paolo vineis imperial college london and hugef foundation torino epigenetics firenze 20 june 2013
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Paolo VineisImperial College London and HuGeF Foundation Torino
EPIGENETICS
Firenze 20 june 2013
“Inheritance” in images, from Darwin’s “tree of life” to DNA’s iconic
crystallography to the epigenetic dynamics
The iconic power of the double helix is related to its heuristic simplicity, particularly as a mechanism to
explain inheritance.
However, the script needs to be interpreted and receives meaning only from the interplay with the
environment (interpretation of the script).
The emphasis has been originally put on “variation” (inherited mutations, common variants …),
i.e. an error in the script is at the basis of disease
Things become more complex with the discovery of DNA’s “metabolism”, with transposable elements,
repair, methylation, miRNA, histone acetylation, i.e. INTERPLAY WITH THE (internal and external)
ENVIRONMENT
What is Epigenetics?
• Epigenetics is the study of inherited changes in phenotype (appearance) or gene expression caused by mechanisms other than changes in the underlying DNA sequence.
• These changes may remain through cell divisions for the remainder of the cell's life and may also last for multiple generations.
• Changes in gene expression that do not involve alterations in DNA base sequence
Epigenetic Modifications
•DNA Methylation
•Histone Modification (e.g. Acetylation, methylation)
•Non-coding RNAs (e.g. microRNA)
•All Regulate Gene Expression
Epigenetic Modifications•DNA Methylation
–C-5 position of cytosine in CpG dinucleotides (Islands)
- When histones are tagged, or acetylated, chromatin is open and genes are potentially active;
- When histones are not chemically tagged, deacetylated, the chromatin condenses and genes silenced.
Epigenetic Modification: Histone Modifications
Epigenetic Modification: Non-coding RNAs
A new mechanism for gene regulation • RNA which is not used for making proteins (non-coding RNA) can be cleaved and used to inhibit protein-coding RNAs
•siRNAs, microRNAs (~22Nucleotides; fine tune geneExpression)
Epigenetic-Regulated Phenomena
•Cellular Differentiation–Totipotent cells become pluripotent cells of the embyro
which differentiate into specific lineages•X-chromosome Inactivation
–Gene expression on one of the female X-chromosomes is downregulated
–DNA methylation and histone modifications
•Imprinting–Epigenetic marking of a locus on the basis of parental origin–Results in monoallelic gene expression
H19
IGF2*
H19*
IGF2
Paternal Imprinting
Maternal Imprinting
Parental Imprinting
Epigenetics and Cancer
1. DNA methylation •Gene specific hypermethylation (eg RASSF1, MLH1) •Genome-wide hypomethylation (4% down to 2-3% of all cytosines)
2.Histone Modifications •Active vs Inactive histone marks •Polycomb group gene silencing (H3-K27-me3)
•Growing data on the importance of epigenetics in the aetiology and pathogenesis of cancer
Cancer Epigenetics Paradox: Global Loss of DNA methylation in addition to locus-specific gain in methylation are causally linked to human cancer
Many cancer risk factors cause epigenetic modifications
DNA Methylation Influences Cancer Processes
DNA Repair
Hormonal Regulation
Carcinogen Metabolism
Apoptosis
DifferentiationCell Cycle
DNA
Methylation
Epigenetics and The Environment
•Epigenetic changes can be inherited mitotically in somatic cells
•Pre-natal and early post-natal exposures can result in changes in risk of developing disease
–Nutrition–Xenobiotic chemicals–Behavioural Factors–Reproductive Factors, Hormonal Exposures
•Epigenetic alterations may also be inherited transgenerationally (developmental origins of adult-onset disease)
Epigenetics and The Environment
• Prenatal environment • Famine exposure, Folic acid use (Tobi et al HMG 2009,Steegers-Theunissen et al Plos One 2009)
• Adult methylome • Smoking, Diet (Breitling AMHG 2011, Zhang Journal of Nutrition
2010) • Cancer methylome • Alcohol and folate (Christensen et al Plos genetics 2010)• Methylation variability between monozygotic twins increases with age (Fraga et al PNAS 2005)
Mapping sequences with differential DNA methylation between MZ twins.
Fraga M F et al. PNAS 2005;102:10604-10609
1- 3 H
-Met
hyl A
ccep
tanc
e (%
)
100
50
0
Day of Depletion6 69
Jacob et al J. Nutr. 128:1204, 1998
Dietary Folate Deficiency Causes Hypomethylation in Human Lymphocytes
In Utero Nutritional Exposure and Changes in Offspring Phenotype
Odds ratio for the metabolic syndrome according to birth weight among 407 men born in Hertfordshire (adjusted for adult body mass index).
