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Subtle killers and sudden death: Genetic variants modulating ventricular fibrillation in thesetting of myocardial infarction

Pazoki, R.

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Citation for published version (APA):Pazoki, R. (2015). Subtle killers and sudden death: Genetic variants modulating ventricular fibrillation in thesetting of myocardial infarction.

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C hapter 4Genome-wide association study identifi es a

susceptibility locus at 21q21 for ventricular

fi brillation in acute myocardial infarction

Nat Genet. 2010 Aug;42(8):688-91.

Connie R. Bezzina*, Raha Pazoki*, Abdennasser Bardai*,

Roos F. Marsman*, Jonas S.S.G. de Jong*, Marieke T. Blom, Brendon P. Scicluna,

J. Wouter Jukema, Navin R. Bindraban, Peter Lichtner, Arne Pfeufer,

Nanette Bishopric, Dan M. Roden, Th omas Meitinger, Sumeet S. Chugh,

Robert J. Myerburg, Xavier Jouven, Stefan Kääb, Lukas R.C. Dekker,

Hanno L. Tan, Michael W.T. Tanck, Arthur A.M. Wilde

*Th ese authors contributed equally to this study.

56 Chapter 4

Abstract

Sudden cardiac death from ventricular fibrillation (VF) during acute myocardial

infarction (MI) is a leading mode of death. We report the first genome-wide associa-

tion study addressing this problem. Genome-wide association analysis in a set of 972

patients with a first acute MI, 515 of whom had VF and 457 did not, identified SNPs at

21q21, in moderate to complete linkage disequilibrium (LD), associated with VF. The

most significant association was found for rs2824292 and rs2824293 in complete LD

(Odds ratio = 1.78; 95% CI: 1.47, 2.13; P = 3.3 × 10−10). The association of rs2824292 with

VF was replicated in an independent case-control set consisting of 146 out-of-hospital

cardiac arrest patients with MI and VF and 391 MI survivor controls (Odds ratio = 1.49;

95% CI: 1.14, 1.95; P = 0.004). The closest gene is CXADR, encoding a viral receptor

implicated in myocarditis and dilated cardiomyopathy, and which has recently been

identified as a modulator of cardiac conduction. This locus has not previously been

implicated in arrhythmia susceptibility.

GWAS identifies a susceptibility locus at 21q21 for VF in MI 57

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Introduction

Sudden cardiac death (SCD) accounts for 15-20% of all natural deaths in adults in

the U.S. and Western Europe, and up to 50% of all cardiovascular deaths 1. Ventricular

fibrillation (VF, uncoordinated contraction of the ventricles) is the most common un-

derlying cardiac arrhythmia 2. It arises through multiple mechanisms, depending on

the underlying cardiac pathology 3. In the last decade, considerable progress has been

made in the understanding of the genetic, molecular and electrophysiological basis

of SCD in the uncommon (monogenic) familial arrhythmia syndromes affecting the

structurally normal heart and the rare inherited structural disorders associated with

increased SCD risk 4,5. However, the overwhelming majority (~80%) of SCDs in adults

are caused by the sequelae of coronary artery disease, namely myocardial ischemia or

acute myocardial infarction (MI) 6,7. Here SCD is the first clinically-identified expres-

sion of heart disease in up to one half of cases 8. Progress in understanding genetic and

molecular mechanisms that determine susceptibility to these common arrhythmias,

affecting a much greater proportion of the population, has been limited 3,9,10. The

identification of genetic variants associated with this phenotype is challenging due to

difficulties in ascertaining these patients.

Epidemiological studies have indicated that sudden death in a family member is

a risk factor, suggesting a genetic component in susceptibility 11,12. Recently, we also

identified family history of sudden death as a strong risk factor for cardiac arrest

during MI in the Arrhythmia Genetics in the NEtherlandS (AGNES) study, in which

we compared patients with and without VF during the early phase of a first acute

MI 13.

Accordingly, in the present study, we sought to identify common genetic factors

underlying susceptibility to VF during MI by conducting a genome-wide association

study (GWAS) in the AGNES case-control set. The strategy of selecting this highly spe-

cific arrhythmia phenotype allowed us to avoid the problem of loss of statistical power

inherent in analyzing cases or controls with multiple pathophysiologies. In particular,

confining the case group to those with a first MI minimizes the inclusion of patients

with extensive pre-existing myocardial scarring from previous MIs, in whom VF may

also arise from other mechanisms such as scar-related reentry.

Methods

Study samples

All individuals studied were of self-declared Caucasian ancestry. Local ethics commit-

tees approved the study protocols, and all participants gave written informed consent.

58 Chapter 4

The AGNES sample: The AGNES case-control set consisted of patients with a first

acute ST-segment elevation MI 13.

AGNES cases had ECG-registered VF occurring before reperfusion therapy for an

acute and first ST- segment elevation MI. AGNES controls were patients with a first

acute ST- segment elevation MI but without VF. All were recruited at seven heart

centers in the Netherlands from 2001-2008. We excluded patients with an actual non-

ST- segment elevation MI, prior MI, congenital heart defects, known structural heart

disease, severe co-morbidity, electrolyte disturbances, trauma at presentation, recent

surgery, previous coronary artery bypass graft (CABG) and class I and III antiarrhyth-

mic drug usage. Patients who developed VF during or after percutaneous coronary

intervention (PCI) were not eligible. Furthermore, since early reperfusion limits the

odds of developing VF, potential control patients undergoing PCI within 2 hours after

onset of symptoms were not included. This time interval was based on the observation

that > 90% of cases developed VF within 2 hours after onset of complaints.

