Articles

A multilocus genetic risk score for coronary heart disease: case-control and prospective cohort analyses
Samuli Ripatti, Emmi Tikkanen, Marju Orho-Melander, Aki S Havulinna, Kaisa Silander, Amitabh Sharma, Candace Guiducci, Markus Perola, Antti Jula, Juha Sinisalo, Marja-Liisa Lokki, Markku S Nieminen, Olle Melander, Veikko Salomaa, Leena Peltonen*, Sekar Kathiresan

Summary
Background Comparison of patients with coronary heart disease and controls in genome-wide association studies has revealed several single nucleotide polymorphisms (SNPs) associated with coronary heart disease. We aimed to establish the external validity of these ndings and to obtain more precise risk estimates using a prospective cohort design. Methods We tested 13 recently discovered SNPs for association with coronary heart disease in a case-control design including participants diering from those in the discovery samples (3829 participants with prevalent coronary heart disease and 48 897 controls free of the disease) and a prospective cohort design including 30 725 participants free of cardiovascular disease from Finland and Sweden. We modelled the 13 SNPs as a multilocus genetic risk score and used Cox proportional hazards models to estimate the association of genetic risk score with incident coronary heart disease. For case-control analyses we analysed associations between individual SNPs and quintiles of genetic risk score using logistic regression. Findings In prospective cohort analyses, 1264 participants had a rst coronary heart disease event during a median 107 years follow-up (IQR 67136). Genetic risk score was associated with a rst coronary heart disease event. When compared with the bottom quintile of genetic risk score, participants in the top quintile were at 166-times increased risk of coronary heart disease in a model adjusting for traditional risk factors (95% CI 135204, p value for linear trend=7310). Adjustment for family history did not change these estimates. Genetic risk score did not improve C index over traditional risk factors and family history (p=019), nor did it have a signicant eect on net reclassication improvement (22%, p=018); however, it did have a small eect on integrated discrimination index (0004, p=00006). Results of the case-control analyses were similar to those of the prospective cohort analyses. Interpretation Using a genetic risk score based on 13 SNPs associated with coronary heart disease, we can identify the 20% of individuals of European ancestry who are at roughly 70% increased risk of a rst coronary heart disease event. The potential clinical use of this panel of SNPs remains to be dened. Funding The Wellcome Trust; Academy of Finland Center of Excellence for Complex Disease Genetics; US National Institutes of Health; the Donovan Family Foundation.
Lancet 2010; 376: 1393400 See Comment page 1366 *Prof Peltonen died in March, 2010 Institute for Molecular Medicine Finland FIMM, University of Helsinki, Helsinki, Finland (S Ripatti PhD, E Tikkanen MSc, K Silander PhD, Prof L Peltonen PhD); National Institute for Health and Welfare, Helsinki, Finland (S Ripatti, E Tikkanen, A S Havulinna MSc, K Silander, Prof M Perola MD, A Jula MD, Prof V Salomaa MD, Prof L Peltonen); Department of Clinical Sciences in Malm, Lund University, Malm, Sweden (Prof M Orho-Melander PhD, A Sharma PhD, O Melander MD); Broad Institute, Cambridge, MA, USA (C Guiducci BS, Prof L Peltonen, S Kathiresan MD); Division of Cardiology, Department of Medicine, Helsinki University Central Hospital (HUCH), Helsinki, Finland (J Sinisalo MD, Prof M S Nieminen MD); Transplantation Laboratory, Haartman Institute, University of Helsinki, Helsinki, Finland (M-L Lokki PhD); Wellcome Trust Sanger Institute, Hinxton, UK (Prof L Peltonen); Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA, USA (S Kathiresan); Department of Medicine, Harvard Medical School, Boston, MA, USA (S Kathiresan); and Cardiovascular Research Center, Massachusetts General Hospital, Boston, MA, USA (S Kathiresan) Correspondence to: Dr Samuli Ripatti, FIMM, P O Box 20, FIM-00014 University of Helsinki, Finland samuli.ripatti@mm.

Introduction
Coronary heart disease is complex in origin, with contributions from lifestyle and genetic factors.1 Family history of premature coronary heart disease is an independent risk factor, suggesting that inherited DNA sequence variants contribute to risk of the disease. Using a case-control design, genome-wide association studies have identied single nucleotide polymorphisms (SNPs) at 13 genomic regions associated (p<510) with coronary heart disease, myocardial infarction, or both.2 In discovery studies, each copy of the risk allele at these loci was estimated to increase risk of myocardial infarction by 1292%. Discovery genome-wide association studies for myocardial infarction or coronary heart disease have ascertained cases on the basis of early age of disease onset or aected family members, and as such the reported eect estimates might not be representative of the general population. Although ecient for discovery, crosssectional and case-control designs have the potential for several types of bias, whereas the prospective cohort study
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is regarded as the gold standard in epidemiological investigations.3 Therefore, we set out to answer two questions: rst, are the reported genetic association ndings externally generalisable in studies diering from the discovery studies; and second, can more precise risk estimates be obtained with a prospective cohort design?

