APPENDIX
PARTICIPATING STUDY DESCRIPTIONS
German Competence Network for Atrial Fibrillation (AFNET/KORA)
The German Competence Network for Atrial Fibrillation (AFNET) is a national registry of AF
patients. In the context of the registry, additional DNA samples have been collected from
patients with AF onset before age 60 years at the Medical Department I of the University
Hospital Munich, Campus Grosshadern of the Ludwig-Maximilians University Munich in
collaboration with the Institute of Epidemiology at the Helmholtz Zentrum Munich. Cases were
selected if the diagnosis of AF was made on an electrocardiogram analyzed by a trained
physician. Patients with signs of moderate to severe heart failure, moderate to severe valve
disease or with hyperthyroidism were excluded from the study. Referent subjects were drawn
from the Cooperative Research in the Region of Augsburg (KORA) S4 study, with ages ranging
from 25-74 years, and had no history of AF, myocardial infarction, heart failure or valve disease
and had documented sinus rhythm at the time of blood draw. The KORA S4 study is a
population-based epidemiological survey of persons living in the city of Augsburg, Southern
Germany, or its two adjacent counties and was conducted between 1999 and 2001. The survey
population consisted of German nationality residents identified through the registration office. A
sample of 6640 participants was drawn with ten strata of equal size according to sex and age, and
4261 individuals (66.8%) agreed to participate.
Age, Gene/Environment Susceptibility Reykjavik Study (AGES)
The original Reykjavik Study, conducted between 1967 and 1996, included approximately 19000
men and women living in the greater Reykjavik area, born between 1907 and 1935 (1). Survivors
of this study were invited to be part of AGES, which recruited 5764 men and women in 20022006. Of these, 5427 had a complete clinic exam, and 4469 met inclusion criteria and were
considered for this analysis.
Atherosclerosis Risk in Communities Study (ARIC)
The ARIC study recruited 15792 men and women, aged 45-64 years, from 4 communities in the
United States (Forsyth County, NC; Washington County, MD; Jackson, MS; and suburbs of
Minneapolis, MN) in 1987-89 (2). Participants were mostly white in the Minnesota and
Washington County field centers, white and African-American in Forsyth County, and
exclusively African-American in the Jackson field center. After study inception, participants had
3 follow-up examinations, each approximately 3 years apart.
Cleveland Clinic Lone AF GeneBank Study (CCAF)
The Cleveland Clinic Lone AF GeneBank Study is comprised of individuals aged ≥ 18 years
with lone atrial fibrillation (AF), defined as that occurring in the absence of significant structural
heart disease. Participants had a history of recurring or persistent lone AF, ≤ 50% coronary artery
stenosis in the coronary arteries (if cardiac catheterization done) or with normal stress test results
(documentation of normal cardiac catheterization or stress test required if age ≥ 50 years), and
had normal left ventricular ejection fraction (LVEF) ≥ 50%. Subjects were excluded if they had
heart failure, history of significant valvular disease (>2+ valvular regurgitation, any valvular
stenosis), significant coronary artery disease (>50% coronary artery stenosis), prior myocardial
infarction, prior percutaneous coronary intervention, or coronary artery bypass graft, or latest
LVEF <50%. Referent subjects were drawn from the Illumina iControlDB online database.
Referent subjects were included if they came from iControlDB Studies 64, 65, 66 or 67. All
referent subjects in those studies were identified as Caucasian. Age at DNA collection, sex, and
race were the only available variables for the referent subjects.
Cardiovascular Health Study (CHS)
In 1989-90, CHS recruited 5201 men and women 65 years or older from 4 communities (Forsyth
County, NC; Washington County, MD; Sacramento County, CA; and Pittsburgh, PA) (3). CHS
participants had annual study exams through 1999; surveillance for cardiovascular events has
been ongoing from baseline through the present. Included in this analysis were CHS participants
of European ancestry who were free of clinically recognized myocardial infarction, stroke, and
heart failure at baseline and who gave consent for research use of their genetic data.
Framingham Heart Study (FHS)
The FHS is a community-based observational, cohort study initiated in 1948 to prospectively
investigate cardiovascular disease and its risk factors. The Original cohort (4) (n=5209) has
received biennial exams. The Original Cohort children (& spouses), termed the Offspring cohort
(5) (n=5214), were recruited in 1971, and are examined every four to eight years. At each FHS
clinic examination, participants’ medical histories, physical examinations, and
electrocardiograms were obtained to ascertain symptoms and findings suggestive of
cardiovascular disease. Records of all interim hospitalizations for cardiovascular disease were
sought for review. Participants were classified as having AF if either atrial flutter or fibrillation
was present on an electrocardiogram obtained at an FHS clinic visit or encounter with an
external clinician, Holter monitoring, or noted in hospital records. Two physicians adjudicated
AF events in FHS (6).
Heart and Vascular Health Study (HVH)
The Heart and Vascular Health Study (HVH) is a study of incident AF in the setting of Group
Health Cooperative, a large integrated health care system in Washington State, USA. All plan
members assigned a new ICD-9 code of 427.31 or 427.32 in the inpatient or outpatient setting
between 1 October 2001 and 31 December 2004 were identified. Incident AF was verified by
review of medical records with the requirement that the AF be documented by 12-lead
electrocardiogram and clinically recognized by a physician, with no previous evidence of AF in
the medical record. Control subjects were identified from the Group Health membership, and had
no history of AF. AF cases included in this analysis had early-onset AF; they were less than 66
years of age at AF diagnosis, without a history of coronary artery disease, valvular disease, heart
failure, poor left ventricular function, chronic obstructive pulmonary disease, active cancer or
hyperthyroidism. Referent subjects were identified from the enrollment of Group Health
Cooperative, were 40-69 years old, and had no history of AF.
