Richard Saffery Cancer and Disease Epigenetics Murdoch Childrens Research Institute and

1. Investigating the utility of archival birth blood spots for (epigenetic) epidemiology- an overview Richard Saffery Cancer and Disease Epigenetics Murdoch Childrens Research Institute and University of Melbourne, Aust

2. Outline • Introduction and definitions • Epigenetics mechanisms and DNA methylation • Epigenetics and complex disease • Challenges for epigenetic epidemiology • Birth blood spots – general • Different measures from archived blood spots • DNA methylation measurements from archived blood spots • The future..........

3. Some definitions • Epigenetics: Literally ‘above DNA’ • Factors ‘imposed’ on DNA that alter gene expression without changing underlying DNA sequence • Heritable through cell division – ‘ cellular memory’ • Multiple Types of epigenetic modification • Epigenome: the total epigenetic state of a cell • Unlike the genome (ie DNA sequence), there is no single epigenome of an individual, organ, or even tissue.

4. A role for epigenetics in complex disease?

5. GEE interaction and Complex Disease GEE interaction Environmental Gene dysregulation Epigenetically induced Genetic Genetic variation Environmentally induced mutation Genetically induced epigenetic Disease Risk Stochastic Epigenetic Epigenetic variation Combined environmental and genetic induced epigenetic change Many components to risk!! Environment Environmentally induced epigenetic

6. DNA Methylation - features 5-Methylcytosine • Very stable covalent modification • Can be measured in any DNA sample • Binary variable equivalent to SNP at specific CpG sites • Each cell has two copies of specific sites each of which can have 2 states - methylated or not (ie. 0,0 ; 1,0 ; 1,1 ) • Each gene can have many sites that act in a coordinated way to regulate gene expression • Each cell in a biospecimen has a specific profile that contributes to measurement output of a biological sample • This reflects the % of specific sites within the sample that are methylated

7. Challenges for epigenetic epidemiology

8. Comment: (1st International Epigenetics and Epidemiology Network Meeting, Stockholm 2009) • Existing archived biospecimens are valuable for a LIMITED range of epigenetic profiling • Several caveats exist including; • Limited amounts of available material • Generally poor quality of archived sample • Relevance to target tissue • Potential heterogeneity in archived specimen • Limited capacity to validate any data obtained • New studies should included appropriate biospecimen collection protocols to “future-proof” research capacity

9. Recurring themes in epigenetic association studies (Cambridge, 2011) FACT 1: Epigenetic profile changes over time, particularly in early development Problem: cause and effect cannot be inferred as per genetic epidemiology Solution: prospective collection of samples in all cohort studies, preferably from birth FACT 2: Epigenetic profile varies between different cell and tissue types Problem: relevance of ‘accessible’ tissues to disease of interest Solution: collect as many tissues as possible; measure correlation of epigenetic marks between tissues

10. FACT 3: Many incredibly rich cohort studies commenced years before epigenetics was considered of relevance to complex disease etiology Problem: Most did not collect ‘pre disease’ biospecimens hindering the examination of the role of epigenetics in the causal pathway to disease Solution: examine the utility of coincident biospecimens collected at birth for primarily non-research purposes

11. Ethical - not given for the purposes of research (consent for use???) 2. Sample quality – generally not stored as per current recommendations for international biospecimens 3. Sample quantity – only limited amount (particularly with blood spots) Archived clinical specimens: issues for consideration

12. Usually 4x 1cm spots maximum Proteins Vitamins, micronutrients, other nutrients Steroids – vitamin D RNA – nc / miRNA DNA – genotyping and epigenetics ISSUES OF STABILITY ARE KEY AND REQ TESTING Potential utility of neonatal archived blood spots

13. Genome-wide genetic analysis from neonatal blood spots • `

15. Measuring vitD in neonatal blood spots

17. DNA methylation analysis from neonatal blood spots??

18. Considerations for blood spot DNA methylation analysis and data interpretation • Age of spots correlates with level of DNA degradation • Storage environment (including temperature) are important • Method of DNA extraction is critical (no second chance!!) • Blood composition is variable and may associate with pregnancy associated factors (maternal/gestational age) – currently no way to check this

20. Study: Investigating the utility of archived blood spots as a source of DNA for epigenetic analysis AIM: To determine whether archived blood spots are compatible with locus specific DNA methylation analysis AVAILABLE SAMPLES: Statewide collection (Victoria) of archived blood spots from 1960s stored at room temperature in the dark (> 1 million samples) - Single 3mm punches available

21. Comparison of different methods VDR gene Upto 100ng from 3mm punch [Upto 250ng from 1cm spot]

22. Microsatellite (genetic) and bisulphite (epigenetic) amplification

23. What about quantitative analysis? H19_DMR

26. However.......

27. Genome-scale analysis from blood spots? - factors for consideration • 1. DNA yield from 3mm punch ~ 2-100ng • 2. Starting material is finite and irreplaceable • 3. Different technologies require different amounts of starting material • - Single locus (>1ng) • Infinium arrays (>200ng) • MeDIP/MBD seq or arrays (>3ug) • RRBS (1.5ug) / WGBS (>5ug) • Developments are moving at a very rapid pace (>2 fold increase in sensitivity and cost reduction per year)

28. Now and the future • Genome-scale methylation from DSB is reality but requires (i) a large proportion of individual blood spot or (ii) amplification of DNA • Over $1 billion is currently being used to develop epigenomic technologies including those related to the use of minute samples Prediction: within 3-5 years, all archival blood spots will be amenable to genome-wide DNA methylation analysis The real question should be....... are you ready for the data??

29. Acknowledgments Epigenetics Dr Nick Wong Dr David Martino Boris Novakovic Anna Czajko Mandy Parkinson-Bates Minhee Halemba Jane Ng Eric Joo Zac Chatterton Leah Morenos Bioinformatics Dr Alicia Oshlak Ms Lavinia Bell

30. A model for allergy and epigenetics Epigenetic disruption ?? (Prescott, et al 2011)

31. Early life blood epigenome is dynamic Unsupervised clustering of 27,000 DNA methylation values from 14,500 genes

32. Epigenetics and disease • Unequivocal Evidence for; • imprinting disorders (BWS, SRS) • ICF syndrome (DNMT3B mutations) • many different cancers • Evidence emerging for; • Immune related (diabetes, MS, atopy, asthma) • Neurological (bipolar, schiz, MD, eating disorders, alzheimers) • Metabolic (type II diabetes, obesity) • Cardiovascular (fetal programming)