105

FPRL1 Fprl1 Fprl1 formyl peptide receptor-like 1, HM63, lipoxin A4-R, FPR2A, FMLP-R-II, Lxa4r

CC-motif chemokine receptors: nomenclatureHuman Mouse Rat Historical names

Chemokines

Chemokine function: classical

Chemokines

Chemokine function: recent

-Intercellular communication (brain)

-Angiogenesis (f.i. tumour related)

-Orchestration of the immune response * Pro- and anti-inflammatory

-Hematopoiesis

-Organogenesis

-Ovulation, luteal regression

Chemokines in pathology: atherosclerosis

Chemokines in pathology: renal disease

J Am Soc Nephrol 11:152-176, 2000

CC-motif chemokine receptor 2 comes in two flavours

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

Ligands:

-MCP-1 (CCL2)-MCP-2-MCP-3-MCP-4-MCP-5

CCR2 64I: a single nucleotide polymorphism (SNP)

Gives rise to a valine to isoleucine substitution

Literature on CCR2 64I and clinical outcome

CCR2 64I is associated with reduced coronary artery calcification suggesting increased risk of unstable plaques (Valdes et al., ATVB 22, 2002)

CCR2 64I is associated with higher prevalence of myocardial infarction (Ortlebb et al., J Mol Med 81, 2003; Petrkova et al., Immunol. Letters, 88, 2003)

The minor allele (I) is associated with delayed progression to AIDS in HIV-1patients (Smith et al., Science 277, 1997)

CCR2 64I shows no protective effect on death after development of AIDS in adults (Ioannidis et al., Ann Intern Med 135, 2001)

CCR2 64I is associated with increased mortality in HIV-1 infected neonates after a survival period of 6 years (Ioannidis et al., J Med Genet. 41, 2004)

Risk of acute renal transplant rejection was reduced in carriers of the CCR2-64I allele (Abdi et al., JASN 13, 2002)

CCR2 64I and receptor function

Initially scientists saw no apparent function difference between CCR2 64Vand CCR2 64I (chemotaxis, intracellular Ca2+ , ligand-binding etc.)

However, classically scientists examined CCR2B, and not CCR2A due to historical results that suggested much lower expression of CCR2A in monocytes

Increasing evidence shows CCR2A and CCR2B expression depends on microenvironment ! For instance:

* CCR2A ++ in myopathy (Bartoli et al., Acta Neuropath. 102, 2001)* CCR2B ++ in monocytic cellines (Tanaka et al., BBRC 290, 2002)

Indeed, Nakayama et al.(2004) showed neatly that the CCR2A and NOT CCR2B functionality is influenced by the valine to isoleucine substitution:

- Increased surface expression of CCR2A- More stable expression of CCR2A- Increased down-regulation of CCR5, the principle co-receptor required by HIV-1 for infection

CCR2 64I and general population?

What about predisposed cardiovascular morbidity?

Previous studies only in patient populations already predisposed with CV-disease

What about mortality?

Populatie Groningen

85000 individuals

40000 urine samples

8500 in preclinic (MA enriched)

Prevention of REnal and Vascular ENd-organ Damage

PhenotypingGenotyping

Predisposition: hypertension in history

Baseline differences between hypertensives and normotensives in the study population

Hypertensives Normotensives

N 890 6529

Age 60.3 ± 9.8 47.7 ± 12.3a

Male (%) 53.26 48.69 b

BMI (kg/m2) 28.51 ± 4.3 25.75 ± 4.1 a

MAP (mmHg) 102.2 ± 11.47 90.97 ± 11.9 a

HDL-c (mmol/L) 1.21 ± 0.35 1.34 ± 0.40 a

(V)LDL-c (mmol/L) 4.70 ± 1.05 4.28 ± 1.22 a

TGL (mmol/L) 1.37 ± 0.99 1.77 - 1.04 a

LLD (%) 17.07 2.96 b

UAE (mg/24h) 14.98 (8.01 - 40.11) 9.08 (6.25 - 16.17) c

BMI, body-mass index; MAP, mean arterial pressure; LLD, lipid lowering drugs; UAE: urinary albumin excretion. Continuous values show mean and standard deviation, except for UAE (median and 25th – 75th percentile). a: ANOVA P-value < 0.05; b: Pearson Chi square P-value < 0.05; c: Mann-Whitney U-test P­-value < 0.05.