Methods
Study populations
We tested the 13 recently discovered SNPs for association with coronary heart disease in two designs: a case-control design including participants diering from those in the discovery samples (3829 participants with prevalent coronary heart disease and 48 897 controls free of the disease); and a prospective cohort design including 30 725 participants free of cardiovascular disease from Finland and Sweden. Coronary heart disease was dened as myocardial infarction, unstable angina pectoris, coronary revascularisation (coronary artery bypass graft or percutaneous transluminal coronary angioplasty), or death due to coronary heart disease. Cardiovascular

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FR 1992 (n=5353) Sex Male Female Age (years) Cholesterol Total (mmol/L) LDL (mmol/L) HDL (mmol/L) Blood pressure Systolic (mm Hg) Diastolic (mm Hg) Body-mass index (kg/m) Current smoker Current drug therapy Lipid-lowering Antihypertensive Diabetes mellitus Prevalent cases CHD CVD MI Incident cases CHD CVD MI 296 (6%) 416 (8%) 157 (3%) 64 (1%) 77 (1%) 50 (1%) 82 (2%) 496 (9%) 198 (4%) 1353 (194) 812 (119) 261 (44) 1498 (28%) 56 (11) 35 (10) 14 (03) 2449 (46%) 2904 (54%) 445 (113)

FR 1997 (n=7939)

FR 2002 (n=7635)

Health 2000 (n=5796)

MDC-CC (n=5104)

COROGENE (n=6015)

MPP (n=14 884)

4006 (50%) 3933 (50%) 49 (134) 55 (11) 35 (09) 14 (04) 1362 (201) 824 (113) 267 (45) 1846 (23%) 280 (4%) 1056 (13%) 481 (6%) 222 (3%) 276 (3%) 161 (2%) 419 (5%) 599 (8%) 230 (3%)

3526 (46%) 4109 (54%) 484 (131) 56 (11) 34 (09) 15 (04) 1350 (200) 791 (114) 269 (47) 1993 (26%) 563 (7%) 1095 (14%) 433 (6%) 198 (3%) 264 (3%) 119 (2%) 132 (2%) 186 (2%) 65 (1%)

2697 (47%) 3099 (53%) 508 (130) 59 (11) 38 (12) 13 (04) 1337 (206) 821 (112) 269 (47) 1728 (30%) 1396 (24%) 1306 (23%) 460 (8%) 59 (1%) 378 (7%) 28 (<1%) 151 (3%) 201 (3%) 112 (2%)

2141 (42%) 2963 (58%) 574 (59) 62 (11) 42 (10) 14 (04) 1411 (189) 869 (95) 259 (49) 1307 (26%) 109 (2%) 641 (13%) 399 (8%) NA 107 (2%) 63 (1%) NA 468 (9%) 266 (5%)

4192 (70%) 1823 (30%) 665 (109) NA NA NA NA NA NA NA NA NA NA NA NA 2101 (35%) NA NA NA

9773 (66%) 5111 (34%) 453 (70) 56 (10) NA NA 1277 (144) 842 (88) 244 (34) 5639 (38%) NA 645 (4%) 617 (4%) NA 1749 (12%) 1122 (8%) NA NA NA

Data are mean (SD) or number (%). FR=FINRISK. MDC-CC=Malm Diet and Cancer Cardiovascular cohort. MPP=Malm Preventive Project. NA=not available. CHD=coronary heart disease. CVD=cardiovascular disease. MI=myocardial infarction.

Table 1: Background characteristics of the seven study populations

See Online for webappendix

disease included coronary heart disease and ischaemic stroke events. Detailed case denitions are described in the webappendix. Participants from seven cohorts were included in our analyses (table 1). The FINRISK 1992, 1997, and 2002 cohorts consist of a representative random sample selected from inhabitants of dierent regions in Finland aged 2574 years. The survey included a mailed questionnaire and a clinical examination at which a blood sample was drawn. The study protocol has been described previously.4 23 036 individuals participated in these cohorts and genotype data was available from 20 927 participants. The Health 2000 study was based on a stratied two-stage cluster sampling from the National Population Register to represent the total Finnish population aged 30 years and older.5 The survey included an interview about medical history, health-related lifestyle habits, and a clinical examination at which a blood sample was drawn. 6200 people participated in the study. After exclusion of individuals older than 80 years and without sucient genotype data, the nal dataset consisted of 5796 participants. A detailed methodology report is available online.6