LURIC
The Ludwigshafen Risk and Cardiovascular Health (LURIC) study is an ongoing prospective
study of more than 3,300 individuals of German ancestry in whom cardiovascular and metabolic
phenotypes (CAD, MI, dyslipidemia, hypertension, metabolic syndrome and diabetes mellitus)
have been defined or ruled out using standardized methodologies in all study participants.
Inclusion criteria for LURIC were: German ancestry (limitation of genetic heterogeneity),
clinical stability (except for acute coronary syndromes) and availability of a coronary angiogram.
Exclusion criteria were: any acute illness other than acute coronary syndromes, any chronic
disease where non-cardiac disease predominated and a history of malignancy within the last five
years. Genome-wide analyses using the Affymetrix 6.0 have been completed in all participants.
A 10-year clinical follow-up for total and cause specific mortality has been completed.
Massachusetts General Hospital Atrial Fibrillation Study (MGH/MIGEN)
The Massachusetts General Hospital Atrial Fibrillation Study (MGH) enrolled serial patients
with lone AF or AF and hypertension referred to the arrhythmia service between July 5, 2001
and February 19, 2008. Inclusion criteria were AF documented by electrocardiography, and age
less than 66 years. Individuals with structural heart disease as assessed by echocardiography,
hyperthyroidism, myocardial infarction, or heart failure were excluded. Each patient underwent a
physical examination and standardized interview. All patients were evaluated by 12-lead
electrocardiogram, echocardiogram, and laboratory studies. Referent subjects were selected from
the control population of the MIGEN study (7), and were healthy patients without a history of
MI.
PROSPER
All data come from the PROspective Study of Pravastatin in the Elderly at Risk (PROSPER) (8).
A detailed description of the study has been published elsewhere. PROSPER was a prospective
multicenter randomized placebo-controlled trial to assess whether treatment with pravastatin
diminishes the risk of major vascular events in elderly. Between December 1997 and May 1999,
we screened and enrolled subjects in Scotland (Glasgow), Ireland (Cork), and the Netherlands
(Leiden). Men and women aged 70-82 years were recruited if they had pre-existing vascular
disease or increased risk of such disease because of smoking, hypertension, or diabetes. A total
number of 5,804 subjects were randomly assigned to pravastatin or placebo. A large number of
prospective tests were performed including BioBank tests and cognitive function measurements.
A whole genome wide screening has been performed in the sequential PHASE project with the
use of the Illumina 660K beadchip. Of 5,763 subjects DNA was available for genotyping.
Genotyping was performed with the Illumina 660K beadchip, after QC (call rate <95%) 5,244
subjects and 557,192 SNPs were left for analysis. These SNPs were imputed to 2.5 million SNPs
based on the HAPMAP built 36 with MACH imputation software.
Rotterdam Study (RS-I, RS-II)
The Rotterdam Study (RS) is a community-based study of elderly individuals from a suburb of
Rotterdam with a focus on identifying determinants of health and cardiovascular, neurogeriatric,
bone, and eye diseases (9). Participants age ≥55 years were examined up to 4 times every 3
years. AF was diagnosed based on study visit electrocardiograms, review of hospital discharge
information, and general practitioner diagnoses. AF was verified by two physicians and
disagreements settled by review of a cardiologist. The first cohort (RS I) was founded in 1990,
and included 7,983 participants. The current analysis included 5,974 participants from RS-I that
met inclusion criteria. The second cohort (RS II) was started in 2000 with the same inclusion
criteria and included 3,011 participants. Of these 1,805 participants met inclusion criteria.
Study of Health in Pomerania (SHIP)
Study of Health in Pomerania (SHIP) is a longitudinal population-based cohort study in West
Pomerania, a region in the northeast of Germany. SHIP was designed to assess prevalence and
incidence of common risk factors, subclinical disorders and clinical diseases and to investigate
complex associations among risk factors, subclinical disorders and clinical diseases. From the
total population comprising 212,157 inhabitants in 1995, a two-stage stratified cluster sample of
adults aged 20 to 79 years was drawn. From the net sample of 6,265 eligible subjects, 4,308
subjects (2,192 women) of European ancestry participated in the baseline examination, SHIP-0
(response 68.8%). During the baseline examination between 1997 and 2001 (SHIP-0) as well as
during the 5-year follow-up examination between 2002 and 2006 (SHIP-1) resting
electrocardiograms were digitally stored (Personal 120LD, Esaote, Genova, Italy) and processed
by the MEANS ECG Interpretation and Measurement software (Welch Allyn, Skaneateles Falls,
NY) according to the method described above for the RS. In addition, a Tele-ECG subproject
was conducted in SHIP-1 to assess the prevalence of symptomatic and asymptomatic cardiac
arrhythmias. Subjects were considered as having AF if it was present in at least one of these
examinations.
Women’s Genome Health Study (WGHS)
The Women’s Genome Health Study (WGHS) is a prospective cohort comprised of over 25,000
initially healthy female health professionals enrolled in the Women’s Health Study, which began
in 1993. All participants in WGHS provided baseline blood samples and extensive survey data.
Women were asked to report diagnoses of AF at baseline, 48 months, and then annually
thereafter. Beginning on September 19, 2006, women enrolled in the continued observational
follow-up who reported an incident AF event after baseline on at least one yearly questionnaire
were sent an additional questionnaire to confirm the episode and to collect additional
information. They were also asked for permission to review their medical records, particularly
available ECGs, rhythm strips, 24-hour ECGs, and information on cardiac structure and function.
