Collaborative research initiatives in cardiovascular disease

1. Collaborative research initiatives in cardiovascular disease J Danesh October 2005

2. Lancet Cytomegalovirus 700 0.9 (0.7-1.2) 1997 H pylori BMJ Mixed strains of 3500 1.2 (0.9-1.5) 1999 H pylori Circulation Cytotoxic strains of 600 1.3 (0.9-1.9) 2000 C pneumoniae BMJ IgG titres 3000 1.2 (1.0-1.4) 2000 C pneumoniae IgA titres Eur Heart J 2300 1.2 (1.0-1.5) 2000 Lancet 2001 Lancet ICAM-1 1400 1.2 (1.0-1.6) Lancet VCAM-1 1300 1.0 (0.8-1.3) 2001 Lancet 800 1.1 (0.7-1.4) 2001 P-selectin Rheology Eur Heart J Viscosity 1300 1.6 (1.3-1.9) 2000 Eur Heart J 2000 Haematocrit 8000 1.2 (1.1-1.3) Eur Heart J 2000 ESR 1700 1.3 (1.2-1.5) Literature-based meta-analyses of some novel blood-based risk factors Type of factor/examples No. of CHD cases Risk ratio Reference (Danesh et al) Acute-phase reactants JAMA Fibrinogen 3000 1.8 (1.6-2.0) 1998 BMJ Albumin 3700 1.5 (1.3-1.7) 2000 Leucocyte count 6000 1.4 (1.3-1.5) BMJ 2000 Serum amyloid A protein 600 1.6 (1.1-2.2) BMJ2000 C-reative protein 7000 1.5 (1.4-1.6) NEJM 2004 In preparation 1500 1.8 (1.3-2.4) Interleukin-6 Haemostatic Eur Heart J von Willebrand factor 1500 1.5 (1.1-2.0) 2002 Circulation Fibrin D-dimer 1300 1.7 (1.3-2.2) 2001 Eur Heart J 2004 tPA antigen 1500 1.5 (1.1-2.0) LP-PLA2 in preparation 1700 1.4 (1.2-1.7) Lipids Circulation 2000 In preparation Lipoprotein(a) Triglycerides 3800 8000 1.7 (1.4-1.9) 1.5 (1.4-1.6) Chronic infections Cell adhesion molecules E-selectin 800 1.2 (0.9-1.6) 2001 Metalloproteins 600 1.0 (0.8-1.3) Circulation 1999 Ferritin 6000 0.9 (0.7-1.1) Circulation 1999 Transferrin Vitamin-related Homocysteine 1000 1.3 (1.1-1.5) J Card Risk 1998

3. Fibrinogen • Large glycoprotein • Usual values, 3.5 vs 2.5 g/L Top and Bottom 1/3 • ~4-fold rise

4. Prospective studies of fibrinogen and coronary heart disease reported by 1998 Danesh et al, JAMA 1998

5. b) Stroke (2775 cases) a) CHD (7118 cases) 64 64 Age at risk: Age at risk: Age at risk: 32 32 70-89 70-89 70-89 16 16 (floating absoule risk (95% CI) (floating absoule risk (95% CI) (floating absoule risk (95% CI) (floating absoule risk (95% CI) 60-69 60-69 60-69 8 8 Hazard ratio Hazard ratio Hazard ratio Hazard ratio 40-59 40-59 40-59 4 4 2 2 1 1 2·5 3·0 3·5 4·0 2·5 3·0 3·5 4·0 Usual Fibrinogen (g/L) Usual Fibrinogen (g/L) c) Other vascular deaths (988 cases) d) Non vascular deaths (7473 cases) - Age at risk: 64 64 70-89 32 32 16 16 60-69 8 8 4 4 40-59 2 2 1 1 2·5 3·0 3·5 4·0 2·5 3·0 3·5 4·0 Usual Fibrinogen (g/L) Usual Fibrinogen (g/L) 1 Fibrinogen Studies Collaboration: individual data on 154 000 participants with 20 000 fatal and non-fatal endpoints • protocol • published in 2004 • 38 studies • 121 co-authors Fibrinogen Studies Collaboration, JAMA 2005

