Genome Wide and Candidate Gene Studies in Cardiovascular Disease. Martin Hersberger Division of Clinical Chemistry and Biochemistry

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1 Genome Wide and Candidate Gene Studies in Cardiovascular Disease Martin Hersberger Division of Clinical Chemistry and Biochemistry

2 Global Risk Assessment for Cardiovascular Events in the Asymptomatic Patient Der Score dient der Schätzung des kardiovaskulären Gesamtrisikos für Männer und postmenopausale Frauen (inkl. Diabetiker Typ 1 ohne Endorganschäden), soweit sie nicht direkt den Risikokategorien «Sehr hohes Risiko» oder «Hohes Risiko» zugeordnet werden. Für prämenopausale Frauen ist der ermittelte Risikowert rund 4-mal zu hoch, so dass er korrigiert werden muss (dividiert durch 4).

3 Strategies for the Prevention of Heart Disease Risk Intervention Risk for myocardial infarction High (7.5%) Intermediary (15%) Low (77.5%) >20 % / 10 years % / 10 years <10 % / 10 years Intensive, and global reduction of risk factors Intensive life style change ± drugs Life style change & control

4 Comparison of Methods for the Estimation of Global Coronary Risk in Middle Aged Men ESC/EAS (high risk) ATP III ITF / IAS Sensitivity (%) Specificity (%) Positive predictive value (%) Negative predictive value (%) Diagnostic efficacy (%) Prevalence of treatment requiring persons (%)

5 Relative Risk for Death from CHD in Twins when One s Twin Died from Premature CHD (N Engl J Med Apr 14;330(15):1041-6) Monozygotic twins Dizygotic twins Females RR = 15 (n = 4012) RR = 2.6 (n = 7730) Males RR = 8.1 (N = 3298) RR = 3.8 (N = 5964)

6 Heritability of CHD Risk Factors in Twin Studies (Heart 2013;99: ) CAD risk factor HDL-C LDL-C Triglycerides Total cholesterol Lp(a) Blood pressure BMI Physical activity Diabetes I & II Heritability (%)

7 Number of Publications Investigating Polymorphisms Associated with CHD and MI

8 Meta-Analyses of Polymorphisms Associated with CHD (Atherosclerosis Dec;207(2):492-5 / Circulation Dec 18;104(25): / Circulation Jan 25;111(3): / Ann Intern Med Jul 20;141(2): / Circulation Mar 23;109(11): / Heart Jan;90(1):82-6 / Thromb Haemost Dec;80(6): ) Polymorphism G20210A prothrombin Factor V Leiden R506Q 4G/5G PAI1 LDLR ins/del Apo E4 CETP TaqIB COX2 G-765C CX3CR1 (fractalkine) enos Glu298Asp Frequency OR for CHD

9 ApoE is Associated with CAD Risk in a Meta-Analysis (n = ) (Ann Intern Med Jul 20;141(2):137-47) E E E3E Relative Risk E4+ genotype frequency: 26%

10 CHD Risk by ApoE Genotype and Smoking in the Northwick Park Heart Study II (n=3052) (Lancet (2001) 358: ) E4+ [163/21] E2+ [98/5] Smokers E3E3 [367/5] 1.68 Never smoked [727/32] Relative Risk

11 Genetic Risk Estimate Including 5 Functional SNPs to Predict CHD in the Northwick Park Heart Study II (n=3052) (Clin Chem Jan;53(1):8-16) Conventional risk factors Genetic risk factors Combination showed significant improvement over the CRF score Additional imoprovement when gene-environment interactions included A set of 5 common SNPs with genotype frequencies >10% was used for this genetic risk score (UCP2, APOE, LPL, and APOA4 )

12 Meta-Analysis Genetic Risk Estimate to Predict CHD (Ann Hum Genet Sep;71(Pt 5):611-9) Distribution in the Population OR for # of Risk Genotypes A set of 10 common SNPs with meta-analysis risk estimates and genotype frequencies >10% was used for this genetic risk score (APOB, NOS3, APOE, ACE, PAI1, MTHFR, ITGA2B, PON1, LPL, and CETP)