Hales C N , Barker D J P Br Med Bull 2001;60:5-20
Some examples from our research
Smoking and epigenetics
AHRR
1x10-5
1x10-7
Smoking intensity is directly correlated with hypomethylation at AHRR
and 2q37.1.
Methylation β-values are presented for non-smokers (0), former smokers (1)
and increasing intensities in current smokers as 2 (1-3 cigarettes per day), 3 (4-
8), 4 (9-13), 5 (14-18), 6 (19-23), 7 (24-28), 8 (29-33) and 9 (>34). The y-axis
represents methylation β-values with box and whisker plots showing the
median line, 25th-75th percentiles and whiskers showing the 95th percentile
range for each probe.
P=0.007P=0.006
Smoking intensity is directly correlated with hypomethylation at AHRR and 2q37.1. Shenker et al, Methylation and smoking in EPIC-Torino (submitted).
The relevance of the findings or environmental epidemiology is due to the key role played by AHR in th metabolism of dioxins, PAHs and other environmental toxicants.
P=0.004
Rischio cumulativo di cancro del polmone nei non-fumatori e in fumatori che hanno cessato di fumare a diverse età o non hanno cessato.
Vineis P et al. JNCI J Natl Cancer Inst 2004;96:99-106
© Oxford University Press
SES and epigenetics
Social inequalities in health
Gallo et al. PLoS One, 2012
Results: Total mortality among men with the highest education level is reduced by 43% compared to men with the lowest
(HR 0.57, 95% C.I. 0.52–0.61); among women by 29% (HR 0.71, 95% C.I. 0.64–0.78).
The risk reduction was attenuated by 7%in men and 3% in women by the introduction of smoking and
to a lesser extent (2% in men and 3% in women) byintroducing body mass index and additional explanatory
variables (alcohol consumption, leisure physical activity, fruit and vegetable intake) (3% in men and 5% in women).
In the last years, research on SES has expanded with the aim of identifying the biological mechanisms through which
socioeconomic status is embedded and eventually “gets under the skin”
In humans, low socioeconomic status across the lifecourse has been associated with greater diurnal cortisol production,
increased inflammatory activity, higher circulating antibodies for several pathogens (suggesting dampened cell-mediated
immune response), reduction in prefrontal cortical grey matter and greater amygdale reactivity to threat, among others.
Human and animal studies have shown that socioeconomic status influences DNA Methylation and gene expression, in
particular across genome regions regulating the immune function.
Social and financial adversities over the entire lifespan (and more critically in early life) would program a “defensive”
phenotype that, through glucocorticoid receptor resistance, would lead to exaggerated glucocorticoid levels and
uncontrolled inflammatory responses to challenges in adult life, both increasing the risk of developing chronic diseases.
Dominance rank and expression level of pro-inflammatory genes (macaques)
Tung et al. Social environment is associated with gene regulatory variation in the rhesus macaque immune system.Proc Natl Acad Sci U S A. 2012 Apr 24;109(17):6490-5.
Unpublished data from the EPIC-Torino cohort (n=830)(analysis by Silvia Stringhini)
Illumina 450K results under investigation
16 candidate genes have been selected by S.S. as being implicated in psycho-social stress
Results after Bonferroni correction:
-1.5
-1
-0.5
0
0.5
1
Met
hyla
tion
diff
eren
ce b
etw
een
SES
extr
emes
(%
)
NFATC1 NFATC1 is one of the three genes whose expression was more strongly associated with social rank in macaques (more expressed in macaques with low social rank)
NFATC1 gene is involved in the expression of cytokine genes in T-cells, especially in the induction of the IL-2 or IL-4 gene transcription
NFATC1 regulates not only the activation and proliferation but also the differentiation and programmed death of T-lymphocytes as well as lymphoid and non-lymphoid cells
SES and DNA methylation – EPIC Turin
Diet and epigenetics
Dutch Hunger Winter 1944-1945
•Famine in The Netherlands towards the end of WWII-caused by Nazi occupation (blockade) and severe winter; led to severe malnutrition in Dutch population
•Dutch Famine Birth Cohort Study-prospectively studies offspring of mothers who were exposed to the famine
•Offspring shown to be increased risk of diabetes, obesity, CVD
•Offspring were smaller than those born to non-exposed mothers and then the children of these offspring were also, on average, smaller
–Suggestive of transgenerational inheritance-epigenetics
Difference in DNA methylation of CpG dinucleotides in siblings discordant for periconceptional exposure to famine.
