PG16: Epigenetics of Lung Disease Designing Studies of DNA Methylation

Transcription

1 Andrea Baccarelli, MD, PhD, MPH Laboratory of Environmental Epigenetics Harvard School of Public Health PG16: Epigenetics of Lung Disease Designing Studies of DNA Methylation

2 Presentation Outline Intro to Epigenetics Design a Study Challenges & Opportunities Applying Epigenetics to Human Studies

3 Intro to Epigenetics What it is, why we care about it

4 Epigenetics Programming of gene expression that: does not depend on the DNA code (relatively) stable, i.e., replicated through: cell mitosis meiosis, i.e. transgenerational (limited evidence in humans) Characteristics of epigenetic programming Modifiable (can be reprogrammed) Active or poised to be activated: Potentially associated with current health states or predict future events

5 A Symphonic Example DNA Phenotype Epigenetics

6 Epigenetics & Music Use the Same Markings

7 markings in ink (permanent) pencil markings (can be erased) Epigenetics & Music Use the Same Markings

8 Epigenetic markings DNA methylation Methyl marks added to certain DNA bases repress gene transcription Histone modifications A combination of different molecules can attach to the tails of proteins called histones. These alter the activity of the DNA wrapped around them

9 DNA methylation suppresses RNA expression (more accurately: it is usually associated with suppressed RNA) DNA methylation DNA methylation inactive DNA demethylation active or poised to be activated

10 Chromosomal structure Nucleosome fundamental unit of chromatin Histone modifications 147 bp DNA wound 1.75 turns around histones histone octamer: 2 x (H2A, H2B, H3, H4)

11 Histone modifications types and functions Histone modifications Ac acetyl (lysine), Me methyl (lysine), P phosphoryl (Ser or Thr)

12 Why Epigenetics in Biomedical Research? The epigenome is environmentally sensitive May provide records of past exposures (including prenatal and transgenerational) and help to reconstruct risk factor experience The epigenome harbors profiles potentially useful to identify at risk individuals May help to predict future risks of disease Epigenomic investigations might bring further mechanistic understanding Challenges in human studies need to be considered

13 Design an epigenetic study sample collection, tissue specificity, and reverse causation

14 Epigenetic history Each living organism has two histories that determine its biology: an evolutionary history whose duration is in the hundreds of thousands of years, and a developmental history that starts at the time of its conception. Ze'ev Hochberg, 2011

15 Before conception Preconceptional exposures Fetal life In-utero exposures Early life exposures Childhood & adult life Later life exposures Exposure of gametes Epigenome at birth Epigenome in childhood Adult/aging epigenome Epigenome (Parental) Genome (parental) Genome (offspring) Programming of disease risks Disease programming throughout the lifecourse Figure adapted from Fleisch, Wright & Baccarelli, J Mol Endocrinol, 2013

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17 2,000 CASES Recruit from 14 hospitals SIBLINGS 2,000 CONTROLS Recruit from census (letters, phone calls) Selfadministered Questionnaire Blood or Buccal Specimens Tissue Collection Fresh frozen Paraffin blocks Tissue slides CAPI Clinical Data Hospitals Homes CAPI Blood or Buccal Specimens Selfadministered Questionnaire Transportation Team Study Documentation Center Scan Data Verify completeness within 30 days Blood Components: Fresh frozen Whole blood PBMC (white cells) RBC (red cells) Serum Plasma Buffy coat DNA RNA Blood cards within 4 hours Central Laboratory Data Processing Center Cluster server with mirrored database weekly within 4 hours ship weekly in: liquid nitrogen dry ice room temp documents are shipped at the end of the study PRINCIPAL INVESTIGATORS

18 Flowchart for blood collection, EAGLE study PBMC 3 vials 5 millions cells/ml in 1.5 ml vials RBC and granulocytes 3 vials 1 ml each in 1.5 ml vials Storage: : vapor phase of liquid nitrogen Shipping: vapor phase of liquid nitrogen ACD 2x7 ml (real volume) spin Plasma 6 vials 1 ml each in 1.5 ml vials 2 Blood cards Storage: - 20 C Shipping: dry ice Storage: -80 C Shipping: dry ice Buffy coat 1ml 7 ml Tube 3 vials 1.5 ml with 0,2 ml buffy Plasma 3 vials,1 ml each in 1.5 ml 125 l in criovials one microtube DNA extractio n 50 g each 9 vials 1.5 ml with 1 g each and 2 vials with the rest (3 in Italy and in USA) EDTA 1x7 ml (real volume) 1x3ml (real vol) RBC and granulocytes 3vials, 1 ml each in 1.5 ml criovials Storage: -80 C Shipping: dry ice 3 ml Tube 3 vials, whole blood,1 ml each in 1.5 ml criovials Storage: - 80 C Shipping: dry ice PreAnalytix Tube 2x2,5 ml (real volume) 1 preanalitix tube Storage: -80 Shipping: dry ice 6 vials 1.5 ml with 3 g RNA each vials Storage: vapor phase of liquid nitrogen Shipping: vapor phase of liquid nitrogen Serum 1x7 ml (real volume) 3 vials 1.5 ml, with 1 ml each Storage: vapor phase of liquid nitrogen Shipping: vapor phase of liquid nitrogen

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20 Six samples from each of three placental areas (A, B, C) left at room temperature for 0 to 24 hours before 80 C freezing Effect of time to storage on DNA methylation Villahur N, Epigenomics 2013

