Transcript Slide 1

PPARGC1β is a Genetic Determinant
of the Cardiovascular Risk Factor,
Thromboxane A2 –
an Anglo-Scandinavian Cardiac
Outcomes Trial (ASCOT) Sub-study
Nina McCarthy1, Ciara Vangjeli1, Praveen Surendran1,4, Achim Treumann1,
Cathy Rooney1, Emily Ho1, Peter Sever2, Simon Thom2, Alun Hughes2,
Patricia Munroe3, Philip Howard3, Toby Johnson3, Mark Caulfield3, Denis
Shields4, Eoin O’Brien4, Desmond Fitzgerald4, Alice Stanton1.
1Royal
College of Surgeons in Ireland, Dublin 2, Ireland
2Imperial College London, London W21LA, UK
3Barts and The London, Queen Mary's School of Medicine and Dentistry,
London EC1M 6BQ, UK
4 University College Dublin, Dublin 4, Ireland
TxA2 Functions
• Platelet activator
ATP
AC
cAMP
• Vascular smooth
muscle cell
constrictor and
mitogen
Thrombin
• Plaque growth
TxA2 Formation
COX-1
COX-2
TxA2 and Atherothrombotic Events
• Target of aspirin – responsible for
both therapeutic and harmful effects
• Independent predictor of
atherothrombotic events (OR=2 for
MI, OR=3.5 for CV-related death)
HACVD, a Substudy of ASCOT
Excluded (urine
sample inadequate)
N=16
Urinary TxA2
measured N=990
HACVD
Substudy
N=1,006
N=19,257
Atenolol
Bendroflumethiazide
N=9,618
ASCOT
BPLA
Amlodipine
Perindropril
(N=9,639)
Prospective,
randomised, open,
blinded end-point
Caucasian
N=859
N=10,305
TC ≤ 6.5 mmol/L
DNA Extracted
N=800
Atorvastatin 10 mg
N=5,618
Genotyped on CVD50K beadchip
N=540
HACVD Substudy
ASCOT
LLA
Placebo
N=5,137
Double-blind
Multinational Randomised Controlled Trial
Methods
• Phenotyping
– 11-dehydro TxB2 (TxM), expressed as pg of TxM/mg creatinine.
– LC-MS-MS
• Genotyping
– CVD50K chip; 2,000 genic regions related to cardiovascular,
inflammatory, and metabolic phenotypes.
• Statistical Analysis:
linear regression analysis, adjusting for the covariates;
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age
sex
smoking habit
diabetes
systolic blood pressure
body mass index
high density lipoprotein
low density lipoprotein
aspirin use at time of TxM measurement
randomized anti-hypertensive regimen
Population Characteristics
Demographic & Clinical Characteristics
TxM (pg/mg creatinine): Median (IQR)
Age (years): Mean ± SD
Male: N (%)
Current smokers: N (%)
Current diabetes: N (%)
SBP (mmHg): Mean ± SD
BMI (kg/m2): Mean ± SD
HDL (mmol/L): Mean ± SD
LDL (mmol/L): Mean ± SD
Randomised to ACE-I: N (%)
All Subjects
(N=540)
482 (290-798)
64.7 ± 8.1
423 (78%)
110 (20%)
93 (17%)
134.8 ± 11.8
Subjects Not On Aspirin
(N= 272)
716 (460-1071)
64.2 ± 8.1
223 (82%)
55 (20%)
35 (13%)
132.1 ± 10.5
Subjects On Aspirin
(N= 268)
323 (211-518)
65.3 ± 8.1
200 (75%)
55 (20%)
58 (22%)
137.5 ± 12.4
P
2E-16*
0.11
0.04*
0.93
7E-03*
9E-08*
29.2 ± 4.5
1.3 ± 0.3
3.0 ± 0.9
275 (51%)
28.9 ± 4.3
1.3 ± 0.3
3.0 ± 0.9
144 (53%)
29.4 ± 4.7
1.3 ± 0.3
3.0 ± 0.8