The Malm Diet and Cancer (MDC) study was a community-based prospective epidemiological cohort of 28 449 people recruited for a baseline examination between 1991 and 1996.7 From this cohort, 6103 people were randomly selected to participate in the Cardiovascular Cohort (MDC-CC), which sought to investigate risk factors for cardiovascular disease. All participants underwent a medical history, physical examination, and laboratory assessment for cardiovascular risk factors, as described previously.8 Final data with genotypes were available for 5104 participants. During follow-up of the FINRISK and HEALTH 2000 cohorts, data for admission to hospital and mortality were obtained from the Finnish National Hospital Discharge Register and the Finnish National Causes-ofDeath Register. These registers have excellent validity and coverage.9,10 Follow-up ended on Dec 31, 2007. Follow-up of the MDC-CC is as previously described.11 The Malm Preventive Project (MPP) is a cohort from southern Sweden that was set up in 1974. 33 346 individuals were screened during 197492. Information concerning lifestyle factors and medical history was obtained from a questionnaire. All participants underwent physical examination and biochemical analyses. Of individuals
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Region Candidate gene(s)

Weight Reference Risk allele

Risk allele Other Coronary heart disease frequency allele (total n=19 790) Pooled HR (95% CI) p value 0854 0431 0512

Cardiovascular disease (total n=24 894) Pooled HR (95% CI) 103 (094112) 097 (087107) 098 (089107) p value 0546 0515 0587

Myocardial infarction (total n=24 894) Pooled HR (95% CI) 103 (091118) 104 (089122) 094 (082108) p value 0624 0636 0378

rs17465637 rs11206510 rs646776

1q41 1p32 1p13

MIA3 PCSK9 CELSR2 PSRC1 SORT1 WDR12 MRAS LPA PHACTR1 CDKN2A CDKN2B CXCL12 HNF1A SH2B3 LDLR SLC5A3 MRPS6 KCNE2

114 115 119

15 15 15

C T T

075 084 079

A C C

099 (087112) 094 (081109) 096 (084109)

rs6725887 rs9818870 rs3798220 rs9349379 rs4977574 rs1746048 rs2259816 rs3184504 rs1122608 rs9982601

2q33 3q22 6q26 6p24 9p21 10q11 12q24 12q24 19p13 21q22

117 115 168 112 129 117 108 113 115 120

15 16 18 15 15 15 16 17 15 15

C T C C G C T T G T

011 010 001 044 043 084 036 040 079 014

T C T T A T G C T C

114 (096135) 088 (073106) 207 (139309) 116 (104129) 121 (108134) 113 (097133) 102 (091114) 103 (092115) 100 (087114) 129 (107157)

0126 0174 38105 0008 0001 0113 0774 0568 0988 0009

100 (089112) 092 (081103) 163 (122217) 110 (102119) 116 (108125) 101 (091112) 100 (092108) 110 (102119) 102 (093112) 111 (098126)

0960 0150 0001 0014 93106 0846 0932 0011 0693 0092

096 (080114) 086 (071103) 180 (119272) 111 (099124) 119 (106133) 111 (095131) 101 (090114) 115 (103129) 098 (086112) 124 (104148)

0629 0097 0005 0075 0002 0198 0836 0012 0774 0016

SNP=single nucleotide polymorphism. HR=hazard ratio. *Association tested with Cox proportional hazards model adjusted for sex, LDL and HDL cholesterol, smoking, body-mass index, systolic and diastolic blood pressure, blood pressure treatment, and diabetes; age was used as the timescale. SNP specic weights for genetic risk score calculation; weights are eect sizes for the SNPs from the studies referenced. Results from FINRISK 1992, 1997, and 2002, Health 2000, and Malm Diet and Cancer Cardiovascular Cohort were combined with xed eects meta-analysis.21

Table 2: Association between SNPs and incident coronary heart disease, cardiovascular disease, and myocardial infarction*

who participated in the baseline examinations, 17 284 were rescreened during 200206. The nal data with genotypes included 14 884 individuals. For the COROGENE cohort, initially, all consecutive Finnish patients undergoing coronary angiogram between June, 2006, and March, 2008 (n=5330), in the Helsinki University Central Hospital were included and a questionnaire, information about previous medical conditions and cardiovascular risk factors, hospital records for patients history, laboratory measurements, electrocardiogram, echocardiogram, and medication were obtained. Of these patients, 2118 (53%) had acute coronary syndrome and were selected as COROGENE cases. The controls for COROGENE cases were selected from the Helsinki-Vantaa region participants of FINRISK 1997, 2002, and 2007 by risk set sampling.12 For each case, two controls matched by sex and birth year and free of acute coronary syndrome were sampled. In total, 2101 cases and 3914 controls (of which 1453 were unique) formed the nal genotyped COROGENE casecontrol sample. The FINRISK 1992, 1997, 2002 and Health 2000 study protocols were approved by the ethics committee of the National Institute for Health and Welfare, the MDC-CC and MPP study protocols by the ethics committee of Lund University, and the COROGENE study protocol by the ethics committee of Helsinki University Hospital, Internal Medicine. All participants provided written informed consent.
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SNP selection and genotyping

