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Common Sequence Variants Associated With

Coronary Artery Disease Correlate With the Extent


of Coronary Atherosclerosis
Eythor Bjornsson, Daniel F. Gudbjartsson,* Anna Helgadottir,* Thorarinn Gudnason,
Tomas Gudbjartsson, Kristjan Eyjolfsson, Riyaz S. Patel, Nima Ghasemzadeh,
Gudmar Thorleifsson, Arshed A. Quyyumi, Unnur Thorsteinsdottir, Gudmundur Thorgeirsson,
Kari Stefansson
ObjectiveSingle-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 ResultsWe 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=7.31017). 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, 1.451.94). The
GRS significantly improved prediction of multivessel disease over traditional cardiovascular risk factors (2 likelihood
ratio 48.1; P<0.0001) and modestly improved discrimination, as estimated by the C-statistic (without GRS versus with
GRS, 64.0% versus 64.8%) and the integrated discrimination improvement (0.52%). Furthermore, the GRS associated
with an earlier age at diagnosis of angiographic CAD. These findings were replicated in an independent sample from the
Emory Biobank study (n=1853).
ConclusionsWhen combined into a single GRS, known genetic risk variants for CAD contribute significantly to the
extent of coronary atherosclerosis in patients with significant angiographic disease.(Arterioscler Thromb Vasc Biol.
2015;35:1526-1531. DOI: 10.1161/ATVBAHA.114.304985.)
Key Words: atherosclerosis coronary disease genetics

oronary artery disease (CAD) is a complex disease with


both environmental and heritable contributions.1 To date,
genome-wide association studies have yielded common single-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 cardiovascular events in several prospective cohorts of European
ancestry.310 GRSs based on common CAD risk variants have
been 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 quantifying 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 predict the extent of angiographic CAD, suggesting a role for
these loci in influencing the development and progression
of coronary atherosclerosis.1315 In this study, we evaluated
the effects of all known common genetic variants associated

Received on: November 14, 2014; final version accepted on: March 30, 2015.
From the Faculty of Medicine (E.B., A.H., T.G., U.T., G.T., K.S.) and Department of Engineering and Natural Sciences (D.F.G.), University of Iceland,
Reykjavik, Iceland; deCODE Genetics, Reykjavik, Iceland (E.B., D.F.G., A.H., G.T., U.T., K.S.); Department of Medicine (T.G., K.E., G.T.), and
Department of Surgery (T.G.), Landspitali University Hospital, Reykjavik, Iceland; Division of Cardiology, Department of Medicine, Emory University
School of Medicine, Atlanta, GA (R.S.P., N.G., A.A.Q.); and Institute of Cardiovascular Sciences, University College London, London, United Kingdom
(R.S.P.).
*These authors contributed equally to this article.
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.114.304985/-/DC1.
Correspondence to Kari Stefansson, MD, PhD, deCODE Genetics, Sturlugata 8, 101 Reykjavik, Iceland. E-mail kstefans@decode.is
2015 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org

DOI: 10.1161/ATVBAHA.114.304985

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Bjornsson et al CAD Variants and Extent of CAD 1527

Nonstandard Abbreviations and Acronyms


CAD
GRS
SNP

coronary artery disease


genetic risk score
single-nucleotide polymorphism

with risk of CAD on the extent of coronary atherosclerosis in


patients with significant CAD on coronary angiography, both
individually and combined in a GRS.

Materials and Methods


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

Results
Characteristics of the Patients
A total of 8622 Icelandic patients with significant angiographic 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 ancestry. Characteristics of the study patients are shown in Table1.
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 current 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 Extent


Among the 50 SNPs tested, rs1333049 at chromosome 9p21
and rs10455872 in LPA associated significantly (P<0.001)

with the number of coronary arteries with at least 50% diameter stenosis in a combined analysis of the samples, adjusting
for traditional cardiovascular risk factors: age, sex, hyperlipidemia, diabetes mellitus, hypertension, current smoking, and
former smoking (Table I in the online-only Data Supplement).
Figure1 illustrates the linear relationship between the magnitude of the effects of individual SNPs on CAD extent and their
respective odds ratio for the risk of CAD, previously reported
in meta-analyses of genome-wide association studies (Table I
in the online-only Data Supplement).
The combined GRS was strongly associated with CAD
extent when adjusting for traditional cardiovascular risk factors (difference per SD increase in GRS, 0.076; P=7.31017).
Estimates in models adjusting for traditional cardiovascular
risk factors did not differ substantially from those in models
adjusted for age and sex only (Table II in the online-only Data
Supplement), and the association remained significant after
further consecutive adjustment for family history of premature
CAD (difference per SD increase in GRS, 0.072; P=3.01015;
Table III in the online-only Data Supplement). To further illustrate this relationship, we divided patients into quintiles based
on the GRS and compared the proportion of patients with
multivessel disease (2 coronary arteries with at least 50%
diameter stenosis) between the top and the bottom quintiles
(Figure2). Roughly 65% of patients in the top quintile had
multivessel disease compared with 56% of patients in the bottom quintile (Table IV in the online-only Data Supplement).
Thus, patients who were in the top quintile of the GRS were
1.67 more likely to have multivessel disease compared with
patients in the bottom quintile (adjusted odds ratio, 1.67; 95%
confidence interval, 1.451.94; Table2).