For all deceased participants who reported AF during the trial and extended follow-up period,
family members were contacted to obtain consent and additional relevant information. An endpoint committee of physicians reviewed medical records for reported events according to
predefined criteria. An incident AF event was confirmed if there was ECG evidence of AF or if a
medical report clearly indicated a personal history of AF. The earliest date in the medical records
when documentation was believed to have occurred was set as the date of onset of AF. Only
confirmed events are included in this analysis. Prevalent AF events self reported at the beginning
of the study were not confirmed by medical record review, although confirmation rates of selfreported AF in this cohort are high.
BioBank Japan
BioBank Japan is a hospital-based disease study of 47 common diseases including AF, in which
66 clinical hospitals in Japan have participated. Cases in this study are participants of BioBank
Japan enrolled between 2003 and 2006. Control subjects included those with 11 diseases (hepatic
cirrhosis, osteoporosis, colorectal cancer, breast cancer, prostate cancer, lung cancer, uterine
myoma, amyotrophic lateral sclerosis, drug eruption, gallbladder and bile duct cancer and
pancreatic cancer) registered in BioBank Japan and 906 healthy volunteers who were recruited
from Osaka-Midosuji Rotary Club.
In this analysis, principal components of ancestry were available for adjustment in a
subset comprised of 844 cases, and all 3,393 control samples.
FUNDING / SUPPORT
AFNET/KORA: German National Genome Research Network NGFN 01GS0838, 01GR0803,
BMBF-01EZ0874, 01GR0803, NGFN 01GI0204, 01GR0103, NGFN-2, NGFNPlus 01GS0823,
and NGFNPlus 01GS0834; German Federal Ministry of research 01EZ0874; German
Competence Network on AF (AFNET) 01 GI 0204/N; Leducq Foundation 07-CVD 03, BMBF
spitzen cluster personalized medicine m4 (01 EX1021E), LMU Excellence Initiative (42595-6);
Munich Center of Health Sciences (MC Health) as part of LMUinnovativ. The KORA research
platform (KORA, Cooperative Research in the Region of Augsburg) was initiated and financed
by the Helmholtz Zentrum München - German Research Center for Environmental Health,
which is funded by the German Federal Ministry of Education and Research and by the State of
Bavaria. Furthermore, KORA research was supported within the Munich Center of Health
Sciences (MC Health), Ludwig-Maximilians-Universität, as part of LMUinnovativ. Dr. Sinner is
supported by the German Heart Foundation.
AGES: The Age, Gene/Environment Susceptibility Reykjavik Study has been funded by NIH
contract N01-AG-12100, the NIA Intramural Research Program, Hjartavernd (the Icelandic
Heart Association), and the Althingi (the Icelandic Parliament).
ARIC: The Atherosclerosis Risk in Communities Study is carried out as a collaborative study
supported by National Heart, Lung, and Blood Institute contracts (HHSN268201100005C,
HHSN268201100006C, HHSN268201100007C, HHSN268201100008C,
HHSN268201100009C, HHSN268201100010C, HHSN268201100011C, and
HHSN268201100012C), R01HL087641, R01HL59367 and R01HL086694; National Human
Genome Research Institute contract U01HG004402; and National Institutes of Health contract
HHSN268200625226C. The authors thank the staff and participants of the ARIC study for their
important contributions. Infrastructure was partly supported by Grant Number UL1RR025005, a
component of the National Institutes of Health and NIH Roadmap for Medical Research. This
work was additionally supported by grants RC1-HL-099452 from the National Heart, Lung, and
Blood Institute and 09SDG2280087 from the American Heart Association.
CCAF: R01 HL090620 from the National Heart, Lung, and Blood Institute (Chung, Barnard, J.
Smith, Van Wagoner); NIH/NCRR, CTSA 1UL-RR024989 (Chung, Van Wagoner); Heart and
Vascular Institute, Department of Cardiovascular Medicine, Cleveland Clinic (Chung); Leducq
Foundation 07-CVD 03 (Van Wagoner, Chung); Atrial Fibrillation Innovation Center, State of
Ohio (Van Wagoner, Chung).
CHS: This CHS research was supported by NHLBI contracts HHSN268201200036C,
HHSN268200800007C, N01HC55222, N01HC85079, N01HC85080, N01HC85081,
N01HC85082, N01HC85083, N01HC85086; and NHLBI grants HL080295, HL087652,
HL105756 with additional contribution from the National Institute of Neurological Disorders
and Stroke (NINDS). Additional support was provided through AG023629 from the National
Institute on Aging (NIA). A full list of CHS investigators and institutions can be found at
http://www.chs-nhlbi.org/pi.htm. The provision of genotyping data was supported in part by the
National Center for Advancing Translational Sciences, CTSI grant UL1TR000124, and the
National Institute of Diabetes and Digestive and Kidney Disease Diabetes Research Center
(DRC) grant DK063491 to the Southern California Diabetes Endocrinology Research Center.
FHS: Our research was conducted using data and resources from FHS of the National Heart
Lung and Blood Institute of the National Institutes of Health and Boston University School of
Medicine based on analyses by Framingham Heart Study investigators participating in the SNP
Health Association Resource (SHARe) project. The work was supported by Contract No. N01HC-25195 and its contract with Affymetrix, Inc for genotyping services Contract No.N02-HL-64278. A portion of this research utilized the Linux Cluster for Genetic Analysis (LinGA-II)
funded by the Robert Dawson Evans Endowment of the Department of Medicine at Boston
University School of Medicine and Boston Medical Center. Other support came from 1R01
HL092577; 1RO1 HL076784; 1R01 AG028321; Evans Center for Interdisciplinary Biomedical
Research ARC on Atrial Fibrillation at Boston University
(http://www.bumc.bu.edu/evanscenteribr/the-arcs/the-arcs/
(Benjamin), 6R01-NS 17950 and American Heart Association 09FTF2190028.
