Placental Epigenetics

Carmen J. Marsit, PhD

Pharmacology & Toxicology

Community and Family Medicine

Geisel School of Medicine at Dartmouth

Developmental Origins

CAN EPIGENETIC MECHANISMS

FILL THE BLACK BOX?

Environmental

Exposure

Metabolic

Cardiovascular

Cancer

Infection/Immunity

Mental Health

Epigenetics

Heritable and Stable Control of Gene

Expression Potential Beyond the DNA

Sequence

Heritable: can pass on to successive cells

Does not alter the genetic sequence

Stable: cannot easily be altered

Potentially reversible

Controls Gene Expression Potential

On, Off, Level?

Modes of Epigenetic Regulation

DNA Methylation

Histone post-translational

modification

Imprinting

RNA-mediated regulation

Transcription

Control

Post-

transcription

Control

Where we study epigenetic

mechanisms matters

Epigenetic Patterning is highly tissue specific

Represents functional alteration

Placenta

First complex organ to form

Regulates intrauterine environment

Transport

Nutrients

Water

Gas

Waste products

Immuno-endocrine

Hormones

Growth factors

• Demonstrate placental molecular features

integrate environmental signals

• Link variability in molecular features to Infant

Outcomes (and beyond!)

MATERNAL

ENVIRONMENT PLACENTAL

EPIGENOME

PLACENTAL

FUNCTION •Growth

•Nutrition

•Drugs

•Stress

• Exposures

Role of Placental Epigenome

Epigenetic Mechanisms in Placenta

Monoallelic expression of a subset of genes

Some tissue specificity

Placenta has unique imprinting profile and function

Parent-of-origin fashion

Proposed as an environmental sensor (Jirtle & Skinner, Nat Rev Genet 2007)

Alterations to imprinting linked to critical children’s health outcomes

Various Alterations Can be Examined

Gene Expression

Loss of imprinting

Methylation of Imprinting Control Regions

Genomic Imprinting DNA Methylation

Mechanism to control gene expression

Highly tissue specific

Placenta has specific DNA methylation profile

Altered Methylation linked to environmental exposures

Variability linked to disease risk

Genome-wide DNA methylation

Opportunity to identify novel genes or pathways targeted by exposures

Can then examine if these alterations predict downstream outcomes

Use Genome-Wide Array Based Approaches

Illumina Infinium Methylation450K Array

Mom

• Medical Chart

Review

• Questionnaire

Pregnancy Delivery

Infant

• Clinical

Characteristics

• NNNS Assessment

Study Population: Rhode Island Child

Health Study

• Maternal blood

• Toenail Samples

• Placenta

• Cord Blood

• Toenails

Linking Molecular

Character with

Exposures and

Outcomes

Hospital-based Birth Cohort

Moms enrolled following delivery at Women & Infant Hospital

WIH sees 75% of deliveries in RI

2009-2014

Total Enrollment n=899

Mothers

18-40 years old

No history of psychological disorders

In good physical health

Infants

Viable

No known genetic disorders

No life threatening illness

Term (≥ 37 weeks)

Oversampled for SGA (small) and LGA (large) infants. Matched to AGA (appropriate)

Gestation time, infant sex, maternal age (±3 yrs)

Outcome: Newborn Neurobehavior

Developed by Lester and Tronick (2004)

Built on backbone of Neonatal Behavioral Assessment Scales (NBAS) developed by Brazelton (1973)

Developed for use in at-risk infant

Specifically substance exposed – used in the Maternal Lifestyle Study

Designed for broad applicability

Generalizable

Reproducible

Sensitive to variety of infant risk factors

Infants from 30 weeks gestation to ~2months post-partum

Approx. 30 minute exam performed after 24 hours of life but prior to discharge

Examine three major area of neurodevelopment

Neurological

Behavioral

Stress/Abstinence

Summarized into 13 Summary Scales reflecting various aspects of neurodevelopment

Can examine individually

Used latent profiling strategy to create profiles of neurobehavior (Liu et al Pediatrics 2010)

NICU Network Neurobehavioral Scales (NNNS)

Exposures of interest: Trace Metals

Arsenic Known carcinogen

Birth consequences

Reduced birthweight

Spontaneous abortion

Neurobehavioral deficits

Increased Placental Oxidative Stress

Exposure through water and food

Manganese Normal dietary constituent

Higher level exposures

Neurological/neurodevelopmental disorders

Effects on growth

Exposure through water, food

Mercury Crosses placenta

Interferes with placental function

Neurobehavioral effects associated with prenatal and childhood exposure

Exposure through diet (seafood), dental amalgams, industrial pollution

Imprinted Genes, Exposures and

Neurobehavioral Outcomes

Gene expression of 22 candidate imprinted genes

Quantitative RT-PCR

106 placenta samples from RICHS study

Associations with arsenic and manganese exposure

Associations of imprinted gene expression profile and NNNS summary scores

Recursively partitioned mixed modeling to develop profiles of imprinted gene expression