Characteristics of hyper- and normotensivesby CCR2-V64I

Hypertensives Normotensives

VV VI + II VV VI + II

N 730 160 5481 1048

Age (yrs) 60.28 ± 9.74 60.57 ± 10.17 48.01 ± 12.32 47.61 ± 12.24

Male (%) 52.33 57.50 48.60 49.14

HDL (mmol/L) 1.22 ± .35 1.17 ± .34 1.34 ± .40 1.32 ± .40

(V)LDL (mmol/L) 4.71 ± 1.04 4.69 ± 1.11 4.28 ± 1.22 4.26 ± 1.24

TGL (mmol/L) 1.74 ± 1.01 1.90 ± 1.18 1.36 ± .93 1.43 ± 1.24 a

BMI (kg/m2) 28.59 ± 4.32 28.20 ± 4.08 25.73 ± 4.11 25.83 ± 3.99

MAP (mmHg) 102.45 ± 11.46 100.84 ± 11.45 90.89 ± 11.78 91.41 ± 12.54

LLD (%) 17.76 14.00 3.13 2.08

UAE (mg/24h) 15.33 (8.02 - 40.87) 14.22 (8.01 - 33.55) 9.05 (6.20 - 16.29) 9.26 (6.46 - 15.77)

Distribution of cardiovascular events

ANormotensives B Hypertensives

Event VV VI + II Event VV VI + II

MI 64 (1.17) 10 (0.95) MI 23 (3.15) 12 (7.50)

PTCA 72 (1.31) 10 (0.95) IHD 25 (3.42) 11 (6.88)

IHD 61 (1.11) 6 (0.57) PTCA 26 (3.56) 8 (5.00)

Angina pectoris 55 (1.00) 5 (0.48) Angina pectoris 22 (3.01) 6 (3.75)

Acute but ill-defined cerebrovascular disease present

11 (0.20) 4 (0.38) Coronary artery bypass grafting 14 (1.92) 6 (3.75)

Ruptured aneurysm 15 (0.27) 4 (0.38) Peripheral artery bypass grafting 5 (0.68) 4 (2.50)

Arterial embolism and thrombosis 7 (0.13) 4 (0.38) Intracerebral hemorrhage 3 (0.41) 3 (1.88)

Coronary artery bypass grafting 34 (0.62) 3 (0.29) Occlusion of cerebral arteries 11 (1.51) 3 (1.88)

Occlusion and stenosis precerebral arteries

9 (0.16) 3 (0.29) Acute but ill defined cerebrovascular disease

2 (0.27) 3 (1.88)

Transient cerebral ischemia 11 (0.20) 3 (0.29) Atherosclerosis of the extremities 4 (0.55) 3 (1.88)

Occlusion of cerebral arteries 17 (0.31) 2 (0.19) Ruptured aneurysm 8 (1.10) 2 (1.25)

Atherosclerosis of the extremities 6 (0.11) 2 (0.19) Arterial embolism and thrombosis 4 (0.55) 2 (1.25)

Intracerebral hemorrhage 5 (0.09) 1 (0.10) Aorta carotics surgery 9 (1.23) 2 (1.25)

Other and unspecified intracranial hemorrhage

2 (0.04) 1 (0.10) Peripheral bypass 2 (0.27) 2 (1.25)

Aorta carotics surgery 13 (0.24) 1 (0.10) Occlusion and stenosis precerebral arteries

3 (0.41) 1 (0.63)

Peripheral artery bypass grafting 12 (0.22) 1 (0.10) Other and unspecified intracranial hemorrhage

1 (0.14) 0 (0)

Peripheral bypass 4 (0.07) 0 (0) Transient cerebral ischemia 3 (0.41) 0 (0)

0

10

20

30

Non-hypertensives Hypertensives

%

VVII+IV

*

Prevalence of cardiovascular events during follow-up

0

2

4

6

8

10

12

1 2 3 4 5Number of CE

% o

f ge

not

ype

grou

p

VI + IIVV

Relative risk of cardiovascular events in hyper- and normotensive

CCR2V64I I-carriers versus VV homozygotes.

