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Coronary artery disease (CAD) is a complex disease withboth environmental and heritable contributions.1 To date,genome-wide association studies have yielded common sin-

gle-nucleotide polymorphisms (SNPs) at 50 chromosomal loci

associated with risk of CAD.2 Multilocus genetic risk scores

(GRSs) combining multiple SNPs with modest effects on

cardiovascular risk have been shown to predict incident car-

diovascular events in several prospective cohorts of European

ancestry.3–10 GRSs based on common CAD risk variants havebeen associated with atherosclerotic phenotypes such as

peripheral artery disease,11 carotid intima-media thickness,12

and coronary artery calcium,6 which is an indirect measure of

atherosclerotic burden.

Coronary angiography remains the “gold standard” in quan-

tifying the extent and severity of CAD and thus atherosclerotic

burden. Previous studies have shown that genetic sequence

variants at chromosome 9p21 and in the apolipoprotein(a)

gene ( LPA) not only associate with risk of CAD but also pre-

dict the extent of angiographic CAD, suggesting a role for

these loci in influencing the development and progressionof coronary atherosclerosis.13–15 In this study, we evaluated

the effects of all known common genetic variants associated

© 2015 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org DOI: 10.1161/ATVBAHA.114.304985

Objective—Single-nucleotide polymorphisms predisposing to coronary artery disease (CAD) have been shown to predict

cardiovascular risk in healthy individuals when combined into a genetic risk score (GRS). We examined whether the

cumulative burden of known genetic risk variants associated with risk of CAD influences the development and progression

of coronary atherosclerosis.

Approach and Results—We investigated the combined effects of all known CAD variants in a cross-sectional study of

8622 Icelandic patients with angiographically significant CAD (≥50% diameter stenosis). We constructed a GRS based

on 50 CAD variants and tested for association with the number of diseased coronary arteries on angiography. In models

adjusted for traditional cardiovascular risk factors, the GRS associated significantly with CAD extent (difference per SD

increase in GRS, 0.076; P=7.3×10−17). When compared with the bottom GRS quintile, patients in the top GRS quintile

were roughly 1.67× more likely to have multivessel disease (odds ratio, 1.67; 95% confidence interval, 1.45–1.94). The

GRS significantly improved prediction of multivessel disease over traditional cardiovascular risk factors (χ2 likelihood

ratio 48.1; P


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Bjornsson et al CAD Variants and Extent of CAD 1527

with risk of CAD on the extent of coronary atherosclerosis inpatients with significant CAD on coronary angiography, both

individually and combined in a GRS.

Materials and MethodsMaterials and Methods are available in the online-only Data Supplement.

Results

Characteristics of the Patients

A total of 8622 Icelandic patients with significant angio-

graphic CAD (≥50% diameter stenosis) were included in the

main analysis. Replication was sought in 1853 patients from

the Emory Biobank. All participants were of European ances-try. Characteristics of the study patients are shown in Table 1.

Diabetes mellitus, hypertension, and hyperlipidemia were more

common in patients from the Emory Biobank, whereas Icelandic

patients tended to be younger and were more likely to be cur-

rent smokers. On average, patients from the Emory Biobank had

more extensive coronary disease and were more likely to have

history of myocardial infarction and coronary revascularization.

Association With CAD ExtentAmong the 50 SNPs tested, rs1333049 at chromosome 9p21

and rs10455872 in LPA associated significantly (P


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1528 Arterioscler Thromb Vasc Biol June 2015

Because variants at chromosome 9p21 and LPA have pre-

viously been reported to associate with the extent of angio-

graphic CAD,13–15 we investigated whether the effect of the

GRS was dominated by these variants. After excluding vari-

ants at chromosome 9p21 (rs1333049) and LPA (rs10455872

and rs3798220) from the GRS, the association remained sig-

nificant (P=8.1×10−8

; Table 2). Similar results were obtainedin models additionally adjusted for family history of prema-

ture CAD and in models adjusted for age and sex only (Table

II and Table III in the online-only Data Supplement).

