The NIEHS Environmental Genome Project: The NIEHS Environmental Genome Project: Enabling Studies of Gene-Environment Enabling Studies of Gene-Environment

InteractionInteraction

Douglas A. Bell, Ph.D.Douglas A. Bell, Ph.D.

Environmental Genomics SectionEnvironmental Genomics Section

National Institute of Environmental Health National Institute of Environmental Health SciencesSciences

Professor, Dept of EpidemiologyProfessor, Dept of Epidemiology

UNC School of Public HealthUNC School of Public Health

NIEHS’s Environmental Genome ProjectNIEHS’s Environmental Genome Project

Resequencing of ~500 Candidate Genes Resequencing of ~500 Candidate Genes Potentially Involved in Environmental DiseasePotentially Involved in Environmental Disease

Concept and rationaleConcept and rationale Examples of gene-environment interactionExamples of gene-environment interaction Resequencing studies, accomplishments, and Resequencing studies, accomplishments, and

accessing data.accessing data.

Modulation of Response to Modulation of Response to ExposureExposure

ExposureExposure Early Early EffectsEffects

Genetic SusceptibilityGenetic Susceptibility

DiseaseDisease

Genetic Modulation of Exposure, Damage, and Biological

Response

DiseaseDisease

Genetic Variation in: Genetic Variation in:

• Metabolism, or distribution, affects dose to the tissueMetabolism, or distribution, affects dose to the tissue• Detection and repair of damageDetection and repair of damage• Differences in growth and recovery from damageDifferences in growth and recovery from damage

Exposure Exposure TargetTargettissuetissue

Biological Response

Genetic Modulation of Exposure Risk Genetic Modulation of Exposure Risk

Exposure

No No ExposureExposure

4-Fold Risk

2-Fold Risk

BackgroundRisk Level

(low)Sensitive

Genotype

Sensitive

Genotype

Resistant

Genotype

Resistant

Genotype

PAH-oxide

GST + Glutathione

Inactive

Benzo[a]pyrene Metabolism Benzo[a]pyrene Metabolism Glutathione

HO

HO

CYP450

DNA ReactiveDNA Reactive

PAH-oxide

GST + Glutathione

Inactive

DNA ReactiveDNA Reactive

Benzo[a]pyrene Metabolism Benzo[a]pyrene Metabolism Glutathione

HO

HO

CYP450

GSTM1 GSTM1 NullNull

Bladder Cancer Risk Associated with Bladder Cancer Risk Associated with Smoking and GSTM1 Null GenotypeSmoking and GSTM1 Null Genotype

*P<0.001; Bell et al, JNCI 85:1559,1993

Nonsmokers Nonsmokers

1- 50 Packyears 1- 50 Packyears SmokingSmoking

>50 Packyears>50 PackyearsSmokingSmoking

1 1.31 1.3

2.2* 4.3*2.2* 4.3*

3.5* 5.9*3.5* 5.9*

GSTM1 GSTM1 (+)(+)

GSTM1 GSTM1 nullnull

Exposure R

iskGenetic Risk

Examples of Gene-Environment Interaction Examples of Gene-Environment Interaction (gene modifies environmental effect)(gene modifies environmental effect)

Malaria and Sickle Cell gene.Malaria and Sickle Cell gene. HIV infection and CCR5 receptor variant.HIV infection and CCR5 receptor variant. LPS sensitivity and Toll Receptor (TLR4) LPS sensitivity and Toll Receptor (TLR4) Adverse drug response and CYP2D6 poor Adverse drug response and CYP2D6 poor

metabolism.metabolism. Alcohol intolerance and aldehyde dehydrogenase.Alcohol intolerance and aldehyde dehydrogenase. Smoking, GSTM1 null, NAT2 slow genotypes, and Smoking, GSTM1 null, NAT2 slow genotypes, and

bladder cancer risk .bladder cancer risk .

