Data Supplement

SUPPLEMENTAL MATERIAL
Supplemental Figures
Figure S1: Position of lead SNP and genes at the associated chromosome 16 locus. Gene coordinate data from UCSC
1
Genome Browser .
Figure S2: Cardiovascular event-free survival by rs4888378 genotype in (a) all subjects, (b) men and (c) women.
Event data is from IMPROVE. a) Overall, each A allele is associated with a significant reduction in HR of 23% (Cox
proportional hazard model; p=0.01). b) In men, no significant difference is seen (reduction in HR 14%, p=0.24) c) In
women, each A allele is associated with a significant reduction in HR of 23% (reduction in HR 36%, p=0.01).
Figure S3: EMSA shows differential protein binding for one of the six tested SNPs. Labelled probes 1-6 refer to SNPs
rs4888378, rs4888379, rs4888392, rs2865530, rs3743609 and rs11643207 respectively; the letter denotes the allele.
Bands show the position of biotin-labelled probe on the gel (direction from top to bottom). Probes unbound by DNA
run fastest and are present at the bottom of the picture; bands above are protein-bound probe which migrates more
slowly. Competitor probes (unlabelled) are added in excess to nuclear extract 30 minutes before labelled probe; hence
a probe-protein band will disappear when the protein binds first to the competitor.
Figure S4: Enhancer fragments with both the A and G allele of rs4888378 show decreased expression compared to
pGL3-promoter control. Luciferase reporter assay data. P-values derive from unpaired t-test. Error bars show SEM.
(12 replicates each.)
Figure S5: Gene expression by rs4888378 genotype in aortic artery and tibial artery for TMEM170A, ZNRF1 and
LDHD. a) TMEM170A expression, found by Gertow et al (2012) to associate with rs4888378 genotype in aortic intimamedia and adventitia, is also nominally associated with rs4888378 in tibial artery. Lower expression was seen with
each high-risk G allele, but this did not remain significant after correction for multiple testing. No association was seen
in aortic artery. b) ZNRF1 expression is associated with genotype in aortic artery, with higher expression seen with
each high-risk G allele; this did not remain significant after correction for multiple testing. No association was seen in
tibial atery. c) LDHD expression, found by Gertow et al (2012) to associate with rs4888378 genotype in aortic intimamedia and adventitia, is not associated with the SNP in aortic or tibial artery. Data and graphs from Gene-Tissue
2
Expression (GTEx) Portal .
Figure S6: Gene expression by rs4888378 genotype in aortic media (a and b) and aortic adventitia (c and d).
Expression data is shown for whole sample, men and women. The gene under study is shown on the y-axis. The genetissue combinations studied here were those in which a significant rs4888378-expression association was found in
Gertow and colleagues' original study, with the exception of BCAR1-plaque, for which we did not have sex-specific
data.
Supplemental tables
Table S1: Characteristics of IMPROVE.
Men (n=1772)
64.0 (0.13)
Women (n=1931)
64.4 (0.13)
BMI (kg/m )
27.4 (0.09)
27.1 (0.11)
Systolic BP (mmHG)
142.4 (0.44)
141.6 (0.42)
Diastolic BP (mmHg)
83.1 (0.24)
80.9 (0.22)
Hypertension
67.9% (1203)
69.9% (1349)
Total cholesterol (mmol/l)
5.25 (0.02)
5.71 (0.03)
LDL-C (mmol/l)
3.39 (0.02)
3.68 (0.02)
HDL-C (mmol/l)
1.14 (0.01)
1.38 (0.01)
Triglycerides (mmol/l)*
Combined vascular event
(cardiac, cerebrovascular or
peripheral)
Diabetes prevalence
1.42 (0.02)
1.29 (0.01)
7.6% (134)
4.2% (81)
29.4% (521)
20.3% (392)
Lipid-lowering therapy
47.6% (827)
50.7% (965)
Age (years)
2
Anti-hypertensive therapy
54.6% (967)
59.1% (1142)
*Log-transformed due to non-normality; SEM is approximate
Values expressed as mean (SEM) or percentage (frequency)
Table S2: Characteristics of PLIC.