Table 1. Characteristics of the Patients


Characteristics
Age, y

Iceland (n=8622)

Emory Biobank (n=1853)

P Value*
<0.001

64.4 (10.7)

65.6 (10.6)

Male sex, %

75.1

73.8

0.24

Diabetes mellitus, %

11.4

31.5

<0.001

Hypertension, %

54.3

70.7

<0.001

Hyperlipidemia, %

50.3

74.6

<0.001

Current smoker, %

27.4

14.7

<0.001

Former smoker, %

47.6

47.9

0.83

Previous MI, %

29.7

49.2

<0.001

Previous PCI, %

4.2

57.5

<0.001

Previous CABG, %

7.6

31.3

<0.001

Family history, %

43.1

44.7

0.21

1.94 (0.88)

2.10 (0.91)

<0.001

1-vessel disease

No. of diseased vessels

39.1

32.0

<0.001

2-vessel disease

30.3

31.6

0.30

3-vessel disease

28.1

31.3

0.007

4-vessel disease

2.5

5.1

<0.001

Data are presented as percentages or means (SD). CABG indicates coronary artery bypass grafting; MI, myocardial infarction;
and PCI, percutaneous coronary intervention.
*P values for continuous variables were calculated using Student t test. P values for categorical variables were calculated
using the 2 test.
Total number of coronary arteries with at least 50% stenosis on coronary angiography (left anterior descending, circumflex,
the right main coronary artery, and the left main coronary artery).

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1528 Arterioscler Thromb Vasc Biol June 2015

Model Performance

Figure 1. The effects of 50 single-nucleotide polymorphisms


(SNPs) on the extent of coronary artery disease (CAD), expressed
as the increase in number of diseased coronary arteries (with
at least 50% stenosis) per SNP risk allele, plotted against their
respective effect on CAD risk (odds ratio), previously reported in
meta-analyses of genome-wide association studies (Table I in the
online-only Data Supplement for references). Combined effect
sizes in the Icelandic and Emory Biobank samples are presented
where available. The solid line denotes best linear fit, the dashed
lines indicate 95% confidence limits.

Because variants at chromosome 9p21 and LPA have previously been reported to associate with the extent of angiographic CAD,1315 we investigated whether the effect of the
GRS was dominated by these variants. After excluding variants at chromosome 9p21 (rs1333049) and LPA (rs10455872
and rs3798220) from the GRS, the association remained significant (P=8.1108; Table2). Similar results were obtained
in models additionally adjusted for family history of premature CAD and in models adjusted for age and sex only (Table
II and Table III in the online-only Data Supplement).

Figure 2. Adjusted odds ratios for multivessel disease by quintiles of the genetic risk score (GRS) in the Icelandic (black) and
Emory Biobank (gray) samples. Odds ratios are referenced to
the bottom GRS quintile and presented with 95% confidence
intervals.

As shown in Table3, the GRS significantly improved prediction 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<0.0001). To estimate the improvement in discrimination, we compared the C-statistics (area under the
receiver-operating-characteristic curve) for models with and
without the GRS. The C-statistics for the models without the
GRS ranged from 62.9% to 64.8% (Table3). Addition of
the GRS to the models resulted in modest increases in the
C-statistic, ranging from 0.6% to 0.8% (Table3). Similarly,
the integrated discrimination improvement ranged from
0.46% to 0.53%, indicating a marginal improvement in discrimination for multivessel disease with the addition of the
GRS (Table3).

Association With Age at Angiography


The GRS associated significantly with age at angiography
when adjusting for sex, hyperlipidemia, diabetes mellitus,
hypertension, current smoking, and former smoking (difference per SD increase in GRS, 0.90 years; P=7.21017).
This association persisted when variants at chromosome
9p21 and LPA were excluded from the GRS (difference per
SD increase in GRS, 0.64 years; P=2.5109). Patients
in the top quintile of the GRS were on average 2.4 years
younger than patients in the bottom quintile (63.6 years compared with 66.0 years), as shown in Table IV in the onlineonly Data Supplement.