Variation in Risk Estimates in Human Variation in Risk Estimates in Human PopulationsPopulations

Phenotypic variation Phenotypic variation in response due to: in response due to:

PhysiologyPhysiology

MetabolismMetabolism

RepairRepair

GrowthGrowth

Timing of ExposureTiming of Exposure

RiskRisk

ExposureExposure

Example: Metabolism PolymorphismsExample: Metabolism Polymorphisms

freq

uen

cy

Activity

No Phenotypic No Phenotypic PolymorphismPolymorphism

Range of Enzyme Activity in Range of Enzyme Activity in Human PopulationsHuman Populations

Distribution of Polymorphic Enzyme Activity Distribution of Polymorphic Enzyme Activity in a Populationin a Population

freq

uen

cyfr

equ

ency

ActivityActivity ActivityActivity

HighHighLowLowHighHighLowLow

+/++/++/++/+

+/-+/-+/-+/--/--/- -/--/-

Examples: N-Acetyltransferase 2, GSTM1, CYP2D6Examples: N-Acetyltransferase 2, GSTM1, CYP2D6

How does frequency of a risk How does frequency of a risk factor impact exposure induced factor impact exposure induced (G x E) risk in the population?(G x E) risk in the population?

freq

uen

cyfr

equ

ency

ActivityActivity

5% 5% 95% 95%

Effects of Exposure in High and Low Risk Effects of Exposure in High and Low Risk Human PopulationsHuman Populations

RiskRisk

ExposureExposurefr

eq

ue

nc

yfr

eq

ue

nc

y

ActivityActivity

5% 5% 95% 95%

100

10

0

High Risk

Low RiskLow Risk

Average

How will genetic data be used in How will genetic data be used in public health risk assessment?public health risk assessment?

Given detailed information on the Given detailed information on the relationship between genotype and relationship between genotype and phenotype, more accurate risk phenotype, more accurate risk assessments may be possible. assessments may be possible.

Risk Management

MoreMore/Less /Less ControlControlHuman

Genetic Susceptibility

ExposureAssessment

Engineering design

Risk Assessment ProcessRisk Assessment Process

Animal toxicology (dose/response)

Risk Model(Extrapolation to

humans)

SS

RR

Effects in Humans ?

Replace default Replace default assumptions assumptions

about variabilityabout variability

Hazard/Risk Assessment

Chemical Chemical XX

Cancer - Yes/NoCancer - Yes/No

Dose ?Dose ?

Extrapolate to Extrapolate to HumansHumans

Susceptible Susceptible human human

subgroup?subgroup?

• BiochemistryBiochemistry

• Mechanism of toxicityMechanism of toxicity

• Genes, pathwaysGenes, pathways

• Human geneticsHuman genetics

Incorporating Human Genetic Polymorphism Incorporating Human Genetic Polymorphism Information Into Risk Assessment Information Into Risk Assessment

Incorporating Genetics Into Risk Incorporating Genetics Into Risk Assessment: IssuesAssessment: Issues

A polymorphism may have different effects A polymorphism may have different effects depending on the chemical, the target organ/ depending on the chemical, the target organ/ disease, and the population being considered. disease, and the population being considered.

Thus, a protective allele for one chemical may Thus, a protective allele for one chemical may

convey risk for a different chemical. Similarly convey risk for a different chemical. Similarly one organ system may be protected at the risk of one organ system may be protected at the risk of another; e.g. immune system response could another; e.g. immune system response could increase DNA damage or neurotoxicity. increase DNA damage or neurotoxicity.

D.A.Bell NIEHS

Ethylene oxide

HCHO

DetoxicationDetoxication

GSTT1 + Glutathione

ActivationActivation

Methylene chloride GSTT1

+ Glutathione

DNA

DNA

GST Theta 1 (GSTT1) - One gene with 2 effects

Glutathione

H2C

HO

CH2

Cl

Glutathione

Cl- CH2

+

(Unstable)

DNA DNA

ReactiveReactive

Inactive Inactive

(also Methyl chloride)

Activation vs. Detoxication Activation vs. Detoxication

Effects of polymorphism dependent on Effects of polymorphism dependent on chemical and toxicity pathway:chemical and toxicity pathway:

ActivationActivation - If the activation pathway is missing (null - If the activation pathway is missing (null genotypes), some individuals may have zero risk genotypes), some individuals may have zero risk even if they have exposure. even if they have exposure.