Tobi E W et al. Hum. Mol. Genet. 2009;18:4046-4053
•60 individuals pre-natally exposed to famine compared with matched, unexposed siblings
•Investigated several genes involved in metabolism
•Positive difference indicates higher methylation level among exposed individuals
Evidence for the effects of maternal malnutrition on offspring comes from a historical cohort of Dutch individuals whose
mothers were exposed during the wartime famine of 1944–1945. Offspring of women exposed during early pregnancy were
more likely to develop the metabolic syndrome in adulthood compared to offspring of women pregnant before or after the
famine.
Epigenetic analyses in these individuals nearly 60 years later show differential methylation in several genes involved in
growth and metabolic control, which are dependent on sex and time of exposure during gestation. Hypomethylation at the
promoter of IGF2, a maternally imprinted gene implicated in growth and development, has also been observed in those
exposed during the peri-conceptional period relative to unexposed siblings.
•1-carbon metabolism is involved in DNA synthesis and methylation key for epigenetics
•1-C metabolism is highly related to dietary intakes (e.g. folate, Methionine…)
study of the association between 1-C metabolism and epigenetics through dietary exposure
Lung cancer and 1-carbon metabolism in EPIC (Johansson, Vineis, Brennan) – JAMA 2010
Analysis by quartiles
Vitamin B6 1·00 (reference)0·76 (0·57 - 1·00)0·54 (0·40 - 0·73)0·30 (0·32 - 0·59)ptrend
5 = 5x10-7
Methionine 1.00 (reference)0·90 (0·69 - 1·18)0·51 (0·38 - 0·69)0·53 (0·40 - 0·72)ptrend
5 = 2x10-6
<= 8 years > 8 years
Controls Cases OR 95% CI Controls Cases OR 95% CI
CDKN2A/P16 (Tumor Suppressor)
0 6
41 1.00 48 8 1.00
>0 9 28 0.42 (0.08-2.19) 35 15 2.02 (0.71-5.77)
RASSF1A (Tumor Suppressor)
<1.82 5 31 1.00 48 7 1.00
>=1.82 10 38 0.51 (0.11-2.39) 35 16 2.91 (0.98-8.61)
Odds ratios (OR) and 95% confidence intervals (CI) for methylation levels (below/above median in controls) and lung cancer risk by time since blood drawing (Vineis et al, Epigenetics 2010)
Methylation patterns in sentinel genes in peripheral blood cells of heavy smokers: influence of cruciferous vegetables in an
intervention study
Scoccianti C, Vineis P et al, Epigenetics 2011
Intervention trial with 3 different diets in heavy smokers: methylation of repeat sequences, tumour suppressor genes,
MTHFR and other genes.
These data suggest that a healthy diet may stabilize global epigenetic (LINE-1 DNA methylation) patterns in peripheral white
blood cells, but do not provide specific evidence for potential prevention of methylation changes in specific genes.
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
Normal Enriched Supplemented
Dietary Groups
Me
an
% m
eth
yla
tio
n
LINE T0
LINE T4
MLH1 T0
MLH1 T4
RASSF1A T0
RASSF1A T4
CDKN2A T0
CDKN2A T4
MTHFR T0
MTHFR T4
ARF T0
ARF T4
Epigenetics and disease prediction
Flanagan J, Vineis P et al. (Cancer Res, 2012).
Hypermethylation of ATM and other DNA repair genes in breast cancer in four independent studies with pre-
clinical samples
Methylation of ATM and breast cancer. Flanagan J, Vineis P et al. (Cancer Res,2012).
Chemicals and epigenetics
There is much more than genotoxicity - need to explore several steps/segments of carcinogenic process
Simplified models of carcinogenesis (Vineis, Schatzkin, Potter, Carcinogenesis 2010)
Model 1 Model 2 Model 3 Model 4“mutational” “DNA instability” “non-genotoxic” “Darwinian”
Chemical carcinogens Familiarity Clonal expansion/ Clonal expansion/Viruses Genome instability Epigenetics Cell selection
Tobacco and lung Colon cancer Diet, hormones Beta-carotene,HPV Retinoblastoma folate,
chemotherapy
DNA adducts CI, MI, MMR, Rb, Methylation Selective advantagemutations BRCA1, TSG histone acetylationoncogenes
Mathematical models: Armitage-Doll Knudson Moolgavkar Nowak
Thank you
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