Men (n= 795)
Women (n= 1076)
54.0 (0.4)
55.0 (0.3)
BMI (kg/m )
27.12 (0.12)
26.07 (0.14)
Systolic BP (mmHg)
134.3 (0.6)
129.3 (0.5)
Diastolic BP (mmHg)
83.4 (0.3)
80.4 (0.3)
Total cholesterol (mmol/l)
5.66 (0.04)
5.79 (0.03)
HDL-C (mmol/l)
1.28 (0.01)
1.53 (0.01)
Triglycerides (mmol/l)
1.39 (0.03)
1.10 (0.01)
LDL-C (mmol/l)
3.74 (0.03)
3.75 (0.02)
apoA-I (mg/dL)
141.50 (0.89)
154.64 (0.79)
apoB (mg/dL)
115.03 (0.98)
112.57 (0.77)
Remnant-C (mmol/L)
0.64 (0.02)
0.51 (0.01)
Glucose (mmol/l)
5.42 (0.03)
4.97 (0.02)
0.663 (0.005)
0.643 (0.004)
Age (years)
2
CCA-IMT (mm)
Values expressed as mean (SEM).
Table S3: Primer sequences for EMSA probes
SNP no
1
SNP name
rs4888378
3
rs4888379
8
rs4888392
9
rs2865530
14
rs3743609
15
rs11643207
Allele
A
A
G
G
A
A
T
T
T
T
C
C
G
G
T
T
G
G
C
C
C
C
T
T
Orientation
For
Rev
For
Rev
For
Rev
For
Rev
For
Rev
For
Rev
For
Rev
For
Rev
For
Rev
For
Rev
For
Rev
For
Rev
Primer sequence
ATAAAACAAATGAATTCTGTGTTTG
CAAACACAGAATTCATTTGTTTTAT
ATAAAACAAATGGATTCTGTGTTTG
CAAACACAGAATCCATTTGTTTTAT
AGTATCACCCTAACCCATTCTGAAA
TTTCAGAATGGGTTAGGGTGATACT
AGTATCACCCTATCCCATTCTGAAA
TTTCAGAATGGGATAGGGTGATACT
TACAGTCTGTTTTTGGGGTCAGCTA
TAGCTGACCCCAAAAACAGACTGTA
TACAGTCTGTTTCTGGGGTCAGCTA
TAGCTGACCCCAGAAACAGACTGTA
AAAAATGGTCTTGAGCTTACAGGCA
TGCCTGTAAGCTCAAGACCATTTTT
AAAAATGGTCTTTAGCTTACAGGCA
TGCCTGTAAGCTAAAGACCATTTTT
CGCACACGCCTCGGGCCGGAGCGGC
GCCGCTCCGGCCCGAGGCGTGTGCG
CGCACACGCCTCCGGCCGGAGCGGC
GCCGCTCCGGCCGGAGGCGTGTGCG
CTCTGCTCTGCCCTTCTCTGCAGCG
CGCTGCAGAGAAGGGCAGAGCAGAG
CTCTGCTCTGCCTTTCTCTGCAGCG
CGCTGCAGAGAAAGGCAGAGCAGAG
Table S4: Consensus competitor sequences for multiplex competitor EMSA.
F and R refer to forward and reverse oligonucleotides respectively.