HVH: The Heart and Vascular Health Study is supported by grant numbers R01 HL 068986,
R01 HL085251, and R01 HL073410 from the National Heart Lung and Blood Institute.
LURIC: LURIC has received funding from the 6th Framework Program (integrated project
Bloodomics, grant LSHM-CT-2004-503485) and from the 7th Framework Program
(Atheroremo, grant agreement number 201668 and RiskyCAD, grant agreement number 305739)
of the European Union as well as from the INTERREG IV Oberrhein Program (Project A28,
Genetic mechanisms of cardiovascular diseases) with support from the European Regional
Development Fund (ERDF) and the Wissenschaftsoffensive TMO.
MGH/MIGEN: This work was supported by NIH grants R01HL092577 (Ellinor and Benjamin),
R01HL104156, K24HL105780 (Ellinor), K23HL114724 (Lubitz), and an American Heart
Association Established Investigator Award 13EIA14220013 (Ellinor) and Fellow to Faculty
Award 12FTF11350014 (Lubitz).
PROSPER: The PROSPER study was supported by an investigator initiated grant obtained from
Bristol-Myers Squibb. Prof. Dr. J. W. Jukema is an Established Clinical Investigator of the
Netherlands Heart Foundation (grant 2001 D 032). Support for genotyping in PHASE was
provided by the seventh framework program of the European commission (grant 223004) and by
the Netherlands Genomics Initiative (Netherlands Consortium for Healthy Aging grant 050-060810).
Rotterdam Study: The Rotterdam Study (RS) is supported by the Erasmus Medical Center and
Erasmus University Rotterdam; The Netherlands Organization for Scientific Research; The
Netherlands Organization for Health Research and Development (ZonMw); the Research
Institute for Diseases in the Elderly; The Netherlands Heart Foundation; the Ministry of
Education, Culture and Science; the Ministry of Health Welfare and Sports; the European
Commission; and the Municipality of Rotterdam. Support for genotyping was provided by The
Netherlands Organization for Scientific Research (NWO) (175.010.2005.011, 911.03.012) and
Research Institute for Diseases in the Elderly (RIDE). This study was supported by The
Netherlands Genomics Initiative (NGI)/Netherlands Organization for Scientific Research (NWO)
project nr. 050-060-810.
SHIP: SHIP is part of the Community Medicine Research net of the University of Greifswald,
Germany, which is funded by the Federal Ministry of Education and Research (grants no.
01ZZ9603, 01ZZ0103, and 01ZZ0403), the Ministry of Cultural Affairs as well as the Social
Ministry of the Federal State of Mecklenburg-West Pomerania. Generation of genome-wide data
has been supported by the Federal Ministry of Education and Research (grant no. 03ZIK012) and
a joint grant from Siemens Healthcare, Erlangen, Germany and the Federal State of
Mecklenburg- West Pomerania. The University of Greifswald is a member of the ‘Center of
Knowledge Interchange’ program of the Siemens AG.
WGHS: The WGHS is supported by HL 099355 (Buring), HL 043851 (Buring) and HL 080467
(Buring) from the National Heart, Lung, and Blood Institute and CA 047988 (Buring) from the
National Cancer Institute, the Donald W. Reynolds Foundation and the Fondation Leducq
(Ridker), with collaborative scientific support and funding for genotyping provided by Amgen.
AF endpoint confirmation was supported by HL-093613 (Albert) and a grant from the Harris
Family Foundation (Tedrow).
BioBank Japan: This study was supported by the Ministry of Education, Culture, Sports,
Science, and Technology, Japan.
Role of the Sponsor: None of the funding agencies had any role in the study design, data
collection or analysis, interpretation of the data, writing of the manuscript, or in the decision to
submit the manuscript for publication.
Supplemental Methods
Phylogenetic conservation
The conservation score (represented as the phyloP score) was obtained from the UCSC Genome
Browser, which was calculated by the multiple sequence alignment of 44 vertebrate species.
More information is available at http://genome.ucsc.edu/cgibin/hgTrackUi?hgsid=342995335&c=chr4&g=cons44way. Regions surrounding the identified
non-redundant SNPs from the conditional analysis and flanked by 5 kb on either side (upstream
and downstream) were identified, and conservation scores across these regions were averaged. In
order to calculate the conservation score at the remainder of the chromosome 4q25 locus, we
averaged the conservation scores across the region centered on SNP rs6817105 and flanked by 1
Mb on either side (upstream and downstream), minus the non-redundant regions described
above. The conservation scores were compared between the non-redundant regions and the
remainder of the chromosome 4q25 locus with the Student’s t-test assuming equality of
variances.
Supplemental Table 1. Details regarding study samples, genotyping and data cleaning.
AFNET /
MGH /
AGES
ARIC
CHS
CCAF
FHS
HVH
LURIC
KORA
MIGEN
CardioCleveland Framingham Heart and Ludwigshafen
Mass.
