Epigenetic Markers for Transplacental Epigenetic Markers for Transplacental Exposure to Airborne Polycyclic Exposure to Airborne Polycyclic

Aromatic Hydrocarbons (Aromatic Hydrocarbons (PAHsPAHs) and ) and Childhood Asthma Childhood Asthma

Winnie WanWinnie Wan--yeeyee Tang, Tang, Ph.DPh.D Department of Environmental Health

University of Cincinnati College of Medicine, Ohio

Epigenetic and Genetic interactions Epigenetic and Genetic interactions influence interinfluence inter--individual disease riskindividual disease risk

Modified from Miller and Ho 2008

Environmental FactorsEnvironmental Factors

Genetic polymorphismsGenetic polymorphisms(born with)(born with)

InterInter--individual variabilityindividual variabilityIn disease risksIn disease risks

EpigeneticsEpigenetics(can be modified after conception)(can be modified after conception)

Quantitative, sensitive to environmental Quantitative, sensitive to environmental stimuli, agestimuli, age--dependentdependent

11

Mechanisms of epigenetic programming Mechanisms of epigenetic programming

Epigenetic changes are reversible, heritable modifications that do not involve alterations in the primary DNA sequence.

Three distinct and intertwined mechanisms :Non-coding RNAs, DNA methylation, and Histone modification

These processes affect transcript stability, DNA folding, nucleosomepositioning and chromatin compaction.

Singularly or conjointly, they determine whether a gene is silenced or activated.

22

Epigenetics, environment and Epigenetics, environment and developmentdevelopment

Modified from Foley et al., 2008

Dysregulation of epigenetic processes -> Disease development

ElderlyElderlyBabyBaby AdolescentAdolescent AdultAdultEmbryoEmbryo FetusFetusZygoteZygoteGametesGametes

Environmental exposureEnvironmental exposure

Maternal FactorsMaternal Factors Diet and Life styleDiet and Life style

Developmental epigenetic reprogrammingSomatic epimutation

Germlineepimutation

Genome-wideDemethylation

33

Epigenetic epidemiologyEpigenetic epidemiology--key pointskey points

Epigenetic epidemiology studies show how the inter- individual epigenetic variation affects disease risk.

Sample selectionSample selection

Study designsStudy designs--DNA methylationDNA methylation

Statistical analysisStatistical analysis

Use of Use of in vitroin vitro and and in vivo in vivo datadata

44

Airborne PAHs, Epigenetics, AsthmaAirborne PAHs, Epigenetics, Asthma

University of Cincinnati, Department of Environmental Health University of Cincinnati, Department of Environmental Health and Center of Environmental Geneticsand Center of Environmental GeneticsDrs. Shuk-mei Ho, Wan-yee Tang, Linda Levin

Columbia Center for ChildrenColumbia Center for Children’’s Environmental Health (CCCEH)s Environmental Health (CCCEH)Drs. Frederica Perera, Julie Herbstman, Deliang Tang, Rachel Miller

Epigenetic reprogrammingEpigenetic reprogramming

Transplacental exposureTransplacental exposure

Childhood asthma Childhood asthma Or airway inflammationOr airway inflammation

Airborne Airborne PAHsPAHs(common traffic-related air pollutants)

55

PAHs and AsthmaPAHs and Asthma

Asthma is the most common chronic childhood disease and its risk may be strongly influenced by prenatal events (Selgrade et al., 2006).

In our longitudinal cohort study of children residing in urban low-income and minority communities of New York City, the asthma rate exceeds 25% and is among the highest in the nation (Perera, Miller et al., 2003, 2004, 2006).

We showed that in utero exposure to the common traffic-related air pollutants, polycyclic aromatic hydrocarbons (PAHs), is a risk factor for development of childhood asthma (Miller et al., 2004).

66

Study PopulationStudy Population

Monitor PAH exposure:Monitor PAH exposure: Personal prenatal air monitoring was conducted for two consecutive days during the 3rd trimester of pregnancy.

Asthma Status:Asthma Status: Based on parental report of a doctor’s diagnosis of asthma or probable asthma before the age of five, children were classified as “asthmatic” or “non-asthmatic.” (“with or without a parental report of asthma”).

