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, Ireland2Imperial College London, London W21LA, UK3Barts and The London, Queen Mary's School of Medicine and Dentistry, London EC1M 6BQ, UK4 University College Dublin, Dublin 4, Ireland

TxA2 Functions

• Platelet activator

• Vascular smooth muscle cell constrictor and mitogen

• Plaque growth

AC

ATP

cAMP

Thrombin

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

HACVD SubstudyN=1,006

Excluded (urine sample inadequate)

N=16

Urinary TxA2

measured N=990

N=19,257

Amlodipine Perindropril (N=9,639)

Atenolol Bendroflumethiazide

N=9,618

N=10,305

TC ≤ 6.5 mmol/L

Atorvastatin 10 mg N=5,618

Placebo N=5,137

ASCOT BPLA

ASCOT LLA

Prospective, randomised, open, blinded end-point

Double-blind

HACVD Substudy

Multinational Randomised Controlled Trial

Caucasian N=859

Genotyped on CVD50K beadchipN=540

DNA Extracted N=800

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;

– 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

Demographic & Clinical CharacteristicsAll Subjects

(N=540)Subjects Not On Aspirin

(N= 272)Subjects On Aspirin

(N= 268) PTxM (pg/mg creatinine): Median (IQR) 482 (290-798) 716 (460-1071) 323 (211-518) 2E-16*Age (years): Mean ± SD 64.7 ± 8.1 64.2 ± 8.1 65.3 ± 8.1 0.11Male: N (%) 423 (78%) 223 (82%) 200 (75%) 0.04*Current smokers: N (%) 110 (20%) 55 (20%) 55 (20%) 0.93Current diabetes: N (%) 93 (17%) 35 (13%) 58 (22%) 7E-03*SBP (mmHg): Mean ± SD 134.8 ± 11.8 132.1 ± 10.5 137.5 ± 12.4 9E-08*

BMI (kg/m2): Mean ± SD 29.2 ± 4.5 28.9 ± 4.3 29.4 ± 4.7 0.23HDL (mmol/L): Mean ± SD 1.3 ± 0.3 1.3 ± 0.3 1.3 ± 0.3 0.49LDL (mmol/L): Mean ± SD 3.0 ± 0.9 3.0 ± 0.9 3.0 ± 0.8 0.69Randomised to ACE-I: N (%) 275 (51%) 144 (53%) 131 (49%) 0.35

Population Characteristics

Results: Manhattan Plots

PPARGC1β Peak

CHR SNP BP Position AF BETA P AF BETA P AF BETA P5 rs10515638 149132724 intron 1 0.07 223.1 7.3E-04 0.07 391.9 1.9E-03 0.07 74.7 0.125 rs2161257 149170190 intron 1 0.44 123.0 2.7E-04 0.43 183.9 2.3E-03 0.45 52.1 0.055 rs4235745 149171304 intron 1 0.30 166.7 4.3E-06 0.29 284.9 2.2E-05 0.31 50.5 0.065 rs17110447 149173039 intron 1 0.28 148.8 6.0E-05 0.27 252.9 2.4E-04 0.29 46.7 0.095 rs251468 149174678 intron 1 0.26 125.2 9.4E-04 0.26 236.6 9.3E-04 0.26 22.8 0.415 rs251464 149176427 intron 1 0.26 125.2 9.4E-04 0.26 236.6 9.3E-04 0.26 22.8 0.415 rs32589 149180082 intron 1 0.12 172.5 7.6E-04 0.11 365.2 2.1E-04 0.12 8.8 0.815 rs32588 149180236 exon 2 0.12 172.5 7.6E-04 0.11 365.2 2.1E-04 0.12 8.8 0.815 rs32586 149181113 intron 2 0.12 172.5 7.6E-04 0.11 365.2 2.1E-04 0.12 8.8 0.815 rs28282 149182399 intron 2 0.31 157.2 1.1E-05 0.31 262.1 6.2E-05 0.31 49.3 0.075 rs32582 149185610 intron 2 0.17 201.1 9.1E-06 0.17 353.1 2.1E-05 0.17 45.4 0.185 rs32581 149185823 intron 2 0.28 159.0 2.1E-05 0.28 259.8 1.4E-04 0.29 52.9 0.065 rs32579 149191041 intron 4 0.30 129.5 3.7E-04 0.30 220.5 9.2E-04 0.30 34.1 0.215 rs32574 149199079 intron 8 0.23 164.7 5.1E-05 0.21 273.4 3.1E-04 0.25 59.8 0.05

PPARGC1B All Subjects (N=540) Subjects Not On Aspirin (N=272)

Subjects On Aspirin (N=268)

r2=0.42

r2=0.07

r2=0.19

PPARGC1β Peak with Imputation

rs32582

rs32587

TxM* All Subjects (N=540)

Haplotype Analysis

HAPLOTYPE FREQUENCY BETA P BETA P BETA PTTA 0.06 382.4 2.1E-06 658.7 9.5E-06 105.5 0.08GTA 0.11 136.2 0.01 259.2 0.01 24.0 0.55GTC 0.14 52.4 0.29 77.8 0.40 27.1 0.46TCC 0.02 -141.1 0.28 -259.3 0.28 -10.1 0.92GCC 0.66 -145.7 5.5E-05 -249.0 2.1E-04 -45.3 0.09

All Subjects (N=540) Subjects Not On Aspirin (N=272)

Subjects On Aspirin (N=268)

Proportion of TxM variation explained by the three genotypes:

All subjects: 5.2%

Subjects not on aspirin: 8.8%

Subjects on aspirin: 1.8%

Minor alleles

Major alleles

PPARGC1β Function

PPARγRXR

prostaglandins

PPRE TATA Target gene

PPARGC1B

RNA polymerase

transcription

Anti-atheroscleroticMMP-9iNOSCOX-2TNF-α, IL-6, IL-1β MCP-1, VCAM, ICAM

PPARGC1β Variants

149.08Mb 149.10Mb 149.12Mb 149.14Mb 149.16Mb 149.18Mb 149.20Mb 149.22Mb

exons

NFκB binding site

rs10515638 rs4235745 rs32582

PPARGC1βgene

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

• Is genotype associated with events in whole ASCOT cohort? (N=~9,000)

• Functional studies

• Validation of SNP as a pharmacogenetic marker

Future work