We selected SNPs from genome-wide association studies published before June, 2009 in which phenotypes studied were myocardial infarction or coronary heart disease, and association between a SNP and myocardial infarction or coronary heart disease exceeded a genome-wide association threshold (p<510). 13 SNPs from seven reports1319 met these criteria, including 1q41 in MIA3, 1p32 near PCSK9, 1p13 near CELSR2PSRC1SORT1, 2q33 in WDR12, 6p24 in PHACTR1, 9p21 near CDKN2ACDKN2B, 10q11 near CXCL12, 19p13 near LDLR, 21q22 near SLC5A3 MRPS6KCNE2, 3q22 in MRAS, 6q26 in LPA, 12q24 near HNF1A, and 12q24 in SH2B3. Samples were genotyped with the Sequenom platform (iPlex MassARRAY, San Diego, CA, USA) at the Institute for Molecular Medicine Finland FIMM (FINRISK 1992 and 2002), the Wellcome Trust Sanger Institute, UK (Health 2000), or the Broad Institute, USA (FINRISK 1997 and MDC-CC), and with Sequenom or Taqman (Applied Biosystems, Foster City, CA, USA) platforms at Lund University, Sweden (MDC-CC and MPP). COROGENE was genotyped with Illumina 610K chip (Illumina HumanHap 610-Quad SNP array, San Diego, CA, USA) at the Sanger Institute. Genotypes were manually curated with call rates above 97%.

Statistical analysis
We tested associations between SNPs and incident cardiovascular events using Cox proportional hazards
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Genetic risk score quintile 1 (reference) HR (95% CI) for CHD (total n=25 243) FR 1992 FR 1997 FR 2002 Health 2000 Pooled FR 1992 FR 1997 FR 2002 Health 2000 MDC-CC Pooled FR 1992 FR 1997 FR 2002 Health 2000 MDC-CC Pooled 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 097 (065145) 102 (072144) 106 (056199) 093 (051168) 100 (080125) 103 (074143) 088 (066118) 118 (070200) 103 (063169) 083 (057119) 095 (080112) 100 (057173) 102 (063165) 122 (050295) 089 (044181) 093 (058151) 099 (076127) 107 (071160) 117 (084162) 118 (065215) 141 (081245) 117 (094146) 110 (079154) 112 (086146) 143 (087236) 131 (081212) 082 (057118) 110 (093129) 100 (057177) 136 (087211) 089 (035226) 168 (089317) 078 (047128) 111 (087143) 160 (110234) 132 (095183) 143 (079258) 113 (062206) 139 (112172) 135 (098187) 120 (092158) 133 (079223) 130 (080212) 075 (051110) 116 (099136) 160 (095269) 132 (084208) 116 (048281) 100 (048205) 089 (055146) 119 (093153) 154 (106225) 176 (128241) 182 (103322) 151 (087262) 166 (135204) 155 (114212) 154 (118199) 201 (124325) 170 (108268) 113 (080158) 150 (129175) 145 (086244) 187 (121287) 205 (092460) 135 (070262) 103 (065164) 146 (115186) 2 3 4 5

p value for trend

0001 11105 0019 0087 731010 0001 11105 0004 0009 0511 191010 0039 0002 0095 0320 0891 28105

HR (95% CI) for CVD (total n=29 318)

HR (95% CI) for MI (total n=29 318)

HR=hazard ratio. CHD=coronary heart disease. FR=FINRISK. CVD=cardiovascular disease. MDC-CC=Malm Diet and Cancer Cardiovascular cohort. MI=myocardial infarction. *Association tested with Wald test with a Cox proportional hazards model adjusted for sex, LDL and HDL cholesterol, smoking, body-mass index, systolic and diastolic blood pressure, blood pressure treatment and diabetes; age was used as the timescale. Results were combined with xed eects meta-analysis.21

Table 3: Association between genetic risk score and incident coronary heart disease, cardiovascular disease, and myocardial infarction*