Replication
In the Emory Biobank sample, the GRS based on 32 SNPs
was significantly associated with CAD extent (difference
per SD increase in GRS, 0.115; P=2.6108; Table2). In the
Emory Biobank sample, 77% of patients in the top quintile
of the GRS had multivessel disease compared with 62% of
patients in the bottom quintile (Table V in the online-only
Data Supplement), corresponding to an adjusted odds ratio
of 2.08 (95% confidence interval, 1.502.90; Table2). As
shown in Table3, The GRS significantly improved prediction of multivessel disease over cardiovascular risk factors
and modestly improved discrimination, as estimated by the
increase in the C-statistics for the models with the addition
of the GRS (ranging from 1.7% to 1.9%) and the integrated
discrimination improvement (1.5% for all models). The GRS
associated with age at angiography when adjusting for sex,
hyperlipidemia, diabetes mellitus, hypertension, current
smoking, and former smoking (difference per SD increase
in GRS, 0.60 years; P=0.011), but the association was not
significant when variants at 9p21 and LPA were excluded
from the GRS (difference per SD increase in GRS, 0.44
years; P=0.062).

Association With CAD Extent When Including


Individuals With Nonsignificant CAD
As expected, the association between the GRS and CAD
extent was even more pronounced when individuals with

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Bjornsson et al CAD Variants and Extent of CAD 1529


Table 2. Association of the GRS With the Number of Diseased Coronary Arteries on Coronary Angiography
Contrast Top vs Bottom GRS Quintile

No. of SNPs

Difference Per SD Increase


(95% CI)

SE

P Value

OR for Multivessel Disease* (95% CI)

Full GRS

50

0.076 (0.0580.094)

0.0091

7.31017

1.67 (1.451.94)

Full GRS excluding 9p21 and LPA

47

0.049 (0.0310.066)

0.0091

8.110

1.32 (1.141.52)

Restricted GRS

32

0.072 (0.0540.089)

0.0091

3.21015

1.55 (1.341.78)

GRS

32

0.115 (0.0750.155)

0.021

2.6108

2.08 (1.502.90)

GRS excluding 9p21 and LPA

29

0.070 (0.0290.110)

0.021

7.310

1.50 (1.092.08)

Iceland (n=8622)
8

Emory Biobank (n=1853)


4

Associations were tested using linear and logistic regression models adjusted for traditional cardiovascular risk factors (age, sex, hyperlipidemia, diabetes mellitus,
hypertension, current smoking, and former smoking). CI indicates confidence interval; GRS indicates genetic risk score; OR, odds ratio; and SNP, single-nucleotide
polymorphisms.
*Multivessel disease was defined as having at least 2 coronary arteries with 50% stenosis on coronary angiography.

nonsignificant CAD (<50% stenosis) were also included


(Table VI and Figure I in the online-only Data Supplement).

Discussion
In this study, we demonstrate that a genetic score based on known
common CAD risk variants is strongly associated with the extent
of coronary atherosclerosis in patients with established angiographic CAD. This association is independent of traditional cardiovascular risk factors and family history of CAD. We found
that the GRS significantly improved prediction of multivessel
disease over established cardiovascular risk factors although
the improvement in discrimination was modest. Compared
with patients in the bottom quintile of the GRS, patients in the
top quintile were roughly 1.67 (Iceland) and 2.08 (Emory
Biobank) more likely to have multivessel disease. Furthermore,
we found that the GRS associated with younger age at angiography, consistent with an earlier disease onset for individuals with
a high burden of common genetic risk variants for CAD.
Previously, a genetic variant at chromosome 9p21 and 2
variants at LPA were shown to influence the extent of coronary

atherosclerosis as determined by coronary angiography.1315


In keeping with these findings, these variants showed the
strongest association with CAD extent in the present study.
Because of their large effect sizes on the risk of CAD, they
were assigned the greatest weights in the GRS. To evaluate
whether the GRS was dominated by these loci, we excluded
them from the GRS in a separate analysis. We found that the
GRS restricted to variants outside chromosome 9p21 and LPA
was also significantly associated with CAD extent, despite
showing a somewhat weaker effect than that of the unrestricted
GRS. These results show that currently known genetic risk
variants for CAD, not previously associated with CAD extent,
collectively associate with extent of coronary atherosclerosis.
This suggests that many of these genetic variants influence the
development of coronary atherosclerosis although the effect
of a single variant is likely to be small.
Previous studies have suggested that some genetic variants
associated with CAD may primarily promote coronary atherosclerosis, whereas other variants may predispose to myocardial
infarction in the presence of coronary atheroma. For example,

Table 3. Model Prediction for Multivessel Disease With and Without the GRS
C-Statistic
Model Covariates

Without GRS, %

With GRS, %

Increase, %

IDI, %

LR 2

P Value*

Iceland (n=8622)
Model 1: age and sex only

62.9

63.7

0.8

0.53

48.7

<0.0001

Model 2: Traditional cardiovascular risk factors

64.0

64.8

0.8

0.52

48.1

<0.0001

Model 3: Traditional cardiovascular risk factors


and family history of premature CAD

64.8

65.4

0.6

0.46

42.7

<0.0001

Emory Biobank (n=1853)