131 (49%)
0.23
0.49
0.69
0.35
Results:
Manhattan Plots
PPARGC1β Peak
PPARGC1B
CHR
5
r2=0.42
5
5
5
r2=0.19 5
5
r2=0.07 5
5
5
5
5
5
5
5
SNP
rs10515638
rs2161257
rs4235745
rs17110447
rs251468
rs251464
rs32589
rs32588
rs32586
rs28282
rs32582
rs32581
rs32579
rs32574
BP
149132724
149170190
149171304
149173039
149174678
149176427
149180082
149180236
149181113
149182399
149185610
149185823
149191041
149199079
All Subjects (N=540)
Position
intron 1
intron 1
intron 1
intron 1
intron 1
intron 1
intron 1
exon 2
intron 2
intron 2
intron 2
intron 2
intron 4
intron 8
AF
0.07
0.44
0.30
0.28
0.26
0.26
0.12
0.12
0.12
0.31
0.17
0.28
0.30
0.23
BETA
223.1
123.0
166.7
148.8
125.2
125.2
172.5
172.5
172.5
157.2
201.1
159.0
129.5
164.7
P
7.3E-04
2.7E-04
4.3E-06
6.0E-05
9.4E-04
9.4E-04
7.6E-04
7.6E-04
7.6E-04
1.1E-05
9.1E-06
2.1E-05
3.7E-04
5.1E-05
Subjects Not On Aspirin
(N=272)
P
BETA
AF
391.9 1.9E-03
0.07
183.9 2.3E-03
0.43
284.9 2.2E-05
0.29
252.9 2.4E-04
0.27
236.6 9.3E-04
0.26
236.6 9.3E-04
0.26
365.2 2.1E-04
0.11
365.2 2.1E-04
0.11
365.2 2.1E-04
0.11
262.1 6.2E-05
0.31
353.1 2.1E-05
0.17
259.8 1.4E-04
0.28
220.5 9.2E-04
0.30
273.4 3.1E-04
0.21
Subjects On Aspirin
(N=268)
P
AF BETA
74.7 0.12
0.07
52.1 0.05
0.45
50.5 0.06
0.31
46.7 0.09
0.29
22.8 0.41
0.26
22.8 0.41
0.26
8.8 0.81
0.12
8.8 0.81
0.12
8.8 0.81
0.12
49.3 0.07
0.31
45.4 0.18
0.17
52.9 0.06
0.29
34.1 0.21
0.30
59.8 0.05
0.25
PPARGC1β Peak with Imputation
TxM* All Subjects (N=540)
rs32587
rs32582
Haplotype Analysis
All Subjects (N=540)
Minor
alleles
Major
alleles
HAPLOTYPE FREQUENCY
TTA
0.06
GTA
0.11
GTC
0.14
TCC
0.02
GCC
0.66
BETA
382.4
136.2
52.4
-141.1
-145.7
P
2.1E-06
0.01
0.29
0.28
5.5E-05
Subjects Not On Subjects On Aspirin
Aspirin (N=272)
(N=268)
BETA
P
BETA
P
658.7
9.5E-06
105.5
0.08
259.2
0.01
24.0
0.55
77.8
0.40
27.1
0.46
-259.3
0.28
-10.1
0.92
-249.0
2.1E-04
-45.3
0.09
Proportion of TxM variation explained by the three genotypes:
All subjects: 5.2%
Subjects not on aspirin: 8.8%
Subjects on aspirin: 1.8%
PPARGC1β Function
prostaglandins
PPARGC1B
PPARγ
RXR
PPRE
RNA
polymerase
TATA
transcription
Target gene
Anti-atherosclerotic
MMP-9
iNOS
COX-2
TNF-α, IL-6, IL-1β
MCP-1, VCAM, ICAM
PPARGC1β Variants
149.08Mb
149.10Mb
149.12Mb
149.14Mb
rs10515638
149.16Mb
149.18Mb
rs4235745
rs32582
PPARGC1β
gene
exons
NFκB
binding site
149.20Mb
149.22Mb
Study Implications
• Suggests PPARγ transcriptional regulation
regulates TxA2 production
• SNPs may be genetic markers of high-risk
patients
• SNPs may be pharmacogenetic markers to
inform use of aspirin
Acknowledgements
• Health Research Board Ireland
• Pfizer
• British Heart Foundation
• ASCOT HACVD participants
Future work
• Is genotype associated with events in whole
ASCOT cohort? (N=~9,000)
• Functional studies
• Validation of SNP as a pharmacogenetic
marker