Study German AF Age, Gene/ Atherosclerosis
Network Environment
Risk in
vascular
Clinic AF Heart Study Vascular
Risk and
General
Susceptibility Communities Health Study
Study
Health Cardiovascular Hospital AF
Study
Study
Study
Health Study Study &
MIGEN
(12)
(13,14)
(15)
(4,16)
Design (10,11)
papers
Illumina
Affymetrix 6.0 Illumina 370
Illumina
Affymetrix
Array Illumina
HumanCN HumanCNV37
CNV
Hap550 v1 or Gene Chip®
V370
0-Duo
v3 and
500K Array
And
BeadChip
Hap610 v1
Set
Illumina
& 50K
Human550
Human
K
Gene
Focused
Panel
Birdseed
BeadStudio BeadStudio Bayesian
Callin BeadStudio BeadStudio
Robust
g
Linear
Algori
Modeling
thm
<98%
<97%
<95%
<97%
<95%
<97%
Per
SNP
Call
rate
<10-5
<10-6
<10-6
<10-5
FDR < 0.20
<10-6
HWE
pvalue
NA
NA
NA
≤2
NA
N>100
Mend
elian
errors
ND
NA
NA
ND
FDR < 0.01
subject
Excess
heterohetero
zygosity >5
zygosi
SD away
ty
from the
mean
<5%
<1%
<1%
Excluded
<5%
<1%
MAF
SNPs with 0
heterozygotes
(17)
(18)
(19)
Illumina Affymetrix 6.0 Affymetrix
370 CNV
6.0
PHASE /
RS-I
PROSPER
PHArmacogenetic Rotterdam
Study-!
study of Statins in
the Elderly at risk /
PROspective Study
of Pravastatin in the
Elderly at Risk for
vascular disease
(8,20)
(9)
Illumina 660K
beadchip
RS-II
Rotterdam
Study-II
(9)
SHIP
WGHS
The Study of Women’s
Health in
Genome
Pomerania Health Study
(21)
(22)
BioBank
Japan
BioBank
Japan
(23)
Illumina
Illumina550K Affymetrix
Illumina
Illumina
Infinium Duo, 610KQuad
6.0
HumanHap3 Human610HumanHap5
00 Duo+
Quad and
50Illumina
chip v3.0
Human
Hap550v3
BeadChip
BeadStudio
Birdseed
Birdseed
Beadstudio
BeadStudio
<97%
< 98%
<97%
<98%
<98%
<98%
ND
<90%
<99%
<10-5
<10-4
<10-6
<10-6
<10-6
<10-6
ND
<10-6
<10-6
≤2
NA
NA
NA
NA
NA
NA
NA
NA
ND
ND
ND
subject heterozygosity >4 SD
away from the
mean
ND
ND
ND
Excluded
SNPs with
0
heterozygot
< 1%
<1%
<1%
ND
<1%
<1%
GenomeStudio
>0.336; n=21 >0.336; n=21
<1%
<1%
BeadStudio BeadStudio BeadStudio
v2
AFNET /
KORA
Selecti
on
criteri
a for
PCs
P<0.05
4
Numb
er of
PCs in
the
model
Numb 315,972
er of
SNPs
used
for
imput
ation
Imput Mach1 v
ation 1.0.10(24)
softwa
re
Imput Build 35
ation
Backb
one /
NCBI
Build
Build 35
SNP
positio
n from
NCBI
AGES
ARIC
CHS
CCAF
FHS
PCs examined P< 0.0005
All PCs
in relation to
unassociated,
incident and
p>0.05
prevalent AF.
Bonferroni
adjusted P
value
threshold to
determine
whether PCs
were
associated
with AF.
6
0
HVH
LURIC
MGH /
MIGEN
es
ND
ND
P<0.05
NA
NA
6
4
NA
NA
0
1 EV for
incident AF,
3 EVs for
prevalent AF
2
305,353
686,195
638,338
557,192
530,683
537,405
869,224
331959
31,927
P<0.05
Eigenstrat:
anyone >8 SD
from top 10
PCs was
removed
(225/9747
individuals)
NA
NA
308,340
602,642
306,655
460,569
Mach1 v
1.0.16(24)
Mach1 v
1.0.16(24)
BIMBAM
Mach1 v
1.0.16(24)
Build 36
Build 36
Build 36
Build 36
Build 36
Build 36
Build 36
Build 36
Build 36
Build 36
Build 36
Build 36
Build 36
Build 36
385,958
Mach1 v BIMBAM(
1.0.15(24)
25)
Mach 1 v
1.0.16(24)
HapMap II
CEU r22
PHASE /
PROSPER
RS-I
RS-II
SHIP
WGHS
BioBank
Japan
Outliers as
Outliers as
Outliers as AF, adjusted Association Within ±
identified by IBS identified by identified by for sex+age,
with AF
0.02 from
clustering were
IBS
IBS clustering
was
(p<0.05)
centroid for
excluded
clustering were excluded associated
both
were
with the PC
components
excluded
(p<0.05),
tested for
first 10 PCs
obtained
from
EIGENSTR
AT
Mach1 Mach1 v 1.0.15(24) Mach1 v Machv1.0.16(24 IMPUTEv0.5 Mach1 v. SHAPEIT v2
v1.0.16(24)
1.0.15(24)
)
.0 against
1.0.16(24)
r71 +
HapMap II HapMap II IMPUTEv2.3
CEU v22
CEU r22
.0against
1000
Genomes
Integrated
Phase 1 v3
Build 36
Build 36
Build 36
Build 36
Build 36
Build 36
Build 37
(GRCh37)
Build 36
Build 36
Build 36
Build 36
Build 36
Build 36
Build 37
(GRCh37)
AFNET /
KORA
AGES
ARIC
CHS
CCAF
FHS
HVH
LURIC
MGH /
MIGEN
build
R(27)
ProbABEL, R
R(27)
GWA ProbABEL ProbABEL(26) ProbABEL(26) R, version 2.7 ProbABEL(2 R packages
(26), R(27)
, R(27)
, PLINK(28),
6), R(27)
kinship,
S
R(27)
GEE,
Statist
COXPH (27)
ical
Analy
sis
2,408,991
2,512,759
I:2,319,581
2,509,367 I: 2501666 2,316,203
2,543,887
2,508,401
Total 2,521,723
P: 2,317,847
P: 2501188
numb
er of
SNPs
used
in the
analys
is
(MAF
>0.005
)
1.02
I: 1.005
1.007
I: 1.045
1.034
I:1.017
1.09
0.997
1.022
Inflati
P:1.062
P: 1.038
P:1.038
on
factor
(λ)
Abbreviations: NA, not available.
