Meeting on Genes, Environments & Human Development, Health & Disease (GEHDHD)

• Additional group of eminent scientists– Many eminent scientists already participated

and we wanted new expertise at this point to build upon what had already been established

• Topics defined by participants’ expertise– Presentations were on scientific topics rather

than about existing set of NSC hypotheses

• Discussion of basic concepts & development– Discussion encouraged focus on GxE

interactions with processes of development

2-Day GEHDHD Meeting:Participants and Topics

• Day 1: Hood (Systems Biology)Wadhwa (Fetal Adaptation)Sing (Analysis of Complexity)Cox (Genetic Epidemiology)Kramer (Cohort Design)Simhan (Fetal Medicine)Ayala (Evolutionary Biology)Wallace (Mitochondria)Feinberg (Epigenetics)Haig (Conflict in Evolution)Moyzis (Positive Selection)

• Day 2:Hartwell (Biotechnology)Gillman (Fetal Origins)Meaney (Maternal Nurturing)Jirtle (Imprinting)Cooper (Exercise)Murray (Clefting & Preterm Birth)Lanphear (Biomarkers)Posner (Attention & Development)Cheverud (Parent of Origin)Templeton (Ancestry)Procaccio (Positive Selection)

National Children’s Study SampleAll Births

in the Nation

Sample of Study Locations

Sample of Study Segments

Sample of Study Households

Sample of Study Women

105 Locations(counties)

Selection of neighborhoods

All or a sample of households within

neighborhoods

All eligible women in the households - ~1.5 million

~4 million births in 3,141

counties

Sample of Study Children

105,000 births

NCS Schedule of Contacts or Visits • 16 face-to-face

contacts over 21 year study period

• Contacts most frequent early in the study

• Between visits: ongoing data collection by phone, PDA, etc.

Enrollment 18 months

1st Trimester 3 years

2nd Trimester 5 years

3rd Trimester 7 years

Delivery 9 years

1 month 12 years

6 months 16 years

12 months 20 years

Theme 1: Technology and Biology

(Hood and Hartwell) • Assume & anticipate technological developments

– Efficient sequencing of each person’s genome will avoid reliance on current sets of markers (SNPs)

– Efficient measurement of all molecules in a blood sample will provide information on gene products from most organs of body

• Store & annotate repeated samples over time– Use nested case-control design and repeated samples to allow

efficient backtracking of changes from healthy to diseased state– Use change over time to reduce dimensionality of the data due to

individual differences in genotype and phenotype

• Use databases & multivariate analyses – Evaluate sets of genes in networks by using large databases of

accumulated information on gene pathways– Evaluate change over time in complex networks that may be

different across individuals

Dynamics of a Brain Perturbed Network:Prion Disease in Mice

Disease Arises from Disease Perturbed Networks

Non-Diseased Diseased

Hood and Hartwell emphasized the importance of within-individual change over time for the reduction of the dimensionality of data and characterization of the fundamental network properties that are similar across individuals but may be the result of different interactions and combinations of factors in different individuals.

Theme 2: Epigenetics & mtDNA(Feinberg, Meaney, Jirtle, Wallace)

• Epigenetics marks are not fixed for lifetime– Methylation & chromatin structure affect what DNA does by

imprinting and other mechanisms– Over the course of development, epigenetic change and mtDNA

change can occur and have large effects• Environmental effects are documented

– Maternal diet affects imprinting of agouti gene and appearance & size of Agouti mouse (Jirtle)

– Maternal-infant interaction affects imprinting of HP gene and affects stress response (Meaney)

– Conditions at conception (in ART), during pregnancy, and in infant development may affect epigenetic status (Feinberg)

– Mt germ mutations associated with diseases and accumulation of somatic mutations affects energy utilization (Wallace)

• Multiple measures over time should be taken– Maternal and paternal epigenotype and mtDNA genotype should

be establish for determination stabile forms– Multiple measures over time should be obtained to document if

and when change occurs

BWS is linked to an imprinted gene domain on 11p15

• LOI of LIT1 (40%) and IGF2 (15%), Paternal UPD (10%)• Mutation of p57 KIP2 (5%). Chromosomal rearrangement

(1%)

Beckwith-Wiedemann Syndrome (BWS) as a Model Disorder for Cancer EpigeneticsPrenatal overgrowth, Macrosomia, organomegaly, Pancreatic islet cell hyperplasia, Neonatal

hypoglycemia, Macroglossia, Abdominal wall defects, Dysmorphic, Embryonal tumors (Wilms)

IGF2

KVLQT1

p57

TSSC5 H19ASCL2

NAP2

TSSC3

TSSC4

TSSC6

LIT1

DNA methylation serves as an interface between the dynamic environment and the fixed genome

C -

meth

yla

tion 5' CpG region of NGFI-

A/RE

HighLow

Days of Age

Lic

kin

g/g

room

ing C T A C G T A C T C G G A A T C T C G

CH3CH3CH3

Meaney: DNA methylation serves to imprint social factors, such as maternal behavior, upon the offspring’s genome

