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Supplementary material online (optional)

Supplemental material: Sedlacek et al.: Common genetic variants in ANK2 modulate
QT interval
SUPPLEMENTAL MATERIAL
Supplemental methods
Detailed information on QT interval correction
In bradycardia and tachycardia, traditional Bazett’s formula does not adequately
correct the QT interval. Furthermore, linear regression formulas that adjust for various
covariates significantly affecting the QT interval amplify the genetic component of the overall
QT interval variance. We therefore calculated sex specific correction formulas for both
populations (KORA S3 and KORA S4) using multivariate linear regression with covariates
including heart rate (RR interval), age (A) and sex (S) resulting in QTc-RAS. The correction
formulas used in this study were as follows:
KORA S3 males:
QTc-RAS = QT – 0.157 * (RR – 1000) – 0.402 * (AGE – 60)
KORA S3 females:
QTc-RAS = QT – 0.157 * (RR – 1000) – 0.402 * (AGE – 60) – 7.44
KORA S4 males:
QTc-RAS = QT – 0.152 * (RR – 1000) – 0.318 * (AGE – 60)
KORA S4 females:
QTc-RAS = QT – 0.154 * (RR – 1000) – 0.207 * (AGE – 60) – 4.58.
Detailed information on genotyping
Genotyping in the KORA S3 sample was performed using 5' exonuclease TaqMan
technology with pre-designed assays (Applied Biosystems, Foster City, CA, USA). The
replication panel (KORA S4 population) was genotyped using the primer extension MALDITOF MS platform (Autoflex HT; Sequenom, San Diego, CA, USA) according to the
manufacturer’s protocol. For each genotyping experiment, 10 ng DNA was used in a total
volume of 5 µl containing 1x TaqMan Universal PCR Master Mix (Applied Biosystems). PCR
reaction and post-PCR endpoint plate read was carried out following the manufacturer’s
instructions using the Applied Biosystems 7900HT Real-Time PCR System. Sequence
Detection System software version 2.2 (Applied Biosystems) was used to assign genotypes
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Supplemental material: Sedlacek et al.: Common genetic variants in ANK2 modulate
QT interval
applying the allelic discrimination test. Duplicates of samples (15%) were employed to
assess intraplate and interplate genotype quality. The overall mean call rate of all used
TaqMan assays was 97.7%. The replication panel (KORA S4 population) was genotyped
using the primer extension MALDI-TOF MS platform (Autoflex HT; Sequenom, San Diego,
CA, USA) according to the manufacturer’s protocol. No genotyping discrepancies were
detected.
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Supplemental material: Sedlacek et al.: Common genetic variants in ANK2 modulate
QT interval
Supplemental Table 1. Description of TaqMan and MALDI-TOF assays used in the
study.
rs ID
(dbSNP128)
Position on
chr. 4 (hg18)
Location
rs11098171
113,929,622
5' intergenic
fine-mapping (KORA S3), replication (KORA S4)
rs6850768
113,938,349
5' intergenic
fine-mapping (KORA S3), replication (KORA S4)
rs1979086
113,956,986
5' intergenic
initial (KORA S3), replication (KORA S4)
rs10026837
113,959,159
5' intergenic
fine-mapping (KORA S3), replication (KORA S4)
rs4834308
114,027,575
5' intergenic
fine-mapping (KORA S3), replication (KORA S4)
rs11098182
114,114,131
5' intergenic
fine-mapping (KORA S3), replication (KORA S4)
rs10433900
114,181,260
5' intergenic
initial (KORA S3)
rs313979
114,201,226
Intron 1
initial (KORA S3)
rs1385662
114,245,968
Intron 1
initial (KORA S3)
rs13101559
114,262,327
Intron 1
initial (KORA S3)
rs313960
114,271,871
Intron 1
initial (KORA S3)
rs413019
114,298,576
Intron 1
initial (KORA S3)
rs404950
114,305,691
Intron 1
initial (KORA S3)
rs2285711
114,336,729
Intron 2
initial (KORA S3)
rs6533673
114,342,328
Intron 4
initial (KORA S3)
rs13118200
114,343,814
Intron 4
initial (KORA S3)
rs362498
114,378,501
Intron 7
initial (KORA S3)
rs29322
114,436,144
Intron 22
initial (KORA S3)
rs29308
114,447,734
Intron 23
initial (KORA S3)
rs11940206
114,469,346
Intron 26
initial (KORA S3)
rs4834330
114,509,359
Intron 42
initial (KORA S3)
rs12508397
114,528,732
3' intergenic
initial (KORA S3)
Genotyping set
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Supplemental material: Sedlacek et al.: Common genetic variants in ANK2 modulate
QT interval
RNA isolation and reverse transcription
samples (fat, coronary arteries excluded) using an RNeasy midi kit (Qiagen, Valencia, CA,
USA). 500 ng of RNA (with a 260/280 ratio of 1.9 to 2.1) was reverse-transcribed into cDNA
d in subsequent PCR reactions.