The ARREST-MI sample: The ARREST-MI sample was drawn from the “AmsteR-dam REsuscitation STudy” (ARREST-11). ARREST-11 is an ongoing prospective

population-based study aimed at establishing the genetic, clinical and environmental

determinants of SCD in the general population. Caucasian out-of-hospital cardiac ar-

rest patients in the ARREST-11 dataset presenting with MI at hospital admission and

with ECG-documented VF were included in the replication analysis. The diagnosis of

MI was established at hospital admission by a cardiologist based on ECG abnormali-

ties and cardiac enzymes, and was retrieved retrospectively from hospital records. At

the time of this study (August 2009), 146 such patients were retrieved and all were

included in the replication study.

The GENDER-MI sample: The GENDER-MI sample included patients with a history

of MI drawn from the GENetic DEterminants of Restenosis study (GENDER) 14, a study

aimed at investigating the association between genetic polymorphisms and clinical

restenosis. The GENDER study comprises consecutive patients who underwent suc-

cessful percutaneous transluminal coronary angioplasty (PTCA) for treatment of

stable angina, non–ST-segment elevation acute coronary syndromes or silent ischemia

in four referral centers for interventional cardiology in the Netherlands. The inclusion

period lasted from March 1999 until June 2001 and in total, 3104 consecutive patients

were included in this prospective, multicenter, follow-up study. Approximately 40% of

subjects included in the GENDER study had a history of MI. A sample of 941 GENDER

subjects was genotyped on the Illumina Human 610-Quad Beadchips 15. The GENDER-

MI sample used in the current study consisted of 391 of these genotyped cases; the 391

were all those with a history of MI.

Random sample of the Dutch Caucasian general population: The random sample

of Dutch Caucasian general population constituted the white Dutch component of the


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“Surinamese in The Netherlands: Study on Ethnicity and Health” study, a cross-sectional

study that aimed to assess the cardiovascular risk profile of three ethnic groups in The

Netherlands: Creole, Hindustani, and individuals of European ancestry 16. It is based on

a sample of 35–60 year-old, non-institutionalized people from south-east Amsterdam,

The Netherlands. A random sample of 2000 Surinamese and approximately 1000 Dutch

individuals of European ancestry were drawn from the Amsterdam population register

between 2001 and 2003. People were classified as Dutch if they and both their parents

were born in The Netherlands. The overall response rate was 61% among these Dutch

individuals. Those who responded to the oral interview were invited for medical exami-

nation, at which blood for DNA extraction was drawn. The response rate at this stage

among the Dutch individuals was 90%, resulting in a total of 508 subjects with DNA.

DNA samples from these 508 subjects were used for genotyping in the current study.

Genotyping

Genome-wide genotyping of SNPs on the 515 AGNES cases and 457 AGNES controls

was done using the Illumina Human610-Quad v1 array. All except 3 DNA samples

passed quality control criteria. Excluding sex chromosome and mitochondrial DNA

left 507,436 single nucleotide polymorphisms (SNPs). The analysis was performed with

the BeadStudio software and the Genotyping module version 3.2.33 using the cluster

file provided by Illumina (Human610-Quadv1_B.egt). The GenCall score cut-off was

0.15 and the average call rate was 99.45%. Multiple quality control measures were

implemented. The estimated (genotyped) sex was compared with the phenotypic sex.

Exclusion criteria included P-values for deviation from Hardy-Weinberg expectation

< 10−4 (in controls), sample call rate < 0.95, and SNP call rate < 0.98. For GENDER-MI,

genotypes at rs2824292, rs1353342 and rs12090554 were extracted from previously

obtained genotypes (Illumina Human 610-Quad Beadchip) 15. Quality control criteria

for genotypic data for the GENDER study were the same as those applied in the AG-

NES study. For ARREST-MI and for the sample of the Dutch Caucasian population,

genotypes at rs2824292, rs1353342 and rs12090554 were determined by means of a

Taqman assay (Applied Biosystems).

Statistical analysis and imputation

Differences in continuous phenotypic variables between cases and controls were

tested using an independent t-test when data were normally distributed or a Mann-

Whitney U test otherwise. Values are presented as means ± standard deviation (SD)

or median and interquartile range (IQR), respectively. Differences in categorical vari-

ables were compared using a Fisher exact test and values are presented as numbers

and percentages. Genotype imputation into the HapMap reference panel (release 22)

was done using Markov chain Monte Carlo methods implemented in MACH1.0 17.

60 Chapter 4

An Rsq threshold of 0.3 is implemented in the program to identify and discard low-

quality imputations. Each SNP was analyzed for association with VF using logistic

regression assuming an additive genetic model with adjustment for age and sex, using

the ProbABEL analysis package 18. SNPs with a minor allele frequency (MAF) > 0.05

were analyzed. The P-value for genome-wide significance was set at P < 5 × 10−8, cor-

responding to a target α (P-value) of 0.05 with a Bonferroni correction for 1 million

independent tests 19. The P-values were corrected for the genomic control factor. A

2-sided P-value of P < 0.017 (Bonferroni-corrected P value for the three independent

SNPs) was considered as significant in the replication study. In the multifactor analy-

sis, we adjusted for age, sex, hypercholesterolemia, diabetes, ST-segment deviation,

maximal CK-MB, body mass index and family history of SCD.