6. Characteristic No. of CHD cases Geographical Location ‡ Western Europe 5369 North America 1718 Study Population Population register 4345 Workforce 974 Prevention trial 1799 Assay Method Clotting time 4382 Clot weight 610 Non-clotting 2126 Assay timing* Within a few days 3611 Within a few weeks 1252 More than a few weeks 1224 Not stated 1031 Sex Male 6171 Female 947 Total Cholesterol (mmol/L) <5.73 2376 5.73-6.73 2378 >6.73 2364 Triglycerides (mmol/L)† <1.29 1175 1.29-2.00 1165 >2.00 1145 SBP (mmHg) <131 2450 131-150 2375 >150 2293 Smoking Status Non current 3784 Current 3334 History of Diabetes No history 4703 Positive history of diabetes 645 Not recorded 1770 BMI (kg/m2) <25 2809 25-28 2300 >28 2009 1.0 1.5 2.0 4.0 Impact of fibrinogen on CHD in different subgroups: individual data on 154 000 participants 2 Fibrinogen Studies Collaboration, JAMA 2005

7. Emerging Risk Factors Collaboration (extension of the Fibrinogen Studies Collaboration) • Factors to be studied: triglycerides, HDL, • C-reactive protein, leucocytes, • lipoprotein(a), apo-A1 –B, • albumin etc • Potential scope: 70+ cohorts with primary data • on >700 000 individuals • (primary data already supplied for 500 000 participants)

8. Literature-based meta-analyses of some genetic factors in CHD Phenotypic pathway Gene Polymorphism No. cases/controls Per allele risk ratio (95%CI) LIPIDS Paraoxonase/HDL PON1 gln192arg 9800/11000 1.12 (1.07-1.17) leu55met 5700/5700 1.01 (0.95-1.07) T(-107)C 1400/1300 1.02 (0.92-1.14) PON2 ser311cys 1500/2100 1.04 (0.93-1.17) Apolipoprotein A1 APO-A1 G(-75)A C*3T Apolipoprotein B APO-B I/D signal peptide Apolipoprotein C-lll APO-Clll C(-482)T C1100T Apolipoprotein E APO E E2/E3 3000/4000 1.18 (1.12-1.24) E2/E4 1600/2000 1.37 (1.26-1.48) Lipoprotein(a) LP-a C93T G121A A11764C Lipoprotein lipase LPL gln188glu 950/9300 4.9 (1.2-19.6) LPL asp9asn 1000/1000 1.4 (0.8-2.4) LPL asn291ser 2300/10200 1.2 (0.9-1.5) LPL ser447ter 1600/1900 0.8 (0.7-1.0) ATP binding cassette ABCA-1 R219K LDL receptor C766T Leptin-like LDL receptor LOX-1 G501C Hepatic lipase C(-514)T C(-480)T G(-250)A Cholesterol transport CETP C(-629)A Taq1B VASCULAR FUNCTION Endothelial nitric oxide NOS3 E298D 8650/5690 1.11 (1.05-1.18) NOS3 4a/b 6950/4950 1.02 (0.94-1.10) NOS3 T(-786)C 1300/1300 1.25 (1.10-1.41) Beta-2 adrenergic receptor gln27glu arg16gly Superoxide production p22 Phox C242T Gap junctions Connexin-37 C1019T continued…..

9. Literature-based meta-analyses of somegenetic factors in CHD contd…. Phenotypic pathway Gene Polymorphism No. cases/controls Per allele risk ratio (95%CI) INFLAMMATION Cytokines CD14 C(-159)T 6400/6400 1.08 (1.01-1.15) IL6 G(-174)C 3800/5100 1.04 (0.96-1.12) IL6 G(-572)C 1200/4200 1.00 (0.80-1.24) TNF alpha G(-308)A 3400/2100 0.98 (0.88-1.09) TNF alpha G(-238)A 1100/1200 1.05 (0.81-1.37) IL10 C-reactive protein CRP G1059C von Willebrand factor VWF A(-1185)G G(-1051)A HAEMOSTASIS Fibrinogen Beta-fib G(-455)A 9500/18500 1.00 (0.95-1.05) Coagulation factor V Leiden G1691A 7800/23000 1.16 (1.04-1.29) Coagulation factor VII Coagulation factor XIII Prothrombin FII G2021A 7000/8200 1.13 (0.85-1.52) Plasmingen inhibitor SERPIN1 4G/5G 6100/7300 Platelet receptor GP la/lla C807T 2600/2500 1.03 (0.91-1.15) Platelet receptor GPIIIa A1/A2 12800/12000 Platelet receptor GP Ib C1018T Hemoglobin binding Haptoglobin HP1/2 700/700 PLAQUE FORMATION Metalloproteinase-1 MMP1 G(-1607)GG Metalloproteinase-9 MMP9 C(-1562)T R279Q Metalloproteinase-12 MMP12 A(-82)G Stromelysin MMP3 5a(-1171)6A OXIDATIVE STRESS Heme oxygenase HO-1 GTn repeat

