This presentation will: - Review the concept of ‘fetal programming’ - Demonstrate that early life nutritional events may serve as molecular memory of individual in utero experiences - Show how changes persist following multiple rounds of cell division - Highlight extrinsic (recapitulation) & Intrinsic (genetic) mechanisms that strongly suggest Intergenerational transmission of traits via epigenetics in humans - Look at how to best move forward as a scientific and clinical community

1. Zach Ferraro, PhD, CEP
CIHR Postdoctoral Fellow
Chronic Disease Program, Ottawa Hospital Research Institute (OHRI)
Clinical Research Associate, Division of Maternal-Fetal Medicine, The Ottawa Hospital
CON Obesity Summit 2015
Toronto, ON
April 29th, 2015
website: www.DrFerraro.ca
twitter: @DrFerraro
email: zach.ferraro@gmail.com
Developmental Origins of Obesity:
Inheritance or recapitulation?

2. Objectives
 Review the concept of ‘fetal programming’
 Early life nutritional events may serve as molecular memory of
individual in utero experiences
 Following multiple rounds of cell division
 Highlight extrinsic (recapitulation) & Intrinsic (genetic) mechanisms
 Intergenerational epigenetics in humans
 Moving forward

3. Birthweight & Metabolic Syndrome Risk
Dutch ‘hunger winter’ 1945
Pettitt DJ. Curr Diab Rep 2001; 1: 78–81; Von Hagens, 2005
Body Worlds; Ong KK. Horm Res 2006; 65: 65–69.
SGA & LGA neonates
Cardiometabolic Disease

4. Developmental Origins of NCDs
 Nutrition
 Weight gain
 Smoking
 Stress
 Toxins
 Physical activity
 Aging

5. Gluckman, PD et al. Nat. Rev. Endocrinol. 5, 401-408 (2009).
Environmental sensitivity of
epigenome throughout life
PA? PA? PA? PA?
 Plasticity has high energetic cost & is limited to early development
 Reengineering tissue/body after phenotype developed is costly

6. DEVELOPMENTAL PLASTICITY
AND CHRONIC DISEASE RISK
Gluckman et al. NEJM 2008 Jul 3;359(1):61-73.
M Desai et al. International Journal of Obesity (2015) 633 – 641
transmission
recapitulation

7. Epigenetics
 “interactions of genes with their
environment which bring the phenotype
into being” - Conrad Waddington
 Mitotic or meiotic heritable alterations in
gene expression potential that occur
without alterations in DNA sequence
Ozanne, S. E. Nat. Rev. Endocrinol. 11, 67–68 (2015)
Waddington, C. H. Organizers and Genes (Cambridge U Press, 1940)
M Desai et al. International Journal of Obesity (2015) 633 – 641

8. Regulatory mechanisms
 DNA methylation: CH3-attached to CpG islands regulate gene activity. Renders
the DNA inaccessible and suppresses gene expression
 Histone (covalent) modifications: methylation (Me) or acetylation (Ac) of
histones determines the activity of the DNA wrapped around them
 microRNA (miRNA): noncoding (19-22 nucleotides) molecules that silence RNA
& post-transcriptional regulation of gene expression, bind to complementary
sequences in the 3′ end of mRNA and reduce the rate of protein synthesis
Gluckman et al. NEJM 2008 Jul 3;359(1):61-73.

9. DNA Methylation
 Dynamic in embryogenesis
 Pre-implantation, DNA hypo-Me, > DNA-Me over time
 Differentiation & organogensis
 Mediated by DNMT
 Silenced expression
 Role of in utero nutrition/CH3 donors
Clarke HJ. Biochem Cell Biol 1992; 70:
856–866; Weaver JR et al. Mamm
Genome 2009; 20: 532–543; Desai et al.
IJO (2015) 633-41; sciblogs.co.nz;
www.discoverymedicine.com