Results

GWAS was performed in 515 AGNES cases (MI with VF) and 457 AGNES controls (MI

without VF), all of Dutch Caucasian ethnicity. The clinical characteristics of AGNES

cases and controls are presented in Table 1.

Table 1 | Baseline Characteristics of the AGNES case-control set

Characteristic Na Total(n = 972)

Cases(n = 515)

Controls(n = 457)

P-valueb

Sex (male) c 783 (80.6) 412 (80.0) 371 (81.2) 0.68

Age at MI (years) d 56.4 ± 11 55.9±11.2 56.9±10.8 0.17

ST segment deviation (mm) e 735/379/356 17 (19) 22 (22) 14 (14) < 0.001

MI location c 962/505/457

Inferior MI 408 (42.4) 203 (40.2) 205 (44.9) 0.14 f

Anterior MI 554(57.6) 302 (59.8) 252 (55.1)

CK-MB (μg/L) e 786/359/427 215(299) 225.6(365) 211.4(258) 0.02

Family history of sudden death c 909/459/450 291 (32.0) 174 (37.9) 117 (26.0) < 0.001

Beta blocker usage c 936/482/454 85 (9.1) 50 (10.4) 35 (7.7) 0.17

Cardiovascular risk factors

Current Smoking c 914/473/441 565 (61.8) 303 (64.1) 262 (59.4) 0.15

Diabetes Mellitus c 893/462/431 62 (6.94) 21 (4.6) 41 (9.5) 0.004

Hypertension c 294 (30.2) 149 (28.9) 145 (31.7) 0.36

Hypercholesterolemia c 297 (30.6) 127 (24.7) 170 (37.2) < 0.001

BMI (kg/m2) d 879/439/440 26.5 ± 3.9 26.1 ± 3.8 26.9 ± 4.0 0.004

MI, myocaridal infarction; CK-MB, creatine kinase-MB; aIn case of missing values, the sample sizes of the total, case and control sets (total, case, control) for whom information was available is given; bP-value for comparison of cases and controls for each item; cNumber (%); dmean ± s.d.; eMedian (interquartile range); fP-value for comparison of inferior and anterior MI between cases and controls.


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The genomic control factor was small (λ = 1.026) indicating that, as expected,

overall inflation of genome-wide statistical results due to population stratification

was minimal. There was a significant excess of SNPs associated with VF compared to

the number expected under the null hypothesis of no association (Supplementary Figure 1).

The distribution of P- values for the association of SNPs with VF is shown in Fig-ure 1 and SNPs displaying the strongest associations (chr. 21q21, chr. 9q21, chr. 1q25)

are presented in Table 2 and Supplementary Table 1.

Eight SNPs on chr. 1q21, all in moderate to complete LD with each other (r2 = 0.4–

1.0), surpassed our preset threshold for genome-wide significance of P < 5 × 10−8. Of

these, the most significant association was found for rs2824292 and rs2824293 (338

bp apart, r2 = 1.0), situated 98 Kb from the gene CXADR (1.78 per G-allele; 95% CI:

1.47, 2.13; P = 3.3 × 10−10). After adjustment for the main association signal (rs2824292)

a significant reduction in the association between VF and the other seven SNPs on

chromosome 21 occurred, indicating that these SNPs do not represent independent

secondary signals.

The genotype frequencies for rs2824292 in AGNES cases and controls as well as

for rs1353342 (9q21) and rs12090554 (1q25) are presented in Supplementary Table 2, together with the genotype frequencies in a sample of the general Dutch Caucasian

population. As expected, the frequencies of the risk and protective alleles for each SNP

in the general population were between those of AGNES cases and AGNES controls.

Since AGNES cases and controls differed in some characteristics, including risk

factors for coronary artery disease and measures of infarct size (represented by CK-

MB), we carried out multifactor analysis to assess whether rs2824292 was an inde-

pendent predictor for VF. Multifactor analysis for the association of rs2824292 with VF

adjusted for those factors that appeared different between AGNES cases and controls

(Table 1) resulted in an odds ratio of 1.51 per G-allele (95% CI: 1.13, 2.01, P = 0.005),

indicating that this SNP is an independent risk factor for VF. This was also the case

for rs1353342 and rs12090554 (respectively, OR = 0.48, 95% CI: 0.30, 0.76, P = 0.002;

OR = 0.58, 95% CI: 0.41, 0.82, P = 0.002). Nevertheless, future research will be required

in order to provide more evidence of the independent effects of these SNPs.

We next proceeded to validate the association of the leading SNPs at the 21q21,

9q21 and 1q25 loci in an independent set of cases and controls (Table 3). Our P-value

threshold for this analysis was set to P < 0.017 (0.05/3).

Cases for the replication study (ARREST-MI, n = 146; 80.1% male, average age

63.12±13.10 years) were Caucasian out-of-hospital cardiac arrest patients from the

ARREST dataset presenting with MI at hospital admission and with ECG-documented

VF. Controls for the replication analysis (GENDER-MI, n = 391; 79.5% male, average

age 61.7±10.1 years) were MI survivors drawn from the GENDER study (see Methods).