Central HarlemCentral Harlem

Washington HeightsWashington Heights

South BronxSouth Bronx

NonsmokingNonsmoking Dominican andDominican and African African American women American women and their childrenand their children

Study SubjectsStudy Subjects

77

Columbia Center for ChildrenColumbia Center for Children’’s Environmental Health (CCCEH)s Environmental Health (CCCEH)

Study PopulationStudy Population

88

- Level and pattern of epigenetic marks is tissue-specific, cell-type specific and may vary over time.

- A wide range of samples, if feasible, have to be collected and the non-invasive sample collection is recommended.

--Sample SelectionSample Selection--

BiospecimensBiospecimens for for epigenetic studiesepigenetic studies

Amniocentesis Umbilical Urine/Stool/Blood/Amniocentesis Umbilical Urine/Stool/Blood/ BuccalBuccal cells/Salivacells/Salivacells cord bloodcells cord blood

99

Maternal and/or umbilical cord blood sample at delivery were collected. DNA (100ng-500ng) was extracted from cord blood white blood cells (UCWBCs).

UCWBC provide a reasonable surrogate for our target organ/tissue (lung/immune cells) because they contain stem cells that can populate in the lungs in later life and also provide a rich source of T cells which are important producers of cytokines andother asthma mediators.

The matched fetal placental tissues (FPTs) provides ample tissue for obtaining fetal RNA to verify correlative changes in gene expression.

Columbia Center for ChildrenColumbia Center for Children’’s Environmental Health (CCCEH)s Environmental Health (CCCEH)

--Sample SelectionSample Selection--

1010

--Study DesignStudy Design--

- DNA methylation is the addition of a methyl group derived from S- adenosyl-L-methionine to the fifth carbon of the cytosine ring to form the fifth base 5-methyl cytosine.

- It occurs predominantly in cytosines located at 5’ of guanines, known as CpG dinucleotides (CpGs). These CpGs cluster as CpG islands (CGIs) in 1-2% of the genome.

SS--adenosylmethioneadenosylmethione SS--adenosylhomocysteineadenosylhomocysteine

Normal

DNMTs

DNMTs

Gene silencingGene silencing

DNMTs

DNMTs

DNA Methylation

MeCPsMeCPs

TFTF

1111

-Few hundred ng levels of DNA are sufficient for DNA methylation studies

-Percent methylation within a population of alleles can be measured with

specific methylation site analyses or in a high-throughput manner.

-DNA methylation pattern is cell-type specific, gene specific and may change over time.

-Epigenetic regulatory regions have to be characterized and validated at all stages for all samples.

--Study DesignStudy Design--

1212

Workflow of DNA methylation studiesWorkflow of DNA methylation studies

Gene expressionGene expressionRealReal--Time PCRTime PCR

DNA DNA methylationmethylation statusstatus-- BisulfiteBisulfite SequencingSequencing-- MethylationMethylation--Specific PCR (MSPCR)Specific PCR (MSPCR)

Data confirmationData confirmation

Methylation profiling toolsMethylation profiling tools(ie. Methylation Sensitive Restriction Fingerprinting)

DNA extracted from gel, reDNA extracted from gel, re--amplified by amplified by PCR and PCR and subclonedsubcloned into vectorinto vector

Isolation of DNA from UCWBC samplesIsolation of DNA from UCWBC samples(*n=10; PAH below cohort median (2.3 ng/m3); n=10; PAH above cohort median)

Sequencing / BLAST / BLAT searchSequencing / BLAST / BLAT search

Promoter / CpG island searchPromoter / CpG island search

1313

Using MSRF, we have analyzed UCWBCs from an initial sample of 20 CCCEH cohort children*.

(*n=10; PAH below cohort median (2.3 ng/m3); n=10; PAH above cohort median)

DNA methylation profilingDNA methylation profiling--MSRFMSRF

1414

Data ConfirmationData Confirmation

Choose potential epigenetic marker(s) Choose potential epigenetic marker(s) and and

test on another 56 samples in the cohorttest on another 56 samples in the cohort

1515

-2000 -1500 -1000 -500 +1 +500 bp5’

TSS

CGI (1058bp)ACSL3A

-2000 -1500 -1000 -500 +1 +500 bp5’

TSS

DUSP22CGI (927bp)B

TSS

-2000 -1500 -1000 -500 +1 +500 bp5’

RAD21CGI 1

(704bp)CGI 2

(272bp)C

BSPCR

BSPCR

BSPCR

SCD5

-2000 -1500 -1000 -500 +1 +500 bp5’

TSS

CGI (1108bp)D

SFMBT2E

-2000 -1500 -1000 -500 +1 +500 bp5’