Relationship to scores on handling, quality of movement, attention, arousal, and excitability

Increased expression of imprinted

genes with As and Mn exposure

Patterning of expression and specific

newborn behavioral outcomes

Ha

nd

ling

Q

ua

lity o

f

Mo

ve

me

nt

Placenta Samples (n=106)

Imp

rin

ted

G

en

e

Exp

ress

ion

Marsit, et al. J Pediatr 2012

Placental Imprinting

Environmental exposures associated with changes of

specific genes

Coordinated regulation of imprinted genes

Related to complex phenotypes at birth

Associations with Quality of Movement and Handling

Suggest possibility that imprinted genes influence

neuromuscular development

Known for a number of specific genes

May influence programming of stress responses

Future work to expand gene profiling and fully link

exposures and outcomes through placental imprinting

Discovery Study of Hg associated

placental DNA Methylation

Loci related to Hg Exposure

212

64

211

40

213

32

214

13

212

40

212

31

212

43

211

75

213

13

212

95

213

94

212

37

213

20

214

29

213

05

212

25

213

26

213

41

213

16

212

32

212

08

211

56

211

58

212

93

213

22

213

30

214

21

213

21

213

19

213

76

213

45

211

64

212

60

211

96

211

53

211

46

211

54

213

11

212

69

213

81

214

18

cg15385386cg10441365cg02589828cg17339147cg00002033cg03494795cg20174000cg11464064cg10288784cg17163751cg03349922cg26166804cg19726179cg07258916cg13687570cg14166009cg24834889cg12024906cg12948621cg05280698cg26734888cg27591450cg24347663cg08857144cg03384579cg23427269cg05659187cg15170605cg03022609cg15351736cg25885322cg24139169cg00543972cg04517722cg16024904cg16744531cg20187719cg12466610cg07021532rs5936512rs6546473cg13092806cg24385580cg20919942cg19497031cg13965724cg05134500cg05454501cg09859398cg15436096cg04186360cg21893185cg21558508cg18260973cg18318704cg26293310cg01948148cg07697895cg27331524cg22821834cg10014563cg16322262cg26910511cg11199014cg12454169cg15652532cg06680397cg05956126cg21158476cg02124514cg00054702cg15454726cg19783435cg20696478cg10156366cg10512745cg03976877cg10485664cg10454766cg04861929cg06719900cg17882660cg24642825cg07015190cg20206437cg01336390cg22655988cg10320659cg08475953cg06183338cg15892115cg08768621cg23318063cg12969170cg02673015cg16063666cg14048874cg13267931cg16662267cg27307781cg08024264cg11144103cg19863210rs264581cg21931717cg24960960cg23490161cg08422420cg08778598cg27378537cg27149073cg06721546cg26472636cg21913319cg03988092cg27639199cg12556569cg22664298cg15845365cg26864826rs6982811rs2385226cg05704942rs739259cg20592836rs2857639rs1416770rs798149rs9292570cg09088988cg21882477cg00332745cg11093548cg23448505cg02511231cg09696939cg11497957cg07229186cg24603464cg15165122cg03609614cg21164300cg01393340cg14056849cg13251750cg06417478cg21488538cg19190016cg03517284cg16511983cg22848598cg17323488cg00988148cg17138852cg04194432cg23892028cg24506221rs951295rs966367cg10172783cg04032226cg24714905cg16519587cg02636041cg12451631cg23136139cg02090762cg07875818cg00321709cg03841977cg00695416cg13401703cg25385940cg26531700cg22455450cg10165894cg26402828cg19080354cg27179533cg14874750cg27528510cg23424003cg13236378cg05062612cg11291313cg18237551cg21228005cg04675542cg02343823cg25420101cg03449867cg05900567cg12568536cg12894709cg11593111cg23160717cg06711418cg08049519rs4331560rs3818562cg07501029rs4742386rs5987737cg27230769cg22851875cg27467876rs9363764cg25465065cg18816122cg14175932cg17240976cg16744961cg12469381cg20673830cg15210622rs939290rs2125573cg05593887cg00980980cg07686394cg02907150cg26846609cg18237047cg21203249cg04453550cg24694833rs654498cg18828306cg04610028cg19245335cg16043227cg02658043cg05712748cg17635970cg04462931cg00146240cg27452255cg22728830cg09481537cg16657538cg11009736cg17974166cg20250269cg09310644cg10376408cg25649515cg24844518cg04990378cg16891431cg05714155cg17128947cg21149357cg14264023cg03224005cg13506281cg18391209cg05237503cg03084184cg21852408cg07180307cg03315469cg09856068cg05115233cg22244940cg12288267cg03920544cg07820332cg05114178cg08858160cg21422164cg09142848cg11484348cg19587838cg01127608cg06430688cg13697578cg02096887cg02720091cg09415955cg13755866cg07374961cg05986417cg02546601cg16100355cg22109827cg12195446cg19653246cg20381372cg00243527cg00695177cg08900396cg13275129cg12624040cg14317384cg12031275cg00689685cg01356752cg21543270cg14562786cg24651215cg05430997cg13590055cg10470368cg27256423cg23169584cg14554788cg03358735cg12386614cg13133387cg20606489cg00303341cg21864016cg14995921cg17951978cg13225272cg24900563cg15425276cg23119063rs10774834rs715359rs1941955cg24836826cg13568515rs1484127rs133860rs10033147cg17662493cg07319199rs1510480cg01891583cg02113055rs2208123cg08603678rs13369115cg05971102cg08849813cg06520095cg05519582cg05279330