PLINK, http://pngu.mgh.harvard.edu/purcell/PLINK/
Eigenstrat, http://genepath.med.harvard.edu/~reich/Software.htm
MACH, http://www.sph.umich.edu/csg/abecasis/MaCH/index.html
BIMBAM, http://stephenslab.uchicago.edu/software.html
PHASE /
PROSPER
RS-I
RS-II
SHIP
WGHS
BioBank
Japan
ProbABEL
Mach2QTL Mach2QTL QUICKTES ProbABEL,
GenABEL + GenABEL +
T v0.94
R
PLINK,
PLINK, R(27),
R(27),
GRIMP(29)
GRIMP(29)
R, version
2.10.0
2,543,887
I: 2,502,002
P: 2,501,903
2,541,494
2,598,639
2,608,508
430,963
I:1.035
P:1.024
1.006
0.998
1.017
1.03
Supplemental Table 2. Construction of genetic risk score categories.
Unweighted
Weighted
Chromosome 4q25
All loci
Chromosome 4q25
All loci
score<0.5 = 0
score<0.5 = 0
score<0.106 = 1
score<0.922 = 1
score≥0.5 & score<1.5 = score≥0.5 & score<2.5
score≥0.106 &
score≥0.922 &
1
=2
score<0.145 = 2
score<1.072 = 2
score≥1.5 & score<2.5 = score≥2.5 & score<4.5
score≥0.145 &
score≥1.072 &
2
=4
score<0.214 = 3
score<1.179 = 3
score≥2.5 & score<3.5 = score≥4.5 & score<6.5
score≥0.214 &
score≥1.179 &
3
=6
score<0.230 = 4
score<1.282 = 4
score≥3.5 & score<4.5 = score≥6.5 & score<8.5
score≥0.230 &
score≥1.282 &
4
=8
score<0.325 = 5
score<1.385 = 5
score≥4.5 & score<5.5 =
score≥8.5 &
score≥0.325 &
score≥1.385 &
5
score<10.5 = 10
score<0.370 = 6
score<1.487 = 6
score≥5.5 & score<6.5 =
score≥10.5 &
score≥0.370 &
score≥1.487 &
6
score<12.5 = 12
score<0.557 = 7
score<1.588 = 7
score≥6.5 & score<7.5 =
score≥12.5 &
score≥0.557 &
score≥1.588 &
7
score<14.5 = 14
score<0.692 = 8
score<1.749 = 8
score≥7.5 & score<8.5 =
score≥14.5 &
score≥0.692 &
score≥1.749 &
8
score<16.5 = 16
score<0.815 = 9
score<1.943 = 9
score≥16.5 &
score≥0.815 = 10
score≥1.943 = 10
score<18.5 = 18
score≥18.5 &
score<20.5 = 20
score≥20.5 &
score<22.5 = 22
score≥22.5 &
score<24.5 = 24
Weights were applied as follows: 0.13*rs3903239_G; 0.17*rs6666258_C; 0.12*rs1448818_C;
0.48*rs6817105_C; 0.25*rs4400058_A; 0.11*rs6838973_C; 0.13*rs3807989_G; 0.12*rs10821415_A;
0.16*rs10824026_A; 0.13*rs1152591_A; 0.15*rs7164883_G; 0.21*rs2106261_T
Supplemental Table 3. Linkage disequilibrium between identified susceptibility signals at chromosome 4q25.
rs1448818* rs2723288 rs6817105* rs4400058* rs4032974 rs17570669 rs3853445 rs6838973*
–
0.523
0.001
0.026
0.026
0.016
0.061
0.014
rs1448818*
–
0.001
0.044
0.044
0.016
0.053
0.001
rs2723288
–
0.017
0.017
0.037
0.009
0.017
rs6817105*
–
1
0.011
0.007
0.001
rs4400058*
–
0.011
0.007
0.001
rs4032974
–
0.114
0.093
rs17570669
–
0.308
rs3853445
–
rs6838973*
All values represent r2. Data from the HapMap CEU panel release 22.
*Carried forward as non-redundant signals as described in the methods.
Supplemental Table 4. Relations between non-redundant signals at the 4q25 locus and phylogenetic conservation.
Start and Stop
Positions (HG 19)
P-value for comparison
to remainder of 4q25
locus
Mean conservation
Chromosome 4q25 Region*
score (± SD)†
110,705,7680.189825 ±
Remainder of locus
–
112,705,768
1.034205
0.2853925 ±
Non-redundant signals
< 2.2x10-16
–
1.159416
111,565,2230.1566212 ±
rs1448818
0.001
111,575,223
1.027322
111,700,7680.1134964
rs6817105
1.4x10-13
111,710,768
±1.027837
111,711,6730.5704372 ±
rs4400058
< 2.2x10-16
111,721,673
1.322774
111,760,4950.3010151 ±
rs6838973
< 2.2x10-16
111,770,495
1.178208
*For non-redundant signals, regions are defined as the SNP ± 5 kb. The remainder of the chromosome
4q25 locus excludes the regions surrounding the non-redundant signals.
†Conservation score represented as phyloP score.
Supplemental Table 5. Associations between non-redundant SNPs and AF in a subset of the
BioBank Japan sample adjusted for principal components of ancestry.
