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
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