the role of epigenetics in the developmental origins...

The role of epigenetics

in the developmental origins of human metabolic disease

Dr Karen A LillycropInstitute of Developmental Sciences

School of Biological SciencesUniversity of Southampton

Genotype

Early life environment

Phenotype+

Adult life style factorsEg

. Energy rich dietphysical inactivity

Chronic disease susceptibility(CVD, obesity, type II diabetes, cancer)

3

Infant mortality rates per 1000 live births during 1901-10

high medium lowBarker DJP (1998) Mothers, babies and health in later life

Standardised mortality ratios for coronary heart diseaseamong men aged 65-74 (1968-78)

The prenatal environment determines future disease risk

Developmental origins of health and disease

hIn 1986 David Barker showed that low birth weight is associated with an increased incidence of CVD and metabolic syndrome in later life

Mortality from coronary heart diseasebefore 65 years in 15,726 men and

women in Hertfordshire

< 5.5

5.5 -

6.56.5

- 7.5

7.5 -

8.58.5

- 9.5 >9

.50

50

100MenWomen

Birth weight (lbs)

Stan

dard

ised

mor

talit

y ra

tio

Prevalence of metabolic syndromeaccording to birth weight

(407 men aged 65yr)

< 5.5

5.5 - 6

.56.5

- 7.5

7.5 - 8

.58.5

- 9.5

>9.5

0

10

20

30

40

Birth weight (lbs)

Prev

alen

ce (%

)

hSubsequent epidemiological studies have confirmed a link between low birth weight and CVD

●and showed that smaller babies have an increased risk of

HypertensionRaised serum cholesterolType 2 diabetesObesityCancer

To study how the intrauterine environment may influence development and later risk of disease a number of animal models have been established

Eg

Low protein diet global dietary restriction

Animal Models

high blood pressureDyslipideamiaImpaired glucose toleranceVascular dysfunction

Early life environment

NutritionHormones

Oxygen

How does this work?

Future Actual PostnatalEnvironment

e.g. Poor Nutrition

Low Disease RiskAppropriate PAR

MATCH

Inc Disease RiskInappropriate PAR

MISMATCH

Future Actual PostnatalEnvironment

e.g. High Calorie Diet

AlternativeDevelopmental Path

ResponseEnvironmental Cue

e.g. Poor Nutrition

●

During development organisms can respond to an environmental stimulus which allows the organism to shifts its developmental path to produce a phenotype which confers a survival/reproductive advantage in postnatal life

Predictive adaptive responses (PAR)

Developmental PlasticStage

“prenatal environment”

Reduce energy demandsIncrease capacity for fat storageLess investment in muscle mass

A second developmental pathway to obesity?

Over-

nutrition in early life can also increase of obesity in later life

●a J/U shaped relationship has been seen between BW and rates of obesity/diabetes

In animal models: High fat feeding during pregnancy

over –feeding in early postnatal life

Birthweight (kg)

0

5

10

15

20

25

30

35

40

45

-2.5 -3.0 -3.5 -4.0 -4.5 >4.5

Type-2 diabetes 1179 Pima Indians

aged 20-39 yrs

Age

adju

sted

pre

vale

nce

(%)

ObesityDiabetesCVD disturbances

Developmental plasticity

Genotype Fixed – Mutations rate and influence populations over evolutionary time scales

Phenotype Phenotype Phenotype Phenotype

Interaction with the (uterine) environment

Epigenetics

Definition: Processes that induced heritable changes in gene expression potential without a change in gene sequence

The major epigenetics

processes are

DNA methylation

Histone

modification

microRNAs

DNA MethylationiCytosine is methylated

to 5-methyl cytosine

i90% of methylated

cytosine is found as a dinucleotide

CpG

iCpGs are not randomly distributed throughout the genome but are clustered at the 5’

ends of genes

= methylation

= no methylation

Gene activity

++++

++

-Exon1 Exon 2

Exon1 Exon 2

Exon1 Exon 2

Exon1 Exon 2

+

methylation

Once established, these methylation patterns are then stably maintained throughout the life of an organism

Methylation patterns are largely establishment during development and early life

Tissue differentiation

muscle

skin

adipocyte

nerve

blood

1-carbon metabolism, which provides the methyl groups for virtually all methylation reactions, is highly dependent upon dietary methyl donors

and cofactors

(adapted from Santos & Dean)

Protein-restricted rat model

50% reduction in maternal protein intake during pregnancy, normal diet during lactation

Hypertension, dyslipidaemia, insulin resistance, altered kidney structure, increased cancer risk

Are epigenetic processes involved in the developmentalorigins of chronic diseases?