models adjusted for traditional risk factors: sex, LDL and HDL cholesterol, current cigarette smoking, body-mass index, systolic and diastolic blood pressure, blood pressure treatment, and prevalent type 2 diabetes. Age was used as the baseline timescale in the Cox models. The proportional hazards assumption was met when tested with scaled Schoenfeld residuals.20 We constructed a multilocus genetic risk score for each individual by summing the number of risk alleles (0/1/2) for each of the 13 SNPs weighted by their estimated eect sizes in the discovery sample (table 2 shows SNP specic weights). Missing genotype values were imputed with the cohort-specic averages of risk allele frequencies. Estimates of association between the genetic risk score divided into quintiles and time to coronary heart disease, cardiovascular disease, and myocardial infarction were calculated with Cox proportional hazards models. For each cohort we calculated 95% CIs for hazard ratios (HRs) and tested the null hypothesis of no linear eect over the quintiles using 1 df Wald test. For prevalent case-control analyses, we analysed individual SNP and quintiles of genetic risk score associations using a logistic regression model adjusted for age and sex. COROGENE data were analysed with conditional logistic regression. Each cohort was analysed separately, and the estimates weighted on the inverse of their standard errors were combined across cohorts with xed eects meta-analysis.21
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To evaluate the potential value of genetic risk score in risk prediction, we used two cohorts (FINRISK 1992 and 1997) and up to 10-year follow-up. First, we compared the receiver operating characteristic (ROC) curves22 of models with and without genetic risk score. The statistical signicance of change in the area under the ROC curve (AUC) between models was tested with the correlated C-index approach.23 Second, we calculated net reclassication improvement (NRI) and clinical NRI24 using the Kaplan-Meier approach with bootstrap-based p values,25 and integrated discrimination improvement (IDI).26 The model calibration was tested with HosmerLemeshow goodness-of-t test. Since each of the 13 reported SNPs has previously been associated with coronary heart disease or myocardial infarction at signicance levels exceeding a stringent genome-wide threshold, in this report we regarded an association to be signicant if a twosided p value was less than 005 (for the same risk allele in the same direction as in the original report). The R statistical package (version 2.11.1) was used for all analyses.

Role of the funding source

The sponsors had no role in the conduct or interpretation of the study. The corresponding author had full access to all data in the study and had nal responsibility for the decision to submit for publication.
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Results
52 726 participants in FINRISK 1992, 1997, and 2002, Health 2000, MDC-CC, MPP, and COROGENE were included in our analysis of prevalent cases versus controls. The total number of prevalent cases of coronary heart disease was 3829 (7%). 30 725 participants from ve cohorts (FINRISK 1992, 1997, and 2002, Health 2000, and MDCCC) were included in the prospective cohort analyses. Median follow-up was 107 years (IQR 67136). 1264 (4%) incident cases of coronary heart disease occurred during follow-up. Table 1 shows background characteristics along with risk factor distributions for the cohorts. In single SNP analyses for prevalent cases, 9p21 near CDKN2ACDKN2B, 21q22 near SLC5A3MRPS6 KCNE2, and 1q41 in MIA3 were associated with coronary heart disease, cardiovascular disease, and myocardial infarction, and 19p13 near LDLR was associated with prevalent coronary heart disease (webappendix p 2). In analysis of incident cases, 6q26 in LPA and 9p21 near CDKN2ACDKN2B were associated with all three endpoints. Additionally, 6p24 in PHACTR1 was associated with incident coronary heart disease and cardiovascular disease, 12q24 in SH2B3 with cardiovascular disease and myocardial infarction, and 21q22 near SLC5A3MRPS6 KCNE2 with coronary heart disease and myocardial infarction (table 2). Overall, seven of the 13 variants were associated in at least one analysis. Genetic risk score was strongly associated with incident coronary heart disease, cardiovascular disease, and myocardial infarction when adjusted for age and sex (webappendix p 3) and traditional risk factors (table 3). Adjustment for traditional risk factors did not substantially change the estimates from the model adjusted for age and sex only. Participants in the top quintile of genetic risk score were estimated to have 166-times increased risk of coronary heart disease compared with those in the bottom quintile (95% CI 135204, p value for linear trend across the quintiles=7310), 150-times increased risk of cardiovascular disease (95% CI 129175, p=1910), and 146-times increased risk of myocardial infarction (95% CI 115186, p=2810). Results were broadly similar when the genetic risk score was divided into tertiles. In models adjusted for traditional risk factors, participants in the top tertile of the genetic risk score were estimated to have a 156-times increased risk of coronary heart disease compared with those in the bottom tertile (95% CI 133183, p value for linear trend across tertiles=2810), 140-times increased risk of cardiovascular disease (95% CI 124158, p=1410), and 134-times increased risk of myocardial infarction (95% CI 112162, p=00015). The genetic risk score conferred risk comparable to other established risk factors such as plasma LDL cholesterol (HR 208, 95% CI 157276, for top vs bottom quintile of LDL cholesterol in FINRISK studies), systolic blood pressure (HR 166, 95% CI 119230, for top vs bottom quintile of systolic blood pressure in FINRISK studies), or
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4 3 Density

Genetic risk score CHD No CHD

LDL cholesterol

04 03 Density 02 01 0

2 1 0 03 04 05 06 07 Weighted score 08

2 4 6 Concentration (mmol/L)