Model PerformanceAs shown in Table 3, the GRS significantly improved predic-

tion of multivessel disease over cardiovascular risk factors

in models including age and sex only (model 1), traditional

cardiovascular risk factors (model 2), and family history

of premature CAD (model 3), as evaluated by likelihood

ratio tests (P


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nonsignificant CAD (


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1530 Arterioscler Thromb Vasc Biol June 2015

chromosome 9p21 has been shown to associate primarily with

coronary atherosclerosis but not myocardial infarction per

se.16,17 Reilly et al18 showed that 12 genome-wide significant

CAD variants did not associate individually with myocardial

infarction among patients with angiographic CAD. Extending

these observations, Patel et al19 showed that a GRS based on 11

CAD risk variants associated with prevalent myocardial infarc-

tion in individuals undergoing coronary angiography but not

when the analysis was restricted to patients with established

angiographic CAD. These studies suggest that genetic risk vari-

ants for CAD, identified in early large-scale genome-wide asso-

ciation studies, relate primarily to coronary atherosclerosis and

may have a minimal role in plaque rupture or thrombosis lead-

ing to acute coronary events. Our findings support the hypothe-

sis that most common CAD variants identified to date influence

the development of coronary atherosclerosis. Although the

extent and overall burden of angiographic CAD unequivocally

increase the risk of adverse cardiovascular events,20,21 it remains

to be established whether genotype scores based on common

CAD variants are predictive of cardiovascular events in patientswith established disease. Large prospective studies are war-

ranted to evaluate the potential clinical use of genomic data as

prognostic factors in patients with established CAD.

Our study should be interpreted in the context of several impor-

tant limitations. First, we used standard coronary angiography to

assess and quantify the extent of coronary atherosclerosis as the

number of coronary arteries with at least 50% diameter steno-

sis. Although angiography is the most widely used and validated

method for CAD assessment, it does not provide information

on the volume or composition of the atherosclerotic plaque.22 In

the Icelandic sample, angiographic data for calculation of more

sophisticated angiographic scoring systems such as the Gensini

score or Duke CAD Severity Index were not available. Second,

the GRS used for replication analyses in the Emory Biobank was

constructed from an available 32 SNP subset of the 50 SNPs and

was therefore not directly comparable with the GRS used for the

main analyses. The main strengths of our study include large

sample sizes and an unbiased nationwide coverage for the selec-

tion of the larger sample of Icelandic patients.

In summary, we have demonstrated that a combined GRS

based on known common genetic risk variants for CAD

is associated with the extent of coronary atherosclerosis

in 2 independent populations of patients with established

angiographic CAD. These findings show that patients with

CAD with a high burden of common genetic variants asso-ciated with CAD risk are more likely to have extensive cor-

onary disease than those who carry a low burden of such

risk variants.

AcknowledgmentsWe thank all the individuals who participated in this study and whosecontribution made this work possible. We also thank our valued col-

leagues who contributed to the data collection and phenotypic char-acterization of clinical samples, as well as genotyping and analysis ofgenome-wide association data.

Sources of Funding

The work was supported by Landspitali University Hospital ResearchFund, Jónína Gísladóttir fund, Bent Scheving Thorsteinsson research

fund, and Research Fund of the Icelandic Society of Cardiology.Emory Cardiovascular Biobank: this work was supported by theAmerican Heart Association (Postdoctoral Fellowship for RSP),National Institutes of Health R01 HL89650-01, Robert W. WoodruffHealth Sciences Center Fund, Emory Heart and Vascular CenterFunds and supported, in part, by National Institutes of Health (NIH)grant UL1 RR025008 from the Clinical and Translational ScienceAward program and NIH grant R24HL085343.

DisclosuresE. Bjornsson, A. Helgadottir, D.F. Gudbjartsson, G. Thorleifsson,U. Thorsteinsdottir, and K. Stefansson are employees of deCODEGenetics/Amgen Inc. The other authors report no conflicts.

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Previous studies have shown that common genetic risk variants for coronary artery disease at chromosome 9p21 and in the lipoprotein(a)

gene associate with angiographic extent of the disease, suggesting a role for these loci in the development of coronary atherosclerosis.

In this study, we show that the cumulative burden of currently known genetic risk variants for coronary artery disease associates signifi-

cantly with the extent of coronary atherosclerosis in 2 independent populations of patients with established angiographic coronary artery

disease. Compared with patients in the bottom quintile of the genetic score, patients in the top quintile were significantly more likely to

have multivessel disease.