PCR amplification of ANK2 cardiac transcripts
PCR products were amplified from reverse-transcribed ventricular mRNA using
Advantage 2 polymerase (Clontech, Mountain View, Ca, USA) at an annealing temperature
of 63°C. Two primer sets were designed to amplify the 5’-sequence of ANK2. The primers
are as follows: E0-5’ (CTGCTGTCTCATAGACATCAGCTTG), E1-3’
(GCAACATGGTGGTCATTTG), and E4-3’ (GACAACTTCTGCTTGTCCAGC). PCR products
were separated on a 1.5% agarose gel.
Statistical analyses
Description and references for software and scripts used in the study:
JMP 5.0.1 (SAS Institute, Cary, NC, USA)
SPSS v. 15.0 (SPSS Inc., Chicago, Illinois, USA).
EM algorithm
Slatkin M, Excoffier L. Testing for linkage disequilibrium in genotypic data using the
Expectation-Maximization algorithm. Heredity 1996; 76:377-383.
HTR (haplotype trend regression)
Zaykin DV, Westfall PH, Young SS, Karnoub MA, Wagner MJ, Ehm MG. Testing
association of statistically inferred haplotypes with discrete and continuous traits in
samples of unrelated individuals. Hum Heredity. 2002; 53:79-91.
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Supplemental material: Sedlacek et al.: Common genetic variants in ANK2 modulate
QT interval
Haploview
Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and
haplotype maps. Bioinformatics. 2005; 21:263-265.
SNPStats
Solé X, Guinó E, Valls J, Iniesta R, Moreno V. SNPStats: a web tool for the analysis
of association studies. Bioinformatics. 2006; 22:1928-1929.
Metal
a script performing sample size weighted and inverse variance weighted metaanalysis; http://www.sph.umich.edu/csg/abecasis/Metal/index.html
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Supplemental material: Sedlacek et al.: Common genetic variants in ANK2 modulate
QT interval
Supplemental Tables
Supplemental Table 2. Statistical association results of the screening set SNPs in
the KORA S3 population.
SNP
MAF
HWE
Best P value
rs1979086
0.49
0.45
0.005 (A)
rs10433900
0.40
0.50
n.s.
rs313979
0.11
0.76
n.s.
rs1385662
0.20
0.16
n.s.
rs13101559
0.33
0.84
0.015 (A)
rs313960
0.12
0.26
0.018 (D)
rs413019
0.34
0.84
0.034 (R)
rs404950
0.35
1.00
n.s.
rs2285711
0.15
0.14
n.s.
rs6533673
0.45
0.77
n.s.
rs13118200
0.49
0.72
n.s.
rs362498
0.28
0.66
n.s.
rs29322
0.19
0.21
n.s.
rs29308
0.23
0.93
n.s.
rs11940206
0.12
1.00
n.s.
rs4834330
0.12
1.00
n.s.
rs12508397
0.48
0.96
n.s.