Systolic blood pressure

C-reactive protein

0015 030 Density Density 100 150 200 Blood pressure (mm Hg) 250 0010

020

0005

010

0 2 0 2 Concentration (mg/L) 4

Figure: Distributions at baseline of genetic risk score, LDL cholesterol, systolic blood pressure, and log-transformed C-reactive protein by 10-year incident coronary heart disease event status in FINRISK 1992 and 1997 cohorts Data for C-reactive protein only available in FINRISK 1997. CHD=coronary heart disease.

plasma C-reactive protein (HR 179, 95% CI 115280, for top vs bottom quintile in FINRISK studies). Although the group means were statistically dierent, the distribution of each quantitative risk factor between those who went on to develop coronary heart disease and those who did not was broadly overlapping (gure). Table 4 shows results for prevalent events. The odds ratio for coronary heart disease between the highest and lowest quintile group was 163 (95% CI 124215, p=4810), for cardiovascular disease was 130 (95% CI 115147, p=2610), and for myocardial infarction was 156 (95% CI 138176, p=1210). Additionally, we investigated whether adjustment for a history of early-onset myocardial infarction among rst-degree relatives changed genetic risk score estimates in the FINRISK studies. Family history was signicantly associated with incident events (HR for coronary heart disease 140, 95% CI 120164; webappendix p 4), but the eect of the genetic risk score did not change after adjustment for family history (webappendix p 5). We then investigated whether the genetic risk score association was dominated by rs4977574 at 9p21 near CDKN2BCDKN2A, which is the strongest myocardial infarction locus reported to date. After adjustment for rs4977574, the HR between the highest and lowest genetic risk score quintile was 151 (95% CI 119191)
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Genetic risk score quintile 1 (reference) OR (95% CI) for CHD (total n=29 318) FR 1992 FR 1997 FR 2002 Health 2000 Pooled FR 1992 FR 1997 FR 2002 Health 2000 MDC-CC MPP Pooled FR 1992 FR 1997 FR 2002 Health 2000 MDC-CC MPP COROGENE Pooled 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 230 (093569) 095 (062147) 101 (060171) 156 (076322) 115 (086153) 17 (076378) 089 (06132) 110 (071169) 097 (071133) 178 (089354) 097 (082115) 101 (089115) 283 (100797) 095 (058156) 097 (049193) 144 (054385) 235 (090618) 104 (085128) 123 (103146) 116 (103131) 122 (043340) 086 (056133) 155 (096251) 167 (080347) 120 (090159) 095 (038238) 087 (059128) 136 (090205) 112 (082153) 232 (120448) 111 (094130) 113 (100128) 107 (030373) 076 (046127) 188 (103343) 128 (045361) 239 (092622) 105 (086129) 136 (114162) 122 (108138) 267 (110646) 086 (055135) 132 (079219) 196 (097399) 127 (095170) 249 (117529) 081 (054121) 117 (076181) 104 (076142) 139 (068287) 134 (114157) 124 (109140) 251 (088716) 108 (067176) 152 (080290) 270 (109666) 151 (053427) 138 (113167) 140 (118167) 140 (124158) 213 (084536) 132 (087201) 203 (126325) 158 (078323) 163 (124215) 169 (076380) 126 (087184) 163 (109246) 116 (086157) 158 (079317) 127 (108149) 130 (115147) 279 (098794) 101 (061168) 182 (098338) 158 (061409) 297 (118750) 133 (110162) 175 (148208) 156 (138176) 2 3 4 5

p value for trend

0117 0341 0002 0184 48105 0090 0396 0022 0275 0502 10105 26108 0120 0807 0023 0142 0092 11105 881012 121015

OR (95% CI) for CVD (total n=47 179)

OR (95% CI) for MI (total n=53 741)

OR=odds ratio. CHD=coronary heart disease. FR=FINRISK. CVD=cardiovascular disease. MDC-CC=Malm Diet and Cancer Cardiovascular Cohort. MPP=Malm Preventive Project. MI=myocardial infarction. *Association tested with Wald test with a logistic regression adjusted for age and sex; COROGENE data with matched case-control design was analysed with conditional logistic regression. Results were combined with xed eects meta-analysis.21

Table 4: Association between genetic risk score and prevalent coronary heart disease, cardiovascular disease, and myocardial infarction*

Genetic risk score 05% Cases, by predicted risk* 05% 510% 1020% >20% 05% 510% 1020% >20% 82 (93%) 10 (8%) 0 0 9224 (99%) 179 (12%) 0 0

Genetic risk score 510% 6 (7%) 100 (84%) 9 (5%) 0 121 (1%) 1149 (79%) 120 (14%) 0

Genetic risk Genetic risk score 1020% score >20% 0 9 (8%) 136 (81%) 9 (7%) 0 131 (9%) 687 (80%) 54 (13%) 0 0 22 (13%) 129 (93%) 0 0 49 (6%) 351 (87%)

Non-cases, by predicted risk*

*10-year predicted risk on the basis of traditional risk factors only.