Significance


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Arshed A. Quyyumi, Unnur Thorsteinsdottir, Gudmundur Thorgeirsson and Kari StefanssonGudbjartsson, Kristjan Eyjolfsson, Riyaz S. Patel, Nima Ghasemzadeh, Gudmar Thorleifsson,

Eythor Bjornsson, Daniel F. Gudbjartsson, Anna Helgadottir, Thorarinn Gudnason, TomasExtent of Coronary Atherosclerosis

Common Sequence Variants Associated With Coronary Artery Disease Correlate With the

Print ISSN: 1079-5642. Online ISSN: 1524-4636Copyright © 2015 American Heart Association, Inc. All rights reserved.

Greenville Avenue, Dallas, TX 75231is published by the American Heart Association, 7272 Arteriosclerosis, Thrombosis, and Vascular Biology

doi: 10.1161/ATVBAHA.114.3049852015;35:1526-1531; originally published online April 16, 2015; Arterioscler Thromb Vasc Biol.

http://atvb.ahajournals.org/content/35/6/1526

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SUPPLEMENTAL MATERIAL

Common sequence variants associated with coronary artery disease correlate with

the extent of coronary atherosclerosis

Eythor Bjornsson et al.


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Supplementary Table I. Associations of the individual SNPs included in the GRS with CAD extent

SNP Chr Nearest Gene(s)Codedallele

Coded allelefrequency

SNP info* Oddsratio forCAD†

Effect on CAD extent‡

Iceland (n=8,622) Emory (n=1,853) Combined

Iceland Emory Iceland Emory Beta P value Beta P value Beta P value

rs599839 1p13 SORT1 A 0.80 0.79 0.997 0.975 1.111 0.014 0.444 0.112 0.002 0.033 0.037

rs4845625 1q21 IL6R T 0.37 0.998 1.052 -0.016 0.278

rs11206510 1p32 PCSK9 T 0.83 0.82 0.995 0.996 1.062 0.043 0.024 -0.050 0.182 0.024 0.156

rs17114036 1p32 PPAP2B A 0.93 0.91 0.998 0.959 1.112 -0.008 0.782 -0.031 0.554 -0.013 0.602

rs17465637 1q41 MIA3 C 0.69 0.75 0.998 0.988 1.141 0.007 0.637 0.012 0.732 0.008 0.568

rs1561198 2p11VAMP5-VAMP8-

GGCX A 0.46 0.999 1.062 0.016 0.268

rs6544713 2q21 ABCG5-ABCG8 T 0.29 0.999 1.062 0.030 0.050

rs2252641 2q22 ZEB2-AC074093.1 G 0.44 0.998 1.042 -0.001 0.966

rs515135a 2p24 APOB G 0.87 0.82 0.999 0.944 1.072 0.013 0.526 0.014 0.718 0.013 0.466

rs6725887 2q33 WDR12 C 0.13 0.13 0.995 0.982 1.122 0.051 0.015 -0.016 0.715 0.038 0.042

rs9818870 3q22 MRAS T 0.16 0.17 1.000 0.982 1.072 0.021 0.261 -0.030 0.454 0.012 0.489

rs1878406b 4q31 EDNRA T 0.15 0.16 0.999 0.967 1.082 0.041 0.034 0.019 0.634 0.037 0.034

rs7692387 4q32 GUCY1A3 G 0.81 0.998 1.072 -0.023 0.200

rs273909 5q31SLC22A4-SLC22A5

C 0.15 0.999 1.092 0.028 0.158

rs17609940 6p21 ANKS1A G 0.75 0.80 0.999 0.996 1.071 0.025 0.124 0.028 0.45 0.025 0.087

rs10947789 6p21 KCNK5 T 0.78 0.999 1.062 0.019 0.252

rs12190287 6q23 TCF21 C 0.66 0.63 0.995 0.982 1.072 0.020 0.185 0.059 0.050 0.027 0.040

rs12526453 6p24 PHACTR1 C 0.70 0.68 0.999 0.985 1.101 0.030 0.049 0.020 0.545 0.028 0.041

rs2048327 6q25SLC22A3-LPAL2-

LPAG 0.41 0.999 1.062 0.032 0.025

rs3798220 6q25SLC22A3-LPAL2-

LPAC 0.02 0.02 1.000 1.000 1.282 0.079 0.087 0.159 0.110 0.093 0.026

rs10455872 6q25SLC22A3-LPAL2-

LPA G 0.07 0.09 0.984 1.000 1.443

0.160 2.1x10-9

0.187 2.7x10-4

0.165 2.5x10-12

rs4252120 6q26 PLG T 0.73 0.998 1.072 0.011 0.472

rs2023938 7p21 HDAC9 G 0.10 1.000 1.082 0.004 0.859

rs10953541 7q22 BCAP29 C 0.76 0.999 1.084 0.028 0.090

rs11556924 7q32 ZC3HC1 C 0.66 0.62 0.996 0.982 1.092 -0.005 0.750 0.022 0.461 0.001 0.967