Unmethylated Methylated

Cp

G L

oci

Placenta Samples 21

26

42

11

40

21

33

22

14

13

21

24

02

12

31

21

24

32

11

75

21

31

32

12

95

21

39

42

12

37

21

32

02

14

29

21

30

52

12

25

21

32

62

13

41

21

31

62

12

32

21

20

82

11

56

21

15

82

12

93

21

32

22

13

30

21

42

12

13

21

21

31

92

13

76

21

34

52

11

64

21

26

02

11

96

21

15

32

11

46

21

15

42

13

11

21

26

92

13

81

21

41

8

cg15385386cg10441365cg02589828cg17339147cg00002033cg03494795cg20174000cg11464064cg10288784cg17163751cg03349922cg26166804cg19726179cg07258916cg13687570cg14166009cg24834889cg12024906cg12948621cg05280698cg26734888cg27591450cg24347663cg08857144cg03384579cg23427269cg05659187cg15170605cg03022609cg15351736cg25885322cg24139169cg00543972cg04517722cg16024904cg16744531cg20187719cg12466610cg07021532rs5936512rs6546473cg13092806cg24385580cg20919942cg19497031cg13965724cg05134500cg05454501cg09859398cg15436096cg04186360cg21893185cg21558508cg18260973cg18318704cg26293310cg01948148cg07697895cg27331524cg22821834cg10014563cg16322262cg26910511cg11199014cg12454169cg15652532cg06680397cg05956126cg21158476cg02124514cg00054702cg15454726cg19783435cg20696478cg10156366cg10512745cg03976877cg10485664cg10454766cg04861929cg06719900cg17882660cg24642825cg07015190cg20206437cg01336390cg22655988cg10320659cg08475953cg06183338cg15892115cg08768621cg23318063cg12969170cg02673015cg16063666cg14048874cg13267931cg16662267cg27307781cg08024264cg11144103cg19863210rs264581cg21931717cg24960960cg23490161cg08422420cg08778598cg27378537cg27149073cg06721546cg26472636cg21913319cg03988092cg27639199cg12556569cg22664298cg15845365cg26864826rs6982811rs2385226cg05704942rs739259cg20592836rs2857639rs1416770rs798149rs9292570cg09088988cg21882477cg00332745cg11093548cg23448505cg02511231cg09696939cg11497957cg07229186cg24603464cg15165122cg03609614cg21164300cg01393340cg14056849cg13251750cg06417478cg21488538cg19190016cg03517284cg16511983cg22848598cg17323488cg00988148cg17138852cg04194432cg23892028cg24506221rs951295rs966367cg10172783cg04032226cg24714905cg16519587cg02636041cg12451631cg23136139cg02090762cg07875818cg00321709cg03841977cg00695416cg13401703cg25385940cg26531700cg22455450cg10165894cg26402828cg19080354cg27179533cg14874750cg27528510cg23424003cg13236378cg05062612cg11291313cg18237551cg21228005cg04675542cg02343823cg25420101cg03449867cg05900567cg12568536cg12894709cg11593111cg23160717cg06711418cg08049519rs4331560rs3818562cg07501029rs4742386rs5987737cg27230769cg22851875cg27467876rs9363764cg25465065cg18816122cg14175932cg17240976cg16744961cg12469381cg20673830cg15210622rs939290rs2125573cg05593887cg00980980cg07686394cg02907150cg26846609cg18237047cg21203249cg04453550cg24694833rs654498cg18828306cg04610028cg19245335cg16043227cg02658043cg05712748cg17635970cg04462931cg00146240cg27452255cg22728830cg09481537cg16657538cg11009736cg17974166cg20250269cg09310644cg10376408cg25649515cg24844518cg04990378cg16891431cg05714155cg17128947cg21149357cg14264023cg03224005cg13506281cg18391209cg05237503cg03084184cg21852408cg07180307cg03315469cg09856068cg05115233cg22244940cg12288267cg03920544cg07820332cg05114178cg08858160cg21422164cg09142848cg11484348cg19587838cg01127608cg06430688cg13697578cg02096887cg02720091cg09415955cg13755866cg07374961cg05986417cg02546601cg16100355cg22109827cg12195446cg19653246cg20381372cg00243527cg00695177cg08900396cg13275129cg12624040cg14317384cg12031275cg00689685cg01356752cg21543270cg14562786cg24651215cg05430997cg13590055cg10470368cg27256423cg23169584cg14554788cg03358735cg12386614cg13133387cg20606489cg00303341cg21864016cg14995921cg17951978cg13225272cg24900563cg15425276cg23119063rs10774834rs715359rs1941955cg24836826cg13568515rs1484127rs133860rs10033147cg17662493cg07319199rs1510480cg01891583cg02113055rs2208123cg08603678rs13369115cg05971102cg08849813cg06520095cg05519582cg05279330