Table 5: Reclassication of individuals in the FINRISK 1992 and 1997 cohorts on the basis of 10-year predicted risk of coronary heart disease with and without genetic risk score

for coronary heart disease. Thus, other variants in the genetic risk score seem to have predictive power beyond the 9p21 locus (webappendix p 5). The AUC estimates for coronary heart disease, cardiovascular disease, and myocardial infarction models with traditional risk factors and genetic risk score were 0872, 0853, and 0881, respectively, and
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they were not signicantly higher than the estimates from the models with only traditional risk factors (0871, p=019; 0853, p=048; and 0880, p=035, for coronary heart disease, cardiovascular disease, and myocardial infarction, respectively). Table 5 and table 6 show risk reclassication results for coronary heart disease. When participants were classied into four risk categories (05%, 510%, 1020%, and >20%) on the basis of their 10-year predicted risk, 22 (13%) participants with coronary heart disease classied at 1020% risk category in the model with traditional risk factors changed their risk category into the greater than 20% category when genetic risk score was included in the model. Similarly, 54 (13%) of the participants without incident coronary heart disease were reclassied from the greater than 20% to the 1020% category. IDI was signicant for coronary heart disease (IDI 0004, p=00006), cardiovascular disease (IDI 0004, p=00004), and for myocardial infarction (IDI 0003, p=003). For coronary heart disease, overall NRI was not signicant (NRI 22%, p=0182), but there was a signicant improvement in reclassication of participants at intermediate risk (clinical NRI 97%, p=310). The calibration of the models with (p=052) and without (p=047) genetic risk score was good.
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Individuals reclassied in table 5 Up Cases Non-cases Total 37 301 Down 28 353

NRI Value 0018 0004 0022 95% CI 0014 to 0051 00001 to 0008 0010 to 0055 p value 0278 0038 0182

Clinical NRI Value 0041 0056 0097 95% CI 0001 to 0081 0044 to 0069 0056 to 0140 p value 0042 51018 3106

NRI=net reclassication improvement. *Comparison of models with and without genetic risk score, adjusted for sex, LDL and HDL cholesterol, smoking, body-mass index, systolic and diastolic blood pressure, blood pressure treatment, and diabetes; age was used as the timescale. NRI for the subset of participants in 520% risk category in the model without genetic risk score, with risk classes 05%, 520%, and >20%.23

Table 6: Net reclassication improvement of genetic risk score for coronary heart disease in the FINRISK 1992 and 1997 cohorts*