13 GWAS for Cardiovascular Disease (Trends in Genetics June 2012, Vol. 28, No. 6) Myocardial infarction Heart faillure

14 GWAS Identified Loci for CAD/MI with a Role in Lipipoprotein Metabolism (Can J Cardiol Jan;29(1):23-9) Locus Lead SNP Risk allele freq OR per risk allele mean (CI) Genes of interest Associated lipoprotein phenotype 1p32.3 rs ( ) PCSK9 LDL-C 1p13 rs ( ) SORT1 LDL-C ABCG5 2p21 rs ( ) ABCG8 LDL-C 6q25.3 rs ( ) LPA Lipoprotein (a) 6q26 rs ( ) LPA Lipoprotein (a) 8q24 rs ( ) TRIB1 Triglycerides APOA5 11q23 rs ( ) APOA1 APOC3 Triglycerides, HDL-C 12q24 rs ( ) SH2B3 LDL-C, BP 19p13 rs ( ) LDLR LDL-C 19q13 rs ( ) APOE LDL-C

15 SORT1 and TRIB1 May be Involved in VLDL- Lipoprotein Secretion from the Liver (J Lipid Res Nov;52(11): )

16 GWAS Identified Loci for CAD/MI Associated with Risk Factors or Related Phenotypes (Can J Cardiol Jan;29(1):23-9) Locus Lead SNP Risk allele freq OR per risk allele mean (CI) Genes of interest Associated CV risk factors or phenotypes 6p24.1 rs ( ) PHACTR1 Coronary calcification 9q34 rs ( ) ABO IL-6, E-selectin 10q11 rs CXCL12 Attenuation of neutrophil migration 10q23 rs ( ) LIPA Endothelial function CYP17A1 10q24 rs ( ) CNNM2 NT5C2 Blood pressure, Intracranial aneurysms 13q34 rs ( ) COL4A1/A2 Type IV collagen chain of basement membrane 15q25 rs ( ) ADAMTS7 Proliferative response to vascular injury

17 GWAS Identified Loci for CAD/MI Not Associated with Risk Factors or Related Phenotypes (Can J Cardiol Jan;29(1):23-9) Locus Lead SNP Risk allele freq OR per risk allele mean (CI) Genes of interest 1p32.2 rs ( ) PPAP2B 1q41 rs ( ) MIA3 2q33.1 rs ( ) WDR12, NBEAL1 3q22.3 rs ( ) MRAS 6p21.33 rs HLA-C, HLA-B 6p21.31 rs ( ) ANKS1A 6p24.1 rs ( ) C60rf105 6q23.2 rs ( ) TCF21 7q22 rs ( ) BCAP29, DUS4L 7q32 rs ( ) ZC3HC1 10p11 rs ( ) KIAA1462 9p21.3 rs ( ) ANRIL, CDKN1, CDKN2 11q22 rs ( ) PDGFD 14q32 rs ( ) HHIPL1 17p11 rs ( ) RASD1, PEMT, RAI1 17p13 rs ( ) SMG6 17q21 rs ( ) UBE2Z 21q22 rs ( ) SLC5A3, MRPS5, KCNE2

18 Consistent Association of 9p21 with CAD/MI (Circ Cardiovasc Genet 2010;3: ) CARDIoGRAM: replication and meta-analysis study for GWAS in European ancestries with > cases and > controls

19 Risk Haplotypes at 9p21 are Located in ANRIL (Nature Feb 10;470(7333):264-8) CAD T2D Genes in region ANRIL (non-coding) CDKN2B

20 Risk Haplotypes at 9p21 May Influence Enhancer Function of this Locus (Nature Feb 10;470(7333):264-8)

21 The Long Non-Coding mrna ANRIL Represses CDKN2B Expression (Cancer Res Aug 15;71(16):5365-9) CDKN2B PRC: polycomb repressive complex

22 Strategies for the Prevention of Heart Disease Risk for myocardial infarction High (7.5%) Intermediary (15%) Low (77.5%) >20%/10 years 10-20%/10 years <10%/10 years Intensive, and global reduction of risk factors Intensive life style change ± drugs Life style change & control

23 No Major Improvement of Cardiovascular Risk Prediction Through Inclusion of 9p21 (Arterioscler Thromb Vasc Biol Feb;32(2): ) CARDIoGRAM: replication and meta-analysis study for GWAS in European ancestries with > cases and > controls

24 Prospective 2-Stage Risk Screening Strategy for Coronary Heart Disease (n=24 124) (Arterioscler Thromb Vasc Biol Sep;33(9):2261-6) 28 genetic variants previously associated with CHD in GWAS used to build the genetic risk score (GRS)

25 Prospective 2-Stage Risk Screening Strategy for Coronary Heart Disease (n=24 124) (Arterioscler Thromb Vasc Biol Sep;33(9):2261-6) Targeted GRS screening of clinically relevant risk group (10% 20%) would reclassify 12% in the intermediate- to high-risk category Statin allocation for reclassified individuals (2144) could prevent 135 CHD cases over 14 years