Model 1: age and sex only

60.9

62.8

1.9

1.5

28.1

<0.0001

Model 2: Traditional cardiovascular risk factors

62.1

63.8

1.7

1.5

28.4

<0.0001

Model 3: Traditional cardiovascular risk factors


and family history of CAD

62.2

63.9

1.7

1.5

28.4

<0.0001

C-statistics and the IDI are reported as percentages. CAD indicates coronary artery disease; GRS, genetic risk score; IDI, integrated discrimination
improvement; LR, likelihood ratio; and SNP, single-nucleotide polymorphisms.
*All P values reported are from likelihood ratio 2 tests for nested models.
GRS based on 50 SNPs.
Traditional cardiovascular risk factors were defined as age, sex, hyperlipidemia, diabetes mellitus, hypertension, current smoking, and former
smoking.
GRS based on 32 SNPs.

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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 infarction in individuals undergoing coronary angiography but not
when the analysis was restricted to patients with established
angiographic CAD. These studies suggest that genetic risk variants for CAD, identified in early large-scale genome-wide association studies, relate primarily to coronary atherosclerosis and
may have a minimal role in plaque rupture or thrombosis leading to acute coronary events. Our findings support the hypothesis 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 patients
with established disease. Large prospective studies are warranted 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 important 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 stenosis. 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 selection 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 associated with CAD risk are more likely to have extensive coronary disease than those who carry a low burden of such
risk variants.

Acknowledgments
We thank all the individuals who participated in this study and whose
contribution made this work possible. We also thank our valued colleagues who contributed to the data collection and phenotypic characterization of clinical samples, as well as genotyping and analysis of
genome-wide association data.

Sources of Funding
The work was supported by Landspitali University Hospital Research
Fund, Jnna Gsladttir fund, Bent Scheving Thorsteinsson research

fund, and Research Fund of the Icelandic Society of Cardiology.


Emory Cardiovascular Biobank: this work was supported by the
American Heart Association (Postdoctoral Fellowship for RSP),
National Institutes of Health R01 HL89650-01, Robert W. Woodruff
Health Sciences Center Fund, Emory Heart and Vascular Center
Funds and supported, in part, by National Institutes of Health (NIH)
grant UL1 RR025008 from the Clinical and Translational Science
Award program and NIH grant R24HL085343.

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

References
1. Marenberg ME, Risch N, Berkman LF, Floderus B, de Faire U.
Genetic susceptibility to death from coronary heart disease in a
study of twins. N Engl J Med. 1994;330:10411046. doi: 10.1056/
NEJM199404143301503.
2. Roberts R. Genetics of coronary artery disease. Circ Res. 2014;114:1890
1903. doi: 10.1161/CIRCRESAHA.114.302692.
3. Morrison AC, Bare LA, Chambless LE, Ellis SG, Malloy M, Kane JP,
Pankow JS, Devlin JJ, Willerson JT, Boerwinkle E. Prediction of coronary
heart disease risk using a genetic risk score: the Atherosclerosis Risk in
Communities Study. Am J Epidemiol. 2007;166:2835. doi: 10.1093/aje/
kwm060.
4. Ripatti S, Tikkanen E, Orho-Melander M, et al. A multilocus genetic
risk score for coronary heart disease: case-control and prospective cohort analyses. Lancet. 2010;376:13931400. doi: 10.1016/
S0140-6736(10)61267-6.
5. Davies RW, Dandona S, Stewart AF, Chen L, Ellis SG, Tang WH, Hazen
SL, Roberts R, McPherson R, Wells GA. Improved prediction of cardiovascular disease based on a panel of single nucleotide polymorphisms
identified through genome-wide association studies. Circ Cardiovasc
Genet. 2010;3:468474. doi: 10.1161/CIRCGENETICS.110.946269.
6. Thanassoulis G, Peloso GM, Pencina MJ, Hoffmann U, Fox CS, Cupples
LA, Levy D, DAgostino RB, Hwang SJ, ODonnell CJ. A genetic risk
score is associated with incident cardiovascular disease and coronary
artery calcium: the Framingham Heart Study. Circ Cardiovasc Genet.
2012;5:113121. doi: 10.1161/CIRCGENETICS.111.961342.
7. Hughes MF, Saarela O, Stritzke J, et al. Genetic markers enhance coronary
risk prediction in men: the MORGAM prospective cohorts. PLoS One.
2012;7:e40922. doi: 10.1371/journal.pone.0040922.
8. Vaarhorst AA, Lu Y, Heijmans BT, et al. Literature-based genetic
risk scores for coronary heart disease: the Cardiovascular Registry
Maastricht (CAREMA) prospective cohort study. Circ Cardiovasc Genet.
2012;5:202209. doi: 10.1161/CIRCGENETICS.111.960708.
9. Ganna A, Magnusson PK, Pedersen NL, de Faire U, Reilly M, Arnlv
J, Sundstrm J, Hamsten A, Ingelsson E. Multilocus genetic risk scores
for coronary heart disease prediction. Arterioscler Thromb Vasc Biol.
2013;33:22672272. doi: 10.1161/ATVBAHA.113.301218.
10. Tikkanen E, Havulinna AS, Palotie A, Salomaa V, Ripatti S. Genetic risk
prediction and a 2-stage risk screening strategy for coronary heart disease. Arterioscler Thromb Vasc Biol. 2013;33:22612266. doi: 10.1161/
ATVBAHA.112.301120.
11. Tragante V, Doevendans PA, Nathoe HM, van der Graaf Y, Spiering W,
Algra A, de Borst GJ, de Bakker PI, Asselbergs FW; SMART Study
Group. The impact of susceptibility loci for coronary artery disease on
other vascular domains and recurrence risk. Eur Heart J. 2013;34:2896
2904. doi: 10.1093/eurheartj/eht222.
12. Hamrefors V, Hedblad B, Engstrm G, Almgren P, Sjgren M, Melander
O. A myocardial infarction genetic risk score is associated with markers of carotid atherosclerosis. J Intern Med. 2012;271:271281. doi:
10.1111/j.1365-2796.2011.02472.x.
13. Dandona S, Stewart AF, Chen L, Williams K, So D, OBrien E, Glover C,
Lemay M, Assogba O, Vo L, Wang YQ, Labinaz M, Wells GA, McPherson
R, Roberts R. Gene dosage of the common variant 9p21 predicts severity of coronary artery disease. J Am Coll Cardiol. 2010;56:479486. doi:
10.1016/j.jacc.2009.10.092.
14. 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. The chromosome 9p21 risk locus is associated with