Jirtle: Methyl Donor SupplementationJirtle: Methyl Donor SupplementationViable yellow Agouti (AViable yellow Agouti (Avyvy) ) LocusLocus

Waterland et al. Mol. Cell Biol. 23: 5293-5300, 2003

Control Diet

Pseudo-agouti80%

0 1 2 3 4 5 6 7

0

10

20

30

40

50

60

70

80

90

100

Cel

ls M

eth

yla

ted

(%

)

CpG Site in Avy PSIA

Heavily Mottled50%

Mottled26%

Slightly Mottled13%

Yellow7%

100

Supplemented Diet

0 1 2 3 4 5 6 7

0

10

20

30

40

50

60

70

80

90

CpG Site in Avy PSIA

Pseudo-agouti

HeavilyMottled

MottledSlightlyMottled

Yellow

50

40

30

20

10

0

Avy

Off

sp

rin

g (

% o

f T

ota

l) Control Diet

Supplemented Diet(Folic acid, Vitamin B12, Choline chloride and Betaine) p = 0.008

Pseudo-agouti

HeavilyMottled

MottledSlightlyMottled

Yellow

3’

4.1 Kb

5‘

1A PS1A 2~100 Kb

Avy

A, a

IAP 5’

~15 Kb3‘

Threshold Expression (CNS, heart, etc.)

High Mutation Rate (germ line & somatic)

100

50

0

Kidney

Endocrine

Heart / muscle

Central nervous system

Normal

Damaged mtDNA% OXPHOS capacity

Normal

Mitochondrialdisease

AGEAGE

100

50

0

Mitochondrialdisease

Principles of Mitochondrial GeneticsMaternal Inheritance

Replicative segregation & heteroplasmy

Theme 3: Maternal-Fetal Environment(Gillman, Wadhwa, Simhan, Cheverud)

• Developmental origins of health and disease– Do events during early development affect health

outcomes over the lifetime• Fetal adaptive response and environment

– Stress during fetal development shapes structure and function that set the stage for long-lasting effects

• Fetal environment and timing of events– Multiple environments play important but different roles in

fetal development and that depend on time in development• Parent of origin effects

– Imprinting effects could be consequence of maternal gene inherited by offspring or maternal environment or both

Developmental Origins of Health and Disease: Gillman

0

0.5

1

1.5

2

2.5

<5.0 5.0-5.5 5.6-7.0 7.1-8.5 8.6-10.0 >10.0

Birthweight (lb)

Rel

ativ

e ri

sk

CHDStroke HypertensionType 2 Diabetes

Bhargava, et al., NEJM 2004 “thrifty genotype & thrifty phenotype

Barker Hypothesis

What is the fetal environment? The importance of perspective

Fetus proper

Maternal milieu

External forces

placenta

Imp 11 12,21 22

11 ---- ---- ----

21 i1 i1 ----

12 ---- i2 i2

22 ---- ---- ----

Dir 11 12,21 22

11 ao ao ----

21 do do ----

12 ---- do do

22 ---- -ao -ao

Mat 11 12,21 22

11 am dm ----

21 am dm ----

12 ---- dm -am

22 ---- dm -am

D M I

21 do am+dm i1

12 do -am+dm i2

D M I

21 do dm i1

12 do dm i2

Mom

Off

Off

Off

Off

Off

All Mothers

Heterozygous Mothers

Imprinting and Maternal Effects: Cheverud

BEST MOTHER POOR MOTHER

Theme 4: Evolutionary Biology(Ayala, Moyzis, Haig, Procaccio, Templeton) • Positive Selection and sets of gene

– Common variants may be at high frequencies in the genome due to positive or balancing selection

• Evolutionary Conflict– Paternally-derived alleles associated with growth enhancers and

maternally-derived alleles with growth inhibitors are in conflict

• Ancestry and Thrifty Genes– Genes selected in ancient environments have effects in current

environments with abundant food and low exercise demands

• Geographical Variation in mtDNA– Neutral, deleterious & advantageous mutations contribute to

signs of drift, purifying and adaptive selection in mtDNA

• Evolutionary Biology and Social Behavior– Principles of evolutionary biology can help understanding

differences in populations and development in individuals

DA ReceptorDA Receptor D4D4 (DRD4) (DRD4)Ding et al, 2002 Wang et al, 2004

Resequencing71 SNPs found in DRD4 gene

Individuals binned on basis of VNTR homozygosity;4R allele: low LD7R allele: high LD

Genome Wide Application Wang et al, 2005

Perlegen 1.6m SNPsHapMap 3.9m SNPs

Sliding 1Mb window for Linkage Disequilib. Decay (LDD) test;Set of 1800 genes identified with sign (high LDD) of positive selection

Signs of Positive Selection: Moyzis

Extensive mtDNA sequence variation between populations

ND2

12srRNA

16srRNA

V

L

F

ND1

I QM

W

A NCY

COI

COII

COIIIS

D K

AT

Pase8

ATPase6

ND4L

ND4

G

RND3

HS

L

ND5

ND6E

Cyt bP

T

D-Loop

Africa L

0

America D

Europe H

Europe J

Europe Uk

Europe T

Europe J1 & Uk

Haplogroup: group of mtDNAs defined by unique set of variants acquired from the same ancient common female ancestor

Adaptive Selection and mtDNA: Procaccio

Jasienska, G., I. Thune, and P. T. Ellison. 2006. Fatness at birth predicts adult susceptibility to ovarian suppression: An empirical test of the Predictive Adaptive Response hypothesis. PNAS 103:12759-12762.