AP1_F
CGCTTGATGACTCAGCCGGAA
AP1_R
TTCCGGCTGAGTCATCAAGCG
AP2a_F
GATCGAACTGACCGCCCGCGGCCCGT
AP2a_R
ACGGGCCGCGGGCGGTCAGTTCGATC
AR_F
GAAGTCTGGTACAGGGTGTTCTTTTTG
AR_R
CAAAAAGAACACCCTGTACCAGACTTC
Brn3_F
CACAGCTCATTAACGCGC
Brn3_R
GCGCGTTAATGAGCTGTG
CBP_F
AGACCGTACGTGATTGGTTAATCTCTT
CBP_R
AAGAGATTAACCAATCACGTACGGTCT
CDP_F
ACCCAATGATTATTAGCCAATTTCTGA
CDP_R
TCAGAAATTGGCTAATAATCATTGGGT
CEBP_F_R
TGCAGATTGCGCAATCTGCA
cMyb_F
TACAGGCATAACGGTTCCGTAGTGA
cMyb_R
TCACTACGGAACCGTTATGCCTGTA
CREB_F
AGAGATTGCCTGACGTCAGAGAGCTAG
CREB_R
CTAGCTCTCTGACGTCAGGCAATCTCT
CTCF_F
GGCGGCGCCGCTAGGGGTCTCTCT
CTCF_R
AGAGAGACCCCTAGCGGCGCCGCC
E2F1_F
ATTTAAGTTTCGCGCCCTTTCTCAA
E2F1_R
TTGAGAAAGGGCGCGAAACTTAAAT
Egr_F
GGATCCAGCGGGGGCGAGCGGGGGCGA
Egr_R
TCGCCCCCGCTCGCCCCCGCTGGATCC
ER_F
GGATCTAGGTCACTGTGACCCCGGATC
ER_R
GATCCGGGGTCACAGTGACCTAGATCC
Ets_F
GGGCTGCTTGAGGAAGTATAAGAAT
Ets_R
ATTCTTATACTTCCTCAAGCAGCCC
Ets1_F
GATCTCGAGCAGGAAGTTCGA
Ets1_R
TCGAACTTCCTGCTCGAGATC
FAST1_F
TGTGTATTCA
FAST1_R
TGAATACACA
GAS_F
AAGTACTTTCAGTTTCATATTACTCTA
GAS_R
TAGAGTAATATGAAACTGAAAGTACTT
GATA_F
CACTTGATAACAGAAAGTGATAACTCT
GATA_R
AGAGTTATCACTTTCTGTTATCAAGTG
Gfi1_F
TAAATCACTGC
Gfi1_R
GCAGTGATTTA
GR_F
AGAGGATCTGTACAGGATGTTCTAGAT
GR_R
ATCTAGAACATCCTGTACAGATCCTCT
HIF1a_F
TCTGTACGTGACCACACTCACCTC
HIF1a_R
GAGGTGAGTGTGGTCACGTACAGA
ISRE_F
AAGTACTTTCAGTTTCATATTACTCTA
ISRE_R
TAGAGTAATATGAAACTGAAAGTACTT
HNF4_F
CTCAGCTTGTACTTTGGTACAACTA
HNF4_R
TAGTTGTACCAAAGTACAAGCTGAG
IRF1_F
GGAAGCGAAAATGAAATTGACT
IRF1_R
AGTCAATTTCATTTTCGCTTCC
MEF1_F
GATCCCCCCAACACCTGCTGCCTGA
MEF1_R
TCAGGCAGCAGGTGTTGGGGGGATC
MEF2_F
GATCGCTCTAAAAATAACCCTGTCG
MEF2_R
CGACAGGGTTATTTTTAGAGCGATC
MIBP1_F
TCTTTTCCCA
MIBP1_R
TGGGAAAAGA
MycMax_F_R
GGAAGCAGACCACGTGGTCTGCTTCC
NF1_F
TTTTGGATTGAAGCCAATATGATAA
NF1_R
TTATCATATTGGCTTCAATCCAAAA
NFE2_F
TGGGGAACCTGTGCTGAGTCACTGGAG
NFE2_R
CTCCAGTGACTCAGCACAGGTTCCCCA
NFATc_F
CGCCCAAAGAGGAAAATTTGTTTCATA
NFATc_R
TATGAAACAAATTTTCCTCTTTGGGCG
NFkB_F
AGTTGAGGGGACTTTCCCAGGC
NF-kB_R
GCCTGGGAAAGTCCCCTCAACT
NR5A2_F
GATCAACGACCGACCTTGAG
NR5A2_R
CTCAAGGTCGGTCGTTGATC
OCT1_F
TGTCGAATGCAAATCACTAGAA
OCT1_R
TTCTAGTGATTTGCATTCGACA
p53_F
TACAGAACATGTCTAAGCATGCTGGGG
p53_R
CCCCAGCATGCTTAGACATGTTCTGTA
Pax5_F
GAATGGGGCACTGAGGCGTGACCACCG