10. Paraoxonase-1 Serum protein found on HDL Breaks down insecticides Variations in PON1 Gene (7q21.3) correlate strongly with PON1 enzyme activity 30Kb 27Kb 110Kb

11. Per-allele relative risk and 99% confidence interval Study Cases/ Genotypefrequencies1 controls RR QR QQ Myocardial infarction Yamada, 2002 1035/1168 13% vs 11% 42% vs 44% 45% vs 45% Cascorbi, 1999 649/983 7% vs 7% 40% vs 40% 53% vs 53% Herrmann, 1996 642/701 10% vs 11% 42% vs 38% 48% vs 52% Gardemann, 2000 1059/535 8% vs 7% 42% vs 40% 50% vs 52% Wang, 2003 330/475 18% vs 11% 47% vs 48% 36% vs 41% Senti, 2001 280/396 11% vs 10% 39% vs 42% 50% vs 49% Sen-Banerjee, 2000 492/518 1% vs 3% 52% vs 44% 47% vs 54% Rice, 1997 173/607 10% vs 9% 39% vs 40% 50% vs 51% Ferre, 2002 215/215 11% vs 7% 40% vs 43% 49% vs 49% Aubo, 2000 156/310 8% vs 11% 38% vs 40% 54% vs 50% Serrato, 1995 134/247 15% vs 11% 57% vs 40% 28% vs 49% Ko, 1998 114/218 46% vs 42% 42% vs 44% 12% vs 14% Suehiro, 1996 91/252 35% vs 37% 49% vs 49% 15% vs 13% Pfohl, 1999 95/170 13% vs 12% 42% vs 45% 45% vs 43% Heijmans, 2000 62/604 6% vs 8% 58% vs 44% 35% vs 48% Ombres, 1998 80/204 6% vs 7% 46% vs 41% 48% vs 52% Salonen, 1999 55/109 7% vs 6% 35% vs 39% 58% vs 54% Ayub, 1999 48/50 6% vs 6% 50% vs 30% 44% vs 64% Turban, 2001 13/301 0% vs 7% 38% vs 48% 62% vs 45% Subtotal 5723/8063 Coronary stenosis Gardemann, 2000 768/535 7% vs 7% 44% vs 40% 49% vs 52% Cascorbi, 1999 324/983 6% vs 7% 41% vs 40% 54% vs 53% Mackness, 2001 417/282 8% vs 10% 41% vs 35% 50% vs 55% Imai, 2000 210/431 53% vs 44% 42% vs 42% 4% vs 14% Sanghera, 1997 246/244 35% vs 33% 48% vs 50% 17% vs 17% Rice, 1997 153/607 9% vs 9% 40% vs 40% 51% vs 51% Chen, 2003 178/411 8% vs 9% 44% vs 40% 48% vs 51% Wang, 2003 144/475 15% vs 11% 44% vs 48% 40% vs 41% Antikainen, 1996 380/169 8% vs 4% 37% vs 44% 56% vs 51% Ruiz, 1995 171/263 10% vs 5% 50% vs 42% 40% vs 53% Osei-Hyiaman, 2001 201/231 11% vs 3% 21% vs 19% 68% vs 78% Hong, 2001 113/191 17% vs 17% 44% vs 44% 39% vs 39% Serrato, 1995 89/247 22% vs 11% 44% vs 40% 34% vs 49% Hasselwander, 1999 103/388 9% vs 8% 43% vs 46% 49% vs 46% Sanghera, 1997 122/165 14% vs 13% 59% vs 40% 27% vs 47% Ko, 1998 104/218 38% vs 42% 52% vs 44% 11% vs 14% Ombres, 1998 95/204 7% vs 7% 46% vs 41% 46% vs 52% Pati, 1998 120/80 17% vs 10% 58% vs 15% 25% vs 75% Pfohl, 1999 75/170 11% vs 12% 49% vs 45% 40% vs 43% Aynacioglu, 2000 96/105 36% vs 49% 52% vs 41% 11% vs 10% Zama, 1997 75/115 52% vs 32% 44% vs 53% 4% vs 15% Suehiro, 1996 43/252 35% vs 37% 51% vs 49% 14% vs 13% Odawara, 1997 42/122 40% vs 36% 57% vs 43% 2% vs 20% Watzinger, 2002 43/260 2% vs 7% 51% vs 37% 47% vs 57% Turban, 2001 35/301 11% vs 7% 14% vs 48% 74% vs 45% Letellier, 2002 36/96 8% vs 3% 31% vs 40% 61% vs 57% Subtotal 4383/7545 Total 10106/11786* 0.5 1.0 4.0 2.0 43 studies of paraoxonase Gln192Arg polymorphisms: 11 000 coronary cases and 13 000 controls Wheeler et al, Lancet 2004