62 Chapter 4

As observed in the AGNES case-control set, the risk G-allele of SNP rs2824292

at chr. 21q21 was more abundant while the protective A-allele was less abundant in

ARREST-MI cases compared to GENDER-MI (Supplementary Table 2). The odds

ratio per G-allele in this case-control set was 1.49 per risk allele (95% CI: 1.14, 1.95:

P = 0.004), comparable to that found in the AGNES discovery set. SNP rs12090554

at chr. 1q25 showed a trend for association with a direction of effect similar to that

found in the AGNES discovery set (OR = 0.70 per copy of A-allele, 95% CI: 0.49, 1.01,

GWAS identifies a locus at 21q21 as a susceptibility locus for VF in acute MI | 65

4

VF and the other seven SNPs on chromosome 21 occurred, indicating that these

SNPs do not represent independent secondary signals.

The genotype frequencies for rs2824292 in AGNES cases and controls as well as

for rs1353342 (9q21) and rs12090554 (1q25) are presented in

Supplementary Table 2, together with the genotype frequencies in a sample of

the general Dutch Caucasian population. As expected, the frequencies of the risk

and protective alleles for each SNP in the general population were between those

of AGNES cases and AGNES controls.

Figure 1 | Results of genome-wide association analysis in the AGNES case-control set. (A) Overview of the genome-wide association scan in the AGNES case-control population. P-values corrected for age, sex and inflation factor are shown for each SNP tested. Within each chromosome, the results areplotted left to right from the p-terminal end. The horizontal red line indicates the preset threshold for genome-wide significance, P < 5 × 10

-8. (B) Locus-specific association map, generated from genotyped as

well as imputed SNPs, centered at rs2824293, located at 338bp from rs2824292 and in complete LD with it. Imputed SNPs are in gray; rs2824293 is depicted in blue; SNPs in red have r2 ≥ 0.8 with rs2824293; SNPs in orange have r2 = 0.5-0.8; those in yellow have r2 = 0.2-0.5; those in white have r2 < 0.2 with the leading SNP. Superimposed on the plot are gene locations (green) and recombination rates (blue). Chromosome positions are based on HapMap release 22 build 36.2. Figure 1B was prepared using SNAP 20.

Chromosome 21 position (kb)Chromosome 21 position (kb)

B

A

P=3.34 10

Figure 1 | Results of genome-wide association analysis in the AGNES case-control set. (A) Over-view of the genome-wide association scan in the AGNES case-control population. P-values corrected for age, sex and inflation factor are shown for each SNP tested. Within each chromosome, the results are plotted left to right from the p-terminal end. The horizontal red line indicates the preset threshold for genome-wide significance, P < 5 × 10−8. (B) Locus-specific association map, generated from geno-typed as well as imputed SNPs, centered at rs2824293, located at 338bp from rs2824292 and in com-plete LD with it. Imputed SNPs are in gray; rs2824293 is depicted in blue; SNPs in red have r2 ≥ 0.8 with rs2824293; SNPs in orange have r2 = 0.5-0.8; those in yellow have r2 = 0.2-0.5; those in white have r2 < 0.2 with the leading SNP. Superimposed on the plot are gene locations (green) and recombination rates (blue). Chromosome positions are based on HapMap release 22 build 36.2. Figure 1B was prepared using SNAP 20.


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Tabl

e 2

| Lea

din

g si

ngl

e n

ucl

eoti

de p

olym

orph

ism

s (S

NP

s) a

t th

e th

ree

loci

mos

t st

ron

gly

asso

ciat

ed w

ith

ven

tric

ula

r fi

bril

lati

on in

th

e A

GN

ES

case

-co

ntr

ol s

et

SNPa

Ch

rom

osom

eP

osit

ion

Pos

itio

nb

Mi/

Ma

Min

or A

llel

e Fr

equ

ency

OR

(95%

CI)

cP

-val

ued

Clo

sest

Gen

ese

Tota

lC

ases

Con

trol

s

rs28

2429

321

q21

1770

9385

G/A

4753

391.

78(1

.47,

2.13

)3.

3 ×

10−1

0C

XA

DR

, BT

G3

rs28

2429

221

q21

1770

9047

G/A

4753

391.

78(1

.47,

2.13

)3.

3 ×

10−1

0C

XA

DR

, BT

G3

rs13

5334

29q

2178

0645

89A

/C23

714

0.46

(0.3

4,0.

63)

3.3

× 10

−7P

CSK

5, R

FK, G

CN

T1

rs12

0905

541q

2518

3818

971

A/G

2319

280.

58(0

.47,

0.72

)7.

9 ×

10−7

HM

CN

1, IV

NS1

AB

P

Ch

r, ch

rom

osom

e; M

i/M

a, m

inor

/maj

or a

llele

; OR

, od

ds

rati

o p

er c

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of m

inor

alle

le; C

I, c

onfi

den

ce in

terv

al; a O

nly

rs2

8242

93 w

as im

pu

ted

, th

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st w

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b Bas

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n B

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36.

2; c O

dd

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ad

just

ed fo

r ag

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-val

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are

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; e Gen

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ith

in a

n

area

of 1

Mb

cen

tere

d a

t th

e SN

P a

re li

sted

.

64 Chapter 4

P = 0.057). On the other hand, rs1353342 on chr. 9q21 was not associated with VF in

the ARREST-MI versus GENDER-MI analysis (OR = 0.84, 95% CI: 0.53, 1.34, P = 0.46).