TSS

CGI 1(335bp)

CGI 2(567bp)

-2000 -1500 -1000 -500 +1 +500 bp5’

TSS

CGI (955bp)WWOX

FBSPCR

BSPCR

BSPCR

CGI 3(229bp)

Six MSRF candidates chosen for validation Six MSRF candidates chosen for validation

1616

Methylation status and gene expressionMethylation status and gene expression14 UCWBC DNA and their fetal placental tissues (FPT) RNA

Hypermethylated at high PAH Hypomethylatedat high PAH

RER: Relative Gene Expression Ratio; Overall Met%: Promoter methylation percent 1717

Evaluation of concordance between degree Evaluation of concordance between degree of CGI methylation in UCWBC and level of of CGI methylation in UCWBC and level of transcript expression in FPTtranscript expression in FPT

Statistical AnalysisStatistical Analysis

*The Kendall coefficient of concordance (tau) measures the strength of relationship between gene methylation and gene expression. Negative sign indicates inverse relationship.

14 UCWBC DNA and their fetal placental tissues (FPT) RNA

1818

It plays an essential role in fatty acid metabolism and is expressed in lung and thymic tissue.

Exposure to the PAH benzo(a)pyrene increased the expression of acyl-COA oxidase, catalase and glutathione peroxidase (Orbea et al., 2002).

Interestingly, ACSL3 is located in 2q36.1 which has recently been shown to be associated with regions of the asthma susceptibility loci, in specific populations (Bouzigon et al., 2007; Choudhry et al., 2008)

AcylAcyl--COA COA synthetasesynthetase long chain long chain family member 3 (ACSL3)family member 3 (ACSL3)

1919

Choosing ACSL3 as targetChoosing ACSL3 as target

2020

ACSL3 promoter ACSL3 promoter hypermethylatedhypermethylated at high PAHat high PAH(Assayed by Bisulfite Sequencing)

UnmetMet

R1 R3R2

Low PAHNo AsthmaOverall Met%:68.3±2.1

High PAHNo AsthmaOverall Met%:89.1±4.0*

ID# PAH755, 0.85

813, 1.71

826, 1.80

886, 1.72

1066, 1.22

70, 6.61

78, 2.43

104, 2.66

120, 4.87

946, 2.84

2121

ID: 755 813 826 886 1066

Low PAH group (No asthma) High PAH group (No asthma)

ACSL3M U M U M U M U M U

70 78 104 120 946M U M U M U M U M U

ID: 14 75 164 468 850 1 34 116 256 404

Low PAH group (Asthma) High PAH group (Asthma)

M U M U M U M U M U M U M U M U M U M U

ACSL3

ACSL3 promoter methylation, PAH and asthmaACSL3 promoter methylation, PAH and asthma

MethylationMethylation--Specific PCRSpecific PCR 2222

--Statistical AnalysisStatistical Analysis--

- Standard epidemiology analytic tools can be applied to examine the association between the outcome and epigenetic factors and the interaction between the environmental factors and epigenetic factors.

- Several important covariates and potential confounders have to be concerned. Statistics algorithms (eg. false discovery rate) should be applied to identify as many true association as possible and minimize the overall portion of false-positive.

2323

ACSL3 methylation is strongly associated with ACSL3 methylation is strongly associated with the levels of PAH exposurethe levels of PAH exposure

56 samples from the cohort

Odds ratio: 13.8 (95% CI’s:3.8, 50.2)

2424

ACSL3 methylation is associated with ACSL3 methylation is associated with asthma at age 5asthma at age 5

56 samples from the cohort56 samples from the cohort2525

TSSSp1 Sp1 AP2 AP2 GCF c-fos junB GCF

DMSO treatedOverall Met%=66.7±0.9

BaP-0.01nMOverall Met%=66.4±0.3

BaP-0.1nMOverall Met%=67.3±0.2

BaP-1nMOverall Met%=76.3±0.8*

UTR exon

Exposure of H1299 to Exposure of H1299 to BaPBaP increased increased methylation of ACSL3 promoter and lowered methylation of ACSL3 promoter and lowered

gene expressiongene expression

0

0.5

1

1.5

B0 B0.01 B0.1 B1

BaP

nM

RER * *

2626

-Animal models or cell line models can show how environmental influences can cause persistent changes in epigenetic gene regulation.