Pax5_R
CGGTGGTCACGCCTCAGTGCCCCATTC
Pbx1_F
CTCCAATTAGTGCATCAATCAATTCG
Pbx1_R
CGAATTGATTGATGCACTAATTGGAG
Pit1_F
TGTCTTCCTGAATATGAATAAGAAATA
Pit1_R
TATTTCTTATTCATATTCAGGAAGACA
PPAR_F
AGGTCAAAGGTCA
PPAR_R
TGACCTTTGACCT
PR_F
GATCCTGTACAGGATGTTCTAGCTACA
PR_R
TGTAGCTAGAACATCCTGTACAGGATC
RAR_F
AGGGTAGGGTTCACCGAAAGTTCACTC
RAR_R
GAGTGAACTTTCGGTGAACCCTACCCT
RXR_F
AGCTTCAGGTCAGAGGTCAGAGAGCT
RXR_R
AGCTCTCTGACCTCTGACCTGAAGCT
SIE_F
GTGCATTTCCCGTAAATCTTGTCTACA
SIE_R
TGTAGACAAGATTTACGGGAAATGCAC
Smad_F
GTCTAGACCA
Smad_R
TGGTCTAGAC
Smad34_F
TCGAGAGCCAGACAAAAAGCCAGACATTTAGCCAGACAC
Smad34_R
GTGTCTGGCTAAATGTCTGGCTTTTTGTCTGGCTCTCGA
Smuc_F
GGATCCCCCAACACCTGCTGCCTGA
Smuc_R
TCAGGCAGCAGGTGTTGGGGGATCC
Sp1_F
ATTCGATCGGGGCGGGGCGAGC
Sp1_R
GCTCGCCCCGCCCCGATCGAAT
SRE_F
GGATGTCCATATTAGGACATCT
SRE_R
AGATGTCCTAATATGGACATCC
Stat1_F
CATGTTATGCATATTCCTGTAAGTG
Stat1_R
CACTTACAGGAATATGCATAACATG
Stat3_F
GATCCTTCTGGGAATTCCTAGATC
Stat3_R
GATCTAGGAATTCCCAGAAGGATC
Stat4_F
GAGCCTGATTTCCCCGAAATGATGAGC
Stat4_R
GCTCATCATTTCGGGGAAATCAGGCTC
Stat5_F
AGATTTCTAGGAATTCAATCC
Stat5_R
GGATTGAATTCCTAGAAATCT
Stat56_F
GTATTTCCCAGAAAAGGAAC
Stat56_R
GTTCCTTTTCTGGGAAATAC
Tbet_F_R
AATTTCACACCTAGGTGTGAAATT
TFE3_F
GATCTGGTCATGTGGCAAGGC
TFE3_R
GCCTTGCCACATGACCAGATC
TFEB_F
CACGTG
TFEB_R
CACGTG
TFIID_F
GCAGAGCATATAAAATGAGGTAGGA
TFIID_R
TCCTACCTCATTTTATATGCTCTGC
TGIF_F
ACTCTGCCTGTCAAGCGAGG
TGIF_R
CCTCGCTTGACAGGCAGAGT
TR_F
AGCTTCAGGTCACAGGAGGTCAGAGAG
TR_R
CTCTCTGACCTCCTGTGACCTGAAGCT
USF1_F
CACCCGGTCACGTGGCCTACACC
USF1_R
GGTGTAGGCCACGTGACCGGGTG
VDR_F
AGCTTCAGGTCAAGGAGGTCAGAGAGC
VDR_R
GCTCTCTGACCTCCTTGACCTGAAGCT
YY1_F
CGCTCCCCGGCCATCTTGGCGGCTGGT
YY1_R
ACCAGCCGCCAAGATGGCCGGGGAGCG
ZEB_F
GATCTGGCCAAAGGTGCAGGATC
ZEB_R
GATCCTGCACCTTTGGCCAGATC
HNF1_F
GTTAATGATTAAC
HNF1_R
GTTAATCATTAAC
ARP1_F
AGGTGACCTTTGCCCA
ARP1_R
TGGGCAAAGGTCACCT
NFY_F
ATCAGCCAATCAGAGC
NFY_R
GCTCTGATTGGCTGAT
HNF3_F
GCCCATTGTTTGTTTTAAGCC
HNF3_R
GGCTTAAAACAAACAATGGGC
BARP_F
TCACTCAAGTTCAAGTTATT
BARP_R
AATAACTTGAACTTGAGTGA
SREBP1_F
TTTGAAAATCACCCCATGCAAACTC
SREBP1_R
GAGTTTGCATGGGGTGATTTTCAAA
HSF1_F
GATCTCGGCTGGAATATTCCCGACCTGGCAGCCGA
HSF1_R
TCGGCTGCCAGGTCGGGAATATTCCAGCCGAGATC
Table S5: Cloning primers for luciferase sequence fragments.