Discussion

The SNPs most strongly associated with VF in this study appear to be located in an

intergenic area which lacks LD to SNPs within genes (Supplementary Figure 2).

These SNPs are not necessarily the functional variants for the observed effect but may

serve as proxies for the actual functional variant(s). Thus, these SNPs, or functional

SNPs in LD with them, are likely to modulate VF risk by exerting long-range effects on

gene expression 21. Inspection of 1 megabase spanning rs2824292 identifies 3 genes

in the region, all located within the 0.5 megabase downstream of this SNP. The gene

closest to rs2824292, located 98 Kb away, is CXADR encoding the Coxsackievirus and

adenovirus receptor (CAR) protein, a type I transmembrane protein of the tight junc-

tion 22,23. CAR has a long-recognized role as viral receptor in the pathogenesis of viral

myocarditis and its sequela of dilated cardiomyopathy 24,25. Notably, active coxsackie B

virus infection has been reported at a high frequency in a group of individuals with MI

who died suddenly 26. More recently, two studies have reported a physiological role for

the receptor in localization of connexin 45 at the intercalated disks of the atrio- ven-

tricular node cardiomyocytes, and its role in conduction of the cardiac impulse within

this cardiac compartment 27,28. Thus, CXADR is a candidate gene for the association

reported here. Two other genes are located within 0.5 megabases of rs2824292. BTG3,

located 179 Kb from rs2824292, encodes B-cell translocation gene 3, a member of the

PC3/BTG/TOB family of growth inhibitory genes 29. Its mRNA expression level is rela-

tively high in testis, lung, and ovary but low in heart 29. C21orf91, located 374 Kb from

rs2824292, encodes the human ortholog of the chicken EURL (early undifferentiated

retina and lens) gene, known to be expressed in the developing chick retina and lens

and suggested to play a role in the development of these structures 30,31.

Table 3 | Replication results for the three strongest AGNES loci

SNP Odds Ratio Per Copy ofMinor Allele (95% CI)a

P-value

rs2824292 1.49 (1.14-1.95) 0.004

rs1353342 0.84 (0.53-1.34) 0.46

rs12090554 0.70 (0.49-1.01) 0.057

ARREST-MI cases, n = 146; GENDER-MI controls, n = 391.aOdds ratios are adjusted for age and sex.


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The AGNES study did not include patients dying prior to hospital arrival and it is

likely that some of these cases died from VF arising immediately or very soon after

initiation of chest pain, before arrival of medical help. Thus, we cannot assert that the

genetic association identified in this study also holds for the group of patients who die

immediately after onset of symptoms. Nevertheless, an initial comparison of patients

presenting with VF occurring within 5 minutes of onset of chest pain (n = 119) versus

those presenting with VF occurring after more than 30 minutes of onset of chest pain

(n = 204) uncovered no significant difference in distribution of genotypes for rs2824292

(Supplementary Table 3).

Further studies, such as exploration of expression of these genes in human heart

as a function of genotype at the rs2824292 SNP (or other linked SNPs), as well as func-

tional studies, are next steps. SNPs in the NOS1AP locus, associated with variability in

QTc duration in the general population 32-34 have recently been implicated as predic-

tors of SCD risk in candidate SNP analyses 35,36. In our GWAS, however, there was no

association between SCD risk and SNPs at this locus. Predicting and preventing SCD

remains a major challenge for which new strategies are needed, and understanding

the underlying molecular mechanisms represents a crucial step in this process. Al-

though much of the focus on genetic influences on SCD risk in common diseases has

been on variants that directly or indirectly influence ion channel function, the results

of this study are hypothesis-generating and support a concept of a novel avenue for

research into alternative and/or complementary biological pathways that may be

implicated in VF.

Acknowledgements

This study was supported by research grants from the Netherlands Heart Foundation

(grants 2001D019, 2003T302 and 2007B202), the Leducq Foundation (grant 05-CVD)

and the Interuniversity Cardiology Institute of the Netherlands (project 27). Dr. C.R.

Bezzina is an Established Investigator of the Netherlands Heart Foundation (grant

2005T024). Dr. H.L. Tan is supported by the Netherlands Organization for Scientific

Research (NWO, grant ZonMW Vici 918.86.616) and the Medicines Evaluation Board

Netherlands (CBG). Dr. A. Bardai was supported by the Netherlands Organization

for Scientific Research (NWO, grant Mozaiek 017.003.084). Prof. A.H. Zwinderman

and E.P.A. van Iperen provided the genotype data of the GENDER study. We thank

Leander Beekman for technical support.

66 Chapter 4

References

1. Myerburg, R. J. & Castellanos, A. in Braunwald’s Heart Disease: ATextbook of Cardiovascular

Medicine (eds P. Libby, R. O. Bonow, D. L. Mann, & D. P. Zipes) (Elsevier, 2007).

2. Huikuri, H. V., Castellanos, A. & Myerburg, R. J. Medical progress: Sudden death due

to cardiac arrhythmias. New England Journal of Medicine 345, 1473-1482, doi: 10.1056/

NEJMra000650 (2001).

3. Arking, D. E., Chugh, S. S., Chakravarti, A. & Spooner, P. M. Genomics in sudden cardiac

death. Circulation Research 94, 712-723, doi: 10.1161/01.res.0000123861.16082.95 (2004).