Downloaded from http://atvb.ahajournals.org/ by guest on March 6, 2016

Bjornsson et al CAD Variants and Extent of CAD 1531


angiographic severity and progression of coronary artery disease. Eur
Heart J. 2010;31:30173023. doi: 10.1093/eurheartj/ehq272.
15. Helgadottir A, Gretarsdottir S, Thorleifsson G, et al. Apolipoprotein(a)
genetic sequence variants associated with systemic atherosclerosis and coronary atherosclerotic burden but not with venous thromboembolism. J Am Coll Cardiol. 2012;60:722729. doi: 10.1016/j.
jacc.2012.01.078.
16. Chan K, Patel RS, Newcombe P, et al. Association between the chromosome 9p21 locus and angiographic coronary artery disease burden: a
collaborative meta-analysis. J Am Coll Cardiol. 2013;61:957970. doi:
10.1016/j.jacc.2012.10.051.
17. Patel RS, Asselbergs FW, Quyyumi AA, Palmer TM, Finan CI, Tragante V,
Deanfield J, Hemingway H, Hingorani AD, Holmes MV. Genetic variants
at chromosome 9p21 and risk of first versus subsequent coronary heart
disease events: a systematic review and meta-analysis. J Am Coll Cardiol.
2014;63:22342245. doi: 10.1016/j.jacc.2014.01.065.
18. Reilly MP, Li M, He J, et al; Myocardial Infarction Genetics Consortium;
Wellcome Trust Case Control Consortium. Identification of ADAMTS7
as a novel locus for coronary atherosclerosis and association of ABO

with myocardial infarction in the presence of coronary atherosclerosis:


two genome-wide association studies. Lancet. 2011;377:383392. doi:
10.1016/S0140-6736(10)61996-4.
19. Patel RS, Sun YV, Hartiala J, et al. Association of a genetic risk score with
prevalent and incident myocardial infarction in subjects undergoing coronary angiography. Circ Cardiovasc Genet. 2012;5:441449. doi: 10.1161/
CIRCGENETICS.111.960229.
20. Ringqvist I, Fisher LD, Mock M, Davis KB, Wedel H, Chaitman BR,
Passamani E, Russell RO Jr, Alderman EL, Kouchoukas NT, Kaiser GC,
Ryan TJ, Killip T, Fray D. Prognostic value of angiographic indices of
coronary artery disease from the Coronary Artery Surgery Study (CASS).
J Clin Invest. 1983;71:18541866.
21. Nicholls SJ, Hsu A, Wolski K, Hu B, Bayturan O, Lavoie A, Uno K, Tuzcu
EM, Nissen SE. Intravascular ultrasound-derived measures of coronary
atherosclerotic plaque burden and clinical outcome. J Am Coll Cardiol.
2010;55:23992407. doi: 10.1016/j.jacc.2010.02.026.
22. Topol EJ, Nissen SE. Our preoccupation with coronary luminology. The
dissociation between clinical and angiographic findings in ischemic heart
disease. Circulation. 1995;92:23332342.