10. Transgenerational mechanisms
Inheritance
 Transmitted through
genes that are
passed from parents
to children
 The reception of
genetic qualities by
transmission from
parent to offspring
Recapitulation
 The repetition of an evolutionary or
other process during development
 Re-occurrence in an individual
organism's development (phenotype)
resembling the series of ancestral
types from which it descended so
offspring retraces the phylogeny of
its group
 Largely environmental
 Nutrition
 Smoking/Alcohol/Drugs
 Physical activity
 Stress
 Exposure to endocrine disruptors
 Etc.
R. Waterland (personal communication); Waterland, Annu. Rev. Nutr. 2014;34:337–55;
http://ghr.nlm.nih.gov/glossary=geneticinheritance

11. Maternal Diet in Pregnancy
Classic example of CH3-dependent
Epigenetic Modification
 BPA ↓ methylation of agouti gene
 When mothers fed BPA their babies were
yellow & obese
 When moms fed BPA + CH3-rich foods the
offspring were brown & healthy
 Supplementation counteracted exposure
 Demonstrates how environmental exposure in
utero can alter phenotypes in isogenetic pairs
Waterland, Annu. Rev. Nutr. 2014;34:337–55
http://learn.genetics.utah.edu/content/epigenetics/nutrition/

12. Inheritance: direct (epigenetics)
vs. indirect (recapitulation)
 ‘Soft’ inheritance or recapitulation operates indirectly,
via re-creation in each generation of the conditions,
which generate certain phenotypic effects in offspring
 Extrinsic process
 For instance, small mothers might generate small
offspring through:
 ↓ uterine size in each generation
 Behaviours (e.g., smoking or food preference)
 Factors that have familial component

13. Examples
Trangenerational Inheritance
 Genetically driven
 Intrinsic process
Recapitulation of Phenotype
 Environmentally driven
 Early acquisition of language
 Extrinsic process
R. Waterland (personal communication); Waterland, Annu. Rev. Nutr. 2014;34:337–55 ; M Desai et al. International Journal of
Obesity (2015) 633 – 641; P. D. Gluckman et al., (2010). Journal of Developmental Origins of Health and Disease, 1, pp 618

14. Bariatric BPD surgery ↓ F1 Obesity
 N= 49 moms who lost 36% body weight
sustained for 12yr & n=111 children (54
BMS and 57 AMS) aged 2.5–26
 AMS children:
↓ birth weight
↓ macrosomia
↔ LBW
3x ↓ severe obesity at f/u
Extrinsic process
 Epigenetic alteration in somatic tissues
with required repeat exposures each
generation
 Altered maternal phenotype via nutrient
restriction
Smith, 2009. J Clin Endocrinol Metab, 94(11):4275–4283

15. Human evidence of transmission:
Famine & Overnutrition
 In the Dutch Famine (1944–1945) cohort, 60 yo
adults prenatal exposure to famine showed hypo-
Me of whole blood IGF2 gene
 Hyper-Me of 2 obesity-related non-imprinted genes
(IL-10, leptin) vs. unexposed, same-sex siblings
 Hypo-He at IGF2 DMR associated
with paternal obesity
 reprogramming of imprint marks during
spermatogenesis

16. Human evidence of transmission: GWG
 (CpG) Dinucleotide site Me in newborn cord blood DNA from 88
participants Avon Longitudinal Study of Parents and Children
 >GWG in T1 (0-18 wks) associated with ↑ DNA-Me in 4 CpG
sites at MMP7, KCNK4, TRPM5 and NFKB1 genes
 Newborns of mom with excess GWG ↑ DNA-Me at MMP7 CpG
site vs. IOM-recommended GWG

17. A clean slate?
 Following fertilisation, global DNA methyl tags are erased
 By blastocyst stage (implantation), the genome is hypomethylated
 After blastocyst hatching, DNA methylation levels are re-established in a lineage
specific manner
 trophectoderm-derived cells remain hypo-Me vs. inner-cell mass-derived cells
 Placenta is likely to show the > evidence of environmental ‘footprint’ or
‘memory’ of environmental insult during pregnancy
 tissue most exposed to environmental factors during pregnancy
Placenta 33 (2012) 959-970

18. Persistent epigenetic marks
 -CH3 marks not completely erased in early
development & gametogenesis
 -CH3 sites preserved, replicated & DNA +
histones passed along during cell division
 Effects stem cell fate & gene expression throughout life
Fan S et al. Biochem Biophys Res Commun 2009; 383: 421–425;
Flanagan JM et al. Am J Hum Genet 2006; 79: 67–84;Trasler JM. Mol
Cell Endocrinol 2009; 306: 33–36; Desai et al. IJO (2015) 633 – 641