Discussion
Using case-control and prospective cohort samples independent from the discovery samples, we sought to validate recently discovered genetic risk factors for coronary heart disease and to estimate the magnitude of risk conferred by these genetic risk factors in the population setting. We found that a genetic risk score including 13 SNPs associated with coronary heart disease or myocardial infarction was associated with risk of prevalent and incident coronary heart disease (even after we accounted for traditional risk factors), and that the 20% of individuals of European ancestry who carry the most risk alleles have a roughly 17-times increased risk of coronary heart disease when compared with those in the lowest quintile. These ndings allow us to draw several conclusions. First, the results from case-control discovery samples do seem to generalise to independent samples, including those from prospective cohorts. Second, the magnitude of eect conferred by genetic risk score (roughly 17-times increased risk in our study) is attenuated when compared with the discovery reports (about 22-times in one report9). Third, even though family history of early-onset cardiovascular disease raised the risk of cardiovascular events by 2540%, adjustment for family history had no eect on the risk estimates because of the genetic risk score. In view of measurement error for family history, and since genetic variants only account for a small proportion of familial risk, this nding might not be unexpected. Finally, although strongly associated with risk of incident coronary heart disease, genetic risk score did not improve risk discrimination when assessed by the C index. This nding of a biomarker being associated with incident disease but yet not improving risk discrimination has been seen with several other biomarkers, including C-reactive protein and B-type natriuretic peptide, among others.27 Since some have argued that the C index might be an insensitive measure of risk discrimination, newer approaches have been developed, including IDI, NRI, and clinical NRI. The tested genetic risk score slightly improved risk prediction for coronary heart disease and myocardial infarction when assessed by IDI and clinical NRI. Overall, these results emphasise the challenge of risk prediction for complex traits on the basis of any single factor.28
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Our combined results show much larger risk dierences between the tails of genetic risk score than in a recently reported follow-up study of women enrolled in a clinical trial.29 The dierence cannot be accounted for by the inclusion of men in our study, since our estimates and predictions are similar for both sexes. Also, nine of the genetic loci in our genetic risk score are the same as in the cardiovascular disease score reported by Paynter and colleagues,29 and the statistical models are comparable. We suggest three potential reasons for the diering results. Paynter and co-workers included a range of endpoints such as strokes (203 of the 777 outcomes) that have not been associated with the genetic variants studied. The inclusion of such outcomes might have diminished their eect estimates. Second, Paynter and colleagues had a small number of myocardial infarction events (199 events) and thus lower statistical power might account for the absence of association in that study. Finally, the women studied were a selected set of health professional participants who volunteered for the clinical trial and as such might not represent the full range of risk seen in the general population. Although we present the largest eort to date to study the association between genetic risk score and risk of incident cardiovascular disease, our results should be interpreted in the context of several potential limitations. Our SNP panel could be incomplete. For example, we did not systematically evaluate SNPs related to cardiovascular risk markers, as Paynter and colleagues did.29 Whereas hundreds of blood biomarkers have been shown to be associated with cardiovascular disease in observational epidemiology research, few have been proven to causally relate to the disease; plasma LDL cholesterol and lipoprotein(a) are notable exceptions. Therefore, we did not consider SNPs that are only associated with cardiovascular risk markers. Our study was undertaken in individuals of Swedish and Finnish descent and hence the results might not be generalisable to others in Europe or to other ancestries. We divided the continuous genetic risk score into ve groups and compared risk between the top and bottom quintiles. Since alternative categorisations are possible we also generated comparisons of the top and bottom third of the distribution, and these results were similar to those seen with quintiles.
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In conclusion, a genetic risk score based on 13 SNPs from genome-wide association studies for myocardial infarction and coronary heart disease was associated with a rst coronary heart disease event, with a relative risk estimate of 17 between the highest and lowest quintiles of genetic risk score. Genetic risk score improved risk reclassication in participants who were at intermediate risk on the basis of traditional risk factors. Whether this genetic risk score will have clinical usefulness remains to be dened in future studies.
Contributors SR, OM, VS, LP, and SK are all senior authors, and planned and managed the project. SR led the data analysis. ET, MO-M, and ASH prepared the data and did the data analyses. CG took part in genotyping. M-LL, JS, and MSN provided COROGENE data. MP and AJ provided clinical guidance and Finnish data. AS took part in data analysis for the Swedish cohorts. KS participated in study planning, did literature searches, and took part in genotyping. SR, ET, ASH, MO-M, VS, and SK wrote the report (with signicant contributions from other authors). Conicts of interest SK has received consultancy fees from Merck and Daiichi Sankyo and grants from Alnylam and Pzer. All other authors declare that they have no conicts of interest. Acknowledgments We wish to dedicate this paper to the memory of Leena Peltonen, who passed away on March 11, 2010. This work was supported by the Wellcome Trust (WT089062/Z/09/Z, WT089061/Z/09/Z), US National Institutes of Health (R01 HL087676), and the Donovan Family Foundation. We thank David Altshuler for guidance and helpful suggestions regarding study design and analyses. LP was supported by the Center of Excellence for Complex Disease Genetics of the Academy of Finland (grants 213506, 129680), the Biocentrum Helsinki Foundation, and The Nordic Center of Excellence in Disease Genetics. VS is supported by the Academy of Finland (grant number 129494), the Finnish Foundation for Cardiovascular Research, and the Sigrid Juselius Foundation. JS was funded by Finnish Foundation for Cardiovascular Research, Special governmental subsidy for health sciences research (EVO). MP was supported by Finnish Foundation for Cardiovascular Research and Finnish Academy Salve Program, grant number 129322 References 1 Kannel WB, Dawber TR, Kagan A, Revotskie N, Stokes J 3rd. Factors of risk in the development of coronary heart diseasesix year follow-up experience. The Framingham Study. Ann Intern Med 1961; 55: 3350. 2 Musunuru K, Kathiresan S. Genetics of coronary artery disease. Annu Rev Genomics Hum Genet 2010; 11: 91108. 3 Healy DG. Case-control studies in the genomic era: a clinicians guide. Lancet Neurol 2006; 5: 70107. 4 Vartiainen E, Laatikainen T, Peltonen M, et al. Thirty-ve-year trends in cardiovascular risk factors in Finland. Int J Epidemiol 2009; 12: 115. 5 Kattainen A, Salomaa V, Hrknen T, et al. Coronary heart disease: from a disease of middle-aged men in the late 1970s to a disease of elderly women in the 2000s. Eur Heart J 2006; 27: 296301. 6 Heistaro S, ed. Methodology report. Health 2000 survey. Helsinki, Finland: National Public Health Institute, 2008. http://www. terveys2000./doc/methodologyrep.pdf (accessed Feb 16, 2010). 7 Berglund G, Elmsthl S, Janzon L, Larsson SA. The Malmo Diet and Cancer Study. Design and feasibility. J Intern Med 1993; 233: 4551. 8 Persson M, Hedblad B, Nelson JJ, Berglund G. Elevated Lp-PLA2 levels add prognostic information to the metabolic syndrome on incidence of cardiovascular events among middle-aged nondiabetic subjects. Arterioscler Thromb Vasc Biol 2007; 27: 141116.

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