Significance
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 significantly 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.

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Common Sequence Variants Associated With Coronary Artery Disease Correlate With the
Extent of Coronary Atherosclerosis
Eythor Bjornsson, Daniel F. Gudbjartsson, Anna Helgadottir, Thorarinn Gudnason, Tomas
Gudbjartsson, Kristjan Eyjolfsson, Riyaz S. Patel, Nima Ghasemzadeh, Gudmar Thorleifsson,
Arshed A. Quyyumi, Unnur Thorsteinsdottir, Gudmundur Thorgeirsson and Kari Stefansson
Arterioscler Thromb Vasc Biol. 2015;35:1526-1531; originally published online April 16, 2015;
doi: 10.1161/ATVBAHA.114.304985
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
Greenville Avenue, Dallas, TX 75231
Copyright 2015 American Heart Association, Inc. All rights reserved.
Print ISSN: 1079-5642. Online ISSN: 1524-4636

The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://atvb.ahajournals.org/content/35/6/1526

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http://atvb.ahajournals.org/content/suppl/2015/04/16/ATVBAHA.114.304985.DC1.html

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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.

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

Coded
Nearest Gene(s)
allele

Effect on CAD extent


Odds
ratio for Iceland (n=8,622) Emory (n=1,853)
Combined
CAD
Iceland Emory Iceland Emory
Beta
P value Beta P value Beta
P value
Coded allele
frequency