4. Wilde, A. A. M. & Bezzina, C. R. Genetics of cardiac arrhythmias. Heart 91, 1352-1358, doi:

10.1136/hrt.2004.046334 (2005).

5. Watkins, H., Ashrafian, H. & McKenna, W. J. The genetics of hypertrophic cardiomyopathy:

Teare redux. Heart 94, 1264-1268, doi: 10.1136/hrt.2008.154104 (2008).

6. Zipes, D. P. et al. ACC/AHA/ESC 2006 Guidelines for Management of Patients With

Ventricular Arrhythmias and the Prevention of Sudden Cardiac Death: a report of the

American College of Cardiology/American Heart Association Task Force and the European

Society of Cardiology Committee for Practice Guidelines (writing committee to develop

Guidelines for Management of Patients With Ventricular Arrhythmias and the Prevention

of Sudden Cardiac Death): developed in collaboration with the European Heart Rhythm

Association and the Heart Rhythm Society. Circulation 114, e385-484, doi: 10.1161/circula-

tionaha.106.178233 (2006).

7. Zipes, D. P. & Wellens, H. J. J. Sudden cardiac death. Circulation 98, 2334-2351 (1998).

8. Myerburg, R. J. Sudden cardiac death: Exploring the limits of our knowledge. Journal of

Cardiovascular Electrophysiology 12, 369-381, doi: 10.1046/j.1540-8167.2001.00369.x (2001).

9. Noseworthy, P. A. & Newton-Cheh, C. Genetic Determinants of Sudden Cardiac Death.

Circulation 118, 1854-1863, doi: 10.1161/circulationaha.108.783654 (2008).

10. Myerburg, R. J. & Castellanos, A. Emerging paradigms of the epidemiology and demograph-

ics of sudden cardiac arrest. Heart Rhythm 3, 235-239, doi: 10.1016/j.hrthm.2005.09.023

(2006).

11. Friedlander, Y. et al. Family history as a risk factor for primary cardiac arrest. Circulation 97,

155-160 (1998).

12. Jouven, X., Desnos, M., Guerot, C. & Ducimetiere, P. Predicting sudden death in the popula-

tion - The Paris Prospective Study I. Circulation 99, 1978-1983 (1999).

13. Dekker, L. R. C. et al. Familial sudden death is an important risk factor for primary ventricu-

lar fibrillation - A case-control study in acute myocardial infarction patients. Circulation

114, 1140-1145, doi: 10.1161/circulationaha.105.606145 (2006).

14. Agema, W. R. P. et al. Current PTCA practice and clinical outcomes in the Netherlands: the

real world in the pre-drug-eluting stent era. European Heart Journal 25, 1163-1170, doi:

10.1016/j.ehj.2004.05.006 (2004).

15. Kleinjan, D. A. & van Heyningen, V. Long-range control of gene expression: Emerging

mechanisms and disruption in disease. American Journal of Human Genetics 76, 8-32, doi:

10.1086/426833 (2005).

16. Bergelson, J. M. et al. Isolation of a common receptor for coxsackie B viruses and adenovi-

ruses 2 and 5. Science 275, 1320-1323, doi: 10.1126/science.275.5304.1320 (1997).

17. Tomko, R. P., Xu, R. L. & Philipson, L. HCAR and MCAR: The human and mouse cellular

receptors for subgroup C adenoviruses and group B coxsackieviruses. Proceedings of the


Ch

ap

ter

4

National Academy of Sciences of the United States of America 94, 3352-3356, doi: 10.1073/

pnas.94.7.3352 (1997).

18. Bowles, N. E., Richardson, P. J., Olsen, E. G. J. & Archard, L. C. Detection of coxsackie-b-

virus-specific RNA sequences in myocardial biopsy samples from patients with myocarditis

and dilated cardiomyopathy. Lancet 1, 1120-1124 (1986).

19. Pauschinger, M. et al. Detection of adenoviral genome in the myocardium of adult patients

with idiopathic left ventricular dysfunction. Circulation 99, 1348-1354 (1999).

20. Hoggart, C. J., Clark, T. G., De Lorio, M., Whittaker, J. C. & Balding, D. J. Genome-wide

significance for dense SNP and resequencing data. Genetic Epidemiology 32, 179-185, doi:

10.1002/gepi.20292 (2008).

21. Andreoletti, L. et al. Active coxsackleviral B infection is associated with disruption of dystro-

phin in endomyocardial tissue of patients who died suddenly of acute myocardial infarction.

Journal of the American College of Cardiology 50, 2207-2214, doi: 10.1016/j.jacc.2007.07.080

(2007).

22. Lim, B.-K. et al. Coxsackievirus and adenovirus receptor (CAR) mediates atrioventricular-

node function and connexin 45 localization in the murine heart. Journal of Clinical Investi-

gation 118, 2758-2770, doi: 10.1172/jci34777 (2008).

23. Lisewski, U. et al. The tight junction protein CAR regulates cardiac conduction and cell-

cell communication. Journal of Experimental Medicine 205, 2369-2379, doi: 10.1084/

jem.20080897 (2008).

24. Strausberg, R. L. et al. Generation and initial analysis of more than 15,000 full-length human

and mouse cDNA sequences. Proceedings of the National Academy of Sciences of the United

States of America 99, 16899-16903, doi: 10.1073/pnas.242603899 (2002).