rs264 8p21 LPL G 0.85 0.995 1.072 0.014 0.490

rs2954029c 8q24 TRIB1 A 0.51 0.53 0.998 0.956 1.052 0.005 0.725 0.003 0.933 0.005 0.723

rs1333049d 9p21 CDKN2BAS1 C 0.48 0.52 0.996 0.972 1.232 0.046 0.001 0.076 0.012 0.051 5.8x10-5


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rs579459e 9q34 ABO C 0.15 0.22 0.995 0.984 1.072 -0.012 0.540 -0.028 0.426 -0.016 0.358

rs2505083 10p11 KIAA1462 C 0.41 0.44 0.995 0.936 1.062 -0.004 0.773 0.023 0.444 0.001 0.945

rs2047009 10q11 CXCL12 C 0.53 0.999 1.052 -0.015 0.292

rs501120f 10q11 CXCL12 A 0.89 0.88 1.000 0.935 1.072 -0.043 0.053 -0.037 0.416 -0.042 0.036

rs1412444 10q23 LIPA T 0.37 0.35 0.997 0.984 1.094 0.014 0.316 0.036 0.242 0.018 0.160

rs12413409g 10q24CYP17A1-

CNNM2-NT5C2G 0.93 0.92 0.998 0.998 1.102 -0.008 0.757 0.092 0.087 0.012 0.621

rs974819 11q22 PDGFD A 0.29 0.997 1.072 0.018 0.252

rs964184 11q23ZNF259-APOA5-

APOA1G 0.13 0.15 0.999 0.985 1.131 0.008 0.715 0.034 0.405 0.013 0.483

rs3184504 12q24 SH2B3 T 0.39 0.49 0.998 0.972 1.072 0.009 0.547 0.045 0.120 0.016 0.220

rs9319428 13q12 FLT1 A 0.34 0.998 1.062 0.020 0.186

rs4773144h 13q34 COL4A1-COL4A2 G 0.44 0.45 0.990 0.983 1.072 -0.003 0.836 -0.005 0.875 -0.003 0.799

rs9515203 13q34 COL4A1-COL4A2 T 0.73 0.992 1.082 0.044 0.005

rs2895811 14q32 HHIPL1 C 0.47 0.44 0.994 0.968 1.062 0.032 0.023 0.031 0.294 0.032 0.012

rs3825807 15q25 ADAMTS7 A 0.58 0.58 0.998 0.962 1.081 0.028 0.052 0.048 0.116 0.031 0.015

rs17514846 15q26 FURIN-FES A 0.47 0.996 1.062 -0.003 0.824

rs12936587 17p11RAI1-PEMT-

RASD1G 0.60 0.56 0.998 0.995 1.062 0.016 0.260 -0.046 0.116 0.004 0.751

rs216172 17p13 SMG6 C 0.39 0.37 0.998 0.968 1.071 0.001 0.957 0.016 0.594 0.004 0.785

rs46522 17q21 UBE2Z T 0.54 0.53 0.999 0.995 1.061 0.000 0.994 -0.017 0.550 -0.003 0.801

rs1122608 19p13 LDLR G 0.79 0.75 0.996 0.970 1.102 0.042 0.015 0.041 0.238 0.042 6.9x10-3

rs2075650 19q13 ApoE-ApoC1 G 0.18 0.15 0.995 0.963 1.112 0.036 0.054 0.125 0.002 0.051 2.5x10-3

rs445925 19q13 ApoE-ApoC1 C 0.92 0.992 1.132 0.002 0.929

rs9982601 21q22Gene desert

(KCNE2 )T 0.14 0.14 0.993 0.951 1.132 0.013 0.534 0.010 0.82 0.012 0.509

*Imputation information score for the imputed SNPs (Iceland) and SNP call rates for the directly genotyped SNPs (Emory).†For each of the SNPs, the odds ratio for the risk of CAD was obtained from a previously published meta-analysis of genome-wide association studies (as

