Epigenetic Mechanisms of Environmentally-mediated Cardiovascular Disease Risk in the WHI

Epigenetic Mechanisms of Environmentally-mediated

Cardiovascular Disease Risk in the WHI

Andrea Baccarelli, MD, PhD, MPHLab of Environmental Epigenetics

Harvard School of Public Health

[email protected]

A. Baccarelli, Harvard School of Public Health

Epigenetics Programming of gene expression

that: does not depend on the DNA code (relatively) stable, i.e., replicated

through: cell mitosis meiosis, i.e. transgenerational (limited

evidence in humans) Characteristics of Epigenetic

Programming Modifiable (can be reprogrammed) Active or poised to be activated:

Potentially associated with current health states or predict future events

A Symphonic Example


DNA Phenotype

Epigenetics


A Symphonic Example

A. Baccarelli, Harvard School of Public Health From the Royal Society of Chemistry website

(www.rsc.org)

DNA Methylation


Changes during mitosis Fidelity of “transcription” of DNA

methylation varies between 97-99.9% De-novo methylation: 3-5% mitosis Much more dynamic compared to DNA

sequence!

DNA methylation is known to be modified: through aging oxidative stress, inflammation,

micronutrients


Triggers of cardiovascular events at the population level

Asymptomatic Angina MI

CardiovascularRisk Factors

Triggers

Prev

entio

nO

ppor

tuni

ties

Primordial Primary Secondary

Subclinical Disease

Racial &

Socioeconomic Disparities

Individual interventions

Societal interventions

Sudden or CHD Death

10 20 30 50 60 7040 80 90Age

Atherosclerosis time-course

• Diabetes mellitus• Hypertension• Smoking• Dyslipidemia• Inflammation• Physical inactivity

• Traffic exposure• Physical exertion• Alcohol• Coffee consumption• Air pollution• Emotions (positive/negative)• Heavy meal• Cocaine abuse• Sexual activity, etc.

Preclinical Clinical

High population attributable fraction (PAF)

High relative or absolute individual risk

Exposure to traffic and air pollution are estimated to trigger more heart attacks than:-Heavy alcohol consumpion (2.5 fold)-Cocaine abuse (10 fold)

Long term exposure to air pollution leads to:-Atherosclerosis-Cardiovascular morbidity and death

Figure from Editorial byBaccarelli & BenjaminThe Lancet 2011

Coding & non-coding DNA in the human genome

Protein coding genes 1-2%Unique non-coding DNA ≈45%

Pseudogenes <1% Repetitive sequences ≈50%LINEs (e.g., LINE-1): 21%SINEs (e.g., Alu): 11%Both LINEs & SINEs are retrotranspons

Heavily methylate

d

Chromosomal stabilityLimits retrotranspositionLimits inflammation



Effects of Air Pollution on Repetitive Element DNA Methylation

Tarantini, et al. Environment Health Perspect, 2009

Effects of PM10 found both at the beginning and at the end of the work week

2525

.526

26.5

2727

.5A

LU D

NA

met

hyla

tion

(%m

c)100 250 500 1500

average PM10 (µg/m³)

7678

8082

84LI

NE

-1 D

NA

met

hyla

tion

(%m

c)

100 250 500 1000 1500average PM10 (µg/m³)

βunadj=-0.30; P=0.07βadj=-0.34; P=0.04

βunadj=-0.18; P=0.04βadj=-0.19; P=0.04

LINE-1 Alu

β for an increment equal to the difference between the 90th and 10th

percentile of PM10Mixed models: unadjusted or adjusted for age, BMI, smoking, cigarettes/day


10

20

30

40

50

60

Cum

ulat

ive

inci

denc

e of

isch

emic

hea

rt di

seas

e or

stro

ke (%

)

24 36 48 60 72 84 96Months from baseline examination

Low (68.1-77.4 %5mC)High (77.5-86.2 %5mC)

LINE-1 methylation

10

20

30

40

50

60

Cum

ulat

ive

inci

denc

e of

isch

emic

hea

rt di

seas

e (%

)


Low (68.1-77.4 %5mC)High (77.5-86.2 %5mC)

LINE-1 methylation

5

10

15

20

25

30

Cum

ulat

ive

inci

denc

e of

stro

ke (%

)