SNP info*

SNP

Chr

rs599839

1p13

SORT1

0.80

rs4845625

1q21

IL6R

0.37

rs11206510

1p32

PCSK9

0.83

0.82

0.995

rs17114036

1p32

PPAP2B

0.93

0.91

rs17465637

1q41

MIA3

0.69

0.75

rs1561198

2p11

VAMP5-VAMP8GGCX

0.46

rs6544713

2q21

ABCG5-ABCG8

rs2252641

2q22 ZEB2-AC074093.1

rs515135a

2p24

rs6725887

0.79

1.111

0.014

0.444

1.052

-0.016

0.278

0.996

1.062

0.043

0.998

0.959

1.112

0.998

0.988

0.997

0.975

0.112

0.002

0.033

0.037

0.024

-0.050

0.182

0.024

0.156

-0.008

0.782

-0.031

0.554

-0.013

0.602

1.141

0.007

0.637

0.012

0.732

0.008

0.568

0.999

1.062

0.016

0.268

0.29

0.999

1.062

0.030

0.050

0.44

0.998

1.042

-0.001

0.966

APOB

0.87

0.82

0.999

0.944

1.072

0.013

0.526

0.014

0.718

0.013

0.466

2q33

WDR12

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

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

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

0.81

0.998

1.072

-0.023

0.200

rs273909

5q31

SLC22A4SLC22A5

0.15

0.999

1.092

0.028

0.158

rs17609940

6p21

ANKS1A

0.75

1.071

0.025

0.124

0.028

0.45

0.025

0.087

rs10947789

6p21

KCNK5

0.78

1.062

0.019

0.252

rs12190287

6q23

TCF21

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

0.70

0.68

0.999

0.985

1.101

0.030

0.049

0.020

0.545

0.028

0.041

rs2048327

6q25

SLC22A3-LPAL2LPA

0.41

1.062

0.032

0.025

rs3798220

6q25

SLC22A3-LPAL2LPA

0.02

0.02

1.000

1.000

1.282

0.079

0.087

0.159

0.110

0.093

0.026

rs10455872

6q25

SLC22A3-LPAL2LPA

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

0.73

0.998

1.072

0.011

0.472

rs2023938

7p21

HDAC9

0.10

1.000

1.082

0.004

0.859

rs10953541

7q22

BCAP29

0.76

0.999

1.084

0.028

0.090

rs11556924

7q32

ZC3HC1

0.66

1.092

-0.005

0.750

0.022

0.461

0.001

0.967

rs264

8p21

LPL

0.85

1.072

0.014

0.490

rs2954029c

8q24

TRIB1

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

0.48

0.52

0.996

0.972

1.232

0.046

0.001

0.076

0.012

0.051

5.8x10-5

0.998

0.80

0.999

0.996

0.999

0.999

0.62

0.996

0.982

0.995

rs579459e

9q34

ABO

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

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

0.53

1.052

-0.015

0.292

rs501120f

10q11

CXCL12

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

0.37

0.35

0.997

0.984

1.094

0.014

0.316

0.036

0.242

0.018

0.160

CYP17A1CNNM2-NT5C2

0.93

0.92

0.998

0.998

1.102

-0.008

0.757

0.092

0.087

0.012

0.621

1.072

0.018

0.252

rs12413409g 10q24

0.999

rs974819

11q22

PDGFD

0.29

0.997

rs964184

11q23

ZNF259-APOA5APOA1

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

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

0.34

1.062

0.020

0.186

rs4773144h 13q34 COL4A1-COL4A2

0.44

1.072

-0.003

0.836

-0.005

0.875

-0.003

0.799

rs9515203

13q34 COL4A1-COL4A2

0.73

1.082

0.044

0.005

rs2895811

14q32

HHIPL1

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

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

0.47

1.062

-0.003

0.824

rs12936587 17p11

RAI1-PEMTRASD1

0.60

0.56

0.998

0.995

1.062

0.016

0.260

-0.046

0.116

0.004

0.751

0.998
0.45

0.990

0.983

0.992

0.996

rs216172

17p13

SMG6

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

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

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

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

0.92

1.132

0.002

0.929

0.992

Gene desert
T
0.14
0.14
0.993 0.951
1.132
0.013
0.534
0.010
0.82
0.012
0.509
(KCNE2)
*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 former
smoking. The outcome variable was the number of diseased coronary vessels with at least 50% stenosis on coronary angiography (range, 1-4). Results
from the Icelandic and Emory samples were combined using fixed-effects inverse variance-weighted meta-analysis.
a
rs562338 is a proxy for rs515135 (r2=0.98) in the Emory Biobank sample
b
rs6842241 is a proxy for rs1878406 (r2=0.91) in the Emory Biobank sample
c
rs2954021 is a proxy for rs2954029 (r2=0.79) in the Emory Biobank sample
d
rs10757278 is a proxy for rs1333049 (r2=0.95) in the Emory Biobank sample
e
rs651007 is a proxy for rs579459 (r2=0.99) in the Emory Biobank sample
f
rs1746048 is a proxy for rs501120 (r2=0.96) in the Emory Biobank sample
g
rs12411886 is a proxy for rs12413409 (r2=1.00) in the Emory Biobank sample
h
rs3809346 is a proxy for rs4773144 (r2=0.99) in the Emory Biobank sample
rs9982601

21q22

Supplementary Table II. Association of the GRS with the number of diseased coronary arteries on
coronary angiography, adjusted for age and sex only

No. of
SNPs

Difference per SD
increase (95% CI)

Standard
error

P value

Contrast top versus


bottom GRS quintile
OR for multivessel
disease* (95% CI)

Full GRS

50

0.077 (0.059-0.095)

0.0091

3.2x10-17

1.67 (1.45-1.93)

Full GRS excluding


9p21 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)

Iceland (n=8,622)

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 9p21


and 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 on
coronary angiography.

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. of
SNPs

Difference per SD
increase (95% CI)

Standard
error

P value

Contrast top versus


bottom GRS quintile
OR for multivessel
disease* (95% CI)

Full GRS

50

0.072 (0.054-0.089)

0.0090

3.0x10-15

1.63 (1.41-1.89)

Full GRS excluding


9p21 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)

0.115 (0.075-0.155)

0.021

2.7x10-8

2.08 (1.50-2.90)

Iceland (n=8,622)

Emory Biobank (n=1,853)


GRS

32

GRS excluding 9p21


29
0.070 (0.030-0.111)
0.021
6.9x10-4
1.51 (1.09-2.09)
and LPA
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 premature
CAD (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 on
coronary angiography.

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


of the GRS
Characteristic

P value*

Q1

Q2

Q3

Q4

Q5

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 as
a continuous variable.

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


quintiles of the GRS
P value*

Characteristic

Q1

Q2

Q3

Q4

Q5

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 a
continuous variable.

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
(<50% stenosis).

No. of
SNPs

Difference per SD
increase (95% CI)

Standard
error

P value

Contrast top versus


bottom GRS quintile
OR for multivessel
disease* (95% CI)

Full GRS

50

0.145 (0.126-0.165)

0.0094

7.0x10-48

2.01 (1.77-2.28)

Full GRS excluding


9p21 and LPA

47

0.105 (0.085-0.124)

0.0100

1.1x10-25

1.60 (1.41-1.82)

Restricted GRS

32

0.139 (0.119-0.158)

0.0099

8.2x10-44

1.87 (1.65-2.12)

0.198 (0.155-0.240)

0.022

1.8x10-19

2.60 (2.01-3.39)

Iceland (n=11,196)

Emory Biobank (n=2,718)


GRS

32

GRS excluding 9p21


29
0.145 (0.102-0.187)
0.022
4.4x10-11
1.69 (1.30-2.19)
and LPA
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.
*Multivessel disease was defined as having at least two coronary arteries with 50% stenosis on
coronary angiography.
8,622 patients with significant CAD (50% stenosis) plus 2,574 individuals with non-significant CAD
(<50% stenosis).
1,853 patients with significant CAD (50% stenosis) plus 865 individuals with non-significant CAD
(<50% stenosis).

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 (<50% stenosis). Odds ratios are referenced to the
bottom GRS quintile and presented with 95% confidence intervals.