Glucocorticoid

receptor (GR)Peroxisome

proliferator

activated receptorα

(PPARα)

Control PR

0

200

400

600

800

1000

1200

1400

Maternal dietary group

mR

NA

con

cent

ratio

n co

mpa

red

to c

ontr

ol (%

)

*

PPARα

expression

Lillycrop et al. 2007

Control PR

0

100

200

300

400

500

*


mR

NA

con

cent

ratio

n co

mpa

red

to c

ontr

ol (%

)

GR

expressionGR

methylation Gluconeogenesis

The PR diet induces altered epigenetic regulation

Β

oxidation

β hy

roxy

buty

rate

con

cent

ratio

n ( μ

mol

/l)

0

100

200

300

400

500

600

700

con PR

maternal dietary group

Glu

cose

con

cent

ratio

n (m

mol

/l)

0

2

4

6

8

10

con PR

maternal dietary group

Control PR

0

100

200

300

400

500


mR

NA

con

cent

ratio

n co

mpa

red

to c

ontr

ol (%

)

*

AOX expression

Control PR0

25

50

75

100

125

150 *


mR

NA

con

cent

ratio

n co

mpa

red

to c

ontr

ol (%

)

PEPCK expression

Control PR

70

80

90

100

110

120


DN

A m

ethy

latio

n co

mpa

red

toco

ntro

l (%

)

*

PPARα

methylation

Control PR

70

80

90

100

110

120

*


DN

A m

ethy

latio

n co

mpa

red

toco

ntro

l (%

)

1 2+1 +251 +944 +1031-306-380

+259-537 CpG

Island

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 170

50

100

150

200

* * * *

CpG

Met

hyla

tion

(%R

elat

ive

toC

ontr

ol)

Maternal diet alters methylation of specific CpGs

in the PPARα

promoter

Day 34juvenile

Day 80adult

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 170

50

100

150

200 Control PR

* * * *

SP1 AHRCREB

SP1 HESFUSBPax9

NRF1ZFSFEGRG

CPBP WHNF

CpG

Met

hyla

tion

(%R

elat

ive

toC

ontr

ol)

Lillycrop et al. 2007

What about other nutritional challenges?

0

50

100

Con PR UN

% In

put

Liver PN84

- Global undernutrition

(UN) also induces GR hypomethylation

E14 fetal

liverE18 fetal

liver

control PR UN0

20

40

60

80

% o

f Inp

ut

0

1

2

3

Con PR UNM t l di t

% In

put

Over feeding in early life also induces altered DNA methylation

Plagemann A et al. 2009

Methylation blocks POMC activationBy hyperleptineamia

and hyperinsulineamia

POMC acts to reduce food intake Normally induced by leptin

and insulin

●

Over-feeding leads to the hypermethylation

of the POMC promoter

Litter size reduced to 3 pups Rapid fat accumulation ObesityCVD disturbances

Detect changes in methylation

Predict metabolic capacity & futuredisease

risk

Altered susceptibility to obesity

Protein restrictionGlobal restriction

Pregnancy

Lactation Weaning

Over-nutrition

Long term changes in gene expressionAltered metabolic capacity

Epigenotype

Altered DNA methylation

Epigenetic mark as predictive biomarkers?

Epigenetic marks as biomarkers?

In humans limited tissue availability?

Available tissues: Umbilical cordCord blood Placentabuccal

cellsBlood

Control PR80

90

100

110

P<0.0001


Prom

oter

met

hyla

tion

rela

tive

toCo

ntro

l die

t (%

; mea

n ±SD

)

Rat umbilical cord PPARα

Control PR

7 0

8 0

9 0

1 0 0

1 1 0

1 2 0

M a te r n a l d ie ta r y g r o u p

DN

A m

ethy

latio

n co

mpa

red

toco

ntro

l (%

)

*

Rat Liver PPARα

Strategy for finding epigenetic biomarkers?