12. Per-allele relative risk and 95% confidence interval Groups of studies Cases/controls Study size >=500 cases: 5 studies 4153/3387 200 to 499 cases: 10 studies 3095/3944 <200 cases: 30 studies 2858/4455 Ethnicity Caucasian: 28 studies 6528/7471 East Asian: 12 studies 2635/3256 Other: 5 studies 943/1059 Blinding of genotyping staff Blinded: 19 studies 5651/5648 Not blinded: 12 studies 2527/3081 Unknown: 14 studies 1928/3057 Source of controls General population: 20 studies 6286/6716 Health check/outpatient: 12 studies 1771/2055 Other patient/negative cardiac: 13 studies 2048/3015 Study population* General population: 38 studies 9383/10904 Diabetic population: 6 studies 620/882 0.5 1.0 2.0 Studies of paraoxonase Gln192Arg polymorphism grouped by study characteristics Wheeler et al, Lancet 2004

13. Studies of paraoxonase Leu55Met polymorphism and coronary heart disease Per-allele relative risk and 99% confidence interval Study Cases/ Genotype frequencies e controls MM LM LL Myocardial infarction Cascorbi, 1999 650/976 11% vs 10% 43% vs 45% 46% vs 45% Gardemann, 2000 1059/535 13% vs 13% 43% vs 46% 45% vs 41% Sen-Banerjee, 2000 492/518 6% vs 8% 40% vs 36% 54% vs 56% Ferre, 2002 215/215 14% vs 18% 50% vs 42% 36% vs 40% Arca, 2002 163/178 12% vs 12% 52% vs 46% 36% vs 43% Yamada, 2002 445/464 0% vs 0% 12% vs 13% 88% vs 87% Heijmans, 2000 62/604 11% vs 11% 48% vs 48% 40% vs 41% Salonen, 1999 55/110 24% vs 10% 40% vs 49% 36% vs 41% Ayub, 1999 48/50 13% vs 12% 33% vs 54% 54% vs 34% Subtotal 3189/3650 Coronary stenosis Gardemann, 2000 768/535 15% vs 13% 48% vs 46% 37% vs 41% Cascorbi, 1999 321/976 14% vs 10% 44% vs 45% 42% vs 45% Mackness, 2001 417/282 12% vs 10% 48% vs 53% 40% vs 37% Chen, 2003 178/411 15% vs 13% 47% vs 48% 38% vs 39% Hasselwander, 1999 103/388 10% vs 14% 51% vs 43% 39% vs 43% Arca, 2002 137/178 18% vs 12% 40% vs 46% 42% vs 43% Blatter Garin, 1997 168/240 0% vs 0% 54% vs 64% 46% vs 36% Sanghera, 1998 114/183 3% vs 5% 35% vs 30% 62% vs 65% Imai, 2000 208/431 0% vs 1% 13% vs 13% 86% vs 86% Watzinger, 2002 43/260 9% vs 15% 28% vs 45% 63% vs 40% Letellier, 2002 36/95 19% vs 11% 39% vs 57% 42% vs 33% Hong, 2001 113/191 0% vs 2% 6% vs 8% 94% vs 91% Zama, 1997 75/115 0% vs 0% 13% vs 18% 87% vs 82% Sanghera, 1998 119/181 0% vs 0% 7% vs 7% 93% vs 93% Subtotal 2800/4466 Total* 5989/6427 0.5 1.0 2.0 4.0 Wheeler et al, Lancet 2004

14. Summary • Collaborative efforts among prospective studies of plasma-based cardiovascular risk factors have demonstrated the value of this approach and have been extended to several hypotheses • Similar collaborative efforts are in progress for genetic factors in cardiovascular disease (eg, literature-based analyses so far include 500+ studies, >100 000 coronary cases and > 100 000 controls) and for integration of plasma and genetic factors • Such initiatives help to: make best use of existing data; clarify evidence; prioritize hypotheses for further investigation; create research frameworks for novel hypotheses