25. Yoshida, Y. et al. ANA, a novel member of Tob/BTG1 family, is expressed in the ventricular

zone of the developing central nervous system. Oncogene 16, 2687-2693, doi: 10.1038/

sj.onc.1201805 (1998).

26. Godbout, R., Andison, R., Katyal, S. & Bisgrove, D. A. Isolation of a novel cDNA enriched

in the undifferentiated chick retina and lens. Developmental Dynamics 227, 409-415, doi:

10.1002/dvdy.10310 (2003).

27. Arking, D. E. et al. A common genetic variant in the NOS1 regulator NOS1AP modulates

cardiac repolarization. Nature Genetics 38, 644-651, doi: 10.1038/ng1790 (2006).

28. Newton-Cheh, C. et al. Common variants at ten loci influence QT interval duration in the

QTGEN Study. Nature Genetics 41, 399-406, doi: 10.1038/ng.364 (2009).

29. Pfeufer, A. et al. Common variants at ten loci modulate the QT interval duration in the

QTSCD Study. Nature Genetics 41, 407-414, doi: 10.1038/ng.362 (2009).

30. Eijgelsheim, M. et al. Genetic variation in NOS1AP is associated with sudden cardiac death:

evidence from the Rotterdam Study. Human Molecular Genetics 18, 4213-4218, doi: 10.1093/

hmg/ddp356 (2009).

31. Kao, W. H. L. et al. Genetic Variations in Nitric Oxide Synthase 1 Adaptor Protein Are Associ-

ated With Sudden Cardiac Death in US White Community-Based Populations. Circulation

119, 940-951, doi: 10.1161/circulationaha.108.791723 (2009).

32. Agyemang, C. et al. Prevalence, awareness, treatment, and control of hypertension

among Black Surinamese, South Asian Surinamese and White Dutch in Amsterdam, The

Netherlands: the SUNSET study. Journal of Hypertension 23, 1971-1977, doi: 10.1097/01.

hjh.0000186835.63996.d4 (2005).

68 Chapter 4

33. Johnson, A. D. et al. SNAP: a web-based tool for identification and annotation of proxy SNPs

using HapMap. Bioinformatics 24, 2938-2939, doi: 10.1093/bioinformatics/btn564 (2008).

34. Sampietro, M. L. et al. A genome wide association analysis in the GENDER study. Nether-

lands Heart Journal 17, 262-264 (2009).

35. Aulchenko, Y. S., Ripke, S., Isaacs, A. & Van Duijn, C. M. GenABEL: an R library for genorne-

wide association analysis. Bioinformatics 23, 1294-1296, doi: 10.1093/bioinformatics/

btm108 (2007).

36. Li, Y. & Abecasis, G. R. Mach 1.0: rapid haplotype reconstruction and missing genotype

inference. Am J Hum Genet S79, 2290 (2006).


Ch

ap

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Supplemental Tables

Supplementary Table 1 | Most significant associations between single nucleotide polymorphisms (SNPs) and ventricular fibrillation in the AGNES case-control set

SNP Chr. Positiona Location Minor/MajorAllele

MAF b Imputed(yes/no)

Rsqc OR(95% CI)d

P-Valuee

rs2824293 21 17709385 Intergenic G/A 47 yes 1.00 1.78 (1.47,2.13) 3.3 × 10−10

rs2824292 21 17709047 Intergenic G/A 47 no 1.00 1.78 (1.47,2.13) 3.4 × 10−10

rs208910 21 17708105 Intergenic C/T 47 yes 0.99 1.77 (1.48,2.13) 4.4 × 10−10

rs12483129 21 17711037 Intergenic C/T 49 yes 0.96 0.58 (0.48,0.70) 6 × 10−9


rs2824294 21 17709789 Intergenic T/C 48 no 1.00 0.60 (0.50,0.72) 3.6 × 10−8

rs13050588 21 17721619 Intergenic T/C 37 yes 0.96 1.69 (1.40,2.04) 4.8 × 10−8

rs4818351 21 17720626 Intergenic C/T 37 yes 0.96 1.69 (1.40,2.05) 4.9 × 10−8


rs8130544 21 17708212 Intergenic C/T 36 no 1.00 1.63 (1.35,1.97) 3.2 × 10−7

rs2824290 21 17707528 Intergenic A/G 64 yes 0.99 0.61 (0.51,0.74) 3.3 × 10−07

rs2824289 21 17706394 Intergenic A/T 36 yes 0.99 1.63 (1.35,1.97) 3.3 × 10−7

rs1353342 9 78064589 Intergenic A/C 11 no 1.00 0.46 (0.34,0.63) 3.3 × 10−07

rs208918 21 17719150 Intergenic T/A 49 yes 0.97 1.59 (1.33,1.92) 3.6 × 10−7

rs2824288 21 17702254 Intergenic G/C 36 yes 0.98 1.61 (1.33,1.96) 4.7 × 10−7

rs208926 21 17722200 Intergenic C/A 48 no 1.00 1.56 (1.31,1.88) 5.1 × 10−7

rs12090554 1 183818971 Intergenic A/G 23 no 1.00 0.58 (0.47,0.72) 7.9 × 10−7

Chr, Chromosome; MAF, minor allele frequency; OR, Odds Ratio Per Copy of Minor Allele; aBased on Build 36.2; bAllele Frequency was based on the total AGNES sample; cRsq estimates the squared cor-relation between imputed and true genotypes; dOdds ratios are adjusted for age and sex; eThe P-values are corrected for genomic control factor.