referenced in the table). In the GRSs, each SNP was weighted using the natural logarithm of the respective odds ratio.‡Association analyses were performed using multiple linear regression adjusting for age, sex, hyperlipidemia, diabetes, hypertension, current and formersmoking. The outcome variable was the number of diseased coronary vessels with at least 50% stenosis on coronary angiography (range, 1-4). Resultsfrom the Icelandic and Emory samples were combined using fixed-effects inverse variance-weighted meta-analysis.ars562338 is a proxy for rs515135 (r 2=0.98) in the Emory Biobank samplebrs6842241 is a proxy for rs1878406 (r 2=0.91) in the Emory Biobank samplecrs2954021 is a proxy for rs2954029 (r 2=0.79) in the Emory Biobank sampledrs10757278 is a proxy for rs1333049 (r 2=0.95) in the Emory Biobank sampleers651007 is a proxy for rs579459 (r 2=0.99) in the Emory Biobank samplef rs1746048 is a proxy for rs501120 (r 2=0.96) in the Emory Biobank samplegrs12411886 is a proxy for rs12413409 (r 2=1.00) in the Emory Biobank samplehrs3809346 is a proxy for rs4773144 (r 2=0.99) in the Emory Biobank sample


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Supplementary Table II. Association of the GRS with the number of diseased coronary arteries on

coronary angiography, adjusted for age and sex only

No. ofSNPs

Difference per SDincrease (95% CI)

Standarderror

P value

Contrast top versus

bottom GRS quintile

OR for multivesseldisease* (95% CI)

Iceland (n=8,622)

Full GRS 50 0.077 (0.059-0.095) 0.0091 3.2x10-17 1.67 (1.45-1.93)

Full GRS excluding9p21 and LPA

47 0.050 (0.032-0.068) 0.0091 3.7x10-8 1.33 (1.16-1.54)

Restricted GRS 32 0.073 (0.055-0.090) 0.0091 2.2x10-15 1.54 (1.34-1.77)

Emory Biobank (n=1,853)

GRS 32 0.113 (0.072-0.153) 0.021 5.0x10-8 2.08 (1.51-2.88)GRS excluding 9p21and LPA

29 0.067 (0.027-0.108) 0.021 0.0011 1.50 (1.09-2.05)

GRS indicates genetic risk score; SD, standard deviation; OR, odds ratio; CI, confidence interval. Associations were tested using linear and logistic regression models adjusted for age and sex.*Multivessel disease was defined as having at least two coronary arteries with ≥50% stenosis oncoronary angiography.


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Supplementary Table III. Association of the GRS with the number of diseased coronary arteries on

coronary angiography, adjusted for age, sex, four cardiovascular risk factors and family history

No. ofSNPs

Difference per SDincrease (95% CI)

Standarderror

P value

Contrast top versusbottom GRS quintile

OR for multivesseldisease* (95% CI)

Iceland (n=8,622)

Full GRS 50 0.072 (0.054-0.089) 0.0090 3.0x10-15 1.63 (1.41-1.89)

Full GRS excluding9p21 and LPA

47 0.046 (0.028-0.064) 0.0090 3.5x10-7 1.30 (1.12-1.50)

Restricted GRS 32 0.067 (0.050-0.085) 0.0090 1.1x10-13 1.51 (1.31-1.75)

Emory Biobank (n=1,853)

GRS 32 0.115 (0.075-0.155) 0.021 2.7x10-8 2.08 (1.50-2.90)

GRS excluding 9p21

and LPA 29 0.070 (0.030-0.111) 0.021 6.9x10-4

1.51 (1.09-2.09)GRS indicates genetic risk score; SD, standard deviation; OR, odds ratio; CI, confidence interval.

Associations were tested using linear and logistic regression models adjusted for age, sex,hyperlipidemia, diabetes, hypertension, current and former smoking and family history of prematureCAD (family history of CAD at any age in the Emory Biobank).*Multivessel disease was defined as having at least two coronary arteries with ≥50% stenosis oncoronary angiography.


