unit presentation dec 08 short
TRANSCRIPT
Epigenetics, Diet and Disease
Giles Elliott
Epigenetics
Disease
Diet
DNA Methylation
Colon Cancer
Overview
What is DNA Methylation?
CC C C C CAA A A AAA AGGGG G G G GT T T T TT
CC C C CC C CAA A A AA G G G G GGT T T T T TT T
Dnmt3
CC C C CC C CAA A A AA G G G G GGT T T T T TT T
Dnmt1
Covalent DNA modificationRetained through mitosis
CC C C C CAA A A AAA AGGGG G G G GT T T T TT
What does DNA methylation do?
Global Methylation
Regional Methylation
Low denisityGenomic stabilityRepression of transposable elements
High Density – CpG Islands (CGIs)Located in promoters and intronsGene Silencing
Promoter Gene
DNA methylation in relation to disease - cancer
Tumour samples exhibited reduced methylation compared to corresponding normal tissue- Feinberg and Vogelstein, 1983
Due to reduction in global methylation - hypomethylation
Increased genomic instability – increased mutation rate
Reactivation of transposable elements – chromosomal rearrangements
Loss of imprinting
DNA methylation in relation to disease - cancer
Hypermethylation of CpG IslandsSilencing of tumour suppressor
genesAffects genes involved in: cell cycle, DNA repair, cell-cell interactions, angiogenisis, proliferation, apoptosis...
Present in both hereditary and sporadic types of cancer
Occurs at all stages of cancer development
Interacts with genetic lesions – 2 hit
Underlying causes inducing hypermethylation unknown
Age related hypermethylation
DNA methylation in relation to disease – Colorectal cancer
Healthy Mucosa AdenomaLifetime risk 50%
CarcinomaLifetime risk 5%
APC K-ras p53Somatic mutation…..
MLH1 p16
Epigenetic deregulation…ESR-1
Tumour Biology
Diet-Related “Field Effects” ?
Dietary and microbiological factorsact on the mucosa fordecades before theemergence of tumours.
Evidence for the Field Effect
Evidence for the Field Effect
canceradenomaNo-neoplasia
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Perc
ent M
eth
yla
tion
APC tissue mean methylation
Healthy Mucosa Adenoma Carcinoma
Diet-Related “Field Effects” ?
Evidence for the Field Effect
CancerAdenomaNo-neoplasia
25
20
15
10
5
Perc
ent M
eth
yla
tion
ESR1 tissue mean methylation
Healthy Mucosa Adenoma Carcinoma
Diet-Related “Field Effects” ?
Evidence for the Field Effect
Multinomial models, based on CGI methylation profiles from normal mucosa correctly identified:
78.9% of cancer patients 87.9% of non- cancer patients informative variables: APC, HPP1, p16, SFRP4, WIF1
and ESR1
CGI methylation of SFRP4, SFRP5 and WIF1 was used to correctly identify:
61.5% of adanoma patients 78.9% of neoplasia-free subjects
This indicates that apparently normal mucosa of patients with neoplasia has undergone significant epigenetic modification associated with the onset of carcinogenesis. Methylation of the genes selected by the models may play a role in the earliest stages of the development of colorectal neoplasia.
What influences the field effect?
Diet?
Age related hypermethylation
Inter-individual variation possibly
due to diet?
Evidence for the involvement of diet
Older twins exhibited more differences in epigenetic status than young twins
Due to environmental not genetic factors
Effect of individual dietary components
Deficiency in methyl donors (folate, choline, vit B12) can induce global
hypomethylation
Treatment of cells with polyphenols can reduce hypermethylatiion by
inhibition of dnmt1
Majority of studies performed in cancer cell lines or animal models
What is going on in humans?
Diet and colon cancer in man
CRC predominantly a western diseaseImpact of western diet highlighted by increased CRC incidence in JapanRisk of CRC increased by obesityInflammatory bowel disease increases risk of CRC
Increased age related hypermethylation in UC
Hypothesis:Inflammatory signals due to dietary (or microbial) factors lead to aberrant DNA methylation resulting in a predisposition to CRC
Aim: To use faecal DNA methylation as a surrogate marker of colonic methylation
Quantify faecal methylation levels
Determine if faecal and tissue methylation correlate?
Are there significant differences in faecal methylation between patient groups?Predicting disease status
Could surrogate markers be used in dietary intervention studies?
Previous work
Summary of early studiesHypermethylated CGIs detected more frequently in cancer than non cancer patients faecal sample
Indicated that a panel of genes may be necessary
Majority of studies performed using non- or semi-quantitative assays
little attempt to correlate to tissue
Study design
Matching tissue and faecal samples from:19 Cancer patients18 Adenoma patients20 Patients with no-neoplasia
Faecal samples from 169 health individuals~10 males and 10 females/decade until 80 years old
Measured methylation of 8 CGIs
Analysis of faecal methylation levels in patients
0
10
20
30
40
50
60
70
80
90
APC Ecad HPP1 ESR1 MLH1 p14 SFRP2 Vim MSP Vim QMSP
% M
e
Normal
Adenoma
cancer
**
***
**
* *
*
CancerAdenomaNo-neoplasia
25
20
15
10
5
Perc
ent M
eth
yla
tion
ESR1 tissue mean methylation
* p ≤ 0.05, ** p ≤ 0.01, ***p ≤ 0.001
Predicting disease status
Multinomial modelling
Age + ESR1Overall = 77%Sensitivity = 63% , Specificity =
84% ESR1 and p14
Overall = 71% Sensitivity = 42%, Specificity =
86%
Correlation between tissue and faecal samples
252015105
25
20
15
10
5
Tissue methylation
Sto
ol m
eth
yla
tion
ESR1 methylation stool vs tissue
ESR1 was the only CGI to correlate
Summary
Some differences in faecal methylation seen between patient groups
Unable to significantly predict disease status from faecal methylation profiles
Faecal methylation profiles do not correlate with corresponding tissue samples
Study limitations
Number of patientsAge difference between patient groupsUse of patients with no-neoplasia as controlsComparison to age and sex matched
volunteers to patient groups
Age and Sex matched Patients vs. Volunteers
No-Neoplasia Adenoma cancer0
5
10
15
20
25
30
35Patient group
Volunteers
Per
cen
t M
eth
ylat
ion
Quantify faecal methylation levels
Determine if faecal and tissue methylation correlate?
Are there significant differences in faecal methylation between patient groups?
Predicting disease statusCould surrogate markers be used in dietary intervention studies?
?
Acknowledgments
IFRNigel BelshawIan JohnsonLiz LundKasia PrzybylskaJack Dainty
Newcastle university
John MathersAmanda CoupeWendy Bal
Wansbeck
Hospital D M Bradburn
TNO NetherlandsRobert Kleeman
FundingFSABBSRC CSGNuGO