70 Chapter 4

Supplementary Table 2 | Genotype distributions in the patient samples studied and in a sample of the general population

Sample N Frequencies (95% CI)

rs2824292

AA AG GG

AGNES cases 513 0.22 (0.19-0.26) 0.48 (0.44-0.52) 0.30 (0.26-0.34)

AGNES controls 457 0.37 (0.32-0.41) 0.48 (0.43-0.52) 0.15 (0.12-0.19)

ARREST-MI (cases) 146 0.25 (0.18-0.32) 0.47 (0.39-0.55) 0.27 (0.20-0.35)

GENDER-MI (controls) 391 0.35 (0.30-0.40) 0.48 (0.43-0.53) 0.17 (0.13-0.21)

General Population 492 0.29 (0.25-0.33) 0.52 (0.48-0.57) 0.19 (0.16-0.23)

rs1353342

GG GT TT

AGNES cases 514 0.86 (0.83-0.89) 0.13 (0.10-0.16) 0.01 (0.00-0.01)

AGNES controls 457 0.73 (0.69-0.77) 0.25 (0.21-0.29) 0.02 (0.01-0.03)

ARREST-MI (cases) 146 0.82 (0.76-0.88) 0.17 (0.11-0.23) 0.01 (−0.01-0.02)



rs12090554

GG AG AA

AGNES cases 515 0.67 (0.63-0.71) 0.29 (0.25-0.33) 0.04 (0.02-0.06)

AGNES controls 455 0.51 (0.46-0.56) 0.42 (0.38-0.47) 0.07 (0.04-0.09)

ARREST-MI (cases) 143 0.66 (0.59-0.74) 0.31 (0.24-0.39) 0.02 (0.0-0.04)



Supplementary Table 3 | Genotype frequencies and odds ratio (95% CI) for rs2824292 Based on time-to-VF from Onset of Complaints

Homozygousfor

major allele

Heterozygous Homozygousfor

minor allele

Odds ratio(95% CI)

additive modela

P valuea P valueb

No VF 168(36.8%) 219(48.0%) 69(15.1%) 1.00 (ref)

Early onset VFc 30(25.2%) 51(42.9%) 38(31.9%) 1.79(1.34-2.40) 7.4 × 10−5

0.76Late onset VFd 51(25.0%) 92(45.1%) 61(29.9%) 1.71(1.35-2.17) 1.0 × 10−5

aCompares early onset or late onset VF against controls adjusted for age and sex; bCompares early onset vs. late onset VF; adjusted for age and sex; cVF occurring within 5 minutes of onset of chest pain; dVF occurring after more than 30 minutes of onset of chest pain.

GWAS identifi es a susceptibility locus at 21q21 for VF in MI 71

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Supplemental Figures

GWAS identifies a region at chr 21q21 as a susceptibility locus for VF in acute MI | 75

4


Supplementary Figure 1 | Quantile-quantile plot of test statistics (P-values) for the associations of single-nucleotide polymorphisms with ventricular fibrillation in the AGNES Population. Under the null hypothesis of no association, the data points would be expected to fall on the black line. P-values corrected for possible population stratification by means of the genomic control method are presented (λ = 1.026).

Supplementary Figure 2 | Haploview plot of linkage disequilibrium (D') for all CEU HapMap SNPs in a 500 Kb region spanning rs2824292. Colour scheme: D′ < 1 and LOD < 2 = white, D′ = 1 and LOD < 2 = blue, D′ < 1 and LOD ≥ 2 = shades of pink/red, D′ = 1 and LOD ≥ 2 = bright red.

Supplementary Figure 1 | Quantile-quantile plot of test statistics (P-values) for the associations of single-nucleotide polymorphisms with ventricular fi brillation in the AGNES Population. Under the null hypothesis of no association, the data points would be expected to fall on the black line. P-values corrected for possible population stratifi cation by means of the genomic control method are present-ed (λ = 1.026).

GWAS identifies a region at chr 21q21 as a susceptibility locus for VF in acute MI | 75

4


Supplementary Figure 1 | Quantile-quantile plot of test statistics (P-values) for the associations of single-nucleotide polymorphisms with ventricular fibrillation in the AGNES Population. Under the null hypothesis of no association, the data points would be expected to fall on the black line. P-values corrected for possible population stratification by means of the genomic control method are presented (λ = 1.026).

Supplementary Figure 2 | Haploview plot of linkage disequilibrium (D') for all CEU HapMap SNPs in a 500 Kb region spanning rs2824292. Colour scheme: D′ < 1 and LOD < 2 = white, D′ = 1 and LOD < 2 = blue, D′ < 1 and LOD ≥ 2 = shades of pink/red, D′ = 1 and LOD ≥ 2 = bright red.

Supplementary Figure 2 | Haploview plot of linkage disequilibrium (D′) for all CEU HapMap SNPs in a 500 Kb region spanning rs2824292. Colour scheme: D′ < 1 and LOD < 2 = white, D′ = 1 and LOD < 2 = blue, D′ < 1 and LOD ≥ 2 = shades of pink/red, D′ = 1 and LOD ≥ 2 = bright red.

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