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Supplementary Table IV. Characteristics of the Icelandic patients (n=8,622) by quintiles

of the GRS

Characteristic Q1 Q2 Q3 Q4 Q5 P value*

n 1725 1724 1725 1724 1724

Age 66.0 64.8 63.6 64.1 63.6 2.5x10-13

Male sex 74.8% 78.2% 77.0% 74.4% 71.0% 8.4x10-5

Diabetes 12.1% 12.3% 9.7% 12.4% 10.6% 0.082

Hypertension 57.4% 53.4% 53.2% 53.8% 53.7% 0.098

Hyperlipidemia 48.8% 47.5% 50.8% 51.6% 52.8% 2.6x10-3

Current smoker 26.3% 28.2% 30.0% 25.9% 26.6% 0.86

Former smoker 50.1% 46.4% 46.0% 49.0% 46.6% 0.051

Prior MI 29.9% 28.5% 29.5% 30.0% 30.7% 0.32

Prior PCI 4.2% 3.6% 3.0% 4.8% 5.2% 0.11

Prior CABG 5.8% 5.7% 7.2% 8.9% 10.6% 4.1x10-11

Family history 36.1% 40.0% 45.0% 45.9% 48.5% 2.3x10-17

Multivessel disease 56.4% 61.8% 60.5% 60.8% 65.1% 1.2x10-6

*Unadjusted P-values calculated using linear and logistic regression considering the GRS asa continuous variable.


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Supplementary Table V. Characteristics of the Emory Biobank patients (n=1,853) byquintiles of the GRS

Characteristic Q1 Q2 Q3 Q4 Q5 P value*

n 372 370 370 371 370

Age 66.8 65.1 66.2 65.2 64.7 0.017

Male sex 74.2% 73.0% 72.4% 71.7% 77.6% 0.49

Diabetes 35.8% 29.7% 35.1% 29.1% 27.6% 0.029

Hypertension 66.9% 73.2% 73.2% 70.6% 69.5% 0.53

Hyperlipidemia 71.8% 73.0% 76.2% 73.3% 78.6% 0.062

Current smoker 14.0% 16.5% 14.9% 14.6% 13.8% 0.94

Former smoker 47.0% 44.3% 45.9% 49.1% 53.2% 0.046

Prior MI 47.3% 44.9% 53.2% 47.7% 53.0% 0.058

Prior PCI 55.6% 51.6% 61.9% 57.1% 61.1% 0.031

Prior CABG 28.0% 24.9% 36.8% 28.6% 38.4% 0.0019

Family history 43.3% 41.4% 46.5% 47.2% 45.1% 0.16

Multivessel disease 61.8% 61.4% 70.8% 69.3% 76.8% 7.2x10-7

*Unadjusted P-values calculated using linear and logistic regression considering the GRS as acontinuous variable.


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Supplementary Table VI. Association of the GRS with the number of diseased coronary arteries

among all individuals undergoing coronary angiography, including those with non-significant CAD

(


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Supplementary Figure. Adjusted odds ratios for multivessel disease by quintiles of the

genetic risk score (GRS) in the Icelandic (black) and Emory Biobank (grey) samples,

including those with non-significant CAD (


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References

1. Schunkert H, König IR, Kathiresan S, et al. Large-scale association analysis identifies

13 new susceptibility loci for coronary artery disease. Nat Genet . 2011;43:333 –338.

2. Deloukas P, Kanoni S, Willenborg C, et al. Large-scale association analysis identifies

new risk loci for coronary artery disease. Nat Genet. 2013;45:25 –33.

3. The IBC 50K CAD Consortium. Large-scale gene-centric analysis identifies novel

variants for coronary artery disease. PLoS Genet . 2011;7:e1002260.

4. The Coronary Artery Disease (C4D) Genetics Consortium. A genome-wide

association study in Europeans and South Asians identifies five new loci for coronary

artery disease. Nat Genet . 2011;43:339 –344.


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Materials and Methods

Icelandic Patients

We identified Icelandic patients with established coronary artery disease (CAD) from

nationwide clinical registries of angiography and percutaneous coronary interventions

(PCI) at Landspitali University Hospital in Reykjavik, the sole center for invasive

cardiology in Iceland. We obtained data for patients undergoing coronary angiography

for any indication from January 1, 1987 to December 31, 2012. The data were obtained

from three registries. First, a prospective registry of all PCI procedures performed in

Iceland between January 1, 1987 and December 31, 2006. Second, the Swedish

Coronary Angiography and Angioplasty Registry (SCAAR), which holds information on

all coronary angiographies and PCI procedures performed in Iceland since January 1,

2007.1

Third, a registry of coronary artery bypass grafting procedures performed inIceland, which holds data on patients undergoing pre-procedural angiography between

January 1, 2002 and December 31, 2011.2 For patients with multiple procedures we

used the earliest record. Patients with significant angiographic CAD (at least 50%

diameter stenosis) and genotype information available were eligible for inclusion. In

total, 12,630 individuals were identified, of which 9,747 had significant angiographic

CAD. Of these patients, 8,662 had genotype information available. Patients with

incomplete or missing data on traditional cardiovascular risk factors (age, sex,

hyperlipidemia, diabetes, hypertension, current and former smoking) were excluded

(n=40). After exclusion, a total of 8,622 patients with genotype information remained

and were included in this study. The study was approved by the National Bioethics

Committee and the Data Protection Authority in Iceland. All subjects provided written

informed consent.