19. Animal evidence of inheritance
Glucose intolerance & Obesity
Jimenez-Chillaron et al. Diabetes 58:460–468, 2009.
 C & UN F1 females were mated at age 2 months with nonsibling F1-C or F1-UN males to
generate 4 experimental groups
 Adverse neonatal exposure (UN) leads to F1, F2 & likely F3 obesity & IGT despite ad
libitum feeding during second pregnancy
 Different aspects of these phenotypes transmitted via maternal lineage (obesity), the
paternal lineage (LBW), or both (glu intolerance)
6mo males 4mo males

20. Animal evidence of inheritance
Glucose intolerance & Obesity
Jimenez-Chillaron et al. Diabetes 58:460–468, 2009.
 C & UN F1 females were mated at age 2 months with nonsibling F1-C or F1-UN males to generate 4
experimental groups
 Adverse neonatal exposure (UN) leads to F1, F2 & likely F3 obesity & IGT despite ad libitum feeding
during second pregnancy
 Different aspects of these phenotypes transmitted via maternal lineage (obesity), the paternal lineage
(LBW), or both (glu intolerance)
6mo males 4mo males
IGT in F1 & F2 generations is linked to impaired
beta-cell function partly explained by
dysregulation of Sur1 expression

21. Germline transmission
 Suboptimal diet (UN) during fetal
development altered germ-cell DNA
methylome of male offspring when
nourished normally from weaning
 Prenatal UN compromises male germline
epigenetic reprogramming & permanently
alters sperm DNA-Me in adult offspring
 DNA-Me in late-gestation somatic tissues of
subsequent generation was not observed
 Altered gene expression in F2
 Gamete methylation may be ‘memory’ early
in utero & developmental exposures
Radford, E. J. et al. Science 345, 1255903 (2014);
Ozanne, S. E. Nat. Rev. Endocrinol. 11, 67–68 (2015)

22. Human evidence of inheritance
Dutch hunger winter
 Offspring born during the famine were smaller than average and
risk of having smaller babies persisted 2 generations (F1 & F2)
Emanuel I, Filakti H, Alberman E, Evans SJ. Intergenerational studies of human
birthweight from the 1958 birth cohort. 1. Evidence for a multigenerational effect.
Br J Obstet Gynaecol 1992; 99: 67–74; Desai et al. IJO (2015) 633-41.

23. Human evidence of inheritance
Dutch hunger winter
 Offspring born during the famine were smaller than average and
risk of having smaller babies persisted 2 generations (F1 & F2)
Emanuel I, Filakti H, Alberman E, Evans SJ. Intergenerational studies of human
birthweight from the 1958 birth cohort. 1. Evidence for a multigenerational effect.
Br J Obstet Gynaecol 1992; 99: 67–74; Desai et al. IJO (2015) 633-41.
 Programmed obesity via alterations in
DNA methylation
 Histone modifications & changes in chromatin
structure not demonstrated in humans

24. Conflicting evidence
 Maternal diet during the F0
pregnancy affected the F2 BW,
independent of the F1 BW
Lumey LH. Paediatr Perinat Epidemiol 1992; 6: 240–253;
Stein & Lumey. Hum Biol 2000; 72: 641–654; Desai et al. IJO (2015) 633-641.
 BW of women with T1 exposure ↑154 g & BW
of women T3 exp ↓ 251 g vs. BW of unexposed
 Maternal prenatal famine exposure does not
affect the association between maternal and
offspring BW
VS.

25. Conflicts cont’d
Maternal Effect
Persists into F2?
 F1 women exposed to famine
as fetuses had F2 babies with
↑ neonatal adiposity & poor
adult health
No Maternal, but
Paternal effect?
 No transgenerational effects
if the grandmother had been
UN
 ↑ adiposity in offspring of
prenatally UN fathers
Painter et al. BJOG 2008; 115: 1243–1249;
Veenendaal et al. BJOG 2013; 120: 548–553; Desai et al. IJO (2015) 633–41.