Are methylation marks at birth associated with later metabolic capacity?

Extracted DNA from umbilical cords from babies within the normal

birth weight range

Genome wide screen of gene promoter methylation using a MeDIP-

promoter array

Associations between methylation of CpGs

and outcome confirmed by bisulfite

sequencing (Sequenom)

Correlated methylation levels to phenotypic outcomes of the children age 9

Stability of methylation patterns?

Methylation in blood samples taken 11-20 years apart

Methylation in recent blood versus recent buccal samples

Heijmans et al., 2010

Can we intervene to prevent or reverse these methylationmarks?

Control Protein restricted Protein restricted + folic acid

Folate

supplementation of the protein restricted diet restores normal phenotype

HypertensionDyslipidemiaVascular dysfunction

Control PR PRF

70

80

90

100

110

120


DN

A m

ethy

latio

n co

mpa

red

toco

ntro

l (%

)

*

Control PR PRF

70

80

90

100

110

120

*


DN

A m

ethy

latio

n co

mpa

red

toco

ntro

l (%

)

PPARα

Glucocorticoid

Receptor

Control PR PRF

0

200

400

600

800

1000

1200

1400


mR

NA

con

cent

ratio

n co

mpa

red

to c

ontr

ol (%

)

*

Control PR PRF

0

100

200

300

400

*


mR

NA

con

cent

ratio

n co

mpa

red

to c

ontr

ol (%

)

Increased maternal folic acid intake prevents induction of altered epigenetic regulation

β-oxidation

Control PR PRF0

100

200

300

400

500

600

700


β h

ydro

xybu

tyra

teco

ncen

trat

ion

( μm

ol/l)

Gluconeogenesis

Control PR PRF0.02.5

5

6

7

8

9


Glu

cose

con

cent

ratio

n(m

mol

/l)

Lillycrop et al., 2005

Day 34

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

0

50

100

150

200 Control PR PRF

* * **

*

*

Sp1 AHRCREB

SP1 HESFPAX9

NRFWHNF

CREB

CpG

Met

hyla

tion

(% R

elat

ive

to C

ontro

l)

Lillycrop

et al. 2008

Increasing maternal folic acid intake induces hypermethylation of specific CpG dinucleotides

1.3%

Gene expression changes in PR and PRF offspring

0.7%

Can supplementation with folic acid after weaning reverse the changes in phenotypes and in gene methylation

induced by the PR diet ?

30

Control PR

d84

1 mg/kg FA

PWD

Pregnancy

Lactation

AIN93G

n = 13 to 18/ group

5 mg/kg FA 1 mg/kg FA 5 mg/kg FA

18% fat (1 mg/kg folic acid)

28d

28d

C/AF C/FS PR/AF PR/FS

β-Hydroxybutyrate

M Control A

F

M PR AFM Contro

l FS

M PR FS

0

100

200

300

400

**

*

Group

Con

cent

ratio

n ( μ

mol

/l, m

ean ±

SD)

Post weaning folic acid supplementation alters the phenotype andepigenotype

NormalFatty

Burdge et al., 2009

Conclusionshincreasing evidence that many chronic diseases originate at least in part in uterohepigenetic mechanisms are likely to play a key role in the developmental origins of disease

●

hInitial studies suggest that these altered epigenetic marks maybe used as predictive markers of future metabolic capacity and potential disease risk

hanimal studies have also show that it is possible to prevent/reversethese epigenetic marks offering the opportunity for intervention

conception birth weaning Growth maturation aging

In vitro cultureMaternal nutrition

Maternal behaviour Folic acid

Southampton

Graham Burdge

Emma GarrettPaula CostelloAbi

LaphamBecky ClarkeAmal

AlenadMark HansonKeith GodfreyTom FlemingCyrus Cooper

Auckland

Allan ShepherdMark VickersPeter Gluckman

Research ManagementCommittee The Gerald Kerkut

Charitable Trust

Acknowledgements

AmsterdamTessa Roseboom

the role of epigenetics in the developmental origins...

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