Low (68.1-77.4 %5mC)High (77.5-86.2 %5mC)

LINE-1 methylation

aFigure 1

b

Adj. Hazard Ratio3.6 (95%CI 1.8-7.0)

p<0.001

HR=2.8 (95%CI 1.3-5.9), p=0.009 for IHDHR=4.3 (95%CI 0.7-25.8), p=0.11 for strokeBaccarelli et al., Epidemiology

2010

DNA Methylation and Incidence of non-fatal IHD or Stroke in the NAS


2

4

6

8

10

12M

orta

lity

(%)

[Dea

ths

from

isch

emic

hea

rt di

seas

e or

stro

ke]

0 12 24 36 48 60 72 84 96Months from baseline examination

Low (68.1-77.4 %5mC)High (77.5-86.2 %5mC)

LINE-1 methylation

Adj. Hazard Ratio2.9 (95%CI 1.4-6.3)

P=0.006

HR=3.5 (95%CI 1.4-8.8), p=0.007 for IHDHR=2.2 (95%CI 0.5-10.0), p=0.30 for strokeBaccarelli et al., Epidemiology

2010

DNA Methylation and Deathfrom IHD or Stroke in the NAS


Potential Roles of Epigenetics

Baccarelli, Rienstra & Benjamin Circulation: Cardiovascular Genetics 2010


Conclusions and Research Needs DNA methylation:

Sensitive to the environment Predicts risk of cardiovascular disease

Limitations: Limited power Unreplicated Limited to repeat elements (or other candidate sequence) environmentally homogeneous, single-city populations of

white men Research needs:

Investigating a diverse population Studies of women Characterizing DNA methylation changes over time and their

association with CVD-related risk factors


R01 ES020836MPIs, Whitsel, Hou, Baccarelli

Epigenetic mechanisms of PM-mediated

cardiovascular risk

Pending (Score=19; Percentile 11%)

Region Type Regions CpG sites covered on 450K BeadChip array

Average # of CpG sites per region

CpG Island 26,153 139,265 5.08N Shore 25,770 73,508 2.74S Shore 25,614 71,119 2.66N Shelf 23,896 49,093 1.97S Shelf 23,968 48,524 1.94

Remote/Unassigned - 104,926 -Total 485,553

CpG shelves, shores & islands classification (UCSC CpGi annotation)

N Shelf N Shore CpG Island

5’ UTR 3’ UTRTSS1500 TSS200

S Shore S Shelf

Illumina 450K BeadChip CoverageThe 450K BeadChip covers a total of 77,537 CpG Islands and CpG Shores (N+S)


Study Population Two-stage, longitudinal study of associations

between PM air pollution, DNA methylation, and CVD risk factors: exam site- and race-stratified, randomly selected

6% minority oversample approximately 4,300 Women’s Health Initiative clinical

trial (WHI CT) women fasting blood draws and resting, standard, twelve-

lead electrocardiograms (ECGs) repeated at three-year intervals from 1993 to 2004.


Study Design Stage 1 (Discovery)

interrogation, discovery and ranking of >450,000 DNA methylation sites potentially sensitive to PM in 1999-2001 blood samples from 800 participants

Stage II (Validation) Longitudinal validation of the 10 most PM-sensitive DNA methylation

sites identified by Stage 1 in up to three blood samples collected serially from the remaining 3,500 participants (1993-2004)

Focus on the temporal relationship between PM and DNA methylation at those sites, and that between site-specific DNA methylation and CVD risk factors.

Other Features: Phenomics framework to incorporate phenotypes Epigenetic data analyses adjusted for both ancestral admixture and

multiple comparisons External validation with cohorts with different participants

characteristics (ARIC, NAS)


DNA methylation in the WHI

The WHI team for the Environmental Epigenomics proposal


Eric Whitsel, University of North Carolina (PI) Lifang Hou, Northwestern University (PI) Andrea Baccarelli, Harvard University (PI) Yun Li, University of North Carolina Duanping Liao, Penn State Simon Lin, Northwestern University Lesley Tinker, Fred Hutchinson Linda Van Horn, Northwestern University

Epigenetic Mechanisms of Environmentally-mediated Cardiovascular Disease Risk in the WHI

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