DetoxicationDetoxication - Since this process will never be - Since this process will never be 100% efficient, both functional and low activity 100% efficient, both functional and low activity genotypes will exhibit risk associated with exposure.genotypes will exhibit risk associated with exposure.

The Effect of GSTT1 Genotype on Metabolism of Methyl Chloride

From Lof, A. et al, Pharmacogenetics 10:645, 2000.

T1 + T1 + Metabolism to Metabolism to DNA reactive DNA reactive formsforms

T1 Null No T1 Null No MetabolismMetabolism

Measure Measure exhaled exhaled methyl methyl chloride chloride

D.A.Bell NIEHS

Smoking, GSTT1 Polymorphism, and Markers Smoking, GSTT1 Polymorphism, and Markers of Genotoxicity in Erythrocytes of Genotoxicity in Erythrocytes

Background: Background: Ethylene oxide –hemoglobin adducts are a Ethylene oxide –hemoglobin adducts are a good measure of smoking exposure in blood.good measure of smoking exposure in blood.

Experiment:Experiment: To test if GST genotypes modulated effects To test if GST genotypes modulated effects of smoking in erythrocytes, we measured ethylene oxide of smoking in erythrocytes, we measured ethylene oxide hemoglobin adducts in freshly collected human hemoglobin adducts in freshly collected human erythrocytes from nonsmokers and smokers.erythrocytes from nonsmokers and smokers.

Results:Results: Ethylene oxide adducts (HEV) were ~50% higher in Ethylene oxide adducts (HEV) were ~50% higher in

GSTT1 null individuals.GSTT1 null individuals.

GSTT1GSTT1 null genotypes have null genotypes have higherhigher levels of levels of smoking-induced hemoglobin adductssmoking-induced hemoglobin adducts

Effect of GSTT1 null Genotype:Ethylene Oxide-Hemoglobin Adducts Vs

Cotinine

0

100

200

300

400

500

600

700

800

0 200 400 600

Plasma Cotinine (ng/ml)

HE

Va

l Ad

du

cts

(fm

ol)

Series1

Series2

Linear (Series1)

Linear (Series2)

GSTT1 null

GSTT1 +

GST T1 Null

GST T1 +

Study Design: Study Design: 16 nonsmokers16 nonsmokers32 smokers 32 smokers

HEVal hemoglobin HEVal hemoglobin adducts measure by adducts measure by mass spectrometrymass spectrometry

P = 0.001 for difference P = 0.001 for difference in slopes;in slopes;Nonparametric analysisNonparametric analysis similar.similar.

Fennel et al CEBP 9:705,2000Fennel et al CEBP 9:705,2000

Incorporating Genetics Into Risk Assessment Incorporating Genetics Into Risk Assessment Needs: Needs: Identify genes involved in toxicological response.Identify genes involved in toxicological response. Detailed population genetic information including:Detailed population genetic information including:

Identify polymorphisms.Identify polymorphisms. Determine frequency in populations.Determine frequency in populations. Population-based risk estimates in large studies (n=2000).Population-based risk estimates in large studies (n=2000).

Determine functional relationship between genotype and Determine functional relationship between genotype and phenotypephenotype BiochemicalBiochemical In vitro, in vivo quantitative measurements of a cellular phenotype In vitro, in vivo quantitative measurements of a cellular phenotype

(tumors, adducts, mutation, cell death, gene expression).(tumors, adducts, mutation, cell death, gene expression).

Consider role of multiple genes, multiple pathways, etc.Consider role of multiple genes, multiple pathways, etc. Incorporate kinetic or other functional data into risk model.Incorporate kinetic or other functional data into risk model.

Environmental GenomicsEnvironmental Genomics

Discovery:Discovery:Phenotype-directedPhenotype-directedGenotype-directedGenotype-directed

FunctionalFunctionalAnalysisAnalysis

Disease Risk Disease Risk CharacterizationCharacterization

PhenotypePhenotypeGenotypeGenotype

CTTATGT A/C GGGTAT

Altered Binding

Effects in Populations

Polymorphism and FunctionPolymorphism and Function

Gene Deletions, DuplicationsGene Deletions, Duplications

Coding region changes:Coding region changes:aa subs, deletions, stops.aa subs, deletions, stops.