Hg tertile Infant gender Maternal ethnicity

Maternal age Birthweight group

339 CpG loci average methylation differed by >0.125 between Hg tertiles

79 increased methylation

34 decreased methylation

54 non-monotonic

58 loci had P<0.001

Many within genes or gene promoters associated with neurologic outcomes

Neurodevelopment

(DIXDC1, NRBP2, KIF26B,

FEZF1, DMRTA2, ACTN1,

MYO10, LRFN1)

Neurobehavior (CPLX1,

LMX1B, ADD2)

Schizophrenia (DIXDC1,

ARVCF, MAGI2, ZIC2)

ADHD (TCERG1L)

Movement disorders (NOL3,

TP53INP2)

Autism (PLXNA4, WNT2)

J.Z.J. Maccani, et al. EHP, accepted

Focused on “At-risk” profile

-1.50

-1.00

-0.50

0.00

0.50

1.00

1.50

Not Profile 7

Profile 7

Me

an

-ce

nte

red

sc

ore

Hg-associated loci are associated with

NNNS High Excitability Profile

Illumina CpG

Designation

Gene Symbol P Value UCSC CpG

Island

Designation

cg13267931 EMID2 8.25x10-6 Island

cg14874750 EMID2 6.06x10-5 Island

cg23424003 EMID2 7.30x10-5 Island

cg27179533 EMID2 5.46x10-5 Island

cg27528510 EMID2 9.00x10-5 Island

cg14048874 EMID2 0.0023 Island

cg14175932 2.84x10-5

cg17128947 CPLX1 0.0054 Island

cg25385940 TTC23 0.0059 N Shore

cg10470368 0.0075

J.Z.J. Maccani, et al. EHP, accepted

Placental hypomethylation of EMID2

associated with Risk Behavioral Profile

Risk Profile

(n=16)

Non-Risk Profile

(n=135)

J.Z.J. Maccani, et al. EHP, accepted

Methylation Correlated with

Expression

0.1 0.2 0.3 0.4 0.5

31

33

35

37

EMID2 Mean Methylation

EM

ID2

Expre

ssio

n

J.Z.J. Maccani, et al. EHP, accepted

EMID2

Collagen protein, unknown placental function

Variant associated with asthma/airway

hyperresponsiveness in nasal passages (Pasaje et al. 2011;

Pasaje et al. 2012)

A SNP within EMID2 mediates side effects on

vision and hearing in response to an

antidepressant (Adkins et al. 2012)

More work needed to understand the

functional role of this gene in placenta

Focus on Placenta

Critical organ involved in regulating intrauterine environment and programming numerous fetal functions

Alterations to genes or pathways can have long-term consequences on development

Genomic Imprinting & DNA methylation are susceptible to environmental signals

Toxicant Exposures

Maternal Factors/Lifestyle

Stress, Psychosocial adversity

Imprinting and DNA methylation can be linked to critical newborn outcomes

Growth

Neurobehavioral development

Ongoing work

Better defining what environment can do

Consequences of these alterations

Various Health Outcomes later in childhood

Acknowledgments

Dartmouth Dave Armstrong

Corina Lesseur Perez

Allison Paquette

Allison Appleton

Dylan Guerin

Brian Jackson

Margaret Karagas &

Children’s Center Team

Oregon State Andy Houseman

Mt. Sinai Jia Chen

Luca Lambertini

Women and Infants Barry Lester

James Padbury

Brown Jennifer Maccani

Karl Kelsey

Funding

NIMH R01MH094609

NIEHS R01ES022223

NIEHS P01 ES022832; EPA RD83544201