MAF, minor allele frequency; HWE, Hardy-Weinberg equilibrium. An exact test was used to
calculate the statistical significance of deviation from the HWE (P<0.05); n.s., not significant;
(A); (D); (R), log-additive, dominant, and recessive model of inheritance, respectively.
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Supplemental material: Sedlacek et al.: Common genetic variants in ANK2 modulate QT interval
ODS Table 3. Statistical association results of studies populations and genetic models.
SNP
KORA S3
Sex
KORA S4
Combined population*
Dominant Recessive Log-additive Dominant Recessive Log-additive Dominant Recessive Log-additive
rs11098171
rs6850768
rs1979086
rs10026837
All
0.57
0.55
0.49
0.65
0.002
0.17
0.51
0.005
0.13
Men
0.77
0.60
0.66
0.40
0.10
0.20
n.p.
n.p.
n.p.
Women
0.60
0.75
0.58
0.85
0.008
0.50
n.p.
n.p.
n.p.
All
0.0008
0.21
0.001
0.006
0.006
0.001
0.0004
0.002
<0.0001
Men
0.001
0.10
0.002
0.14
0.05
0.04
n.p.
n.p.
n.p.
Women
0.10
0.91
0.19
0.02
0.05
0.007
n.p.
n.p.
n.p.
All
0.04
0.01
0.005
0.51
0.52
0.11
0.53
0.48
0.97
Men
0.09
0.005
0.006
0.50
0.93
0.71
n.p.
n.p.
n.p.
Women
0.25
0.47
0.25
0.04
0.33
0.06
n.p.
n.p.
n.p.
All
0.08
0.04
0.02
0.06
0.48
0.11
0.45
0.67
0.83
Men
0.08
0.005
0.005
0.53
0.79
0.58
n.p.
n.p.
n.p.
Women
0.25
0.70
0.25
0.04
0.47
0.08
n.p.
n.p.
n.p.
Data are P values for the specified genetic model. * Analysis of a combined KORA S3 and S4 population in this table was performed using data
transformation into their corresponding z scores, in order to standardize the raw QTc-RAS values in both populations. P values < 0.05 are
written in bold to highlight their clustering.
Dominant, dominant model of inheritance; Recessive, recessive model of inheritance; Log-additive, log-additive model of inheritance; n.p.,
analysis not performed.
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Supplemental material: Sedlacek et al.: Common genetic variants in ANK2 modulate
QT interval
Supplemental Figures and Figure Legends
Supplemental Figure 1. Annotation of the ANK2 genomic region
Description of the annotation data is given on the left margin of the figure. Explanation to
annotation datasets:
Chr4,
physical distance on the chromosome 4 (in base-pair, bp)
Original set,
primary set of markers that were used in the KORA S3 population
Replication set, fine-mapping and replication set of markers genotyped in KORA S3 and
KORA S4 populations.
USCS gene predictions, based on RefSeq, UniProt, GenBank databases, and comparative
genomics data
RefSeq genes, reference genes in the RefSeq database
Conservation, sequence conservation among different species
Unphased CEU R^2, pair-wise linkage disequilibrium based on HapMap data of the
European panel of individuals, displayed as r2
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Supplemental material: Sedlacek et al.: Common genetic variants in ANK2 modulate
QT interval
Supplemental Figure 2. Detailed view and annotation of the distant 5’ ANK2
genomic region
Description of the annotation data is given on the left margin of the figure. Explanation to
annotation datasets:
Chr4,
physical distance on the chromosome 4 (in base-pair, bp)
Original set,
primary set of markers that were used in the KORA S3 population
Replication set, fine-mapping and replication set of markers genotyped in KORA S3 and
KORA S4 populations.
USCS gene predictions, based on RefSeq, UniProt, GenBank databases, and comparative
genomics data
Conservation, sequence conservation among different species
Unphased CEU R^2, pair-wise linkage disequilibrium based on HapMap data of the
European panel of individuals, displayed as r2
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