Transcription Transcription FactorsFactors

Effects of Polymorphism:

Altered function

Quantity of protein

Regulatory polymorphisms alter Regulatory polymorphisms alter transcription factor binding and transcription factor binding and mRNA/protein level.mRNA/protein level.

Exon 1 Exon 2Promoter 3’ UTR

e.g. GSTM1, CYP2D6e.g. GSTM1, CYP2D6

C TGGGCCCCGCCCCCTTATGTAGGGTATAAAGCCC …. CCCGTCACC ATG   SP1/Oct

Phenotype—Directed Approach to Find SNPs Phenotype—Directed Approach to Find SNPs That Alter Gene Expression LevelThat Alter Gene Expression Level

Liu, X. et al Liu, X. et al

Sequence-Directed Approaches to Sequence-Directed Approaches to Catalogue All Significant SNPs In The Catalogue All Significant SNPs In The Human PopulationHuman Population

Resequencing Projects: Describing Resequencing Projects: Describing candidate gene polymorphisms in diverse candidate gene polymorphisms in diverse populations.populations.

~9 million SNPs in dbSNP now,~9 million SNPs in dbSNP now, by 2006, expect ~20 million human SNPs.by 2006, expect ~20 million human SNPs.

A SNP every ~100 bases.

Haplotype Map: Describing which SNPs Haplotype Map: Describing which SNPs occur together on chromosomes in occur together on chromosomes in populations (haplotypes).populations (haplotypes).

SNP Discovery ProjectsSNP Discovery Projects

The SNP Consortium – ~1 million SNPs The SNP Consortium – ~1 million SNPs across genome across genome

NIEHS – Environmental/toxicology NIEHS – Environmental/toxicology genesgenes

NHLBI – Heart disease genes, NHLBI – Heart disease genes, inflammation inflammation

NIGMS – Pharmacogenetic genesNIGMS – Pharmacogenetic genes

SNP data is entered into the NCBI dbSNP database SNP data is entered into the NCBI dbSNP database

HapmapHapmap

UCSCUCSC

U Wash EGP U Wash EGP WebsiteWebsite

Characterize the large scale genetic Characterize the large scale genetic structure across the genome.structure across the genome.

Genotyping SNPs at 1 kb interval across Genotyping SNPs at 1 kb interval across the genome in European, African, and the genome in European, African, and Asian populations.Asian populations.

HapMap WebsiteHapMap Website

HapMap WebsiteHapMap Website Seattle SNPs or EGP websiteSeattle SNPs or EGP website Many other freely available programsMany other freely available programs

Bioinformatic Tools Available For Bioinformatic Tools Available For Picking Haplotype Tagging SNPs Picking Haplotype Tagging SNPs

NIEHS Environmental Genome NIEHS Environmental Genome ProjectProject

Resequencing of candidate Resequencing of candidate environmental disease genesenvironmental disease genes

Accomplishments:Accomplishments: Total genes sequenced = 437Total genes sequenced = 437 Total kilobases sequenced = 11,001 kbTotal kilobases sequenced = 11,001 kb Total SNPs identified = 59,475 Total SNPs identified = 59,475

NIEHS’s Environmental Genome ProjectNIEHS’s Environmental Genome ProjectSummary: Summary:

Gene-environment interaction affects disease risk.Gene-environment interaction affects disease risk. Effects of G x E interactions can be complex.Effects of G x E interactions can be complex. Resequencing projects are providing many new Resequencing projects are providing many new

candidate gene polymorphism.candidate gene polymorphism. Determining the important functional SNPs that affect Determining the important functional SNPs that affect

disease risk is a difficult challenge.disease risk is a difficult challenge.

Strategies For Incorporating SNPs Strategies For Incorporating SNPs Into Epidemiology StudiesInto Epidemiology Studies

1.1. Whole genome association studies Whole genome association studies

Test 10,000-100,000 SNPs in case control studies.Test 10,000-100,000 SNPs in case control studies. Identify candidate regions, genes, followup with candidate Identify candidate regions, genes, followup with candidate

gene studies.gene studies.