Gene-Environment Interaction: Thrifty GenesTempleton

Theme 5: Design and Analysis (Sing, Cox, Murray, Kramer)

• Genetic heterogeneity will be great– Genotypes with large effects are rare and with small

effects are common• Genetic epidemiology and reciprocity

– Environmental effects feedback and affect gene-environment interaction

• Optimal cohort design includes relatives– Information on relatives allow for additional analyses of

affected relatives and maternal effects• Size of cohort depends on burden & resources

– Large/thin & small/thick designs balance these factors to address different questions

GENOTYPE(Initial Condition)

Two Possible Environmental Histories in theTime-Space Continuum Encountered by a Genotype

HEALTHY

DISEASED

Potential toreact

Potential toreact

DISEASED

Age 63

0

0.1

0.2

0.3

10 20 30 40 50 60 70 80

0

0.1

0.2

0.3

10 20 30 40 50 60 70 80

Females

Males

Context Dependent ApoE EffectsRochester, MN

Age window midpoint (years)

Pro

po

rtio

n o

f p

lasm

a A

po

Eva

rian

ce a

ttri

bu

tab

le t

o A

po

E g

ene

h2B

(An/The) Optimal Study DesignAllows for case control, affected family member,

assessment of maternal effects and others

Cohort Study Attributes and Two Contrasting Approaches: Kramer

Study Attribute Large/Thin Small/Thick

Type of Exp measurement simple, inexpensive complex, expensive

Freq of Exp measurement infrequent frequent

Type of outcome simple, inexpensive complex, expensive

Sample size >100,000 <30,000

Cost per subject low high

Precision/Power high low

Exp/Outcome misclassification high low

Theme 6: Infant & Child Development(Cooper, Lanphear, Posner)

• Exercise and growth– Physical activity has effects on inflammation and growth

factors that affect health and development

The new morbidities and low exposures– Low levels of toxic substances such as lead and smoke

may be etiologic factors in ADHD & LD

• Development of brain networks and attention– Development of executive control of emotion & cognition

depends on environmental and genetic factors

ExerciseGrowth Hormone

IGF-I

Stimulates local tissues (?IGF-I) directly

Target Tissues

? GH binding

proteins

? IGF binding proteins

? BP Proteolysis

? mechanisms

Physical Physical InactivityInactivity

++Diet high in fat Diet high in fat

and caloriesand calories++

Maturational Maturational StatusStatus

++Environmental Environmental

FactorsFactors++

Genetic FactorsGenetic Factors==

Obesity, Obesity, Metabolic Metabolic Syndrome, Syndrome,

Asthma Asthma Adult CVD, Adult CVD,

CancerCancer

Exercise and Inactivity: Cooper

Gene-Environment Interactions for Prenatal Tobacco Exposure

Lanphear BP, Bearer CF. ADC 2005;90:594-600.

Group Recognized Strengths of NCS

• Representative & large sample – Provides power to assess GxE & GxG interactions in complex disorders

• Prospective assessment starting early – Assess adaptation of structures that have long-lasting effects

• Repeated observations over time– Phenotypes defined by trajectories over time and individual as control

• Reciprocal relationship based on feedback– Observe environmental effects that then affect environment

• Comprehensive exposure measures in all participants– In separate cohorts chance to evaluate interactions is lost

• Comprehensive outcome measures in all participants– Assess risks, precursors, & origins of later disease and GxE interaction

Group Recognized Opportunities

• Genetics should be part of every hypothesis– In example 29 only two were “genetic” but all are “GxE”

• Great repository of stored samples for future use– GxE & GxG models using appropriate complex assumptions– Considering genetics in two entities & genomes that interact– Adding genetics to developmental origin hypothesis– Providing evolutionary context for gene sequence– Set of genes based on concepts as well as pathways

• Opportunity to test “proof of principle” studies– Use of molecules in blood to evaluate body & brain– Epigenetic & mitochondrial genetic change over time

Conclusions of GEHDHD Meeting

• Geneticists are interested in the NCS– We invited 25 scientists & almost all decided to

participate

• Geneticists are excited by potential of NCS– With prospective, repeated measures from before

birth & large sample critical GxE by Time issues can be evaluated

• Geneticists can advance areas by using NCS – Ideas generated from developing areas

(complexity of genome, systems biology, epigenetics, mtDNA, evolutionary biology & others now emerging) can be evaluated