Hypertensives Normotensives Total

Model RR P RR P RR P

Crude 1.85 0.005 0.83 0.292 1.14 0.331

Age 1.85 0.006 0.85 0.364 1.15 0.321

Full 1.70 0.028 0.87 0.472 1.14 0.361

RR: relative risk, P: P-value of the genotype term in the logistic regression analyes. The full model includes, besides subject age, significant contribution of: usage of lipid-lowering drugs, sex, HDL-cholesterol, LDL+VLDL-cholesterol, mean arterial pressure and urinary albumin excretion..

Conclusion: CCR2 64I associates with CV-events when subjects already suffer from hypertension

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)

COD Genotype VV VI II OR (VI)

Total 6343 1177 57

Female 3226 583 27

Male 3117 594 30

Malignant

Respiratory system 17 (0.27) 7 (0.59) 0 (0) 2.22

Breast 4 (0.12) 3 (0.51) 0 (0) 4.15

Digestive system 27 (0.43) 5 (0.42) 0 (0) 1.00

Urinary system 7 (0.11) 5 (0.42) 0 (0) 3.85

Female reproductive system 3 (0.09) 0 (0) 0 (0) -

Male reproductive system 4 (0.06) 1 (0.08) 0 (0) 1.35

Neoplasm (unspecified site) 14 (0.22) 3 (0.25) 0 (0) 1.15

Rest (non-related) 16 (0.25) 0 (0) 0 (0) -

Overall 92 (1.45) 27 (2.03) 0 (0) 1.41

Cardiovascular

Ischemic heart disease 25 (0.39) 7 (0.59) 0 (0) 1.51

Brain located 8 (0.13) 1 (0.08) 0 (0) 0.67

Aneurysm 6 (0.09) 1 (0.08) 0 (0) 0.90

Rest (non-related) 18 (0.28) 1 (0.08) 0 (0) 0.29

Overall 58 (0.91) 10 (0.85) 0 (0) 0.93

Other

Rest (not related) 21 (0.33) 10 (0.85) 0 (0) 2.56

All 171 (2.70) 47 (4.00) 0 (0) 1.48

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

Cox competing risk models

ModelCR HR 95% CI P PCR

Crude CV 0.93 0.48 - 1.82 0.84 0.157

non-CV 1.63 1.11 - 2.39 0.013

Age + Sex CV 0.93 0.48 - 1.83 0.84 0.159

non-CV 1.63 1.11 - 2.39 0.013

Full CV 0.81 0.37 - 1.78 0.59 0.098

non-CV 1.71 1.14 - 2.58 0.009

Crude CV 0.93 0.48 - 1.82 0.84 0.312

Malignancy 1.41 0.90 - 2.21 0.13

Age + Sex CV 0.94 0.48 - 1.83 0.84 0.316

Malignancy 1.41 0.90 - 2.21 0.13

Full CV 0.81 0.37 - 1.78 0.59 0.212

Malignancy 1.45 0.90 - 2.35 0.13

Conclusions: 1. CCR2 64I associates with CV-events when subjects already suffer from hypertension

2. CCR2 64I associates with all-cause mortality, but does not predict CV-mortality

3. Results suggest an association of CCR2 64I with cancer-related mortality

Acknowledgements

Medical Biology

Gerrit van der Steege Elvira OosteromMarcel MulderRon Korstanje

Trial Coordination Centre

Hans Hillege

Internal Medicine, Nephrology

Gerjan Navis Paul E. De Jong