21 Time to storage and DNA methylation Repeated elements: CV=2.6% LUMA: CV=9.3% Villahur N, Epigenomics 2013

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23 Cross sectional correlation between Body Mass Index (BMI) and DNA methylation Manhattan plot showing the distribution of p values of the association of methylation probes with body mass index in the discovery cohort The red dots indicate probes that fall within KLF13 (chromosome 15), CLUH (chromosome 17), and HIF3A (chromosome 19). Data from the Dick et al., Lancet 2014

24 The ideal world Epigenetic Inheritance Systems Stochastic Events Germ line Genetic Variation Epigenome Intermediate Phenotypes / Biomarkers Disease Environment Relton and Davey Smith Int J Epidemiol 2012

25 The real world Epigenetic Inheritance Systems Stochastic Events Germ line Genetic Variation Epigenome Intermediate Phenotypes / Biomarkers Disease Environment Relton and Davey Smith Int J Epidemiol 2012

26 The real world? Epigenetic Inheritance Systems?? Stochastic Events Germ line Genetic Variation Epigenome Intermediate Phenotypes / Biomarkers Disease Environment Relton and Davey Smith Int J Epidemiol 2012

27 Reverse Causation In reverse causation: Cause and effect are reversed BMI Study: methyla on BMI BMI methyla on Either is equally probable Study design Cross sectional and case control studies are susceptible to reverse causation Longitudinal studies should be preferred in epigenetic epidemiology Two step mendelian randomization can provide an analytical approach to test for causality (Relton and Davey Smith Int J Epidemiol 2012)

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29 Tissue specificity Epigenetic markings are Tissue Specific. Potentially each tissue or cell type has a specific methylation profile. Epigenetics contribute to tissue differentiation

30 Blood Counts and Methylation (combined analysis of 5 studies) Alu LINE 1 Beta * P value * Beta * P value * White blood cells, 10 3 cell/mm Neutrophils, % Lymphocytes, % Monocytes, % Eosinophils, % Basophils, % * Adjusted for age, gender and study. Zhu et al., Int J Epidemiology 2012 Need to account for signals from cell type differences Adjust for cell type in multivariate analysis Normalize methylation data for cell types before data analysis

31 DNA methylation arrays as surrogate measures of cell mixture distribution Houseman et al, BMC Bioinformatics 2012

32 Predicting DNA methylation level across human tissues Two datasets: 450K Illumina data (480,000 CpGs, n=14) on PBLs, atrium, and internal mammary artery (IMA). HumanMethylation27 data (27,000 CpGs, n=39) on peripheral blood leukocytes (PBLs) and Epstein Barr Virus (EBV) transformed lymphoblastoid cell lines (LCLs) Between tissue patterns: Relatively high background correlations between tissues Differences between tissues highly consistent and reliably reproducible across multiple individuals Linear regression and Support Vector Machine (SVM) models for each CpG site to predict methylation in target tissues based on surrogates. PBLs Atrium, Raw R 2 =0.83; calibrated R 2 =0.99 PBLs IMA, Raw R 2 =0.81; calibrated R 2 =0.94 LCLs PBLs, Raw R 2 =0.92; calibrated R 2 =0.99 Ma B et al. Nucleic Acid Research 2014 (Epub ahead of print)

33 DNA methylation and asthma related inflammation Nasal brush in 36 Children with asthma (studied twice, n=72) Baccarelli et al, Epigenomics, 2012

34 Piko FEV 1 (obstruction) FeNO (inflammation)

35 DNA Methylation in Nasal Epithelial Cells vs. Exhaled Nitric Oxide and FEV1 Exhaled Nitric Oxide (log scale) p=0.001 FEV-1 (log scale) inos promoter methylation (%5mC) p= IL-6 promoter methylation (%5mC) Baccarelli et al. Epigenomics, 2012

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37 MT-TF & MT-RNR1 Methylation (%) Mitochondrial mtdna methylation in steel workers exposed to metal rich air particles (PM1) P=0.002 Controls (n=20) High-exposed steel workers (n=20) Byun et al, Particle Fib Tox, 2013

38 Other cytosine modifications Relation between 5 hydroxy methyl cytosines and 5 methylcytosine in human blood DNA N=237 R=0.21 p=0.001 Hou et al, in preparation

39 Different mechanisms, same design issues? Interest in other epigenetic modifications is growing mtdna methylation 5 hydroxy methylcytosine, 5 formylcytosine and 5 carboxylcytosine Non CpG DNA methylation Others (including histone modifications, mirnas, etc) Most of the same design considerations will apply Sample collection Time to storage Reverse causation Tissue specificity

40 Challenges & Opportunities Problems or resources?

41 Challenges in epigenetics How many epigenomes in one body? Tissue specificity Most studies in humans are on blood DNA Need to investigate tissues relevant for the exposure disease of interest (challenging in epidemiology) How many epigenomes in one lifetime? The epigenome changes over time Reverse causation is always a potential issue Need for longitudinal studies How many epigenomic markings in one epigenome? DNA methylation, histone modifications, others Which is most informative?

42 Opportunities in epigenetics How many epigenomes in one body? Opportunities for screenings of multiple epigenomes: Multiple tissues Multiple cell types (e.g., blood subpopulations) How many epigenomes in one lifetime? Opportunities for lifecourse epigenetics: The epigenome might record recent or past experiences The epigenome might predict future risk of disece How many epigenomic markings in one epigenome? Integrate multiple epigenomic markings Coordinated and complementary mechanisms

43 Environmental Epigenetics Lab Thanks!