Emory Biobank Patients

Participants in the Emory Cardiovascular Biobank Study were enrolled at Emory

University Hospital and its affiliated centers in Atlanta, Georgia, USA. The Emory

Cardiovascular Biobank Study was designed to investigate the association of

biochemical and genetic factors with CAD in subjects undergoing cardiac

catheterization. Full details have been published previously.3 After restricting the study

to patients of self-reported Caucasian ancestry with significant angiographic CAD

(≥50% stenosis), 1,853 patients were included. The study was approved by the

Institutional Review Board at Emory University, Atlanta, GA, USA. All subjects provided

written informed consent.

Coronary Angiography

All study patients had previously undergone coronary angiography performed for


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clinical indications. Angiographic data were obtained from aforementioned registries.

In these registries, coronary angiograms were evaluated and scored by interventional

cardiologists at the time of procedure without knowledge of patients’ genotype status.

Significant angiographic CAD was consistently defined as luminal diameter stenosis of

at least 50% in any of the major coronary arteries (the left main coronary artery, the

left anterior descending artery, the circumflex artery or the right coronary artery). In

both samples, the extent of CAD was determined using a modified version of the

Coronary Artery Surgery Study (CASS) score4 which has been described previously3.

It is defined as the number of major coronary arteries with at least 50% stenosis; left

main stenosis of at least 50% is scored as a single-vessel disease. The total score

ranges from 1 to 4 and corresponds to one-, two-, three- or four-vessel coronary

disease. Multivessel disease was defined as at least 50% stenosis in two or more major

coronary arteries. Patients with non-significant angiographic CAD (


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were used as weights (odds ratios ranged from 1.04 to 1.44). For SNPs reported in

more than one study, the effect size reported by the study comprising the largest

number of study participants was chosen.

Genotyping of all samples was carried out at deCODE Genetics in Reykjavik,

Iceland. In the Icelandic sample, whole-genome sequencing of 2,230 Icelanders was

used to inform the imputation of the 50 SNPs into 95,085 Icelanders genotyped with

Illumina SNP chips, using long-range phasing-based imputation.5,10 Additionally,

familial imputation methods were used to impute the variants into un-genotyped

relatives of chip-genotyped Icelanders, yielding a total sample size of 8,622 Icelandic

study patients with genotype information. The imputation information score for the

SNPs ranged from 0.984 to 1.000.

In the replication sample (Emory Biobank), analyses were based on an availablesubset comprising 32 SNPs previously genotyped as part of ongoing research

collaborations (Table I in the online-only Data Supplement). In this subset, proxy SNPs

were used for SNPs at 2p24, 4q31, 8q24, 9p21, 9q34, 10q11, 10q24 and 13q34 (r 2

between 0.79 and 1.00). Information on the remainder of SNP genotyped in the

Icelandic sample was not available for the Emory Biobank sample. Samples from the

Emory Biobank were genotyped using the Centaurus (Nanogen) platform at deCODE

genetics in Reykjavik, Iceland. SNP call rate ranged from 0.935-1.000.

Genetic Risk Scores

For each patient, we calculated a weighted genetic risk score (GRS) by adding the

product of the number of risk alleles for each of the SNPs and the natural logarithm of

the effect size (odds ratio for CAD) reported in the reference studies. As 1 SNP at

chromosome 9p21 and 2 SNPs LPA gene have previously been reported to associate

with extent of angiographic CAD3,11,12, we additionally examined a GRS restricted to

SNPs with no reported association with CAD extent. In the Icelandic sample, the

weighted GRS was calculated based on 50 SNPs whereas, in the Emory Biobank

sample, the GRS was calculated based on the available subset of 32 SNPs (Table I in

the online-only Data Supplement). For comparison, we generated a GRS restricted to

these 32 SNPs for the Icelandic sample. In all analyses, GRSs were standardized to a

mean of 0 and a standard deviation of 1.

Statistical Methods

We used linear regression to test the association of individual SNPs with CAD extent.