Entire sample
N=7,916 cases /
3,393 controls
Age- and sexadjusted
AF risk /
referent allele
RR (95% CI)
Subset
N=844 cases /
3,393 controls
Age-, sex- and
principal
componentadjusted
RR (95% CI)
Adjusted for SNPs on
chromosome 4q25
rs1448818
C/A
0.98 (0.90-1.06)
1.09 (0.95-1.25)
rs6817105
C/T
1.92 (1.76-2.10)
1.91 (1.63-2.24)
rs4400058
A/G
1.43 (1.30-1.58)
1.53 (1.29-1.83)
rs6838973
C/T
1.12 (1.04-1.20)
1.09 (0.95-1.22)
Adjusted for SNPs at all
loci
rs6666258
C/G
1.23 (0.91-1.67)
1.35 (0.81-2.24)
rs3903239
G/A
1.12 (1.04-1.20)
1.18 (1.05-1.34)
rs1448818
C/A
0.97 (0.89-1.06)
1.09 (0.95-1.26)
rs6817105
C/T
1.90 (1.74-2.08)
1.92 (1.64-2.25)
rs4400058
A/G
1.42 (1.29-1.57)
1.56 (1.31-1.87)
rs6838973
C/T
1.11 (1.03-1.20)
1.08 (0.95-1.22)
rs3807989
G/A
1.25 (1.16-1.35)
1.35 (1.18-1.53)
rs10821415
A/C
1.08 (0.99-1.17)
1.12 (0.97-1.28)
rs10824026
A/G
0.94 (0.88-1.01)
0.95 (0.84-1.07)
rs1152591
A/G
1.02 (0.94-1.10)
1.11 (0.98-1.27)
rs7164883
G/A
0.97 (0.86-1.08)
0.99 (0.82-1.20)
rs2106261
T/C
1.28 (1.19-1.38)
1.38 (1.21-1.57)
Principal components of ancestry were available for the subset of 844 cases and 3,393 controls
with genome-wide data available.
Supplemental Table 6. Characteristics of the survival sample.
Time to Death (Survival)
N total
N events
Mean follow-up (years)
Male
Age
25,007
8,444
10.6 (5.4)
45%
69.0 (8.9)
Time to Disease or
Death (Survival Free
of Major Disease)
16,995
7,314
8.8 (5.7)
44%
67.7 (8.5)
Supplemental Table 7. Associations between AF associated SNPs and survival or survival free of major disease or mortality.
Survival
Survival free of major disease or mortality
SNP and AF
Chromosomal
AF risk allele
AF risk allele
risk allele
locus
frequency
RR (95% CI) P value
frequency
RR (95% CI)
P value
1q21 / KCNN30.99 (0.95rs6666258_C
PMVK
0.29
1.02)
0.47
0.29
0.99 (0.96-1.03)
0.75
1.04 (1.016.6x10
3
rs3903239_G
1q24 / PRRX1
0.44
1.08)
0.44
1.04 (1.01-1.08)
0.02
0.99 (0.95rs1448818_C
4q25 / PITX2
0.25
1.02)
0.41
0.25
0.98 (0.95-1.02)
0.44
0.99 (0.94rs6817105_C
4q25 / PITX2
0.12
1.04)
0.62
0.13
0.97 (0.92-1.02)
0.26
1.05 (0.99rs4400058_A
4q25 / PITX2
0.10
1.10)
0.09
0.10
1.00 (0.95-1.06)
0.95
1.00 (0.97rs6838973_C
4q25 / PITX2
0.56
1.03)
0.81
0.57
1.00 (0.96-1.03)
0.83
1.02 (0.99rs3807989_G
7q31 / CAV1
0.59
1.05)
0.26
0.59
1.02 (0.98-1.05)
0.31
1.00 (0.97rs10821415_A
9q22 / C9orf3
0.42
1.04)
0.80
0.42
1.02 (0.99-1.06)
0.20
0.98 (0.94rs10824026_A 10q22 / SYNPO2L
0.84
1.02)
0.35
0.84
1.01 (0.96-1.06)
0.68
1.02 (0.99rs1152591_A
14q23 / SYNE2
0.47
1.05)
0.24
0.47
1.00 (0.97-1.04)
0.82
1.00 (0.96rs7164883_G
15q24 / HCN4
0.15
1.04)
0.96
0.15
1.02 (0.97-1.07)
0.38
1.00 (0.96rs2106261_T
16q22 / ZFHX3
0.17
1.05)
0.88
0.17
1.01 (0.96-1.05)
0.76
Analyses adjusted for age, sex, and principal components of ancestry.
Supplemental Figure 1. Location of atrial fibrillation susceptibility signals and at the chromosome 4q25 locus and relations with
phylogenetic conservation and regulatory elements.
Each susceptibility signal is marked by a vertical hash and the SNP rsID tagging the signal. Regions containing SNPs with an r2≥0.9
with each susceptibility signal are highlighted with a black bar above the respective SNP. Select tracks from the UCSC genome
browser are also shown and include RefSeq genes, nucleotide conservation across non-placental vertebrate and mammalian species,
and several ENCODE tracks including H3K27Ac marks from 7 cell lines (a marker for the H3 histone protein acetylation which may
be associated with enhanced transcription), CpG methylation sites (a marker for promoter sites at which methylation may regulate
transcription), DNase I hypersensitivity areas across 125 cell types (markers for regulatory regions), and transcription factor sites
as assayed by ChIP-seq. Full details are available at http://genome.ucsc.edu.
Supplemental Figure 2. Weighted genetic risk models in the AFGEN and BioBank Japan samples.
Associations between categories of weighted risk scores with AF relative to the lowest category are displayed for scores comprised of
independent SNPs from A) chromosome 4q25 and B) all loci. Score categories were defined based upon deciles of score distributions
in the MGH sample.