Sequence
Forward/
Sequence
Restriction
reverse
site
1
Forward
AATCGATAAGGATCCATGTCAGTACAAGGCGAGCA
BamHI
1
Reverse
AAGGGCATCGGTCGACGGGTTCCCAGATACAGGTTG
SalI
2
Forward
AAATCGATAAGGATCCCTGAAGTCCTCCAAAACCAGA
BamHI
2
Reverse
AAGGGCATCGGTCGACTGCTTTTACGTCATCAGCAATCT
SalI
3/4
Forward
AAATCGATAAGGATCCCCTCCAGGCTTTGTGTATAAGG
BamHI
3
Reverse
AAGGGCATCGGTCGACATGCTTGGGACTGGAAGTGT
SalI
4
Reverse
AAGGGCATCGGTCGACATTTGCTTTTACGTCATCAGCA
SalI
Primers were designed to incorporate restriction sites for BamHI and SalI; the pGL3-promoter vector was and
sequence fragments were cut with these enzymes to allow ligation.
Table S6: Cardiovascular risk factors and CCA-IMT by rs4888378 genotype in PLIC – men only.
Parameter
P (post-hoc)
GG
GA
AA
P (trend)
BMI (Kg/cm )
27.2
(0.23)
27.3
(0.17)
26.5
(0.23)
0.139
0.757
0.118
0.050
Waist (cm)
96.8
(0.64)
97.6
(0.72)
96.2
(0.50)
0.331
0.382
0.541
0.154
Hip (cm)
104.7
(0.45)
104.6
(0.37)
101.7
(0.86)
0.001
0.833
0.001
0.001
Waist/hip ratio
0.92
(0.01)
0.93
(0.01)
0.93
(0.01)
0.150
0.218
0.055
0.316
Triglycerides (mmol/L)
1.35
(0.05)
1.43
(0.04)
1.34
(0.06)
0.572
0.289
0.719
0.602
Glucose (mmol/L)
5.34
(0.05)
5.48
(0.05)
5.35
(0.08)
0.234
0.105
0.727
0.298
Remnants-C (mmol/L)
0.62
(0.02)
0.66
(0.02)
0.59
(0.03)
0.571
0.298
0.719
0.602
Basal CCA-IMT (mm) *
0.649
(0.014)
0.655
(0.010)
0.633
(0.021)
0.934
0.500
0.473
0.852
Annual ΔCCA-IMT (mm) *
0.019
(0.001)
0.020
(0.001)
0.019
(0.002)
0.519
0.829
0.943
0.733
2
GG vs GA
GG vs AA
GA vs AA
P(trend) values were adjusted for age and smoking. *Log-transformed due to non-normality; SEM is
approximate.
Table S7: Cardiovascular risk factors and CCA-IMT by rs4888378 genotype in PLIC – women only.