The outcome variable was the number of diseased coronary arteries with at least 50%

stenosis on coronary angiography. Estimates were adjusted for traditional

cardiovascular risk factors (age, sex, hyperlipidemia, diabetes, hypertension, current


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and former smoking). Results for SNPs genotyped in both the Icelandic and Emory

Biobank samples were combined using fixed-effects inverse variance-weighted meta-

analysis. In single-SNP analyses, we considered a P value of less than 0.001 to be

significant to account for the testing of 50 SNPs.

Association analyses of the GRS with extent of CAD were performed using

multivariate linear regression, adjusting for traditional cardiovascular risk factors. In a

further analysis, family history of CAD was included as a covariate. To further illustrate

the association with CAD extent, we divided the study patients into quintiles according

to the GRS and compared the association of the top versus the bottom quintiles with

multivessel disease (≥2 diseased coronary arteries) in multivariate logistic regression

models adjusted for traditional cardiovascular risk factors. In addition, as early age at

angiography may be an indicator of accelerated development of coronary

atherosclerosis, we tested whether the GRS associated with age at angiography. We

tested whether the GRS provided incremental value to prediction of the presence of

multivessel disease over known cardiovascular risk factors using the likelihood-ratio χ2

test for nested models. We evaluated improvement in discrimination by comparing the

areas under the receiver-operating characteristic curve (C-statistic) with and without

the GRS. An increase in the C-statistic reflects improved discrimination between

patients with and without multivessel disease. In addition, we calculated the integrated

discrimination improvement13.

The main analyses were restricted to patients with significant angiographic CAD.

In a separate analysis, we also included data from individuals with non-significant CAD

(


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3. Helgadottir A, Gretarsdottir S, Thorleifsson G, et al. Apolipoprotein(a) geneticsequence variants associated with systemic atherosclerosis and coronaryatherosclerotic burden but not with venous thromboembolism. J Am Coll Cardiol .2012;60:722 –729.

4. Ringqvist I, Fisher LD, Mock M, Davis KB, Wedel H, Chaitman BR, Passamani

E, Russell RO, Alderman EL, Kouchoukas NT, Kaiser GC, Ryan TJ, Killip T, FrayD. Prognostic value of angiographic indices of coronary artery disease from theCoronary Artery Surgery Study (CASS). J Clin Invest. 1983;71:1854 –66.

5. Kong A, Masson G, Frigge ML, et al. Detection of sharing by descent, long-rangephasing and haplotype imputation. Nat Genet . 2008;40:1068 –1075.

6. Schunkert H, König IR, Kathiresan S, et al. Large-scale association analysisidentifies 13 new susceptibility loci for coronary artery disease. Nat Genet .2011;43:333 –338.

7. The Coronary Artery Disease (C4D) Genetics Consortium. A genome-wide

association study in Europeans and South Asians identifies five new loci forcoronary artery disease. Nat Genet . 2011;43:339 –344.

8. Deloukas P, Kanoni S, Willenborg C, et al. Large-scale association analysisidentifies new risk loci for coronary artery disease. Nat Genet . 2013;45:25 –33.

9. The IBC 50K CAD Consortium. Large-scale gene-centric analysis identifiesnovel variants for coronary artery disease. PLoS Genet . 2011;7:e1002260.

10. Styrkarsdottir U, Thorleifsson G, Sulem P, et al. Nonsense mutation in the LGR4gene is associated with several human diseases and other traits. Nature.2013;497:517 –520.

11. Dandona S, Stewart AFR, Chen L, Williams K, So D, O’Brien E, Glover C, LemayM, Assogba O, Vo L, Wang YQ, Labinaz M, Wells GA, McPherson R, RobertsR. Gene dosage of the common variant 9p21 predicts severity of coronary arterydisease. J Am Coll Cardiol . 2010;56:479 –86.

12. Patel RS, Su S, Neeland IJ, Ahuja A, Veledar E, Zhao J, Helgadottir A, Holm H,Gulcher JR, Stefansson K, Waddy S, Vaccarino V, Zafari AM, Quyyumi AA. Thechromosome 9p21 risk locus is associated with angiographic severity andprogression of coronary artery disease. Eur Heart J . 2010;31:3017 –3023.

13. Pencina MJ, D’Agostino RB, Vasan RS. Evaluating the added predictive abilityof a new marker: from area under the ROC curve to reclassification and beyond.

Stat Med . 2008;27:157 –72.

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