26. Conflicting results
 Different data collection methods
 Early studies based on record
retrieval and relied on parents'
recall of their offspring's size at
birth and later health status
 Phone surveys
 Questionnaires
 Interviews
 Newer studies, the offspring
were directly contacted to assess
their body composition & health
Desai et al. IJO (2015) 633–641.

27. Human evidence lacking
 “True transgenerational transmission [in humans] should be
demonstrable by effects [+/-] induced in F0 persisting to the
F3 generation, but such long-term studies are expensive and
not frequently performed”
Sir Peter Gluckman
Gluckman, PD et al. (2009). Nat. Rev. Endocrinol. 5, 401-408.
Gluckman, PD et al. (2010). J DOHD, 1, p 6-18.
PA? PA? PA? PA?

28. In utero milieu, fetal plasticity & chronic
disease risk
Gluckman et al. NEJM 2008 Jul 3;359(1):61-73.
M Desai et al. International Journal of Obesity (2015) 633 – 641
Role of nutrition

29. In utero milieu, fetal plasticity & chronic
disease risk
Gluckman et al. NEJM 2008 Jul 3;359(1):61-73.
M Desai et al. International Journal of Obesity (2015) 633 – 641
transmission
recapitulation
Maternal nutrition affects F1 gene
methylation
a. Immune response
b. Adipogenesis
c. Lipogenesis
= metabolic abnormalities

30. In utero milieu, fetal plasticity & chronic
disease risk
Gluckman et al. NEJM 2008 Jul 3;359(1):61-73.
M Desai et al. International Journal of Obesity (2015) 633 – 641
transmission
recapitulation

31. Predictive adaptive response
 Human example
 Thickened heel pads on the feet of infants at birth
 prediction is reliable & assimilated into genomic determinants
 No evolutionary explanation for these which does not
involve an anticipatory component
 Classic example of a developmental process fixed in the
genome, yet it is a predictive response
 Exact mechanisms remains to be establish
P. D. Gluckman, M. A. Hanson and T. Buklijas (2010). A conceptual framework for
the developmental origins of health and disease. Journal of Developmental
Origins of Health and Disease, 1, pp 618

32. Moving forward…
 Quantify the influence of genotype, ENV, & intxn with human
epigenome
 Most compare DNA methylation to phenotype independently of genotype
 Genotype is an essential factor in these relationships
 Assess the degree to which env influences are moderated by
genotype
Teh, 2014 Genome Research 24:1064–1074

33. Acute exercise remodels
promoter–Me in human muscle
 Effects of chronic
exercise?
 Muscle
 Germ cells
 Behavioural-induced
changes persistent
across generations?
 Effect of postnatal
intervention on
offspring ‘exposed’ to
suboptimal env?
Barres et al., 2012. Cell Metabolism 15, 405–411.

34. Conclusions
 Phenotype transmission demonstrated
 Animals (DNA-Me & Histone Mods)
 Humans (DNA-Me + familial components)
 How does under- & over-nutrition produce similar phenotypes?
 Predictive adaptive response?
 How does an acute insult persist across generations?
 Need to establish causal relationships between loci-specific epigenetic
marks in response to adverse ENV & metabolic adult disease phenotypes
 Are epigenetic marks ‘erasable’ with intervention?
 Can the beneficial effects be inherited?
 Similar or different mechanisms?
 Developmental origins of health and disease

35. Thank you.
Acknowledgements:
 CIHR Allied care provider Fellowship, Human Development, Child & Youth Health
 Dr. Erin Keely – The Ottawa Hospital Endocrinology/Metabolism
 Dr. Kristi Adamo & Lab group – CHEO
 Dr. Laura Gaudet – The Ottawa Hospital MFM
 Dr. Mark Walker – The Ottawa Hospital MFM
 Dr. Karen Fung Kee Fung – The Ottawa Hospital MFM
 Dr. Felipe Moretti – The Ottawa Hospital MFM
 The late Dr. Andree Gruslin – The Ottawa Hospital MFM/OHRI
 Adamo Lab Staff & Students – CHEO
 CON & CON-SNP staff, students, & volunteers
 Everyone in attendance
For more discussion follow me on twitter: @DrFerraro