2. High resolution candidate gene studies.2. High resolution candidate gene studies. Test functional SNPs and additional haplotype tagging SNPs in Test functional SNPs and additional haplotype tagging SNPs in

case/control or other design.case/control or other design. Bioinformatics to identify 1500 SNPs, 150 genes (10 SNPs/gene). Coding SNPs, regulatory SNPs, haplotype tag SNPs.

Application to p53 response elementsApplication to p53 response elements Application to NRF2 response Application to NRF2 response

elementselements

Bioinformatic Identification of SNPs Bioinformatic Identification of SNPs That Affect Gene ExpressionThat Affect Gene Expression

p53p53

p53 inducible genes contain p53 p53 inducible genes contain p53 RResponse esponse EElements.lements.

RRRCWWGYYY RRRCWWGYYY

RNA PolRNA Pol

Using bioinformatic methods, identify SNPs that Using bioinformatic methods, identify SNPs that

disrupt p53 response elements.disrupt p53 response elements.

SEI1SEI1mRNA mRNA

ATGATG

p53p53 p53p53 p53p53

SEI1 gene SEI1 gene

Following UV exposureFollowing UV exposurep53 binds RE of target gene.p53 binds RE of target gene.

RRRCWWAYYY

Test SNPs Against p53 Response Element Consensus

Filter:Filter:Best HitsBest Hits

Access Access databasedatabase

Build Table of Build Table of All Promoter SNPsAll Promoter SNPs

RRRCWWGYYYRRRCWWGYYYAAAGGACAAGTTGAAACTTGCACAAGCAGCCTCCATTCTG

DNA ambiguity codeDNA ambiguity code

R = A or GR = A or G

Y = C or TY = C or T

W = A or TW = A or T

dbSNP dbSNP DataData

Binding SiteBinding SiteConsensusConsensus

NCBI/EnsembleNCBI/EnsembleGenome DataGenome Data

Dan TomsoDan Tomso

Mismatch Mismatch with with

consensusconsensusCCWWWWGG

motifmotif

Dan TomsoDan Tomso

Do SNPs in putative p53 response elements Do SNPs in putative p53 response elements affect p53 induced expression in Saos2 affect p53 induced expression in Saos2 cells? cells?

0

5

10

15

20

25

p21-5' ADARB1 DCC ARHGEF7 RRM1 TLR8 EOMES SEI-1 SCGB1D2

REL

ATI

VE IN

DU

CTI

ON

Strong

Weak

WeakWeak

StronStrongg

Mike Resnick, Alberto Inga, Daniel Menendez

Saos2 Osteosarcoma Cells (p53 null)Saos2 Osteosarcoma Cells (p53 null)

Environmental Genomics Environmental Genomics SectionSection

Douglas A. BellDouglas A. BellGary S. PittmanGary S. Pittman

Merrill ‘Chip’ Miller, IIIMerrill ‘Chip’ Miller, IIIDaniel J. TomsoDaniel J. Tomso

Michelle R. CampbellMichelle R. CampbellXuemei LiuXuemei Liu

Xuting WangXuting WangMonica HorvathMonica Horvath

~4000 Human ~4000 Human ARE containing ARE containing

genesgenes

Phylogenetic Footprinting of NRF2/ARE GenesPhylogenetic Footprinting of NRF2/ARE Genes

~2100 Rat ARE ~2100 Rat ARE containing genes containing genes

~4000 Mouse ~4000 Mouse ARE containing ARE containing

genesgenes

Human/ Human/ mouse/ratmouse/rat

~380 ~380

1000 1000 human/mouse human/mouse

Gene x Environment Interaction Gene x Environment Interaction

Pharmacogenetics: Pharmacogenetics: Adverse drug reactions (toxicity)Adverse drug reactions (toxicity) Reduced efficacy Reduced efficacy

Environmental diseaseEnvironmental disease Modification of exposure-induced toxicityModification of exposure-induced toxicity Modification of exposure-induced diseaseModification of exposure-induced disease

Can we generalize about risk associated with a Can we generalize about risk associated with a specific gene?specific gene?