-They are used to discover the epigenetic signatures directly associated with various environmental factors.

PAH/Diesel exposurePAH/Diesel exposure Asthma phenotypesAsthma phenotypes

--Use of in vitro/in vivo modelUse of in vitro/in vivo model--

Epigenetic Epigenetic

ReprogrammingReprogramming

2727

Conclusions

Over 30 DNA sequences were identified Over 30 DNA sequences were identified whosewhose methylation status was methylation status was dependent on the level of maternal PAH exposuredependent on the level of maternal PAH exposure by using Methylation by using Methylation Sensitive Restriction Fingerprinting on 20 cord blood white blooSensitive Restriction Fingerprinting on 20 cord blood white blood cells samples. d cells samples.

Methylation of the Methylation of the ACSL3ACSL3 55’’--CGI was found to be significantly associated with CGI was found to be significantly associated with maternal airborne maternal airborne PAH when exposure exceeding 2.41 ng/m3 (OR=13.8; PAH when exposure exceeding 2.41 ng/m3 (OR=13.8; pp<0.001<0.001; sensitivity=75%; specificity=82%) and with a parental report o; sensitivity=75%; specificity=82%) and with a parental report of f asthma asthma symptoms in children prior to age 5 (OR=3.9; symptoms in children prior to age 5 (OR=3.9; pp<0.05).<0.05).

Thus, if validated, Thus, if validated, methylatedmethylated ACSL3 ACSL3 55’’CGI in UCWBC DNA may be CGI in UCWBC DNA may be a surrogate a surrogate endpoint for assessing endpoint for assessing transplacentaltransplacental PAH exposurePAH exposure and/or predicting and/or predicting childhood asthma risk. childhood asthma risk.

This proofThis proof--ofof--principle study provides a principle study provides a new blueprint for the discovery of new blueprint for the discovery of epigenetic biomarkersepigenetic biomarkers relevant to other exposure assessments and/or relevant to other exposure assessments and/or investigations of exposureinvestigations of exposure--disease relationships in birth cohortsdisease relationships in birth cohorts.. 2828

Future ProspectsFuture Prospects

Can the markers be validated in other independent Can the markers be validated in other independent cohort studies?cohort studies?Can we identify a specific Can we identify a specific signature for asthmasignature for asthma ??

Can the markers predict asthma caused by other risk Can the markers predict asthma caused by other risk factors, e.g. ozone, tobacco smoke, allergens?factors, e.g. ozone, tobacco smoke, allergens?

Characteristics of good biomarkers:Characteristics of good biomarkers:SensitiveSensitiveSpecificSpecificEarly stage indicatorEarly stage indicator

How do polymorphisms in asthmaHow do polymorphisms in asthma--related genes interact related genes interact with epigenetic mechanisms to confer higher with epigenetic mechanisms to confer higher susceptibility to environmental influences?susceptibility to environmental influences?

Use of HighUse of High--throughput platform for discovery:throughput platform for discovery: Methylation Microarray, Methylation Microarray,

ChIPChIP--sequencing, Transcriptome Profilingsequencing, Transcriptome Profiling

Use of HighUse of High--throughput platform for validation:throughput platform for validation: MassARRAYMassARRAY, , MethyLightMethyLight

2929

University of Cincinnati University of Cincinnati College of MedicineCollege of Medicine

ShukShuk--mei Ho, PhDmei Ho, PhDLinda Levin, PhDLinda Levin, PhDOther HoOther Ho’’s lab memberss lab members

AcknowledgementAcknowledgement

University of ColumbiaUniversity of Columbia(CCEH)(CCEH)

Frederica Perera, PhDFrederica Perera, PhDRachel Miller, MDRachel Miller, MDJulie HerbstmanJulie Herbstman, , PhDPhDDeliangDeliang Tang, PhDTang, PhD

Research supported byResearch supported by•• Department of Defense postdoctoral trainee award DAMD Department of Defense postdoctoral trainee award DAMD

W81XWHW81XWH--0606--11--0373 (W. Tang)0373 (W. Tang)•• NIEHSNIEHS--1K99ES168171K99ES16817--1 (W. Tang)1 (W. Tang)•• NIEHSNIEHS--ES015905 (F. Perera and S.M. Ho)ES015905 (F. Perera and S.M. Ho)•• NIEHSNIEHS--P30ES006096,R01ES015584 (S.M. Ho)P30ES006096,R01ES015584 (S.M. Ho)

3030