Supplemental References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
Harris TB, Launer LJ, Eiriksdottir G et al. Age, Gene/Environment SusceptibilityReykjavik Study: multidisciplinary applied phenomics. Am J Epidemiol 2007;165:10761087.
The ARIC Investigators. The Atherosclerosis Risk in Communities (ARIC) study: design
and objectives. Am J Epidemiol 1989;129:687-702.
Fried LP, Borhani NO, Enright P et al. The Cardiovascular Health Study: design and
rationale. Ann Epidemiol 1991;1:263-276.
Dawber TR, Meadors GF, Moore FE, Jr. Epidemiological approaches to heart disease:
the Framingham Study. Am J Public Health Nations Health 1951;41:279-81.
Feinleib M, Kannel WB, Garrison RJ, McNamara PM, Castelli WP. The Framingham
Offspring Study. Design and preliminary data. Prev Med 1975;4:518-25.
Fuster V, Ryden LE, Cannom DS et al. ACC/AHA/ESC 2006 Guidelines for the
Management of Patients with Atrial Fibrillation: a report of the American College of
Cardiology/American Heart Association Task Force on Practice Guidelines and the
European Society of Cardiology Committee for Practice Guidelines (Writing Committee
to Revise the 2001 Guidelines for the Management of Patients With Atrial Fibrillation):
developed in collaboration with the European Heart Rhythm Association and the Heart
Rhythm Society. Circulation 2006;114:e257-354.
Kathiresan S, Voight BF, Purcell S et al. Genome-wide association of early-onset
myocardial infarction with single nucleotide polymorphisms and copy number variants.
Nat Genet 2009;41:334-41.
Shepherd J, Blauw GJ, Murphy MB et al. Pravastatin in elderly individuals at risk of
vascular disease (PROSPER): a randomised controlled trial. Lancet 2002;360:1623-30.
Hofman A, Breteler MM, van Duijn CM et al. The Rotterdam Study: 2010 objectives and
design update. Eur J Epidemiol 2009;24:553-72.
Holle R, Happich M, Lowel H, Wichmann HE, Group MKS. KORA--a research platform
for population based health research. Gesundheitswesen 2005;67 Suppl 1:S19-25.
Wichmann HE, Gieger C, Illig T. KORA-gen--resource for population genetics, controls
and a broad spectrum of disease phenotypes. Gesundheitswesen 2005;67 Suppl 1:S26-30.
Harris TB, Launer LJ, Eiriksdottir G et al. Age, Gene/Environment SusceptibilityReykjavik Study: multidisciplinary applied phenomics. Am J Epidemiol 2007;165:107687.
The Atherosclerosis Risk in Communities (ARIC) Study: design and objectives. The
ARIC investigators. Am J Epidemiol 1989;129:687-702.
Alonso A, Agarwal SK, Soliman EZ et al. Incidence of atrial fibrillation in whites and
African-Americans: the Atherosclerosis Risk in Communities (ARIC) study. Am Heart J
2009;158:111-7.
Fried LP, Borhani NO, Enright P et al. The Cardiovascular Health Study: design and
rationale. Ann Epidemiol 1991;1:263-76.
Kannel WB, Feinleib M, McNamara PM, Garrison RJ, Castelli WP. An investigation of
coronary heart disease in families. The Framingham offspring study. Am J Epidemiol
1979;110:281-90.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
Heckbert SR, Wiggins KL, Glazer NL et al. Antihypertensive treatment with ACE
inhibitors or beta-blockers and risk of incident atrial fibrillation in a general hypertensive
population. American journal of hypertension 2009;22:538-44.
Winkelmann BR, Marz W, Boehm BO et al. Rationale and design of the LURIC study--a
resource for functional genomics, pharmacogenomics and long-term prognosis of
cardiovascular disease. Pharmacogenomics 2001;2:S1-73.
Ellinor PT, Yoerger DM, Ruskin JN, MacRae CA. Familial aggregation in lone atrial
fibrillation. Hum Genet 2005;118:179-84.
Trompet S, de Craen AJ, Postmus I et al. Replication of LDL GWAs hits in
PROSPER/PHASE as validation for future (pharmaco)genetic analyses. BMC Med Genet
2011;12:131.
Volzke H, Alte D, Schmidt CO et al. Cohort profile: the study of health in Pomerania. Int
J Epidemiol 2011;40:294-307.
Ridker PM, Chasman DI, Zee RY et al. Rationale, design, and methodology of the
Women's Genome Health Study: a genome-wide association study of more than 25,000
initially healthy american women. Clin Chem 2008;54:249-55.
Nakamura Y. The BioBank Japan Project. Clin Adv Hematol Oncol 2007;5:696-7.
Li Y, Willer CJ, Ding J, Scheet P, Abecasis GR. MaCH: using sequence and genotype
data to estimate haplotypes and unobserved genotypes. Genetic epidemiology
2010;34:816-34.
Guan Y, Stephens M. Practical issues in imputation-based association mapping. PLoS
Genet 2008;4:e1000279.
Aulchenko YS, Ripke S, Isaacs A, van Duijn CM. GenABEL: an R library for genomewide association analysis. Bioinformatics 2007;23:1294-6.
R Development Core Team. R: A language and environment for statistical computing.
Vienna, Austria: R Foundation for Statistical Computing, 2006.
Purcell S, Neale B, Todd-Brown K et al. PLINK: a tool set for whole-genome association
and population-based linkage analyses. Am J Hum Genet 2007;81:559-75.
Estrada K, Abuseiris A, Grosveld FG, Uitterlinden AG, Knoch TA, Rivadeneira F.
GRIMP: a web- and grid-based tool for high-speed analysis of large-scale genome-wide
association using imputed data. Bioinformatics 2009;25:2750-2.