26 Mendelian Randomisation Studies Compared to a Randomised Intervention Trial (BMJ 2012;345:bmj.e7325)

27 Mendelian Randomisation Studies to Show Causality of an Intermediate Risk Factor (TCM Vol. 19, No. 6, 2009) Genetic variation at the LDLR locus YES Increased LDL levels Coronary heart disease Intermediate phenotype Disease

28 Circulating CRP is not a Causal Factor in CHD: Mendelian Randomisation Meta-Analysis (n= ) (BMJ 2012;345:bmj.e7325) Other risk factors Environment Genetic variation at the CRP locus NO Increased CRP levels Coronary heart disease Intermediate phenotype Disease

29 Mendelian Randomisation Studies for CHD Risk Factors (Hypertension May;61(5): / PLoS One Aug 20;3(8):e2986 / Lancet Mar 31;379(9822): / Lancet Aug 11;380(9841): / Lancet May 8;375(9726): / N Engl J Med 2009; 361: / J Clin Endocrinol Metab Feb;97(2):E / BMJ Feb 15;342:d548 / JACC Vol. 62, No. 21, 2013 / Circulation Sep 17;128(12): ) Polymorphism in gene LDLR Lp(a) ApoA5 ApoA1 Endothelial Lipase LCAT spla2 IL-6R CRP Fibrinogen Hypertension GRS Intermediate phenotype LDL Lp(a) Triglycerides HDL HDL HDL spla2 IL-6R signaling CRP Fibrinogen Hypertension Intermediate phenotype causal for CHD Yes Yes Yes No No No No Yes No No Yes

30 12/15-LOX: Janus Enzymes (Prostaglandins Leukot Essent Fatty Acids Aug;77(2):67-77) Lipoxins, 12-HETE, 15-HETE, 13-HODE 12-HETE, 15-HETE, 13-HODE Antiinflammatory mediators Increased monocyte adhesion Increased angiotensin II signalling Ox-LDL formation ANTI-

31 ALOX15 c.1693c>t (Met560Thr) Abolishes ALOX15 Activity (Atherosclerosis May;198(1):136-44) Activity of recombinant proteins from E. coli

32 Association of ALOX15 c.1693c>t (Met560Thr) with CAD (Atherosclerosis Jul;205(1):192-6 / Atherosclerosis May;198(1):136-44) ADVANCE (n = 3169) OR p-value ALOX15 Met560Thr Case-control: symptomatic early onset CAD, older subjects presenting with stable angina or AMI ARIC (n = 11567) OR p-value ALOX15 Met560Thr Prospective: AMI, coronary artery bypass surgery, unstable angina, or coronary-related death KORA (n = 2629) OR p-value ALOX15 Met560Thr Case-control for MI MAF: 8 % in Hispanics, 1.2 % in Caucasians and absent in East Asians

33 Conclusions Risk prediction using the GWAS SNPs or meta-analysis genetic risk estimates for functional SNPs may improve risk prediction for CHD or MI above the traditional risk factors in the future The GWAS SNPs explain 10%-25% of the genetic risk for cardiovascular disease and intermediate phenotypes GWAS identified several loci associated with CAD or MI which encode genes or enhancers not previously implicated with atherosclerosis Novel targets for drug intervention will eventually emerge from the GWAS studies Mendelian randomisation studies seem powerful in dissecting causality of an intermediate phenotype with CAD Association of functional polymorphisms with CAD can dissect the role of genes with no intermediate phenotype

34 In Collaboration with University Children s Hospital Zurich Clinical Chemistry and Biochemistry C. Gemperle Dr. M. Herova J. Marti-Jaun M. Schmid M. Rösinger Dr. V. Waechter A. Weber Dr. A. Weiss Dr. J. Wittwer Dr. S. Wüst University of Zurich Cardiovascular Center Division of Cardiology Prof. Dr. Thomas F. Lüscher PD Dr. Jörg Muntwyler Johann Wolfgang Goethe University Frankfurt am Main Institute of Food Chemistry Prof. Dr. Armin Mosandl Dr. Mathias Bayer University of Innsbruck Division of Genetic Epidemiology Prof. Dr. F. Kronenberg GSF - National Research Center for Environment and Health Munich Institute of Epidemiology Prof. Dr. I. Heid Prof. Dr. Th. Illig

35 Besten Dank für Ihre Aufmerksamkeit!