References
1.

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


13 new susceptibility loci for coronary artery disease. Nat Genet. 2011;43:333338.

2.

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


new risk loci for coronary artery disease. Nat Genet. 2013;45:2533.

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;43:339344.

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 in
Iceland, 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

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 (<50% stenosis)
were not included in this study.
Cardiovascular Risk Factors
Information on cardiovascular risk factors was obtained from aforementioned clinical
registries. Hypertension was defined as a previous diagnosis of hypertension or
treatment with anti-hypertensive drugs. Diabetes mellitus was defined as a previous
diagnosis of diabetes or treatment with antidiabetic medications. Hyperlipidemia was
defined as a previous diagnosis of hyperlipidemia or treatment with lipid-lowering
therapy. For the Icelandic patients, family history of premature CAD was ascertained
for each participant based on a genealogy database covering the entire Icelandic
population5 and linkage with nationwide clinical registries. Family history of premature
CAD was defined as the proportion of first-degree relatives (parents or siblings) with a
history of premature CAD defined as myocardial infarction (MI), surgical or
percutaneous coronary revascularization or death attributed to CAD occurring before
the age of 55 years in males and 65 years in females. In the Emory Biobank sample,
family history of CAD was self-reported and defined as having any first degree relative
(parents or siblings) with history of CAD at any age.
SNP Selection and Genotyping
We selected all SNPs reported to be associated with CAD or myocardial infarction in
several large-scale meta-analyses of genome-wide association studies in European
populations published before December 2012. A review of meta-analyses reported by
the CARDIoGRAM6, C4D7, CARDIoGRAMplusC4D8 and the IBC 50K CAD9 consortia
yielded 50 independent SNPs (pairwise r2<0.2) reported at genome-wide significance
(Table I in the online-only Data Supplement). These SNPs were included for calculation
of the GRSs where the effect sizes (odds ratio) obtained from the reference studies

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 available
subset 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 (r2
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

and former smoking). Results for SNPs genotyped in both the Icelandic and Emory
Biobank samples were combined using fixed-effects inverse variance-weighted metaanalysis. 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 2
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
(<50% stenosis) and genotype information available.
All statistical analyses were performed using R, version 3.1.0 (www.rproject.org). Unless otherwise noted, a two-sided P-value less than 0.05 was
considered statistically significant.

References
1.

Gudnason T, Gudnadottir GS, Lagerqvist B, Eyjolfsson K, Nilsson T,


Thorgeirsson G, Andersen K, James S. Comparison of interventional cardiology
in two European countries: A nationwide internet based registry study. Int J
Cardiol. 2012;2; 1237-1242.

2.

Biancari F, Gudbjartsson T, Heikkinen J, et al. Comparison of 30-Day and 5Year Outcomes of Percutaneous Coronary Intervention Versus Coronary Artery
Bypass Grafting in Patients Aged 50 Years (the Coronary aRtery diseAse in
younG adultS Study). Am J Cardiol. 2014;114:198205

3.

Helgadottir A, Gretarsdottir S, Thorleifsson G, et al. Apolipoprotein(a) genetic


sequence variants associated with systemic atherosclerosis and coronary
atherosclerotic burden but not with venous thromboembolism. J Am Coll Cardiol.
2012;60:722729.

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, Fray
D. Prognostic value of angiographic indices of coronary artery disease from the
Coronary Artery Surgery Study (CASS). J Clin Invest. 1983;71:185466.

5.

Kong A, Masson G, Frigge ML, et al. Detection of sharing by descent, long-range


phasing and haplotype imputation. Nat Genet. 2008;40:10681075.

6.

Schunkert H, Knig IR, Kathiresan S, et al. Large-scale association analysis


identifies 13 new susceptibility loci for coronary artery disease. Nat Genet.
2011;43:333338.

7.

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;43:339344.

8.

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


identifies new risk loci for coronary artery disease. Nat Genet. 2013;45:2533.

9.

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


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

10.

Styrkarsdottir U, Thorleifsson G, Sulem P, et al. Nonsense mutation in the LGR4


gene is associated with several human diseases and other traits. Nature.
2013;497:517520.

11.

Dandona S, Stewart AFR, Chen L, Williams K, So D, OBrien E, Glover C, Lemay


M, Assogba O, Vo L, Wang YQ, Labinaz M, Wells GA, McPherson R, Roberts
R. Gene dosage of the common variant 9p21 predicts severity of coronary artery
disease. J Am Coll Cardiol. 2010;56:47986.

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. The
chromosome 9p21 risk locus is associated with angiographic severity and
progression of coronary artery disease. Eur Heart J. 2010;31:30173023.

13.

Pencina MJ, DAgostino RB, Vasan RS. Evaluating the added predictive ability
of a new marker: from area under the ROC curve to reclassification and beyond.
Stat Med. 2008;27:15772.

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