Genes, Environments and Human Development, Health and Disease (GEHDHD)

1. Genes, Environments and HumanDevelopment, Health and Disease (GEHDHD) • 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 and large sample critical GxE by Time issues can be evaluated • Geneticists can advance new areas by using NCS • Ideas generated from developing areas (complexity of genome, systems biology, epigenetics, mtDNA, evolutionary biology, and others now emerging) can be evaluated

2. Organization of GEHDHD Meeting • 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

3. 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) 2-Day GEHDHD Meeting:Participants and Topics

4. 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

5. 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

6. Class http://nationalchildrensstudy.gov/research/hypotheses/hypotheses_list.cfm Pregnancy Outcomes Birth defects from impaired glucose metabolism Increased risk of preterm birth from intrauterine exposure to mediators of inflammation Plus GxE Increased risk of birth defects and developmental disabilities in children born through ART Plus GxE Neuro-development & Behavior Non-persistent pesticides and poor neurobehavioral and cognitive skills Plus GxE Prenatal infection and neurodevelopmental disabilities Plus GxE Prenatal and perinatal infection and schizophrenia Plus GxE Injury Repeated head trauma and neurocognitive development Plus GxE Behavioral exposures, genetics, and early onset physical aggression Plus GxE Behavioral exposures and adolescent-onset physical aggression Plus GxE Plus GxE Asthma Impact of maternal stress during pregnancy and risk of asthma Plus GxE Environmental exposures and genetic variation interactions and asthma Plus GxE Infection expos early in life and increased risk of asthma Plus GxE Indoor, outdoor air pollution and asthma risk Plus GxE Dietary antioxidants and asthma risk Plus GxE Plus GxE Disparities in asthma and physical environmental risk factors, psychosocial stress, and HRBs Plus GxE Early exposure to bacterial and microbial product decreases risk of asthma Plus GxE Obesity & Growth Obesity and insulin resistance from impaired maternal glucose metabolism Plus GxE Obesity and insulin resistance from intrauterine growth restriction Plus GxE Breastfeeding associated with lower rates of obesity and lower risk of insulin resistance Plus GxE Fiber, whole grains, high glycemic index and obesity, insulin Plus GxE Impact of neighborhood environment on risk of obesity and insulin resistance Plus GxE Chemical environmental agents and the endocrine system and age at puberty Genetics Gene Environment Interactions and Behavior Plus GxE Genetics, environmental exposures, and Type I Diabetes Plus GxE Psychosocial Exposure Family influences on child health and development Plus GxE Impact of neighborhood and communities on child health Plus GxE Social institutions and child health and development Plus GxE Impact of media exposure on child health and development Impact of public policy and programs on child health

7. Use of molecules in blood to evaluate body & brain: 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

9. Dynamics of a Brain Perturbed Network:Prion Disease in Mice

10. Disease Arises from Disease Perturbed Networks 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. Non-Diseased Diseased

11. Epigenetic & mitochondrial genetic changeFeinberg, Meaney, Jirtle, Cheverud, Wallace • Epigenetics marks and mtDNA 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 genes and affects stress response (Meaney) • Conditions at conception (in ART), during pregnancy, and in infant development may affect epigenetic status (Feinberg) • 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 used to evaluate parent of origin effects (Cheverud) • Multiple measures over time should be obtained to document if & when change (methylation) or mutations (mtDNA) occur

12. 5' CpG region of NGFI-A/RE 40 High Low 30 Licking/grooming 1.0 C - methylation 0.8 20 0.6 CH CH CH 10 0.4 3 3 3 C T A C G T A C T C G G A A T C T C G 0.2 Days of Age 0 0.0 9-10 1-2 3-4 5-6 7-8 E20 1 6 21 90 Meaney: DNA methylation serves to imprint social factors, such as maternal behavior, upon the offspring’s genome DNA methylation serves as an interface between the dynamic environment and the fixed genome

13. Avy Yellow Slightly Mottled Mottled Heavily Mottled Pseudo- agouti IAP 3’ 5’ A, a 5‘ 3‘ ~15 Kb 1A PS1A 2 4.1 Kb ~100 Kb Control Diet Supplemented Diet 100 100 90 90 50 Pseudo-agouti 80% Control Diet 80 80 Supplemented Diet (Folic acid, Vitamin B12, Choline chloride and Betaine) p = 0.008 70 70 40 60 60 30 Heavily Mottled 50% 50 50 Avy Offspring (% of Total) Cells Methylated (%) 40 40 20 30 30 Mottled 26% 20 20 10 Slightly Mottled 13% 10 10 Yellow 7% 0 0 0 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 Yellow Slightly Mottled Mottled Heavily Mottled Pseudo- agouti CpG Site in AvyPSIA CpG Site in AvyPSIA Jirtle: Methyl Donor Supplementation Viable yellow Agouti (Avy) Locus Waterland et al. Mol. Cell Biol. 23: 5293-5300, 2003

14. NAP2 TSSC5 KVLQT1 TSSC4 ASCL2 H19 LIT1 p57 TSSC3 TSSC6 IGF2 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) 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%)

15. 100 100 50 50 Normal Mitochondrial disease Normal Central nervous system Heart / muscle Endocrine Kidney 0 0 Mitochondrial disease AGE AGE % OXPHOS capacity Damaged mtDNA Principles of Mitochondrial Genetics Maternal Inheritance Replicative segregation & heteroplasmy Threshold Expression (CNS, heart, etc.) High Mutation Rate (germ line & somatic)

16. Considerations for Design and AnalysisSing, Cox, Murray, Kramer, Wadhwa, Gillman, Lanphear • Genetic heterogeneity will be great • Genotypes with large effects are rare & with small effects are common • Genetic epidemiology and reciprocity • Environmental effects feedback & affect gene-environment interaction • Phenotype is defined over time as a trajectory • Developmental origins and fetal adaptive response initiates journey • Optimal cohort design includes relatives • Information on relatives allow for additional analyses of affected relatives & evaluation of parent of origin effect (maternal G & E) • Size of cohort depends on burden & resources • Large/thick & small/thin cohorts have been established to address different questions about change over time

17. DISEASED HEALTHY GENOTYPE (Conception) Potential to react DISEASED PHENOTYPE (Age 63) Developmental Origins & Fetal AdaptationWadhwa, Gillman, Lanphear, Sing, Cox, Templeton Bhargava, et al., NEJM 2004 “thrifty genotype & thrifty phenotype Barker Hypothesis Nested Case-Control Comparisons Early environment Later outcome Asthma & GSTMI ADHD & DAT Birth Weight & GSTTI

18. Genes, Environments and Human Development, Health and Disease:Issues and Challenges • Which genetic factors should be evaluated? • Which environments should be targeted? • Which developmental processes are most critical? • at what periods of time? • in which tissues?