Parameter
P (post-hoc)
GG
GA
AA
P (trend)
BMI (Kg/cm )
25.4
(0.20)
26.2
(0.20)
26.8
(0.30)
0.001
0.020
0.001
0.052
Waist (cm)
84.30
(0.61)
86.4
(0.55)
88.2
(0.97)
0.003
0.035
0.002
0.082
Hip (cm)
102.9
(0.48)
104.3
(0.43)
104.7
(0.72)
0.012
0.015
0.009
0.614
Waist/hip ratio
0.82
(0.01)
0.83
(0.01)
0.84
(0.01)
0.023
0.340
0.006
0.036
Triglycerides (mmol/L)
1.05
(0.02)
1.11
(0.02)
1.16
(0.04)
0.075
0.110
0.039
0.330
Glucose (mmol/L)
4.92
(0.03)
4.95
(0.03)
5.07
(0.05)
0.025
0.361
0.007
0.037
Remnants-C (mmol/L)
0.48
(0.01)
0.51
(0.01)
0.52
(0.02)
0.075
0.111
0.039
0.321
Basal CCA-IMT (mm) *
0.620
(0.011)
0.627
(0.010)
0.629
(0.016)
0.274
0.799
0.962
0.868
Annual ΔCCA-IMT (mm) *
0.026
(0.001)
0.020
(0.001)
0.017
(0.002)
0.246
0.405
0.040
0.201
2
GG vs GA
GG vs AA
GA vs AA
P(trend) values were adjusted for age and smoking. *Log-transformed due to non-normality; SEM is approximate.
Table S8: IMT phenotypes by rs4888378 genotype in IMPROVE
Overall
Variable
Mean common carotid
Mean common carotid
(cm closest to
bifurcation)
Mean internal carotid
arteries
GG
0.749
(0.004)
GA
0.745
(0.003)
AA
0.729
(0.005)
Men
Effect
size
p
-0.005
5.71E-03
GG
0.772
(0.007)
GA
0.775
(0.006)
AA
0.758
(0.008)
Women
Effect
size
p
-0.004
0.185
GG
0.726
(0.005)
GA
0.717
(0.004)
AA
0.705
(0.006)
Effect
size
-0.006
0.828
0.823
0.824
0.808
0.796
0.789
-0.005 1.29E-02
-0.002 0.380
0.787
0.772
0.760
(0.007) (0.006) (0.010)
(0.005) (0.004) (0.006)
(0.007) (0.005) (0.007)
-0.006
0.903
0.870
0.840
0.973
0.948
0.907
0.832
0.799
0.784
-0.012 5.06E-04
-0.013 0.013
(0.011) (0.008) (0.012)
(0.017) (0.013) (0.020)
(0.014) (0.010) (0.015)
-0.011
1.167
1.150
1.096
1.240
1.226
1.182
1.093
1.080
1.025
-0.013 5.58E-05
-0.012 0.012
Mean bifurcations
(0.012) (0.010) (0.015)
(0.017) (0.015) (0.023)
(0.015) (0.012) (0.019)
-0.014
0.907
0.890
0.862
0.954
0.943
0.917
0.859
0.842
0.817
-0.010 1.97E-06
-0.009 4.01E-03
Mean whole carotid tree (0.006) (0.005) (0.007)
(0.009) (0.007) (0.011)
(0.008) (0.006) (0.009)
-0.010
Maximum whole carotid 2.113
2.029
1.919
2.264
2.184
2.093
1.957
1.888
1.776
-0.020 1.34E-07
-0.017 1.29E-03
tree
(0.025) (0.019) (0.030)
(0.037) (0.029) (0.047)
(0.031) (0.025) (0.038)
-0.022
Mean of maximal values 1.279
1.253
1.210
1.345
1.333
1.297
1.211
1.180
1.138
-0.011 1.57E-07
-0.008 0.014
in each segment
(0.009) (0.007) (0.011)
(0.013) (0.011) (0.017)
(0.011) (0.009) (0.013)
-0.013
0.734
0.685
0.620
0.789
0.772
0.715
0.677
0.607
0.542
-0.283 1.88E-07
-0.221 0.010
-0.304
Presence of plaque
(0.013) (0.011) (0.019)
(0.017) (0.014) (0.026)
(0.020) (0.016) (0.026)
Phenotype values shown are mean (standard error). P values calculated by linear or logistic regression as appropriate; adjusted for age, MDS coordinates and sex (where
applicable).
p
5.06E-03
6.48E-03
0.014
1.32E-03
9.47E-05
2.42E-05
7.96E-06
2.29E-05
References
1.
Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM, et al. The Human Genome
Browser at UCSC. Genome Res. 2002;12:996–1006.
2.
Lonsdale J, Thomas J, Salvatore M, Phillips R, Lo E, Shad S, et al. The Genotype-Tissue
Expression (GTEx) project. Nat Genet. 2013;45:580–585.